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).