Cyp 5

Wound tissues (0.2 g) were harvested and lysed in 2 mL of ice-cold lysis buffer with 2 mmol/L PMSF, 100 mmol/L Na₄P₂O₇, 50 mmol/L HEPES, 10 mmol/L EDTA, 100 mmol/L sodium fluoride, 10 mmpl/L sodium orthovanadate, and protease inhibitor mixture for 15 min. Lysate was centrifuged at 13,000 g for 20 min at 4°C. Proteins denatured in supernatants were electrophoretically separated on an 8% stacking gel and following 12% sodium dodecyl sulfate-polyacrylamide gel and then transferred to 0.2-μm nitrocellulose membranes. The membranes were blocked in TBS-T (10 mmol/L Tris, 150 mmol/L NaCl, and 0.1% Tween 20, pH 7.5) with 5% nonfat milk for 2 hours at room temperature and incubated with rabbit polyclonal antibodies β-actin (1 : 1000), CYP-5 (1 : 1000) (Bio Basic Inc., Canada), HO-1 (1 : 300), TNF-α (1 : 400), IL-6 (1 : 400), CBS (1 : 400), and CSE (1 : 400) (Wuhan Boster Bioengineering Limited Company, China) in TBS-T with 5% nonfat milk overnight at 4°C. After the membranes were extensively washed, a horseradish peroxidase-conjugated secondary anti-rabbit antibody (1 : 10 000 dilution; Sigma Chemical Co., St. Louis, MO, USA) was added and incubated for 2 hours. After being rinsed three times, the membranes were visualized by DAB (Bio Basic Inc., Canada).

Chloral hydrate, streptozotocin, hemin, and a horseradish peroxidase-conjugated secondary anti-rabbit antibody were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA). Rabbit polyclonal antibodies β-actin, CYP-5 were purchased from Bio Basic Inc. (Canada), and HO-1, TNF-α, IL-6, CBS, and CSE were obtained from Wuhan Boster Bioengineering Limited Company (Wuhan, China).