Ripa protein lysate

Total protein of IPEC-J2 cells were extracted with RIPA protein lysate (Beyotime Technology, Shanghai, China) containing 1% protease inhibitor. Protein concentrations were measured by a bicinchoninic acid (BCA) protein assay kit (#A045-4-2, Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). Equivalent amounts of protein with 4 × loading buffer were denatured at 98°C for 10 min, followed by cooling on ice. The denatured protein was separated by 12% SDS-PAGE gel and transferred to the PVDF membrane (Millipore, Bedford, MA, USA). Then, the PVDF membrane was blocked with 5% skim milk for 1 h at room temperature, and incubated with the primary antibodies PCNA (#GB11010, Servicebio Technology Co., Ltd., Wuhan, China), Cyclin-D1 (#GB111372, Servicebio Technology Co., Ltd.), AHR (#A00225-4, BOSTER Biological Technology Co., Ltd.), and β-actin (#20536-1-AP, Proteintech Group, Inc., IL, USA) at 4°C overnight. After being washed three times with TBST, the PVDF membrane was incubated with the secondary antibody for 1 h at room temperature. Similarly, the PVDF membrane was washed three times with TBST. Finally, the target protein bands were visualized by using a chemiluminescence system (Tanon, Shanghai, China). Band intensities were quantified using ImageJ 1.53 m software, and all results were expressed as target protein/β-actin protein ratio.

As previously described [23 (link),24 (link)], the RIPA protein lysate (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China) was employed to extract the total proteins from fresh colon tissue homogenates. The proteins were quantified with the bicinchoninic acid (BCA) protein assay kit (Biosharp, Wuhan, China) and denatured at 95 °C for 10 min, then separated by 12% SDS-PAGE gel electrophoresis, and transferred to the nitrocellulose membrane by electroblotting. The membrane was blocked with 5% skim milk, then incubated with primary antibodies against TLR4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and cleaved Caspase 3, Bcl-2, Bax, NF-κB p65, p-STAT3 (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Proteintech, Wuhan, China) overnight at 4 °C. The HRP-labeled goat anti-rabbit or mouse secondary antibodies (Servicebio, Wuhan, China) were added to incubate for 1 h. The gray value of each strip was measured after ECL (Wuhan Kerui Biotechnology Co., Ltd., Wuhan, China) luminous development. β-actin was utilized as the control in the same sample.

Proteins were obtained from cells lysed in RIPA protein lysate (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors (Roche). Quantification of protein was conducted with Bicinchoninic Acid Protein Assay Kit (Thermo Scientific, USA). Protein samples were separated by 4–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Blocked in 5% skim milk for 2 h at room temperature, the membrane was in diluted primary antibody overnight at 4°C. Washing with TBST, the membrane was then incubated with a secondary antibody. The band signals were visualized and quantified using the Quantity One system (Bio-Rad, Hercules, CA, USA). GAPDH and β-actin were selected to be the loading controls. All the antibodies employed in this study were listed in Table. S2.

Western blotting was conducted as described previously (Kim et al., 2018 (link)). The cell extracts were prepared using RIPA protein lysate (Beyotime, P0013) and measured by a Bio-Rad protein assay. The proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), blotted onto PVDF membranes, and the membranes were immediately blocked with 5% skim milk/bovine serum albumin (BSA) in tris-buffered saline and Tween 20 (TBST) for 1 h at room temperature, followed by incubation with primary antibodies at 4°C overnight. Following incubation, the blots were incubated with the corresponding horseradish peroxidase secondary antibody. The target proteins were visualized with an enhanced chemiluminescence system using ECL Advance Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, United Kingdom). Densitometric analysis for the quantification of the band intensities was performed using ImageJ software.

Tissue specimens were collected from 43 cases of HCC specimens surgically resected and their adjacent normal tissues from February 2018 to October 2018 in our hospital. The study was approved by the medical ethics committee of the hospital, and all patients and their families have signed informed consent; Dulbecco's Modified Eagle Medium (DMEM), 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT), fetal bovine serum, and trypsin were purchased from GIBCO; Lipofectamine ^TM 2000, Trizol, and TaqMan miRNA reverse transcription kit were purchased from Thermo Fisher; polyvinylidene difluoride (PVDF) membrane was purchased from Roche (Basel, Switzerland); ECL luminescent solution and RIPA protein lysate were purchased from Beyotime Biotechnology Co., Ltd.; The luciferase reporter gene detection kit was purchased from Promega; Trans‐well chambers, Matrigel were purchased from Costar; semi‐dry film converters were purchased from BIO‐RAD.