Pvdf membrane

Cells transfected for 48 h were harvested from all groups, followed by lysis using RIPA lysis buffer (Beyotime, Shanghai, China). The cells were then centrifuged for 15 min at 12,000 g and 4°C to collect protein supernatants, and the BCA kit (Thermo Fisher Scientific, Inc.) was used to measure protein content. Later, proteins were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) as well as 2 h of blocking using 5% nonfat milk at ambient temperature. The primary antibodies (Abcam, Shanghai, China), anti-HMGB2 (ab124670, 1: 10,000), and anti-GAPDH (ab9485, 1:2500) were added, followed by an overnight incubation at 4°C. The next day, the membrane was rinsed with Tris-buffered saline with Tween-20 (TBST) and incubated with horseradish peroxidase (HRP)-labeled secondary antibody (ab205718, 1:5000) for 2 h at 37°C. After the PVDF membranes were washed with TBST three times, bands were visualized using ECL chemiluminescence (Beyotime, Shanghai, China) [40 (link)].

Cellular protein were extracted from RCC tissues and cells using RIPA buffer (Thermo Scientifc, Rockford, IL, USA) supplemented with protease inhibitor (Beyotime Institute of Biotechnology, Haimen, China) and PMSF (Beyotime Institute of Biotechnology). Thirty micrograms of protein lysate were separated in 4-20% SDS-PAGE gels and transferred onto PVDF membranes (Beyotime Institute of Biotechnology). The membranes were incubated with primary antibodies including XIAP and GAPDH at 4°C overnight. Secondary antibodies conjugated with horseradish peroxidase (Boster Bio-engineering Limited Company, Wuhan, China) were incubated with the membranes for 2 hrs at room temperature. The protein signals were dectected using ECL kit (Beyotime Institute of Biotechnology).

Protease inhibitors (Beyotime Biotechnology, Nanjing, China) and radio immunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Nanjing, China) were used to distill total protein from Jurkat cells. Total protein concentration was measured using the Bicinchoninic Acid Protein Assay Kit (Beyotime Biotechnology, Nanjing, China). Protein extracts were then dissolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA) by a semidry transfer way. Next, the membranes were blocked by a sealed liquid at room temperature for 0.5 h and then washed with Tris-buffered saline-Tween-20 (TBST). Then, the membranes were incubated with MAPK1 (1:1,000) and GAPDH (1:1,000) primary antibodies at 4°C overnight. The following morning, the membranes were washed with TBST and incubated with horseradish peroxidase-labeled secondary antibody immunoglobulin G (IgG) (1:1,000) for 2 h at room temperature. The PVDF membranes were then rewashed in TBST, and the Enhanced Chemiluminescence (ECL) Kit (Beyotime Biotechnology, Nanjing, China) was used to visualize positive immunobinding. GAPDH was selected as the internal control. These experiments were replicated three times in total.

GC (PubChem CID: 161120) and PQ were obtained from Sigma-Aldrich (Sigma, MO, USA). Anti-Nrf2, anti-HO-1, anti-NQO-1, anti-GCLM, anti-ICAM-1, anti-VCAM-1, anti-iNOS, anti-IKK-β, anti-IκB-α, anti-NF-κB p65, anti-phosphorylated (p)-IκB-α, anti-Bcl-2, anti-Bcl-xl, anti-Bax, anti-Caspase-3, anti-Caspase-9, anti-GAPDH, anti-β-actin, anti-histone, and IgG-HRP antibodies were products of Santa Cruz Biotechnology (Santa Cruz, Texas, USA). BCA protein concentration assay kit, PVDF membranes, and SDS-PAGE gel preparation kit were purchased from Beyotime Institute of Biotechnology. ECL plus kit was obtained from Nanjing KeyGen Biotech Co., Ltd. (KeyGen, Nanjing, CN). TNF-α, IL-1β, and IL-6 ELISA kits were obtained from Abcam (Cambrige, UK). GSH, NADPH, SOD, CAT, MDA, CK, and LDH kits were products of Nanjing Jiancheng Engineering Institute (Nanjing, CN).

We extracted total protein using SDS lysis buffer (Beyotime) supplemented with protease and phosphatase inhibitor cocktail (Beyotime). Total proteins were quantified using the Enhanced BCA Protein Assay Kit (Beyotime). Protein (30 μg per lane) was loaded into 12% SDS‐PAGE gel and run at 120 V for 2 h. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, Massachusetts, USA) at 250 mA for 1 h. PVDF membranes containing proteins were blocked by QuickBlock Blocking Buffer for Western Blot (Beyotime) at room temperature for 20 min and cut horizontally to examine multiple proteins of different sizes. Then, membranes were incubated with diluted primary antibodies (Supporting information Table S3) at 4°C overnight, followed by washing for three times. Membranes were subsequently soaked with horseradish peroxidase (HRP)‐conjugated secondary antibodies at room temperature for 1 h. Finally, blots were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore).

Cells were washed twice in PBS and lysed in RIPA buffer (Solarbio, China) with 1 mM phenylmethanesulfonyl fluoride (PMSF). A BCA protein kit was used to quantify protein concentrations (Beyotime, China). Total proteins (30 μg) were separated on 15% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Beyotime, China). The membranes were then blocked in TBST with 3% non-fat milk (Sangon Biotech, China) at 25°C for 2 h and incubated with anti-survivin (1:1,000), anti-caspase-3 (1:1,000), anti-Bcl-2 (1:3,000), anti-P-gp (1:3,000), and anti-β-actin (1:5,000) primary antibodies (Proteintech, USA) overnight at 4°C. Next, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies (1:10,000) (Proteintech, USA) at 25°C for 1 h. Protein bands were visualized by ECL chemiluminescence kit (Sangon Biotech, China), and the gray values of the protein bands were analyzed by Image J.