Fig. 4
From: Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly
a HEK293 cells were transfected with either MOCK (empty vector), 4EBP1 WT (wild-type) or negative control 4EBP1 YLM construct. Cells were analysed 48 h post transfection for eIF4ES209 phosphorylation using the AlphaScreen Surefire assay. 4EBP1 constructs were also FLAG tagged. Inset western blot analysis of whole cell lysate derived from equivalent HEK293 cell transfections probed for phosphorylated eIF4E, endogenous 4EBP1, FLAG-tagged constructs and β-actin. b PC-3 cells transfected with the NanoBit eIF4E:eIF4G606–646 system and treated with compound titrations of either the mTORC1/2 active site inhibitors, Torin and PP242, or the MNK1/2 kinase inhibitor CGP57380. c PC-3 cells were treated with titrations of either Torin, PP242 or CGP57380, and eIF4E phosphorylation was determined using the AlphaScreen Surefire eIF4E209 phosphorylation assay. The signal was normalised to GAPDH levels, which were also determined using an AlphaScreen assay. d PANC-1 cells transfected with the NanoBit eIF4E:eIF4G606–646 system and treated with compound titrations of either the mTORC1/2 active site inhibitors, Torin and PP242, or the MNK1/2 kinase inhibitor CGP57380. e PANC-1 cells were treated with titrations of either Torin, PP242 or CGP57380, and eIF4E phosphorylation was determined using the AlphaScreen Surefire eIF4E209 phosphorylation assay. The signal was normalised to GAPDH levels, which were also determined using an AlphaScreen assay. f Table containing IC50 values determined for eIF4E S209 phosphorylation and NanoBit eIF4E:4G disruption. IC50 values were derived from fitting the relevant titration curves with four parameter models using non-linear regression (Prism, GraphPad Ltd.). All values represent mean ± SD (n = 3). The molecular mass of the protein marker is indicated in kilodaltons