Birb796

Stock (10 mM) of BIRB796 (Tocris, USA) was made in dimethyl sulfoxide (DMSO) solution. The stock solution was diluted with tick cell media to a final concentration of 10 μM. Mock solution was prepared in a similar way but without BIRB796. Tick cells (1 × 10⁵) were seeded onto 12 well plates and incubated for 16–20 h. Following incubation, tick cells were treated with 10 μM BIRB796 for 4 h followed by A. phagocytophilum (isolated from 2 × 10⁵ NCH-1 infected HL-60 cells) infection. Equal volumes of mock solution (corresponding to 10 μM volume) was added to control cells. The cells were then incubated for 24 h and processed further for RNA and protein extractions to measure p38 mapk transcripts and protein levels, respectively. In other experiments, after 16–20 h post plating, tick cells were treated with 100 μM XA and 20 μM BIRB796 for 4 h followed by A. phagocytophilum infection. Equal volume of mock solutions (corresponding to 100 μM XA and 20 μM BIRB796 vol) were added to control cells. The cells were incubated for 24 h and processed for DNA extractions to measure bacterial loads.

Recombinant human IL1β (R&D Systems) and TNF-α (R&D Systems) were dissolved in PBS containing 0.1% bovine serum albumin (Sigma-Aldrich). Budesonide (gift from AstraZeneca), dexamethasone (Sigma-Aldrich), SB203580 (Calbiochem), JNK-IN-VIII (Calbiochem), U0126 (Calbiochem), BIRB796 (Tocris), and VX745 (Tocris) were dissolved in dimethyl sulfoxide as stocks of 10 μM. Toll-like receptor 2 agonists, Pam2CSK4, and Pam3CSK4 (both Invivogen) were prepared as stocks of 1 mg/ml in sterile water.