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Fig. 5. CD81 depletion diminishes POS binding. RPE-J cells were transfected with either control siRNA (si-control), CD9-specific or CD81-specific siRNA (si-CD9 and si-CD81 respectively). 48 hours after transfection, protein expression (A-D) or phagocytic activity (E,F) of transfectants were evaluated. (A,B) Whole cell lysate representing equal numbers of cells were analyzed by western blotting with antibodies as indicated. (C) Quantification of western blots shows that CD81 and CD9 protein levels were reduced to 0.48±0.11-fold and 0.33±0.02-fold, respectively, by si-CD81 or si-CD9 transfection compared to si-control (mean ± s.d., n=3). (D) Transfectants, as indicated, were fixed with 4% paraformaldehyde before labeling with CD81 or CD9 antibodies (green). X-y maximal projections of representative images were acquired as described for Fig. 1. (E) Transfected cells were challenged with FITC-POS for 3.5 hours. X-y maximal projections of representative images show total FITC-labeled POS (green) taken up by the cells. Cell nuclei are shown in red in D and E. (F) Live transfectants were labeled on ice with 
v
5 antibody P1F6 and fixed before labeling with ZO-1 antibodies. X-z scans of representative areas show ZO-1 only or overlay of v5 and ZO-1 as indicated. Bars, 10 µm. (G) POS phagocytosis assays were analyzed by fluorescence scanning to quantify bound and internalized POS. Bars represent mean ± s.d., n=3, bound and internalized POS per RPE cell as indicated (white bars: si-control; gray bars: si-CD9; black bars: si-CD81). CD81 siRNA significantly reduced the numbers of bound POS compared to si-control (asterisk, Student's t-test, P<0.02). CD9 siRNA had no significant effect (P>0.05).
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