Overview

  • Product name

    FOXO1 Transcription Factor Assay Kit (Colorimetric)
    See all FOXO1A kits
  • Detection method

    Colorimetric
  • Sample type

    Nuclear Extracts
  • Assay type

    Semi-quantitative
  • Sensitivity

    < 5000 ng/well
  • Assay time

    3h 30m
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Product overview

    FOXO1 Transcription Factor Assay Kit (Colorimetric) (ab207204) is a high throughput assay to quantify FOXO1 activation in nuclear extracts. This assay combines a quick ELISA format with a sensitive and specific non-radioactive assay for transcription factor activation.


    A specific double stranded DNA sequence containing the FOXO1 consensus binding site (5’ –TTGTTTAC– 3’) has been immobilized onto a 96-well plate. Active FOXO1 present in the nuclear extract specifically binds to the oligonucleotide. FOXO1 is detected by a primary antibody that recognizes an epitope of FOXO1 accessible only when the protein is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides sensitive colorimetric readout that at OD 450 nm. This product detects human, mouse and rat FOXO1.


    Key performance and benefits:



    • Assay time: 3.5 hours (cell extracts preparation not included).

    • Detection limit: < 5 µg nuclear extract/well.

    • Detection range: 5 – 20 µg nuclear extract/well.

  • 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). AFX mRNA is expressed at high levels in heart and skeletal muscle, and moderately in brown and white adipose tissue. FKHR mRNA is detected at its highest levels in brown adipose tissue, white adipose tissue and spleen, with lower levels in liver and skeletal muscle. FKHRL1 mRNA is expressed at its highest levels in brain, heart, kidney and spleen, with moderate expression in white adipose tissue and testis.

    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.

    While Forkhead transcription factors do not bind to a clear consensus sequence, the subclasses have been shown to bind to specific sequence elements. For example, FOXO and HNF subclasses of Forkhead proteins have been shown to bind to the insulin response elements (IREs) of insulin-like growth factor-binding protein-1 (IGFBP-1). FOXO transcription factors bind to a consensus core sequence of 5´-TTGTTTAC-3´, which includes a sequence TRTTTAY (with R a purine base and Y a pyrimidine base) conserved among various Forkhead members.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests 5 x 96 tests
    10X Antibody Binding Buffer 1 x 2.2ml 1 x 22ml
    10X Wash Buffer 1 x 22ml 1 x 110ml
    96-well FKHR assay plate 1 unit 5 units
    Anti-rabbit HRP-conjugated IgG (0.25 μg/μL) 1 x 11µl 1 x 55µl
    Binding Buffer 1 x 10ml 1 x 50ml
    Developing Solution 1 x 11ml 1 x 55ml
    Dithiothreitol (DTT) (1 M) 1 x 100µl 1 x 500µl
    FKHR (FOXO1) antibody 1 x 12µl 1 x 60µl
    Herring sperm DNA (1 μg/μL) 1 x 100µl 1 x 500µl
    Lysis Buffer 1 x 10ml 1 x 50ml
    Mutated oligonucleotide (10 pmol/µL) 1 x 100µl 1 x 500µl
    Plate sealer 1 unit 5 units
    Protease Inhibitor Cocktail 1 x 100µl 1 x 500µl
    Raji nuclear extract (2.5 µg/µL) 1 x 40µl 1 x 200µl
    Stop Solution 1 x 11ml 1 x 55ml
    Wild-type oligonucleotide (10 pmol/µL) 1 x 100µl 1 x 500µl
  • 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

Images

  • Different amounts of nuclear extracts from untreated HeLa (grey) and Raji (black) cells were tested for FOXO1 activity. These results are provided for demonstration purposes only.

Protocols

References

ab207204 has not yet been referenced specifically in any publications.

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