Mapping of domains important in the interaction of RUNX1 with GATA-1. Protein interactions were analyzed using mammalian GST pull-down. Vectors expressing GST alone, GST-GATA-1 full length, or GST-RUNX1 full length were cotransfected with expression vectors for RUNX1 and GATA-1 mutants into 293T cells. (A) Diagram of RUNX1 proteins analyzed, indicating the following domains: runt, negative regulatory domain for DNA binding (NRBD), transcriptional activation domain (TAD), and WRPY sequence, which binds TLE/groucho corepressors. (B) Absence of RUNX1 and GATA-1 binding to GST (left panel). Interaction of RUNX1 mutants with GST–GATA-1; where indicated, CBFβ (Cb) was coexpressed (right panel). (C) Diagram of GATA-1 proteins analyzed, indicating TAD and zinc fingers (left panel). (D) Interaction of GATA-1 mutants with GST-RUNX1; where indicated, CBFβ (Cb) was coexpressed (right panel). The Δ1-85 GATA-1 mutant was expressed with an amino terminal FLAG tag because of loss of epitopes recognized by the N1 and N6 monoclonal antibodies. (E) The Δ1-85 GATA-1 mutant shows loss of cooperation with RUNX1 plus CBFβ. Transactivation of αIIb-598-Luciferase reporter by FLAG-Δ1-85 GATA-1 (F-Δ1-85), RUNX1 (R1), and CBFβ (Cb). K562 cells were transiently cotransfected with the indicated vectors. Results represent the mean ± SEM of 3 independent experiments. Whole cell lysates from duplicate transfections were immunoblotted with the indicated antibodies.