Key features and details
- Assay type: Semi-quantitative
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 3 hr 30 min
- Sample type: Nuclear Extracts
- Sensitivity: 5000 ng/well
Product nameFOXO1 Transcription Factor Assay Kit (Colorimetric)
See all FOXO1A kits
Sample typeNuclear Extracts
Sensitivity< 5000 ng/well
Assay time3h 30m
Species reactivityReacts with: Mouse, Rat, Human
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.
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.
Storage instructionsPlease 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
- Signal Transduction
- Signaling Pathway
- Nuclear Signaling
- Nuclear Hormone Receptors
- Epigenetics and Nuclear Signaling
- Nuclear Signaling Pathways
- Nuclear Receptors
FunctionTranscription 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.
Involvement in diseaseDefects 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 similaritiesContains 1 fork-head DNA-binding domain.
modificationsPhosphorylated 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 localizationCytoplasm. Nucleus. Shuttles between cytoplasm and nucleus.
- Information by UniProt
- FKH 1
ab207204 has been referenced in 2 publications.
- Kapetanou M et al. FoxO1 Is a Novel Regulator of 20S Proteasome Subunits Expression and Activity. Front Cell Dev Biol 9:625715 (2021). PubMed: 33634126
- Majumder S et al. Hydrogen sulphide mitigates homocysteine-induced apoptosis and matrix remodelling in mesangial cells through Akt/FOXO1 signalling cascade. Cell Signal 61:66-77 (2019). PubMed: 31085234