20μg of protein was electrophoresed on 4-12% Nu-Page gel (Life Technologies) and transferred to a nitrocellulose membrane. Membranes were blocked in 5% milk in TBST at room temperature for 30 minutes. Membranes were incubated overnight at 4 degrees Celsius with the primary antibodies: anti-GCN5 (1:1000), anti-MYC (1:1000), anti-AKT (1:1000), anti-phospho-AKT (1:1000), anti-SYK (1:1000), anti-BTK (1:1000), anti-FOXO1 (1:1000), anti-phospho-FOXO1 (1:1000), and anti-PARP (1:1000) (Cell Signaling); anti-Ada2b (1:500); anti-Beta Actin (1:5000) (Santa Cruz), anti-USP22 (1:1000) (homemade); H3 acetyl lysine 9 (1:500) (Millipore); H3(1:10,000) (Abcam). Membranes were incubated in secondary-horseradish peroxidase (HRP) conjugated antibodies (GE Healthcare, Cat. # NA934V for Rabbit, NA931V for Mouse) for 1 hour at room temperature. Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) was used for chemiluminescent protein detection.
Anti phospho foxo1
Manufactured by Cell Signaling Technology
Sourced in United States, China
Anti-phospho-FOXO1 is a primary antibody that specifically recognizes the phosphorylated form of the FOXO1 transcription factor. FOXO1 is a key regulator of cellular processes such as apoptosis, cell cycle progression, and metabolism. This antibody can be used to detect and quantify the phosphorylation state of FOXO1 in various experimental systems.
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Lab products found in correlation
Anti foxo1, by Cell Signaling Technology (5 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti phospho akt, by Cell Signaling Technology (3 mentions)
Anti 4e bp1, by Cell Signaling Technology (2 mentions)
Anti phospho 4ebp1, by Cell Signaling Technology (2 mentions)
Anti syk, by Cell Signaling Technology (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Anti gcn5, by Cell Signaling Technology (1 mentions)
Anti btk, by Cell Signaling Technology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti myc, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Anti phospho p44 42, by Cell Signaling Technology (1 mentions)
Anti p70s6k, by Cell Signaling Technology (1 mentions)
Anti phospho p70s6k, by Cell Signaling Technology (1 mentions)
Anti phospho tsc2, by Cell Signaling Technology (1 mentions)
Anti phospho mtor, by Cell Signaling Technology (1 mentions)
Anti phospho mek1 2, by Cell Signaling Technology (1 mentions)
Anti p21cip1, by Santa Cruz Biotechnology (1 mentions)
8 protocols using anti phospho foxo1
1
Western Blot Analysis of Signaling Proteins
2
Signaling Pathway Protein Analysis Protocol
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The following reagents and antibodies were obtained commercially: MG-132 (Calbiochem), anti-Akt1, anti-phospho-Akt1 (Thr308, Ser473), anti-p44/42 (Erk1/2), anti-phospho-p44/42 (Erk1/2) (Thr202/Tyr204), anti-MEK1/2, anti-phospho-MEK1/2 (Ser 217/221), anti-mTOR, anti-phospho-mTOR (Ser2448), anti-p70S6K, anti-phospho-p70S6K (Thr389), anti-Rictor, anti-Raptor, anti-PRAS40, anti-phospho-PRAS40 (Thr246), anti-4E-BP1, anti-phospho-4E-BP1 (Thr 70), anti-FoxO1, anti-phospho-FoxO1 (Thr24 and Ser256), anti-FoxO3a, anti-phospho-FoxO3a (Ser253 and Thr32), anti-TSC2, anti-phospho-TSC2 (Thr1462), anti-SGK1, anti-SGK3 (Cell Signaling Technology), anti-p21Cip1 (Santa Cruz Biotechnology); anti-p27Kip1 (BD Biosciences), anti-cyclin D1 (MBL), and anti-β-tubulin (Sigma). anti-phospho-FoxO3a Ser314 antibodies were a generous gift from Dr. Michael E. Greenberg (Harvard Medical School, Boston).
3
Western Blot Analysis of Embryonic and Tumor Samples
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Zebrafish embryos were collected at the indicated stage and directly lysed in SDS sample buffer. For nuclear FOXO1 detection, cytoplasmic and nuclear protein samples were obtained from 48 hpf zebrafish embryos using Nuclear and Cytoplasmic Protein Extraction Kit (Thermo fisher) according to the manufacturer’s instruction. Tumor tissue lysates were prepared in RIPA buffer for 30 min on ice. HUVEC samples were directly harvested into SDS sample buffer. Proteins were separated by SDS-PAGE and blotted onto polyvinylidene fluoride membranes. The following antibodies were used: anti-MYC (Genomics Technology, SG4110-18, 1:1000), anti-a-Tubulin (Sigma, T9026, 1:1000), anti-GAPDH (Absci, 21612-2, 1:2000), anti-HIF1a (Ruiying Biological, RLT2133, 1:500), anti-phospho-FOXO1 (Cell Signaling Technology, 9464, 1:500), anti-FOXO1 (Cell Signaling Technology, 2880, 1:500) and anti-Histone-3 (Cell Signaling Technology, 9715, 1:2000).
4
Antibodies for Cellular Signaling Analysis
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The antibodies used were: anti-phospho JNK (Thr183/Tyr185) (#4668), anti-phospho STAT3 (Tyr705) (#9131), anti-STAT3 (#8719), anti-phospho p38 MAPK (Thr180/Tyr182) (#9211), anti-p38 MAPK (#9212), anti-phospho Foxo1 (#9461) and anti-Akt (#9272) from Cell Signaling Technology (MA, USA); anti-phospho IGFIR (Tyr1165/1166) (sc-101704), anti-JNK (sc-571), anti-phospho-Akt1/2/3 (Ser473) (sc-7985-R), anti-caspase 1 (sc-514), anti-Nrf2 (sc-722) and anti-Keap1 (sc-33569) from Santa Cruz (Palo Alto, CA); anti-phospho IRS1 (Tyr1179) (07-844), anti-phospho IRS1 (Ser 307) (07-247), anti-IRS1 (06-248), anti-p85α (06-195) and anti-HO1 (AB1284) antibodies from Merck Millipore (Merck KGaA, Darmstadt, Germany); anti-β-actin (A-5441) antibody from Sigma Chemical Co. (St Louis, MO); anti-Lamin B (aB16048) and FasL (aB68338) from Abcam (Abcam, Cambridge, UK). Anti-IGFIR antibody was a gift of S. Pons (CSIC, Spain).
5
Protein Extraction and Western Blotting
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The isolated liver samples were homogenized in ice-cold RIPA buffer containing protease and phosphatase inhibitors (Beyotime Biotechnology, China) to extract total protein, which concentration was determined with the BCA Protein Assay Kit (Beyotime Biotechnology). The aliquots of protein samples (20μg) were separated by 10% SDS-PAGE gel and then transferred to activated polyvinylidene fluoride membranes (Merck Millipore, Germany). After being blocked with 5% skimmed milk in TBST buffer (Sangon Biotech, China), the membranes were incubated with the primary antibody overnight in a refrigerator at 4°C and then with secondary antibody for 2h. The immune complexes were visualized with a Beyo ECL Plus kit (Beyotime Biotechnology) and quantified using ImageJ software (Bethesda, United States). The specific primary antibodies are anti-FoxO1 (ET1608-25, HuaBio, China), anti-phospho-FoxO1 (#9461, Cell Signaling Technology, United States) anti-Akt1 (#4691, Cell Signaling Technology, United States), anti-phospho-Akt1 (#4060, Cell Signaling Technology, United States), anti-Sirt1 (sc-74,465, Santa Cruz Biotechnology), anti-Nrf2 (ab62352, Abcam, China) and anti-β-tubuliin (#2146, Cell Signaling Technology, United States) antibodies. The secondary antibodies are anti-mouse antibody (Solarbio, China) and anti-rabbit antibody (SongonBiotech, China).
6
Fractionation and Immunoblotting of Cellular Proteins
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Proteins from liver lysates, cell lysates, cell nucleus and cell cytoplasm were extracted by previous methods53 or kits (Beyotime Biotechnology, Shanghai, China). Primary antibodies were used at the dilution of 1:1000. Anti-phospho-FoxO1 (#9461), FoxO1 (#2880), phospho-ERK1/2 (#4370), ERK1/2 (#4695), phospho-Akt (Thr308) (#9275), phospho-Akt (Ser473) (#9271), Akt (#4685), ATGL (#2439), PPARγ (#2435), lamin B1 (#13435), β-actin (#3700) and horseradish peroxidase-conjugated anti-mouse or rabbit IgG were purchased from Cell Signaling Technology. Anti-SRAP antibody (#A300-743A) was purchased from Bethyl Laboratory, Inc.
7
Immunoblotting Analysis of Autophagy and Signaling Pathways
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Mouse skin samples and THP-1 cells were washed with PBS and lysed in RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1%TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) on ice for 30 min. Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose (NC) membranes. The membranes were incubated with the appropriate primary antibodies: anti-LC3B (1:1000, Cell Signaling Technology), anti-Beclin1 (1:1000, Cell Signaling Technology), anti-P62 (1:1000, Cell Signaling Technology), anti-pan-Akt (1:1000, Cell Signaling Technology), anti-Phospho-Akt (1:1000, Cell Signaling Technology), anti-FoxO1 (1:1000, Cell Signaling Technology), anti-Phospho-FoxO1 (1:1000, Cell Signaling Technology), anti-Phospho-ERK1/2 (1:2000 Cell Signaling Technology), anti-Erk1/2 (1:1000, Cell Signaling Technology), anti-Phospho-STAT3 (1:2000, Cell Signaling Technology), anti-STAT3 (1:2000, ABclonal), and anti-α-tubulin (1:2000, Cell Signaling Technology). After incubation with secondary antibodies for 1 h at room temperature, the blotted membranes were visualized using an Odyssey two-color infrared laser imaging system (LI-COR Biosciences) and quantified using ImageJ software and normalized to α-tubulin.
8
Analyzing Muscle Hypertrophy Markers
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We performed Western blotting on the overloaded muscles to assess whether there were different expression patterns of the hypertrophic markers between the different groups. For that purpose, equal concentrations of protein (15-30 µg) were separated under reducing conditions using 12% SDS-PAGE (Biorad mini-PROTEAN) and transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked in 5% nonfat dry milk or 5% bovine serum albumin in TBS-0.1% Tween 20 and then incubated overnight at 4°C with primary antibody. The following primary antibodies from Cell Signaling Technologies (Leiden, The Netherlands) were used: anti-phospho-Akt (No. 4060S, 1:2,000), anti-Akt (No. 9272S, 1:2,000), anti-phospho-4EBP1 (No. 2855S, 1:1,000), anti-4EBP1 (No. 9644S, 1:1,000), anti-phospho FoxO1 (No. 9461S, 1:1,000), and anti-FoxO1 (No. 2880S, 1:1,000). After incubation with the horseradish peroxidase (HRP)-conjugated secondary antibody (P0217, Sigma), protein bands were detected using an enhanced chemiluminescence system (Sigma-Aldrich, Belgium and Thermo Fisher Scientific) and analyzed using the software package (Bio 1D) of the blot imaging system (Photo print, Vilber, France), and phosphorylation status (activity) was determined as the ratio of phosphorylated to total protein.
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