Fig. 3

EGFR signaling cascade in the presence of CSMD1. A Representative western blots of total lysates CTRL and CSMD1 MDA-MB-231 cells immunodetecting phosphorylated EGFR at the residue Y1068 (pEGFR Y1068) and total EGFR with β-tubulin used as a loading control upon stimulation with EGF (25 ng/mL), TGF-α (25 ng/mL) and AREG (100 ng/mL) for 30 min. CTRL and CSMD1 MDA-MB-231 cells were serum starved for 2 h and then treated with 25 ng/mL EGF for the indicated time points (5, 15 and 30 min). Unstimulated cells were also included in the experiment. B Immunoblot analysis of phosphorylated EGFR at the residue Y1068, total EGFR and β-tubulin used as a loading control. C Densitometry of pEGFR Y1068 normalized to total EGFR. D Densitometry of pEGFR Y1068 normalized to β-tubulin, and (E) densitometry of total EGFR normalized to β-tubulin. F Immunoblot analysis of phosphorylated Akt at the residues Ser473 (pAkt Ser473) as well as Thr308 (pAkt Thr308), total Akt and GAPDH used as a loading control. G Densitometry of pAkt Ser473 normalized to total Akt. H Densitometry of pAkt Ser473 normalized to GAPDH, (I) densitometry of pAkt Thr308 normalized to total Akt and (J) densitometry of pAkt Thr308 normalized to GAPDH. All experiments were repeated at least 3 times with bars indicating mean ± SD, black and 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 3 or more groups with 2 variables (* < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001)