Bicinchoninic acid (bca)

Cells or ground tissue were lysed using RIPA lysis buffer (Cat: P0013C, Beyotime, China) supplemented with proteasome inhibitors (Cat: ST507, Beyotime, China) for 30 min, during which time they were sonicated three times 10 s each. bicinchoninic acid (Cat: P0010S, Beyotime, Cina) was added to the lysate for 30 min at 37 °C. Then, the protein concentration of the lysate was measured and mixed with 5X loading buffer (Cat: BL502B, biosharp, China). After boiling at 100 °C for 10 min, the mixture was used for electrophoresis for 70 min, followed by transfer onto 0.45um PVDF membrane (Cat: IPVH00010, Merck, German). After that, 5% skimmed milk (Cat: GC310001, Servicebio, China) was soaked close to the PVDF membrane for 90 min. Next, the PVDF membrane was incubated with primary and secondary antibodies. After being washed with TBST (Cat: G0001, Servicebio, China) three times, the PVDF membrane was subjected to Electrochemiluminescence (ECL). The detailed information on primary and secondary antibodies employed in this study is presented in Additional file 1: Table S1.

Brain tissues were harvested and homogenized on ice before centrifugation at 13,000×g at 4 °C for 5 min. The supernatants were collected and saved at −20 °C. Total protein concentration was measured with the bicinchoninic acid method (Beyotime Biotechnology, Co., Ltd., Jiangsu, China). The samples were separated using 12% gel electrophoresis (90 V for 25 min, then 120 V for 1 h) and then transferred to a nitrocellulose membrane at 200 mA for 1 h. The blots were blocked in 5% milk Tris-buffered saline and Tween-20 (TBST) buffer for 1.5 h at 25 °C, and then incubated with a monoclonal antibody against caspase-3 (1:1000; Cell Signaling Technology, Inc. #9662, Danvers, MA, USA) or β-actin (1:1000; Cell Signaling Technology) in 5% milk TBST buffer at 4 °C overnight. After washing in TBST, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:6000 dilution for β-actin and caspase-3, Wanleibio Co., Ltd., Shenyang, China) for 2 h at RT. Following the ECL reaction, the membranes were subjected to autoradiography and films were scanned using Tanon Imager 5200 software v2.03 (Tanon Co., Ltd., Shanghai, China). Western blot band intensity was quantified by the mean pixel intensity using Gel-Pro Analyzer 4 software (Media Cybernetics Inc., Bethesda, MD, USA).

The right cortex and heart tissue proteins were extracted as the method described (Mlyniec et al. 2014a , b (link)). Eight-month-old mice were sacrificed and the proteins from fresh cortex and heart tissue were prepared. The tissues were homogenized using RIPA buffer (20 mg of tissue with 200 μl of RIPA), and the tissue lysates were centrifuged at 12,000 rpm for 5 min and the supernatants were collected to determine the protein concentrations using a bicinchoninic acid (Beyotime Institute of Biotechnology, Shanghai, China).

Cells and tissues were treated with radioimmunoprecipitation assay lysis buffer (Abcam) to extract total protein and treated with bicinchoninic acid (Beyotime) to quantify protein concentration. After 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis-dependent separation, protein samples were transferred to polyvinylidene fluoride membranes (Bio-Rad, China) and blocked with 5% bovine serum albumin. After these steps, the membranes were treated with primary antibodies diluted with blocking buffer in a shaker at 4 ^∘C overnight. The next day, the primary antibodies were washed away with Tris-Buffered Saline Tween (TBST), followed by incubation of the membranes with specific secondary antibodies in a shaker for 2 h. After another wash with TBST, signaling was detected with the enhanced chemiluminescence kit, using NIH Image J software (version 1.52a; National Institutes of Health, Bethesda, MD, USA) to analyze gray-scale values. The antibodies used in this assay were as follows: HDAC9 (1:10000, ab109446, Abcam), VEGFA (1:450, ab183100, Abcam), GAPDH (1:2500, ab9485, Abcam), and secondary antibody (1:2000, ab205718, Abcam).