Anti bcl 2 antibody

The protein was extracted from fresh-frozen hippocampal tissue using RIPA lysate. The BCA method was used to measure protein concentration. The protein solution and SDS-PAGE loading buffer (5x) were mixed at 1 : 4, then heated at 100°C for 10 min to denature the protein. After this production, equal amounts of proteins were separated using SDS-PAGE gel and transferred onto the PVDF membrane. Then, PVDF membranes were blocked with 10% skim milk and incubated with anti-GRP78 antibody (1 : 5000, Proteintech, China), anti-ATF4 antibody (1 : 2000, Proteintech, China), anti-Bax antibody (1: 5000, Proteintech, China), anti-Bcl2 antibody (1 : 2000, Immunoway, China), anti-CHOP antibody (1 : 1000, Proteintech, China), caspase9 (1 : 1000, CST, USA), TNF-α (1 : 1000, Proteintech, China), IL-6 (1 : 1000, Proteintech, China), IL-10 (1 : 1000, Proteintech, China), and anti-β-actin antibody (1 : 10000, Santa Cruz, USA) overnight at 4°C. Afterwards, the bolts were incubated for 1 hour at 37°C using the corresponding anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (1 : 10000, Proteintech, China). Finally, the protein bands were identified using the ECL Chemiluminescence Kits (Millipore, USA). Protein levels were normalized to β-actin as reference. The relative density of the protein level was quantitated by the ImageJ software.