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. Author manuscript; available in PMC: 2012 Nov 15.
Published in final edited form as: Cancer Cell. 2011 Nov 15;20(5):606–619. doi: 10.1016/j.ccr.2011.09.012

Figure 3. H2O2 Treatment Induces the Formation of a Large Complex(es) Containing DNA Methyltransferases, SIRT1, and Polycomb Group Proteins.

Figure 3

(A) Nuclear extracts from untreated NCCIT cells or cells treated with 2 mM H2O2 for 30 minutes were added to a 15 to 60% sucrose gradient and fractions were assayed by immunoblotting. Fraction numbers and 650 kDa molecular mass standard are across the top. Larger fraction numbers indicate smaller molecular weight of the complex(es). (B) Fractions from (A) were pooled into 5 groups as indicated at the bottom of (A). Co-immunoprecipitations for control IgG or anti-DNMT3B (3B) antibodies were performed from the pooled fractions. Right panels are inputs from the pooled fractions. # isoform 2 of EED. (C) HCT116 DNMT1 hypomorph cells expressing FLAG-DNMT1 were untreated or treated with 8 mM H2O2 for 30 minutes. Nuclear extracts, sucrose gradients, and pooling of fractions were performed as in (A). Co-immunoprecipitations for control IgG or anti-FLAG (Fl) antibodies were performed from pooled fractions. Right panels are inputs from the pooled fractions. (D) Flag co-immunoprecipitations were performed in HCT116 DNMT1 hypomorph cells expressing FLAG-DNMT1 that were either untreated (U) or treated with 8 mM H2O2 for 30 minutes (T). After elution with flag peptide (elute) a second immunoprecipitation was done using IgG or EZH2 antibodies. # isoform 2 of EED.