Western blot analysis of intestine HO-1

Total cell proteins from intestinal grafts were released using Complete Lysis M reagent (Roche, Germany) according to the manufacturer’s instructions. Then, 50 μg protein samples were boiled for 5 min in loading buffer containing 4% sodium dodecyl sulfate (SDS), 20% glycerol, and bromophenol blue. Proteins were separated by 10% and 8% sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) and transblotted onto a polyvinylidene fluoride membrane (Roche, Germany). Nonspecific reactivity was blocked using 5% nonfat dry milk in TBST (10 mmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 0.05% Tween-20) for 1 h at room temperature. The membrane was then incubated with polyclonal rabbit anti-rat HO-1 antibody (Stressgen Biotechnologies) or β-actin (Sigma, USA) at 4 °C overnight. After three washes with TBST, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody for 2 h at room temperature. Reactive protein was detected using the ECL chemiluminescence system.