Figure 4

BCR signaling in B cells from WT, ERα-deficient and ERβ-deficient mice. (A) Splenic cells from WT, ERα-deficient and ERβ-deficient mice treated with E2 or placebo were incubated with or without 20 µg/mL anti-IgM F(ab)′₂ antibody, and Erk phosphorylation was determined by flow cytometry. Transitional B cells from E2-treated WT and ERβ-deficient mice displayed a decrease in pErk after anti-IgM stimulation compared with transitional B cells from placebo-treated mice (stimulated/unstimulated (Stim/Unstim)) as determined by flow cytometry. There was no reduction in anti-IgM-induced pErk in B cells of E2-treated ERα-deficient mice compared with B cells of placebo-treated mice. (B) Total splenic B cells from WT, ERα-deficient and ERβ-deficient mice were stimulated with 20 µg/mL anti-IgM F(ab)′₂ antibody for 0, 5 and 15 min at 37°C, and 20 µg protein at each time point was subjected to Western blotting. Erk phosphorylation was determined by probing the blots with antibodies to Erk and phospho Erk1/2. To normalize for protein levels, the blots were probed with antibodies to HPRT. (C) The blots were scanned to quantitate pErk1:Erk1 as well as pErk2:Erk2 ratio at 0, 5 and 15 min of stimulation with anti-IgM F(ab)′₂ antibody and were expressed as arbitrary units.