Tetracycline-inducible expression of mouse MtFt in H1299 cells. (A) Human tTA-H1299 lung cancer cells were stably transfected with HA-epitope–tagged wild-type mouse MtFt. Two representative MtFt transfectants (clones A6 and B9) were grown for 48 hours with 2 μg tetracycline/mL (+) or without tetracycline (-) and analyzed for expression of HA-tagged the MtFt by Western blotting. The hybridization membrane was probed with antibodies to HA (bottom) and stripped and reprobed with β-actin (top). The apparent size of the MtFt band was 22 kDa. (B) Time course of MtFt expression in clone B9. The figure shows that the amount of MtFt expression increased with time from 24 to 96 hours. (C) Expression of MtFt mutant without mitochondrial leader sequence. H1299 cells were transiently transfected (48 hours) with either full-length MtFt (WT) or a mutant lacking the leader sequence (Δ56-MtFt). Full-length MtFt transfection yielded an unprocessed 28-kDa band in addition to the mature 22-kDa form. The mutant protein migrated as a single band of almost identical size to the mature WT.