ActD induces ubiquitin-independent but proteasome-dependent degradation of MCL-1. (A) Representative western blot analysis of Trp53^+/+;Eμ-Myc lymphoma cells treated for 4 hours with 5 nM ActD, with or without 20 μM quinoline-Val-Asp-difluorophenoxymethylketone (QVD-OPh; QVD). Expression of antiapoptotic MCL-1 (arrowhead), determined as in Figure 3A, is shown as relative to MCL-1 levels in NT cells in the absence of QVD (n = 3). (B) Co-IP of NOXA, PUMA, and BAK with MCL-1 in Trp53^+/+;Eμ-Myc lymphoma cells treated for 4 hours with 5 nM ActD compared with NT cells (n = 3). Indicated band (arrowhead) corresponds to the immunoglobulin light chain (IgL) of the antibodies used for IP. (C) Ubiquitination assay in MCL-1 immunoprecipitates from Trp53^+/+;Eμ-Myc and Trp53^–/–;Eμ-Myc lymphoma cells treated for 6 hours with 5 nM ActD compared with NT cells (n = 2). (D-E) FLAG-tagged MCL1 stability of either FLAG-tagged MCL-1^wt- (D) or FLAG- (E) tagged MCL-1^KR-overexpressing Eμ-Myc lymphoma cells determined by CHX chase after treatment with or without 5 nM ActD for 4 hours. Line graphs display FLAG-tagged MCL1 expression (arrowhead) over time determined from MCL-1 immunoblots (supplemental Figure 4F-G) as in Figure 3E (n = 2). (F) Representative western blot analysis of Trp53^+/+;Eμ-Myc lymphoma cells treated for 30 minutes with 1.25 µM MG132 before treatment with ActD for 4 hours. Expression of antiapoptotic MCL-1 (arrowhead), calculated as in Figure 3B, is shown as relative to MCL-1 levels in NT cells in the absence of MG132 (n = 4). Data are presented as mean ± SEM. *P < .05; **P < .01 (2-way ANOVA test).