Figure 3. Expression, and Raft Distribution of CD4 Mutant RA5 in B16 Cells.

RA5 mutant (RA5-GFP) was expressed in B16-CXCR4 cells by transient transfection, examined for cell surface expression, analyzed for raft location by detergent insolubility and for HIV-1 envelope glycoprotein-mediated fusion. The results were compared with wt-CD4 (wt-CD4-GFP) under identical conditions, using GM95-CXCR4 cells as controls. Figure 3A shows the site of mutation in RA5 in the membrane proximal amino acid region (from RHRRR to AAAAA) (adapted from [25]). Figure 3B shows cell surface expression of RA5 and wt-CD4 in B16-CXCR4 or GM95-CXCR4 cells by immuno-staining with RPA-T4 Mab.

Figure 3C-E show results of CD4 and RA5 distribution in sucrose density gradient fractions of TX100-solubilized membranes of B16 cells. Fig. 3C and D show quantification of relative grey levels in various fractions normalized to the 60kD M.W. standards (see Methods section). We used GM3 as and internal raft marker in our gradient analysis that was quantified by dot-blot analysis using the GMR6 Mab. Figure 3C and D, solid bars, wt-CD4 and RA5, respectively. Figure 3C and D, diagonal bars, GM3 levels. The protein content for wt-CD4 and RA5 in various cell fractions is shown in Figure E.