β-complex assembly is restricted to cells induced to erythroid differentiation. (A) The β-complex assembles in CD34⁺ cells induced to erythroid, but not to granulocytic/monocytic differentiation. A [³²P]-labeled RNA, corresponding to the polyadenylated human β-globin 3′UTR, was incubated with cytoplasmic extract prepared from human UBC CD34⁺ cells that were induced along either the erythroid (Ery) or the GM lineages. GAPDH and β-globin immunoblots of extracts used in EMSA analyses control for extract integrity and erythroid differentiation, respectively. (B) β-complex assembly is reduced in K562 cells induced to megakaryocytic differentiation. β-complex formation was assessed in cytoplasmic extracts prepared from untreated, DMSO-treated, and PMA-induced K562 cells. The integrity of the extracts was validated by an immunoblot using control β-actin Ab. (C) Steady-state β-globin mRNA levels are reduced in PMA-induced K562 cells. Levels of β-globin mRNA were assessed in K562β cells relative to endogenous control β-actin mRNA using quantitative RT-PCR. The level of β-globin mRNA in untreated (−) cells was set as 100%. Bars depict the mean of 5 independent experiments; error bars indicate 1 SE.