Il 1alpha

Paw tissue was retrieved at 96 h post CFA injection and minced in ice cold lysis buffer (20 mM imidazole hydrochloride, pH 6.8; 100 mM potassium chloride, 1 mM magnesium chloride, 10 mM ethylene glycol tetraacetic acid, 1.0% Triton X-100, 10 mM sodium fluoride, 1 mM sodium molybdate, 1 mM ethylenediaminetetraacetic acid (Sigma-Aldrich, Munich; Merck, Darmstadt; Carl Roth GmbH, Karlsruhe, all Germany) with complete Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany). The homogenate was frozen at −80°C. Before experiment, the homogenate was thawed, incubated at 4°C overnight, centrifuged at 14,000 g for 10 min and the supernatant was used for ELISA [13] (link). ELISA kits were used according to the manufacturer's instructions: IL-1alpha, IL-1beta and interferon (IFN)-gamma (R&D systems, London, UK); CXCL10 (Peprotech, Hamburg, Germany); TNF-alpha and IL-4 (Invitrogen, Life Technologies, Darmstadt, Germany) and IL-13 (Abcam, Cambridge, UK).

A549 cells were purchased from American Type Culture Collection (ATCC) and cultured in DMEM (Invitrogen), supplemented with 10% FBS and antibiotics (Invitrogen). Dexamethasone sodium phosphate 4 mg/ml was from Simplist/USA. Dilutions were made in cell culture medium to reach a final concentration of 100 nM treatments. For western blots, cells were directly lysed in 1 × SDS sample buffer (Invitrogen). Lysates were sonicated to shear DNA, and equivalent lysate levels were loaded onto SDS PAGE gel, blotted to nitrocellulose membrane, and probed with antibodies to ZFP36, and GAPDH. Affinity-purified ZFP36 rabbit polyclonal antibody was custom-made with Genescript and was used previously^{5 (link)}. GAPDH, Phspho-P38 antibody was purchased from Cell Signaling. P38 antibody was purchased from Santa Cruz. IL1 alpha was from R&D systems, and was used to treat the cells at a 3ng/ml concentration.