Phosphorylation of cellular proteins by IL-3 restoration. FL5.12 cells transfected with the empty vector, pMT-TEF, pMT-TEF/BX, or pMT–E2A-HLF were cultured in IL-3–containing medium in the presence or absence of Zn for 16 hours, and then the cells were transferred to IL-3–deficient medium in the presence or absence of Zn for 12 hours. Thereafter, cells were restimulated with IL-3 for the indicated periods of time. (A) Immunoblot analysis of FL5.12 cells that had been transfected with the empty vector (lanes 1-4) or pMT-TEF (lanes 5-8) and then cultured in IL-3–containing medium in the presence of Zn using an antiphosphotyrosine monoclonal antibody. (B) Immunoblot analysis for the phosphorylated (p) and nonphosphorylated forms of STAT5, p44/42 MAPK, p70S6K, and Akt in transfectants that had been restored with IL-3 for 5 minutes in the absence (lanes 1-8) or the presence (lanes 9-16) of Zn. The blots were probed with antiphospho-STAT5, antiphospho-p44/42 MAPK, antiphospho-p70 S6K, and antiphospho-Akt as well as anti-STAT5, anti-p44/42 MAPK, anti-p70S6K, and anti-Akt antibodies. (C) Activation of STAT5 DNA-binding by IL-3 restoration. EMSA was performed using nuclear lysates from FL5.12 cells that had been transfected with either the empty vector (lanes 1-6) or pMT-TEF (lanes 7-10) with a β-casein sequence as a probe. In the competition inhibition, an approximately 100-fold molar excess of the unlabeled β-casein sequence oligonucleotide (C; lane 5) or β-casein sequence oligonucleotide with mismatches (M; lane 6) was added in the reaction mixture. A bracket indicates the mobility of specific DNA-protein complexes and • indicates unbound, labeled oligonucleotide probes.