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. 2017 Dec 11;128(1):517–530. doi: 10.1172/JCI95410

Figure 5. PRMT5 increases SOX10 and PAX3 expression by arginine methylation–dependent inhibition of SKI.

Figure 5

(A) Luciferase reporter assay of TGF-β signaling in WM793 cells expressing scrambled shRNA or HOIP-, HOIL-1L–, or SHARPIN-specific shRNAs. Cells transfected with TGF-β reporter (CAGAx9-luc) and pCMV-Cypridina-luc (internal control) reporter plasmids were treated with TGF-β (20 ng/ml) for 8 or 24 hours. (B) qPCR analysis of SOX10, PAX3, and MITF expression in WM115 cells expressing empty vector (control), SHARPIN, or PRMT5 expression plasmids and treated with TGF-β (20 ng/ml) for 0, 24, or 48 hours. (C) Immunoblot and qPCR analysis of WM115 cells transfected with scrambled (control) siRNA or SKI-specific SMARTpool siRNA. (D) ChIP analysis of WM115 cells expressing empty vector, SHARPIN, or PRMT5 and treated with vehicle (Veh) or TGF-β (20 ng/ml) for 18 hours. ChIP analysis was performed with anti-SKI antibody, and coimmunoprecipitated SOX10 or PAX3 promoter sequences were quantified by qPCR. (E) Immunoblot analysis of SKI and p53 in WM115 cell lysates immunoprecipitated with normal rabbit serum (NRS) or SYM10 antibody. (F) Analysis as in E except WM115 cells expressed scrambled or PRMT5-specific shRNA. (G) Immunoblot analysis of anti-Flag immunoprecipitates of A375 cells coexpressing WT, Arg8 mutated (R8K), or Arg658/660 mutated (RR/KK) SKI with either PRMT5 or SHARPIN. Asterisks and arrowhead indicate SHARPIN and tubulin, respectively. (H) Immunoblot analysis of anti-Myc immunoprecipitates of HEK293T cells expressing Myc-tagged SHARPIN and Flag-tagged WT, R8K mutant, or RR/KK mutant SKI proteins. (I) Immunoblot analysis of IgG or anti-SKI immunoprecipitates of SK-Mel-28 cells. (J) ChIP analysis as in D of WM115 cells expressing WT or R8K mutant SKI proteins. Lysates were immunoprecipitated with indicated antibodies. For immunoprecipitation analyses, input indicates 5% of lysates. Statistical significance was calculated using 2-way ANOVA (Dunnett’s test, A and B) or 2-tailed Student’s t test (C, D, J). Data are presented as mean ± SD (n = 3). *P < 0.05; **P < 0.005; ***P < 0.0005.