P0033

Cells were cultured in 6-well plates and washed with PBS after absorbing the medium. The cells were treated with a cellular digest containing EDTA but without trypsin so that they did not stick to the wall and then dispersed the medium by pipetting over the cell layer surface several times. Cells were collected by centrifugation, supernatant was removed, and cell precipitation was left for later use. Steps followed manufacturer's instructions (Beyotime, P0033).

The hippocampus and prefrontal cotex were weighed and homogenized in the lysis buffer (P0033, Shanghai Beyotime Biotechnology Co., Ltd, China) at a concentration of 160 μl/mg. The homogenate was placed on ice for 40 min and then centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was transferred into a new centrifuge tube for later use and the excess samples were stored at −80°C.

Membrane proteins, mitochondrial proteins, nuclear proteins, and cytoplasmic proteins were isolated using subcellular fractionation protein extraction kits (P0033, C3601, P0028, Beyotime, Shanghai, China) according to the manufacturer's instructions. Proteins were analyzed by SDS‒PAGE and immunoblotting.

The macrophage membrane was isolated from the murine macrophages RAW 264.7 cell line. The cells were washed three times with sterile PBS, and then lysed in buffer A (P0033, Beyotime, China) supplemented with phenylmethanesulfonyl fluoride (PMSF, ST506, Beyotime, China) in an ice bath for 10 min. The cell suspension was homogenized in a glass homogenizer for 25 times to disrupt the cell membrane. After centrifuging at 700g, 4 °C (Centrifuge 5418 R, Eppendorf, Germany) for 10 min, the supernatants were collected and further centrifuged at 14,000g for 30 min at 4 ℃. Macrophage membrane was obtained by collecting the bottom sediment.

Total protein was extracted from the myocardial tissue in the infarct area and the corresponding surviving myocardial tissue or HCMECs by using RIPA lysis buffer. Membrane and cytosol proteins were extracted according to the manufacturer’s instructions (P0033, Beyotime). The bicinchoninic acid (P0011, Beyotime) kit was used to determine the protein concentration. Then, 10% SDS-PAGE was used to separate the cell or tissue lysate. Once the protein transfer step was completed, the PVDF membrane (ISEQ00010, Millipore) was sealed with 5% skimmed milk and incubated with anti-rabbit VE-cadherin (ab205336, Abcam), anti-rabbit PI3K (4257, Cell Signaling Technology), anti-rabbit Phospho-PI3K (17366, Cell Signaling Technology), anti-rabbit AKT (4691, Cell Signaling Technology), anti-rabbit Phospho-AKT (4060, Cell Signaling Technology), and anti-mouse β-tubulin (66240-1-Ig, Proteintech) antibodies overnight at 4 °C. After the samples were washed three times in tributyltin compound buffer, they were incubated at room temperature for 1 h. Finally, the proteins were visualized using the ECL solution (KGP112, Jiangsu Kechuang Biotechnology Co., LTD.). Blot analysis was performed using the Tanon image system (TANON-1600, Tanon).