Pfoxo1 s256

Manufactured by Cell Signaling Technology

Sourced in United States

PFoxO1(S256) is a phospho-specific antibody that detects endogenous levels of FoxO1 when phosphorylated at serine 256. This antibody is designed for use in western blotting applications.

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5 protocols using pfoxo1 s256

Immunoblotting for Insulin Signaling Pathway

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Specific antibodies for pAkt(Ser473)(#4060, 1:5000), pAkt(Thr308)(#4056, 1:2500), Akt(#4691, 1:5000), pINSR(#3024, 1:5000), INSRβ(#3025, 1:5000), p-p70-S6K(#97596, 1:2000), S6K(#9202, 1:5000), pFoxO1(S256)(#9461, 1:2000), FoxO1(#2880, 1:5000), pGSK3β(#5558, 1:5000), GSK3α/β(#5676, 1:5000), p-ERK1/2(T202/Y204)(#9101, 1:5000), ERK1/2(#4695, 1:5000), and Hsp70(#4872, 1:2000) were obtained from (Cell Signaling Technology Inc., Danvers, MA, USA). pIRS1(#09432, 1:2500) and IRS1(#06248, 1:2500) were acquired from (Merck KGaA, Darmstadt, Germany). β-Actin(#A228, 1:5000) was procured from (Sigma-Aldrich). Peroxidase-conjugated secondary antibodies Anti-Rabbit IgG (#NA934, 1:5000) and Anti-Mouse IgG (#NA931, 1:5000) were purchased from (GE Healthcare BioSciences AB, Chicago, IL, USA). Anti-Rabbit IgG Secondary Antibody (Alexa Fluor™ 568, 1:5000) was obtained from (Thermo Fisher Scientific, Waltham, MA, USA).

Dong Q., Endo M., Kawamura G, & Ozawa T. (2022). Systematic Interrogation of the Temperature Perturbation in the Insulin Signaling Pathway for Optogenetic Stimulation. Cells, 11(19), 3136.

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Western Blot Analysis of Hepatic Proteins

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As described previously,^{16 (link)} liver tissues from mice fasted for 16 h were extracted, quantified via BCA assay, and equivalent quantities of protein were separated via SDSPAGE (Thermo Scientific, Waltham, MA) and transferred to nitrocellulose membranes. The primary antibodies used were p-AKT, AKT, p-Foxo1 S256, Foxo1, and actin from Cell Signaling Technology (Danvers, Massachusetts) and PI3K-p85, AR (N20) from Santa Cruz Biotechnology (Dallas, Texas), Glucose-6-phosphatase catalytic subunit (G6PC) from Novus Biotechnologicals (Centennial, CO), and phosphoenolpyruvate carboxykinase (PEPCK) from Abcam (Cambridge, MA) all 1:1000 dilutions. The blots were then placed in secondary antibodies (goat anti mouse or goat anti-rabbit, BioRad), and detected using enhanced chemiluminescence (Perkin Elmer Life Sciences, Boston, MA) or Odyssey CLx Imaging System (LI-COR Biosciences, Lincoln, NE).

Andrisse S., Feng M., Wang Z., Awe O., Yu L., Zhang H., Bi S., Wang H., Li L., Joseph S., Heller N., Mauvais-Jarvis F., Wong G.W., Segars J., Wolfe A., Divall S., Ahima R, & Wu S. (2021). Androgen-induced insulin resistance is ameliorated by deletion of hepatic androgen receptor in females. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 35(10), e21921.

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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).

Fjällström A.K., Evertsson K., Norrby M, & Tågerud S. (2014). Forkhead box O1 and muscle RING finger 1 protein expression in atrophic and hypertrophic denervated mouse skeletal muscle. Journal of Molecular Signaling, 9, 9.

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Insulin Signaling Pathway Characterization

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Insulin, wortmannin, PD98059, SP600125 and β-MHC antibody (cat#M8421) were purchased from Sigma. Antibodies against Foxo1 (cat# 9454), pFoxo1-S²⁵⁶(cat#9461), Akt (cat#9272), pAkt-S⁴⁷³(cat#9271), and α-actinin (cat#6487) were from Cell Signaling Technology (Danvers, USA). IRS1 (cat#06-248) and IRS2 (cat#MABS15) were from EMD Minipore. Foxo1 antibody used for chromatin immunuprecipitation was from Santa Cruz Biotech Inc. (cat#sc11350, Dallas, USA).

Qi Y., Zhu Q., Zhang K., Thomas C., Wu Y., Kumar R., Baker K.M., Xu Z., Chen S, & Guo S. (2014). Activation of Foxo1 by Insulin Resistance Promotes Cardiac Dysfunction and β-Myosin Heavy Chain Gene Expression. Circulation. Heart failure, 8(1), 198-208.

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Western Blot Analysis of Cell Signaling

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Cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were probed with antibodies against cyclin D1, p21, p27, FOXO1, p-FOXO1 (S256), AKT, p-Akt (T308) and p-Akt (S473) (Cell Signaling Technology, Beverly, MA, USA), and GSK3β, p-GSK3β (S9), INPP4A and INPP5J (Abcam, Cambridge, MA, USA), and PHLPP1 and PHLPP2(Abnova, Taipei, Taiwan, China) overnight at 4°C, and then incubated with horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology) for 1 h at room temperature. α-tubulin (Abcam) was used to correct for differences in protein loading from the control and experimental groups.

Jiang J., Zhang Y., Guo Y., Yu C., Chen M., Li Z., Tian S, & Sun C. (2015). MicroRNA-3127 promotes cell proliferation and tumorigenicity in hepatocellular carcinoma by disrupting of PI3K/AKT negative regulation. Oncotarget, 6(8), 6359-6372.

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