Blocking buffer: 5% NFDM/TBST Primary ab dilution: 1:5000 Primary ab incubation condition: 2 hours at room temperature Secondary ab: Goat Anti-Rabbit IgG H&L (HRP) Lysate: Mouse brain, Rat brain Protein loading quantity: 20 μg Exposure time: 60 s Predicted MW: 80 kDa Observed MW: 80 kDa

The Hela (H) cells were fixed with 4% PFA (10 min at r.t.) and then permeabilized with 90% ice-cold methanol for 20 min at -20℃,the cells then were incubated in 5%BSA to block non-specific protein-protein interactions (30 min at r.t.).Primary Antibody (green):Rabbit Anti-GRK2 antibody (bs-60564R,1:100); Secondary Antibody (white blue): Goat anti-Rabbit IgG-BF488 (bs-60295G-BF488): 1 μg/test. Isotype Control (orange): Rabbit IgG (bs-0295P). Blank control (black): PBS. Acquisition of 20,000 events was performed.