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. 2017 Dec 20;293(6):2125–2136. doi: 10.1074/jbc.M117.811463

Figure 4.

Figure 4.

Pulldown assays show that BCL11A(2–16) peptide is sufficient to interact with PRC2, NuRD, and SIN3A epigenetic complexes in SUM149 cell lysate. A, amino acid sequences of the biotin-labeled BCL11A(2–16) WT and scramble (SCR) peptides used in our pulldown studies. B, Western blot analysis of RBBP4, RBBP7, HDAC1, HDAC2, and other components of PRC2 (EZH2 and SUZ12), NuRD (MTA1), SIN3A (SIN3A), and CoREST (CoREST) complexes following BCL11A(2–16) WT or scramble peptide pulldown. Ten micrograms of SUM149 whole-cell lysate (∼4.4% of the lysate used in the pulldown lanes) were loaded as a control for detection of the proteins of interest. C, HDAC activity assay following BCL11A(2–16) WT or scramble peptide pulldown was done using the HDAC fluorometric activity assay kit (Cayman Chemical). To determine the background fluorescence level, the pulldown samples were treated with 1 μm TSA, an HDAC inhibitor, before the assay. Results shown are mean ± S.D. (****, p < 0.0001, one-way analysis of variance). D, HDAC activity (nmol/min/ml) following BCL11A(2–16) WT or scramble peptide pulldown was calculated as described under “Materials and methods.” Results shown are mean ± S.D. (***, p < 0.001, Student's t test).