Fig. 5
Kinases Lyn and Syk selectively regulate Ras isoform’s activation through Ras-GEFs. a CD40 activates the upstream kinases Lyn and Syk in a dose-dependent manner, with lower doses activating Syk and higher doses activating Lyn. b Immunoblot analysis of total Ras and activated Ras isoforms in the lysates of un-transfected or Syk- or Lyn-specific siRNA transfected, anti-CD40 antibody (3 μg/ml) treated P388D1 cells. c Immunoblot analyses of phospho-tyrosine (p-tyr-Vav)), and Vav translocated Sos-1/2 (Tr-Sos-1/2), translocated Ras-GRP (Tr-RasGRP), CD71, syk, lyn and β-actin in the lysates of untreated or Syk siRNA or Lyn siRNA or anti-CD40 antibody (3 μg/ml) treated P388D1 cells. d Immunoblot analysis of phospho-tyrosine (p-tyr Vav) and Vav translocated Sos-1/2, translocated Ras GRP, CD71, Lyn and phospho-Lyn, Syk and phospho-Syk (p-syk) in the lysates of untreated or Syk inhibitor (Syk Inh, 3 μM; Calbiochem, San Diego, CA) or PP-1 (340 nM; BIOMOL International, PA) treated and/or anti-CD40 antibody (3 μg/ml) treated macrophages. e Analysis of the association of Syk or Lyn with Sos-1/2, Vav, and Ras-GRP in the co-immunoprecipitates from the lysates of anti-CD40 antibody (1 μg/ml, 3 μg/ml and 6 μg/ml) treated macrophages.