Figure 5 : The Sema4AF350C proteins exhibit severe structural defects and impaired function.

From: A point mutation in Semaphorin 4A associates with defective endosomal sorting and causes retinal degeneration

Figure 5

(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 Sema4AF350-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 Å3 (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 Sema4AF350C/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).