Western Blot Analysis of Cellular Proteins

Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100) with protease- and phosphatase inhibitors (Roche), except for the WABS fibroblasts (Fig. 3a, c), which were directly scraped in sample buffer. Proteins were separated by 3–8%, 4–15% or 8–16% SDS-PAGE (NU-PAGE or BioRad) and transferred to immobilon-P membranes (Millipore). Membranes were blocked in 5% dry milk in TBST-T (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.04% Tween-20), incubated with primary and peroxidase-conjugated secondary antibodies (DAKO Glostrup, Denmark; 1:10000) and bands were visualized by chemoluminescence (Amersham). Antibodies used for detection are mouse-anti-DDX11 (B01P, Abnova; 1:250–1:1000), goat-anti-β-actin (I-19, Santa Cruz; 1:1000), mouse-anti-α-tubulin (B-5-1-2, Santa Cruz #sc-23948; 1:2000), mouse-anti-CDC6 (Santa Cruz #sc-9964; 1:500), mouse-anti-p62 (D5L7G, cell signaling; 1:1000), mouse-anti-Flag (M2, Sigma; 1:10000), mouse-anti-p53 (DO-1, Santa Cruz #sc-126; 1:1000), mouse-anti-vinculin (H-10, Santa Cruz #sc-25336; 1:2000), guinea pig anti-ESCO2 (1:500^{83 (link)}). Uncropped western blots are provided in Supplementary Fig. 13.