Description
Key facts
Detection method:Colorimetric,
Sample types:Nuclear Extracts,
Reacts with:Mouse, Rat, Human,
Assay type:Semi-quantitative,
Sensitivity:< 5000 ng/well,
Assay time:3h 30m,
Assay Platform:Microplate reader
Product details:
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.
Properties and Storage Information:
Shipped at conditions-Dry Ice, Appropriate short-term storage conditions-Multi, Appropriate long-term storage conditions-Multi, Storage information-Please refer to protocols
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
FOXO1A also known as forkhead box O1 FOXO1 or FOXO1 transcription factor is a protein with a molecular weight of approximately 70 kDa. It is an important transcription factor in the FOXO family responsible for regulating gene expression in response to environmental and physiological stimuli. FOXO1A predominantly localizes in the nucleus but can translocate to the cytoplasm under certain conditions. Expression of FOXO1A occurs in various tissues including liver adipose tissue and pancreatic islets indicating its broad functional roles in different organs.
Biological function summary
FOXO1A acts as a regulator of metabolic pathways apoptosis and cell cycle regulation. Its role in glucose metabolism stands out as a critical function where it influences insulin signaling and glucose homeostasis. FOXO1A can also form complexes with other transcription factors like PPARγ enhancing its regulatory capacity. Interactions within these complexes dictate the transcriptional outcomes influencing cellular responses to stress and growth signals.
Pathways
The FOXO1 transcription factor plays a critical role in the insulin signaling and PI3K-Akt pathway. These pathways are essential for maintaining energy balance and cellular survival. FOXO1A interacts with proteins like Akt which phosphorylates FOXO1A resulting in its cytoplasmic sequestration and subsequent inactivation. This regulation is vital for insulin-mediated glucose uptake and overall metabolic homeostasis preserving cellular functions under fluctuating nutrient conditions.
Aberrant FOXO1A activity is associated with type 2 diabetes and certain cancers. Dysregulation leads to impaired insulin signaling and glucose metabolism contributing to insulin resistance and hyperglycemia in diabetes. In cancer altered FOXO1A expression affects apoptosis and cell proliferation influencing tumor progression and survival. Associations with proteins such as Akt and PPARγ in these conditions further exemplify its network in disease mechanisms highlighting its potential as a therapeutic target.
