Hi,

Did you run the assay on SDS-PAGE?

I have no experience with FoxO1, but it often happens if the sample preparation was not sufficiently denatured.

Note that some types of primary antibodies often show nonspecific binding, and one way to confirm this is to change the antibody.

In addition, is it insulin receptor, not IRS-1?

I think immunoprecipitation will tell you whether the binding is to the insulin receptor or not. In that case, try to conduct it with Naive protein extracts without SDS contained buffer.

https://academic.oup.com/endo/article/154/8/2600/2423638

Good luck.

FOXO1 can act both as coactivator or corepressor. When it comes to insulin signalling, insulin suppresses the FOXO1 activity through PI3K/AKT-pathway (also known as the mTOR pathway). The mediator protein Akt phosphorylates FOXO1 at multiple sites.

The band detected around 170 - 190 kDa is probably insulin receptor substrate 2 (IRS2) that mediates the effect of some cytokines, including insulin and insulin-like growth factor (IGF).

Antibodies are a class of immunoglobulins that specifically bind to corresponding antigens. Under ideal conditions, antibodies can specifically recognize the protein of interest. However, for proteins with high homology, which may have similar antibody recognition epitopes, it is very likely that the antibodies will cross-react with different immunogenic proteins. Therefore, the phospho-Foxo1 antibody has the potential to recognize other proteins with high homology to its antigen.