Fig. 3

EMP3 restricts RAB7 shuttling and ligand-induced degradation of EGFR in a TBC1D5-dependent manner. A Western blot showing the kinetics of EGFR phosphorylation (p-EGFR Tyr1068) and degradation (total EGFR) in U-118 control and EMP3 KO cells over the course of 2 h after treatment with 100 ng/mL EGF (n = 3). B Quantification of EGF-induced degradation of EGFR in U-118 control and EMP3 KO cells after treatment with 100 ng/mL EGF (n = 3). Band intensities of EGFR were normalized to β-actin, and log2-fold changes (mean + S.E.M.) were calculated relative to the EGFR level at t = 0 and plotted over time. (Welch’s two-tailed t-test; *P < 0.05). C Representative PLA images testing the association between p-EGFR and RAB7 after 30-min EGF treatment of U-118 control and EMP3 KO cells (n = 3). D Quantification of p-EGFR-RAB7 PLA signals from EGF-treated U-118 control and EMP3 KO cells. PLA signals were derived from 30 random fields from n = 3 independent experiments (10 fields/experiment). Signals were normalized to nuclei count per field (Welch’s two-tailed t-test; ****P =  < 0.0001). E Western blot showing TBC1D5 WT-mediated rescue of enhanced EGFR degradation in U-118 EMP3 KO cells (n = 3). F Quantification of EGF-induced EGFR degradation in U-118 control, EMP3 KO, EMP3 KO + TBC1D5 WT, and EMP3 KO + TBC1D5 R169A/Q204A (n = 3). Log2-fold changes (mean + S.E.M.) were calculated and plotted as described (Welch’s ANOVA with Dunnett’s T3 multiple comparisons test; *P < 0.05)