BIRB 796 and dasatinib influence TNFα gene expression posttranscriptionally. Means ± SD are shown. All P values were calculated using the Student t test. (A) BIRB 796 effectively inhibited secreted TNFα in monocytes. T-shNT and T-shFC cells were treated with BIRB 796 (0-500nM) for 6 hours, followed by treatment with R848 (30μM) for 24 hours. Supernatant media were then collected and TNFα was quantified by ELISA. Low doses of BIRB 796 (50nM) suppressed secreted TNFα protein by 20-fold. (B) T-shNT and T-shFC cells were treated with BIRB 796 (0-500nM) for 6 hours, followed by treatment with R848 (30μM) for 24 hours. mRNA was then prepared and TNFα transcripts measured by real-time qRT-PCR. Data are expressed as the fold change normalized to 18S rRNA and are relative to mRNA levels in unexposed (control) T-shNT cells. TNFα mRNA is reduced modestly in the presence of BIRB 796 and minimally at the 50nM dose that was fully suppressive of TNFα protein production (A). One of 2 identical experiments is shown. (C) Low doses of BIRB 796 (50nM) suppress R848-induced MK2 phosphorylation in whole-cell lysates from T-shFC and T-shNT cells. One of 2 identical experiments is shown. (D) Immunoblot analysis for p65 and c-Jun phosphorylation in nuclear extracts of T-shFC and T-shNT cells revealed that low doses of BIRB 796 had no effect on p65 or c-Jun phosphorylation in R848-induced T-shFC and T-shNT cells. One representative immunoblot of 3 identical experiments is shown.