Fig. 1. Effect of bound antibodies, GA and CPZ on ErbB2 localization in SK-BR-3 cells. (A) ErbB2 in fixed, permeabilized cells detected by immunofluorescence. (B) Cells were warmed for 2 hours after binding Fl-anti-ErbB2 before fixation. (C) Cells were treated with GA for 2 hours before detecting ErbB2 by immunofluorescence. (D,E) Cells were pre-treated for 45 minutes at 37°C with GA, with (D) or without (E) 12 µg/ml CPZ, before binding anti-ErBb2 antibodies (left, green) and Alexa-Fluor-594-Tf (middle, red) for 1 hour on ice and warming for 2 minutes with the same drugs. Cells were acid-washed and processed for immunofluorescence, detecting ErbB2 with the Alexa-Fluor-488 Zenon mouse IgG labeling kit. Merged images are shown on the right. Scale bar: 10 µm. (F,G) Internalization of biotinylated Tf (F) or biotinylated anti-ErbB2 antibodies (G) was measured by CELISA after treatment with GA (circles) or both GA and CPZ (squares). Values shown are the mean ± s.e.m. of three experiments.