Ba3257

Total protein was extracted from cells or tissues using RIPA buffer (Beyotime) and quantified with an Easy II Protein Quantitative Kit (TransGen Biotech). The protein samples were subjected to SDS‐PAGE and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk for 1 hour and incubated with primary antibodies against PARP1 (1:1000, PB9309, Boster Biological Technology), HMGB1 (1:1000, A00066‐1, Boster Biological Technology), Bcl‐2 (1:1000, A00040‐2, Boster Biological Technology), Bax (1:500, bs‐0127R, Bioss, Beijing, China), cleaved caspase‐3 (c‐Caspase3; 1:500, BA3257, Boster Biological Technology), acetylated lysine (1:500, MA1‐2021, Thermo Fisher), cytochrome C (1:500, bs‐0013R, Bioss), COX IV (1:2000, bsm‐52750R, Bioss) and GAPDH (1:1000, BA2913, Boster Biological Technology) overnight at 4°C. Then, HRP‐conjugated AffiniPure Goat Anti‐rabbit/mouse IgG secondary antibody (1:5000, Boster Biological Technology) was incubated for 1 hour. The EasySee^® Western Blot Kit (TransGen Biotech) was used to detect the protein bands.

Expression levels of IL-6, caspase-3, and cleaved-caspase-3 proteins, extracted from the left hemisphere of the cerebral cortex of rats in the five groups, were assessed using western blotting. Equal quantities (10 μg) of the desaturated sample mixture from the five groups were added into different wells on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Subsequently, the total protein was fully transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, Massachusetts, USA). The membrane with total protein was blocked using bovine serum albumin (5%) for 2 h at 18–22°C and then incubated overnight with antibodies against NeuN (1:2000, ab177487, Abcam, Cambridge, MA, USA), beta-actin (1:2000, ab197277, Abcam), IL-6 (1:500, BA4339, Boster, Wuhan, China), or caspase-3 (1:500, BA3257, Boster, Wuhan, China). The blots were washed with TBST (1% Tween in Tris-buffered saline) on a shaker. Finally, the polyvinylidene fluoride membrane was incubated with HRP-conjugated goat anti-rabbit (H+L) cross-absorbed secondary antibody (1:10000, SA00001-2, Thermo Fisher Scientific, Waltham, MA USA) for 1 h, washed with TBST, and visualized with enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA USA).