The phospho-FoxO1 kit is designed to monitor the inactive form of FoxO1 modulation, phosphorylated on Ser256.
See more
  • Ease-of-use Ease-of-use
  • Highly specific Highly specific
The phospho-FoxO1 kit is designed to monitor the inactive form of FoxO1 modulation, phosphorylated on Ser256.
-
In stock

Overview

The phospho-FoxO1 (Ser256) assay is designed for the robust quantification of FoxO1 modulation, phosphorylated on Ser256, as an indicator of its inactivation. FoxO1 is a member of the Forkhead transcription factor FOXO subfamily, and is highly expressed in insulin-responsive tissues where it regulates glucose/lipid metabolism and stress resistance. It also functions as a tumor suppressor by inhibiting cell proliferation. FoxO1 deregulation plays a critical role in the development of metabolic disorders such as diabetes and NAFLD. Its dysfunction is also linked to various types of cancer.

Benefits

  • COMPATIBLE WITH MANY CELL TYPES
  • SPECIFICITY

Phospho-FoxO1 (Ser251) assay principle

The Phospho-FoxO1 (Ser251) assay measures FoxO1 when phosphorylated at Ser251. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-FoxO1 (Ser251) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
Phospho-FoxO1 (Ser251) assay principle

Phospho-FoxO1 (Ser251)  2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Phospho-FoxO1 (Ser251) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-FoxO1 (Ser251) 2-plate assay protocol

Phospho-FoxO1 (Ser251) 1-plate assay protocol

Detection of Phosphorylated FoxO1 (Ser251) with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Phospho-FoxO1 (Ser251)) 1-plate assay protocol

FoxO1 Inactivation following insulin treatment

Huma HepG2 and mouse C2C12 cells were plated and cultured in a high-glucose medium prior to treatment with increasing concentrations of insulin for 1 hour. After lysis, cells were transferred twice over into a low volume white microplate finally before before the additon of the HTRF phospho-FoxO1 or total FoxO1 detection antibodies. The HTRF signal was recorded after 4 hours of incubation. In both cell lines, insulin treatment induces FoxO1 phosphorylation on Ser256 (inactivation), while its expression level remains stable.
FoxO1 Inactivation following insulin treatment

FoxO1 activation in HepG2 cells with cytokines and oxidative stress

Human HepG2 cells were plated and cultured in high-glucose culture medium before being treated with a cocktail of TNF-a/IL-1ß/IL-6 or H2O2. Following lysis, soluble cell lysates were transferred twice over into a low volume white microplate before finally adding HTRF phospho-FoxO1 or total FoxO1 detection antibodies. Results indicate pro-inflammatory cytokines and ROS (reactive oxygen species) are factors driving metabolic deregulation by inducing FoxO1 dephosphorylation (activation) in HepG2 cell line, while its expression level remains stable.
FoxO1 activation in HepG2 cells with pro-inflammatory cytokines
FoxO1 activation in HepG2 cells with oxiditative stress

FoxO1 Activaiton using PI3K inhibitor Wortmannin in pancreatic ß-cells

Mouse pancreatic ß-cell line Min-6 was plated and cultured in high-glucose culture medium for 3 days prior to treatment with increasing concentrations Wortmannin for 1 hour. Following lysis, cell lysates were transferred twice over into a low volume white microplate before finally adding HTRF phospho-FoxO1 or total FoxO1 detection antibodies. Results showed Wortmannin induces PI3K/AKT pathway inhibition, leading to FoxO1 dephosphorylation (activation) in Min-6 cell line. The FoxO1 expression level remains stable, demonstrating that there is no cytotoxic effect of the compound on the cells.
FoxO1 Activaiton using PI3K inhibitor Wortmannin in pancreatic ß-cells

Inactivation of FoxO1 using IGF-1 in cancer cervical cell line HeLa

Human HeLa cells were plated and cultured in complete medium and incubated for 24. They were then incubated overnight in serum-free medium before treatment with increasing concentrations of IGF-1. Folowing lysis, cell lysates were transferred twice over into a low volume white microplate before the addition of phospho or total FoxO1 antibodies. Results show that the growth factor IGF-1 induces FoxO1 inactivation by phosphorylation on Ser256, leading to tumor cell proliferation and that it's expression level remains stable.
Inactivation of FoxO1 using IGF-1 in cancer cervical cell line HeLa

HTRF phospho- & total FoxO1 assays vs WB on human HEK293 cells

HEK293 cells were seeded and cultured in complete medium until 90% confluency was reached. Following lysis, soluble supernatants were collected via centrifugation. Serial dilutions of the cell lysate were performed and transferred into a low volume white microplate before the addition of HTRF phospho- or total detection antibodies. Equal amounts of lysates were used for a side by side comparison between WB and HTRF. The HTRF assay is shown to be 2-fold more sensitive than the Western Blot technique. Using the HTRF Total FoxO1 kit the HTRF assay is 8-fold more sensitive than the WB.
HTRF phospho-FoxO1 assay vs WB on human HEK293 cells
HTRF Total FoxO1 assay vs WB on human HEK293 cells

FoxO1 Simplified Pathway

The Insulin/IGF-1 signaling pathway activates the AKT kinase which in turn phosphorylates FoxO1 on Ser256, leading to its cytoplasmic sequestration and the inhibition of its transcriptional activity. In these conditions, hepatic glucose production is inhibited, lipogenesis increases, and cells proliferate. Conversely, the dephosphorylated form of FoxO1 can translocate to the nucleus and induce the transcription of genes involved in glucose production, lipolysis, inhibition of lipogenesis, stress resistance, apoptosis, autophagy and inflammation. The stress proteins JNK and p38 activate FoxO1 in the presence of pro-inflammatory cytokines as well as oxidative stress, fasting and exercise.
FoxO1 signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

FOXO1: a role in non-alcoholic fatty liver disease NASH

FOXO1 use in a fatty liver disease model - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Webinar: NASH - Fibrosis Research and Drug Discovery

Featuring a panel of experts - Videos

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

tab6