Bca protein assay kit

The EMT-6 cells were incubated with or without CpG ODN-conditioned supernatant for 24 hours, washed with PBS, harvested and lysed in ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS). The cell lysate were centrifuged (14,500g for 20 min at 4°C) and the supernatant was collected, quantified using a BCA protein assay kit (Wanleibio, Shenyang, China) and separated by 12% SDS-PAGE. Then the separated protein bands were transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked overnight at 4°C with Tris-buffered saline containing 5% nonfat dried milk and then incubated with anti-H60 mAb (R&D system, USA) or anti-GAPDH mAb (Proteintech, USA). Blots were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for 1 h at room temperature (Jackson immunoresearch laboratories, USA). Immunoreactive bands were visualized with SuperSignal West Pico chemiluminescent substrate (Thermo, USA).

Total protein was extracted from HRECs or retinal tissues using a Whole Cell Lysis Assay (Wanleibio, Shenyang, China), and the protein concentration was detected using the BCA Protein Assay kit (Wanleibio). Forty micrograms of total protein were separated by 6%, 8%, or 12% SDS-PAGE and were transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) at 80 V for 90 min. After blocking with 5% skimmed milk for 1 h, the membranes were incubated with primary antibodies, anti-ROBO4 (1:1000; 2022-1-AP, Proteintech, Wuhan, China), anti-DNMT1 (1:1000; 24206-1-APm, Proteintech), anti-TET2 (1:1000; A5682, ABclonal, Wuhan, China), anti-ZO-1 (1:500; WL03419, Wanleibio), anti-occludin (1:500; WL01996, Wanleibio), or anti-β-actin (1:1000; WL01845, Wanleibio) overnight at 4 °C. The membranes were then incubated at 37 °C for 45 min with a horseradish peroxidase-conjugated secondary antibody (1:5000; WLA023, Wanleibio) and developed using enhanced chemiluminescence substrate. All bands were quantified using a Gel-Pro Analyzer, and band densities were normalized to β-actin values.

RAW264.7 cells were seeded in dishes with 6 cm at 2 × 10⁶ cells/well in RPMI 1640 complete medium in a humidifying incubator filled with 5% CO₂ at 37°C overnight. Cells were processed with 500 ng/ml LPS, and indicated three concentration of SOG (25, 50, 100 μM) after changing of medium. After washing three times with ice-cold PBS, cells were lysed in RIPA buffer consisted of 150 mM NaCl, 50 mM Tris, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS. The supernatant was collected after being centrifuged at 14,500 g at 4°C for 20 min. A BCA-protein assay kit (Wanleibio, Shenyang, China) was employed to determine contents of protein of samples. Fifty micrograms of protein were isolated using 12% SDS-PAGE and electro-transferred to a PVDF membranes (Millipore, U.S.A.). The membranes were blocked by TBST containing 3% BSA for 2 h at room temperature, and probed with various primary antibodies at 4°C overnight. After being washed three times with TBST at intervals of 10 min, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After three times of washing, the immunoreactive bands were visualized using chemiluminescent substrate of SuperSignal West Pico (Thermo, U.S.A.) and were further quantified using ImageJ software (National Institutes of Health, U.S.A.).

NIH/3T3 cells were cultured and treated as described above. After protein extraction, the target protein was quantified by the BCA protein assay kit (Wanleibio, China), heated at 100°C for 5 min to denature, and stored at −20°C. The protein was separated in 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) (Wanleibio, China) and then transferred onto a 0.45-μm PVDF membrane via electrophoresis in Tris-glycine buffer (Solarbio Biotechnology, China). The PVDF membrane was blocked in Tris-buffered saline containing 0.05% Tween-20 (TBST) with 5% skimmed milk powder at room temperature for 1 h, followed by incubation with AKT, p-AKT, mTOR, p-mTOR, GAPDH, Beclin-1, p62, LC3-I, and LC3-II primary antibodies for overnight at 4°C. Then, the PVDF membrane was washed with TBST and incubated with a secondary antibody at room temperature for 1 h. Finally, after washing with TBST, the membrane was exposed and imaged using the ECL chemiluminescence kit (Beyotime, China). HRP-conjugated affinipure goat antirabbit IgG was sourced from Wanleibio (China).

Western blotting was used to measure the IL-17 pathway-related target proteins. The total proteins of lung tissue were extracted by whole-cell lysis assay, and the supernatant protein content was determined using a BCA protein assay kit (Wanlei, Liaoning, China). The membranes were incubated overnight on a shaker at 4°C with primary antibodies against β-actin (1:5000 dilution), TRAF6 (1:500 dilution), CIKS (Act1), NFκB (p65), AP1 (c-jun), and C/EBPβ (1:1000 dilution). All the primary antibodies were purchased from Bioss Bioscience Inc., Beijing, China. Secondary anti-mouse and anti-rabbit IgG peroxidase was used for 1 h, and then bound immune complexes were detected using enhanced chemiluminescence (ECL) detection. The protein bands were analyzed by densitometry using Image J (Ver 1.42, National Institutes of Health, United States).