HER2 internalizes through a clathrin-coated mechanism. (A) Effect of HER2 and epidermal growth factor receptor (EGFR) trafficking by vaccine-induced anti-HER2 antibodies (HER2-VIA) and epidermal growth factor (EGF). HEK 293 cells transiently expressing HER2-RFP and EGFR-GFP were (a to c, g to i, m to o) left untreated, or were stimulated with (d to f) HER2-VIA, (j to l) EGF and (p to r) HER2-VIA/EGF at 37°C for 30 minutes. Confocal images from the same cells were taken before and after HER2-VIA and/or EGF ligand stimulation. Arrowheads indicate internalized receptors. Representative images from three experiments are presented. (B) Co-localization of HER2-VIA-stimulated HER2-YFP with the β2-adrenergic receptor (β2AR). Confocal images of unstimulated HEK293 cells expressing both (a) HER2-YFP and (b) β2AR-RFP. (c) Merged image of (a) and (b). ( d to f) Confocal images of cells simultaneously stimulated with 0.1 μM isoproterenol (Iso) and 100 μg/ml HER2-VIA at 37°C for 1 hour. (f) Merged image of (d) and (e). Arrowheads indicate co-localized vesicles. (C) Co-localization of HER2-VIA-stimulated HER2-YFP with transferrin. ( a to c) Confocal images of cells expressing the HER2-YFP and treated simultaneously with Alexa-546 transferrin (Tf) (100 μg/ml) and HER2-VIA (100 µg/ml) for 1 hour at 37°C. (c) Merged picture. Arrowheads indicate co-localized vesicles.