Blocking buffer: 5% NFDM/TBST Primary Ab dilution: 1:2000 Primary Ab incubation condition: 2 hours at room temperature Secondary Ab: Goat Anti-Rabbit IgG H&L (HRP) Lysate: 1: HeLa, 2: HepG2, 3: HEK-293, 4: NIH/3T3, 5: PC-12 Protein loading quantity: 20 μg Exposure time: 60 s Predicted MW: 12 kDa Observed MW: 12 kDa

4% Paraformaldehyde-fixed Hela (H) cell; Triton X-100 at r.t. for 20 min; Antibody incubation with (RBX1) monoclonal Antibody, unconjugated (bsm-60704R) 1:100, 90 min at 37°C; followed by conjugated Goat Anti-Rabbit IgG antibody (green, bs-60295G-BF488) at 37°C for 90 min, DAPI (blue, C02-04002) was used to stain the cell nuclei. PBS instead of the primary antibody was used as the blank control.

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-RBX1 antibody (bsm-60704R): 1 μg/10^6 cells; Secondary Antibody (white blue): Goat anti-Rabbit IgG-BF488 (bs-60295G-BF488): 1 μg/test. Blank control (black): PBS. Acquisition of 20,000 events was performed.