Immunoblotting Protein Isolation and Detection

Proteins were isolated in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100) with protease- and phosphatase inhibitors, separated by 3–8% or 8–16% SDS-PAGE (NU-PAGE), blotted onto polyvinylidene fluoride transfer membranes, incubated with the appropriate primary and secondary antibodies, and bands were visualized by chemoluminescence (Amersham). Antibodies used for detection are mouse anti-DDX11 (Abnova #H00001663-B01P, dilution 1:1,000), mouse anti-vinculin (H-10, Santa Cruz #sc-25336, dilution 1:1,000), rabbit anti-APC2 (kind gift from J. Pines, dilution 1:5,00), mouse anti-α-tubulin (B-5-1-2, Santa Cruz #sc-23948, dilution 1:5,000), mouse anti-APC3 (BD Transduction laboratories, #610454, dilution 1:1,000), goat anti-APC4 (C-18, Santa Cruz #SC-21414, dilution 1:500), rabbit anti-p31^comet (Abcam #ab150363, dilution 1:1,000), guinea pig anti-ESCO2 (ref. 40 (link)) (dilution 1:1,000), mouse anti-Rad21 (Oncogene #NA80, dilution 1:1,000) and rabbit anti-WAPL (Bethyl #A300-268 A, dilution 1:1,000). Uncropped images of western blots are provided in Supplementary Fig. 9.