Figure 5: The Sema4A^F350C proteins exhibit severe structural defects and impaired function.

(a) BN-PAGE and SDS–PAGE with an anti-Sema4A antibody were performed using cell lysates derived from COS-7 cells transfected with constructs expressing Sema4A-EGFP mutant proteins, or pEGFP vector (negative control). In BN-PAGE, the 222-kDa bands represent Sema4A dimers, while the 111-kDa bands represent Sema4A monomers. SDS–PAGE was performed after immunoprecipitation with an anti-Sema4A antibody, using the same lysate as BN-PAGE. (b) Representative images (top) and enlarged images (bottom) obtained by confocal microscopy. ARPE-19 cells were transfected with the plasmid constructs expressing Sema4A^F350-EGFP mutant proteins, incubated for 48 h and stained with phalloidin. Scale bar, 10 μm. (c) COS-7 cells were transfected with plasmid constructs and incubated for 48 h, stained with an anti-Sema4A antibody and analysed by flow cytometry. Structural diagrams for amino-acid residue replaced with F350 in each mutant are shown below the histograms, together with their apparent volume per molecule in ų (ref. 34). (d,e) Structural modelling of mouse Sema4A ectodomain, which was built using the structure of human Sema4D structural model previously reported¹⁵. (f) Immunofluorescent images of mouse RPE cells after H₂O₂ treatment (250 μM). Prosaposin (red) was peripherally distributed in wild-type (Sema4A^+/+) RPE cells but not Sema4A^−/− or Sema4A^F350C/F350C RPE cells. Scale bar, 5 μm. (g) Quantitative analysis of the normalized fluorescence intensity of prosaposin at the surface or perinuclear area of the respective RPE cells with (after 60 min) or without (0 min) H₂O₂ treatment (±s.e.m.; n=10). To quantify the intensity, we calculated the mean normalized intensity within the square with its side having an outer 1/6 of radius (‘surface’) or inner 1/6–3/6 of radius (‘perinuclear’). *P<0.01 (Student’s t-test). (h) Immunofluorescent images of mouse RPE cells using an anti-CRALBP antibody. Scale bar, 5 μm. (i) Quantitative analysis of the normalized fluorescence intensity of CRALBP on the surface or perinuclear area of RPE cells (±s.e.m.; n=10–15). *P<0.01 (Student’s t-test).