Bicinchoninic acid protein assay kit

Frozen colonic and hepatic tissues as well as serum were homogenized with radioimmunoprecipitation assay lysis buffer (Solarbio, Beijing, China) to extract total proteins. Total protein level was quantified with a bicinchoninic acid protein assay kit (Solarbio, Beijing, China). The concentrations of IL-6, IL-1β, and IL-10 were measured by ELISA kits (R&D Systems, Minneapolis, MN, USA). The levels of total superoxide dismutases (T-SOD), catalase (CAT), glutathione peroxidase (GSH-px), and malondialdehyde (MDA) were quantified using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s directions.

After indicated treatment, cells were harvested and washed twice with PBS. Then they were lysed in RIPA buffer (Solarbio, Beijing, China) containing complete protease and phosphatase inhibitor (Solarbio, Beijing, China). The level of protein concentrations in cells was measured with a bicinchoninic acid protein assay kit (Solarbio, Beijing, China). The proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes by a wet electrophoretic transfer method. The membrane was blocked with TBST containing 5% non-fat milk for 2 h at room temperature followed by incubation overnight at 4 °C with primary antibodies. After overnight incubation, the blots were washed three times with TBST and incubated with secondary antibodies for 1 h at room temperature. Finally, the membranes were scanned using a Bio-Rad Gel imaging system (Bio-Rad, Berkeley, CA, USA) after visualization treatment with the ECL reagent. The primary antibodies, rabbit anti-PBK, rabbit anti-KIAA0101, rabbit anti-TOP2A, rabbit anti-IL-1β, and rabbit anti-β-actin, were purchased from Cell Signaling Technology (Boston, MA, USA). The rabbit anti-ASPM and rabbit anti-MCM10 were obtained from Proteintech (Rosemont, Chicago, IL, USA). Quantitative assessment of the and intensity was performed using Image Lab statistical software.

The cells and tissue specimens were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing phenylmethanesulfonyl fluoride (Beyotime) and a protease inhibitor (CoWin Biosciences). The protein concentration was determined using a bicinchoninic acid protein assay kit (Beijing Solarbio Science and Technology Co., Ltd. Beijing, China). Each sample with equal amounts of protein were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After running 1–2 h under 120V, the protein was transferred onto a polyvinylidene difluoride membrane. Next, the membranes were blocked with 5% not-fat milk for 1 h at room temperature and incubated with primary antibodies c-Myc, HIF-1α, CXCR4, SDF-1, vimentin, E-cadherin, and β-actin overnight at 4°C (Affinity Biosciences). Subsequently, the membranes were washed 3 times with tris-buffered saline and Tween-20 and incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology, Beverly, MA, USA). Finally, the membranes were treated with a chemiluminescence reagent and exposed to X-ray. The band intensities were quantified using image J software (National Institutes of Health, Bethesda, MD, USA). Relative protein expression was quantified using control protein β-actin.