Dasatinib and BIRB 796 do not inhibit R848-induced NF-κB p65 or c-Jun levels in FANCC- or FANCA-deficient mononuclear phagocytes, but do inhibit MK2 phosphorylation. T-shNT cells and T-shFC cells were treated with BIRB 796 (500nM) or dasatinib (500nM) for 6 hours and then stimulated with R848 (30μM) for 24 hours. (A) Western blot analyses of nuclear extracts were performed using Abs for total p65, phospho-p65(Ser536), and acetylated p65(Lys310). Whereas both BIRB 796 and dasatinib suppressed p65 phosphorylation and acetylation in T-shNT cells, neither agent suppressed p65 phosphorylation in T-shFC cells, and only BIRB 796 inhibited p65 acetylation. One representative example of 3 identical experiments is shown. (B) Western blot analyses of nuclear extracts were performed as above, but Abs to total c-Jun and phosphorylated (Ser73) c-Jun were used. BIRB 796 did not suppress c-Jun phosphorylation and the suppressive effect of dasatinib was minimal (lane 6 vs lane 4). One representative example of 3 identical experiments is shown. (C) Immunoblot analysis of whole-cell lysates of shFC cells exposed to R848 with and without either dasatinib or BIRB 796 demonstrated marked suppression of MK2 phosphorylation by both small molecules (lanes 4 and 8 vs lanes 3 and 7). One representative example of 3 identical experiments is shown. (D) T-shFA and T-shNT cells were exposed to R848 alone for 24 hours or to BIRB 796 for 6 hours, followed by R848 for 24 hours. Whole-cell lysates were used in immunoblot assays for total and phosphorylated p65, c-jun, p38, and MK2. BIRB 796 had no influence on p65 or c-jun phosphorylation, but profoundly suppressed p38 and MK2 phosphorylation. One representative experiment of 2 is shown.