Cocrosslinking the BCR and FcγRIIB inhibits BCR-induced glucose utilization. (A) B cells were cultured in medium alone (Med) or 10 μg/mL F(ab′)₂ fragments of anti–mouse IgG (αIg) for 15 minutes. Parallel B cells were cultured with 10 μg/mL intact anti–mouse IgG (wIg) in the presence or absence of 10 μg/mL 2.4G2 mAb. Cells were then evaluated for phosphorylation of Akt on Ser473 and β-actin by Western blot. (B) B cells were cultured in medium alone (Med), 10 μg/mL F(ab′)₂ fragments of anti–mouse IgG (IgG), or 10 μg/mL intact anti–mouse IgG (wIg) for 12 hours. Cells were then assayed for 2-[³H]deoxyglucose uptake as described in Figure 1. (C) Glut1 expression was monitored by flow cytometry in B cells cultured in medium alone (Med) and stimulated with 10 μg/mL F(ab′)₂ fragments of anti–mouse IgG (anti Ig) for 12 hours or with 10 μg/mL intact anti–mouse Ig (wIg) for 12 hours. Control Ab indicates an isotype-matched control staining of B cells stimulated with 10 μg/mL F(ab′)₂ fragments of anti–mouse IgG for 12 hours. In data not shown, the staining of B cells with an anti-MHC Ab was similar in all conditions. (D) Glycolysis was measured in B cells cultured as described in panel B. In panels B and D, error bars reflect standard deviation from the mean of triplicate measurements. The data are representative of 3 independent experiments. (E) Incorporation of [1-¹³C]glucose into metabolites in B cells stimulated with 10 μg/mL F(ab′)₂ fragments of anti–mouse IgG (control) for 8 hours as described in Figure 4E. In parallel, B cells were cultured with 10 μg/mL intact anti–mouse Ig (FcR). The increases in ¹³C content (compared with the same metabolites in quiescent B cells where the ¹³C content is 1.0) are shown for the lactate methyl group (black bars), glutamate methylenes (open bars) (the average increase in ¹³C uptake into each glutamate methylene carbon is shown), and the citrate methylenes (gray bars).