Aldo, rapamycin (RP), chloroquine (CQ), 3-methyladenine (3-MA), tunicamycin (Tun), tauroursodeoxycholic acid (TAUDC), and anti-β-actin antibody were purchased from Sigma (St Louis, MO). Antibodies against LC3, Akt, p-Akt, mTOR, p- mTOR, S6K1, p-S6K1, 4EBP1, p-4EBP1, GRP78, GRP94, CHOP, FOXO1, p-FOXO1, Rab5, and Rab7 were purchased from Cell Signaling Technology (Beverly, MA). Anti-Podocin, anti-Nephrin, and anti-p62 antibodies were obtained from Abcam (Cambridge, MA). P300, Ac-FOXO1, and nestin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Ac foxo1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Ac-FoxO1 is a laboratory reagent used for research purposes. It is a recombinant protein that represents the acetylated form of the FoxO1 transcription factor. FoxO1 is a key regulator of cellular processes such as metabolism, cell cycle, and apoptosis. The Ac-FoxO1 product provides researchers with a tool to study the acetylation-mediated regulation of FoxO1 function in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Sirt1, by Santa Cruz Biotechnology (6 mentions)
Foxo1, by Cell Signaling Technology (4 mentions)
Ac p53, by Cell Signaling Technology (3 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (3 mentions)
Bcl 2, by Santa Cruz Biotechnology (3 mentions)
β actin, by Bioworld Technology (3 mentions)
β actin, by Merck Group (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Enhanced chemiluminescence solution, by Thermo Fisher Scientific (2 mentions)
Un scan it 6, by Silk Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Tauroursodeoxycholic acid, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Grp78, by Cell Signaling Technology (1 mentions)
P foxo1, by Cell Signaling Technology (1 mentions)
Nestin, by Santa Cruz Biotechnology (1 mentions)
P s6k1, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti podocin, by Abcam (1 mentions)
10 protocols using ac foxo1
1
Podocyte Autophagy Regulation Mechanisms
2
Western Blot Analysis of Protein Expression
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After washing, cold RIPA with protease inhibitors (Roche) was used to lyse cells for 15 min. Then, after centrifugation, the cell supernatant was kept frozen at −80°C until use. Bradford assays (Bio-Rad Laboratories; CA, USA) were employed to assess protein concentrations. Samples (20 μg) were separated via SDS-PAGE, after which dry transfer to nitrocellulose membranes was conducted. After being blocked in 5% BSA for 1 h, primary antibodies were used to probe the membranes overnight at 4°C, including antibodies targeting human SIRT1, acetylated-p53 at K382 (Ac-p53), p21, forkhead box O1 (FoxO-1) (Cell Signaling Technology; USA), Ac-FoxO-1, total p53 (1: 2000 dilution), and senescence marker protein-30 (SMP-30) (all from Santa Cruz Biotechnology). β-actin served as an internal control and was purchased from Sigma Aldrich. After incubation with the appropriate secondary antibodies (1: 10 000 dilution; GE Healthcare, Buckinghamshire, UK), the immunostained protein bands were visualized with an ECL system (ProteinSimple; Santa Clara, USA), followed by quantification using ProteinSimple image software. All samples had 3 biological replicates, and each biological replicate was assayed in duplicate; therefore, the data for each time point are an average of 6 individual replicate runs. Representative images of the immunostained bands are presented in the figures.
3
Immunohistochemical Analysis of Ovarian Tissue
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Ovarian tissue sections were immunostained with primary antibodies against FoxO1 (1 : 250, Cell Signaling Technology), SIRT1 (1 : 250; Santa Cruz Biotechnology), Ac-FoxO1 (1 : 400; Santa Cruz Biotechnology), and cleaved caspase-3 (1 : 200; Cell Signaling Technology) overnight at 4 °C. The following day, the sections were incubated with a goat anti-rabbit secondary antibody (rabbit ABC detect kit, ZSBio, Beijing, China) at 37 °C for 30 min. Next, the sections were stained with 3, 3'-diaminobenzidine (DAB) and counterstained with hematoxylin. Control sections were similarly pretreated and processed concurrently with the experimental sections using nonspecific rabbit IgG. Nonspecific staining was not detected in the controls.
4
Protein Extraction and Immunoblotting in Granulosa Cells
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Total protein was prepared from cultured GCs or ovarian tissue as previously described.19 (link) Nuclear and cytoplasmic proteins were extracted using the NE-PER nuclear and cytoplasmic extraction reagents (Thermo) according to the manufacturer’s instructions. Immunoblotting was performed with primary antibodies against caspase-3 (1 : 1000; Cell Signaling Technology, #9662s, Danvers, MA, USA), cleaved caspase-3 (1 : 500; Cell Signaling Technology, #9661s), FoxO1 (1 : 1000; Cell Signaling Technology, #2880), Ac-FKHR (1 : 500; Ac-FoxO1, Santa Cruz Biotechnology, sc-49437), phospho-FoxO1 (Ser 256, 1 : 1000; Cell Signaling Technology), phospho-FoxO1 (Ser 319, 1 : 1000; Bioworld Technology, BS4712, MN, USA), FasL (1 : 500; Bioworld Technology, BS1122), Bcl-2 (1 : 1000; Proteintech, 12789-1-AP, Wuhan, China), Bax (1 : 500; Bioworld Technology, BS6420), and SIRT1 (1 : 500; Santa Cruz Biotechnology, sc-15404). β-actin (1 : 10000; Abcam, Cambridge, CA, USA), HSP60 (1 : 1000; Bioworld Technology), and Lamin B (1 : 1000; Santa Cruz Biotechnology, sc-6216) served as internal controls for detecting the expression levels of total protein lysates, cytoplasmic protein lysates, and nuclear protein lysates, respectively.
5
Protein Extraction and Western Blot Analysis
6
Western Blot Analysis of FoxO1 and MuRF1 Proteins
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Western blots were prepared essentially as described in [50 (link)]. Fifteen to thirty μg protein were reduced, denatured and electrophoretically separated on a 12% polyacrylamide gel with a 5.2% polyacrylamide stacking gel on top. Gels were electroblotted onto PVDF Plus transfer membranes (Amersham Hybond-P, GE Healthcare, Buckinghamshire, England) and the membranes were blocked and then incubated with antibodies. Primary antibodies for detecting total-FoxO1 (rabbit monoclonal) (C29H4) [2880] and pFoxO1 S256 (rabbit polyclonal) [9461] were obtained from Cell Signaling Technology (Beverly, CA), MuRF1 (goat polyclonal) [AF5366] was obtained from R&D systems (Abingdon, England) and Ac-FoxO1 (rabbit polyclonal) (FKHR D19) [49437] from Santa Cruz Biotechnology (Santa Cruz, CA). All primary antibodies were used at a dilution of 1/800 – 1/1500. Antibodies were visualized with horseradish peroxidise conjugated secondary immunoglobulin diluted 1/1000 goat anti-rabbit IgG [P0448] and 1/1000-1/10000 rabbit anti-goat IgG [P0449] (Dako, Glostrup, Denmark). The bound immune complexes were detected using the ECL Plus Western blotting detection system and Hyperfilm ECL (Amersham International and Amersham Pharmacia Biotech, Buckinghamshire, England).
7
Antibody Analysis of Cellular Proteins
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Antibodies against the following proteins were used: HSP90 (Enzo Life Sciences, Plymouth Meeting, PA, USA), FoxO1, Sirt6, Ac-Lys (Cell Signaling Technology), Ac-FoxO1, ubiquitin (Santa Cruz Biotechnology), CCR3, FoxO3a, FoxO4 (Abcam), lamin B (Bioworld Technology, St Louis Park, MN, USA) and β-actin (Sigma-Aldrich, St. Louis, MO, USA).
8
Western Blotting Analysis of SIRT1, FoxO1, and NLRP3
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Western blotting was performed according to our previous study (18 (link)). After sample preparation, equal amounts of protein samples were separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was then incubated with primary antibodies against SIRT1 (1:200, cat# SC-15404, Santa Cruz), ac-FoxO1 (1:200, cat# sc-49437, Santa Cruz), NLRP3 (1:200, cat# SC-66846, Santa Cruz Biotechnology), adaptor apoptosis-related speck-like protein (ASC) (1:200, cat# SC-22514, Santa Cruz Biotechnology), caspase-1 (1:200, cat# SC-56036, Santa Cruz Biotechnology), caspase-1 p20 (1:200, cat# SC-398715, Santa Cruz Biotechnology), and β-actin (1:3000, cat# AP0060, Bioworld Technology, Minneapolis, MN, USA) overnight at 4°C. Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-conjugated IgG for 2 h at room temperature. Protein bands were incubated with enhanced chemiluminescence solution (Thermo Fisher Scientific, Waltham, MA, USA). Band density was analyzed using Image J software.
9
Oxidative Stress and Apoptosis Evaluation
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BBR, Evans blue (EB), triphenyltetrazolium chloride (TTC), 4′,6-diamino-2-phenylindole (DAPI), and protease inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium carboxymethylcellulose (CMC-Na) was purchased from Solarbio Technology (Beijing, China). Lactate dehydrogenase (LDH) assay kit, creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), superoxide generation, IL-6, and myeloperoxidase (MPO) assay kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). TNF-α ELISA kit was purchased from R&D Corporation (Minneapolis, USA). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kit was obtained from Roche Molecular Biochemicals (Mannheim, Germany). BCA protein quantification kit was purchased from Merck Millipore Technology (Darmstadt, Germany). The primary antibodies against SIRT1, Ac-Foxo1, gp91phox, caspase-3, Bcl-2, Bax, β-actin, and SIRT1 siRNA were purchased from Santa Cruz Biotechnology (CA, USA). Rabbit anti-goat, goat anti-rabbit, and goat anti-mouse secondary antibodies were purchased from the Zhongshan Company (Beijing, China).
10
Western Blot Analysis of Protein Expression
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For western blot analysis, equal protein amounts per lane were separated by 10% SDS-polyacrylamide gels and transferred to a polyvinylidenedifluoride membrane. The membrane was blocked in 5% skim milk for 2 h at room temperature and incubated overnight at 4 °C with primary antibodies against SIRT1 (cat# sc-15404, 1:200, Santa Cruz, CA, USA), Iba-1 (cat# sc-98468, 1:200, Santa Cruz, CA, USA), ac-FoxO1 (cat# sc-49437, 1:200, Santa Cruz, CA, USA), ac-p53 (cat# 2570, 1:400, Cell Signaling, MA, USA), ac-NF-кB (cat# 12629 S, 1:400, Cell Signaling), Bcl2 (cat# sc-492, 1:200, Santa Cruz, CA, USA), Bax (cat# sc-493, 1:200, Santa Cruz, CA, USA), caspase-3 (cat# 9661, 1:400, Cell Signaling), histone 3 (cat# BS7416, 1:2000, Bioworld, MN, USA) and β-actin (cat# AP0060, 1:5000, Bioworld) in tris-buffered saline with Tween-20 containing 5% skim milk. After the membrane was washed, it was incubated with horseradish peroxidase-conjugated IgG for 2 h at room temperature. The blotted protein bands were visualized by enhanced chemiluminescence western blot detection reagents (Thermo Scientific, Kalamazoo, MI, USA) and were exposed to X-ray film. The quantification of band density was performed using the UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA).
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