Anti sirt1

Homogenized renal tissues, cells, and exosome lysates were separated via 8% or 12% SDS-PAGE, transferred to nitrocellulose membranes (Millipore, Jaffrey, NH, USA), and probed with the following antibodies: anti-CD9 (#20597-1-AP, 1:500; Proteintech), anti-CD63 (#ab216130, 1:500; Abcam), anti-CD81 (#18250-1-AP, 1:500; Proteintech), anti-VEGF (#19003-1-AP; 1:1000, Proteintech), anti-HIF-1α (#AF1009, 1:1000; Affinity), anti-SIRT1 (#13161-1-AP, 1:500; Proteintech), anti-eNOS (#AF0096; 1:1000, Affinity), anti-p-eNOS (#AF3247, 1:1000; Affinity) and anti-β-actin (#4970, 1:1000; Cell Signaling Technology).

Male Sprague-Dawley (SD) rats were purchased from Sina-British Sippr/Bk Lab Animal Ltd. (Shanghai, China). Tetramethylpyrazine (TMP), Crocin, Ferulic acid and Chlorogenic acid, acetylcholine, phenylephrine (PE) and D-glucose were purchased from Sigma-Aldrich Co. (St Louis, Missouri, USA). Dulbecco's modified Eagle's medium (DMEM) supplemented with 5.6 mmol/L glucose was obtained from Hclone (Logan, UT, USA). DMEM without red phenol, fetal bovine serum (FBS) and Trizol were purchased from Invitrogen (Carlsbad, CA, USA). CM-H₂DCFDA, DAF-FM diacetate and MitoSOX™ Red mitochondrial superoxide indicators were obtained from Invitrogen (Carlsbad, CA, USA). JC-1 indicator was bought from Molecular Probes (Eugene, OR, USA). Antibodies including anti-complex III, anti-PGC-1α, anti-SIRT1 and anti-β-actin were purchased from Proteintech (ProteinTech Group, Chicago, IL, USA). Primers for PGC-1α, NRF-1, TFAM, SIRT1 and 18s were provided by Sangon Biotech CO. Ltd. (Shanghai).

The instruments and reagents used in these experiments were as follows: a 7.0T animal magnetic resonance instrument (Bruker, Germany); novel object recognition and test system (Boster Bioengineering, China); Y labyrinth video analysis system (Shanghai Xinruan Information Technology, China); high-fat feed (21% fat, 0.15% cholesterol, Jiangsu Medisen Biomedicine, China); isoflurane (Shenzhen Reward, China); immunohistochemistry kit and DAB staining kit (Boster Bioengineering, China); and haematoxylin staining solution and eosin staining solution (Beijing Solebao Technology, China). The primary antibodies in this study were as follows: anti-SIRT1 (Proteintech, USA), anti-TNF-α (Proteintech, USA), anti-β-actin (Proteintech, USA), anti-IBA1 (Proteintech, USA), anti-GFAP (Proteintech, USA), anti-PGC-1α (Abcam, USA), anti-BDNF (Abcam, USA), anti-IL-1β (Abcam, USA), NF-κBp65 antibody (Cell Signaling Technology, USA), goat anti-mouse/anti-rabbit secondary antibody (Proteintech, USA), and antibody diluent (Beijing Biyuntian, China).

NaHS was purchased from Sigma-Aldrich (St Louis, MO, USA), and the cigarettes were purchased from Guangdong Tobacco Industry Co., Ltd. (Guangzhou, China). SRT1720 and EX 527 were purchased from Selleck Chemicals (Houston, TX, USA). The TRIzol Reagent was purchased from Ambion (Life Technologies, CA, USA). The PrimeScript RT reagent Kit with gDNA Eraser was from Takara Bio Inc. (Takara, Shiga, Japan), and the SsoFast EvaGreen Supermix was obtained from Bio-Rad Laboratories, Inc. (CA, USA). The primary and second antibodies described in this study include: anti-Bcl-2 and anti-β-actin polyclonal antibodies were purchased from Proteintech (Chicago, IL, USA); anti-MFN1, anti-SIRT1, anti-FOXO3 and anti-Bax antibodies were purchased from Abclonal (Wuhan, China); anti-p21 and anti-p53 antibodies were purchased from Cell Signaling Technology (CA, USA); anti-FIS1 antibodies, and the horseradish peroxidase (HRP)-labeled Goat Anti-Rabbit/Mouse IgG (H+L) were purchased from Abcam Biotechnology (Cambridge, MA, USA). The poly-vinylidene fluoride (PVDF) membranes were from Millipore Corporation (Billerica, MA, USA). ECL-Plus detection kit probed was purchased from Tanon Science and Technology Co., Ltd. (Shanghai, China). Other reagents were all purchased from GBCBIO Technologies Inc. (Guangzhou, China) unless otherwise indicated.

As previous describe [21 (link)], briefly a total of 100 porcine oocytes per group were lysed with 1 × sodium dodecyl sulfate sample buffer by heating at 98°C for 10 min. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Next, the membranes were blocked in 5% (w/v) skim milk for 1 h and then incubated at 4°C overnight with anti-SIRT1 (Cat #60303-1-Ig, Proteintech), anti-ubiquitin (Cat #ab19247, Abcam), or GAPDH (Cat #sc-365062, Santa Cruz), followed by incubation at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1:20000; Santa Cruz Biotechnology). The membrane was detected using Pierce ECL substrate (Thermo Fisher Scientific). Blots were visualized using a CCD camera and UVISoft software (UVITEC, Cambridge, UK).

PC12 cells were seeded in 6-well plates at 4.0×10⁵ cells per well, treated with X5 for 18 h, and sampled for total proteins on ice using the culture cell total protein extraction reagent (Boster Biological Technology Co. Ltd., China). Total protein was then separated by SDS-PAGE electrophoresis and the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was then incubated with primary antibodies (Anti-SIRT1, Proteintech, 13161-1-AP, China, 1:1000; anti-beclin-1, Proteintech, 11306-1-AP, China, 1:1000; anti-LC3-β, Proteintech, 18725-1-AP, China, 1:1000; anti-GAPDH, Proteintech, 10494-1-AP, China, 1:1000) at 4°C for overnight. The following day, the membrane was incubated with secondary antibodies (Anti-Rabbit igg (H + L), Proteintech, SA00001-2, China, 1:2000) at room temperature for 90 min, and the immunofluorescent bands were imaged using the ChemiDoc XRS+ system (Bio-Rad, USA). Finally, the images were quantitatively analyzed using Image J (NIH, USA).