Nmnat2

For total protein extraction, the contusion-side cortex of animals was lysed completely in ice-cold radioimmunoprecipitation lysis buffer (Beyotime, Shanghai, China) that contained protease inhibitor with centrifugation at 12000 g at 4°C for 20 minutes. The supernatant was collected, and then protein concentration was estimated using a bicinchoninic acid assay kit (23227; Thermo Fisher Scientific). After denaturing at 95°C for 5 minutes in 5× loading sodium dodecyl sulfate (SDS) buffer (Invitrogen), a total of 30 µg protein of each sample was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). Blocking buffer containing 5% non-fat milk was used to block the membranes for 1 hour at room temperature. Then the membranes were incubated with primary antibodies against β-actin (1 : 10000), NMNAT2 (Abcam, Cambridge, UK; 1 : 1000), BCL-2-associated X protein (Bax; 1 : 500; Protein Tech, Rosemont, IL, USA) at 4°C overnight. The next day, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase. Finally, the immunoblots were detected by enhanced chemiluminescence reagents (Millipore) in an imaging system (BioRad, Hercules, CA, USA). The relative protein intensity was analyzed using Image-J software (National Institutes of Health, Bethesda, MD, USA).