Bax rabbit mab

Cells were lysed in RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% NP-40) supplemented with protease inhibitor cocktail. Cell lysates were centrifuged at 12,000 rpm for 30 min at 4 °C, supernatants were saved, and protein concentrations were determined by BCA protein assay (Thermo Scientific, Rockford, Illinois, USA). Equal amounts of total cell lysates or CM of cell culture were subjected to western blot assays as we described previously [41 (link), 42 (link)] to measure protein expression. Antibodies for western blot analyses were from the following sources: NNMT rabbit mAb (E6N2Z, #33361), SIRT1 mouse mAb (1F3, #8469), Hexokinase II rabbit mAb (C64G5, #2867), LDHA rabbit mAb (C4B5, #3582), c-myc rabbit mAb (E5Q6W, #18583), BAX Rabbit mAb (D2E11, #5023), Bim Rabbit mAb (C34C5, #2933), Bcl2 Rabbit mAb (D55G8 #4223), FOXO3a rabbit mAb (D19A7, #12829), p53 Mouse mAb (1C12, #2524), Acetylated-Lysine Antibody (#9441), and β-actin mouse mAb (8H10D10, #3700) (Cell Signaling Technology, Beverly, MA, USA).

For Western blots, 40 μg of protein extracted from the mouse hippocampal tissue was mixed with 5× SDS-PAGE loading dye and boiled at 95°C for 5 min in a shaking incubator (KTM-100, KBT, Seongnam, Korea). Proteins were then run at 80 V for 20 min across a 14% polyacrylamide gel, followed by 120 V for 1 hr at RT. Protein in the gel was transferred to a methanol-activated PVDF membrane at 400 mA for 40 min on ice. The membrane was blocked in 5% bovine serum albumin (BSA) (160069, MP Biomedicals, Santa Ana, CA) diluted in 1x PBS for 1 hr at RT, and then incubated with primary antibodies (Bax rabbit mAb, 14796, Cell Signaling Technology, Danvers, MA; Recombinant TNF alpha antibody, ab183218, Abcam, Cambridge, UK; β-tubulin mouse mAb, 86298, Cell Signaling Technology) diluted in 2.5% BSA solution with 0.2% Tween-20 at 4°C overnight. The next day, the membrane was incubated with secondary antibodies (goat anti-rabbit HRP conjugate, 170-6515, Bio-Rad, Hercules, CA; goat anti-mouse HRP conjugate, STAR207P, Bio-Rad). The conjugates were activated with enhanced chemiluminescence substrates (#1705061, Bio-Rad) and visualized under a LuminoGraph chemiluminescent imaging system (WSE-6200H, ATTO, Daejeon, Korea).

Anhydrous cobalt chloride (CoCl₂) was obtained from Aladdin (Shanghai, China). Acriflavine (ACF) was purchased from MCE (Monmouth Junction, NJ, USA). Polyethylene glycol diacetate (COOH-PEG-COOH) was sourced from Melo PEG (Shanghai, China), and the CCK8 kit was acquired from ZOMANBIO (China, Beijing). PBS and Dulbecco’s modified Eagle’s medium (DMEM)were obtained from Gibco (Grand Island, NY, USA). Coomassie Blue Fast Staining Solution, PH fluorescent probe (BCECF AM), cell activity and cytotoxicity detection kit (CalceinAM/PI), reactive oxidative species (ROS) detection kit, and SDS-PAGE protein sample buffer (5×) were purchased from Beyotime (Shanghai, China). HIF-1α Rabbit mAb (RRID: AB_2799095), MMP-2 Rabbit mAb (RRID: AB_2799191), Bax Rabbit mAb (RRID: AB_10557411), and Caspase-3 Antibody (RRID: AB_331439) were obtained from Cell Signaling Technology (Danvers, MA, USA). N-Hydroxy succinimide (NHS) was sourced from ThermoFisher Scientific (Waltham, MA, USA). Tris-Glycine Running Buffer, Western Transfer Buffer, and 4% paraformaldehyde fixative solution were purchased from biosharp (Hefei, China). Lastly, the Cell-Light EdU Apollo567 In Vitro Kit was acquired from RIBOBIO (Guangzhou, China).