Fig. 4.

EGFR endocytic trafficking in the presence of CSMD1. A Detection of EGFR dimers upon EGF (25 ng/ml for 15 min) and GEF (10 μM) stimulation followed by crosslinking with BS³. B Densitometry of dimers of EGFR normalized to βtubulin (C) CSMD1 triggers EGFR ubiquitination which was detected using EGFR immunoprecipitation followed by immunoblotting with anti-Ub antibody. Representative blots from three independent experiments are shown. D Representative immunoblots of EGFR levels in lysates of MDA-MB-231 CTRL and CSMD1 overexpressing clonal cells, pre-treated with translation inhibitor cycloheximide (CHX) (100 μg/mL) for 2 h and treated with EGF (25 ng/ml) for 0, 4, 8, 12 h (h). E Quantification of EGFR levels in MDA-MB-231 cells plotted against time. Representative immunoblots of EGFR levels in CTRL and CSMD1 overexpressing cells, pre-treated with CHX (100 μg/mL) for 2 h followed by treatment with 25 ng/mL EGF in the presence of (F) NH₄Cl and (G) MG132 for 12 h. H Quantification of EGFR recovery in both NH₄Cl or MG132 treated CSMD1 and CTRL cells. I Representative confocal images of CTRL and CSMD1 BCCs co-stained for EGFR (red), CSMD1 (blue), EEA1 (green), nucleus (SYTOX orange). Quantification of colocalization of (J) EGFR-EEA1, (K) EEA1-CSMD1 and (L) CSMD1-EGFR complexes. M Representative confocal images of CTRL and CSMD1 BCCs co-stained for EGFR (red), CSMD1 (blue), LAMP1 (green), nucleus (SYTOX orange). Quantification of colocalization of (N) EGFR-LAMP1 and (O) LAMP1-CSMD1 complexes. Scale bars 5 μm. All experiments were repeated at least 3 times with bars indicating mean ± SD, grey circles correspond to independent data points for CTRL and CSMD1 groups, respectively. A two-way ANOVA Bonferroni’s multiple comparisons test was used when comparing CTRL and CSMD1 groups (* < 0.05, **** < 0.0001). A one-way ANOVA Dunnetts’s multiple comparisons test was used when comparing CSMD1 cells in different time-points (* < 0.05, **** < 0.0001)