Establishment of FL5.12 cells conditionally expressing TEF, TEF/BX, or E2A-HLF. (A) Immunoblot analysis of FL5.12 cells using HLF(C) antiserum. Representative pools of FL5.12 cells transfected with the empty vector (lanes 1-2), pMT-TEF (lanes 3-4), pMT-TEF/BX (lanes 5-6), or pMT–E2A-HLF (lanes 7-8) were cultured in the presence (even lanes) or absence (odd lanes) of 100 μM ZnSO4 for 16 hours. (B) Time course analysis of TEF expression in pMT-TEF–transfected FL5.12 cells. FL5.12 cells transfected with pMT-TEF were cultured in IL-3–containing medium in the presence of 100 μM Zn for the indicated periods of time and subjected to immunoblot analysis using the HLF(C) antiserum. (C) TEF protein expression in pMT-TEF–transfected FL5.12 cells cultured in the presence of different concentrations of Zn. FL5.12 cells transfected with pMT-TEF were cultured in the presence of Zn at the indicated concentrations for 16 hours and subjected to immunoblot analysis using the HLF(C) antiserum. The expression of tubulin was also analyzed as control. (D) Antibody-perturbed electrophoretic mobility shift analysis (EMSA) with the HLF-CS probe. FL5.12 cells transfected with the empty vector (lanes 1-2), pMT-TEF (lanes 3-4), pMT-TEF/BX (lanes 5-6), or pMT–E2A-HLF (lanes 7-8) were cultured with 100μM of Zn for 16 hours. Nuclear extracts from these cells were incubated with either preimmune (P; odd lanes) or anti-HLF(C) (H; even lanes) antiserum. Brackets show the mobility of DNA-protein complexes containing the indicated proteins and the • indicates unbound, labeled oligonucleotide probes.
