Western Blot Analysis of DT40 Cell Extracts

Whole-cell extracts were prepared from the DT40 cell lines, and were separated by SDS-PAGE. The gels were transferred onto PVDF membranes (FluoroTrans, PALL). The membranes were blocked with 5% skim milk (BD), and then probed with the following antibodies: rabbit anti-GFP (1:5000; MBL), rabbit anti-chicken CENP-A (1:5000)^{73 (link)}, mouse anti-H4K20 monomethylation (1:5000; #CMA421)^{28 (link),49 (link)}, or Rat anti-pan H3 (1:5000; #140-1G1)^{74 (link)} as the primary antibody. HRP-conjugated anti-rabbit IgG (1:15,000; Jackson ImmunoResearch; 111-035-144) and HRP-conjugated anti-mouse IgG (1:15,000; Jackson ImmunoResearch; 115-035-003) were used as secondary antibodies. Signals were developed using ECL Prime (GE Healthcare), and were detected by a ChemiDoc Touch imaging system (Bio-Rad). The uncropped images of all blots are presented in the Supplementary Figures 12 and 13.