Fig. 6

a HEK293 cells were transfected with the NanoBit eIF4E:eIF4G^606–646 system and treated either with titrations of the AKT inhibitor Ipasertib or the ERK 1/2 inhibitor SCH-772984. Additionally, transfected HEK293 cells were also treated with a titration of Ipasertib with SCH772984 present throughput at constant concentration of 1 μM. Data was normalised to DMSO controls and equivalent cell viability experiments. b Western blot analysis of selected titration points in a probed for TSC2, ERK, eIF4E^S209 and 4EBP1 phosphorylation status (T37/T46). c Western blot analysis of a wider range of titration points in a using anti-phospho Serine 65 4EBP1 antibody. Beta-actin was used as loading control. See “Materials and methods”. d Transfected HEK293 cells were treated with either titrations of Ipasertib, the MNK 1/2 inhibitor CGP57380, or with titrations of Ipasertib with CGP57380 kept at different constant concentrations. Treatments were performed for 4 h and then transfected HEK293 cells were assayed for luciferase activity. All values represent mean ± SD (n = 3). The molecular mass of the protein marker is indicated in kilodaltons