Effects of the PI-3K inhibitor, Ly294002, on activation of Rac-1 and p38 MAPK induced by IL-3 or cross-linking of the BCR.
(A) BMMCs from Balb/c mice or (B) LPS-stimulated B lymphoblasts from BDF1 mice were pretreated with Ly294002 or with ethanol (solvent), and subsequently stimulated for 5 minutes with PBS (con), IL-3 (1 μg/mL), or F(ab′)2 fragments of antimouse IgM (40 μg/mL). Activated, endogenous Rac-1 (Rac-1 GTP) was precipitated by GST-PAK bound to GT beads. Immunoblots were performed using a Rac-1–specific monoclonal antibody. Activation levels of endogenous Akt, Erk, and p38 MAPK were measured from WCLs using antibodies capable of specifically recognizing the phosphorylated form of these molecules (p-Akt, p-Erk, p-p38 MAPK). Equivalency of loading was confirmed by reprobing phosphoblots with antibodies capable of recognizing both phosphorylated and nonphosphorylated proteins (Akt, Erk, p38 MAPK).
