Ligation of CD19 on mouse and human B cells results in Akt activation.

Treatment with 50 nM wortmannin was conducted 10 minutes before stimulation. Akt1 was immunoprecipitated, and the precipitates were divided. Half of them were used for Western blot analysis using anti-Akt1 Ab (lower panels). The remaining half were used for in vitro kinase assay using histone H2B as an exogenous substrate. The kinase reaction products were resolved on 15% SDS-PAGE, and their phosphorylation was quantified by autoradiography (upper panels). (A) Purified splenic B cells from C57BL/6 mice (1 × 10⁷) were incubated with 1 μg/mL biotin-conjugated antimouse CD19 mAb 1D3 or biotin-conjugated control IgG2a for 3 minutes and subsequently cross-linked by using streptavidin (20 μg/mL) at 37°C for indicated times. (B) Human Raji B cells (5 × 10⁶) were incubated with 1 μg/mL antihuman CD19 mAb B4 or control IgG1 for 3 minutes, followed by cross-linking with anti–mouse IgG F(ab′)₂fragment (20 μg/mL) at 37°C.