FIGURE 5.

DUB activity of USP19 and E2-repressed myogenesis. A, C2C12 cells were transfected with pCAGGS-Myc-USP19, pCAGGS-Myc-USP19(C548A), or pCAGGS-Myc-USP19(C548S) for 24 h, followed by culturing in differentiation medium in the presence or absence of 10 nm E2 for an additional 4 days. Cell lysates were analyzed by Western blotting with anti-Myc, anti-MHC, anti-tropomyosin, and anti-GAPDH antibodies. B, pcDNA3.1-HA-Ub was transfected into C2C12 cells with pCAGGS-Myc-USP19, pCAGGS-Myc-USP19(C548A), or pCAGGS-Myc-USP19(C548S) for 24 h, followed by induction of differentiation for an additional 4 days. Cell lysates were analyzed by Western blotting with anti-Myc and anti-HA antibodies. C, C2C12 myoblasts were cultured in differentiation medium containing 5 μm MG132 in the presence or absence of 10 nm E2. Cell lysates were analyzed by Western blotting with anti-ubiquitin antibody. D, C2C12 cells were transfected with USP19 siRNA (siUSP19-1) or control siRNA (siCTL) for 24 h. Cells were induced to differentiate in the presence or absence of 5 μm MG132 and/or 10 nm E2 for 12 h, and cell lysates were subjected to Western blot analysis with anti-ubiquitin antibody. The graph is representative of three independent experiments.