Figure 6

A) Schematic representation of the C-terminally biotin-V5-tagged GATA-1. NZF and CZF: N-terminal and C-terminal zinc fingers, respectively. V5 and biotin (BIO) tags are not drawn to scale. B) Western blot detected with anti-GATA-1 N6 antibody showing the relative amounts of biotin-V5-GATA-1 and endogenous GATA-1 in the cells used for ChIP. C) Comparison of V5, M280 and two different anti-GATA-1 antibodies, N6 and M20, tested for the binding of GATA-1 to the EKLF promoter. The enrichment is calculated over a BirA-only transfected control or over IgG negative control, respectively. V5 ChIP gives the highest yield in the EKLF enhancer and promoter elements, streptavidin ChIP with M280 Dynabeads gives comparable yield in the upstream enhancer element as M20 anti-GATA-1 antibody. The M20 antibody can enrich for more GATA-1 bound to the EKLF promoter than the M280 Dynabeads. The N6 antibody precipitates the least amount of GATA-1 bound to EKLF promoter elements. D) Comparison of V5, M280 and two different anti-GATA-1 antibodies N6 and M20, tested for the binding of GATA-1 to the EKLF promoter. Enrichment of the specific binding to EKLF promoter and enhancer was calculated over the negative primer set (-1.35 kb element in EKLF promoter). V5 agarose and M280 Dynabeads bring down comparable amounts of GATA-1 bound to EKLF enhancer and promoter sequences. The M20 antibody enriches the most for GATA-1 bound to the EKLF upstream enhancer, though this also included sequences in vivo bound by endogenous GATA-1 protein which can not be bound by M280 or anti-V5 beads. Rat and goat IgGs as well as BirA control show similarly low enrichments of specific primer sets.