Fig. 1

eIF4A2 is not a canonical component of eIF4F, but instead interacts with the Ccr4-Not complex. a Schematic of Flag-tagged proteins expressed. b Immunoprecipitation of Flag-tagged proteins expressed in HEK293 cells transfected with the indicated constructs. IPs were performed 48 h after transfection, and Western blots were probed with eIF4G antibody to show interaction. Western blot shows a representative experiment of 5. c HEK293 cells were transfected with constructs depicted in a and a Renilla luciferase reporter plasmid. Cells were harvested after 24 h, luciferase activity was measured and reporter mRNA was quantified by qPCR. Translational efficiency denotes luciferase activity over RNA abundance, graph represents 3 independent experiments, and significance calculated from unnormalized data using Student’s t test, *p < 0.05, **p < 0.01. Western blot represents Flag-protein expression levels in one of the replicates . d LC-MS/MS analysis of endogenous eIF4A1 and eIF4A2 IPs from HeLa cytoplasmic extract. Table shows quantitation of proteins using emPAI [41] specifically enriched in the eIF4A2 IP. Table shows results from two experiments, one with two technical replicates. e Western blot confirmation of selected LC-MS/MS hits with and without RNaseA digestion. IPs were performed for endogenous proteins. The pan-eIF4A antibody recognizes both eIF4A1 and eIF4A2. f. RNaseA-treated IPs using indicated antibodies from gel filtration fractions of HeLa lysate enriched in CNOT1 and eIF4A2. The interaction between CNOT1 and DDX6 is not as clear because of high background in the IgG IP (right panel). Asterisk denotes non-specific band from IgG