Fig 2.

Ikaros-GATA protein interactions in vitro. (A) Schematic view of Ik1, Ik2, Ik4, Ik6, GATA1, GATA2, and GATA3 protein structures and deletion mutants. (B) Coomassie staining of bacterially purified Ik1-, Ik4-, and Ik6-GST proteins. (C) GST pulldown assay results: [³⁵S]methionine-labeled GATA1 (G1), GATA2 (G2), and GATA3 (G3) proteins were tested for interactions with GST, GST-Ik1, GST-Ik4, GST-Ik6, and GST-PU.1 proteins. Filled circles, nonspecific bands. (D) GST pulldown assay results: [³⁵S]methionine-labeled Ik1 proteins were tested for interactions with GST or GST-GATA1 (GST-G1) proteins. Filled circle, nonspecific band. (E) GST pulldown assay results: [³⁵S]methionine-labeled truncated GATA1, GATA2, and GATA3 proteins, as indicated above each panel, were tested for interactions with GST or GST-Ik1 proteins. Full, full length; NF, N-terminal zinc finger; CF, C-terminal zinc finger; ZnF, C- and N-terminal zinc fingers; N-ter, constructs lacking both zinc fingers. (F) GST pulldown assay results: [³⁵S]methionine-labeled truncated GATA1 fragments were tested for interactions with GST or GST-Ik2 proteins. Full, full length; NF, N-terminal zinc finger; CF, C-terminal zinc finger; ZnF, C- and N-terminal zinc fingers; N-ter, constructs lacking both zinc fingers. (G) In vitro immunoprecipitation assay results: GST-Ik1 and GST-GATA1 proteins were purified from bacteria, and the GST moiety was eliminated by thrombin digestion. Co-IP experiments were carried out with Ikaros (Ik) or GATA1 (G1) antibodies, and co-IP samples were analyzed by Western blotting with Ik or GATA1 antibodies. Filled circle, nonspecific band. (H) GST pulldown assay results: [³⁵S]methionine-labeled wild type or phosphomimetic mutants of GATA1 protein were tested for interaction with GST or GST-Ik1 proteins.