Apoptosis inducing factor

Briefly, heart tissues were homogenized with RIPA lysis buffer (Santa Cruz Biotechnology, CA). Total proteins were extracted and separated on SDS-PAGE gels, and then proteins were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% nonfat dried milk for 1 h and then incubated overnight at 4°C with the following primary antibodies: intercellular adhesion molecule 1 (ICAM-1, Santa Cruz Biotechnology, CA), tumor necrosis factor alpha (TNF-α, Abcam, Cambridge, MA), plasminogen activator inhibitor-1 (PAI-1, BD Bioscience, Sparks, MD), 3-nitrotyrosine (3-NT, Millipore, Billerica, MA), 4-hydroxy-2-nonenal (4-HNE, Alpha Diagnostic International, San Antonio, TX), B-cell lymphoma 2 (BCL-2) and BCL2-associated X protein (BAX) (Cell Signaling, MA), cleaved-caspase-8 (Cell Signaling, MA), apoptosis inducing factor (AIF) (Cell Signaling, MA), caspase-12 (Exalpha Biologicals, MA), C/EBP homologous protein (CHOP; Santa Cruz Biotechnology, CA) GAPDH (Santa Cruz Biotechnology, CA), and cleaved-caspase-3 (Cell Signaling, MA). After three washes with Tris-buffered saline (pH 7.2) containing 0.1% Tween 20 (TBST), membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. Antigen-antibody complexes were then visualized using an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL) [22 (link)].