Monensin-treatment increases the accumulation of mutant EGF receptors in perinuclear endocytic vesicles. Cells were growth factor-deprived in D3 medium (HBE135) or 0.1% FBS-containing growth medium (all other cell lines) for 48 hr and then preincubated with DMSO or 10 μM monensin for 3 hr. Cells were loaded with 10 μg/ml AF546-Tf (Tf) (red) for 45 min at 37°C (with continuation of DMSO or monensin treatment). This was followed by incubation with (+ EGF) or without (- EGF) 10 ng/ml EGF for 30 min with continued DMSO or monensin treatment. Cells were fixed, permeabilized, and immunostained with anti-EGFR antibody 528 (green). Images were acquired under a confocal microscope at the medial plane. Bars represent 20 μm.