Figure 4.

D box but not KEN motif is required for HPIP degradation by APC-C/Cdc20. A, physical map of HPIP displaying the location of various D boxes and the KEN box present in it. B, HPIP domain conservation in human (h) and mouse (m). Conserved arginine and leucine residues at respective D box were replaced with alanine. Similarly, conserved lysine, glutamic acid, and asparagine residues at KEN box were replaced with alanine. C, HEK293T cells were co-transfected with various HPIP (T7 tag) mutant plasmid constructs and HA–Cdc20. 48 h post-transfection, the cell lysates were blotted as indicated. D, HEK293T cells were co-transfected with HA–ubiquitin and various combinations of plasmid constructs including wtHPIP or mtHPIP–D4 (T7 tag) and with or without GFP–Cdc20 constructs. 48 h post-transfection, T7 immunoprecipitates were blotted as indicated. E, T7–HPIP or mtHPIP–D4 transfected HeLa cells were synchronized by thymidine–nocodazole (Thy-Noc) block and released into fresh medium as indicated and blotted. IP, immunoprecipitation; MW, molecular weight; Vec, vector.