FIG. 1.

ICBP90 interacts specifically with di- and trimethyl K9 H3 in vitro. (A) Pull-down assays were performed with HeLa cell nuclear extracts and unmodified (unmod.) or dimethyl K9 H3 peptides. Bound polypeptides were analyzed by SDS-PAGE and stained with Coomassie. Arrows indicate polypeptides enriched in the H3K9me2 pull-down. A polypeptide of ∼90 kDa enriched in the dimethyl K9 reaction was identified by mass spectrometry as ICBP90. (B) Pull-down assays were performed as described for panel A and analyzed by Western blotting (WB) with anti-ICBP90 antibody. (C, D, E, and F) Western blotting with anti-ICBP90 following pull-down of HeLa cell nuclear extracts with various histone tail peptides, as indicated. (G) Competition assays using pull-down of HeLa cell nuclear extracts with immobilized trimethyl K9 H3 and increasing amounts of free unmodified or trimethyl K9 H3 peptides. Bound polypeptides were analyzed by Western blotting with anti-ICBP90. (H) Pull-down of baculovirus-expressed ICBP90 with various histone tail peptides. Excess BSA was included as a blocking medium.