FIGURE 2.

SKI enhances MDM2-mediated p53 ubiquitination. A, SKI increases p53 ubiquitination by MDM2. Various plasmids were introduced into H1299 cells, and the amount of plasmids was equalized with empty vector. 40 h after transfection, the cells were treated with 20 μm MG-132 for 4 h, lysed with 2% SDS buffer, and heated at 95 °C for 5 min followed by dilution with regular buffer to 0.1% SDS and centrifugation. The supernatant was immunoprecipitated (IP) with anti-p53 antibody, followed by the immunoblot (IB) analysis with anti-HA antibody. The blot was stripped and probed again with anti-FLAG antibody to gauge the p53 level. A separate portion of the lysate was used to analyze the expression level of MDM2 and SKI with immunoblot analysis. B, wild type but not mutant SKI enhances p53 ubiquitination by MDM2. The experiment was done essentially as in A in H1299 cells. C, endogenous SKI was crucial for MDM2-mediated p53 ubiquitination. The experiment was done essentially as in A except with introduction of siRNA for SKI.