Bca assay kit

The protein in cells or ectosomes was extracted by RIPA lysate (Fdbio Science, Guangzhou, China) and ultrasonic lysis of cells and ectosomes, and protein degradation was inhibited by protein phosphatase inhibitor. The total protein levels in these samples were measured by BCA assay kit (Fdbio Science). After separation of samples by SDS-PAGE, they were transferred to a 0.22um PVDF membrane in the wet transfer method. At 4 °C on a shaker, blocking was performed on each membrane using 5% whole milk. After blocking, the membranes were further processed for immunoblotting using specific primary antibodies. Subsequently, the protein levels were measured by Fdbio-Dura ECL (Fdbio-Science) after incubation with specific secondary antibodies in the membrane for 1 h. The antibodies used in this study for western blot include: HSP70 (1:1000, Abcam, ab181606), CD81 (1:1000, Ptoteintech, 66866-1), Calnexin (1:500, Ptoteintech, 10427-2), COL2 (1:1000, Abclonal, A1560), Tsg101 (1:1000, Abcam, ab125011), MMP13 (1:1000, Abclonal, A11148), p16^INK4a (1:2000, Abcam, ab211542), NSD1 (1:500, Abclonal, A9981), Mono Methyl-Histone H3-K36 (1:2000, Abclonal, A22863), DiMethyl-Histone H3-K36 (1:10000, Abclonal, A22087), Histone H3 (1:2000, Abclonal, A17562), HRP-labeled goat IgG H&L (Jackson ImmunoResearch), GAPDH (1:8000, Proteintech, 60004-1-Ig), and P21 (1:1000, Abcam, ab109199).

Mouse hippocampal tissue was lysed using a denaturing cell lysis buffer (Invent, SD-001) supplemented with a protease inhibitor (Fdbio Science, FD1001) and a phosphatase inhibitor (Fdbio Science, FD1002). The protein concentration was quantified using a bicinchoninic acid (BCA) assay kit (Fdbio Science, FD2001). Subsequently, fifty micrograms of protein per sample were resolved on an SDS–polyacrylamide gel for electrophoresis and then electroblotted onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% bovine serum albumin (BSA, ZOSEN, N0008-100) at room temperature for one hour, followed by incubation with the primary antibody at 4 °C overnight. This was succeeded by incubation with the appropriate secondary antibody. Detection of protein bands was accomplished using an Odyssey imaging system (LI-COR, USA), and the densities of the protein bands were quantitatively analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).