Mechanisms of reciprocal inhibition, GATA-1–PU.1, and GATA-1–c-Myb.

(A) GATA-1 and c-Myb form the complex in vivo. 293T cells were transfected with GATA-1 or PU.1, and total cellular lysates were prepared. Formation of the GATA-1–c-Myb complex was examined by coimmunoprecipitation method. (B-D) The influence of the interaction GATA-1–PU.1 and GATA-1–c-Myb on the respective DNA-binding activities. Nuclear extracts were isolated from 293T cells transfected with various amounts of GATA-1, PU.1, or c-myb expression vectors as indicated. DNA-binding activities of GATA-1 and PU.1 were examined with the probe of Mα and MHC, respectively. In competition assays, nuclear extracts were preincubated with a 200-fold molar excess of unlabeled competitor oligonucleotides before the binding reaction. In a supershift assay, the nuclear proteins were preincubated with 1 μg anti-GATA-1 antibody or anti-PU.1 antibody for 30 minutes at 4°C and subjected to the binding reaction (B, DNA-binding complex; SS, supershifted DNA-binding complex). Expression levels of GATA-1 and PU.1 were also examined by Western blot analysis in each transfectant.