Bicinchoninic acid protein assay kit

The protein lysates from the HK-2 cells were prepared, and the protein content was determined using the bicinchoninic acid protein assay kit (Nanjing KeyGen Biotech, Nanjing, China). The nuclear protein was extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). The protein samples (40 μg/per lane) were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) using a wet transfer system. Membranes were then blocked in 5% bovine serum albumin in Tris-buffered saline with Tween 20 (TBS-T) buffer for 1 h at room temperature, followed by incubation with primary antibodies against ANGPTL2 (1:1000), bcl-2 (1:1500), bax (1:1500), TLR4 (1:800), p65 (1:1000), p-p65 (1:2000), Nrf2 (1:1500), lamin B1 (1:800), HO-1 (1:1000), and β-actin (1:1500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. Subsequently, membranes were washed by TBS-T buffer for three times and incubated with secondary horseradish peroxidase-conjugated antibodies (1:2000; Santa Cruz Biotechnology) for 2 h at room temperature. The blots were detected using an ECL advanced system (GE Healthcare, Piscataway, NJ, USA), and the signals were analyzed by ImageJ software (National Institutes of Health, NIH, Bethesda, MD, USA). The experiment was performed in triplicate.

Cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology) supplemented with a protease inhibitor cocktail (Roche Diagnostics), and the extracted protein was quantified using a bicinchoninic acid protein assay kit (Nanjing KeyGen Biotech Co., Ltd.). Equal amounts of protein (30 µg per well) were separated by electrophoresis on 10% SDS-PAGE gels, and transferred onto polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked for 2 h at room temperature with 5% nonfat dry milk in Tris-buffered saline (0.1% Tween-20). After blocking, the membranes were incubated with primary antibodies [anti-HDAC9 (cat. no. ab109446; Abcam) and anti-GAPDH (cat. no. ab128915; Abcam), both 1:1,000] at 4°C overnight, and further incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. ab205718; Abcam). The blots were developed using the Immobilon Western Chemilum HRP substrate (EMD Millipore) and the assay was repeated three times. Quantity One software version 4.62 (Bio Rad Laboratories, Inc.) was used for densitometric analysis.

Precooled RIPA lysis buffer (Beyotime; Shanghai, China) supplemented with protease inhibitors (Beyotime) was used to isolate total proteins from the cells. The total-protein concentrations were determined using the Bicinchoninic Acid Protein Assay Kit (Nanjing KeyGen Biotech Co., Ltd.). Equal amounts of protein were subjected to sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis and subsequent electrophoretic transferred onto polyvinylidene fluoride membranes. Next, the membranes were blocked in 5% nonfat milk diluted in Tris-buffered saline containing 0.1% Tween 20 (TBST). The membranes were then incubated with primary antibodies specific for HMGB1 (1:1000 dilution in TBST; cat. No. ab79823; Abcam, Cambridge, UK) or GAPDH (1:1000 dilution in TBST; cat. No. ab128915; Abcam). Following an overnight incubation at 4 °C, the membranes were rinsed thrice with TBST and probed with horseradish peroxidase-conjugated secondary antibody (1:5000 dilution in TBST; cat. No. ab205718; Abcam) at room temperature for 1 h. The protein immunoblots were visualized using the Immobilon Western Chemilum HRP substrate (Millipore). GAPDH acted as the loading control.

Following transfection for 48 h, Kyse410 and Kyse520 cells were harvested and lysed with RIPA buffer (KeyGEN BioTECH); protein concentration was quantified using a bicinchoninic acid protein assay kit (Nanjing KeyGen Biotech Co., Ltd.). The samples were boiled with 5X loading buffer, and 20-µg aliquots of total protein were separated using 8% SDS-PAGE gels, prior to transfer onto PVDF membranes (EMD Millipore). The membranes were blocked at room temperature in 5% nonfat milk for 2 h and incubated with GAPDH (1:500; cat.no. GB11002; Wuhan Servicebio Technology Co., Ltd.) or hTERT (rabbit anti-human TERT, polyclonal; 1:1,000; cat. no. ab183105; Abcam) primary antibodies overnight at 4°C. The membranes were than washed, and subsequently incubated with goat anti-rabbit IgG (H+L) secondary antibody (1:10,000; cat. no. SA00001-2; ProteinTech Group, Inc.) for 1 h at room temperature. Proteins were visualized using the FluorChem M System (ProteinSimple).