Anti mef2c

Whole tissue proteins from the hippocampus were homogenized using the pre-cold radioimmunoprecipitation (1 ×) assay buffer (Boster, China) mixed with inhibitors of protease and phosphatase (Boster, China). And lysates were further depolymerized with an ultrasonic cell disrupter (Scientz, Ningbo, China). The homogenized lysates were centrifuged (12,000 g, 15 min, 4 ℃) and the resulting supernatant was kept. Concentrations of total protein were detected according to the instructions of the bicinchoninic acid protein assay kit (Boster, China). Next, Supernatants were mixed with 5 × protein loading buffer from Boster (China) and boiled for 5 min.
Following this, hippocampal protein lysates of protein (30 μg) were loaded onto 10% or 12% sodium dodecyl sulfate–polyacrylamide gels for electrophoresis. The separated proteins were then transferred to 0.45 μm polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked using a solution of 5% bovine serum albumin (Biofroxx, German) dissolved in Tris-buffered saline (Servicebio, China) with 0.1% Tween 20 (TBST). The blocking step was carried out at room temperature for 1 h. Then membranes were incubated with corresponding primary antibodies including anti-HDAC4 (1:1000; Affinity, China), anti-MEF2C (1:1000; Proteintech, China), anti-β Actin (1:3500; ABclonal, China), anti-Bcl2 (1:1000; Proteintech, China), anti-Bax (1:1000; Cell Signaling Tech, USA), anti-cleaved caspase 3 (1:1000; Cell Signaling Tech, USA) and anti-AKT3(1:1000; ABclonal, China) overnight at 4 ℃. The next day, stripes were washed with TBST (3 times, 10 min each time) and incubated with goat anti-rabbit or goat anti-mouse IgG horseradish peroxidase antibody (1:5000, proteintech, China) at room temperature (1 h). Subsequently, stripes were also washed three times for 10 min within TBST. Finally, protein bands were exposed with enhanced chemiluminescence (Abbkine Scientific, China), and photographed using the ChemiDoc XRS chemiluminescence imaging system (Bio-Rad, Hercules, USA).

The total protein of cells was lysed in a RIPA buffer containing 1% protease inhibitors (PMSF) and collected. After SDS-PAGE, the proteins were transferred onto polyvinylidene fluoride membranes. The primary antibodies were anti-PKNOX2 (Proteintech, Wuhan, China), anti-MyoG (Abcam, Cambridge, UK), anti-MEF2C (Proteintech, Wuhan, China), anti-MyHC (Millipore, Billerica, MA, USA), and anti-β-tubulin (Servicebio, Wuhan, China). The HRP-conjugated secondary antibodies HRP-labeled goat anti-mouse IgG (Servicebio, Wuhan, China) and HRP-labeled goat anti-rabbit IgG (Servicebio, Wuhan, China) were used.