FIGURE 3.

USP13 knockdown decreases Siah2 protein levels and increases Siah2 activity. A, HeLa cells were transfected with Myc-USP13 and endogenous levels of Siah2 and Sprouty2 from total cell lysates were detected by Western blot analysis. B, HeLa and melanoma Lu1205 cells were transfected with control shRNA or shRNA targeting USP13. Endogenous Siah2 and Spry2 from total cell lysates were detected by Western blot analysis. C, cycloheximide (CHX) chase of Siah2 in cells with USP13 knocked down. Following the addition of cycloheximide cells were harvested after 1, 2, and 3 h, and cell lysates were subjected to Western blot to detect Spry2 and Siah2. β-Actin served as the loading control in A–C. D, degradation curves of Spry2 by cycloheximide chase in control or USP13-KD. The graph represents the mean value of band density in 3 experiments..