Fig. 1: HSP90 inhibitor-incurred ErbB2 ubiquitylation leads to its internalization and degradation. | Cell Death & Differentiation

Fig. 1: HSP90 inhibitor-incurred ErbB2 ubiquitylation leads to its internalization and degradation.

From: The deubiquitylase USP2 maintains ErbB2 abundance via counteracting endocytic degradation and represents a therapeutic target in ErbB2-positive breast cancer

Fig. 1

a ErbB2-positive breast cancer cells were treated with the HSP90 inhibitor 17-AAG (500 nM) for 4 h or left untreated (control) before immunofluorescence assays to examine ErbB2 localization. Images show representative confocal sections, with triangles pointing to intracellular ErbB2 punctae. Scale bar = 10 μm. b Cultured ErbB2-positive breast cancer cells were subjected to 17-AAG treatment at 500 nM for indicated times and lysed for immunoblotting using two clones of anti-ErbB2 antibodies. Tubulin was probed to show equal loading. Degradation curves show correlating quantification of relative ErbB2 intensities (0 h set as 100%). c SKBR3 cells were treated with 500 nM of 17-AAG for indicated times and harvested for flow cytometric analysis to examine surface levels of ErbB2. d SKBR3 cells were treated with 500 nM of 17-AAG for indicated times and lysed. ErbB2 proteins were immunoprecipitated using mouse anti-ErbB2 antibody (clone 9G6) and analyzed by immunoblotting with anti-ubiquitin antibody, with immunoblotting analysis of cell lysates performed in parallel to examine cellular levels of ErbB2 and Tubulin. e Cultured HeLa cells were treated with 20 ng/ml of EGF for indicated times and lysed. EGFR was immunoprecipitated from each sample and analyzed by immunoblotting with ubiquitin antibody to examine the ubiquitylation of EGFR. Cell lysates were also analyzed by immunoblotting to probe for EGFR and Tubulin. f Relative quantification data of ubiquitin signal after ErbB2 and EGFR normalization from (d) and (e), respectively. The samples showing highest intensities were considered as 100%, with 0.5 h and 1 h set for EGFR and ErbB2, respectively. All error bars represent standard error of the mean (n = 3).

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