Effect of IL-12 and IFN-γ on T-bet mRNA expression and STAT1 activation in B cells. (A-C) T-bet mRNA expression. In panels A and B, purified primary B cells were activated for up to 72 hours with IL-12, IFN-γ, or IL-12 plus anti-IFN-γ and then lysed at various time points. mRNA was extracted and reverse transcribed. Panel A shows the PCR bands for the 24-hour time point with T-bet-specific primers. In panel B, T-bet and GAPDH cDNAs were quantified as described in “Materials and methods.” Results (means ± SEMs) are expressed as -fold increases in normalized T-bet cDNA values versus untreated cells. (C) B cells were activated for 48 hours in various conditions as indicated. T-bet mRNA expression was quantified as described. (D) STAT1 activation. Purified primary B cells were treated with IL-12 or IFN-γ for 15 and 45 minutes and then lysed. Western blotting was performed on whole-cell lysates by using antiphospho-STAT1. Membranes were reprobed with anti-STAT1.