Anti bnp

The tissue was homogenized in 10 mM Tris-HCl buffer, pH 7.4 containing 0.5 mM EDTA, pH 8.0, 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₄VO₃, and a protease inhibitor cocktail (Roche-Boehringer-Mannheim, Mannheim, Germany). After centrifugation, the supernatant was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples equivalent to 25 µg of protein content were loaded and size-separated in 8%–12% SDS-PAGE. The proteins on the acrylamide gel were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford MA, USA) at 400 mA in a transfer buffer containing 25 mM Tris and 192 mM glycine, pH 8.4. The nitrocellulose membranes was blocked in tris-buffered saline with 5% non-fat dry milk at room temperature for 1 hour in 0.1% Tween 20 and incubated with the appropriated primary antibodies, including anti-BNP (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and anti-actin (Santa Cruz Biotechnology Inc.), at 4℃ overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology Inc., Danvers, MA, USA) for 1 hour at room temperature. After washing, the membranes were visualized by a chemiluminescent ECL-detection kit from GE-Healthcare (Piscataway, NJ, USA).