Ecl chemiluminescence reagent

The Western blot assay was processed as described previously (Wang et al., 2018). Briefly, the cells were broken down using a lysis buffer supplemented with 1 mM PMSF and then homogenized for 10 min (11,000× g, 4 °C). The supernatants were collected and then denatured with 4× loading buffer at 95 °C for 10 min. The BCA protein assay kit was used to determine the protein concentration. Subsequently, the proteins were separated by 10–15% SDS-PAGE, and then transferred onto a PVDF membrane, which was blocked with 5% nonfat milk dissolved in TBST at room temperature for 2 h. Next, they were incubated with primary antibodies (1:1000), including anti-NDUFA4, anti-SDHA, anti-UQCRQ, anti-COX17, anti-ATP6V1B2, anti-ATP8B2, and anti-β-actin. After incubation overnight at 4 ℃, the blots were washed three times for 20 min before incubating with the secondary antibody at room temperature for 2 h. The blots were detected using a hypersensitive ECL chemiluminescence reagent (Beyotime) and exposed to a Chemiluminescent imaging system (Bio-Rad). Image J software was used to quantitate the protein intensity.

The Hip and mPFC tissues were dissociated by radio-immunoprecipitation assay solution containing protease inhibitors and phosphatase inhibitors. The proteins in the samples were run on 10% SDS polyacrylamide gel and then transferred to polyvinylidene fluoride membrane (Millipore, USA). The proteins were incubated with Rabbit anti-Sirt1 (1:1000, Abcam, ab189494), Rabbit anti-Nrf2 (1:1000, Proteintech, 16396-1-AP), Rabbit anti-HO-1 (1:1000, Proteintech, 10701-1-AP), Rabbit anti-Gpx4 (1:3000, Abcam, ab125066), Rabbit anti-TLR4 (1:1000, Abcam, ab217274), Rabbit anti-NF-κB p65 (phospho S536) (1:1000, Abcam, ab76302), Rabbit anti-NF-κB p65 (1:1000, Cell Signaling, 8242) and Mouse anti-GAPDH (1:10,000, Abcam, ab8245) overnight at 4 °C, and secondary anti-mouse HRP-conjugated (1:10,000, Bio-rad, 170-6516) or secondary anti-rabbit HRP-conjugated antibodies (1:10,000, Bio-rad, 170-6515) were incubated for 2 h at room temperature. The signals were visualized using ECL chemiluminescence reagent (Beyotime; US Everbright Inc.).

The protein lysate was analyzed by SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% fat-free milk in PBST for 30 min, followed by incubation overnight at 4 °C with final dilution of primary antibodies against CXCL12 (Cat No. #3530), IL8 (Cat No. #94407), TGF-β (Cat No. #3711), HGF (Cat No. #52445), mTOR (Cat No. #2983), AKT (Cat No. #4685), AMPK (Cat No. #5831), LC3II/I (Cat No. #4108), ATG5 (Cat No. #12994), p-mTOR (Cat No. #5536), p-AMPK (Cat No. #50081), p-AKT (Cat No. #4060) or GAPDH (Cat No. #5174) all from CST (Beverly, MA, USA). Antibodies on membranes could be stripped using stripping buffer (Cat No. ab270550, Abcam, Cambridge, MA, USA) with gently shaking at 52 °C for 30 min for other blotting examination. Protein bands hybridized with primary and secondary antibodies on membranes were detected using ultrasensitive ECL chemiluminescence reagent (Beyotime Biotechnology, Shanghai, China) and exposed to film. Band intensity was quantified as the mean ± SD of three independent experiments.

The collected platelet or cell sample was added with strong RIPA lysate (Wuhan Boster Biological Technology Co., Ltd., China) protein (including the phosphoric acid protease inhibitor and PMSF protease inhibitor); then, it was oscillated intensely for 5 min, and it had lysis on ice for 1 h. Next, it was centrifuged for 5 min under 12000 r/min, and the supernatant was collected, which was the total protein. The BCA Protein Quantitation Kit (Shanghai Beyotime Biotechnology Company, China) was used to measure the protein concentration, and after dilution, the 4x SDS loading buffer was used to boil and process the sample. The SDS-PAGE protein electrophoresis was conducted, and then, the protein was transferred to the PVDF membrane (Millipore, USA). It was added with 5% skim milk and sealed under 37°C for 2 h. Next, the primary antibodies were added for overnight incubation under 4°C (antibodies GAPDH, ERK1/2, P38, JNK, and PAR-1, and phosphorylated antibodies ERK1/2, P38, and JNK). After it was thoroughly washed using TBST, second antibodies were added for incubation under 37°C for 1 h. Then, the ECL chemiluminescence reagent (Beyotime, China) was used for color development, and the chemiluminescence imaging system (Bio-Rad, USA) was used to collect and analyze the images.

DMEM (high sugar) was obtained from Invitrogen Gibco (Grand Island, NY, USA), FBS from Science Cell Research Laboratories (Carlsbad, CA, USA), and CCK8 cell viability test kit from Nanjing Enogene Biotech Co., Ltd. (Nanjing, China). Asiatic acid was supplied by the client. Transwell chambers with a pore size of 8.0 μm were purchased from Corning Incorporated (Corning, NY, USA), with REF 3422 and LOT 14416045, and BD Matrigel Matrix (Basement Membrane) from BD Biosciences (San Jose, CA, USA), and anti-WAVE3 rabbit anti-human/mouse antibody E 20–74899 from Nanjing Enogene Biotech. Anti-P53 rabbit anti-human/mouse antibody, E11-10276C; EnoGene anti-NF-κB rabbit anti-human antibody, E10-20406; EnoGene anti-p-PI3K rabbit anti-human/mouse antibody, E011508-2 and E1A7005A; and EnoGene anti-GAPDHrabbit anti-human/mouse antibody, E90062, were obtained from Nanjing Enogene Biotech Co., Ltd. (Nanjing, China). The hydrophobic PVDF membrane was purchased from Merck Millipore (Billerica, MA, USA). ECL chemiluminescence reagent was obtained from Beyotime Biotechnology (Shanghai, China), and P0018A, RIPA lysate, BSA, prestaining protein marker, HRP labeled goat anti-rabbit secondary antibody, developer, fixing solution, and BCA protein concentration test kit were purchased from Nanjing Enogene Biotech. Co., Ltd (Nanjing, China).