Sample: Lane 1: Mouse Spleen tissue lysates Lane 2: Mouse Colon tissue lysates Primary: Anti-phospho-E2F1 (Ser337) (bs-5305R) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 46 kDa Observed band size: 53 kDa

Sample: Lane 1: Skin (Mouse) Lysate at 30 ug Lane 2: Spleen (Mouse) Lysate at 40 ug Lane 3: Large intestine (Mouse) Lysate at 40 ug Primary: Anti-phospho-E2F1 (Ser337) (bs-5305R) at 1/1000 dilution Anti-beta-Actin (bs-0061R) at 1/2000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 55-60 kD Observed band size: 58 kD

Paraformaldehyde-fixed, paraffin embedded (human colon carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (E2F1 (Ser337)) Polyclonal Antibody, Unconjugated (bs-5305R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

HepG2 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (phospho-E2F1 (Ser337)) polyclonal Antibody, Unconjugated (bs-5305R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Blank control(blue): TM4 cells(fixed with 2% paraformaldehyde (10 min) , then permeabilized with 90% ice-cold methanol for 30 min on ice). Primary Antibody:Rabbit Anti-E2F 1 antibody(bs-5305R), Dilution: 1μg 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 . Primary antibody (bs-5305R, 1μg /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.

Blank control:Mosue spleen. Primary Antibody (green line): Rabbit Anti-phospho-E2F1 (Ser337) antibody (bs-5305R) Dilution: 2μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-FITC Dilution: 1μg /test. Protocol The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃. 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.