Figure 7. Ras-GTP and Raf-1 activation is required for gp120 to implement its downregulatory effect on Nef induced IL-6.

(A) Immunoblot analysis for the detection of Ras-GTP in immDCs. Ras-GTP activation was measured in presence of gp120; Nef, gp120 and kinase inhibitor (PP2); Nef, gp120 and DC-SIGN siRNA; Nef and gp120. The upper panel of the western blot analysis reveals that Ras-GTP gets activated in the presence of gp120, both in the presence (5^th lane) and absence (2^nd lane) of Nef. This shows that Nef has no role in the activation process. However, presence of kinase inhibitor (3^rd lane) and knock down of DC-SIGN (4^th lane) inhibit the activation of Ras-GTP. The lower panel of the blot denotes the total Ras in the cells. The densitometric analysis corresponds to the respective bands obtained in immunoblotting. Data represent the mean±SEM (n = 3). Statistical analysis was performed using Student's t-test, with the levels of significance defined as p*<0.05. (B) Western blot analysis of Raf-1 phosphorylation at ser338 residue in presence of gp120; Nef and gp120; Nef; Nef, gp120 and kinase inhibitor (PP2); Nef, gp120 and GW5074. The upper panel of the blot clearly shows that Raf-1 phosphorylation is facilitated by gp120. The lower panel denotes the total Raf-1 content of the cell. Data represent the mean±SEM (n = 3). (C) FACS analysis of Raf-1 phosphorylation at ser338 residue in presence of gp120; Nef and gp120; Nef; Nef, gp120 and kinase inhibitor (PP2); Nef, gp120 and GW5074 (Raf-1 inhibitor). The percentage on the upper right hand side of each histogram denotes the cell percentage positively stained.