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Fig. 2 | BMC Biology

Fig. 2

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

Fig. 2

a Titration of the dual MTORC1/2 active site inhibitor PP242 onto HEK293 cells co-transfected with the NanoBit eIF4E:eIF4G604–646 system. b Western blot analysis of endogenous level of eIF4E, eIF4G and 4EBP1 in non-transfected 293FT extracts and associated m7GTP pulldowns of eIF4E containing complexes with differing treatment concentrations of PP242. c Titrations of the mTORC1 allosteric inhibitor Rapamycin and the potent mTORC1/2 active site inhibitor Torin onto HEK293 cells co-transfected with the NanoBit eIF4E:eIF4G604–646 system. Western blot analysis of endogenous level of eIF4E, eIF4G and 4EBP1 in non-transfected 293FT extracts and associated m7GTP pulldowns of eIF4E containing complexes with differing concentrations of d Rapamycin or e Torin. f HEK293 cells were co-transfected with the NanoBit eIF4E:eIF4G604–646 system together with either siRNA Ctrl (siCtrl) or si4EBP1 and PP242 titration was performed again as in a. The inset shows the level of 4EBP1 siRNA-mediated knockdown by western blot using anti-4EBP1 antibody. Beta-actin was visualised as loading control. IC50 values were determined for the titrations described in sections a, c and f using four parameter curve fits. Non-linear regression analysis was performed in GraphPad (Prism). All values represent mean ± SD (n = 3). The molecular mass of the protein marker is indicated in kilodaltons

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