Western blotting analysis using anti-Phospho-FOXO1 (S256) antibody (Cat#U0739). Total lysates (30 μg) were loaded and separated by SDS-PAGE. The blot was incubated with anti-Phospho-FOXO1 (S256) antibody (Cat#U0739, 1:2,500) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using FeQ™ ECL Substrate Kit (Cat#226).

Immunocytochemical analysis of FOXO1 (Phospho-S256) staining in MCF7 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with the primary antibody in 3% BSA-PBS and incubated overnight at 4 °C in a hidified chamber. Cells were washed with PBST and incubated with a DyLight 594-conjugated secondary antibody (red) in PBS at room temperature in the dark.

Immunohistochemical analysis of FOXO1 (Phospho-S256) staining in human lung cancer formalin fixed paraffin embedded tissue section. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0). The section was then incubated with the antibody at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.