Biomax l film

The procedure and protocol for assessing the protein expression of oxidative stress have been described in details in our previous reports [24 (link)–28 (link)]. The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon, Billerica, MA, USA (S7150). DNPH derivatization was carried out on 6 μg of protein for 15 minutes according to the manufacturer’s instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution (anti-DNP 1: 150) for 2 hours, followed by incubation in secondary antibody solution (1:300) for 1 hour at room temperature. The washing procedure was repeated eight times within 40 minutes. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Amersham, UK) which was then exposed to Biomax L film (Kodak, Rochester, NY, USA). For quantification, ECL signals were digitized using Labwork software (UVP, Waltham, MA, USA). For oxyblot protein analysis, a standard control was loaded on each gel.

Western blot analysis was performed according to the previous published paper.²⁰ Equal amounts (50 µg) of protein extracts were resolved using 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). During post‐resolution of the proteins, they were transferred into polyvinylidene difluoride (PVDF) membranes. Membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti‐α‐SMA (Cat. No ab5694; Abcam; 1:500), mouse anti‐col1a1 (Cat. No sc‐59772; 1:500), mouse anti‐fibronectin (Cat. No sc‐8422; 1:500), rabbit anti‐STAT3 (Cat. No ab31370; Abcam; 1:500), rabbit anti‐STAT3 (phospho Y705) (Cat. No ab76315; Abcam; 1:2000), rabbit anti‐PTEN (Cat. No 51‐2400; Thermo Fisher Scientific, the least detectable dose: 1 µg/mL), rabbit anti‐Spry1 (Cat. No12993; Cell Signaling Technology; 1:1000), rabbit anti‐Smurf2 (Cat. No ab94483; Abcam; 1:500), rabbit anti‐PDCD4 (Cat. No ab51495; Abcam; 1:5000) and rabbit anti‐GAPDH (Cat. No ab9485; Abcam; 1:1000). Post‐incubation with HRP‐conjugated anti‐rabbit IgG or antimouse IgG, the immune‐labelled proteins were detected through enhanced chemiluminescence (ECL; Merck Millipore), and the signal was captured on Biomax L film (Kodak). The intensity of the indicated bands was estimated using ImageJ software.

The procedure for assessing the protein expression of oxidative stress was based on our previous reports [37 (link)–40 (link)]. The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon (S7150). DNPH derivatization was carried out on 6 μg of protein for 15 min according to the manufacturer’s instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution (anti-DNP 1: 150) for 2 h, followed by incubation in the secondary antibody solution (1:300) for 1 h at room temperature. The washing procedure was repeated eight times within 40 min. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) which were then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP). For oxyblot protein analysis, a standard control was loaded on each gel.