Quick start bovine serum albumin standard

Extracellular proteins were obtained from culture supernatants. For isolation of intracellular proteins cell pellets were resuspended in 50 mM Tris, pH 7,5 and disrupted by two passages through a French Pressure cell press (2 kbar). The soluble proteins were collected by centrifugation (15.000 rpm, 45 min, 4°C). Intra- and extracellular protein concentrations were quantified by Roti-Nanoquant Kit (Roth) in microtiterplates according to the manufacturer's instructions. Colorimetric reactions were measured with Infinite F200 reader (Tecan) at wavelengths of 610/450 nm. Protein concentrations were determined by quotient of optical densities OD₆₁₀/₄₅₀ referred to internal standard generated by Quick Start Bovine Serum Albumin Standard (Biorad).

Cardiac strips isolated from mice were stimulated with HTK cardioplegia solution and oh8dG. Proteins were isolated from cardiac strips using lysis buffer containing (mM) 20 Tris, 150 NaCl, 1% Triton X-100, 2 EDTA, and a protease inhibitor cocktail; a Bradford assay (Quick Start Bovine Serum Albumin standard, 5000207, Bio-Rad, Hercules, CA, USA) was used to quantify the concentration. Protein samples were denatured in sodium dodecyl sulfate (SDS) sample buffer at 37 °C for 30 min. Denatured protein samples (30 μg) were subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to PVDF membrane and incubated primary antibodies (1:1000 dilution), NBC1e1 (ab187511, Abcam, Cambridge, UK), NBCn1 (ab82335, Abcam), connective tissue growth factor (ac365970, CTGF, Abcam), and β-actin (A3854, Sigma, St. Louis, MO, USA) and then visualized with horseradish-conjugated secondary antibodies (1:2000 dilution for mouse and rabbit IgG) using an enhanced chemiluminescence solution (Thermo Fisher Scientific, Waltham, MA, USA).

The extracellular protein contents were determined from B. cereus culture supernatants. Protein amounts were quantified by Roti-Nanoquant Kit (Roth, Karlsruhe, Germany) in microtiter plates according to the manufacturer's instructions. Colorimetric reactions were measured with Infinite F200 reader (Tecan) at wavelengths of 610/450 nm. Protein concentrations were determined by quotient of optical densities OD 610/450 referred to an internal standard generated by Quick Start Bovine Serum Albumin Standard (Biorad).