Interaction between PU.1 and GATA-1 in vitro.

(A) The indicated fragments of the GATA-1 protein were fused to the C-terminus of GST, expressed in E. coli, and purified on glutathione-sepharose beads. (B) Full-length and 3 N-terminal deletion mutants of PU.1 (the name indicates the first amino acid still present) were translated in vitro in the presence of ³⁵S-labeled methionine and cysteine. The positions of the transactivation domain, PEST domain, and DNA-binding, or ETS, domain are shown above the coding sequence. The translation products are shown in (C), as detected by phosphorimaging after electrophoresis on a 12% SDS-PAGE gel. (D) The³⁵S-labeled PU.1 proteins were subjected to GST pulldown assays using GST alone or fused to the GATA-1 fragments from (A). GST fusion protein, 500 ng, was used in each assay, along with 4 μL of in vitro–translated protein. After electrophoresis of the³⁵S-labeled bound proteins, they were detected by phosphorimaging as in (C). About 10% to 20% of the input protein was retained for all PU.1 derivatives.