Fig. 3

Ras isoforms differentially associate with Ras-guanine nucleotide exchange factor (Ras-GEF) in CD40 signaling. a, b CD40 activates the GEFs in a dose-dependent manner. Higher dose activates Ras-GRP and Vav whereas lower dose activates Sos-1/2. Immunoblot analysis of Vav or phospho tyrosine, CD71, translocated (Tr) Sos-1/2 or RasGRP in the lysates of macrophages treated with the indicated concentrations of anti-CD40 antibody (a). Densitometric quantifications of the blots are shown in the bottom panel (b). c Analysis of the association of Sos-1/2, Ras-GRP and Vav after immunoprecipitation with H-Ras, K-Ras and N-Ras in the lysates of macrophages treated with the indicated doses of anti-CD40 antibody. H-Ras and K-Ras associate with Ras-GRP whereas N-Ras associates with Sos-1/2 in macrophages stimulated with the indicated doses of anti-CD40 antibody. Also shown is the association of Ras-GRP with Vav by co-immunoprecipitation in the above-mentioned macrophage lysates. Vav associated with Ras GRP. Cells were stimulated for 3 min. d The siRNA for GEFs (Sos-1/2, Vav, and Ras-GRP) silenced respective GEFs in siRNA treated-inset. e-g Immunoblot analysis of total Ras and activated Ras isoforms in the lysates of un-transfected or Sos-1/2-, Vav-, Ras-GRP-specific siRNA transfected P388D1 cells stimulated with anti-CD40 (3 μg/ml) for 7 min. Sos-1/2 siRNA reduced N-Ras activation (e) whereas Vav siRNA and Ras-GRP siRNA reduced H-Ras and K-Ras activation (f, g).