Splenic B and T cells were purified by negative selection using CD43 beads or mouse pan T cell isolation kit II (Miltenyi Biotecc), respectively. Purified B cells were stimulated with 10 μg/ml F(ab′)2 fragment of anti-mouse IgM (Jackson ImmunoResearch Laboratories) for the indicated times. B and T cell lysates were prepared and immunoblotting performed as described (21 (link)). All immunoblotting antibodies were from Cell Signaling Technology, except for actin (Santa Cruz Biotechnology), Igα (Abcam), phosphotyrosine (pY) and ezrin (EMD Millipore). To measure intracellular-free calcium levels, purified splenic B cells were loaded with Fluo-3 AM (Molecular Probes) at 37°C for 20 min. Cells were washed, resuspended in DMEM supplemented with 1% BSA (Sigma) and 20 mM HEPES (Sigma), warmed to 37°C for 5 min, and analyzed by flow cytometry. After the baseline was established for 30–40 sec, cells were stimulated with 10 μg/ml F(ab′)2 fragment of anti-mouse IgM for the indicated duration.