Fig. 4: The USP2 inhibitor ML364 accelerates ErbB2 turnover via inducing its ubiquitylation and potentiates HSP90 inhibitors to degrade ErbB2.

a, b SKBR3 and HCC1954 cells were treated with cycloheximide (100 μg/ml) in the absence or presence of 10 μM of ML364 for indicated times and lysed. Samples were analyzed by immunoblotting with indicated antibodies. Actin blots show equal loading. Turnover curves show relative quantification of ErbB2 expression at corresponding time points. c AU565 cells were treated with 10 μM of ML364 for indicated times. Cell lysates were prepared and analyzed by immunoblotting using indicated antibodies. Actin was probed to show equal loading. d AU565 cells were treated as in (c), and ErbB2 was immunoprecipitated using mouse anti-ErbB2 antibody (clone 9G6) from cell lysates, before immunoblotting assays to probe for ubiquitin and ErbB2. Column chart on the right shows the quantification of relative ubiquitin signal after ErbB2 normalization. e AU565 cells were treated with 17-AAG at 80 nM in the absence or presence of 10 μM of ML364 as indicated for 12 h. ErbB2 was immunoprecipitated using mouse anti-ErbB2 antibody (clone 9G6) from cell lysates and analyzed by immunoblotting to examine the ubiquitylation status of ErbB2 along with cell lysates. Column chart on the right shows the relative quantification of ubiquitin signal detected from immunoprecipitations after ErbB2 normalization. f–h SKBR3, AU565, and HCC1954 cells were treated with ML364, ganetespib, PU-H71, or various combinations at indicated concentrations for 12 h and lysed. Cell lysates were subjected to immunoblotting analysis with indicated antibodies. Actin blots are shown to confirm equal loading. The column charts show corresponding quantification of relative ErbB2 levels. Error bars represent standard error of the mean (n = 3), with *p < 0.05 and **p < 0.01.