Rabbit anti bax n20

Total lysates were extracted using 1% NP40 (150 mM NaCl, 50 mM Tris HCl pH 7.4, 10 mM EDTA) supplemented by a cocktail of antiproteases and antiphosphatases. A total of 20 μg of proteins from each point were resolved by SDS-NuPAGE (4–12% Bis-Tris gels, Novex, Les Ulis, France) and transferred to nitrocellulose membranes (Amersham Biosciences, Les Ulis, France). Nonspecific sites were blocked by incubation with 5% milk for 1 h at room temperature, and the membranes were then incubated with mouse monoclonal anti-OPA1 (BD Biosciences Pharmingen, Le Pont de Claix, France; clone 18), anti-Parkin (Millipore, Le Pont de Claix, France), and rabbit anti-Bax (N-20, from Santa-Cruz, Le Pont de Claix, France). Antibodies specific for IKKα, IKKβ, and NEMO were purchased from Cell Signaling (St Quentin Fallavier, France). Equal protein loading was assessed by probing the membranes with anti-actin (Millipore) and anti-HSP60 monoclonal antibodies (Stressgen, St Quentin Fallavier, France). Membranes were treated with horseradish peroxidase-linked goat anti-mouse or anti-rabbit secondary antibodies (Amersham Biosciences). Immunoreactive proteins were detected and quantified by enhanced chemiluminescence (Amersham Biosciences) using a CCD camera (GBOX, SYNGENE, Ozyme, France).