FIGURE 2.
Active PKD isoforms phosphorylate and inactivate SSH1L. A, schematic shows how PKD regulates SSH1L and cofilin activity (from Ref. 28). B, HeLa cells (0.5 × 106, 6-cm dish) were transfected with tagged, constitutively active alleles of PKD1, PKD2, or PKD3, as indicated. SSH1L was immunoprecipitated and analyzed for PKD-mediated phosphorylations at Ser-978 using a phospho-specific antibody. Samples were then stripped and reprobed for total SSH1L. Samples were also analyzed for co-immunoprecipitated 14-3-3 protein using a pan-14-3-3 antibody. For input control, Western blot analysis was performed on lysates to determine expression of active PKD1 (α-HA staining), PKD2 (α-FLAG staining), and PKD3 (α-GST staining).