Flag ub

Ubiquitination reactions were performed in 20 µL ubiquitin assay buffer (50 mM Hepes at pH 7.5, 66 mM NaCl, 2.5 mM MgCl₂, and 2.5 mM DTT) for 20 min at room temperature (RT), unless otherwise indicated for time course reactions (Fig. 3B: 1, 5, 15, 30 min; SI Appendix, Fig. S3A: 1, 3, 5, 15, 30, 60, 90, 120 min). The ubiquitin machinery, including 50 nM E1 (UBA1), 667 nM E2 (UBCH5A), 1.5 µM E3 (UHRF1), and 5 µM ubiquitin (FLAG-ub, BostonBiochem or TAMRA-ub, BioVision), was charged with the addition of ATP (8 mM). Next, 440 nM peptide (H3_(1-32)K9me2) or 220 nM nucleosomes (HeLa mononucleosomes and dNucs) were added. Where indicated, duplex DNA oligonucleotides were added to the reaction (5′-CCATGXGCTGAC-3′ and 5′-GTCAGYGCATGG-3′; UnDNA: X is cytosine and Y is cytosine; HeDNA: X is 5-methylcytosine and Y is cytosine) at the concentrations listed: Fig. 1C, 6.25 µM; Fig. 1D, 1, 6, 20, 80 µM. Reactions were quenched by the addition of SDS loading buffer to a final concentration of 1×. Fresh beta-mercaptoethanol was added to the SDS loading buffer to reduce E1-ub and E2-ub conjugates for all reactions. Reactions were separated by SDS/PAGE and either imaged directly by fluorescence detection of TAMRA-ub (Azure c400) or immunoblotted for FLAG-ub. Gel images and blots shown are representative of at least two independent experiments (separate protein preps and nucleosome reconstitutions).

The His and GST antibodies were purchased from Abmart (Beijing, China). The GAPDH antibody was purchased from Sanjian (Tianjin, China). The FLAG and RGS-His antibodies were purchased from Abmart and Qiagen, respectively. The E1, FLAG-Ub, WT Ub, Lys48-linked Ub2, Ub3, Ub4 and Ub2-7 proteins were purchased from Boston Biochem (Cambridge, MA, USA).