Signalfire enhanced chemiluminescence reagent

MSCs APOPTIN or control cells were washed with cold PBS and immediately lysed in Laemmli buffer (Nanjing Keygen Biotech. Co. Ltd., Nanjing, China). Cell lysates were denatured at 100°C for 5 min and centrifuged at 10,000 × g for 5 min at 4°C. Supernatants were recovered, separated on 12% SDS-PAGE and transferred onto a 0.45-μm polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Following blocking the membrane with Tris-buffered saline-Tween-20 containing 5% non-fat milk (Mengniu Dairy, Inner Mongolia, China) for 1 h at room temperature, the membrane was incubated with the appropriate primary antibodies: Anti-apoptin polyclonal antiserum prepared in the present study (1:500) and monoclonal anti-GAPDH antibody (cat. no. ab8245; 1:10,000; Abcam, Cambridge, MA, USA), overnight at 4°C. The membrane was incubated with horseradish peroxidase-conjugated secondary antibody (cat. no. sc-2005; 1:10,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature and the bands were detected using the SignalFire™ Enhanced Chemiluminescence reagent (Cell Signaling Technologies, Beverly, MA, USA) in a dark room.