BCR and FcγRIIB coligation reduces cellular responsiveness to BLyS. (A) Annexin V staining of C57BL/6 CD23⁺ B cells cultured for 24 hours with various stimuli (concentrations of stimuli are indicated in “B-cell subset isolation, culture, and stimulation”). B cells were stained with annexin V and analyzed by flow cytometry. (B) Proliferation and death profiles of CD23⁺ B cells cultured with and without rBLyS for 72 hours. B cells were loaded with CFSE and cultured with indicated stimuli. One minute before FACS analysis, TO-PRO-3 was added as a vital dye. Plots are representative of at least 3 independent experiments. (C) Pim 2 message levels after 36-hour culture with the indicated stimuli, with and without the addition of rBLyS at time 0 hour (hr) or time 18 hours (concentrations of stimuli are indicated in “B-cell subset isolation, culture, and stimulation”). (D) Top blot shows p100 processing and Bcl-xL levels, as assessed by Western blot after 24 hours of culture with the indicated anti-IgM stimuli and the addition of recombinant human BLyS at 0 hours. NE indicates nuclear extract. Bottom blot and densitometry show nuclear p52 levels after the same culture period, normalized to histone H3. Data are representative of at least 3 independent experiments.