Anti-inflammatory Signaling Pathway Modulation

The following antibodies were used: rabbit anti-HO-1 (1:2000 for western blot, Abcam); rabbit anti-COX-2 (1:1000, Cell Signaling Technology); rabbit polyclonal anti-AKT and anti-pAKT (1:1000, Cell Signaling Technology); rabbit anti-actin (1:5000, Santa Cruz); anti-rabbit-HRP (1:2000, Santa Cruz). Pseudomonas aeruginosa (PA) LPS (Sigma-Aldrich) was prepared in PBS at 100X stock solution and used at a concentration of 100 ng/mL. Flagellin (Imgenex, San Diego CA) was used at a concentration of 100 ng/ml). CFTR inhibitor CFTR_inh172, a kind gift from Dr. Alan Verkman)^{32 (link)}, was freshly prepared in DMSO and used at concentration of 20μM. The PI3K/AKT inhibitor LY94002 (Cell Signaling Technology) was prepared in DMSO and used at a concentration of 20μM. The AKT Activator II, SC79 (Merck Millipore) was dissolved in DMSO and used at the final concentrations indicated. Celecoxib (Sigma-Aldrich, St Louis MO) was dissolved in DMSO (stock solution 20 mM) and used at a final concentration of 25μM. For the in vivo study we used the FDA approved branded Celecoxib (Celebrex) at concentration of 25mg/kg/mouse/day. Ibuprofen (Sigma-Aldrich, St Louis MO) was dissolved in DMSO (stock solution 50 mM) and used at a final concentration of 25μM or 100μM. For the in vivo study we used Walgreens Children’s Ibuprofen Suspension (20mg/ml) at a final concentration of 50 mg/kg/mouse/day.