FIGURE 6.

SUMOylation by hDREF promotes release of Mi2α from chromatin. A, HeLa cells were transfected with FLAG-hDREF, HA-Mi2α (WT or K1971R mutant), or Myc-SUMO-1 as indicated. At 24 h after DNA transfection, cells were treated with or without 40 μm digitonin for 6 min at 20 °C, fixed with 2% PFA, and stained with anti-FLAG and anti-HA antibodies followed by Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies. In the case of no treatment with digitonin, cells were permeabilized with 0.3% Triton X-100 after fixation. Signals of FLAG-hDREF and HA-Mi2α were shown in confocal images using Zeiss LSM 510 microscope. Scale bars, 5 μm. The images are representative of cells (n > 50) examined in three independent experiments. B, HeLa cells simultaneously expressing FLAG-hDREF, HA-Mi2α (WT), and Myc-SUMO-1 were treated with 40 μm digitonin for 7 min at 20 °C, fixed with 2% paraformaldehyde, and immunostained with anti-FLAG and anti-PML antibodies (top) or with anti-FLAG and anti-Myc antibodies (bottom) as indicated. Confocal images of signals were obtained using a Zeiss LSM 510 microscope. Scale bars, 5 μm. The images are representative of cells (n > 50) examined in three independent experiments. C, 293FT cells were transfected with FLAG-hDREF or HA-Mi2α (WT or K1971R mutant) with or without CFP-SUMO-1 plasmid as indicated. At 24 h after DNA transfection, soluble and chromatin-bound fractions were prepared by biochemical fractionation as described under “Experimental Procedures.” hDREF and Mi2α in each fraction were detected by immunoblotting analysis (IB) using anti-FLAG and anti-HA antibodies, respectively. Data are representative of three independent experiments. D, signals of Mi2α in the soluble and chromatin-bound fractions were quantified by densitometry scanning of immunoblots. Graphs show the percentage of Mi2α in the soluble fraction and in the chromatin-bound fraction recovered from total cell lysates. Error bars, S.E. *, p < 0.05, n = 3.