Figure 3. The Sema4A^F350C proteins are mis-localized in RPE cells.

(a) Representative images of immunostaining of EGFP-tagged Sema4A proteins in RPE cells in Sema4A^WT/WT, Sema4A^D345H/D345H, Sema4A^F350C/F350C and Sema4A^R713Q/R713Q retinas using DAB (top) or fluorescence (bottom). EGFP is shown in brown (top) or green (bottom), and nuclei were visualized with SYTO 62 staining as shown in blue (bottom). Fluorescent signal of EGFP was enhanced by immunostaining using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies (bottom). Scale bar, 2 μm. (b) Immunofluorescent images of primary cultured RPE cells derived from Sema4A^WT/WT, Sema4A^D345H/D345H, Sema4A^F350C/F350C and Sema4A^R713Q/R713Q mice. EGFP was stained with anti-GFP and Alexa Fluor 488-conjugated secondary antibodies to enhance the GFP signals (green), and the cytoskeleton was visualized by staining with Alexa Fluor 546-conjugated phalloidin as shown in red. Scale bar, 5 μm. (c) Expression of Sema4A on the plasma membrane. COS-7 cells were transfected with plasmid constructs encoding Sema4A^WT-EGFP, Sema4A^D345H-EGFP, Sema4A^F350C-EGFP and Sema4A^R713Q-EGFP and incubated for 48 h. Subsequently, the cells were stained with an anti-Sema4A antibody and analysed by flow cytometry. Data are representatives of three experiments. (d) ARPE-19 cells were transfected with plasmid constructs expressing Sema4A^WT-EGFP, Sema4A^D345H-EGFP, Sema4A^F350C-EGFP and Sema4A^R713Q-EGFP, incubated for 48 h, fixed, stained with Alexa Fluor 546-conjugated phalloidin, and then examined by confocal microscopy. Representative (top) and enlarged images (bottom) are shown. Scale bar, 10 μm.