FOXO1 Transcription Factor Assay Kit (Colorimetric)

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.