Ab124773

For Western blot, hippocampus tissues were flash frozen and then stored at –80°C until homogenization. Tissues were homogenized for 30 min in ice-cold RIPA lysing buffer [25 mM Tris–HCl (pH 7.6), 150 mM NaCl; 1% NP40; 1% sodium deoxycholate; 0.1% sodium deoxycholate, 0.1% SDS] for a radioimmunoprecipitation assay (Redwood City, California, USA). Protein levels were tested using the bicinchoninic acid method. We prepared a 10% separation gel and a 5% stacking gel. Proteins were separated by SDS-PAGE (140 V), then transferred to a polyvinylidene fluoride membrane (150 mA, constant current, 2 h) and incubated for 1 h at room temperature with 5% skim milk in tris-buffered saline/t solution and then with primary antibodies overnight at 4°C. The membranes were washed with a tris-buffered saline/t buffer three times (5 min/time) and then incubated for 1 h with secondary antibodies. Proteins were exposed by electrochemiluminescence; membranes were washed three times at 5 min intervals and analyzed using a PharosFX Plus Molecular Imager (Bio-Rad). The antibodies were anti-DRP1 (8570, 1 : 1000; Cell Signaling Technology, Danvers, Massachusetts, USA), anti-FIS1 (10956-1-Ap, 1 : 500; Proteintech, Wuhan, P,R,C), anti-MFN2 (ab124773, 1:1000; Abcam, Cambridge, Massachusetts, USA), anti-OPA1 (ab157457, 1 : 1000; Abcam), and anti-β-actin (20536-1-Ap, 1 : 2000; Proteintech).