Fig. 2

CSMD1 interacts with EGFR. A Representative confocal z-stacked maximum projection images of PLA using MDA-MB-231 CTRL and CSMD1 cells. B Quantification of the dots per cell showing number of interactions between CSMD1 and EGFR. Binding between CSMD1 and EGFR was confirmed using co-immunoprecipitation (C) Cell lysates were immunoprecipitated using antibodies against EGFR and corresponding IgG control followed by immunodetection of CSMD1 protein. D Cell lysates were immunoprecipitated using antibodies against CSMD1 and corresponding IgG control followed by immunodetection of EGFR protein. E In ELISA setup an anti-EGFR antibody was used to capture the EGFR protein complexes lysed under non-denatured conditions and an anti-CSMD1 antibody applied to detect the EGFR-CSMD1 interactions. F MDA-MB-231 cell expressing CSMD1 and the control cells were transiently transfected with plasmid constructs that express extracellular domain of EGFR (ECD) and intracellular domain of EGFR (ICD) tagged with c-Myc. Representative blot detecting c-myc and confirming efficient transfection of the cells is shown. G The anti-c-myc capturing antibody was immobilized in ELISA plates, incubated with cell lysates followed by an anti-CSMD1 antibody to detect the myc-ECD-EGFR-CSMD1 and myc-ICD-EGFR-CSMD1 complexes. H Colocalization of CSMD1 and EGFR. MDA-MB-231 CSMD1-expressing cells (c1 clone) were fixed and stained with anti-EGFR (red) and anti-CSMD1 (green). DAPI (blue) was used to stain the nuclei. Scale bars 10 μm. 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. Unpaired t-test was used when comparing 2 samples, one-way ANOVA Dunnett’s multiple comparisons test was used when compared 3 or more samples, and 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)