Effects of MtFt overexpression on the IRE-IRP system, TfR levels, and ferritin synthesis. (A) MtFt overexpression increases IRP-binding activity. Stably transfected H1299 cells (clone B9) were seeded in 100-mm dishes and grown for 24 hours (lanes 1-2, 7-8), 48 hours (lanes 3-4, 9-10), and 72 hours (lanes 5-6, 11-12) without (-) or with (+) 2 μg/mL tetracycline. Cytoplasmic extracts (10 μg) were assayed for their ability to retard the migration of a ³²P-labeled IRE probe in the absence (lanes 1-6) or presence (lanes 7-12) of 2% β-ME. (B) Stimulation of TfR expression by MtFt overexpression. Clone B9 cells were seeded (25% confluence) in 100-mm dishes and grown for 24, 48, and 72 hours without (-) or with (+) tetracycline. The cell lysates were analyzed for expression of MtFt, TfR, and actin by Western blotting. The filters were probed with antibodies against HA-tag (MtFt, upper panel), stripped, and reprobed with TfR (middle panel) and actin (lower panel), respectively. (C-D) Effect of MtFt overexpression on cytosolic ferritin synthesis. In panel C, cells treated without or with tetracycline for 24 hours (lanes 1-2), 48 hours (lanes 3-4), and 72 hours (lanes 5-6) were metabolically labeled with [³⁵S] methionine for 1 hour and cell extracts were subjected to immunoprecipitation with an antiferritin antibody. The immunoprecipitated proteins were separated by SDS-PAGE on 12.5% gels and autoradiographed. In panel D, the same treatment of the cells was used to measure the steady-state amount of ferritins (lower bands) by Western blotting (top band is MtFt that is recognized by the antiferritin antibody).