Impaired T-bet up-regulation in Ets-1^−/− B cells. (A) Semiquantitative RT-PCR analysis of T-bet expression in in vitro–stimulated B cells purified from spleens of Ets-1^−/− (KO) and wild-type (WT) chimera activated B cells. RNA were harvested from cells activated for 2 days under the indicated conditions or left untreated (NA, not activated). Shown are the 10-fold serial dilutions of template cDNA amplified with primers specific for the T-bet gene and Hprt gene used as a control. Results are representative of 5 independent experiments. (B) Western analyses of T-bet in activated B cells. Freshly isolated wild-type (WT) and Ets-1^−/− (KO) B cells were treated for 48 hours under the following conditions: top panel, 25 μg/mL of LPS, 20 ng/mL of IFN-γ, or untreated; bottom panel, 10 μg/mL of anti-CD40 Abs, 20 ng/mL of IFN-γ, or untreated. Whole-cell lysates were subjected to Western analysis with Abs recognizing T-bet or actin as loading control. Results are representative of 3 independent experiments.