(A) COS-1 cells were transfected with Flag-HDAC3 alone or together with Flag-Siah2. After 24 hrs transfection, cells were treated with MG132 for 5 hrs as indicated in the figure, and the levels of HDAC3 were detected by western blot analysis using an anti-Flag antibody. Tubulin was used as a loading control. (B) COS-1 cells were transfected with Flag-Siah2 or Flag-Siah2 (RM), and treated with MG132 after 24 hrs transfection as indicated in the figure. Flag-Siah2 and Flag-Siah2 (RM) were detected by western blot analysis using an anti-Flag antibody. Siah2 (RM): A ring finger mutant, H99A/C102A. (C) COS-1 cells were transfected with Flag-Siah2 (RM) and Myc-HDAC3. Whole cell lysates were prepared 24 hrs post-transfection for the co-immunoprecipitation assay. Flag-Siah2 (RM) was immunoprecipitated with a rabbit anti-Flag antibody, and a rabbit normal IgG was used as a control. The immunocomplexes and 10% input were analyzed by western blot analysis using a mouse anti-Myc antibody to detect Myc-HDAC3 or a mouse anti-Flag antibody to detect Flag-Siah2 (RM).