GATA1/FOG1 and GATA1/DNA interaction studies.

(A) Different in vitro transcription/translated FOG1 fingers (finger 1, finger 9, fingers 5 to 7, and fingers 5 to 9) were incubated with various GST fusion proteins (GATA1 Nf-D218G, GATA1 Nf, and GATA1 Nf-V205M) or with GST immobilized on glutathione agarose beads. After washing the beads, bound proteins were eluted and resolved by SDS-PAGE. The autoradiogram in the top panel shows the amount of retained S³⁵-labeled FOG1. The lower panel shows the Coomassie blue staining of the gel confirming equal GST fusion protein sample loading. (B) DNA binding competition assay (representative for 3 experiments). The amount of biotinylated DNA (100 μM) bound by GST/GATA1 fusion proteins was plotted against the concentration of nonlabeled competitor DNA. Control GST-bound beads (▴) gave no DNA binding. ♦, WT-NfGATA1; ▪, D218G-NfGATA1; ●, V205M-NfGATA1.