Effects of vaccine-induced anti-HER2 antibodies on HER2 internalization. (A) Internalization of HER2-YFP by vaccine-induced anti-HER2 antibodies (HER2-VIA). HEK 293 cells transiently expressing HER2-YFP were stimulated with (a, b) HER2-VIA, (c, d) LacZ-VIA, or (e, f) trastuzumab, respectively for 1 hour. Confocal images from the same cells were taken before and after antibody incubation. Formation of vesicles in the cells indicated receptor internalization. The experiment was repeated three times. NS, non-stimulated. (B). Imaging endogenous HER2 internalization by HER2-VIA. SK-BR-3 and HCC1569 cells were allowed to grow for 24 hours and were treated as described in Materials and methods. Confocal images of cells that (a, e) were left untreated, or were treated with (b, f) HER2-VIA, (c, g) LacZ-VIA or (d, h) trastuzumab. (C) Endogenous HER2 internalization by HER2-VIA as assessed by cell surface Biotin-labeling. Upper panel: immunoblot (IB) for protected (internalized) biotin-labeled HER2 in cells treated with indicated agents/conditions. Lower panel: β-actin serves as loading control to ensure equal amounts of cell lysate. EGF, epidermal growth factor. (D) Effect of lapatinib on HER2-YFP internalization induced by HER2-VIA. Confocal images of HEK293 cells expressing HER2-YFP that (a) were left untreated, or were treated with (b) HER2-VIA, (c) lapatinib or (d) lapatinib then HER2-VIA for 1 hour. (E) The same transfected cells were immunoblotted (IB) for HER2 tyrosine phosphorylation (upper panel) and total HER2 expression (lower panel).