(A, B, E, G) Mammalian two-hybrid assays. The luciferase activity of the reporter with 5×Gal4-binding sites was measured. The interaction between various Gal4-fusion fragments of CoCoA (A, B, E, G) and various VP16-fusion fragments of CCAR1 (A, B) or GATA1 (E, G) were tested. Values (mean ± SD) of a representative experiment performed in duplicate are shown. The results were reproducible in three independent experiments.
(C, D, F) GST-pull down assays. HA-CoCoA (C, D) or FLAG-GATA1 (F) was pulled down by various GST-fusion fragments of GATA1 (C, D) or CoCoA (F). HA-CoCoA and FLAG-GATA1 were visualized using an anti-HA or -FLAG antibody, respectively.
(H) Diagram of CoCoA showing the activation domains AD1 and AD2, the coiled-coil domain, and the leucine zippers. Transcription factors that interact with these domains are also shown.