β2 receptor internalization in cells depleted of clathrin. A, after preincubation at 4 °C for 1 h with the mouse anti-HA antibody (IgG1) to label the β2 receptor on the plasma membrane, HeLa cells, mock-treated (Control) or depleted of clathrin (Clathrin siRNA), were incubated at 37 °C for 30 min in the absence of the ligand. After fixation, the cells were incubated with 488-GAM to label the remaining surface receptor followed by 594-GAM with saponin to label the internalized receptor. The images were taken with the pinhole completely open (the optical section was about 12 μm), and images shown are representative of experiments that were repeated three times. B, same as A but in the presence of ligand (Iso, 1 mm). C, quantification of the β2 receptor internalization at 30 min, with or without depletion of clathrin, in the absence or in the presence of ligand, using a single cell-based method (see “Experimental Procedures” for details). The bar graph shows the percentage of the internalized cargo compared with the total (surface and internalized) of between 50 and 100 cells ± S.D. for each treatment of a representative experiment that was repeated two additional times. The difference between β2 receptor internalization in cells, with or without depletion of clathrin, was significant in both cases, in the absence and in the presence of ligand (*, p < 0.01, and **, p < 0.001).