Sodium salicylate

Rabbit antibodies to C/EBPβ (C-19), ERK1 (C-19), NF-κB p65 (C-20), CaMKKβ (H-95) and actin (I-19-R), mouse antibodies to HuR (3A2), C/EBPβ (47A1), C/EBPβ (H7), p-ERK (E-4), Importin α1(B-9), and goat antibody to AMPKα2 (A-20) were purchased from Santa Cruz Biotechnology. Rabbit antibodies to AMPKα (#2532), pAMPKα (#2531), and p-ERK (#4377) were purchased from Cell Signaling Technologies. Rabbit antibody to p-C/EBPβ (T188) was from Abcam (pT235). Rabbit antibody to AMPKα2 (#21494) was from ThermoFisher. Anti-mouse (W4028) and anti-rabbit (W4018) HRP conjugated secondary antibodies were from Promega. Anti-Mouse Alexa® Fluor 488 and anti-rabbit Alexa® Fluor 594 conjugated secondary antibody was from ThermoFisher. Anti-rabbit Alexa® Fluor 647 conjugated secondary antibody was from Abcam. X-tremeGENE HP DNA transfection reagent was from Roche Diagnostics. AICAR was obtained from Cell Signaling Technology, metformin was from Calbiochem, sodium salicylate was from ThermoFisher, STO-609 was from Sigma, and BAPTA was from Abcam.

Blood eosinophils were activated for 30 min at 37°C with lysine aspirin (LysASA) (10 mM: Sanofi-Aventis; Athens, Greece), ketorolac (10 mM: Hospira; Lake Forest, IL), sodium salicylate (NaSal) (10 mM: Acros Organics; Geel, Belgium) or the combination of phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) with ionomycin (2 μg/ml (Sigma)). For studies with PGE₂, butaprost, EP3 antagonist (L-798106) or the EP1 antagonist (SC-51089) (Cayman Chemical; Ann Arbor MI), these compounds were preincubated with eosinophils for 30 min prior to stimulation with LysASA for an additional 30 min at 37°C. Supernatants were collected and mediators quantified by enzyme immunoassay (EIA) as described below.