FIGURE 1.

USP13 binds to and stabilizes Siah2. 293T cells were transfected with FLAG-Siah2, USP13, and a GFP expression vector. Total cell lysates were analyzed for GFP expression to assess transfection efficiency. A, cells were kept 6 h in normoxia or in hypoxia (2% O₂) before cell lysis. FLAG-Siah2 was immunoprecipitated (IP) with FLAG antibody, and immunocomplexes were subjected to immunoblotting with the indicated antibodies. B, cycloheximide (CHX) chase of Siah2 in the absence and presence of USP13. 293T cells transfected with FLAG-Siah2 and USP13 were treated with the protein synthesis inhibitor cycloheximide (40 μg/ml) for 1, 2, 4, and 6 h, and cell lysates were subjected to Western blot to detect FLAG-Siah2. β-Actin served as the loading control. C, degradation curves of Siah2 by cycloheximide chase in the presence or absence of USP13. The graph represents the mean value of band density in 3 experiments.