Luminol enhancer solution

Cells were mixed with radioimmunoprecipitation assay (RIPA) buffer (Solarbio), phenyl methyl sulfonyl fluoride (PMSF) buffer (Solarbio), and phosphatase inhibitors (Glpbio) for protein extraction. Protein was electrophoresed using SDS–PAGE (10%, 12%, or 15%) and transferred to nitrocellulose membranes. The membranes were blocked with skim milk (5%) in TBST for 1 h at room temperature and incubated with goat anti-rabbit polyclonal β-catenin, TCF4, AFP, ALB, CK19, β-actin (diluted 1:1000–1:10,000) (Proteintech) and p-β-catenin and HNF4A antibodies (diluted 1:1000–1:5000) (Abcam) and CDX2 (diluted 1:1000) (Cell Signal Technology) at 4 °C overnight. After washing with TBST three times, the nitrocellulose membranes were incubated with a 1:1000 dilution of horseradish peroxidase (HRP) goat anti-rabbit IgG (Cell Signalling Technology) for 1 h at room temperature. After washing with TBST three times, the membranes were incubated with a mixture of peroxide solution (Bio-Rad) and luminol enhancer solution (Bio-Rad) (1:1). Finally, the results were detected by a Tanon 5500 chemiluminescent imager system (Tianneng) and standardiz to β-actin protein. Western blotting experiments were repeated three times.

Colon tissues were weighted and put into homogenizer to grind into tissue homogenate with mixture phenylmethylsulfonyl fluoride (PMSF) (Besbio, China) and radioimmunoprecipitation assay (RIPA) (Besbio, China). Protein concentration was measured by bicinchoninic acid assay (BCA) protein detection kit (Biyuntian, China) and micro-spectrophotometer. Proteins were electrophoresed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and transferred to nitrocellulose membranes. Membranes were blocked with skim milk (5%) in TBST for 1 h at room temperature and incubated with a rabbit anti-mouse polyclonal occludin antibody (diluted 1:1,000) (Proteintech, United States) at 4°C overnight (Zacharek et al., 2007 (link)). After being washed thrice in TBST, the membranes were reacted with a 1:1,000 dilution of horseradish peroxidase (HRP) goat anti-rabbit IgG (Dingguo Biotechnology Co., Ltd., China) for 1 h at room temperature. After being washed thrice in TBST, membranes were reacted with the mixture of peroxide solution (Bio-Rad, United States) and Luminol enhancer solution (Bio-Rad, United States) (1:1). Finally, the results of WB were detected by Tanon 5500 chemiluminescent imager system (Tianneng, China), referencing by β-actin protein as standardization. The process was repeatedly thrice for WB experiments.

HEK293T cells (Cat# GNHu43, obtained from Stem cell Bank of Chinese Academic Science, the identity has been authenticated using Short Tandem Repeat profiling) were tested for mycoplasma contamination and showed negative result. Fresh mouse adult testes or HEK293T cells were collected, and proteins were extracted by using RIPA (Radioimmunoprecipitation assay) buffer (Beyotime, P0013J, China). In total, 40 μg of protein lysates were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel, proteins were transferred to PVDF (Polyvinylidene Fluoride) membranes (Bio-Rad) and the membranes were blocked in 5% non-fat milk (blocking solution) for 1 hr. Primary antibodies were incubated overnight at 4°C after blocking. The membranes were washed with TBST three times and then incubated with a secondary antibody for 1 hr before using Luminol/enhancer solution and Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc US). Western blot images were scanned by using Gel Doc XR system (Bio-Rad Laboratories, Inc US).

The Western blotting protocol follows a previous work [18 (link)]. Infective HBMECs were collected with a mixture of phenylmethylsulfonyl fluoride (PMSF) (Besbio, Shanghai, China), radioimmunoprecipitation assay (RIPA) (Besbio), and phosphatase inhibitors (Glpbio, Montclair, CA, USA). PVDF membranes were blocked with skim milk (5%) in TBST for 1 h at room temperature and incubated with a rabbit anti-mouse polyclonal JAK2, p-JAK2 antibody (diluted 1:3000) (Abcam or Proteintech, Rosamond, CA, USA); STAT5b, p-STAT5b and occludin antibody (diluted 1:2000) (Proteintech, Rosamond, IL, USA); and CISH (diluted 1:1000) (Abclone Technology, Wuhan, China) antibody at 4 °C overnight. The membranes were reacted with a 1:1000 dilution of horseradish peroxidase (HRP) goat anti-rabbit IgG (Dingguo, Beijing, China) for 1 h at room temperature. After being washed three times in TBST, the membranes were reacted with a mixture of peroxide solution (Bio-Rad, Hercules, CA, USA) and luminol enhancer solution (Bio-Rad) (1:1). Finally, the WB results were detected by a Tanon 5500 chemiluminescent imager system (Tianneng, Huzhou, China), referencing β-actin protein for standardization.