CTxB does not affect CLIC endocytosis. (A) Cav1^−/− MEFs were incubated with HRP in the presence or absence of CTxB for 15 s. Cells were cooled and DAB reaction perfomed in the presence of ascorbic acid (AA) before fixation. Bars, 200 nm. (B) Quantitation of the number of HRP-filled carriers per cell across 10–12 cells treated as in A. CLICs were defined by their characteristic ring-shaped morphology, CCVs were defined as coated vesicular carriers. Error bars show SEM. (C) Cav1^−/− MEFs were grown in normal media (plus energy), media containing 2-deoxyglucose (no energy), or 2-deoxyglucose media for 1 h followed by a 1-h washout in normal media (recovery) before CTxB-HRP internalization and DAB reaction. Labeled structures were counted across 10 cells. Error bars show SEM. (D and E) Cav1^−/− MEFs were incubated with CTxB-HRP for 5 min before the DAB reaction for either 5 min at 37°C or 10 min at 4°C, followed by fixation at 37°C. All CTxB-HRP–positive structures were counted in 10–12 cells across two independent experiments. Error bars show SEM. Bar, 200 nm. (F) NIH3T3s were treated for 20 min with vehicle (Untreated) or Dyngo4a before internalization of CTxB-555 (left panels) and Tf-647 (right panels) for 5 min. Cells were placed on ice and acid stripped to remove surface labeling. Cells were then bound with CTxB-488 (middle panels). Bar, 10 µm. (G) NIH3T3 cells were left untreated or were treated with Dyngo4a before internalization of CTxB-HRP, followed by DAB reaction. Arrows show CTxB-HRP–laden CLICs, double arrowheads show internalized, labeled CCVs, arrowheads show surface connected unlabeled CCPs. Bars, 200 nm.