Figure 1. Tetracycline-regulated ectopic expression of H-Ras protein and cellular transformation. (A) Cultures of 10T1/2-TR-H-ras were treated with tetracycline at concentrations of 0 (lane 1), 1 (lane 2), 10 (lane 3), and 100 (lane 4) ng/ml for 48 hrs. (B) Cultures of 10T1/2-TR-H-ras cells were treated with tetracycline at a concentration of 100 ng/ml for 0 (lane 1), 24 (lane 2), 48 (lane 3), and 72 (lane 4) hrs. (C) Cultures of 10T1/2-TR-H-ras cells were treated with 100 ng/ml tetracycline for 48 hrs designated as 0 hour (lane 1), followed by removal of tetracycline from cultures for 24 (lane 2), 48 (lane 3), and 72 (lane 4) hrs. Thirty μg of cellular proteins in lysates were analyzed by Western immunoblotting using specific antibodies to H-Ras. Arrows indicate ectopically expressed H-Ras protein. (D) Morphological changes of 10T1/2-TR-H-ras cells were induced with 100 ng/ml tetracycline for 0 (panel a), 24 (panel b), 48 (panel c), and 72 (panel d) hrs. After treatment of 10T1/2-TR-H-ras cultures with tetracycline for 48 hrs, tetracycline was removed from cultures by washing with culture medium. Morphological changes were detectably reversed in tetracycline-free medium for 24 (panel e), 48 (panel f), and 72 (panel g) hrs.