FIG 5.

Inhibition of JAK3 decreases COX-2 protein levels and phosphorylation of cPLA₂ in macrophages infected with F. tularensis. (A) BMDMs were either untreated or pretreated with 50 μM JANEX-1 (JAK3 inhibitor) for 1 h prior to inoculation with LVS at an MOI of 200:1. At 1 h postinoculation, whole-cell lysates were collected and analyzed for cPLA₂ phosphorylated on Ser⁵⁰⁵ by Western blotting (left). The band intensities (right) were measured in each experiment using LiCor Odyssey infrared imaging system software and were normalized to the loading control (tubulin). The graph presents data from eight independent experiments. (B and C) RAW264.7 macrophages were either untreated or pretreated with 50 μM JANEX-1 (JAK3 inhibitor) for 1 h prior to inoculation with LVS at an MOI of 200:1. After 2 h, the cells were incubated in RPMI containing penicillin-streptomycin. At 4 h (B) and 18 h (C) postinoculation, whole-cell lysates were collected and analyzed for COX-2 protein levels by Western blotting (left). The band intensities (right) were measured in each experiment and were normalized to the loading control (tubulin). The graphs present data from seven (B) and six (C) independent experiments. Statistical differences (**, P ≤ 0.01; ***, P ≤ 0.001) compared to infected untreated controls were determined by Dunnett's multiple comparison test.