Western blot analysis of Phospho-PKA R2 (S99) on different lysates with Rabbit anti-Phospho-PKA R2 (S99) antibody (ET1610-29) at 1/2,000 dilution. Lane 1: PC-12 cell lysate Lane 2: PC-12 treated with 100nM Calyculin A for 30 minutes cell lysate Lysates/proteins at 20 µg/Lane. Predicted band size: 46 kDa Observed band size: 50 kDa Exposure time: 20 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 (ET1610-29) 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.