Rabbit anti enos

Total protein was obtained from left ventricular myocardial tissues by sonication, centrifugation and heat denaturation. The protein lysates were electrophoresed and separated on 6–12% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skim milk at room temperature for 1 h, and then incubated overnight at 4°C with primary antibodies, including rabbit anti-Akt (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-phospho Akt (1:1,000; Cell Signaling Technology, Inc.), rabbit anti-eNOS (1:1,000; Sigma), rabbit anti-phospho-eNOS (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-NF-κB (1:800; Cell Signaling Technology, Inc.), rabbit anti-Bcl-2 (1:800; BioWorld, Inc., Visalia, CA, USA), rabbit anti-Bax (1:800; BioWorld, Inc.), and rabbit anti-GAPDH (1:1,000; Cell Signaling Technology, Inc.). The membranes were then incubated with HRP-conjugated secondary antibodies (1:500; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. The SuperSignal ECL kit (Thermo Fisher Scientific, Rockville, MD, USA) was used to detect the antigen-antibody complexes in a western blotting detection system (Bio-Rad). The results were expressed as density values normalized to GAPDH.

Isolated PTC were mixed 1:1 with lysis buffer (1.0% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 80 mM Tris; pH 7.5) containing enzyme inhibitors (Complete Mini; 1 tablet/3 mL; Roche Diagnostics, Mannheim, Germany). Total homogenates from rat brain were used as positive controls. Samples were run on 7.5% Tris-HCl gels with Tris/glycine/SDS buffer. Proteins were detected, after transfer to nitrocellulose membranes, using rabbit anti-nNOS (2 μg/mL; Zymed Laboratories, Invitrogen, Carlsbad, CA, USA), rabbit anti-eNOS (1:2000; Sigma Aldrich), and HRP-conjugated secondary goat anti-rabbit antibody (1:5000; Sigma-Aldrich) by an ECL-camera (Kodak image station 2000; New Haven, CT, USA).