Fig. 8. CD81 antibody inhibits POS binding by 5^+/+ mouse RPE and RCS (MerTK-deficient) rat RPE but not by 5^–/– mouse RPE, whereas CD9 antibody has no effect. (A-F) Live, polarized, primary RPE from 5^+/+ mice, 5^–/– mice, and RCS rats were labeled with CD81 (A-C) or CD9 (D-F) antibody, fixed, and labeled with ZO-1 antibody to visualize tight junctions. A-F show x-y maximal projections acquired by confocal microscopy as described for Fig. 1. CD81 and CD9 apical surface labeling did not differ between samples prepared from animals of different genotype. (G,H) Total FITC-POS uptake by 5^+/+ primary RPE after 1 hour of POS challenge was visibly reduced by CD81 antibody but not by non-immune (n.i.) antibody at 50 µg/ml. Representative fields show maximal x-y projections of FITC-POS, nuclei, and ZO-1 tight junctions. Bars, 10 µm. (I) Primary RPE cells were challenged with FITC-POS for 1 hour in the presence of non-immune (n.i.) IgG (white bars), CD81 (black bars) or CD9 (gray bars) mAbs at 20 or 50 µg/ml as indicated below each bar. Antibodies used were CD81 mAb clone Eat2 and 2F7, anti-mouse CD9 mAb KMC, anti-rat CD9 mAb RPM.7. Fluorescence scanning quantification of bound FITC-POS shows that CD81 antibodies inhibited POS binding by 5^+/+ RPE and RCS rat RPE, but had no effect on the residual POS binding by 5^–/– RPE (black bars). By contrast, CD9 antibodies did not change POS binding by any of the three types of RPE (gray bars). Bars represent mean number of POS bound per RPE cell ± s.d., n=3. Asterisk denotes significant inhibition of POS binding compared to appropriate non-immune antibody control at the same dose (P<0.01, Student's t-test). None of the conditions affected POS internalization (data not shown).