Fig. 2. Endocytosis of LDLR, but not the TfnR, is inhibited in the absence of both ARH and Dab2. (A-F) LDLR antibody internalization. siRNA-treated cells on coverslips were incubated at 4°C with anti-LDLR for 1 hour, followed by internalization for 0 or 10 minutes at 25°C. Non-internalized antibody was removed by acid stripping and cells were fixed and permeabilized. Internalized antibody was detected by immunofluorescence using a DeltaVision microscope. A 4 µm flattened Z-projection of a representative cell is shown for each condition. Bar, 15 µm. (G) The internalization experiment was repeated three times and for each experiment approximately 100 cells were assessed for internalization for each condition. Percent of cells internalizing antibody was averaged for each experiment. Depletion of both ARH and Dab2 or CHC caused a significant reduction in LDLR endocytosis (**P<0.001, as determined by t-test). (H) On separate coverslips, knockdown efficiency was evaluated for each target by immunofluorescence. Approximately 100 cells were analyzed for each siRNA. (I-Q) LDLR antibody and Alexa Fluor-488–Tfn internalization. LDLR internalization was analyzed as for A-F with the addition of labeled-Tfn to assess TfnR endocytosis. A 4 µm Z-stack projection is shown for a representative field for control (I-K), Dab2/ARH-depleted (L-N), and CHC-depleted cells (O-Q). LDLR endocytosis was intact in control cells (I), but was inhibited by depletion of ARH/Dab2 (L) and CHC (O). Tfn was internalized in control (J) and ARH/Dab2-depleted cells (M), but not in CHC-depleted cells (P). Bar, 15 µm.