EGF enhances direct interaction between EGFR and TRPM7 in a c-Src-dependent manner
(A) EGFR was detected when TRPM7 was immunoprecipitated in rVSMCs using anti-TRPM7 antibody (left panel) and TRPM7 was detected when EGFR was immunoprecipitated using anti-EGFR antibody (right panel) (n=3–4). (B) Representative confocal microscopy images of rVSMCs co-immunostained for TRPM7 (Alexa 488, green) and EGFR (Alexa 555, red). Nuclei were stained with DAPI (n=3). (C,D) PLA was used to visualize and quantify TRPM7-EGFR interaction in rVSMCs stimulated with vehicle (veh), and EGF (50 ng/ml) in the presence and absence of gefitinib (1 µM) and PP2 (10 µM). WGA (Alexa 488, green) was used to stain cell membranes. Red fluorescence is identified as PLA positive signals. Orange fluorescence in the merged figure identifies co-localization. PLA signals were quantified using the Analyse Particles plugin of ImageJ software. For each condition, quantifications were performed from at least 100 cells and expressed as the mean number of signals per cell (percentage of control). Scale bar =10 µm. Arrows indicate co-localization of EGFR and TRPM7. *P<0.05 compared with vehicle control (veh, PBS) and †P<0.05 compared with EGF-treated cells.