Puma was not required for B-cell activation. (A) Wild-type splenic B cells (2 × 10⁶ cells/mL) were cultured for 48 hours in the presence or absence of LPS (10 μg/mL), or Fab'2 anti–mouse μ antibody fragments (10 μg/mL) plus anti–mouse CD40 antibodies (5 μg/mL). Expression levels of Puma and tubulin (loading control) were assessed by immunoblotting. (B) Splenic B cells (2 × 10⁶ cells/mL) from wild-type mice were either left untreated or stimulated with Fab'2 anti–mouse μ antibody fragments (10 μg/mL) plus anti–mouse CD40 antibodies (5 μg/mL) for 48 hours. Cells were then labeled with FITC–anti-CD69 and PE–anti-CD19 antibodies and analyzed by flow cytometry. Enlarged activated B cells (R1) with increased forward scatter (FSC) were quantified by flow cytometric analysis and CD19⁺/CD69⁺ cells were quantified within the R1 fraction. (C) Splenic B cells (2 × 10⁶ cells/mL) from wild-type (Wt) or Puma-deficient (Puma^−/−) mice were either left untreated or stimulated with LPS (10 μg/mL) or the combination of Fab'2 anti–mouse μ antibody fragments (10 μg/mL) plus anti–mouse CD40 antibodies (5 μg/mL) for 48 hours. The percentages of activated B cells were determined by flow cytometric analysis and B-cell proliferation was quantified by measuring ³H-thymidine incorporation into cellular DNA during the last 16 hours of culture. Data represent mean (± SEM). (D) Frozen spleen sections from wild-type and Puma^−/− mice that had been immunized 14 days earlier with 100 μg NP coupled to KLH, were stained with antibodies to B220 to identify follicles (red) and GL7 for germinal centers (green; ×20 magnification). Results are representative of 3 independent experiments.