β actin

Total cellular protein was extracted using RIPA lysis buffer (Aspenbio, Wuhan, China) and were measured by the BCA protein assay kit (Aspenbio, Wuhan, China). The primary antibodies including WNT2 (1:500 dilution, Proteintech, Wuhan, China), WNT4 (1:1000 dilution, Abgent, San Diego, CA, USA), and c-myc (1:500 dilution, Proteintech, Wuhan, China) were incubated in 4°C overnight. Goat anti-rabbit IgG (1:4000 dilution, SAB, College Park, MD, USA) and Goat anti-mouse IgG (1:4000 dilution, Aspenbio, Wuhan, China) were used as secondary antibodies. The protein expression levels were normalized to β-actin (1:4000, Sungenebiotech, Tianjin, China).

The primary antibodies and their dilution ratio in western blot analysis were as follows: p-mTOR-S2448 (1:1,000, Cell Signaling Technology); mTOR (1:1,000, Cell Signaling Technology); p-p70S6K-T389 (1:1,000, Cell Signaling Technology); p-p70S6K-T421/S424 (1:1,000, Cell Signaling Technology); p70S6K (1:1,000, Cell Signaling Technology); p-S6 Ribosomal Protein -S235/S236 (1:1,000, Cell Signaling Technology); RPS6 (1:1,000,Abcam); β-Actin (1:1,000, Tianjin Sungene Biotech Co., Ltd., China) and GAPDH (1:1,000, Good Here, China). DHA was purchased from Tokyo Chemical Industry (Cat #D3793, Japan). MK-2206 (AKT inhibitor, Cat #A10003) was purchased from Selleckchem. LY2584702 (p70S6K inhibitor, Cat #1082949–68–5) was purchased from Med Chem Express.

The harvested cells were lysed with radioimmunoprecipitation assay buffer (Beyotime, P0013B) for the extraction of total protein. Protein concentration was quantified with enhanced bicinchoninic acid protein assay kit (Beyotime, P0010). Then, lysate protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, ISEQ00010). The membranes were sequentially blocked and incubated overnight with the following primary antibodies: HK2 (Cell Signaling Technology, 2106), platelet-type phosphofructokinase (Cell Signaling Technology, 8164), phosphoglycerate kinase 1 (Abcam, ab38007), PKM2 (Cell Signaling Technology, 4053), LDHA (Cell Signaling Technology, 3582), c-Myc (Cell Signaling Technology, 13987), B-cell lymphoma-2 (Bcl-2) (Cell Signaling Technology, 4223), Bcl-XL (Proteintech, 26967-1-AP), Bad (Cell Signaling Technology, 9239), Bax (Cell Signaling Technology, 9292) and β-actin (Sungene Biotech, KM9006). The protein bands were acquired as an electronic images format using a ChemiDocTM XRS+ System (Bio-Rad) with immobilon western chemiluminescent horseradish peroxidase substrate (Millipore, WBKLS0100) and quantified the intensities by Image Lab software.

Protein extraction and western blot were performed as previously described [40 (link), 41 (link)]. Protein concentration was determined using a BCA Protein Assay Kit (Cowin Biotech, Beijing, China). Equivalent amounts of proteins were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidenedifluoride membranes (Millipore, Atlanta, GA, USA). The membranes were blocked for 2 h at 25°C± 2°C in Tris-buffered saline containing 0.05% Tween® 20 and 5% nonfat milk, and incubated with specific primary antibodies raised against p53 (1C12; Cell Signaling Technology, Beverly, MA, USA), phospho-p53(Ser15) (Cell Signaling Technology), p21Waf1/Cip1 (12D1; Cell Signaling Technology), cyclin E1 (HE12; Cell Signaling Technology), CDK2 (M2; Santa CruzBiotechnology, Santa Cruz, CA, USA), and β-actin (Tianjin Sungene Biotech, Tianjin, China) at 4°C overnight, and with the corresponding secondary antibody conjugated to horseradish peroxidase at appropriate dilutions for 1 h at 37°C. Images were captured using a GeneGnome XRQ chemiluminescence detector (Syngene, Cambridge, UK); the densities of the protein bands were normalized to the β-actin signal and quantified using GeneSys software (VilberLourmat, France). The abundance of the proteins of interest in the various treatments was expressed relative to the abundance under control conditions.

Protein extraction and SDS-PAGE were performed as described previously (14 (link)). Rabbit polyclonal antibody, β-actin and GAPDH were purchased from SunGene Biotech (SunGene, China). LepA-specific rabbit polyclonal antibody was obtained from HuaAn Biotech (HuaAn, China). Signal transducers and activators of transcription 3 (STAT3) and phosphorylation of STAT3 (p-STAT3) rabbit polyclonal antibodies were purchased from Elabscience Biotech (Elabscience, China).

The intestinal samples were lysed in radio immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) with protease and phosphatase inhibitor cocktails (Sigma Co., St. Louis, MO, United States) for 10 min on a rocker at 4°C. Proteins of samples were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (0.45 μm, Millipore, Billerica, MA, United States). Each PVDF membrane was blocked with tris buffered saline tween (TBST; 100 mM Tris-HCl,150 mM NaCl, 0.05% Tween 20, pH 7.5) with 5% non-fat dried milk for 2 h, and then incubated with the following primary antibodies: NLRP3 (1:1,500), ASC (1:100), Caspase-1 (1:800), IL-1β (1:400), and β-actin (1: 5,000, Sungene biotech, China) overnight at 4°C on a shaker (SC390C, Shanghai Brave Construction Development co., Ltd., Shanghai, China). The goat anti-rabbit of 1:3000 (ZSGE-BIO, China) horse radish peroxidase (HRP) conjugate secondary antibody was then added and the WB results was observed through electrochemical luminescence (ECL) imaging system after adding the enhanced ECL reagent (Beyotime, Shanghai, China). The β-actin was used as a protein loading control. Quantitative analysis was carried out using Amersham Imager 600 (Cytiva, Montana, United States).