Fig. 6. CD81 overexpression increases POS binding by RPE cells. RPE-J cells were transfected with plasmids encoding mouse CD81 (mCD81), CD9 (mCD9), or empty vector as indicated. 48 hours after transfection, tetraspanin expression (A-D) and phagocytic activity of transfectants (E) were evaluated. (A) RPE proteins, as indicated, were analyzed by immunoblotting of transfectant lysates from equal numbers of cells. CD81 antibody Eat2 recognized both endogenous rat and transfected mouse CD81 protein. Mouse CD9 antibody KMC (mCD9) and rat CD9 antibody RPM.7 (rCD9) recognized exogenous mouse and endogenous rat CD9, respectively. (B) Quantification of western blots shows that CD81 transfection increased CD81 protein level to 2.1±0.4 fold of empty vector transfected cells (mean ± s.d., n=3). mCD9 plasmid transfection did not change CD81 expression. (C) Transfected CD81 and CD9 localized to the apical surface of transfected RPE-J cells. Cells were fixed with 4% paraformaldehyde before labeling CD81 and mouse CD9 (mCD9) as indicated (green). Panels show x-y maximal projections acquired as described for Fig. 1 of representative fields. (D) Transfected cells were challenged with FITC-POS for 3.5 hours. Maximal projections of representative fields show total (bound plus internal) FITC-POS (green) taken up by the cells. Cell nuclei were stained with DAPI (red) in C and D. Bars, 10 µm. (E) 3.5-hour POS phagocytosis assays were analyzed by fluorescence scanning to quantify bound and internal POS. Bars represent mean ± s.d., n=3, bound POS and internalized POS per RPE cell transfected with empty vector (white bars), CD9 (gray bars), or CD81 (black bars) expression plasmids. CD81 overexpression significantly increased the numbers of bound POS but not of internal POS as compared to vector control (asterisk, Student's t-test, P<0.03). CD9 transfection had no effect (P>0.05).