Fig. 7. Dab2-mediated receptor traffic requires binding to clathrin, LDLR and phosphoinositides. (A) Drawing of p96 and p67 LDLR constructs, different motifs, binding sites, PtdIns(4,5)P₂ (PIP₂) and FxNPxY sites are indicated. (B-G') Surface distribution of miniLDLR for cells depleted of both Dab2 and ARH, re-expressing vector alone, wild-type, or mutant T7-tagged mouse Dab2. miniLDLR was detected in non-permeabilized cells, and then cells were permeabilized and stained for T7-Dab2. Shown are 0.2 µm sections at the bottom surfaces of representative cells. Bar, 15 µm. (H,I) As in Fig. 6, a 100x100 pixel area was selected for approximately ten cells of each condition, and the average histogram for miniLDLR intensity was plotted for each siRNA (not shown). A treshhold (TH) was determined based on the distribution of miniLDLR in control cells, and the mean value above (H) this TH and mean value below (I) this TH was calculated for each condition. Overall pixel intensities were decreased compared with those in Fig. 6 owing to use of isotype-specific secondary antibodies that produce a less intense signal. Receptor remained clustered in control cells and double knockdown cells re-expressing T7-p96, as indicated by the higher mean (above TH) and lower mean (below TH), whereas receptor was not clustered in double knockdown cells re-expressing vector, T7-p67, T7-p96S122T or T7-p96K53Q (**P<0.001; *P<0.01; P<0.05). Bar, 15 µm.