FIGURE 8.

Lack of effect on TGF-β and mTOR signaling. A, LT-hVam6p binding has no significant effect on the TGF-β pathway. HT1080 cells were transfected with TGF-β inducible reporter p3TP-Lux and empty vector, wild-type LT, hVam6p binding mutant LT, alone or together with hVam6p. 24 h after transfection, cells were replaced with fresh DMEM with 0.2% FBS, left non-treated or stimulated with recombinant human TGF-β1 at 5 ng/ml for 18 h. Reporter activities were measured using the Dual Luciferase Reporter Assay System (Promega) and normalized to non-treated empty vector-transfected conditions. Independent experiments were performed in triplicate three times. Error bars and p values are shown. B, LT-hVam6p binding has no effect on the mTOR pathway. 293 cells were transfected with empty vector, wild-type LT, hVam6p binding LT mutant, alone or together with hVam6p, left in serum starvation (0.3% FBS) overnight. On the second day cells were starved in amino acid-free medium for 2 h and replenished with amino acid solution for 1 h. Non-stimulated and stimulated lysates were subjected to a Western blot analysis to probe for mTOR downstream of the target 4EBP-1 phosphorylation.