Figure 4.
The 5′ UTRS confers rapamycin resistance. (A) Monocistronic FLuc constructs were constructed in the plasmid pEBS-PL with the 5′ UTRs indicated. These constructs were transfected in triplicate into 293T cells along with a reporter plasmid expressing LacZ that served as an internal control. At 4 h post-transfection, triplicate transfections were either treated or not treated with Rap. (B) The effect of Rap on downstream mTOR signalling was confirmed by immunoblots on cell extracts using 4E-BPI and phospho-4E-BPI antibodies. The blot for actin served as a loading control. (C) Cytoplasmic extracts were prepared at 24 h post-transfection. FLuc activity was measured and normalized to the internal LacZ control. The values obtained in the Rap-treated cells were then subsequently normalized to the values obtained in the non-treated control transfections. The graph depicts these FLuc activities (a value of ⩾1 indicates Rap insensitivity). The SEMs for each construct are also indicated.