Overview

  • Product name

    Phospho-FOXO1 (T24) + Total In-Cell ELISA Kit (Chemiluminescent)
    See all FOXO1A kits
  • Detection method

    Luminescent
  • Sample type

    Adherent cells, Suspension cells
  • Assay type

    Cell-based (quantitative)
  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Product overview

    Phospho-FOXO1 (T24) + Total In-Cell ELISA Kit (Chemiluminescent) (ab207472) provides a simple, efficient, cell-based method to monitor proteins activated by phosphorylation. The kit is designed specifically to quantify phosphorylated FOXO1 and/or total FOXO1. Cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are rapidly fixed to preserve activation-specific protein modifications. Each well is then incubated with a primary antibody that recognizes either phosphorylated FOXO1 or total FOXO1. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides an easily quantified chemiluminescent readout. The relative number of cells in each well is then determined using the provided Crystal Violet solution. The 96-well plate format is suitable for high-throughput screening applications.


    Phospho-FOXO1 (T24) + Total In-Cell ELISA Kit (Chemiluminescent) (ab207472) contains 96-well plates and two primary antibodies. The phospho-FOXO1 antibody is specific for phosphorylated FOXO1 and was raised against a synthetic phospho-peptide corresponding to residues surrounding Thr24 of human FOXO1. This antibody recognizes FOXO1 only when phosphorylated at Thr24 and also recognizes FKHRL1 when phosphorylated at Thr32 and AFX when phosphorylated at Thr28. The total-FOXO1 antibody recognizes FOXO1 proteins regardless of the phosphorylation state. The kit can be used to study phosphorylated FOXO1 relative to cell number or to determine FOXO1 phosphorylation relative to the FOXO1 protein found in the cells. Once the phospho-FOXO1 and total-FOXO1 signals have been normalized for cell number, a comparison of the ratio of phosphorylated FOXO1 to total FOXO1 for each of the cell growth conditions can be made. The provided total-FOXO1 antibody can be used as a positive control to demonstrate that the cells contain FOXO1, the kit reagents are functional and that the protocol is performed correctly. Also, because fixed cells are stable for several weeks, many plates can be prepared simultaneously and then the assay performed when desired.

  • Notes

    The Forkhead family of transcription factors are involved in regulation of the cell cycle, cell death, cell metabolism and oxidative stress. The FOXO (Forkhead Box, class O) proteins form a subfamily of Forkhead transcription factors that are direct targets of phosphoinositide 3-kinase (PI3K) mediated signal transduction. In the presence of growth factor/survival signals, PI3K activation leads to PDK mediated downstream activation of PKB/c-AKT. Phosphorylation and activation of PKB causes nuclear translocation of PKB, which phosphorylates and inactivates nuclear FOXO. FOXO then binds 14-3-3 proteins, and the FOXO/14-3-3 complex is exported to the cytoplasm. Phosphorylated, inactive FOXO proteins remain bound to 14-3-3 proteins in the cytoplasm, thereby preventing nuclear import of FOXO. In the absence of survival signals, cytoplasmic FOXO is dephosphorylated, causing dissociation from 14-3-3 proteins and allowing nuclear import of FOXO to activate gene expression.

    There are three AKT phosphorylation sites in the FKHR protein: Thr24, Ser256 and Ser319. Specifically, phosphorylation at Ser256 is thought to play a role in masking a FKHR nuclear localization signal. In addition, phosphorylation at Ser256 may also mediate the effects of insulin on gene expression. Phosphorylation at Thr24 is critical for FKHR interaction with 14-3-3 proteins.

    The most well studied FOXO members include acute-lymphocytic-leukaemia-1 fused gene from chromosome X (AFX/FOXO4), Forkhead in rhabdomyosarcoma (FKHR/FOXO1) and FKHR-Like 1 (FKHRL1/FOXO3a). In different cell types, FOXO proteins modulate various cellular activities. In hepatocytes, FOXO proteins regulate the expression of factors involved in gluconeogenesis, such as peroxisome proliferators-activated receptor-g coactivator-1, glucose-6-phosphate and phosphoenolpyruvate carboxykinase. Using genetic gain and loss of function analysis in mice, FKHR has also been shown to control b cell compensation for insulin resistance and glucose production in type 2 diabetes. In addition, FOXO proteins such as FKHRL1 have been shown to regulate catalase and superoxide dismutase gene expression that protect cells from oxidative stress, suggesting that FOXO factors act to control the mammalian lifespan.

  • Tested applications

    Suitable for: In-Cell ELISAmore details
  • Platform

    Microplate

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests 5 x 96 tests
    1% SDS Solution 1 x 22ml 5 x 22ml
    10% Triton X-100 1 x 10ml 5 x 10ml
    10X PBS 1 x 120ml 5 x 120ml
    1X Antibody Blocking Buffer 1 x 22ml 5 x 22ml
    1X Antibody Dilution Buffer 1 x 30ml 5 x 30ml
    96-well tissue culture plate 2 units 10 units
    Anti-rabbit HRP-conjugated IgG 2 x 11µl 10 x 11µl
    Chemiluminescent Reagent 2 x 2ml 10 x 2ml
    Crystal Violet Solution 1 x 22ml 5 x 22ml
    Phospho-FKHR (FOXO1) antibody 1 x 9µl 5 x 9µl
    Plate sealer 2 units 10 units
    Reaction Buffer 2 x 4ml 10 x 4ml
    Total-FKHR (FOXO1) antibody 1 x 9µl 5 x 9µl
  • Research areas

  • Function

    Transcription factor which acts as a regulator of cell responses to oxidative stress. In the presence of KIRT1, mediates down-regulation of cyclin D1 and up-regulation of CDKN1B levels which are required for cell transition from proliferative growth to quiescence.
  • Tissue specificity

    Ubiquitous.
  • Involvement in disease

    Defects in FOXO1 are a cause of rhabdomyosarcoma type 2 (RMS2) [MIM:268220]. It is a form of rhabdomyosarcoma, a highly malignant tumor of striated muscle derived from primitive mesenchimal cells and exhibiting differentiation along rhabdomyoblastic lines. Rhabdomyosarcoma is one of the most frequently occurring soft tissue sarcomas and the most common in children. It occurs in four forms: alveolar, pleomorphic, embryonal and botryoidal rhabdomyosarcomas. Note=Chromosomal aberrations involving FOXO1 are found in rhabdomyosarcoma. Translocation (2;13)(q35;q14) with PAX3; translocation t(1;13)(p36;q14) with PAX7. The resulting protein is a transcriptional activator.
  • Sequence similarities

    Contains 1 fork-head DNA-binding domain.
  • Post-translational
    modifications

    Phosphorylated by AKT1; insulin-induced (By similarity). IGF1 rapidly induces phosphorylation of Ser-256, Thr-24, and Ser-319. Phosphorylation of Ser-256 decreases DNA-binding activity and promotes the phosphorylation of Thr-24, and Ser-319, permitting phosphorylation of Ser-322 and Ser-325, probably by CK1, leading to nuclear exclusion and loss of function. Phosphorylation of Ser-329 is independent of IGF1 and leads to reduced function. Phosphorylated upon DNA damage, probably by ATM or ATR.
  • Cellular localization

    Cytoplasm. Nucleus. Shuttles between cytoplasm and nucleus.
  • Information by UniProt
  • Alternative names

    • FKH 1
    • FKH1
    • FKHR
    • Forkhead (Drosophila) homolog 1 (rhabdomyosarcoma)
    • Forkhead box O1
    • Forkhead box protein O1
    • Forkhead box protein O1A
    • Forkhead in rhabdomyosarcoma
    • Forkhead, Drosophila, homolog of, in rhabdomyosarcoma
    • FoxO transcription factor
    • foxo1
    • FOXO1_HUMAN
    • FOXO1A
    • OTTHUMP00000018301
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab207472 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
In-Cell ELISA Use at an assay dependent concentration.

Images

  • Measurement of phosphorylated and total FOXO1. CHO-K1 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 20% FBS for 30 minutes and fixed. Total and phospho FOXO1 were each assayed in triplicate using the phospho and total FOXO1 antibodies included in the kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Protocols

References

ab207472 has not yet been referenced specifically in any publications.

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