FIG. 1.

G9a associates with SHP in mouse liver and HepG2 cells. (A and B) SHP and G9a (A), but not SUV39H1 (B), colocalize in the nucleus. Cos-1 cells were cotransfected with expression plasmid DNA for GFP-SHP and either G9a or HA-SUV39H1. SHP was detected by GFP fluorescence (green), and G9a and SUV39H1 were detected by immunofluorescence using rhodamine-conjugated secondary antibody (red) as described in Materials and Methods. Representative confocal microscopic images for G9a or SUV39H1 immunofluorescence, green fluorescent images for GFP-SHP, and merged images are shown. The yellow regions in the merged images indicate colocalization of G9a and SHP. (C) Nuclear extracts (NE) were prepared from mouse liver or HepG2 cells and incubated with GST or GST-SHP. The GST proteins were bound to glutathione-Sepharose, and after a washing step, proteins associated with the GST proteins were detected by SDS-PAGE and Western blotting using G9a antibody. The position of G9a is indicated, and positions of molecular weight markers are indicated at left. The input contains 10% of the amount of extract used in the binding reactions. (D) Nuclear extracts prepared from HepG2 cells were immunoprecipitated using IgG as a control or with antibody (Ab) against SHP or G9a, and association of endogenous G9a or SHP in the immunoprecipitates was detected by SDS-PAGE and Western blotting using G9a or SHP antibodies, respectively. The diffuse bands for G9a compared to those in panel C result from the large amount of protein from the precipitating antibody in the sample, which reduces the resolution of the bands. (E) Schematic diagrams of SHP and full-length (FL) GST-SHP and GST-SHP deletion mutants. RID and RD refer to receptor interacting and intrinsic repression domains, respectively. (F) ³⁵S-G9a was synthesized in vitro, and GST pull-down assays were performed as described in Materials and Methods. The input represents 20% of the ³⁵S-labeled G9a used in the binding reactions. The position of G9a is indicated. (G) A schematic diagram of the GST-G9a deletion mutant (residues 621 to 1000). SET indicates a domain common to Su(var), Enhancer of Zeste, and Trithorax. (H) ³⁵S-SHP was synthesized, and GST pull-down assays were performed as described in panel F. The input represents 20% of the ³⁵S-labeled SHP used in the reactions.