Figure 5

Sox6 is degraded by proteasome in C2C12 cells. (A) Time course of Trip12, Sox6, and Tbp protein levels in C2C12 cells in the presence of cycloheximde (CHX) analyzed by Western blotting using a 4-15% gradient gel (n = 3). (B) Densitometric analysis of the Western blot in (A). Band intensity of each protein was normalized to that of 0 h in CHX and represented as mean ± SD (n = 3). (C) Validation of Psmd1 knockdown by siRNA. Total RNA was extracted from mock- or siRNA-transfected C2C12 cells, and the mRNA level of Psmd1 (the gene encoding a regulatory subunit of the 26S proteasome) was quantified by RT-qPCR. Data are normalized to EGFP siRNA-transfected cells and represented as mean ± SD (n = 3). (D) A Western blot using a 7.5% gel showing an increase in the Sox6 protein level in Psmd1 siRNA-transfected C2C12 cells. (E) Densitometric analysis of Western blotting results shows a ~4 fold increase in the Sox6 protein level in Psmd1 siRNA-treated C2C12 cells. A smaller increase was observed for Tbp protein. Data are normalized for those from EGFP siRNA-transfected cells and represented as mean ± SD (n = 3). (F) Psmd1 knockdown reduced the mRNA level of Myh7, a known target of Sox6. Data are normalized to EGFP siRNA-transfected cells and represented as mean ± SD (n = 3). **p < 0.005.