Erythropoietin promotes dissociation of GATA1-HDAC5 complexes and acetylation of GATA1. (A) Analysis of GATA1 interaction with HDAC5 by GATA1 immunoprecipitation (IP). Extracts from G1ER cells ± erythropoietin (Epo) stimulation underwent IP with anti-GATA1 (G1) or control (Ctrl) antibodies followed by immunoblotting (IB) for HDAC5, HDAC4, and GATA1. “G1er” refers to the position of the GATA1-ER fusion. *Immunoglobulin heavy chain. See also supplemental Figure 4A. (B) Analysis of HDAC5 interaction with GATA1 by HDAC5 immunoprecipitation (IP). Extracts from G1ER cells expressing human HDAC5 ± Epo treatment underwent IP with anti–human-HDAC5 (H5) or control (Ctrl) antibodies followed by immunoblotting (IB) for GATA1 and HDAC5. “G1er” refers to the position of the GATA1-ER fusion. *Immunoglobulin heavy chain. (C-D) Analysis of GATA1 acetylation. (C) Extracts from G1ER cells ± erythropoietin stimulation underwent immunoprecipitation (IP) with anti–acetyl-lysine (AcK) or control (Ctrl) antibodies followed by immunoblotting. (D) Input levels of GATA1. (E-F) Contribution of PKD3 to erythropoietin–induced GATA1 acetylation. G1ER cells transduced with the sh 186 (PKD3 sh) and sh 187 (Control sh) constructs (see Figure 1B) were analyzed for GATA1 acetylation as in panels B and C. See also supplemental Figure 4.