In addition, there are likely to be other mechanisms of resistance to asTORi that are unrelated to the 4EBP:eIF4E ratio. Further investigations should focus on combination approaches that maximize the pro-apoptotic potential of mTOR inhibitors. Full length rat wild type 4EBP1 was obtained from Dr. John Lawrence. This construct was cloned into the pLVX-puro Dox inducible system using Not1 and EcoR1 restriction sites. High titer lentivirus was produced using the lentiviral packing and envelope constructs mentioned above. VAL and OCI-LY7 cell lines expressing the tetracycline transactivator protein were infected with the lentivirus and selected in IMDM culture medium containing Blasticidin and Puromycin. Analysis of the cap-binding complex was performed by adapting a previously described protocol. Following incubation with inhibitors, 4-56106 cells were lysed by three freeze�Cthaw cycles in freeze�Cthaw lysis buffer. In all, protein from the lysates was then incubated with a suspension of 7 methyl-GTPSepharose 4B beads, and placed on a rocker at room temperature. After 1 h, the beads were pelleted and washed twice with lysis buffer. The beads were then boiled in running buffer for 5 min, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and subjected to Western analysis. 1009820-21-6 luciferase reporter construct pRSTF-CVB3 containing the NCR of the Coxsackie B3 virus. cloned between a firefly and renilla luciferase was used to measure cap dependent translation. The construct was electroporated in a FBS free media and cells were allowed to recover in complete IMDM media for 2 hrs followed by inhibitor treatment for 16 hrs. Following treatment, cells were lysed and renilla and firefly luciferase expression was measured using the Dual luciferase assay kit using a luminometer. Cap dependent renilla luciferase expression was normalized to cap independent firefly luciferase expression and results were expressed relative to MCE Chemical R547 untreated control. Since the identification and the characterization of DGAT1 mice, multiple pharmaceutical companies have been actively pursuing the discovery of small molecule DGAT1 inhibitors to reproduce the beneficial metabolic phenotypes of these mice. Recent e
