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 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 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.

 

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.

 

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.