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Figure 4


Fig. 4. Immuno-blocking CD81 mainly decreases surface-binding of POS by RPE cells. (A-D) Rat RPE-J cells were challenged with POS for 3.5 or 5 hours as indicated, in the presence of rat CD9 mAb RPM.7 (gray bars), rat and mouse CD81 mAb 2F7 (black bars), or non-immune IgG (white bars). (E) Human D407 RPE cells were challenged with POS for 5 hours in the presence of human CD81 mAb clone I.3.3.22 (black bars). (F) D407 cells were challenged with POS for 3.5 hours in the presence of human CD9 mAb MM2/57 (gray bar), human CD81 mAb clone I.3.3.22 or JS-81 (black bars a, b, respectively) or non-immune IgG (white bar). Antibody concentrations are indicated in µg/ml below each bar. Pre-incubation with antibody before addition of POS had no additional effect on POS binding (data not shown). Results are presented as mean ± s.d., n=3, of bound (A,C,E,F) and internalized (B,D) POS per RPE cell. In A and B Student's t-test was used to compare samples receiving tetraspanin antibody with samples receiving appropriate non-immune control antibody at the same concentration. Asterisks denote significant differences with P<0.05. (A,B) In 5-hour assays, CD81 antibody significantly inhibited both POS binding (P<0.01) and internalization (P<0.05) by RPE-J cells. CD9 antibody had no significant effect (P>0.05). (C,D) In 3.5-hour assays, CD81 antibody significantly inhibited only POS binding by RPE-J cells (P<0.01). CD9 antibody had no effect (P>0.05) and internalization was unaffected by either antibody (P>0.05). (E,F) Two different CD81 antibodies significantly inhibited POS binding by D407 cells (P<0.02). All other conditions did not cause significant changes (P>0.05). RPE-J cells without antibody bound 2.5 POS and internalized 1.1 POS per cell on average during a 5 hour assay. D407 cells without antibody bound 2.4 POS and internalized 1.4 POS per cell during a 5-hour assay. Hence, non-immune IgG did not significantly alter control uptake.





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