Beyo enhanced chemiluminescence reagent kit

MRS broth, TSB, TSA, MRS agar, 4% cell tissue fixative and Beyo Enhanced Chemiluminescence Reagent Kit were all purchased from Solarbio Life Science (Beijing, China). DMEM-F12 medium, FBS, 1% nonessential amino acids, 1% insulin-transferrin-selenium, PBS, BSA, and mouse TNF-α, IL-6, and IL-1β ELISA kits were all purchased from ThermoFisher Scientific (Waltham, MA, USA). 1% penicillin and streptomycin, Total Protein Extraction Kit, and BCA Protein Assay Kit were purchased from Beyotime Biotechnology (Shanghai, China). Bovine TNF-α, IL-6, and IL-1β ELISA kits was purchased from DG Biotech (Beijing, China), 15 ng/mL EGF was purchased from PeproTech (Cranbury, NJ, USA), and TBST was purchased from Nobleryder (Beijing, China). Primary antibodies for western blot (ZO-1, claudin-1, claudin-4, and occludin for BMECs) were purchased from Bioss Antibodies (Beijing, China), and ZO-1, claudin-3, and occludin for mice mammary tissue were purchased from Abcam, (Cambridge, UK). All secondary antibodies for western blot were purchased from CoWin Biosciences (Beijing, China).

Both BMECs and mammary tissue samples were obtained after treatment, and whole protein was extracted using a Total Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China). The protein concentrations were determined using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Each sample (40 μg of total protein) was separated by 12% SDS polyacrylamide gels and transferred onto PVDF membranes (Bio-Rad, CA, USA). The membranes were blocked with 5% BSA in 0.05% Tris-buffered saline with Tween (TBST, Nobleryder, Beijing, China) overnight at 4. The membranes were then incubated with primary antibodies (ZO-1, claudin-1, claudin-4, and occludin for BMECs, 1:500 dilution, Bioss Antibodies, Beijing, China; ZO-1, claudin-3, and occludin for mice mammary tissue, 1:2000 dilution, Abcam, Cambridge, UK) at room temperature for 3 h. After washing with TBST, incubation with secondary antibodies (1:1000 dilution, CoWin Biosciences, Beijing, China) was performed for 1 h at room temperature. Protein bands were visualized using a Beyo Enhanced Chemiluminescence Reagent Kit (Solarbio Life Science, Beijing, China). The volume of the protein bands was measured by MultiImager (Bio-Rad Gel Doc XR, CA, USA) and ImageJ software (National Institutes of Health, Bethesda, MD, USA).