Characterization of vaccine-induced anti-HER2 antibodies by flow cytometry and western blot analysis. (A) Recognition of cell-surface HER2 by vaccine-induced anti-HER2 antibodies (HER2-VIA). HCC1569 cells were incubated with a mouse anti-human-HER2 mAb (HER2-phycoerythrin (PE)) or isotype control (Becton Dickinson (BD), San Jose, CA, USA), or with diluted (1:200) mouse serum antibodies (HER2-VIA or LacZ-VIA) for 1 hour at 4°C. Samples were then analyzed by BD LSRII flow cytometry, with results represented as histograms. (B) Recognition of total HER2 but not epidermal growth factor receptor (EGFR)-GFP by HER2-VIA. HER293 cells expressing vector (lane 1), HER2-FLAG (lane 2) or EGFR-GFP (lane 3) as well as SK-BR-3 cells lysates were western blotted with HER2-VIA (top panel), anti-FLAG antibody (upper middle panel), and anti-GFP antibody (lower middle panel). β-actin was used as a loading control (bottom panel). IB, immunoblot.