FIG. 4.
Role of clathrin in endocytosis of CNF1 and CNFY. HeLa cells were preincubated with chlorpromazine (CP) (15 μM) for 1 h at 37°C. Cells were then treated with GST-CNF1 (1 nM) for 1 h, with GST-CNFY (1 nM) for 2.5 h, or with diphtheria toxin (10 nM) for 5 h at 37°C in the presence of chlorpromazine. To analyze the effect of chlorpromazine on intoxication after cell lysis, a Rho shift assay was performed (A). Additionally, HeLa cells were cotransfected with a dominant negative form of Eps15 (Eps15mut) or a control plasmid (Eps15ctrl) and a His-tagged form of RhoA. After 48 h, cells were intoxicated with GST-CNF1 (1 nM) for 1 h or with GST-CNFY (1 nM) for 2.5 h at 37°C. Cells were then lysed, and a Rho shift assay was performed (B). (C) Cell rounding of HeLa cells by diphtheria toxin (DT) and inhibition of rounding in the presence of chlorpromazine as an inhibitor control. ctrl, control.