Purified B cells were left at 37°C for 10 min in chamber buffer (PBS, 0.5% FCS, 1 g/liter d -Glucose, 2 mM MgCl2, and 0.5 mM CaCl2) to equilibrate before stimulation. They were then stimulated for various times with 10 µg/ml anti-IgM F(ab′)2 fragment (Jackson ImmunoResearch Laboratories), 10 µg/ml anti-κ (HB558; American Type Culture Collection), or 1 µg/ml CD40L (R&D Systems).
For Cdc42-GTP pull-down experiment, a commercial kit (Cytoskeleton Inc.) was used following the manufacturer’s instructions, specifically using 107 B cells for 15 µg of beads. For immunoblotting, stimulated cells were then lysed in lysis buffer (20 mM Tris-HCL, pH 8.0,150 mM NaCl, 5 mM EDTA, Protease Inhibitor cocktail [Roche], 10 mM NaF, 1 mM Na3VO4, and 1% NP-40) for 15 min on ice, and samples were loaded into 4–20% (for Cdc42-GTP pull-down) or 12% (for all other applications) for electrophoresis. Proteins were detected with antibodies against pErk, pAkt (S473), pCD19, pSyk, pSrc, totalErk, pSTAT3 (Ser), and Cdc42 (all from Cell Signaling Technology) using the secondary HRP-conjugated anti–rabbit or anti–mouse antibodies (Jackson ImmunoResearch Laboratories).
Blot densitometry analysis was performed using the ImageJ (National Institutes of Health) software.
For Cdc42-GTP pull-down experiment, a commercial kit (Cytoskeleton Inc.) was used following the manufacturer’s instructions, specifically using 107 B cells for 15 µg of beads. For immunoblotting, stimulated cells were then lysed in lysis buffer (20 mM Tris-HCL, pH 8.0,150 mM NaCl, 5 mM EDTA, Protease Inhibitor cocktail [Roche], 10 mM NaF, 1 mM Na3VO4, and 1% NP-40) for 15 min on ice, and samples were loaded into 4–20% (for Cdc42-GTP pull-down) or 12% (for all other applications) for electrophoresis. Proteins were detected with antibodies against pErk, pAkt (S473), pCD19, pSyk, pSrc, totalErk, pSTAT3 (Ser), and Cdc42 (all from Cell Signaling Technology) using the secondary HRP-conjugated anti–rabbit or anti–mouse antibodies (Jackson ImmunoResearch Laboratories).
Blot densitometry analysis was performed using the ImageJ (National Institutes of Health) software.