Skip to main content
. 2024 Jun 12;300(7):107463. doi: 10.1016/j.jbc.2024.107463

Figure 6.

Figure 6

USP36 promotes survivin and cIAP1 stability by inhibiting ubiquitination.A, HEK-293 cells were transfected with an empty vector or Myc-USP36 for 48 h. Cell lysates were harvested and analyzed for protein and mRNA levels of endogenous cIAP1 and survivin. Error bars denote s.e.m. (n = 3). B, HEK-293 cells were transfected with control or USP36-specific siRNAs. Western blot analysis of the protein levels using USP36, PARP, cIAP1, survivin, and GAPDH-specific antibodies. C, HCT 116 cells transfected with control or USP36 siRNA were treated with 20 μg/ml cycloheximide (CHX) for the indicated periods of time and analyzed via Western blot. D, HCT 116 cells were transfected with empty vector only, myc-cIAP1, or myc-cIAP1 plus HA-USP36. Cell lysates were subjected to Western blotting (left panel) or qRT-PCR analysis (right panel). E, HCT 116 cells were transfected with empty vector only, HA-survivin, or HA-survivin plus Myc-USP36. Cell lysates were subjected to Western blotting (left panel) or qRT-PCR analysis (right panel). F, Myc-cIAP1 was transfected into HEK-293 cells together with control or HA-USP36. Myc-tagged proteins were immunoprecipitated from cell lysates and blotted with the Myc monoclonal antibody. G, HEK-293 cells were transfected with Flag-cIAP1. After 24 h, the cells were treated with MG-132 (20 μM) for 1 h before being harvested. Ubiquitinated Flag-cIAP1 protein was purified with anti-Flag magnetic beads and eluted with 3×Flag peptide, followed by incubation with purified USP36 WT or USP36 C131A. Reaction mixtures were blotted with the anti-ubiquitin antibody. H, HEK-293 cells were cotransfected with the indicated plasmids. Cells were treated with nocodazole 400 nM (lanes 1 and 2) or 0.4 μg/ml (lanes 3 and 4) for 16 h before harvesting. Proteins were extracted, immunoprecipitated with HA beads, and blotted with the anti-ubiquitin (Ub) antibody. I, purified ubiquitinated survivin protein was incubated with purified proteins of either WT USP36 (lane 2) or USP36 C131A (lane 3) at 30 °C for 1 h, and the mixtures were blotted with the anti-ubiquitin antibody. HA, hemagglutinin; HEK, human embryonic kidney; IAP, inhibitor of apoptosis protein; USP36, ubiquitin-specific protease 36.