Figure 3. PPEF-1 antagonizes CK2 action on PDCD5 stabilization.

(a) PPEF-1 overexpression induced PDCD5 degradation, but the inactive PPEF-1^D172N mutant did not affect ET-induced stabilization. A549 cells were transfected with the indicated constructs, and then treated with ET for 6 h. Fixed cells were stained with the indicated immunofluorescent antibodies. Bar scale = 10 μm (b) Active PPEF-1, but not the inactive PPEF-1^D172N mutant, antagonizes CK2-induced PDCD5 stabilization. A549 cells were transfected with Myc-CK2α or Flag-PPEF-1 as indicated. Total cell extracts were immunoblotted with the indicated antibodies. (c) Inactive PPEF-1^D172N, but not wild-type PPEF-1, fails to antagonize CK2-induced reduction of PDCD5 ubiquitination. HCT116 cells were transfected with HA-tagged ubiquitin, Myc-tagged CK2, PPEF-1, PPEF-1^D172N, or Flag-tagged PDCD5 as indicated. Cells were treated with MG132 (10 μM) for 6 h before harvesting. Total cell extracts were immunoprecipitated using Myc antibody and immunoblotted with HA antibody. Full-length blots are presented in Supplementary Figure 3b-c.