Fig. 6. Surface distribution of miniLDLR is altered in the absence of Dab2. (A-E) Surface miniLDLR distribution in non-permeabilized siRNA-treated cells. The miniLDLR was detected with anti-HA antibody and visualized using a DeltaVision microscope. Single 0.2-µm sections at the bottom surfaces of representative cells are shown. Dab2 and ARH immunofluorescence (not shown) was used to confirm depletion. (F) To quantify receptor distribution, a 100x100 pixel area was analyzed for 10-14 cells of each condition. The average histogram values for these images were plotted. Punctate staining (as in control and ARH-depleted cells) results in a high number of black pixels (arrowhead) and bright white pixels spread out over a broad range of intensities (arrow, magnified region). Diffuse staining (as in Dab2, Dab2/ARH, and CHC siRNA cells) results in fewer black pixels, more grey pixels (open arrowhead) and fewer white pixels. Based on the histogram for control cells, a threshold (TH) was set. Pixels above this TH indicate clustered receptor and below this threshold indicate diffuse receptor. Similar results were obtained in independent experiments. (G) A plot of the mean pixel intensity above and below the TH shows that in Dab2, ARH/Dab2, and CHC siRNA-treated cells, there is less clustered receptor and more diffuse receptor compared with control cells and ARH siRNA treated cells (**P<0.001). (H-L) Surface TfnR distribution was compared in siRNA-treated cells. Only in CHC-depleted cells is there a change in receptor localization, where there is more diffuse receptor accumulating on the surface. Bars, 15 µm.