Cooperation of GATA-1, RUNX1, and CBFβ in transcriptional activation of the αIIb promoter. (A) Transactivation of αIIb-598-Luciferase reporter by GATA-1, RUNX1, and CBFβ. K562 cells were transiently cotransfected with the indicated vectors. Results were expressed as fold activation relative to empty expression vector control and were normalized for transfection efficiency with β-gal assays. Results represent the mean ± SEM of 3 independent experiments. To confirm transgene expression, whole cell lysates from duplicate transfections were subjected to immunoblot with the indicated antibodies. (B) Transactivation of αIIb-598-Luciferase reporter by GATA-1, RUNX1, and CBFβ in transiently transfected C3H10T1/2 fibroblasts. Results are expressed as fold activation relative to empty expression vector control and are normalized for transfection efficiency with β-gal assays. Results represent the mean ± SEM of 3 independent experiments. (C) Cis-acting sequences involved in transcriptional activation by GATA-1 plus RUNX1/CBFβ. Shown in the upper panel are αIIb 5′ promoter truncations: αIIb-348-Luciferase and αIIb-98-Luciferase were compared with αIIb-598-Luciferase in cotransfection assays, as above. Positions of RUNX sites, as determined by the TFSEARCH and TESS programs, are indicated by ovals. Shown in the lower panel are αIIB-598 promoter mutants in which individual RUNX sites have been mutated to the non-RUNX–binding sequence TGTTAG²⁹  (hollow ovals). These Luciferase reporters were cotransfected into K562 cells with expression vectors for GATA-1 and RUNX1/CBFβ. Results are expressed as fold activation relative to empty expression vector control, normalized with β-gal assays. Results represent the mean ± SEM of 3 independent experiments.