Gly208Ser substitution affects GATA-1 interaction with finger 9 of FOG.

(A) GST pull-down assay showing retention of wild-type GATA-1 and the Gly208Ser mutant by different FOG fingers. On the left, 2 control lanes demonstrate that equal amounts of wild-type and Gly208Ser GATA-1 protein were used. Finger 7 has previously been shown not to bind GATA-1 and is included as a negative control. (B) Coomassie-stained gel indicating that approximately equal amounts of GST–FOG fusion proteins were used in the GST pull-down assay. (C) Yeast 2-hybrid experiment was used to study the interaction between wild-type or mutant GATA-1 and FOG fingers 1, 6, and 9. All yeast contain pGBKT7.GATA-1 bait vector and a pGAD10.FOG target vector as indicated. In the left panel, yeast are spread onto -Leu,-Trp media as a control for yeast growth. In the right panel, yeast are spread onto -Leu,-Trp,-His media. In this case growth depends on the physical interaction of the bait and target fusion proteins.