Western blot determination of iNOS, COX‐2 and pSMAD3 level expression

Tissue samples (30 μg protein per lane) were subjected to 8% SDS‐PAGE, transferred to nitrocellulose membranes and incubated overnight (4°C) with polyclonal antibody anti‐iNOS1 raised in rabbit, diluted 1:1000 (Merck‐Millipore, Darmstadt, Germany) in PBS‐T (20 mM Tris‐HCl buffer, 150 mM NaCl and 0.05% Tween 20), and with anti‐COX‐2 rabbit polyclonal antibody (Cayman Chemical Co., Ann Arbor, MI, USA) diluted 1:150 in PBS‐T. After several rinses with PBS‐T, membranes were incubated with anti‐rabbit IgG conjugated with horseradish peroxidase, diluted 1:5000 in PBS‐T at RT for 1 hr. The bands were visualized by enhanced chemiluminescence (ECL) and quantified by densitometric analysis. For SMAD3 signalling pathway analysis, 150 μg of total proteins was loaded onto 10% SDS‐PAGE gel, transferred to nitrocellulose membranes, incubated overnight (4°C) with pSMAD3 and SMAD3 [1:1000 in 5% bovine serum albumin (BSA), TBS‐T; Cell Signaling Technology, Danvers, MA, USA] and successively, incubated with anti‐rabbit secondary antibody (1:2000 in 5% BSA in TBS‐T).