Western blot analysis
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).