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

FIGURE 5.

FIGURE 5.

ZBP-89 physically interacts with GATA-1 zinc finger regions. A, binding of GATA-1 (left panel) and ZBP-89 (right panel) to the G1HE chromatin region was examined by ChIP assays. Cross-linked chromatin from MEL cells was immunoprecipitated using anti-GATA-1 or anti-ZBP-89 antibody and was quantified by real time PCR (black bars). Rat IgG and rabbit IgG were used as controls for anti-GATA-1 and anti-ZBP-89 antibodies, respectively (gray bars). A primer set that amplifies the Gata1 sixth exon coding region was used as a negative control. Values from PCR amplicon using immunoprecipitated chromatin relative to those of input are shown. Results are shown as averages ± S.E. of the data obtained from 6 independent assays. *, p < 0.01; **, p < 0.05 compared with control IgG. B, binding of GATA-1 and ZBP-89 to the chromatin region of G1HE was examined by ChIP-reChIP assay. Cross-linked chromatin from MEL cells was immunoprecipitated with anti-ZBP-89 antibody and the second IP was performed using anti-GATA-1 antibody. Rat IgG was used as a control for anti-GATA-1 antibody (gray bars). A primer set that amplifies the Gata1 sixth exon was used as a negative control. Values from PCR amplicon using immunoprecipitated chromatin relative to those of input are shown. Results are shown as averages ± S.E. of the data obtained from three independent assays. C and D, interaction of ZBP-89 and GATA-1 tested by a GST pull-down assay. C, full-length ZBP-89 GST fusion protein was incubated with GATA-1 proteins fused to MBP. The full-length (lanes 1 and 2), the zinc finger region (amino acids 200–322; lanes 3 and 4), and the C-terminal region (amino acids 323–424; lanes 5 and 6) of the GATA-1 proteins were examined in the assay. As input, 10% of the MBP-GATA-1 proteins tested in the interaction assay were loaded onto the gel (lanes 7–9). D, mapping of the GATA-1-interacting domain of ZBP-89. The zinc finger region of GATA-1 fused to MBP (upper panel) or MBP alone (lower panel) was tested for interaction with various GST-ZBP-89 proteins. Input of MBP protein (lane 1), GST alone (lane 2), the full-length (lane 3), and 3 deletion mutants (lanes 4–6) of ZBP-89 were examined in the assay. White arrowhead corresponds to MBP-GATA-1 (amino acids 200–322). Asterisks indicate the undefined proteins that appeared only in the presence of GST-ZBP-89 (amino acids 282–552).