Download rapid alkaline extraction procedure for screening recombinant

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SUPPLEMENTARY MATERIALS AND METHODS
Cell culture, transfection, siRNAs and shRNAs
The human neuroblastoma cell line SK-N-SH was maintained in MEMα (Invitrogen) containing
10% fetal bovine serum. Mouse P19 cells were obtained from ATCC and maintained in MEMα
containing 7.5% bovine calf serum and 2.5% fetal bovine serum. Neural differentiation of P19
cells was induced by retinoic acid. Briefly, 1x106 cells were cultured in a bacterial grade Petri
dish in 10 ml of media containing 5x10-7 M all-trans retinoic acid (Sigma) for 4 days. The cell
aggregates were resuspended by mild pipetting and trypsin/EDTA treatment. The resuspended
cells were then transferred to a poly-D-lysine-coated tissue culture dish and cultured in retinoic
acid-free media for an additional 4 days. Establishment of the clonal P19 cell lines expressing
shRNAs T-2, T-3 and GFP was described previously (1). The sense sequence of siRNA against
human and mouse Fox-2 is 5’-GGGAUUCGGGUUCGUAACU-3’ (Dharmacon). A mixture of
siRNAs against human PSF (sc-38304) and mouse PSF (sc-38305) were obtained from Santa
Cruz Biotechnology and a non-targeting control siRNA from Dharmacon. Either the Effectene
transfection reagent (Qiagen) or Amaxa Nucleofector (Amaxa Biosystems) was used for
transfection of the plasmid constructs and siRNAs.
RNA preparation, RT-PCR and RNA blot analysis
Total RNA was isolated from mouse tissues and cultured cells using an RNeasy mini kit
(Qiagen). RT-PCR was performed using Superscript III RNase H− reverse transcriptase
(Invitrogen) and Pfu Turbo DNA polymerase (Stratagene). The PCR primers used for the
endogenous NMHC II-B and minigene mRNAs analysis were described previously (2). The
primers used for the GAPDH mRNA were 5’-CCATGGAGAAGGCTGGGG-3’ (forward) and
5’-CAAAGTTGTCATGGATGACC-3’ (reverse). The ethidium bromide-stained bands were
quantified using Quantity One software (Bio-Rad). Real-time PCR was performed using a SYBR
Green PCR kit (Qiagen) in an Applied Biosystems 7500 Real-Time PCR system. RNA blot
analysis was performed using Mouse Multiple Tissue Northern Blots obtained from Clontech.
The full-length coding sequences of Fox-1, 2 and 3 were used as probes. The probe was labeled
using the Ready-To-Go DNA Labeling Beads (Amersham Biosciences) and [α-32P]dCTP
(PerkinElmer Life Sciences). Hybridization was carried out in the ExpressHyb-Hybridization
Solution (Clontech) at 68°C for 18 h. The membrane was washed in 0.5X SSC plus 0.2% SDS at
55°C and exposed to x-ray film.
Construction of expression plasmids and minigenes
The expression plasmids for Fox-1, 2 and 3 in the pCS3+MT vector, which contains the
cytomegalovirus promoter, the SP6 promoter and a myc epitope, have been described previously
(1,2). The expression plasmids for PSF and NonO were obtained from Open Biosystems.
Defined cDNA fragments encoding various regions of Fox-3 and PSF were generated by PCR
with appropriate sets of primers and cloned in the pCS3+MT vector or the pFN2A(GST)Flexi
vector (Promega) which had been modified to delete the Barnase sequence. Fox-3 expression
constructs with silent mutations (myc-smFox-3-L and S) were generated by recombinant PCR.
The
mutations
were
introduced
in
the
primer
5'TACACACCGGCTCAAACACACCCAGAACAACCTGGCACT-3' and a primer with the
complementary sequence. The NMHC II-B minigenes were described previously (2). For
preparation of antigens, Fox-1 and Fox-2 cDNA fragments encoding amino acids 1-116 and 1112, respectively were cloned in the pGEX-5X-1 vector (GE Healthcare).
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Preparation of extracts, immunoprecipitation and immunoblot analysis
Single cell suspensions were prepared by passing the tissue and cultured cells through a cell
strainer. A NE-PER kit (Pierce) was used to prepare nuclear extracts of mouse brain and P19
cells. For immunoprecipitation and co-immunoprecipitation, the nuclear extracts containing 1-2
mg proteins in 5-10 ml of Co-IP buffer (Pierce) were incubated with the primary Abs overnight
at 4°C. The immuno-complexes were collected by incubation with Protein A/G PLUS-Agarose
(Santa Cruz Biotechnology) and washed in Co-IP buffer. For RNase treatment, the immunocomplexes on the beads were incubated in 10 μg/ml of RNase A (Qiagen) at room temperature
for 15 min, and washed again with Co-IP buffer. The complexes were solubilized in SDS sample
buffer and subjected to SDS-PAGE using a 4-12% gradient polyacrylamide NuPAGE Bis-Tris
gel and NuPAGE MOPS SDS running buffer (Invitrogen). ProSieve color protein markers
(Lonza) were used as Mr standards. Tissue and cell extracts were prepared using a radioimmune
precipitation assay buffer (Sigma) supplemented with a protease inhibitor cocktail (Sigma). Total
cell lysates from cultured cells were prepared by direct addition of SDS-sample buffer to whole
cells. Samples that required both protein and RNA analyses were split upon harvesting. For
immunoblot analysis, the protein samples were transferred to a nitrocellulose membrane
following SDS-PAGE. Following incubation with primary Abs, horseradish peroxidaseconjugated goat Abs against rabbit or mouse IgG were used as secondary Abs. Binding of Abs
was detected by the SuperSignal system (Pierce).
Immunofluorescence microscopy
SK-N-SH cells transfected with a Fox-3 expression plasmid were fixed with 4%
paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Mouse brains and spinal
cords were processed as described previously (3). Briefly, paraformaldehyde-fixed tissues were
embedded in paraffin and cut into 4 µm sections. Antigen retrieval was performed on hydrated
sections by immersing them in 10 mM sodium citrate (pH 6.0) and microwaving for 10 min. The
primary Abs are described above. Alexa-488 and Alexa-594 conjugated goat Abs against mouse
IgG and rabbit IgG were used as secondary Abs. Nuclei were counter-stained with 4, 6diamidino-2-phenylindole (DAPI). Specimens were examined using a Zeiss LSM 510 Meta
confocal laser-scanning microscope.
SUPPLEMENTARY REFERENCES
1.
2.
3.
Kim, K.K., Adelstein, R.S. and Kawamoto, S. (2009) Identification of neuronal nuclei
(NeuN) as Fox-3, a new member of the Fox-1 gene family of splicing factors. J. Biol.
Chem., 284, 31052-31061.
Nakahata, S. and Kawamoto, S. (2005) Tissue-dependent isoforms of mammalian Fox-1
homologs are associated with tissue-specific splicing activities. Nucleic Acids Res., 33,
2078-2089.
Ma, X., Kawamoto, S., Uribe, J. and Adelstein, R.S. (2006) Function of the neuronspecific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain
development. Mol. Biol. Cell, 17, 2138-2149.
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Supplementary Figure S1
Figure S1. Tissue-dependent expression of the Fox-1 gene family. (A) RNA blot analysis of
mouse tissues. (B) Immunoblot analysis of mouse tissues. (C) Specificity of antibodies against
each Fox protein. The myc tagged Fox-1, 2 or 3-S construct was transfected into SK-N-SH cells.
Whole cell lysates were subjected to immunoblot analysis using the indicated antibodies. Antimyc verifies equal expression of the Fox proteins. (D) Nuclear localization of Fox-3. The mycFox-3-L is exogenously expressed in SK-N-SH cells. The cells were immuno-stained for Fox-3
(red) using anti-myc and for NMHC II-B (green). DAPI (cyan) stains nuclei.
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Supplementary Figure S2
Figure S2. Endogenous Fox-3 expression is associated with N30 inclusion in P19 cells. (A)
Neural differentiation of P19 cells triggered by retinoic acid (RA) treatment. Phase contrast
microscope images are shown. -, untreated cells; RA, cells treated with RA. Note that RA treated
cells extend axon-like processes. (B) Exogenous expression of Fox-3 with silent mutations
relieves the inhibitory effect of the Fox-3 shRNA on N30 splicing. The T-2 shRNA-expressingP19 cell line was treated with RA and transfected with the NMHC II-B minigene and the
expression constructs indicated at the top. RT-PCR (upper panel) and immunoblots using the
indicated Abs (lower panel) are shown. The expression constructs for myc-Fox-3-L and S (see
Fig. 7B) contain the wild-type nucleotide sequences, whereas the constructs for myc-smFox-3-L
and S contain the silent mutations at the region targeted by the T-2 shRNA. Fox-3-L and S with
silent mutations are expressed at a high level, and this Fox-3 expression is accompanied by an
increase in N30 inclusion.
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Supplementary Figure S3
Figure S3. Tryptic peptide sequences identified as PSF (A) and NonO (B) by mass
spectrometry analysis. Both the N-terminal and C-terminal sites of tryptic cleavage are
designated by a period.
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Supplementary Figure S4
Figure S4. Co-localization of Fox-3 and PSF in the mouse spinal cord. Transverse section of
the spinal cord was co-stained with monoclonal anti-Fox-3 (anti-NeuN, green), polyclonal antiPSF (red) and DAPI for nuclei (cyan). Arrows point to motor neurons. Bar, 50 μm.