αBTX binding induces internalization of AChR. (A and B) CHO-K1/A5 (A) or C2C12 cells (B) were incubated on ice without (−αBTX) or with αBTX (+αBTX) and chased at 37°C for 0 or 6 h in the absence or presence of the toxin. At the end of the incubation, surface levels of AChR were quantified by measuring the extent of anti-AChR mAb 210 binding to surface receptors. The bars in the top (CHO-K1/A5) and bottom histograms (C2C12) represent normalized fluorescence intensity of secondary AlexaFluor568 antibody directed against the primary anti-AChR monoclonal antibody mAb 210 versus that measured in untreated (toxin free) controls. In the case of C2C12 cells, AlexaFluor488-labeled αBTX was used for inducing internalization, and the quantification represents the ratio of mAb 210/αBTX fluorescence at 0 and 6 h, further normalized to ratio at 0 h. Each bar corresponds to weighted mean ± SEM (error bars) obtained from two independent experiments, with 150 cells for CHO cells and 25 cells for C2C12 cells. *, P > 0.1; **, P < 0.001; ***, P < 0.0001. Bars, 10 μm.