Btk−/−PLCγ2−/− pre–B cells have reduced intracellular calcium signaling in response to pre-BCR engagement. (A) Analysis of pre-BCR–induced calcium flux in WT and various mutant pre–B cells. Cultured pre–B cells were loaded with Indo-1 and treated with high (10 μg/mL) and low (2 μg/mL) doses of anti-Igμ F(ab′)2 antibody. Intracellular calcium influx in gated B220+IgL− cells was shown as a ratio of Indo-1 violet/blue fluorescence versus time. (B-C) Western blot analysis of pre-BCR–induced activation of PLCγ2 and PLCγ1. Cultured pre–B cells were stimulated for various periods of time with anti-Igμ F(ab′)2 antibody, and whole-cell lysates were probed with anti–phospho-PLCγ2(Y759) and anti–phospho-PLCγ1(Y783) antibodies. Blots were reprobed with anti-PLCγ2 and anti-PLCγ1 antibodies to determine protein loading. Results shown are representative of 5 and 3 independent experiments for panels A and B, respectively.
