Fig. 5. Dab2-mediated endocytosis does not require AP-2. (A,B) Treatment with siRNA to µ2 adaptin causes loss of adaptin from coated pits. Single 0.2-µm sections at the bottom surfaces of representative cells are shown. (C-F) Steady-state surface levels of TfnR and miniLDLR were measured in control and siRNA-treated cells. Cell surface proteins were biotinylated. For TfnR analysis, lysates were immunoprecipitated using a mouse anti-TfnR antibody, and precipitated proteins were analyzed by western blot analysis first using streptavidin-HRP to detect biotinylated receptor (C, top), and then with rabbit anti-TfnR to detect total cellular TfnR (C, bottom). (E) Ratios of surface to total TfnR were determined for all conditions and compared with control cells. Depletion of AP-2 µ2 in combination with any other protein caused an increase in TfnR at the surface. For the miniLDLR, biotinylated proteins were precipitated with streptavidin-agarose beads and analyzed by western blotting using anti-HA antibody to detect the receptor (D, top). Total cell lysates were also analyzed to determine the total amount of miniLDLR (D, bottom). (F) Ratios of surface to total miniLDLR were determined for each condition and compared with controls. Depletion of AP-2 µ2 caused a large increase in miniLDLR at the surface only if Dab2 was also depleted. Error bars in E and F represent range of duplicate experiments. (G-M) Endocytosis of miniLDLR was measured in siRNA-treated cells. 4 µm Z-stack projections are shown for a representative field from each condition. No internal receptor was detected at 0 minutes (G). Following a 10-minute incubation, miniLDLR was internalized in control cells (H) but not in ARH/Dab2-depleted cells (I). Depletion of AP-2 µ2 alone (J) or in combination with ARH (K) did not affect miniLDLR endocytosis, but in combination with Dab2 (L) or Dab2/ARH (M), endocytosis was completely blocked. Bars, 15 µm.