Biochemical characterization of mutant GATA1^R307H. K562 cells were transfected with wild-type and mutated (c.920G>A) GATA1 cDNA constructs and stimulated for 3 days with 0.05 mM bovine hemin. (A) Nuclear extracts of wild-type and GATA1^R307H-transfected cells were incubated with a ³²P-labeled, double-stranded oligonucleotide probe (5′-CAC TTG ATA ACA GAA AGT GAT AAC TCT-3′). EMSA shows reduced DNA binding of GATA1^R307H. (B) Immunoblot analysis of total and phosphorylated GATA1 was performed. Phosphorylation of Ser310 was totally absent in GATA^R307H. (C) Immunoprecipitation of lysates with FOG1 antibody followed by western blot of GATA1. Input control of actin and FOG1 is shown. GATA1^R307H showed reduced FOG1 affinity.