Fig. 5. Atr2 interacts with histone deacetylase 1 in vivo. (A) The domain structure of Atr2 and the fragments used in transfection experiments are illustrated. N-Atr2 encodes the N-terminal BAH, ELM2 and SANT domains. C-Atr2 encodes the rest of the protein, from the GATA domain through the C terminus. (B) Each indicated protein was flag-tagged at the N terminus, overexpressed in 293 cells, then immunoprecipitated using anti-flag beads. Western blots show immunoprecipitated proteins [IP] run next to total soluble extract [E]. Hdac1 is associated with full-length Atr2, N-Atr2 and Mta2, but not with C-Atr2. The NuRD core subunits RbAp48 and RbAp46 are only found in association with Mta2. Extract lanes show that the total protein concentrations in each extract were similar. (C) Extracts were made from wild-type E9.5 embryos, and endogenous Atr2 was immunoprecipitated using the 286-1 antibody linked to protein G sepharose. 286-1 beads immunoprecipitated the full-length Atr2 as well as Atr2S. Endogenous Atr2 associates with Atr1 and Hdac1 in vivo, but not with RbAp46. Protein G beads did not pull down any of these proteins. (D) Endogenous Atr2 was immunoprecipitated from wild-type and om MEFS with 286-1 beads. Full-length Atr2 is present in wild-type cells, but is missing in om cells; Atr2S was present in both. Atr1 was pulled down with Atr2 in both cell types.