Skip to main content
Fig. 7 | BMC Biology

Fig. 7

From: Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

Fig. 7

a Basal NanoBit eIF4E:eIF4G606–646 luciferase activity in cells both co-transfected with GFP or GFP-Myc and the NanoBit eIF4E:eIF4G606–646 system. b Western blot analysis of HEK293 cells co-transfected with the NanoBit eIF4E:eIF4G606–646 system and either GFP or GFP-MYC post 48 h transfection. c HEK293 cells were co-transfected with the NanoBit eIF4E:eIF4G606–646 system with either GFP or GFP-MYC. 48 h post-transfection HEK293 cells were titrated with BEZ235 and then assayed for luciferase after 4 h after treatment. d A bicistronic luciferase reporter, which measures the relative amount of cap-dependent translation (Renilla) to cap-independent translation (Firefly), was co-transfected with either empty vector (MOCK), GFP or GFP-Myc into HEK293 cells. Renilla and Firefly luciferase activity was measured 48 h post transfection and plotted as a ratio-metric value. The Renilla luciferase is under the control of a GC-rich 5′UTR whose activity correlated to cap-dependent translation, whilst the Firefly luciferase is regulated by an internal ribosomal site (IRES). e Western blot analysis of the BEZ235 titrations shown in c performed under identical co-transfection and treatment conditions (SE = short exposure, LE = long exposure). f Comparative plot of PANC1 and PC-3 cells co-transfected with the NanoBit eIF4E:eIF4G606–646 system and treated with the dual mTOR/PI3K inhibitor BEZ235. Cells were assayed 4 h post treatment. g After 48 h co-transfection with NanoBit eIF4E:eIF4G606–646 system and siRNA Ctrl or siRNA 4EBP1, PC-3 cells were treated with BEZ235 as for f and then assayed for luciferase activity. The level of 4EBP1 siRNA knockdown was demonstrated by western blot using anti-4EBP1 antibody. Beta-actin was visualised as a loading control. All values represent mean ± SD (n = 3). The molecular mass of the protein marker is indicated in kilodaltons

Back to article page