Western Blot Analysis of TNF-α and COX-2 in Skin Tissues

Protein levels of TNF-α and COX-2 in the dorsal skin (n = 10 at each point in time) were analyzed according to our published procedure [17 (link)]. After sacrificing the animals, the tissues were homogenized in 50 mM PBS (pH 7.4) containing 0.1 mM ethylene glycol-bis (2-aminoethyl ether)-N,N,N′,N′ tetraacetic acid (pH 8.0), 0.2% nonidet P-40, 10 mM ethylenediaminetetraacetic acid (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium or thovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol (DTT). The skin tissues were separated using 4‒20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, and the resolved proteins were transferred to a nitrocellulose membrane for 2 h at 40 V. Each membrane was incubated separately with the primary antibodies: rabbit anti-TNF-α (1:1000, Abcam) and rabbit anti-COX-2 (1:1000, Abcam) overnight at 4 °C. The membranes were washed with a washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.05% Tween-20) and exposed to peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:1000 dilution at room temperature for 2 h. The membrane blots were developed using an enhanced luminol-based chemiluminescent (ECL) kit (Pierce Chemical, TX, USA).