FIGURE 2.

USP13 decreases Siah2 ubiquitin ligase activity. A, 293T cells were transfected with an expression vector encoding GFP and Myc-Spry2 alone or with FLAG-Siah2 and or USP13. Total cell lysates were analyzed for GFP expression to assess transfection efficiency. The cells were treated with the protein synthesis inhibitor cycloheximide (CHX, 40 μg/ml) for 2, 4, and 6 h, and the cell lysates were subjected to Western blotting for detection of Myc-Spry2, FLAG-Siah2, and USP13. exp., exposure. B, degradation curves of Spry2 by cycloheximide chase in the presence of Siah2 alone or with USP13. The graph represents the mean value of band density in 3 experiments. C, 293T cells were transfected with Myc-PHD3 and increasing amounts of FLAG-Siah2 in the presence or absence of USP13. Total cell lysates were probed for the presence of USP13, Myc-PHD3, FLAG-Siah2, and β-actin.