Proteasome Inhibitors Regulate ORC1 Proteins

To evaluate the effects of proteasome inhibitors on the regulation of ORC1 proteins, nuclei were extracted from 7 dps whole seedlings (ORC1b-GFP) or roots (ORC1a-GFP) treated as indicated, using Honda Buffer (0.44 M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 20 mM Hepes HOK pH7.4, 10 mM MgCl₂, 0.5% Triton X-100). 70 μg of nuclear proteins were loaded in a 6% Tris-glycine polyacrylamide gels to run SDS-PAGE and subsequent Western Blot. The proteins were transferred to a membrane, blocked 5% non-fat milk and then incubated with the primary antibody overnight at 4 °C (anti-GFP (Abcam ab5450) diluted 1:2000). After three washes the membrane was incubated with the secondary antibody for 1 h at room temperature (Anti-goat IgG -Peroxidase (Sigma A-5420) diluted 1:10000), washed again three times and proteins were detected using the kit Immobilon WB Chemiluminescent for HRP substrates (Millipore).