Fig. 6

Ubiquitin chain length and composition of ligand-activated EGFR. a Anti-EGFR blot of HeLa cells stably expressing EGFR-EGFP-3×FLAG. b Ubiquitylation of EGFR. After 100 ng ml⁻¹ EGF treatment, EGFR-EGFP-3×FLAG was immunoprecipitated with anti-FLAG antibody and subjected to immunoblotting with indicated antibodies. Parental wild-type HeLa cells were also analyzed (ctrl). Degraded EGFR is indicated by an asterisk. c Absolute quantification of ubiquitin linkages in immunoprecipitated EGFR-EGFP-3×FLAG. The gel region above 120 kDa was subjected to Ub-AQUA/MS analysis (mean ± s.d.; n = 2 biological replicates; Supplementary Data 7). d Ub-ProT analysis of ubiquitylated EGFR combined with UbiCRest assay. Immunoprecipitated EGFR was treated with K63 linkage-specific AMSH, K48 linkage-specific OTUB1, or nonspecific USP2, followed by Ub-ProT. e Time-dependent changes of ubiquitin chains on EGFR. After EGF treatment, EGFR-EGFP-3×FLAG was immunoprecipitated at the indicated times and analyzed by Ub-ProT. f A possible model for ubiquitin chain architecture of ubiquitylated EGFR