Validation of FOXO1 knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HeLa cells were stained with FOXO1 antibody (R020990, 1:2,000) and analyzed using CytoFLEX.

Immunocytochemical staining of C2C12 cells with FOXO1 antibody (R020990, 1:1,000). Nuclei were stained blue with DAPI; FOXO1 was stained magenta with Alexa Fluor® 647. Images were taken using Leica stellaris 5. Protein abundance based on laser Intensity and smart gain: Very low. Scale bar: 20 μm.

Western blotting analysis using FOXO1 antibody (R020990). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with FOXO1 antibody (R020990,1:5,000) and HRP-conjugated goat anti rabbit secondary antibody (1:50,000) respectively. Image was developed using ECL Substrate Kit.

Western blotting analysis using FOXO1 antibody (R020990). FOXO1 expression in wild type (WT) and FOXO1 shRNA knockdown (KD) HeLa cells with 30 μg of total cell lysates. GAPDH serves as a loading control. The blot was incubated with FOXO1 antibody (R020990,1:5,000) and HRP-conjugated goat anti rabbit secondary antibody (1:50,000) respectively. Image was developed using ECL Substrate Kit.

Immunocytochemical staining of HeLa cells using FOXO1 antibody (R020990, 1:1,000), Top panel: wild-type (WT); Bottom panal: FOXO1 shRNA knockdown (KD). Nuclei were stained blue with DAPI; FOXO1 was stained magenta with Alexa Fluor® 647. Scale bar, 20 μm. Permeabilization: Triton.