Additional file 3: of Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

Figure S3. (A) Western blot analysis showing endogenous level of eIF4E and 4EBP1 in HEK293, PC-3 and PANC-1 cells. Actin was visualised as a loading control. (B) Western blot analysis of eIF4E phosphorylation at serine 209 in HEK293 cells transfected with NanoBit eIF4E:eIF4G606–646 system and either treated with CGP57380 or DMSO vehicle control. Both short (SE) and long (LE) exposure of the western blot probed with anti phospho-eIF4ES209 are displayed. eIF4E levels were visualised as a loading control. Black arrows indicate SmBit fused eIF4E. (C) PC-3 and (D) PANC-1 cells transfected with the NanoBit eIF4E:eIF4G606–646 system were treated with titrations of CGP57380, Torin and PP242, respectively, for 4 h and assessed for effects on their cell viability as assessed by intracellular ATP levels (CellTiter-GLO, PROMEGA). Luminescence signals were normalised with those obtained from DMSO-treated cells. All values represent mean ± SD (n = 3). The molecular mass of the protein marker is indicated in kDa. (PNG 164 kb)