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Sample: Lymph node (Mouse) Lysate at 40 ug Primary: Anti-IL2RA (bs-0577R) at 1/300 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/10000 dilution Predicted band size: 28 kD Observed band size: 55 kD

Blank control (Black line): Jurkat (Black). Primary Antibody (green line): Rabbit Anti-IL2RA/CD25 antibody (bs-3152R) Dilution: 3μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE Dilution: 1μg /test. Protocol The cells were fixed with 4% PFA (10min at room temperature)and then were incubated in 5%BSA to block non-specific protein-protein interactions for 15 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.

Blank control: U937 (blue). Primary Antibody:Rabbit Anti- CD25 antibody(bs-0577R), Dilution: 0.2μg in 100 μL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit IgG(orange) ,used under the same conditions ); Secondary Antibody: Goat anti-rabbit IgG-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA. Protocol The cells were fixed with 2% paraformaldehyde (10 min) . Primary antibody (bs-0577R, 0.2μg /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/PE antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.