Fig. 3: USP2 precludes endocytic degradation of ErbB2 triggered by HSP90 inhibition in a DUB activity-dependent manner.

a SKBR3 cell lines with stable knockdown of USP2, AMSH, AMSHLP, and USP8 (two shRNAs per target and empty vector pLKO.1 as control) were treated with 80 nM of 17-AAG or DMSO for 10 h. Cell lysates were subjected to immunoblotting analysis using anti-ErbB2 antibody. GAPDH or Tubulin was probed to confirm equal loading. b quantification data of relative ErbB2 intensities from (a). c SKBR3 cells were transfected with constructs expressing GFP, GFP-AMSH, GFP-AMSHLP, GFP-USP8, GFP-USP2, and GFP-USP2(C276A) as indicated. Cells were treated with DMSO or 17-AAG (500 nM) for 4 h and then processed for immunofluorescence analysis using anti-ErbB2 antibody. Nucleus was stained with DAPI. Images show representative confocal sections. GFP-USP2 expressing cell was indicated with solid box. Scale bar = 10 μm. d Intensities of the membrane and intracellular pools of ErbB2 were quantified using results from C with Image Studio software (Version 4.0) and plotted. e SKBR3 cells were treated with 500 nM of 17-AAG for indicated times and lysed. ErbB2 was immunoprecipitated from cell lysates using mouse anti-ErbB2 antibody (clone 9G6). Cell lysates (input) and immunoprecipitation (IP) samples were analyzed by immunoblotting with indicated antibodies. Error bars represent standard error of the mean (n = 3), with n.s. not significant, *p < 0.05, and **p < 0.01.