Ablation of Noxa results in the generation of low-affinity antibody responses. (A-C) Mice were immunized with PE, and 12 days after immunization antigen-specific B cells were visualized by FACS staining (n = 5 per genotype). One of 2 independent experiments with similar results is shown. (A) FACS plots after ex vivo staining with 25 μg/mL PE. Arbitrary gating is applied to distinguish between PE^bright and PE^−/low cells. (B) Fraction of PE^bright (gated as indicated in panel A) cells within GC cells. (C) Quantification of MFI data shown in panel A (stained with 25 μg/mL PE) shows significantly reduced PE-binding of Noxa^−/− B cells. Normalization for differences in background staining between GC and FB cells was applied, based on signal within the PE⁻ cells. This allows for direct comparison of PE staining-intensity of B-cell subsets. (D) Mice were immunized with TNP-KLH and TNP-specific antibody responses were assessed by ELISA under limiting serum dilutions with fixed TNP-BSA coating (5 μg/mL). Shown are IgM (left), IgG1 (middle), and IgG2b (right) responses (n = 5 per genotype; 1 of 2 independent experiments with similar results is shown). X-axis represents the percentage of antiserum in the dilution buffer used for detection of antibodies. (E) Assessment of the K_D of TNP-specific IgG1 antibodies by biacore analysis 14 days after TNP-KLH immunization. (F) Mice were infected intranasally with Influenza A. Virus-specific IgM and IgG1 antibody responses were assessed after 2 weeks by ELISA under limiting antigen dilutions with fixed serum concentration (n = 8 per genotype; 1 of 2 independent experiments with similar results is shown). Serum of mice before infection was used as a negative control (triangles). In all panels mean and SEM are shown (*P < .05, **P < .005; Student t test).