Interaction of GATA-1 with cyclin T1. (A) 293T cells were transfected with constructs encoding either FLAG-RUNX1 (Flag-R1) plus or minus untagged GATA-1 (G1) or FLAG-GATA-1 (Flag-G1) plus or minus untagged RUNX1 (R1). FLAG-immunoprecipitation (IP) was followed by immunoblotting (IB) of immunoprecipitates and inputs with the indicated antibodies. (B) Specificity of interaction. 293T cells transfected with expression vectors for FLAG tagged GATA-1 (G1), Ets1, or C/EBPα (C/α) were subjected to immunoprecipitation and immunoblotting as in panel A. (C) Inducible interaction of endogenous GATA-1 and cycin T1 proteins. Extracts from K562 cells either untreated or treated 48 hours with 25 nM of TPA underwent immunoprecipitation with equal amounts of control rat IgG or N6 monoclonal rat anti-GATA-1 antibody. Immunoprecipitates and inputs underwent immunoblotting with the indicated antibodies. (D) Requirement of P-TEFb kinase activity for inducible recruitment of GATA-1. Coimmunoprecipitations were conducted as in panel C on cells treated 48 hours with 25 nM of TPA plus or minus 100 nM of flavopiridol (FP). (E) Global remodeling of P-TEFb during megakaryocytic induction. Coimmunoprecipitations were conducted as in panel C on cells treated 48 hours with 25 nM of TPA. Immunoprecipitating antibodies consisted of control rabbit IgG or rabbit anticyclin T1 (T1). Immunoblotting was performed for GATA-1 (G1), HEXIM1 (H1), Cdk9, and cyclin T1.