ChIP was performed using the EZ ChIPTM Chromatin Immunoprecipitation kit (Millipore, Burlington, MA, USA) according to the manufacturer's instructions. Briefly, cross-linked chromatin was sonicated into 200–1,000 bp fragments. The chromatin was immunoprecipitated using anti-FOXO1 antibodies (#2880, 1:1,000; Cell Signaling Technology, Beverly, MA, USA). RT-qPCR was conducted to detect the relative enrichment according to the method described above.
Anti foxo1 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-FoxO1 antibody is a research tool used to detect and study the FoxO1 protein, which is a member of the Forkhead box O (FoxO) family of transcription factors. The antibody is designed to specifically recognize and bind to FoxO1, allowing researchers to investigate its expression, localization, and interactions within various cellular and biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt antibody, by Cell Signaling Technology (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Alexa fluor 488 green conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Chip it high sensitivity kit, by Active Motif (2 mentions)
Ez chip chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Vectastaine life abc rabbit igg kit, by Vector Laboratories (1 mentions)
Bz 9000 microscope, by Keyence (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Alexa fluor 594 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence, by Bio-Rad (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Immobilon fl transfer membrane, by Merck Group (1 mentions)
Beta tubulin antibody, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Complete mini, by Roche (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
25 protocols using anti foxo1 antibody
1
FOXO1 Chromatin Immunoprecipitation Protocol
2
Pancreatic Immunohistochemistry and Immunofluorescence
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Pancreas tissues were fixed in 4% paraformaldehyde and paraffin sections
(5 μm) were prepared. For immunohistochemistry, sections were stained
with anti-FAM3A (Sigma-Aldrich, SAB1102488), anti-insulin (Abcam, ab181547), and
anti-PCNA (Cell Signaling Technology, 2586) primary antibodies. After antibody
incubation overnight, diaminobenzidine staining was performed. For
immunofluorescent staining, HIT-T15 cells were fixed, permeabilized, blocked,
and incubated with anti-FOXO1 antibodies (Cell Signaling Technology, 2880) at 4
°C overnight. On the next day, cells were stained with goat anti-rabbit
Alexa Fluor 594. After nuclear staining with DAPI, images were observed under a
confocal laser scanning microscope, and images were acquired.
(5 μm) were prepared. For immunohistochemistry, sections were stained
with anti-FAM3A (Sigma-Aldrich, SAB1102488), anti-insulin (Abcam, ab181547), and
anti-PCNA (Cell Signaling Technology, 2586) primary antibodies. After antibody
incubation overnight, diaminobenzidine staining was performed. For
immunofluorescent staining, HIT-T15 cells were fixed, permeabilized, blocked,
and incubated with anti-FOXO1 antibodies (Cell Signaling Technology, 2880) at 4
°C overnight. On the next day, cells were stained with goat anti-rabbit
Alexa Fluor 594. After nuclear staining with DAPI, images were observed under a
confocal laser scanning microscope, and images were acquired.
3
Immunohistochemical Analysis of Ovarian Tissue
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The ovaries were fixed in 4% (w/v) paraformaldehyde/PBS overnight, dehydrated in 70% (v/v) ethanol, and embedded in paraffin. The paraffin‐embedded fixed sections (4 μm) were deparaffinized in xylene washes and quenched with 3% hydrogen peroxide in methanol. The sections were incubated with 20% (v/v) nonimmune goat serum/PBS to block nonspecific sites, followed by incubation with primary antibodies overnight at 4°C (1:100 of anti‐FOXO1 antibody (catalog #2880; Cell Signaling) or 1:100 of anti‐CD31 antibody (catalog #ab28364; Abcam). The positive signals were visualized using a VECTASTAINE Life ABC rabbit IgG kit (Vector Laboratories) according to the manufacturer's recommendations. Digital images were captured using a Keyence BZ‐9000 microscope.
4
Immunofluorescence Analysis of FoxO1 in GCs
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GCs were cultured in eight-well chambers (Millipore, Billerica, MA, USA) and either infected with Ad-miR-181a/Ad-flag-FoxO1 for 48 h or treated with miR-181a inhibitor/Ad-flag-FoxO1 for 36 h, followed by H2O2 treatment for another 12 h. The cells were then washed with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.5% Triton X-100 in PBS, and incubated with anti-FoxO1 antibody (Cell Signaling Technology) at 4 °C overnight. The following day, the cells were incubated with Alexa Fluor 594-conjugated donkey-anti-rabbit IgG (Life Technology) for 1 h at 37 °C in the dark. The cell nuclei were stained with DAPI (5 μg/ml). Finally, the images were visualized using a laser-scanning confocal microscope (Leica, Germany).
5
Whole-tissue lysate preparation and Western blot
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Whole-tissue lysates were prepared from a piece of QF homogenized in ice-cold PBS using a Precellys homogenizer and lysed in RIPA buffer with protein inhibitor (Complete Mini, Roche), followed by incubation on ice for 20 min. Protein concentration was measured using the Bradford method (Protein Assay, Bio-Rad). Ten micrograms of protein was loaded per well in SDS–PAGE and transferred to Immobilon-FL transfer membrane (Millipore) using semi-dry transfer unit (Hoefer TE70, Amersham Biosciences). The membranes were blocked in 5% milk in TBS 0.1% Tween. Antibody detection reaction was developed by enhanced chemiluminescence (Bio-Rad). The anti-FOXO1 antibody was from Cell Signaling, and the beta-tubulin antibody was from Sigma; HSP70 and mitochondrial respiratory complex antibodies were from Abcam, against CI, respiratory chain complex I, 39-kDa subunit; CII, complex II, 70-kDa subunit; CIV, complex IV, cytochrome c oxidase, COI subunit, 40 kDa; CV, complex V, ATPase, alpha subunit, 55 kDa, ClpP antibody from Proteintech, and antibodies for HSP60, acetylated-FOXO1 (sc-43497), and beta-actin were from Santa-Cruz.
6
Protein Extraction and Western Blot Analysis
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The samples were lysed by RIPA lysis buffer (Beyotime Biotechnology, Nanjing, Jiangsu, China) which contains a protease inhibitor cocktail (EMD Millipore, Billerica, MA, USA) on ice for 30 minutes. Samples should be shaken slightly during lysis to ensure reacting sufficiently. All subsequent protein extracting procedures were performed in a standard manner. Then, standard electrophoresis procedure was performed and the proteins were transferred to nitrocellulose membranes, followed by blocking for 1 h. Subsequently, the membranes were treated with anti-FOXO1 antibody (1 : 1000; Cell Signaling Technology, Danvers, USA), β-actin antibody (1 : 1000; Cell Signaling Technology, Danvers, USA), and LC3B-I/II antibody (1 : 1000; Cell Signaling Technology, Danvers, USA) overnight at 4°C and then with secondary antibody (1 : 1000; Cell Signaling Technology, Danvers, USA) for 1 h at room temperature. The results were obtained by the Odyssey Infrared Laser Imaging system (LI-COR Corporate, NE, USA).
7
Analysis of mTOR and Foxo1 Signaling
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A total of 1 × 106 cells were lysed in 1× Cell Lysis Buffer (Cell Signaling Technology) to obtain whole-cell lysates. Equalized whole-cell lysates were used for immunoblot analysis as previously reported (60 (link)). Antibodies were used for immunoblot analysis as follows: anti–p-mTOR (Ser2448), anti-mTOR, anti-Foxo1, anti–p-Foxo1 (Ser256), anti–4E-BP1, anti–p-4E-BP1 (Thr37/46), anti–p-p70S6K (Thr421/Ser424), anti-p70S6K, anti–p-Stat3 (Tyr705), anti-Stat3, anti–p-Smad2 (Ser465/467), anti-Smad2, anti–p-Stat5 (Tyr694), anti-Stat5, anti-Lamin A/C (4C11), and anti–PP2A-A were obtained from Cell Signaling Technology; anti–glyceraldehyde phosphate dehydrogenase and anti-GSTM1 were from ProteinTech; anti–β-actin was from Santa Cruz Biotechnology. The nucleus and cytoplasm proteins were extracted according to the manufacturer’s instructions by NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific).
For immunoprecipitation, 5 × 106 T cells were harvested and then lysed in phosphatase extraction buffer [20 mM imidazole-HCl, 2 mM EDTA, and 2 mM EGTA (pH 7.0)]. Foxo1 was immunoprecipitated using anti-Foxo1 antibody (Cell Signaling Technology) and protein A agarose. Beads were washed four times in 1× tris-buffered saline, and the proteins were determined by immunoblot analysis.
For immunoprecipitation, 5 × 106 T cells were harvested and then lysed in phosphatase extraction buffer [20 mM imidazole-HCl, 2 mM EDTA, and 2 mM EGTA (pH 7.0)]. Foxo1 was immunoprecipitated using anti-Foxo1 antibody (Cell Signaling Technology) and protein A agarose. Beads were washed four times in 1× tris-buffered saline, and the proteins were determined by immunoblot analysis.
8
Mitochondrial Protein Analysis by Western Blot
9
RIP Assay for FOXO1 Interaction
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According to the manufacturer’s instructions, RIP assay kit (Millipore, USA) was used for RIP assay. Briefly, HEK-293 cell suspension was prepared in RIP buffer. Anti-FOXO1 antibody (Cell Signaling Technology, 5 μg) was incubated with the cell suspension at 4 °C overnight. Then, the precipitated RNA was purified and analyzed by qRT-PCR. Isotype-matched IgG (5 μg) was used as a negative control.
10
Western Blot Analysis of Osteogenic Markers
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Total protein was extracted from tissues or cells using RIPA Lysis Buffer (Beyotime, Beijing, China). The protein was then separated by 12% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% skim milk and incubated with primary antibodies, including anti-FOXO1 antibody (1:400, Cell Signaling Technology), anti-ALP antibody, anti-Runx2 antibody (1:300, Cell Signaling Technology), anti-Ostx antibody (1:300, Cell Signaling Technology), anti-OCN antibody (1:300, Cell Signaling Technology), anti-Skp2 antibody (1:500, Santa Cruz, USA) and anti-GAPDH antibody (1:800, Santa Cruz). Then, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugate secondary antibody (1:2000, Santa Cruz) at room temperature for 1 h. ECL development solution (Thermo, USA) was used to visualize the proteins.
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