Biochemical effects of dasatinib and other src inhibitors. (A) Src inhibitors enhance IL-12p40 production in LPS/IFNγ–matured DCs. Different src kinase family inhibitors were tested in combination with LPS/IFNγ maturation. DCs were matured with LPS/IFNγ in the presence of the respective inhibitor (1.25 μM) and brefeldin A for 16 hours. Subsequently, Intracellular cytokine staining was performed. Data were pooled from 3 independent experiments. (B) Dasatinib added at the time of the activation stimulus leads to the highest synergistic activity. Dasatinib was added to DCs matured with LPS/IFNγ at the indicated time points, followed by IL-12p40 staining 16 hours after stimulation. Pooled data from 3 experiments. (C) Effects of dasatinib on mitogen-activated protein kinase phosphorylation. Dasatinib was added together with the maturation stimulus LPS/IFNγ. Six hours later, cell lysates were obtained and phosphorylation-specific western blots were carried out. Std.: standard maturation conditions with LPS/IFNγ. Mix: use of IL1β, TNFα, and PgE₂ for stimulation. β-Actin served as a loading control (for quantitative evaluation, see supplemental Figure 4A). (D) Dasatinib enhances NF-κB signaling. Dasatinib was added together with the maturation stimulus LPS/IFNγ. Six hours later, cell lysates were obtained and phosphorylation of IκBα/β as well as degradation of IkBα was assessed. β-Actin served as a loading control (for quantitative evaluation, see supplemental Figure 4B). (E) Dasatinib enhances phosphorylation of IRF3. Dasatinib was added together with the maturation stimulus LPS/IFNγ. At the indicated time points (minutes), cell lysates were obtained and phosphorylation of IRF3 as well as degradation of IRAK1 was assessed (for quantitative evaluation, see supplemental Figure 4B).