Rabbit anti caspase 3

Neuronal proteins were isolated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 10% acrylamide gels (Solarbio, Beijing China). The proteins were transferred to polyvinylidene di uoride (PVDF) membranes (Millipore, Burlington, Massachusetts, USA) using electroblotting. Then the membranes with the transferred proteins were blocked in phosphate-buffered saline with 0.1% Tween (PBST) containing 10% bovine serum albumin (BSA) at room temperature for 2h. The membranes were incubated for 2h in Tris-buffered saline with 0.1% Tween (TBST) with primary antibodies, including rabbit anti-BDNF (1:800), rabbit anti-RhoA (1:1000), rabbit anti-Caspase-3 (1:1000), and rabbit anti-GAPDH (1:1000) (Boster, Wuhan, China ). After three washes for 10 min each in TBST, the membranes were incubated for 1h at 37℃ with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG and diluted at 1:800 (Beyotime, Beijing, China). Then the membranes were washed three times in TBST and processed using the enhanced chemiluminescence (ECL) detection system. An antibody to GAPDH was used as the loading control. All experiments were repeated three times. The expression level of proteins was calculated as an average of the densities per band area from each group.