Phospho-FoxO1 (Ser256) and Total FoxO1 Assay Kits

Phospho-FoxO1 (Ser256) and Total FoxO1 assays to study the regulation of glucose/lipid metabolism in insulin-responsive tissues

These cell-based assays are designed to monitor the expression level and the phosphorylation of FoxO1 on Ser256, which is a hallmark of its inactivation.

FoxO1 (Forkhead box protein O1) is a member of the Forkhead transcription factor FOXO subfamily, which includes three other isoforms (FoxO3, FoxO4 and FoxO6).

FoxO1 is highly expressed in insulin-responsive tissues (liver, adipose tissue, skeletal muscle and pancreas) where it regulates glucose/lipid metabolism and stress resistance. It also functions as a tumor suppressor by inhibiting cell proliferation.

Its transcriptional activity is inhibited by the Insulin/IGF-1 signaling pathway, which leads to its phosphorylation on Ser256 by AKT and its cytoplasmic sequestration. Conversely, the AMPK/JNK/p38 pathways activates FoxO1 (dephosphorylated form) which then translocates to the nucleus.

FoxO1 deregulation plays a critical role in the development of metabolic disorders such as diabetes and NAFLD (Non-Alcoholic Fatty Liver Diseases). Its dysfunction is also linked to various types of cancer (cervical, gastric, breast & hellip;).

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Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total FoxO1 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-FoxO1 antibodies, one labeled with Eu3+cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with FoxO1, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

Assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-FoxO1 can be quantitatively detected using the HTRF phospho-FoxO1 (Ser256) and total FoxO1 cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

For added flexibility, the assays can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred into a 384-well low volume white microplate where the HTRF® reagents are added. This also enables monitoring of the cells'viability and confluence in an appropriate cell culture plate.

Product performances

1. Inactivation of FoxO1 following insulin treatment in liver and skeletal muscle cell line models

Human hepatoma cell line HepG2 and mouse myoblast cell line C2C12 were plated in 96-well plates (25,000 and 100,000 cells/well respectively) in a high-glucose culture medium, and incubated at 37°C - 5% CO2. The following day, the cells were incubated in serum-free low-glucose medium for 24 hours before treatment with increasing concentrations of insulin for 1 hour. After medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the additon of 4 µL 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.

2. Activation of FoxO1 by pro-inflammatory cytokines or oxidative stress in liver HepG2 cell line

Human hepatoma cell line HepG2 was plated in a 96-well plate (100,000 cells/well) in high-glucose culture medium, and incubated for 24 hours at 37°C - 5% CO2. The cells were then treated with a cocktail of TNF-α/IL-1β/IL-6 (30 minutes) or with H2O2 (1h). After medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the addition of 4 µL of the HTRF® phospho-FoxO1 or total FoxO1 detection antibodies. The HTRF signal was recorded after 4 hours of incubation.

Pro-inflammatory cytokines and ROS (reactive oxygen species) are factors driving metabolic deregulation. They both induce FoxO1 dephosphorylation (activation) in HepG2 cell line, while its expression level remains stable. 

3. Activation of FoxO1 using the PI3K inhibitor Wortmannin in pancreatic β-cell line Min-6

Mouse pancreatic β-cell line Min-6 was plated in a 96-well plate (50,000 cells/well) in high-glucose culture medium and cultured for 3 days at 37°C - 5% CO2. The cells were then treated with increasing concentrations of the PI3K inhibitor Wortmannin for 1 hour. After medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the addition of 4 µL of the HTRF® phospho-FoxO1 or total FoxO1 detection antibodies. The HTRF signal was recorded after 4 hours of incubation.

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.

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

Human cancer cervical cell line HeLa was plated in a 96-well plate (100,000 cells/well) in complete culture medium and incubated for 24 hours at 37°C - 5% CO2. The cells were then incubated overnight in serum-free medium before treatment with increasing concentrations of IGF-1 for 10 minutes. After medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the addition of 4 µL of the HTRF® phospho-FoxO1 or total FoxO1 detection antibodies. The HTRF signal was recorded after 4 hours of incubation.

The growth factor IGF-1 induces FoxO1 inactivation by phosphorylation on Ser256, leading to tumor cell proliferation. Its expression level remains stable.

5. HTRF phospho- & total FoxO1 cellular assays compared to Western Blot on human HEK293 cells

Human embryonic kidney cell line HEK293 was seeded in a T175 flask in complete culture medium, and incubated for 2 days at 37°C, 5% CO2 until 90% confluency was reached. The cells were then lysed with 3 mL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking. Soluble supernatants were collected after a 10-minute centrifugation.

Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF® phospho- or total detection antibodies. Equal amounts of lysates were used for a side by side comparison between Western Blot and HTRF.

Using the HTRF® Phospho-FoxO1 kit, just 2,000 cells were sufficient for minimal signal detection whereas 4,000 cells were needed for a Western Blot signal. The HTRF assay is therefore 2-fold more sensitive than the Western Blot technique. Using the HTRF® Total FoxO1 kit, only 1,000 cells were sufficient for minimal signal detection while 8,000 cells were needed for a Western Blot signal, demonstrating that the HTRF assay is 8-fold more sensitive than the Western Blot.

Simplified pathway

FoxO1, a key regulator of glucose/lipid metabolism, stress resistance and cell apoptosis

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. FoxO1 can be activated by several signaling pathways, depending on the type of stimuli. The stress proteins JNK and p38 activate FoxO1 in the presence of pro-inflammatory cytokines, oxidative stress and saturated FFAs (free fatty acids), while the AMPK/FoxO1 pathway is activated during fasting or exercise.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
FoxO1 phospho-S256 kit - 500 tests64FOXPEG
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FoxO1 phospho-S256 kit - 10,000 tests64FOXPEH
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FoxO1 total kit - 500 tests64NFOPEG
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FoxO1 total kit - 10,000 tests64NFOPEH
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Companion products

DescriptionCat. noProduct insertMSDS
FoxO1 phospho-S256 kit control lysate64FOXTDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
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Phospho-total protein blocking reagent - 6 ml64KB1AAD
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Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF
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FoxO1 total kit control lysate64NFOTDA
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