Sample: Lane 1: Human Jurkat cell lysates Lane 2: Human MOLT4 cell lysates Lane 3: Human HepG2 cell lysates Primary: Anti-CAMKK2 (bs-6253R) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 55 kDa Observed band size: 70 kDa

Paraformaldehyde-fixed, paraffin embedded (Rat brain); Antigen retrieval by microwave in sodium citrate buffer (pH6.0) ; Block endogenous peroxidase by 3% hydrogen peroxide for 30 minutes; Blocking buffer (3% BSA) at RT for 30min; Antibody incubation with (CAMKK2) Polyclonal Antibody, Unconjugated (bs-6253R) at 1:400 overnight at 4°C, followed by conjugation to the secondary antibody (labeled with HRP)and DAB staining.

Molt-4 cells were fixed with 4% PFA for 10min at room temperature,permeabilized with 0.1% PBST for 20 min at room temperature, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with CAMKK2 Antibody(bs-6253R)at 1:100 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).

Blank control (Black line): Molt4 (Black). Primary Antibody (green line):Rabbit Anti-CAMKK2 antibody (bs-6253R) Dilution: 1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647 Dilution: 1μg /test. Protocol The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with PBST for 20 min at room temperature. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.