Cleaved caspase 3

Western blotting was performed using a gel documentation system (iBrightCL1000, Invitrogen and Image Lab Software version 3.0), and a standard protocol as previously described (Zhang et al., 2019 (link)). The primary antibodies were anti-cyclin dependent kinase inhibitor 2A (P16) (80772, Cell Signaling Technology, United States), anti-cyclin dependent kinase inhibitor 1A (P21) (2947, Cell Signaling Technology), P53 (21083, Signalway Antibody, United States), PTEN (ab31392, abcam, United Kingdom), anti-phosphorylated-AKT (p-AKT) (4060, Cell Signaling Technology) and anti-AKT (4691, Cell Signaling Technology); Cleaved caspase-3 (29034, Signalway Antibody), Bcl-2 (ab196495, abcam), vascular endothelial growth factor (VEGF, ab52917, abcam), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174, Cell Signaling Technology); TSG101 (14497, Proteintech, United States), CD63 (25682, Proteintech), CD81 (66866, Proteintech) and horseradish peroxidase-conjugated were secondary antibodies (Biosharp, China).

Protein extraction and western blot (WB) analysis were performed as previously described [14 (link)]. Briefly, cells were washed with PBS and lysed with lysis buffer on ice for 20 min. The total cell protein concentration was detected using the BCA Protein Assay Kit. The total protein (20 μg) was separated using SDS-PAGE (Invitrogen) and transferred to a PVDF membrane (Roche). The membrane was blocked with 5% bovine serum albumin (0.1%) in TBS-Tween and incubated against the required antibody.
The primary antibodies Bax (5023, Cell Signaling Technology, USA), Bcl2 (ab196495, Abcam, USA), cleaved caspase-3 (29034, Signalway Antibody, USA), BTG2 (A9848, ABclonal), GAPDH (5174, Cell Signaling Technology), TSG101 (14497, Proteintech, United States), CD63 (25682, Proteintech), CD81 (66866, Proteintech), and horseradish peroxidase-conjugated secondary antibody (Santa Cruz) were used. Bands were visualized using enhanced chemiluminescence reagents and analyzed using a gel documentation system (Bio-Rad Gel Doc1000 and Multi-Analyst version 1.1).

Western blotting was performed as described in a previous study with some modifications [20 (link)]. Protein extracts from cells or hippocampi were prepared in a modified RIPA buffer supplemented with protease inhibitors (200612, Signalway Antibody, Greenbelt, MD, USA). The BCA method was used to determine the concentration of protein. The protein extracts were boiled after being diluted in SDS-PAGE protein loading buffer (5×) (Beyotime, Shanghai, China) at a ratio of 4:1. Following separation, the proteins were transferred to polyvinylidene difluoride membranes and blocked in TBST buffer (20 mM Tris–HCl, pH 7.4, 137 mM NaCl, and 0.1% Tween-20) with 5% non-fat milk at 37 °C for 1 h, and incubated at 4 °C with primary rabbit polyclonal antibodies (cleaved caspase-3, 49500, 1:500, Signalway Antibody; BCL-W, 40641, 1:1000, Signalway Antibody; SAPK/JNK (pThr183), 11249, 1:500, Signalway Antibody; SMAC/DIABLO, 39330, 1:500, Signalway Antibody; GAPDH, 21612, 1:3000, Signalway Antibody) overnight. After extensive rinsing, the membranes were incubated with the appropriate HRP-conjugated secondary antibody, then visualized using Super ECL Plus reagents. The gray values of the protein bands were quantified by the optical density using ImageJ software (1.41v, US National Institutes of Health, Bethesda, MD, USA).