Sample: Brain (Mouse) Lysate at 40 ug Primary: Anti-RNF123 (bs-9249R) at 1/300 dilution Secondary: HRP conjugated Goat-Anti-rabbit IgG (bs-0295G-HRP) at 1/5000 dilution Predicted band size: 149 kD Observed band size: 149 kD

Tissue/cell: mouse embryo tissue; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-RNF123 Polyclonal Antibody, Unconjugated(bs-9249R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: mouse brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-RNF123 Polyclonal Antibody, Unconjugated(bs-9249R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Blank control（black line）:HepG2. Primary Antibody (green line): Rabbit Anti-RNF123 antibody (bs-9249R) Dilution:1ug/Test; Secondary Antibody（white blue line）: Goat anti-rabbit IgG-AF488 Dilution: 0.5ug/Test. Isotype control（orange line）: Normal Rabbit IgG 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.