Ruvbl1

Equal amounts of cell lysate were mixed with SDS sample buffer (4×) and heated at 90°C for 5 min. After centrifugation at 3000×g for 30 s, sample (25 μg) was loaded on a 4–12% or 4–20% SDS-PAGE. Proteins were transferred to nitrocellulose membranes and stained with Ponceau S. The nitrocellulose membranes were blocked with 5% milk/TBST for 5 min to remove Ponceau S and for additional 30 min. Primary antibodies were prepared in 3% milk/TBST and incubated for 1 hr at room temperature on a shaker. After removing the primary antibody, membranes were washed three times with 3% milk/TBST for 5 min each. Primary antibodies were used including rabbit polyclonal antibodies against human RUVBL1 and RUVBL2 (Proteintech), anti-β-tubulin and anti-β-actin. Secondary antibodies were incubated for 1 hr at room temperature on a shaker. Membranes were washed three times with TBST with 5 min each time. ECL Plus (GE healthcare) was used to detect signal.