Bca kit

Total protein was extracted from cultured cells using RIPA Lysis Buffer (Beyotime, #P0013B) and the protein concentration was determined by BCA kit (Genstar, #E162). The total protein was loaded onto a sodium dodecyl sulfate-polyacrylamide gel and ran at 120 volts for 2 hours. After electrophoresis, the proteins were transferred to a polyvinylidene fluoride membrane and treated with 5% skim milk TBST (50 mM Tris; 150 mM NaCl; 0.05% Tween 20) closed for one h. The PVDF membrane was then incubated at 4°C overnight with primary antibody and at room temperature with enzyme-labeled secondary antibody for 1 hour. Super-sensitive ECL chemiluminescent substrate (Biosharp, #BL520A) was used to reveal protein bands. The antibodies used are: ZO-1 (1:6000, Proteintech, # 21773–1-AP), HIF-1α (1: 6000, Proteintech, #20960–1-AP), Occludin(1:6000, Proteintech, # 27260–1-AP), alpha-tubulin (1:2000, Proteintech, #11224–1-AP), Goat Anti-Rabbit IgG (H + L)-HRP Conjugate(1:4000, Bio-Rad, #1706515). ImageJ is used for grayscale measurement.

The transfected cells were added to RIPA lysate and protease inhibitor to extract total protein (EpiZyme, Shanghai, China). After lysis on ice for 30 min, the proteins were centrifuged at 12000 rpm for 15 min at 4°C. The loading volume of each sample was measured using a BCA kit (GenStar, Beijing, China). Identical masses of proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were transferred to PVDF membranes (Bio-Rad, UK) and sealed with skim milk powder. PVDF membranes were incubated overnight at 4°C with primary antibodies against VCAM-1 (1:500, Proteintech, USA), ICAM-1 (1:2500, Proteintech, USA), CDH2 (1:1500, Proteintech, USA), and GAPDH (1:10000, Proteintech, USA). Finally, the HRP-labeled goat anti-mouse secondary antibody (1:5000, Proteintech, USA) was incubated with the filter membrane for 1 h. GNG12 protein expression levels were detected using an imager and a chemiluminescent substrate (ECL) kit (Thermo Fisher Scientific, USA).

The GMECs and mammary tissues were lysed using high-efficiency RIPA lysate (Solarbio, Co., Ltd., Beijing, China) or a nuclear protein extraction kit (BestBio, Co., Ltd., Nanjing, China) to obtain total proteins, nuclear proteins and cytoplasmic proteins. The concentration of protein samples was determined using a BCA kit (Genstar). For Western blotting, samples with a loading buffer (CWBIO, Co., Ltd., Beijing, China) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis on gels (Jingcai Biological, Co., Ltd., Xi’an, China) and blocked in Tris-buffered saline Tween (TBST) with 5% non-fat milk (BBI, Co., Ltd., Shanghai, China) for 2 h at 25 °C after being transferred to a polyvinylidene fluoride membrane (Millipore, Sigma-Aldrich). The membranes were then incubated with specific primary antibodies at 4 °C overnight and with horseradish peroxidase-conjugated secondary antibodies (1:4000, diluted in TBST) at 25 °C for 2 h. The dilution ratios of the primary antibodies are listed in Table A2. After antibody incubation, the membrane was washed with TBST and photographed using a G:BOX Chemi XRQ (Syngene, Co., Frederick, MD, USA) imaging system.