Fig. 6

Ubiquitylation governs the endocytic degradation of mutant EGFR. a, serum-starved A549 and HCC827 cells were treated with EGF for indicated times. EGFR was immunoprecipitated and analyzed by immunoblottings along with input samples using indicated antibodies. The ubiquitin signal on EGFR was quantified compared to 0 h of each group and plotted. All error bars represent the standard error of the mean (n = 3), and * indicates p < 0.05. b, HCC827 cells were treated with lapatinib (0.5 μM), gefitinib (0.5 μM), or DMSO as control for 6 h in the presence of chloroquine (100 μM). Then EGFR distribution was examined by immunofluorescence. c, HCC827 cells were treated with lapatinib, gefitinib, or DMSO as control for 12 and 24 h, before processed for immunofluorescence analysis of EGFR staining. Images were captured using a fluorescent microscope (Olympus BX63, 40X objective). Scale bar = 10 μm. d, HCC827 cells were treated with DMSO, lapatinib, or gefitinib for 2 h and lysed. EGFR was immunoprecipitated and analyzed by immunoblottings with indicated antibodies. The column chart shows the quantification data of ubiquitin signal from immunoprecipitated EGFR. e, HEK293T cells were transiently transfected with control, EGFR, and exon 19 deletion mutant-expressing pCDH plasmids. EGFR proteins were immunoprecipitated and ubiquitin signal was detected by immunoblotting. f, EGFR proteins were immunoprecipitated from H1299 cell lines stably expressing wild-type or the exon 19-deleted EGFR before analyzed by immunoblotting with ubiquitin antibody