Figure 1. Characterization of EGFR:GRB2 PLA in cultured cell lines.

(A) Images of PC9 NSCLC cells, which have activating mutations in EGFR, fluorescently labeled by EGFR:GRB2 PLA (red), with an antibody targeting cytokeratin (green), and with DAPI (blue). (B) Images of PC9 or H520 cells labeled as in A. Immunoblot shows that H520 cells have little to no detectable EGFR. (C) Images of PC9 cells exposed to the EGFR inhibitor erlotinib and fluorescently labeled by EGFR:GRB2 PLA (red) and DAPI (blue). Images in A-C are representative of three independent experiments. Scale bars = 20μm. (D and E) Western blots of PC9 cells exposed to the indicated concentrations of erlotinib. In D, lysates were probed for activated EGFR with an antibody against Tyr¹⁰⁶⁸ phosphorylated EGFR (pEGFR). β-actin was used as a loading control. In E, lysates were subjected to coimmunoprecipitation with an antibody to GRB2 and blotted for EGFR. Blots in D and E are representative of three independent experiments.