Fig. 6

EGFR is accumulated in Hrs-depleted cells and its activation partially correlates with induction of the MEK1/2/ERK1/2-pathway. a Left panel: representative immunofluorescence images of pulse-chase experiments: shCT and shHrs#3 C2C12 were stimulated for 5 min with 50 ng/mL of EGF-488 (green) and after removing unbound ligand-chased for the indicated amount of time. Cells were probed with the anti-EEA1 to visualize EE and trafficking of EGF-488/EGFR was followed through the EE pathway. White arrows indicate colocalization of EGF-488 (in green) with the EEA1 EE marker (red) and DAPI (blue). Note the accumulation of EGF-488 in EEA1-positive compartments (yellow merge) in shHrs#3-depleted cells. Right panel: mask of colocalization between EGF-488- and EEA1-positive compartments. Scale bars, 20 μm. b Quantifications of colocalization of EGF-488 in EEA1 compartments. Analysis shows the percentage of EGF-488 overlapping with EEA1 and establishes the endocytic trafficking of EGF ligand through the EE pathway (EEA1 compartment). Open circles correspond to shCT and black squares to shHrs#3 conditions. The data represent mean +/− SEM. Analyses were done on 5 fields per condition on n = 3 experiments. Significance was assessed using a Mann–Whitney U test; ***p < 0.001; **p < 0.005. ns not significant. c Degradation of EGFR. shCT and shHrs#3 cells were stimulated with 50 ng/mL of EGF and with 10 μg/mL of cycloheximide to inhibit de novo synthesis of EGFR. Cells were recovered 15 (lanes 1,4), 60 (lanes 2,5), and 120 (lanes 3,6) min after stimulation and cells extracts were analyzed by Western blotting using anti-EGFR, -pY1068-EGFR, -ERK1/2, and -pT202/Y204ERK1/2. GAPDH was used as a loading control. d Quantifications of EGFR (left panel) and pY1068-EGFR (right panel) protein levels. Data are presented as the ratio of EGFR/GAPDH and pY1068-EGFR/GAPDH. Data are mean +/− SEM, n = 3–4 experiments. Significance was assessed using a two-way ANOVA test; **p < 0.01, ****p < 0.0001; ns not significant. e Representative Western blotting of protein extracts from shCT and shHrs#3 C2C12 collected in Pro (lanes 1 and 7) and at 7, 24, 48, 72, and 96 h of differentiation (lanes 2–6 and 8–12) and probed with anti-MHC, -EGFR, -pY1068-EGFR, and -GAPDH. f Representative immunofluorescence images of shCT and shHrs#3 C2C12 at 24 h of differentiation and probed with anti-pY1068-EGFR (green), -Rab5a (red) for EE and DAPI (blue). White arrows showed the pY1068-EGFR signal associated with Rab5a vesicles. Scale bar, 10 μm