Anti ho 1 5853s

Total proteins, from 3 wells of a 6-well plate, were lysed in radioimmunoprecipitation assay buffer (Sigma) containing protease and phosphatase inhibitors cocktail (Roche, Meylan, France) and were sonicated for 20 seconds. Lysates were quantified for protein concentration using the BCA protein assay kit (Pierce, Waltham, MA). Twenty mg of proteins were separated by electrophoresis on a 4% to 12% BisTris NuPAGE gel (Thermofisher) or 7% Tris acetate NuPAGE gel (Thermofisher) and were transferred onto nitrocellulose membrane using the Trans-Blot Turbo Transfer System (Biorad). After blocking, the membranes were incubated with their respective primary antibodies (anti-caspase 3 [sc7148; Santa Cruz, Dallas, TX; 1/1,000], anti-cleaved caspase 3 [9661; Cell Signaling (Danvers, MA); 1/1,000], anti-Poly(ADP-Ribose) Polymerase 1 [PARP-1, 9542P; Cell Signaling; 1/1,000], anti-HO-1 [5853 S, Cell Signaling; 1/1,000], and anti-HSP60 [sc-1052; Santa Cruz; 1/4,000]) overnight at 4 C. Membranes were further incubated with horseradish peroxidase-conjugated secondary antibody (Sigma, Saint Louis, MO; 1/10,000) for 45 minutes at room temperature. Protein bands were visualized with the Amersham ECL Western blotting detection reagents (GE Healthcare, Chicago, IL) using a Fusion FX Spectra imager (Vilber Lourmat, Collégien, France). Image analysis was performed using ImageJ.