Phosphorylation enhances GATA-1 transactivation activity. (A) GATA-1-deficient G1E cells were transduced with an empty retroviral vector (MIG) alone or vectors containing the indicated inserts, and 48 hours later the differentiation was analyzed by diaminobenzidine staining of hemoglobin in total cells in the absence of cell sorting. Results are expressed as percentages of positive control GATA-1 WT-transduced cells. P values determined by Student t test (GATA-1S310A versus wild type, n = 6; GATA-1 A310S versus Dephos, n = 3). (B) Real-time PCR analysis of gene expression in total cells from panel A. Results shown are normalized to both Gata1 and Hprt mRNA expression. (C) Western blot analysis of GATA-1 protein expression in G1E cells after 48 hours of retroviral transduction. (D) Experiments performed as in panel A, using GATA-1 phosphomimetic mutants to transduce G1E cells cultured in 0.1 U/mL Epo. Real-time PCR analysis, representative Western blot analysis of GATA-1 mutant protein expression in transduced G1E cells. UNT indicates untransduced. In panels A, B, and D, values shown are mean ± SEM.