Western blot analysis of Phospho-Raf1 (S259) on different lysates with Rabbit anti-Phospho-Raf1 (S259) antibody (ET1612-87) at 1/2,000 dilution. Lane 1: HEK-293 cell lysate Lane 2: HeLa cell lysate Lane 3: NIH/3T3 cell lysate Lane 4: C6 cell lysate Lane 5: HEK-293 cell lysate, the membrane treated with λpp for 1 hour Lysates/proteins at 20 µg/Lane. Predicted band size: 73 kDa Observed band size: 65 kDa Exposure time: 59 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 (ET1612-87) at 1/2,000 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.