Constitutively endocytosed mutant EGF receptors colocalize with early/recycling endosomal marker transferrin and late endosomal/lysosomal marker LAMP1. NSCLC cell lines H1650 (A), HCC827 (B), HCC4006 (C) or H1975 (D) were serum-starved for 48 hr and incubated with 10 μg/ml of AF546-transferrin (Tf) (red) for 30 min at 37°C. Cells were fixed, permeabilized and immunostained with anti-EGFR antibody 528 (green), followed by anti-LAMP1 antibody H4A3 (blue). Images were acquired under a confocal microscope at the medial plane. Arrows indicate EGFR and LAMP1 colocalization, and arrowheads indicate EGFR and labeled-transferrin colocalization. Bars represent 10 μm. (E) Colocalization coefficients for the green channel (EGFR) from (A), (B), (C), and (D) were obtained using LSM510 Image Examiner software, and depicted as percentages of EGFR colocalizing with Tf or LAMP1.