Endocytosis via DC-SIGN neck domain is clathrin-independent. (A) Clathrin or control siRNA knockdown immature DCs were incubated with anti-CRD (AZN-D1) or anti-neck (H200) antibody, and endocytosis was induced. Cells were analyzed by flow cytometry, and the percentage of internalization was determined. Experiments were performed in triplicate. Data represent mean values of 3 independent experiments ± SD. *P < .02. For H200: P > 0.5 (unpaired t test). (B) Immature DCs were incubated with antineck antibody (green) at 4°C, washed, and either kept on ice (steady state, 0 minutes) or shifted to 37°C for the indicated time points to trigger endocytosis. After fixation and permeabilization, clathrin was labeled (red), and the samples were analyzed by confocal microscopy. The pictures are enlarged areas taken from the cells shown in the small insets. Scale bar represents 2 μm. (C) Pearson colocalization coefficient plot of anti-CRD or anti-neck antibodies and clathrin of multiple cells in steady state (0 minutes) and after triggering endocytosis (≤ 5 minutes). One representative experiment of 3 is shown. *P < .01 (t test).