Figure 7.

USP36 disassembles K11-linked polyubiquitin chains from cIAP1 and K48-linked polyubiquitin chains from survivin. A, schematic diagram of ubiquitin mutants. B, HEK-293 cells were cotransfected with the indicated plasmids. After 20 h, the cells were treated with 20 μM MG-132 for 4 hours. Proteins were extracted, immunoprecipitated with Myc beads, and blotted with anti-Myc antibody. C, HEK-293 cells were cotransfected with the indicated plasmids. After 20 h, the cells were treated with 20 μM MG-132 for 4 h. Proteins were extracted, immunoprecipitated with Myc beads and blotted with anti-Myc antibody. D, HEK-293 cells were cotransfected with the indicated plasmids. Cells were treated with 0.4 μg/ml nocodazole for 16 h before harvesting. Proteins were extracted, immunoprecipitated with HA beads, and then analyzed for ubiquitination levels by Western blotting with an anti-histone (His) antibody. E, wildtype (WT) or K90R/K91R mutant survivin was cotransfected with ubiquitin (lanes 1 and 3) or in combination with Myc-USP36 (lanes 2 and 4). Proteins were extracted, immunoprecipitated with HA beads, and then analyzed for ubiquitination levels using Western blotting with the anti-ubiquitin antibody. F, HEK-293 cells were transfected with wildtype cIAP1 (lanes 1 and 3) or H588A mutant cIAP1 (lanes 2 and 4). Proteins were extracted, immunoprecipitated with Myc beads, and blotted with the Myc antibody. HA, hemagglutinin; HEK, human embryonic kidney; IAP, inhibitor of apoptosis protein; USP36, ubiquitin-specific protease 36.