Western blot analysis of FOXO1 was performed by loading 25 µg of Hela (lane 1), 293T (lane 2) and rat heart (lane 3) onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a FOXO1 polyclonal antibody (Product # PA1-804) at a dilution of 1:500 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Images were taken at an exposure time of 2 min (lanes 1 and 2) and 1 min (lane 3). Results show a band at ~66 kDa.