Figure 3: Sorting of EGF-bound and stress-internalized EGFR onto separate MVB subsets is WASH dependent.

(a) Control flox/flox and WASH knock-out (WASHOUT) MEFs transfected with human EGFR were exposed to UVC and incubated for 1 h to allow stress-induced EGFR internalization in the presence of anti-EGFR 108 antibody. Cells were washed, incubated with EGF-AlexaFluor 488 for 30 min (red), and processed for immunofluorescence with an AlexaFluor 555 secondary antibody to label EGFR (green). Both EGF+ve and EGF-ve EGFR-containing endosomes are present in control flox/flox, but these are largely merged in WASHOUT MEFs. (b) Control flox/flox and WASHOUT MEFs were co-transfected with human EGFR and OA1-myc or PMEL, and either treated with EGF for 30 min or exposed to UVC and incubated for 1 h. Cells were co-stained for EGFR (green) and the following markers (in red): myc (top panels), fibrillar PMEL (central panels) or non-fibrillar PMEL (bottom panels). (c) Quantification of co-localization between EGFR and expressed markers for the different conditions. Data are mean±s.e.m. of three independent experiments, ***P<0.001 (Student’s t-test). Scale bars, 10 μm; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.