Jnk1 2

The hippocampus was homogenized in a lysis buffer containing a protease inhibitor cocktail (10 μL/ml, GE Healthcare Biosciences, PA, United States). The complex was centrifuged at 12,000 rpm/min at 4°C, and supernatants were removed to determine protein concentrations, calculated according to the standard curve. Proteins were separated on 10 or 12% SDS-polyacrylamide gels at 80 V for 2 h. After separation at 300 mA for 1.5 h, the protein bands were transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk for 1.5 h. After washing three times with Tris-buffered saline with 0.05% Tween 20, the membranes were incubated with the primary antibodies overnight: JNK1/2, p-JNK1/2, GAPDH, superoxide dismutase 2 (SOD2), IL-6 (Proteintech, Chicago, IL, United States), Bax, Bcl-2, C-caspase3, postsynaptic dense protein 95 (PSD95), Nrf2, p-CREB133, BDNF, IL-1β, synapsin-1 synaptophysin, GRP78, ATF, protein kinase R-like endoplasmic reticulum kinase (PERK), p-PERK, p-IRE1α, CREB, p-CREB129, PKA, and IL-10 (Abcam, Cambridge, United Kingdom). The following day, membranes were incubated in corresponding secondary antibodies for 1 h at room temperature. Signals were visualized using the ChemiDocXRS + Imaging System (Bio-Rad). Blot bands were quantified using ImageJ software densitometry and expressed as relative density to GAPDH.