Figure 5

Mutant EGF receptors colocalize with Src in NSCLCcell lines. Cells were growth factor-deprived in 0.1% FBS-containing growth medium for 48 hr and then either left unstimulated (- EGF) or stimulated (+ EGF) with 100 ng/ml of EGF for 10 min. Cells were fixed, permeabilized, and immunostained with (A) anti-EGFR antibody 528 (green) followed by anti-Src antibody SRC2 (red) staining or (B) anti-phospho-EGFR antibody (red) followed by anti-phospho-Src antibody (green). Images were acquired under a confocal microscope at the medial plane. Arrows indicate EGFR and Src colocalization. Bars represent 20 μm. (C) Colocalization coefficients for the green channel (EGFR) from (A), and (D) colocalization coefficients for the red channel (pEGFR) from (B) were obtained using LSM510 Image Examiner software and normalized to the colocalization coefficient from unstimulated (- EGF) H1666 cells.