Phosphatase inhibitor cocktail 100

Lumbar 4–5 spinal cord segments were collected as mentioned above. Tissue samples were homogenized with radioimmunoprecipitation (RIPA) buffer (Thermo Fisher Scientific, MA, USA) plus phosphatase inhibitor cocktail (100×, Thermo Fisher Scientific, MA, USA). After homogenization, tissues were incubated for 15 min on ice and centrifuged at 13,000× g at 4 °C for 20 min. The supernatant was assayed using the Bradford protein assay (BIO-RAD, CA, USA). Thirty micrograms of protein samples were loaded and run on a 8% Tris-glycine sodium dodecyl sulfate-polyacrylamide gel followed by electrophoresis, then were transferred to a nitrocellulose membrane (BIO-RAD, CA, USA). The membranes were blocked with 5% skim milk in 0.05% Tris Buffered Saline with Tween 20 (TBS-T). After blocking, incubated with primary antibody for overnight at 4 °C with rabbit anti-GAD65 (Cell Signaling Technology, MA, USA, 1:1000) in 5% skim milk. After rinsing the membrane with TBS-T, it was incubated with secondary antibody for 2 h at room temperature with Goat anti-rabbit Ig G/ HRP antibody (Solarbio, Beijing, China, 1:2000) in 5% skim milk. Bands were detected using enhanced chemiluminescence (ECL) solution (Donginbio, Seoul, Korea, A:B = 1:1) and imaged with davinch. Density image was quantified by using image J. GAD65 bands were normalized using the amount of β-actin.