Rabbit anti calcineurin

Four groups of rat hippocampus tissues were used to extract the total protein, and NFAT3/c4 protein was extracted using a Nuclear and Cytoplasmic Extraction Kit (Cwbio, China). All the experiments were conducted on ice. Protein concentration measurements were collected using the BCA (Biomiga, China) method. The samples were incubated at 99°C for 10 min. Then, 30 μg of total protein were was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in TBST for 2 h at room temperature and were incubated overnight at 4°C with the proper primary antibodies. The antibodies were purchased and diluted as follows: rabbit anti-calcineurin (1:10 000, Abcam), rabbit anti-NMDAR (1:1000, Abcam), rabbit anti-NFAT3/c4 (1:500, Sigma), rabbit anti-GSK-3β (1:1000, Cell Signal), rabbit anti-Bax (1:1000, Cell Signal), rabbit anti- Bcl-2 (1:1000, Cell Signal), rabbit anti-GAPDH (1:1000, Goodhere), rabbit anti-Histon3 (1:500, Sigma). The membranes were then incubated with an HRP-conjugated secondary antibody (Beyotime, Nantong, China) for 1.5 h at room temperature. The membranes were immunolabeled. After washing the samples with TBST buffer, the membranes were subsequently assayed using the Thermo Scientific Pierce Femto ECL (Rockford, IL, USA).