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Supplementary information for Liu et al., “Oncogenic BRAF regulates -Trcp expression and NF-B activity in human melanoma cells” Materials and Methods Plasmids and chemicals Constructs for mammalian expression of Flag-tagged IB (Scherer et al., 1995), and luciferase reporters driven by B- (DiDonato et al., 1996) and Trcp2 promoters (Spiegelman et al., 2002a) were previously described. Conditionally active forms of BRAF-V600E (BRAFVE) were generated by fusion to modified forms of the hormone binding domain of the human estrogen receptor (hbER, (Littlewood et al., 1995)). To facilitate the generation of such fusion constructs a full length human BRAF cDNA encoding a 766 amino acid form of human BRAFVE was subjected to PCR using the following primers; BRAF1: 5’-CACCGTCGACACCATGGCGGCGCTGAGCGGTGGC-3’ BRAF2: 5’-CCCTAGTCGACTCACTACCTAGGCCCGTGGACAGGAAACGCACC-3’ These primers introduce a unique SalI site upstream of the codon for the initiator methionine and a unique AvrII site between the final codon of BRAF (His766) and the normal stop codon of the mRNA. This PCR product was cloned into a TOPO PCR fragment cloning vector. Next sequences encoding the T2 form of the hormone binding domain of the human estrogen receptor- [hbERT2] were subjected to PCR using the following PCR primers. HBER1: 5’-CACCGGATCCACCATGGGGAACTAGGTCCGATCCATCTGCTCCAG-3’ HBER2: 5’-CCCCTAGTCGACTGTGGCAGGGAAACCCTCTGCCTCCCCCG-3’ These primers introduce a unique AvrII site upstream and a unique SalI site downstream of the sequences encoding hbER. -TOPO cloning vector. Sequences encoding PCR amplified BRAFVE were fused in frame to sequences encoding hbERT2 by subcloning a NotI-AvrII fragment of BRAFVE into the appropriate sites of pENTR/D-TOPO:hbERT2 to generate pENTR vectors encoding BRAFVE:ERT2, in which ERT2 is fused to the C-terminus of activated BRAF. Retrovirus vectors for expression of BRAFVE:ERT2 in mammalian cells were generated by Gateway mediated subcloning into a variety of Gateway compatible retrovirus vectors. Mouse Melan-a cells expressing BRAFVE:ERT2 were generated by infection with a retrovirus vector encoding BRAFVE:ERT2 and resistance to Puromycin (gLXSP3). In this vector expression of BRAFVE:ERT2 is promoted by the MuLV LTR and resistance to puromycin by the SV40 early promoter. Retrovirus infected cells were isolated by selection in Puromycin for 7-10 days. Activity of BRAFVE:ERT2 was induced by adding 4-hydroxytamoxifen (4-OHT, 250nM) to the growth medium for 16hr to allow a robust induction of -Trcp. MEK inhibitor PD 098059 and U0126 (Calbiochem), 4-hydroxytamoxifen (4-OHT) and cycloheximide (Sigma), as well as tumor necrosis factor alpha (TNF, R&D Systems) were purchased. BAY 43-9006 was a kind gift from Dr. Charles Smith (Hershey Medical Center, Pennsylvania State University). Cells Normal human melanocytes and human melanoma cells were cultured as previously described (Satyamoorthy et al., 2003). Melan-A mouse melanocytes (Bennett et al., 1987) or their derivatives that were engineered to express BRAFVE:ERT2 were cultured in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum, 200nM tetradecanoyl phorbol-13-acetate, glutamine, penicillin and streptomycin. Cells were transfected using Lipofectamine Plus (Invitrogen) following the manufacturer’s recommendations. Luciferase assays in melanoma cells transfected with either B- or Trcp2 promotor-driven luciferase reporter constructs and renilla luciferase plasmid were performed using Dual Luciferase kit (Promega). For RNAi approach, the shRNA that specifically target V600E mutant allele of BRAF were described before (Hingorani et al., 2003); cells were harvested 24hr after transfection with these shRNA plasmids. The rate of apoptosis was measured by flow cytometric analysis upon the cell staining with allophycocyanin conjugate (APC) labeled-Annexin V with or without 7Amino-actinomycin D (7-AAD) using Annexin V kit (BD Biosciences Pharmingen). Immunotechniques Monoclonal antibodies against phospho-ERK1/2 or total ERK1/2 (Cell Signaling), Flag tag (M2, Sigma), IKK (PharMingen), BRAF, IKK and IB (Santa Cruz), as well as -actin and -tubulin (Sigma) were purchased. Polyclonal antibody against -Trcp2 (HOS-N) was described earlier (Spiegelman et al., 2002b). An IKK immunokinase assay was carried out with IKK antibody or Flag antibody as described elsewhere (Dejardin et al., 2002). Immunoprecipitation and immunoblotting procedures were described earlier (Spiegelman et al., 2002a). References for Supplementary Information: Bennett, D.C., Cooper, P.J. & Hart, I.R. (1987). Int J Cancer, 39, 414-8. Dejardin, E., Droin, N.M., Delhase, M., Haas, E., Cao, Y., Makris, C., Li, Z.W., Karin, M., Ware, C.F. & Green, D.R. (2002). Immunity, 17, 525-35. DiDonato, J., Mercurio, F., Rosette, C., Wu-Li, J., Suyang, H., Ghosh, S. & Karin, M. (1996). Mol Cell Biol, 16, 1295-304. Hingorani, S.R., Jacobetz, M.A., Robertson, G.P., Herlyn, M. & Tuveson, D.A. (2003). Cancer Res, 63, 5198-202. Littlewood, T.D., Hancock, D.C., Danielian, P.S., Parker, M.G. & Evan, G.I. (1995). Nucleic Acids Res, 23, 1686-90. Satyamoorthy, K., Li, G., Gerrero, M.R., Brose, M.S., Volpe, P., Weber, B.L., Van Belle, P., Elder, D.E. & Herlyn, M. (2003). Cancer Res, 63, 756-9. Scherer, D.C., Brockman, J.A., Chen, Z., Maniatis, T. & Ballard, D.W. (1995). Proc Natl Acad Sci U S A, 92, 11259-63. Spiegelman, V.S., Tang, W., Chan, A.M., Igarashi, M., Aaronson, S.A., Sassoon, D.A., Katoh, M., Slaga, T.J. & Fuchs, S.Y. (2002a). J Biol Chem, 277, 36624-30. Spiegelman, V.S., Tang, W., Katoh, M., Slaga, T.J. & Fuchs, S.Y. (2002b). Oncogene, 21, 856-60.