Cst 13663

Colon tissues were lysed in 1 mL RIPA lysis buffer containing 1 mM PMSF and 1% phosphatase inhibitor cocktail (Applygen Technologies Inc., Beijing, China). Protein concentrations were measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Samples (25–50 μg) were separated using 10% SDS–PAGE and then wet transferred to polyvinylidene difluoride membranes (PVDF, EMD Millipore, Burlington, USA). Membranes were blocked with 5% non-fat milk and incubated with primary antibodies against occludin (1:1000; ab167161, Abcam), claudin-1 (1:1000; ab180158, Abcam), ZO1 (1:1000; CST#13663,Cell Signaling Technologies, Darmstadt, Germany), TLR4 (1:500; sc-293072, Santa Cruz), NF-κB (1:1000; CST#8242, Cell Signaling Technologies), phosphorylated (p) NF-κB (1:1000; CST#8214, Cell Signaling Technologies), MyD88 (1:1000; ab2064, Abcam), or β-actin (1:1000; CST#4970, Cell Signaling Technologies) and incubated overnight at 4°C with gentle shaking. The membranes were washed three times and then incubated with a horseradish peroxidase–linked secondary antibody (1:1000; Beyotime, Jiangsu, China). Immunoreactivity was detected using an enhanced chemiluminescence reagent (WBKLS0500; EMD Millipore) and quantified using Image Lab 5.2.1 software (Bio-Rad, Hercules, CA, USA).

Proteins were extracted from solid tumours and colon tissues with 1% phosphatase inhibitor cocktail, quantified by BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, United States). 10% SDS–PAGE were chose to resolve samples and then transferred to PVDF membranes (EMD Millipore, Burlington, United States). After blocking with 5% BSA for 2 h, the membrane was incubated with primary antibodies: occludin (1:1000; ab167161, Abcam, Cambridge, United Kingdom), SDF-1 (1:1000; ab9797, Abcam), CXCR4 (1:1000; ab124824, Abcam), ZO-1 (1:1000; CST#13663, Cell Signaling Technology, Darmstadt, Germany), cyclin D1 (1:500; CST#2978, Cell Signaling Technology) and c-myc (1:500; CST#5605, Cell Signaling Technology), then gentle shaked overnight at 4°C. After washing, the membranes were incubated with the second antibodies for 1 h (goat anti-rabbit or IgG anti-mouse, 1:5,000; Cell Signalling Technology). Immunoreactivity was visualized with an enhanced chemiluminescence system (ECL kit; Santa Cruz Biotechnology, United States) and quantified using Image Lab 4.0 (Bio-Rad, Hercules, CA, United States).