Quantity 5.2 software system

The spinal cords tissues were lysed in ice-cold RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration was determined using a BCA reagent kit (Beyotime Biotechnology, Shanghai, China). Total protein (30 μg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in tris-buffered saline with 5% non-fat milk and 0.5% bovine serum albumin for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies (1:1,000). After washing, the membranes were incubated with secondary antibodies (1:5,000) for 1 h at 37°C. Blots were visualized with the Chemiluminescent HRP substrate (Millipore) and quantified with the Quantity 5.2 software System (Bio-Rad).

Liver tissues and cells were lysed in ice-cold RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). Protein concentrations were determined using a BCA reagent kit (Beyotime Biotechnology, Shanghai, China). Total protein (30 μg) was separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). The membranes were blocked in Tris-buffered saline with 5% non-fat milk and 0.5% bovine serum albumin for 2 h at room temperature and then incubated overnight at 4 C with primary antibodies (1:1,000). After washing, the membranes were incubated with secondary antibodies (1:5,000) for 1 h at 37 C. Blots were visualized with the chemiluminescent HRP substrate (Millipore) and quantified using the Quantity 5.2 software System (Bio-Rad).

Western blot was performed as described in a previous study.¹¹ Wound skin tissues and harvested cells were lysed in ice‐cold RIPA lysis buffer (Santa Cruz Biotechnology). The protein concentration was determined using a Bradford protein assay kit (Bio‐Rad). The total proteins were separated on 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to PVDF membranes (Millipore). The membranes were blocked in tris‐buffered saline with 5% non‐fat milk and 0.5% BSA for 1 hour, and then incubated with primary antibody overnight at 4°C, followed by incubation with the secondary antibodies for 1 hour at room temperature after standard washing procedures. The primary antibodies against different target proteins were purchased from commercial companies listed below: β‐actin (1:3000, Bioss Biotechnology), Nrf2 (1:1000, Abcam), NAD(P)H dehydrogenase quinone 1 (NQO‐1, 1:1000, Abcam), catalase (1:1000, Cell Signaling Technology); 3‐nitrotyrosine (3‐NT, 1:2000, Millipore), and 4‐hydroxynonenal (4‐HNE, 1:2000, Alpha Diagnostic International). All horseradish peroxidase (HRP)‐conjugated secondary antibodies were purchased from Bioss Biotechnology. Blots were visualized with Chemiluminescent HRP substrate (Millipore) and quantified with Quantity 5.2 software System (Bio‐Rad).