Pf 431396 hydrate

RAW 264.7 cells were plated in a 24-well plate at a density of 2×10⁵ cells-per-well. Cells were incubated (37 °C, 5 % CO₂) for 6 h resulting in adherence to plates. Before infection, cells were activated by adding 1 µg ml⁻¹ of LPS 12 h prior to infection. Activated cells were treated with the indicated concentrations of PF-431396 hydrate (Sigma-Aldrich) for 1 h. PF-431396 hydrate inhibits activity of both Pyk2 and FAK, suppressing the phosphorylation of Pyk2 at its active tyrosine phosphorylation site (Y402) without decreasing total protein level of Pyk2, thus functionally blocking multiple cellular signalling pathways [35 (link)]. Supernatants were removed and cells were infected with a m.o.i. of 100 in 200 µl of RPMI-1640 media containing 3 % FBS and 1 % l-glutamine. Cells were left for 1 h to allow bacterial phagocytosis to occur, before the supernatant was removed and 1 ml of fresh media containing gentamicin (50 µg ml⁻¹) was added for 1 h to kill any extracellular bacteria. Post-gentamicin treatment, supernatants were removed, cells were lysed with 200 µl PBS containing 2 % Triton X-100 for 5 min and bacteria numbers calculated as described above.