Constitutive intracellular vesicular localization and phosphorylation of mutant EGF receptors in NSCLC cell lines. (A) Cells were growth factor-deprived by culturing in D3 medium (HBE135) or 0.1% FBS-containing growth medium (all other cell lines) for 48 hr and then either left unstimulated (- EGF) or stimulated (+ EGF) with 100 ng/ml of EGF for 30 min. Cells were fixed, permeabilized, and immunostained with anti-EGFR antibody 528. Images were acquired under a confocal microscope at the medial plane. Bar represents 20 μm. (B) Cells were growth factor-deprived as in (A) for 48 hr and either left unstimulated (-) or stimulated (+) with 10 ng/ml EGF for 10 min. The indicated amounts of whole cell lysate protein were used for immunoblotting with antibodies against the indicated proteins.