αBTX-induced AChR endocytosis is mediated by Src phosphorylation. (A) CHO-K1/A5 cells were cotransfected with dominant-negative Src K297R and GFP, and the internalization of AChR induced by αBTX was assessed in cells positive for Src K297R. Note the absence of punctate structures in cells transfected with dominant-negative Src K297R. (B) CHO-K1/A5 cells were labeled with b-αBTX in the presence or absence of 10 μM PP2 and chased for 6 h. Surface AChR levels were determined by measuring the accessibility of b-αBTX to SA-PE. The histogram shows the quantification of surface-accessible AChR in the presence or absence (αBTX alone) of PP2. Each bar represents the weighted mean of internal AChR as estimated using flow cytometry data from 10,000 cells (see Materials and methods) normalized to cells labeled with b-αBTX on ice. Error bars represent SEM. *, P < 0.001. (C) CHO-K1/A5 cells treated without or with PP2 were incubated with αBTX for 2 h, and cell lysates from the same cells were analyzed by Western blotting for phosphorylated Src (antiphospho–Tyr418-Src) and total Src. Note that αBTX induces Src phosphorylation, which, in turn, is inhibited by pretreatment with PP2. Bar, 10 μm.