5 bromo 4 chloro 3 indolyl phosphate nitro blue tetrazolium substrate solution

Lung or liver tissue was homogenized (10% w/v) in a lysis buffer (100 mM tris [pH 7.4], 150 mM sodium chloride, 1% triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 5 mM ethylenediaminetetraacetate, 1 mM phenyl-methylsulfonyl fluoride, 5 µg/mL protease inhibitor cocktail) using a Dounce homogenizer at 4°C. The homogenates were spun at 15,000× g for 15 minutes. The supernatant cytosolic fractions were mixed with Laemmli dye, boiled for 5 minutes, and run on sodium dodecyl sulphate-12.5% polyacrylamide gels at 100 V, followed by electrophoretic transfer to polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA) at 15 V for 20 minutes in a Trans-Blot semi-dry blotting apparatus (Bio-Rad Laboratories Inc., Hercules, CA, USA). The membrane was blocked with BSA in PBS and incubated with the primary anti-TNF-α or TGF-β antibody (1:500 dilution) and secondary alkaline phosphatase-conjugated rabbit anti-goat IgG antibody (1:10,000 dilution). Protein bands were visualized by using 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium substrate solution (Sigma-Aldrich Co.) and the intensities were expressed as relative pixel densities using the Image J software system (National Institutes of Health, Bethesda, MD, USA).