Western blot analysis of DENN on different lysates with Rabbit anti-DENN antibody (HA721022) at 1/1,000 dilution. Lane 1: HeLa cell lysate (negative) (20 µg/Lane) Lane 2: Mouse brain tissue lysate (40 µg/Lane) Predicted band size: 183 kDa Observed band size: 250 kDa Exposure time: 46 seconds; ECL: K1801; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721022) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.