BCR crosslinking increases Glut1 expression. (A) B cells were cultured in the absence (Med) or presence of 10 μg/mL anti-Ig (αIg) for 12 and 24 hours. Cell lysates were prepared, and equivalent amounts of total protein were examined by Western blot for Glut1 and Mek-1 protein levels; the latter serves as a loading control. Brain and kidney lysates serve as tissue sources expressing relatively high and low levels of Glut1, respectively. (B) B cells were cultured in the absence (Med) or presence of 10 μg/mL anti-Ig (αIg) for 6 and 12 hours, and then Glut1 expression was determined by flow cytometry. Parallel B cells were pretreated for 30 minutes with 10 μM LY294002 and then stimulated with anti-Ig for 12 hours (αIg 12 h + LY). B cells were also isolated from p85α-deficient mice, cultured in the absence (Med) or presence of 10 μg/mL anti-Ig (αIg) for 12 hours, and then Glut1 expression was determined by flow cytometry. In both the wild-type and p85α-deficient analyses, control Ab indicates an isotype-matched control staining of B cells stimulated with 10 μg/mL anti-Ig for 12 hours. In data not shown, the staining of B cells with an anti-MHC Ab was similar in all conditions. (C) B cells were cultured in medium alone (Med) or stimulated with 10 μg/mL anti-Ig (αIg) for 12 hours. Cells were stained with Alexa Fluor 488–conjugated CT-B and anti-Glut1 Ab as described in “Materials and methods.” The data are representative of 3 independent experiments.