Bicinchoninic acid protein assay reagent kit

Mouse cortical proteins were extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Solarbio, China). The protein concentration was quantified by a bicinchoninic acid protein assay reagent kit (Thermo Fisher Scientific, USA). Then, equivalent amounts of protein (50 μg) with buffer were loaded in gels and separated by SDS-PAGE. Next, proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Roche, USA) and blocked with 5% nonfat milk for 1 h at room temperature, followed by incubation with primary antibodies specific for occludin (Invitrogen, US), claudin5 (Bioworld Technology, USA), TNFα (Santa Cruz, USA), Histone H3 (Abcam, UK), citH3 (citrulline R2+R8+R17) (Abcam, UK), and β-actin (Cell Signaling Technology, USA) overnight at 4°C. After being washed three times with TBST, the membrane was incubated with anti-rabbit IgG secondary antibody (Rockland, USA) or anti-mouse IgG secondary antibody (Rockland, USA) for 1 h at room temperature and then was washed with TBST for another three times. Finally, the membrane was scanned by an infrared scanner (LICOR Bioscience, USA). The intensity of the bands was calculated by the ImageJ software.

Dense and sparse cultures of bovine aortic endothelial cells were lysed in sodium dodecyl sulfate sample buffer (50 mM Tris-HCl buffer, pH 6.8, containing 2% sodium dodecyl sulfate and 10% glycerol) and incubated at 95 C for 5 min. Separately, the nuclear fraction of bovine aortic endothelial cells was prepared using the NE-PER^® Nuclear and cytoplasmic extraction reagents, according to the manufacturer’s protocol. Protein concentrations were determined using a bicinchoninic acid protein assay reagent kit (Thermo Fisher Scientific). 2-Mercaptoethanol and bromophenol blue (1.67% each) were added to samples and incubated at 95 C for 3 min. Cellular proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 8%, 10%, or 12% polyacrylamide gels and electrotransferred onto polyvinylidene difluoride membranes (0.2 µm) at 2 mA/cm² for 1 h. Membranes were blocked with 5% skim milk in 20 mM Tris-HCl buffer solution (pH 7.5), containing 15 mM NaCl and 0.1% Tween 20 (TTBS) or 2% serum albumin-TTBS solution, then incubated with a primary antibody at 4 C overnight. After washing with TTBS, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence using Chemi-Lumi One L and scanned with a LAS 3000 Imager (Fujifilm, Tokyo, Japan).

The concentration of malondialdehyde was analyzed using the thiobarbituric acid colorimetric method [24 (link)]. The antioxidant enzyme activity of RBCs, including catalase, glutathione peroxidase, and superoxide dismutase, was determined by calculating the changes in the optical density of the enzyme activity reaction over one minute [25 (link)–27 (link)], and the data are shown as units/mg protein. RBC protein was analyzed using the bicinchoninic acid protein assay reagent kit (Thermo Scientific, Rockford, IL, USA).

Tissue samples for immunoblotting were placed in 10 ml ice-cold isolation solution, containing 250 mM sucrose, 10 mM triethanolamine (Sigma-Aldrich, St. Louis, MO, USA), 1 mg/ml leupeptin (Sigma-Aldrich) and 0.1 mg/ml phenylmethylsulfonyl fluoride (Sigma-Aldrich) titrated to pH 7.6, and the mixture was homogenized at 13,600 × g with three strokes for 15 sec using a tissue homogenizer (PowerGun 125; Thermo Fisher Scientific, Pittsburgh, PA, USA). Following homogenization, the total protein concentration was measured using a bicinchoninic acid protein assay reagent kit (Thermo Fisher Scientific, Rockford, IL, USA), which was adjusted to 2 mg/ml with isolation solution. Equal amounts of protein and sample buffer were separated using 12% gradient SDS-PAGE, stained with Coomassie Brilliant Blue and transferred to a polyvinylidene fluoride membrane. The blotted membrane was blocked with Tris-buffered saline containing 5% milk, and incubated with HIF-1α, VEGF or CD34 rabbit polyclonal antibodies (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), followed by incubation with a HRP-coupled secondary antibody (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA). The proteins were detected using enhanced chemiluminescence (Thermo Fisher Scientific). All immunoblots were representative of at least three independent experiments.

Epithelial Breast Cancer Cells (T47-D cells, ATCC) were plated and grown Roswell Park Memorial Institute medium (RPMI) without phenol red, supplemented with 10% of fetal bovine serum, 0.2 Units/ml of bovine insulin, 1 mM of HEPES, 2.4 μg/mL of glucose and 1 mM of sodium pyruvate (all from ThermoFisher Canada, Mississauga, ON). Cells were kept in a humidified incubator with 5% CO₂ at 37 °C until they reached ≈90% confluence. The cells were rinsed twice with ice-cold PBS and then harvested in 500 µl of lysis buffer added directly to the plate with scraping. The homogenates were transferred to 1.5 ml microtubes and incubated on ice for 15 min. The cell homogenates were then centrifuged for 5 min at 1000 g at 4 °C. The resulting supernatants were aliquoted and kept at -80 °C.
Lysate protein concentrations were measured using the Bicinchoninic acid protein assay reagent kit (Thermo scientific). Before being used for western blotting, the lysates were mixed with a solution containing 950 µL 4 × LDS Sample Buffer (GenScript – M00676) with 50µL of 8 M DTT to a working concentration of 3 to 4 µg/µL of total protein.

Malondialdehyde (MDA) in plasma was analyzed by the thiobarbituric acid reactive substance method [19 (link)]. Protein carbonyl groups in plasma were measured by condensation reaction with 2,4-dinitrophenylhydrazine [20 (link)]. The protein content in plasma was analyzed with a bicinchoninic acid protein assay reagent kit (Thermo Scientific, Rockford, IL, USA). Protein carbonyl is expressed as nmol/mg protein. Total antioxidant capacity (TAC) in serum and RBC was measured by a Trolox equivalent antioxidant capacity assay, and the wavelength was set at 730 nm [21 (link)].