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. 2009 Sep 1;284(44):30187–30199. doi: 10.1074/jbc.M109.026948

FIGURE 6.

FIGURE 6.

GATA-1 affects the binding of ZBP-89 to the G1HE region. A, 100 pmol of GATA-1 or control siRNA was transfected into K562 cells. The expression of GATA-1 protein was determined by immunoblotting. Ten μg of nuclear extracts were resolved by SDS-PAGE. The anti-lamin B antibody was used for loading control. Lane 1, no transfection; lane 2, control siRNA-transfected cells; lane 3, GATA-1 siRNA-transfected cells. Black arrowheads indicate the bands of the expected size. White arrowhead corresponds to GATA-1s, an alternative translation product of GATA-1 (48). B, formation of a nuclear protein complex on G1HE-(201–235) was examined by EMSA. Nuclear extracts (3 μg) were prepared from K562 cells transfected with control (lanes 1 and 3) or GATA-1 (lanes 2 and 4) siRNA and incubated with probe. Antibodies for ZBP-89 were added to nuclear extracts from untransfected K562 cells (lane 5). DNA-protein complexes 1–3 correspond to those shown in Fig. 3C. S, a complex supershifted by anti-ZBP-89 antibody. C, densitometry measurements of the ZBP-89 containing complex (2) shown in B. Upper panel shows the magnified view of B, lanes 1–5. Measurements of number 2 density were performed by using this image and two additional data of EMSA. These samples were prepared from independent siRNA transfections (n = 4). The densities of number 2 in the GATA-1 siRNA-transfected cells relative to those in the control siRNA-transfected cells are shown as averages ± S.E. *, p < 0.05. D, DAPA on the G1HE-(131–230) region. Biotinylated G1HE-(131–230) oligonucleotide (wild type, GATA site mutant, and G5 string mutants) was incubated with nuclear extracts from K562 cells transfected with the ZBP-89-Myc expression plasmid. DNA-protein complexes were recovered with NeutrAvidin-agarose beads, denatured, and resolved by SDS-PAGE. The GATA-1 (upper panel) and ZBP-89-Myc (lower panel) in these complexes was detected by immunoblotting. E, densitometry measurements of GATA-1 (left panel) and ZBP-89-Myc (right panel) proteins shown in D. Data were obtained from four independent experiments. The amount of GATA-1 or ZBP-89-Myc protein bound to the wild type probe was set at 100. *, p < 0.05. Error bars are 1 S.E.