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Vol. 2. 1 15-127, February Cell Growth 1991 Simian Virus 40 Large Microtubule-associated Transformed Cells1 T Antigen Proteins Division of Molecular Virology Department of Microbiology [S. A. M., S. K. A., E. T. S., J. S. B.] and and Immunology [R. G. C.], Baylor College of Medicine, Houston, Texas 77030, and Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [G. L. D.] possesses is a prototype potent its oncogenic types (2-4). DNA transforming activity virus oncogene potential, in a wide (1). It as evidenced by of cultured cell variety and in different target organs in transgenic T-ag is a remarkably multifunctional protein, activities ranging cellular Abstract The cellular proteins that interact with simian virus 40 large T antigen (T-ag) must be identified in order to understand T-ag effects on cellular growth control mechanisms. A protein extraction procedure utilizing single-phase concentrations of 1-butanol recovered a complex composed of T-ag, p53, and other Mr 35,000 60,000 proteins from suspension cultures of the simian virus 40-transformed mouse cell line mKSA. Partial protease mapping showed each of the associated proteins to be unique. Automated microsequence analysis of the NH2-terminal 30 amino acids of the M 56,000 protein purified after coprecipitating with T-ag and p53 identified it as the subunit of mouse tubulin. The existence of a complex containing tubulin, T-ag, and p53 was confirmed by reciprocal immunoblotting experiments. Both T-ag and p53 were coprecipitated by three different monoclonal antibodies directed against tubulin, and conversely, monoclonal antibodies specific for T-ag or p53 coprecipitated tubulin. Mixing experiments and extractions in the presence of purified tubulin indicated that the complex existed in situ prior to cell lysis. Both p53 and T-ag copurified with microtubules through two cycles of temperaturedependent disassembly and assembly. Both T-ag and p53 were localized to microtubules in the cytoplasm of mKSA cells by immunoeledron microscopy. Treatment of mKSA cells with 10 iM colchicine followed by lysis in 0.1% Nonidet P-40 resulted in increased amounts of solubilized T-ag and p53. Both T-ag and p53 were also associated with microtubules in three other simian virus 40-transformed mouse cell lines growing as monolayers, confirming the generality of the association. An interaction of T-ag and p53 with microtubules may be important in the intracellular transport of these proteins and may affect cellular signal transduction or growth control. 115 and p53 Are in Introduction SV404 T-ag Steve A. Maxwell,2 Sharla K. Ames, Earl T. Sawai, Glenn L Decker, Richard G. Cook, and Janet S. Butel3 & Differentiation from replication transformation (1, of the viral 5-8). The proteins with the molecular oncogenesis. The intracellular to path- role(s) in cell effects their Identification which T-ag interacts should basis of T-ag function in distribution in its multifunctionality. genome regulatory way(s) subverted by T-ag must play central growth control in view of the dramatic redirection may have on cell phenotype. of the cellular help elucidate mice with of T-ag Although T-ag may be involved is found predom- mnantly (‘95%) in the nucleus of infected and transformed cells, a portion gets transported to the plasma membrane (5, 6). Evidence for functionality of the nonnuclear population has been provided by SV4O variants that encode nuclear transport-defective forms of T-ag. Nonkaryophilic T-ag mutants were able to transform established cell lines but not primary cells (9-11) and cooperated with the ras oncogene and polyoma middle T-ag in transformation of primary cells (12, 13). The elevated expression of pmT-ag in actively dividing cells as compared to quiescent cells vation that transformation inhibited in the presence lymphocytes (16), (14, 15), plus the obser- of primary cells by SV4O is of SV4O-specific cytotoxic T- substantiate a functional role for cy- toplasmic T-ag or pmT-ag or both. One cellular target for T-ag is cellular protein p53, an apparent tumor suppressor gene product (17-21) intimately involved in T-ag-mediated 22, 23). A membrane form transformation of p53 has been the surface of SV4O-transformed cells by surface radioiodination experiments (24-26) sensitive 3H-labeled protein A binding assay plasma mouse membrane cells and in the by reactivity during of mitosis both plasma membrane with antiidiotypic detergent and on situ cell and by a (27), at the in transformed by immunocytochemistry At least two subpopulations guished based upon differences of pmT-ag can be extracted nonionic normal (1, 6, detected (28), of Raji B lymphoma anti-CR2 antibodies cells (29). of pmT-ag can be distinin solubility. One class from membranes by the NP4O, whereas a second form, which is modified by palmitylation, can only be extracted from NP4O-insoluble material with the zwitterionic detergent Received 1 This work HD21483 CA08564 10/18/90. was and from supported by National in part by Research Research Service Grants CA22555 and Awards CA09197 and Empigen BB (25, 30, 31). This latter form is believed to be associated with the plasma membrane lamina, a struc- the NIH. Present address: Department of Thoracic Surgery, M. D. Anderson Hospital Cancer Center, Houston, TX 77030. 3 To whom requests for reprints should be addressed at Division of Molecular Virology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. 2 The abbreviations used are: SV4O, simian virus 40; T-ag, large T antigen; pmT-ag, plasma membrane-associated fraction of T-ag; NP4O, Nonidet P-40; BSA, bovine serum albumin; IEM, immune electron microscopy; 4 SDS, sodium dodecyl sulfate. 1 16 SV4O Tag and p53 Bind Microtubuk’s A 3 2 1 B 4 124 w : Tag , 95k ii - - 6ok 56k 5Ok “45k i:.. p53) “35k Fig. 1 Coprecipitation of M, .35,000-60,000 proteins from butanol extracts of sSlabeled mKSA cells using monoclonal antibodies against T.ag or p53. A, mKSA ((115 metabolically labeled with [‘5S]methionine were lysed in 1% NP4O (Lanes I and 2) or were extracted with 2.5% butanol at 37’C (Lanes 3 and 4). Proteins were immunoprecipitated with p53 monoclonal antibody 200.47 (Lanes 2 and 4) or null antibody m73 (Lanes 1 and 3). B, ‘5S-labeled mKSA ((‘115 were extracted with 2.5% butanol at 37’C, and solubilized proteins were precipitated with monoclonal antibody m73 (Lane 1), T-ag mono(lonal antibody SDS.polya rylamide 45,000, and 35,000 ture that directly PAb43O gels. and proteins. underlies (Lane 2), or p53 labeled bands the lipid monoclonal were antibodies detected bilayer proteins 200.47 autoradiography. of the plasma membrane and connects it to the cytoskeleton These subpopulations of pmT-ag may interact tinct membrane or cytoskeletal ing biological effects. by (30, with and exert 32). dis- differ- (Lane 3) and PAb421 Arrowheads, (Lane 4). positions Precipitated of T.ag, p53, proteins and M, were 60,000, separated in 8% 56,000, 50,000, of proteins, ranging in size from M, 35,000 to M, 60,000, differs from that routinely obtained from NP4O detergent extracts of [35S]methionine-labeled mKSA cells using the same p53 monoclonal antibody (Fig. 1A, Lane 2). Identical proteins were precipitated from the butanol extracts identify other proteins that interact and membrane-associated T-ag and a protein extraction technique utilizing using another p53 monoclonal antibody, PAb421 (Fig. 1B, Lane 4), and a T-ag monoclonal antibody, PAb43O (Fig. 18, Lane 2). Specificity of recognition of the Mr 1-butanol. Single-phase concentrations of butanol facilitated solubilization of a complex of 1-ag with cellular proteins ranging in size from M, 35,000 to Mr 60,000 (31, 33). We identify here the M, 56,000 component of the complex as the fi subunit of tubulin and describe a series tibodies was demonstrated by lack of precipitation of the complex using a null antibody (Fig. 1A, Lane 3; Fig. 1B, Lane 1 ). Similar patterns of the M, 35,000-60,000 com- of experiments bodies In a program with cytoplasmic PS3, we adapted both to that document 1-ag and p53 with a specific association of microtubules in viva and in vitro. Results Coprecipitation of Mr 35,00060,000 Cellular Proteins with Monoclonal Antibodies against 1-ag or p53 from Butanol Extracts of mKSA Cells. We previously reported the coprecipitation of at least five cellular proteins with 1-ag and p53 in monoclonal antibody immunoprecipitates from butanol extracts of mKSA cells (31, 33). These proteins were coprecipitated with monoclonal antibodies against either the NH2 or COOH terminus of 1-ag as well as with several monocbonal antibodies against p53. Typical profiles of proteins precipitated with the p53 monocbonal antibody 200.47 from butanob extracts of mKSA cells metabolically labeled with [‘5Sjmethionine are shown (Fig. in, Lane 4; Fig. 1B, Lane 3). The pattern 35,000-60,000 ponents proteins were observed by the reactive using different monoclonal an- monoclonal anti- against 1-ag or p53 (Fig. 1A, Lane 4; Fig. 1 B, Lanes 2-4) (31). The M, 95,000 protein present in the complexes was identified as 1-ag anti-I-ag serum monoclonal by immunoblotting (33). antibody p53 was assays usually immunoprecipitates not using rabbit apparent from in butanol extracts of 55S-labeled mKSA cells, although it could be observed in immunoprecipitates from butanol extracts of 32P-labeled mKSA cells. Immunoblotting analyses using rabbit anti-p53 serum of total protein extracts of unla- beled mKSA cells showed a significant solubilized by butanol treatment (data amount of p53 not shown). The other M, 45,000-60,000 proteins did not appear to be related to I-ag or to p53, based upon immunoblotting assays (31, 33). Partial protease mapping experiments using V8 protease also indicated no similarity with p53 (Fig. 2; compare Lanes 1 and 2 with Lanes 3-10). Each of the M, 45,00060,000 proteins appeared to be unique, Cell 1 2 3 4 5 6 7 Growth 8 & Differentiation 117 910 6Ok 45k)’ Es , 2 a .i Ip I ‘*‘ Fig. 2. Partial protease mapping of 35S-labeled M, 45,000-60,000 proteins coprecipitating 5, 8, and 9) or 1.0 g (Lanes 2, 3, 6, 7, and 10). Authentic p53 (Lanes 1 and 2), M, 56,000 and 8), and M, 45,000 (Lanes 9 and 10) proteins were digested as described in “Materials SDS-polyacrylamide gel. Molecular weight markers are indicated on the left. as partial structural V8 protease mapping similarity among them revealed (Fig. 2). no apparent Identification of the Mr 56,000 Coprecipitating Protein as the fi Subunit of Tubulin. We initiated attempts to identify the Mr 35,00060,000 proteins coprecipitating with 1-ag and p53 by microsequencing. Monocbonal antibody 200.47 immunoprecipitates from butanol extracts of mKSA cells were disrupted in NP4O-urea buffer (34), and eluted proteins were purified by two-dimensional electrophoresis. Only the Mr 56,000 and 45,000 proteins were efficiently solubilized and resolved under these conditions (Fig. 3A). Purified proteins were electro- phoretically blotted from Immobibon PVDF paper automated microsequence the and two-dimensional were subjected analysis. A blocked gel onto directly to NH2 ter- minus prevented microsequencing of p45. However, 30 amino-terminal residues of p56 were sequenced. A search of GenBank sequences identified strong homology of the p56 sequence with the fi subunit of tubulin isoforms 3 and 4 (Fig. 3B). A Complex Present tification bulin monoclonal the nature Reciprocal of 1-ag, from antibodies p53, anti-p53, butanol extracts and Tubulin Is The iden- from mKSA Cells. and the availability allowed and antitubulin of mKSA of antitu- us to investigate of tubulin interaction with immunobbotting experiments out. Anti-I-ag, itates Composed in Butanol Extracts of p56 as tubulin cells 1-ag and p53. were carried immunoprecipwere separated with T-ag and p53. V8 protease was used at 0.1 pg (Lanes 1, 4, (Lanes 3 and 4), M, 60,000 (Lanes 5 and 6), M, 50,000 (Lanes 7 and Methods,” and partial fragments were resolved on a 15% by gel electrophoresis, and the proteins were analyzed by immunobbotting using rabbit anti-I-ag serum (Fig. 4A) or a monocbonal antibody (N.356) specific for the fi subunit of tubulin (Fig. 48). The I-ag monoclonal antibodies PAb43O and PAb419 precipitated I-ag (Fig. 4A, Lanes 4 and 5), and the p53 monoclonal antibodies 200.47 and PAb421 coprecipitated I-ag due to complex formation with p53 (Fig. 4A, Lanes 6 and 7). Three different monoclonal antibodies against tubulin coprecipitated I-ag (Fig. 4A, Lanes 8-JO). Conversely, monocbonal antibodies against either I-ag or p53 coprecipitated tubulin (Fig. 4B, Lanes 2 and 3). Control tubulin was precipitated by a monoclonal antibody against the a subunit of tubulin (Fig. 4B, Lane 4). Null antibodies did not precipitate 1-ag (Fig. 4A, Lanes 1-3) or tubulin (Fig. 4B, Lane 1). No quantitative comparisons could be made from these precipitation experiments, because the tubulin antibody was not used in excess. However, it was evident that complex composed of I-ag, p53, and tubulin was present in butanol extracts from mKSA cells. Mixing experiments were performed to ascertain whether the complex existed in situ or was formed postlysis in the butanol. If complex formation occurred postlysis, excess unlabeled proteins should compete with the labeled proteins and diminish the amount tubulin and other proteins coprecipitated antibodies. mKSA cells were metabolically [35Sjmethionine. Labeled cells were then of labeled by p53 or I-ag labeled with mixed in vary- a 1 18 SV4O T-ag and p53 A ,- Bind 5.9 Microtubules 6.87.2 V V 9.3 V presence of 1 or 3 tg of tubulin or 1 tg of BSA (Fig. 5). Although a decrease in recoverable complex was apparent from samples prepared in the presence of 10 ag of tubulin, a similar decrease occurred when 10 tg of BSA were present in the extraction buffer (Fig. 5), so this was presumably a nonspecific effect. These results strongly suggest that the protein complex exists prior to cell lysis and does not form during the extraction process by nonspecific adherence to tubulin. V P56 60b. - - 5Ok’ 4-4p45 . 45k Localization IEM. As tubulin pS(. luhulun 3suhunii: M _X_E_1_V_H_tLI_Q_A_G_Q_C_G_N_Q_I_G_.A_K_F 51 -R-E-I-V-tl- 21 i)c(, I uhui,n isuhunit [.1 W L -Q-A-G-Q-C-G-N-Q-I-(-.\--K- :iii -E-V-I-S-D-E-X-G-l -E-V-I-S-D-E-H-G-I fig. 3. Purification and amino acid sequencing of M, 45,000 and 56,000 proteins coprecipitating with T-ag and p53. A, butanol-solubilized pro. teins from mKSA cells were immunoprecipitated with monoclonal antibody 200.47, and Precipitated proteins were eluted with urea-NP4O buffer. Soluble proteins were purified by two-dimensional gel electrophoresis and were blotted onto Immobilon PVDF membranes. Membrane strips containing blotted M, 45,000 and 56,000 proteins were subjected to microsequence analysis. B, NH2-terminal sequence comparison of the l subunit of tubulin and M, 56,000 proteins. X. unidentified brackets, tentative identification. C, one-dimensional gel pattern munoprecipitate from which p56 was purified. residue; of rn- ing ratios with unlabeled mKSA cells, the mixtures were extracted with butanol, and the extracts were immunoprecipitated using excess antibody (PAb43O or 200.47). The amount of labeled complex remained the same when cell lysates were prepared in the presence of equal numbers or a 5-fold excess of unlabeled cells (data not shown). The absence of any competition by unlabeled cell proteins of the recovery of labeled complex by immunoprecipitation strongly suggested that the complex existed prior to cell lysis. In a second experiment, we used partially purified tubulin as a specific competitor for the M, 56,000 component of the complex. The tubulin was prepared by two cycles of repolymerization from suspension human lung carcinoma cells of line H69. Methionine-labeled mKSA cells were extracted in 2.5% butanol containing 0, 1, 3, or 10 tg of tubulin. Duplicate samples were extracted in the presence of 1 or 10 of BSA to control for any nonspecific effects that might be encountered due to the presence of extraneous protein in the extraction mixture. This experiment was designed to determine whether the components in the complex were nonspecifically sticking to tubulin or were associating with tubulin during the extraction procedure. If the complex were generated during extraction, a decrease in the recovery of labeled tubulin in the complex would be expected as more competitor unlabeled tubulin was added. As seen in Fig. 5, no decrease in the recovery of labeled components in the complex was observed in either anti-I-ag or anti-p53 immunoprecipitates when extracts were prepared in the of SV4O 1-ag and p53 to Microtubules by appeared to be complexed with I-ag or p53 or both in extracts of transformed cells, we examined whether those proteins were associated with intact microtubules. EM was performed to visualize the cytoskeletal components to which 1-ag and pS3 might be bound. A number of permeabilization and fixation conditions were evaluated in an attempt to balance acceptable retention of ultrastructural integrity with optimal immunolabeling of I-ag. Under conditions that favored ultrastructural preservation and labeling of tubulin by the YLY2 antibody (35), I-ag did not retain adequate antigenicity. As I-ag is predominantly a nuclear antigen with only a small plasma membrane-associated fraction (6), the subpopulation available for microtubule binding is expected to be of low abundance. Similarly, p53 is found mainly in nuclei and also would not be expected to exhibit an abundant microtubule-associated subpopulation. Therefore, conditions were used that produced optimal I-ag labeling with an observable cytoskeleton. The antitubulin antibody did not react well under these conditions but was included in double-labeling reactions to verify that the observed structures were microtubules. mKSA cells were treated with 0.1% NP4O in a harsh permeabilization procedure to remove all soluble and membrane components of the cytoplasm. Cytoskeletal preparations were then processed for IEM and embedded in Lowicryl as described in “Materials and An irrelevant primary antibody, m73, was used as a control for specificity and failed to bind to any of the microtubule subsets, such as centriolar, mitotic spindle, or cytoplasmic microtubules, in immunolabeling experiments performed on thin sections (Fig. 6A). Double Iabeling of thin sections of mKSA cells with mouse antip53 and rat antitubulin antibodies was detected with 15nm gold-goat anti-mouse and 10-nm gold-goat anti-rat secondary antibody probes. The probes for p53 and tubulin cobocalized to microtubules of the centriolar region (Fig. 6B) as well as to mitotic spindles and unassigned microtubules, such as those in Fig. 6A. Similar experiments were done using anti-I-ag and antitubulin primary antibodies. Cobocalization of both probes to microtubular structures of many types indicated that I-ag was associated with cytoplasmic microtubules (Fig. 6, C and D), as well as with the mitotic spindle. The low levels of label over the microtubules are believed to be significant, considering that I-ag and p53 are present in the cytoplasm in very small amounts and that the negative controls displayed very rare labeling reactions. Little extranuclear binding ofT-ag or tubulin antibodies occurred on thin sections of mKSA cells that, prior to permeabilization, were cultured in the presence of 10 LM colchicine for 3 h followed by incubation on ice for 1 5 mm (data not shown). Cytoskeletons were not readily visible in sections of nonpermeabilized cells. Such sam- Cell A I 2 3 4 5 Growth & Differentiation B 6 7 8 9 I 10 234 p Tag I - . , Fig. 4. Coprecipitation of T-ag, p53, and tubulin from butanol extracts of suspension cultures of mKSA . cells. .::=: ttuIin A, immunoblot analysis using rabbit anti-T- ag serum of proteins in immunoprecipitates from butanol extracts of unlabeled mKSA cells. Antisera used for immunoprecipitation were the following: monoclonal antibodies against rotavirus VP7 (Lanes 1 and 2) or m73 (Lane 3), T-ag monoclonal antibodies PAb43O (Lane 4) and PAb419 (Lane 5), p53 monoclonal antibodies 200.47 (Lane 6) and PAb421 (Lane 7), and monoclonal antibodies against the o subunit oftubulin (Lane 8), the $ subunit of tubulin (Lane 9), or one cross-reactive with both subunits (YLY2; Lane 10). Note the coprecipitation of T-ag by pS3 and tubulin antibodies. Bands corresponding to mouse lgG chains from the immunoprecipitates are evident in some lanes. B, proteins precipitated with m73 (Lane 1), PAb43O (Lane 2), 200.47 (Lane 3), and anti-a tubulin subunit (Lane 4) monoclonal antibodies were immunoblotted using monoclonal antibody N.356 against the subunit of tubulin. Note the coprecipitation of tubulin by T-ag and p53 antibodies. The positions of 1-ag (M, 95,000) and tubulin (M, 56,000) are indicated. pIes, however, did show I-ag label in the nucleus (data not shown). Thus, IEM provided ultrastructural evidence that both 1-ag and p53 are associated with a variety of microtubules in mKSA cells. Temperature-dependent Cycle Purification of Tubulin and Associated Proteins. Temperature-dependent cycle purification bule-associated to quantitate of tubulin is a means of identifying microtuproteins. This approach was performed the interaction of 1-ag and p53 with tubulin and to further rule out artifactual be induced by butanol. Two associations that might cycles of temperature- dependent polymerization and depolymerization were carried out as described in “Materials and Methods.” Approximately 45% of tubulin polymerized into microtubules in the first cycle of temperature-dependent purification (Fig. 7A; compare Lanes 1 and 2; see quantitation data in Table 1, step 1). From the microtubules that polymerized in the first cycle, only a portion depolymerized during incubation on ice, as evidenced by the large amount of tubulin (‘-60%) that remained in the cold-stable fraction (pellet) after the depolymerization step (Fig. 7A, Lane 3; Table 1, step 2). Approximately 30% of the depolymerized tubulin repolymerized into microtubules 1, step 3). in the second cycle (Fig. 7A, Lane 5; Table ulin Both I-ag and pS3 that polymerized and second cycles 1). The stoichiometry label in the polymerized remained associated with the tubinto microtubules during the first (Fig. 7, B and C, Lanes 2 and 5; Table of I-ag tubules label (pellet relative to tubulin fractions) through the two cycles of purification varied by less than 2-fold (2.3 versus 4.4; Table 1), whereas the ratio of tubulin:p53 labels did not change (14.3 versus 13.8). This relatively constant stoichiometry of association through two rounds of polymerization suggests a specific interaction of I-ag and p53 with microtubules. It is striking that about 80% of I-ag and p53 was associated with polymerized microtubules after the first cycle, although only 45% of the tubulin polymerized. Similar to tubulin, only a fraction of I-ag and p53 was released from the microtubule pellet upon incubation in the cold (Fig. 7, B and C, Lanes 3; Table 1). Samples were taken prior cycle in vitro repolymerization to and following reaction and med by EM in an effort to visualize dried onto Parlodion-coated nickel 1-ag. grids, were immunolabeled for I-ag and tubulin negatively stained for electron microscopy. taken 3 mm after initiation of repolymerization re-formed microtubules that immunolabeled the secondwere exam- Samples were and the grids and were Samples contained for I-ag 119 120 SV4O T-ag and p53 Bind Microtubules Antibody M73 tgTubulin(BSA) Lane PAb43O 0 1 3 1O Q 1 1 2 3 4 5 3 7 6 200.47 10 (1)(1Q)H 1 3 1O 8 9 10 11121314 *, 97 - 80 - 55 43 . - - - $Sa - - 36- . Fig. S. La k of competition by unlabeled purified tubulin during hutanol extractions. Spinner niKSA cells were metabolically labeled with 1 mCi of [355] methionine or 1 .5 h at 37’C. Aliquots of 3 X 106 labeled cells were pelleted and resuspended in 2.5% butanol or 2.5% butanol plus various concentrations of unlabeled competitor protein. Cells were extracted as described (31). Extracts were immunoprecipitated with monoclonal antibodies, and the imrnunopre(ipitates were washed with buffer [150 mxi NaCI, 1% NP4O, 0.5% sodium deoxycholate, 0.1% SDS, 50 rnsi Tris (pH 8.0)], were disrupted, and were analyzed by SDS-polyacrylarnide gel electrophoresis. Extractions made with 2.5% butanol in the absence of any competitor protein are shown in Lanes 1, 5, and 1 1. Extractions were made with 2.5% butanol in the presence of 1 ig of unlabeled tubulin (Lanes 2, 6, and 12), 3 ig of unlabeled tubulin (Lanes 3, 7, and 1 3), or 10 g of unlabeled tubulin (Lanec 4, 8, and 14). Butanol extractions were made in the presence of 1 and 10 g, respectively, of BSA, a nonspecific competitor protein (Lanes 9 and 10). Extracts were immunoprecipitated with control monoclonal antibody m73 (Lanes 1-4), antiSV4O T-ag rnonodonal antibody PAb43O (Lanes 5-10), or anti-p53 monoclonal antibody 200.47 (Lanes 11-14). Molecular weight markers are shown on the left. (Fig. 8A), as well yet repolymerized. as some tubulin (see inset) that had not Control antibody m73 failed to label the tubular structures (Fig. 8B). Samples taken prior to repolymerization showed cobocalization of I-ag and tubulin probes to small globular structures, presumably the tubulin heterodimer complexes (Fig. 8B, inset). Therefore, ultrastructural studies confirmed that I-ag copurified with tubulin through two cycles of purification and revealed that I-ag was associated with both the depolymerized form of tubulin and intact microtubules. Effect of Colchicine on Extraction of 1-ag and p53. Chemicals such as colchicine, which affect the formation of microtubules in vivo, would he predicted to alter the solubility of cytoplasmic I-ag and p53. Thus, an increase in the amount of soluble I-ag and p53 might be observed in colchicine-treated cells, as compared to control cells, due to the disruption of microtubules. Cultures of mKSA cells were incubated in 10 iM colchicine for 3 h at 37#{176}C. Colchicine-treated and untreated mKSA cells then were incubated at 37#{176}Cin 0.1% NP4O in microtubule stabilization buffer for 15 mm, conditions that would leave nuclei and cytoskeletal structures intact and would solu- bilize predominantly membrane and cytosolic compo- nents. Total soluble proteins in the extracts were precipitated with trichloroacetic acid and analyzed by immunobbotting. More tubulin, I-ag, and p53 were extracted with 0.1% NP4O from colchicine-treated mKSA cells than from untreated cells (Table 2). The biggest change was observed with p53 (2.4-fold more p53 was recovered after colchicine treatment). Colchicine-treated cells observed by IEM using double immunogold labeling showed a loss of microtubular structures and of 1-ag and tubulin labels (data not shown). T-ag Tubulin and p53 Are Associated with Microtubules and in Other SV4O-transformed Cell Lines. It was important to establish p53 with microtubules mouse fibroblast cell that the association of I-ag and was not limited to mKSA cells. A line (WIB1a) and a mouse mam- Cell fig. 6. Localization of SV4O 1ag and p53 to mKSA cytoskeletons by EM using double immunogold labeling. Cytoskeletons prepared from mKSA cells by extraction in 0.1% NP4O were processed for electron microscopy and were embedded in Lowicryl, as detailed in “Mate- ,: .-,...,I,’ Growth & Differentiation ,, fr . : rials and Methods.” Thin sections were immunolabeled. Fifteennrn gold beads linked to an antimouse secondary antibody mark the presence of anti-T-ag or antipS3 beads tubulin antibodies; denote the antibodies. 10-nm gold presence of A, control Ia- beling experiments using the m73 antibody. Microtubules were not labeled. Bar, 0.2 gm. B, double labeling with anti-p53 (PAb421, arrowhead) and antitubulin (YL’/2, arrow) antibodies demonstrated p53 associated with microtubules. Bar, 0.1 zm. C and 0, anti-T-ag )PAb4O5, arrowhead) and antitubulin (YLY2, arrow) antibodies localized 1-ag to microtubules. Bars, 0.1 zm. Cytoplasmic (A), centriolar (B and C), and rnitotic spindle (0) microtubules are shown. ‘1 B A 123 12345 C 12345 4_5 I’b’Pp. tubulifl’1 r T1 .5. Fig. 7. Analysis of temperature-dependent cycle-purified microtubules for the presence of 1-ag and p53. Total protein in the supernatant (Lanes 1 ( and pellet (Lanes 2) from the high-speed centrifugation of the first-cycle polyrnerized microtubules, the cold-stable pellet (Lanes 3), and the supernatant (Lanes 4) and pellet (Lanes 5) from the second polymerization cycle were irnmunohlotted using monoclonal antibody YLY2 against tubulin (A), rabbit anti1-ag serum (B), or rabbit anti-p53 serum (C). The positions oftuhulin )M, 56.000). intact 1-ag )M, 95.000), and p53 are indicated. Some degradation of Tag is evident in B. 121 122 SV4O T-sg Table 1 and p5 Bind Association Mi r(itul)ules 1)1 tuhulin, T-ag, and 3 through two cycles of temperature-dependent (I(’polymerizatiOn and polymerization of mk rotubules About 6 X 10#{176} niKSA ( (‘(IS Wi.’Ii.’ Soni(,lt(’(l in nii rotubule stabilization buffer and were incubated on ice. Clarified supernatant plus GTP (unassembled tubulin( was in(uhated at .37#{176}C or 30 mm. Polyrnerized microtul)lIk’s were l)elleted (step 1). The pellet from step 1 was sonicated and incubated on ice, followed 1)5 C (‘ntntugatR)n at .50,000 X g for 3() nlin. The pelleted ( old-stable microtubules (step 2) represent the population that failed to dissociate when the l)ell(’t obtained miii the first cy Ic of polymerization was in uhated on ice. Clarified supernatant from step 2 was incubated at 37’C for 30 mm, and repolynlerize(l mi( rotubules were f)(’llet(’(l (step 3). For analysis of total proteins by immunoblotting, pellets were solubilized in SDS disruption I)utter; supernatant tra( finns were’ first pr(’( il)itate(l with trichloroa (‘ti( a( id and then dissolved in SDS disruption buffer. Purifi(ati(in Step 1 : First- ycle polynierii,i- tii)ii Step Fr 1( tiiin analyze(l of nli(rotuhuk’s 2: Cold-stable riii r(itu- (iiantitation Tubulin Ratios )cpni)” T-ag” p53 Sux’rnatant P(’ll(’t Pellet 8937 7402 45 16 646 3218 2688 Supernatant Pellet 2 1 28 992 157 226 of cpm Tubulin/T-ag Tubulin/p53 1 30 518 39 1 1 3.8 2.3 1 .7 68.7 14.3 1 1.5 72 1 3.6 4.4 I)Ul(’S St(’1) 3: Se ond-yck’ ization l)o)YIller- ,‘ Protein l)ands from the ininsunoblots were (letermined by liquid S( intillation I) The majority of T-ag in the transformed 1 in the ( ( le purifi(afii)n )r’dUr#{128}’). shown in Fig. 7 were spectros opy. Total cells (>90%) was excised ,mnd solubilized in 0.1 N KOH cpm per immunoblot band are shown. pelleted with other particulate materials for 1 h with when the microtubules. mKSA cells, Following microtubules fied through one cycle 13.8 shaking initial at room cell lysate the from temperature, and 251 cpm was (prior to step described line were for pun- clarified protocol each cell of depolymenization and polym- enization. Total proteins in the supennatant and pellet fractions of the polymerization reaction mixture were analyzed by immunobbotting for tubulin, I-ag, and p53 (Fig. 9). Results similar to those observed for suspension mKSA cells were obtained with the two transformed cell lines growing as monolayers. Both I-ag and p53 copunified with tubulin. These data show that the association of 1-ag and p53 with microtubules is not restricted to growth of cells in suspension culture. It was noted that the amount of I-ag cosedimenting with in vitro polymenized microtubules was significantly greater from the 19C and WIB1a SV4O-transformed cell lines than from the mKSA cells. Another SV4O-transformed mouse cell line, analyzed by IEM using double labeling as above for mKSA cells. Gold beads were rarely on sections I ig. 8. Association i)f SV4O 1-ag with iiie rotubules two cycles of l)urif ation by in vitro assembly-disassembly. we’re l)rePIrt’d for in sitre polymerization experiments ‘Materials and Methods.’ Samples were withdrawn during stained the second ( ycle, with arnrnonium f)olynlerized after mKSA cells as descrilx’d in at various finies were imniunolaheled, and were niolybdate. .-\, a sample removed negatively from the repolymerization reaction after 3 mm of assembly at 37’C contained microtubuk’s as svell as unassembled tuhulin. lnimuncabeling was done using PAb4O5 anti-I-ag nionoclonal antibody (arrowhead). Note labeling of 1-ag on reassembled tul)ules. Bar, 0. 1 m. B, immunogold labeling of a duplicate sample (sanie as in A) was carried out using an irrelevant antibody, ni7 3, as a ( ontrol. Bar, 0.2 pm. Inset, depolymerized tuhulin withdrawn immediately prior to initiation of the second cycle of polyrnerization was labeled with anfi-T-ag (PA64OS, ,irrowhe,id) and antitul)ulin )YL’/2, arrow) aiitibodit’s. Bar, 0.1 Mm. mary were epithelial analyzed cell line (19C), for copurification both transformed of I-ag and by SV4O, p53 with treated with irrelevant control F9I, was described observed antibodies; the m73 control failed to react with all subsets of microtubules, showing the specificity ofthe procedure (Fig. bA). Antibodies for 1-ag (Fig. lOB) and for p53 (Fig. 1OC) cobocalized with tubulin antibodies on microtubules (Fig. 10, B and C). IEM observations, coupled with cycle purification of microtubules, suggest that the association ofT-ag and p53 with microtubules is a common phenom- enon in SV4O-transformed origin or culture cells, regardless of tissue of conditions. Discussion Single-phase concentrations of butanol have facilitated the recovery of a complex composed of I-ag, p53, tububin, and several unidentified proteins from SV4O-transformed mouse cells. The M, 56,000 component of the complex was identified as tububin in this study by amino acid sequence analysis and by immunological reactions using antitubulin monoclonal antibodies. The I-ag/p53/ tububin complex is not recovered using conventional methods of protein extraction that utilize detergents (31). The less deleterious effect of single-phase concentrations Cell Table 2 p53 from Effect mKSA of colchicine cells treatment mKSA cells were incubated colchicine for 3 h at 37#{176}C.Cells microtubule stabilization buffer NP4O extracts were precipitated by immunoblofting as described .. on solubility of tubulin, 1-ag, and inthe presence or absence of 10 MM were then extracted with 0.1% NP4O in for 15 mm. Total soluble proteins in the with trichloroacetic acid and analyzed in “Materials and Methods.” Quantitation (cpm)’ Ratios: treated Solubilized Untreated cells protein Tubulin 1-ag p53 a Protein cpm 3274 944 182 bands were Colchicine-treated cells from determined immunoblots cells/untreated cells 4804 1152 443 were as described 1.47 1.22 2.40 excised in Table and 1, footnote solubilized, and 251 a. of butanol on native protein conformation (36) may allow for recovery of protein complexes that are usually disrupted by detergent during initial cell extraction procedures. The precise origin of the protein complex recovered by butanob from the transformed cells is not known. Previous experiments (31) found that at 37#{176}Cbutanol sobubilized pml-ag as well as a number of proteins resistant to extraction by detergent. Optimal conditions for release of the complex resulted in the leakage of some cytoplasmic proteins (31). It is possible that the complex may reside within or on the periphery of the plasma membrane, in association with cytoplasmic structures, or may reflect transient interactions among proteins being shuttled within the cell. Identification of other cellular A . [)ifferentiation 123 components present within the complex may help clarify its subcellular origin. Deppert and his colleagues (30, 32, 37) have reported that a fraction of pml-ag is tightly bound to an NP4O-insobubbe framework in the plasma membrane, designated the plasma membrane lamina. We have confirmed the existence of an NP4O-insobuble, Empigen BB-sobubbe population of pml-ag (31). Butanob extraction recovered a species of T-ag that was positioned so as to be less accessible to surface iodination than NP4O-sobubbe pml-ag (31). It may be that the butanol-solubbe fraction of I-ag is related to the plasma membrane bamina-associated fraction of I-ag that is predominantly interior in the cell (37). The multimenic protein complex appears to exist in situ prior to cell bysis. The possibility that the observed complex was an artifact induced by butanob was ruled out previously by various control experiments (31). The coprecipitating M, 35,000-60,000 proteins remained relatively constant to one another under various conditions (31, 33). Competition experiments performed here showed that extraction of labeled cells in the presence of either unlabeled cells or unlabeled tububin did not diminish the recovery of the labeled complex. Additional approaches that did not involve butanol (also reported here) substantiated a specific interaction of I-ag and p53 with microtububes. Both 1-ag and p53 copunified with microtubules through two cycles of temperature-dependent depolymenization and polymerization, and treatment of mKSA cells with colchicine prior to extraction under conditions that preserve nuclei and cytoskebetal filaments other than microtubules resulted in increased release of I-ag and p53. Thus, both I-ag and C B 12 Growth 34 12 34 tubulin’ .. . 9. Copurification of 1-ag and p53 with single-cycle purified microtubules from 5V40-transformed pelleted (Lanes 2 and 4) microtubule fractions from the first cycle of temperature-dependent purification fibroblast cell line WIB1a (Lanes 1 and 2) and SV4O-transformed mammary epithelial cell line 19C immunoblotted with antitubulin monoclonal antibody YLY2 (A), rabbit anti-I-ag serum (B), or rabbit 56,000), 1-ag )M, 95,000), and p53 are indicated. Fig. mouse cell lines. Supernatant (Lanes I and 3) and of tubulin were obtained from SV4O-transformed (Lanes .3 and 4). Total protein in the samples was anti-p53 serum (C). The positions of tubulin (M, 124 SV4O I-ag md Bind Mi rotuhules fit the criteria of microtubule-associated proteins. IEM corroborated these biochemical analyses and established an association of I-ag and p53 with intact microtubules in situ. We do not yet know the precise relationship between the T-ag/p53/tubulin complex recovered by butanol and the T-ag/p53/microtubule interactions visualized by IEM. A specific interaction in vitro between SV4O small antigen and tubulin has been observed (38). We have not rigorously ruled out the presence of small t antigen in the butanob extracts, but so far we have not detected it. Small t antigen was not evident in immunobbots of solubilized microtubule-punified proteins using polycbonab rabbit anti-I-ag serum, nor was it observed in immunobbots of protein complexes immunoprecipitated from butanol extracts of transformed cells (33). Small antigen is not responsible for the protein interactions described here. The T-ag/tububin complexes can be immunoprecipitated using monocbonal antibodies directed against COOH-terminal sequences of I-ag not shared by small t antigen (33). Furthermore, the EM studies reported here that revealed I-ag associated with microtubules utilized a COOH-terminal-specific anti-I-ag antibody. The observed association of p53 with cellular microtubules is intriguing. Wild-type p53 exhibits transcniptional activating ability (39, 40). As a negative growthregulatory protein, p53 must exert some influence on the cell cycle. It has been suggested (41) that cell cycle transitions in eukaryotic cells are controlled by p34, the product of the cdc2 gene. The activity of p34, the catalytic subunit of a protein kinase, is regulated by phosphorybation. Microtubube dynamics during the interphase-metaphase transition in Xenopus eggs have been shown to be regulated by cdc2 kinase (42). Human p53 is phosphorylated by p34f(2 (43 44) and it has recently p53 been reported that p53 can associate with p34k2 (44 45). It is possible that the protein complex described here, which contains a M, 35,000 member is important in cell growth control. The association of I-ag and p53 may be functionally important in the transport of these proteins within the cell and to the plasma membrane. Transport motor proteins such as kinesin and dynein facilitate movement of vesicles and proteins on microtubules (46). This mechanism provides an attractive explanation for the transport of I-ag to the plasma membrane. Previous studies have shown that I-ag does not enter the cellular secretory pathway (47-49). Nothing is known about the intraceblular transport of p53. It is possible that 1-ag is following a pathway fig. 10. Denionstration of microtubule association SV4O 1-ag and p53 in SV4O-translormed F9T cells by IEM. F9T cells expressing 1-ag were extracted s ith 0. l% NP4O and were’ prepared for ele tron microscopy. Thin sections of cytoskelefons were inimunolabeled with antitubulin and anti-T-ag, with anti-tubulin and anfi-p53, or with a control antibody. Bars, 0.1 m. A, (ontrol experiments, in which samples were labeled with ii73 as the primary anfil)ody, showed no labeling of microtubules. B, anfi-Tag (PAb4OS. ,irr(iesheaci) and antitubulin (YL’/2, arr(iw) antibodies colocalized to nix rotubules after simultaneous double immunogold labeling. C, anti-p53 (PAb42 1 , arrowhead) and antitubulin (YLV2, ,irrow( antibodies colocalized to rnicrofuhules by double labeling. Mitotii. spindle (A ( and niicrotuhuk’s near the spindle (B and C) are shown. utilized by cellular growth-regulatory proteins such as p53. Other enzymes and proteins bound to microtububes and functioning in some aspect of cell metabolism (50) may be targets affected by I-ag or p53. It is interesting that a correlation has been observed between the association of Rous sarcoma virus p60vsr( with cytoskebetal structures and transforming activity (51). It will be important to define the domain(s) on I-ag and p53 that interact either with tububin or with some microtubule-associated protein. This will help determine the functional importance of the microtubube association. Mapping studies using deletion mutants of I-ag are in progress, as are attempts to identify other members of Cell Growth the T-ag/p53/tubulmn complex SV4O-transformed recovered by butanol Partial compared from cells. partial Materials and Methods Cell Lines. SV4O-transformed cell lines mKSA (14, 25, SV4O-transformed BALB/c 52) and BALB/c mouse WTB1a mouse mammary Rabbit polyclonal antisera 53), the epithelial cell line 1 9C (54, 55), and SV4O-transformed carcinoma cells were used for metabolic protein extraction experiments. Antisera. fibroblast (14, F9T teratolabeling and against gel-pun- fied SV4O I-ag (56) and p53 (31) have been described. Monoclonal antibodies recognizing sites on the NH2 tenminus of 1-ag are designated PAb419 and PAb43O (57, 58). Those specific for carboxy-terminal epitopes on Iag are PAb4O5 and PAb423 (57, 58). Monoclonal antibodies used to detect p53 were PAb421 (57) and 200.47 (59). Monoclonal antibody against tubulin (YL#{189})was obtained commercially from Seralab (Accurate Chemical and Scientific Corp., Westbury, NY), and monoclonal antibodies specific for either the a subunit (N.357) or the 3 subunit (N.356) of tubulin were purchased from Amensham (Arlington Heights, IL). Control monoclonal antibodies were directed against adenovinus E1A protein (m73) (60) or against rotavinus SAl 1 VP7 (provided by M. K. Estes, Baylor College of Medicine). Metabolic Labeling, Protein Extraction, and Protein Analyses. Approximately 5-7 x 10 cells were incubated for 3 h in 1-2 ml of methionine-free minimal 125 Proteolytic Mapping. Complexed proteins were for structural relatedness by the V8 protease peptide mapping technique of Cleveland et a!. (64). [35S]Methionine-Iabeled partial peptides were nesolved in a 15% SDS-polyacrylamide gel (65). Temperature-dependent Cycle Purification of Tubulin. Tubulin was purified by a modification of the temperatune-dependent depolymenization and polymerization method of Vallee (66). Approximately 6 x 108 cells were sonicated in 2-3 ml of microtubule stabilization buffer [0.1 M 2-(N-morpholino)ethanesulfonic acid (pH 6.9), 1 mM ethylenebis(oxyethylenenitnilo)tetraacetic acid, 0.5 mM MgCl2, 1 mM GTP, 200 tM leupeptin, 1% Inasylol, 1 mM phenylmethylsulfonyl fluoride] with two 15-s bursts on ice. After incubation on ice for 15 mm, the lysate was clarified of particulate debris by centnifugation at 50,000 x g for 1 h at 2#{176}C. The supernatant plus GTP (containing unassembled tubulin) was then incubated at 37#{176}C for 30 mm to allow polymerization of tubulin into microtubules (first cycle). The polymenized microtubules were pelleted at 50,000 x g at 25#{176}C for 1 h. The pellet was solubilized in SDS disruption buffer [0.125 M Iris hydrochloride (pH 6.8), 2% SDS, and 5% fl-mercaptoethanol] for electrophoresis and immunoblotting. The supernatant (containing tubulin that failed to polymerize during the incuba- tion at 37#{176}C) was adjusted to 20% tnichloroacetic acid and was incubated on ice for 30 mm; aggregated, insoluble protein was then pelleted at 23,000 x g for 20 mm. The essential & Differentiation protein pellet was washed 95% For proteins, ization, a pellet from the first-cycle polymerization reaction was sonicated in microtubule stabilization buffer and phosphorylated cells were in- cubated for 3 h in phosphate-free minimal essential medium containing 2% dialyzed bovine serum and 500 zCi of 32P (ICN Biomedicals) per ml. Radiolabebed suspension cells were pelleted and completely drained of radioactive medium. Cells were lysed immediately in NP4O lysis buffer [1% NP4O, 0.05 M Iris hydrochloride (pH 8), 1%apnotinin,and 200 IzM Ieupeptin] (61)on were treated with 2.5% butanol in 0.01 M phosphate-buffered saline at 37#{176}C for bO mm (at 4 x b0 cells/mi) as described previously (31, 33). Proteins were then immunoprecipitated and analyzed by SDS gel electrophoresis (31, 56, 62). Immunoblot Assays. Immunobbotting was performed using a modification of the procedure of SlagIe et a!. (63). Immunoprecipitated proteins on total solubilized proteins were resolved in 10% polyacrylamide gels and were electrophonetically blotted onto nitnocellulose. Blots were preincubated for 1 h at 37#{176}C in blocking buffer (0.1% NP4O, 1% dried skim milk, and 0.25% gelatin in Iris-buffered saline). Treated blots 37#{176}C with shaking in a 1 :500 dilution or monoclonal antibody in blocking serum diluted in blocking buffer nitrocellulose strips (treated least 1 h at 37#{176}C to reduce were rinsed incubated gel lane Vigorously sodium saline well with at 37#{176}C with in blocking three deoxycholate, at 37#{176}C. incubated was preincubated buffer After in 1% and with with blocking buffer) nonspecific binding.] blocking 1 at of rabbit antiserum buffer. [Rabbit anti- and 1 h, blots for at Blots then 0.5 jzCi of 125l-Iabeled buffer. times were were were protein A/ washed NP4O, 0.1% SDS, 0.5% NaCI in Iris-buffered M For a second was cycle incubated disruption ethanol, dried, and disrupted immunoblotting. of SDS with fetal bovine serum and (1000-1150 Ci/mmol; Inc., Irvine, CA) per ml. analysis in twice medium containing 2% dialyzed 200 zCi of [355]methionine Tran35S-Iabel; ICN Biomedicals, of depolymenization on ice for 30 mm buffer and for polymer- to depolymenize micro- tubules. Aggregates of cold-insoluble material were removed by centnifugation at 50,000 x g for 30 mm at 2#{176}C. GTP was added to 1 mii to the clarified supennatant, which was then incubated at 37#{176}Cfor 30 mm. Microtu- bule pellets and the supennatants were analyzed for total protein as described for the first-cycle purification. Purification of Proteins for Automated Microsequence Analysis. 35S-Iabeled protein complex components were immunoprecipitated with monoclonal antibody 200.47 and were resolved by two-dimensional electrophoresis (34). Precautions were taken during preparation of immunoprecipitates and gel electrophonesis to minimize possible NH2-terminal blocking on denivatization of amino acid side chains. Immunoprecipitates were disrupted in NP4O-unea buffer (34) and were incubated at room temperature for 2 h. First-dimension separation was performed in the presence of 0.1 mrs’i sodium thio- glycolate to sequester any oxidants on free radicals generated during gel polymerization (67). For second-dimension analysis, isoelectnic focusing gels were equilibrated in sample buffer at room temperature for 30 mm. Focused proteins were then resolved on 10% gels that had been allowed to polymerize overnight in the presence of 0.1 mM sodium thioglycolate. Electrophonesis running buffers also contained sodium thioglycolate. Electrophoresis was performed at 45 V constant voltage overnight. Proteins were then blotted overnight at 0.5 A onto an Immobilon toradiognaphy. PVDF membrane and The area containing were localized pnctein was by auexcised, 126 SV4O 1-ag and p53 Bind Microtubules and the blotted protein was sequenced directly on a PVDF membrane in an Applied Biosystems model 477A protein sequencer. Approximately 50 pmol of purified protein were recovered from butanol extracts of 8 x i0 mKSA cells. Lowicryl with K4M Embedding. Cells stabilization buffer microtubule were washed once followed by a 15- References 1 . Levine, A. J. Oncogenes 496, 1988. of DNA tumor viruses. J. M. Transgenic 2. Cory, S., and Adams, Rev. Immunol., 6: 25-48, mice Cancer Res., 48: 493- and oncogenesis. Annu. 1988. 3. Hanahan, D. Transgenic mice as probes (Wash. DC), 246: 1265-1275, 1989. into complex systems. Science mm treatment with 0.1% NP4O in the same buffer. Cells were washed twice with either 0.1 M sodium phosphate buffer or 0.1 M piperazine-N,N’-bis(2-ethanesulfonic acid) monosodium salt buffer and were then fixed in buffer containing 2% glutaraldehyde and 2% formaldehyde for 30 mm at room temperature. Further processing was done as described by Decker et a!. (68). Briefly, cells were pelleted in a microcentnifuge immediately after addition to fixative. Fixed cell pellets were washed in buffer and dehydrated in dimethylformamide in steps of 50, 70, and 95% for 5 mm each and 100% for 15 mm. The pellets were then transferred to a 1:1 mixture of 4. Sepulveda, A. R., Finegold, M. j., Smith, B., Slagle, B. L., DeMayo, j. L., Shen, R-F., Woo, S. L. C., and Butel, I. S. Development of a transgenic mouse system for the analysis of stages in liver carcinogenesis using dimethylformamide-Lowicryl 8. Stahl, Biochim. for 15-60 mm and were infiltrated with Lowicryl for 60 mm, all at room temperature. Samples were rinsed twice with Lowicryl, embedded in Beem capsules, and polymenized at room temperature via UV light (69). IEM. Fifteen-nm gold-goat anti-mouse and 10-nm goldgoat anti-rat conjugates were purchased from Janssen Life Science Products (Piscataway, NJ). For immunolabeling experiments, the antitubulin antibody was diluted 1:2, and all other antibodies were diluted 1 :20 in phosphate-buffered saline containing 0.1% Triton X-100 and 0.5% Tween 20. Thin sections of Lowicryl-embedded cell pellets were collected on Panlodion-coated nickel grids. Immunolabeling was performed according to the procedure of Decker et a!. (68). Grids were floated on a drop of phosphate-buffered saline with 0.1% Triton X100 and 0.5% Tween 20 for 5-15 mm to block nonspe- cific reactions. All antibody dilutions and the first wash also were performed using this buffer. After blocking, sections were incubated for 60 mm with a primary antibody or, for double-labeling experiments, with a mixture of mouse and rat primary antibodies. Grids were jet washed with 10-15 ml of buffer/grid and were drained on filter paper. Incubations with secondary antibodygold conjugates also were carried out for 60 mm. For double-labeling experiments, a 10-nm gold-goat anti-nat probe was mixed with a 15-nm gold-goat anti-mouse probe for simultaneous reactions. After incubation with probe, grids were jet washed with water. Immunolabeled grids were stained either with osmium tetroxide and tannic acid (70) on with 4% ethanolic unanyl acetate and tissue-specific elements 1989. 6. 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