Polyclonal anti caspase 3 antibody

Cells were lysed for 30 min on ice in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 0.1% sodium dodecyl sulfate (SDS) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Lysates were mixed with SDS-PAGE sample buffer and boiled for 5 min. Protein concentrations were determined with a BCA protein assay kit (Pierce, Rockford, IL) using bovine serum albumin as a standard. Equal amounts of total protein were examined by Western blot. The following antibodies were used: anti-Flag MAb (M2) and anti-βnactin MAb (Sigma-Aldrich), anti-cleaved caspase-3 (Asp175) polyclonal antibody, anti-caspase-3 polyclonal antibody (Cell Signaling Technology, Beverly, MA), anti-GFP MAb (MBL, Nagoya, Japan), horseradish-peroxidase (HRP)-conjugated goat anti-mouse IgG (Amersham Bioscience, Uppsala, Sweden), and HRP-conjugated goat anti-rabbit IgG (Amersham Bioscience). Signals were visualized after the treatment of the membrane with SuperSignal West Pico chemiluminescent substrate (Pierce).

Western blot analysis of total protein isolated from NRCMs was performed as previously described 13. Briefly, protein samples (30 or 50 μg) were separated in 10% and 15% SDS‐PAGE and blotted to PVDF membrane. The membranes were incubated with the primary antibodies including rabbit anti‐SCAD monoclonal antibody (diluted 1:1000; Abcam, Cambridge, UK), anti‐Caspase‐3 polyclonal antibody (diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), Bcl‐2 polyclonal antibody (diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), Bax polyclonal antibody (diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), AMPK monoclonal antibody (diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), p‐AMPK monoclonal antibody (diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA) and mouse anti‐PPARα monoclonal antibody (diluted 1:500; Sigma‐Aldrich, St.Louis, MO, USA), anti‐α‐tubulin monoclonal antibody (diluted 1:5000; Sigma‐Aldrich, St.Louis, MO, USA), and then the HRP conjugated goat‐antimouse or rabbit IgG1 secondary antibody (1:10,000; Sigma‐Aldrich, St.Louis, MO, USA) were used. Finally, the protein expression levels were detected with chemiluminescent substrate (Thermo, Waltham, MA, USA). Results were analysed with the Image J system.