P erk1 2

Granulation tissues were homogenized and lysed in ice-cold lysis buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, and 1% Triton X-100) containing protease inhibitors (2 mmol/L sodium orthovanadate, 0.2 mmol/L PMSF, 2 μg/mL leupeptin, and 2 μg/mL aprotinin). After centrifuged at 13 000 g for 15 minutes at 4°C, the supernatant was collected. Equal amounts of denatured proteins in the supernatants were separated by 12% SDS-PAGE, and transferred to nitrocellulose membranes. Then the membranes were blocked in TBS-T (10 mmol/L Tris, 150 mmol/L NaCl, and 0.1% Tween20, pH 7.5) containing 5% skim milk for 2 hours. The membranes were incubated with rabbit polyclonal antibodies β-actin, COX-2, VEGF, FGF-2, ERK1/2, p-ERK1/2, Akt, and p-Akt (1: 500) (Bio Basic Inc., Canada) in TBS-T with 5% nonfat milk overnight at 4°C. After the membranes were incubated with anti-rabbit HRP-conjugated antibody (1: 10 000) in TBS-T, target proteins were determined via visualization with DAB (Bio Basic Inc., Canada).

Liver tissues (0.2 g) were harvested, lysed and homogenized in 2 ml lysis buffer with 10 mM Tris-buffered saline, 1 mM EDTA, 1 mM EGTA, 2 mM PMSF and 1% Triton X-100 (v/v) for 20 min. Lysates were centrifuged at 13,000 × g for 15 min at 4°C. Protein concentration was measured using a Quick Start™ Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Denatured proteins in supernatants were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in TBS with Tween-20 (10 mM Tris-HCl, 150 mM NaCl and 1% Tween-20) for 2 h. The membranes were subsequently incubated with primary polyclonal antibodies against β-actin (1:1,000), heme oxygenase-1 (HO-1; 1:1,000), cyclooxygenase-2 (COX-2), nuclear factor (NF)-κB, phosphorylated (p)-NF-κB, extracellular signal-regulated kinase (ERK) and p-ERK1/2 (Bio Basic Inc., Markham, ON, Canada) overnight at 4°C. Following an extensive wash with TBST, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000; cat. no. A9169; Sigma-Aldrich; Merck KGaA) for 2 h at room temperature. The membranes were washed three times and visualized with 3,3′-diaminobenzidine (Bio Basic Inc.).