Defective expression and secretion of IgG2a in Ets-1^−/− B cells. (A) Serum Ig isotype levels. ELISA quantification of serum Ig in 5- to 6-week-old viable Ets-1^−/− (KO, dark bar graphs) and littermate controls (WT, open bar graphs). Bar graphs represented the average of 7 mice of each genotype. Error bars indicate ±1 SD. The statistical significances were calculated with the Student t test. *P < .005. (B) In vitro Ig secretion after B-cell stimulation. ELISA quantification of IgG1, IgG2a, IgG2b, IgG3, and IgM produced in the supernatant of B splenocytes from Ets-1^−/− (dark bar graphs) and wild-type (open bar graphs) chimeras cultured for 6 days. CD43⁻ B cells were activated in medium containing 25 μg/mL of LPS, LPS plus 5 ng/mL of IL-4 (LPS-IL-4), or LPS plus 50 ng/mL of IFN-γ (LPS-IFN-γ). Bar graphs represent the average of 5 independent experiments. Error bars indicate ± 1 SD. *P < .005. (C) CD43⁻ purified B cells from wild-type (WT) and Ets-1^−/− (KO) were activated in the indicated conditions and CSR was analyzed by FACS. Numbers represent the percentages of cells falling in each gate. Results are representative of 3 independent experiments. (D) Semiquantitative RT-PCR analysis of γ2a and γ3 germline transcripts in activated B cells. RNA was isolated from B cells purified from spleens of Ets-1^−/− (KO) and wild-type (WT) chimeras either untreated (NA, not activated) or after day 2 of in vitro stimulation. Cells were activated in medium containing 25 μg/mL of LPS, 10 μg/mL of anti-CD40 Abs, 5 ng/mL of IL-4, 50 ng/mL of IFN-γ, or 100 ng/mL of IL-27; 10-fold serial dilutions of template cDNA are indicated. Transcription of the Hprt gene was used as a control. Results are representative of 4 independent experiments.