Figure 5.

Cdc20 interacts with HPIP during mitosis and ubiquitinates at lysine 274. A, HEK293T cells were co-transfected with either wtHPIP or mtHPIP–D4 (T7 tag) and HA–Cdc20 plasmid constructs. 48 h post-transfection, cell lysates were immunoprecipitated using T7 antibody and blotted as indicated. B, thymidine–nocodazole (Thy-Noc) synchronized HeLa cells were released into fresh medium and lysed, and cell lysates were immunoprecipitated using HPIP antibody and blotted as indicated. C, HPIP protein sequence at indicated region from human, mouse, and rat showing conservation of lysines at 274 and 634. D, HEK293T cells co-transfected with T7–HPIP, mtHPIP-K274R, or mtHPIP-K634R and HA–Cdc20 were harvested 48 h post-transfection, and cell lysates were blotted as indicated. E, HEK293T cells were co-transfected with either T7–HPIP or mtHPIP-K274R with or without GFP–Cdc20 and HA–Ub constructs. 48 h post-transfection, cell lysates were T7-immunoprecipitated and blotted with indicated antibodies. F, WST-1 assay demonstrating the effect of HPIP silencing followed by ectopic expression of T7–HPIP, mtHPIP–D4, or mtHPIP-K274R on HeLa cell proliferation. G, Western blotting demonstrating the knockdown of HPIP (lanes 2–5) and ectopic expression (rescue) of various HPIP mutants (lanes 3–5) in HeLa cells. The quantified results are presented as means ± S.D. using analysis of variance. **, p < 0.001; ***, p < 0.0001 were considered significant. Ctrl, control; IP, immunoprecipitation; sh, short hairpin; MW, molecular weight; WB, Western blot.