Additional file 1: of Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly

Figure S1. (A) eIF4E and SmBIT-eIF4E (indicated with black arrows), eIF4G or eIF4G604–646-LgBiT level were detected in whole cell lysate (Left, WCL) and m7GTP pulldown (right) from transfected cells in Fig. 1d. (B) Anti-FLAG immunoprecipitation of HEK293 cells transfected only with 4EBP1 constructs followed by western blot analysis with anti-eIF4E and anti-FLAG antibody. (C) 4EBP1 mutants were expressed and purified (Inset: Coomassie stain analysis of purified protein) from E. coli and assessed for their ability to bind recombinant eIF4E. Their dissociation constants (Kd) were determined using a competitive fluorescent anisotropy (FP) assay. (D) Lysates from cells transfected with the 4EBP1 constructs and anti-FLAG immunoprecipitated were additionally probed for 4EBP1 phosphorylation status at threonine 37 and serine 46. eIF4E was also visualised as a loading control. 4EGi1 and 4E1RCat compound titrations on HEK293 cell co-transfected with (E) NanoBit eIF4E:eIF4G604–646 PPI system or (F) NanoLuc full length plasmid. (G) Viability of HEK293 cells treated as in (E) and (F) were assessed by measuring intracellular ATP concentrations (CellTiter-GLO, PROMEGA). Luciferase activity was measured as described in “Materials and methods”. The molecular mass of the protein marker is indicated in kDa. All values represent mean ± SD (n = 3). (PNG 408 kb)