Fig 1: eIF2β and eIF3G knockdown in human cells suppresses both general and RAN translation. (A) Schematic of AUG initiated [AUG-nLUC-3xFLAG] and + 1CGG RAN translation [+1CGG (100)-nLuc-3xFLAG] initiated reporters along with AUG-Firefly (FF) transfection control reporter. (B) Representative western blot shows confirmation of EIF2S2 (eIF2β) knockdown. GAPDH serves as a loading control. # Denotes the siRNA concentration used in the following experiments. (C) Relative expression of AUG-nLUC and + 1 CGG (100)-nLUC reporters along with co-transfected AUG-FF reporters following knockdown of EIF2S2. (D) Western blot confirmation of EIF3G knockdown. GAPDH is used as a loading control. (E) Relative expression of AUG-nLUC and + 1 (CGG)100-nLUC reporters along with co-transfected AUG-FF reporters following knockdown of EIF3G. (F) Western blot confirmation of EIF3F knockdown. GAPDH is used as a loading control. (G) Relative expression of AUG-nLUC and + 1 (CGG)100-nLUC reporters along with co-transfected AUG-FF reporters following siRNA-mediated knockdown of EIF3F. Statistical analysis: (C, E and G) Two-way ANOVA with Sidak’s multiple comparison test, (n = 9–12/condition). **** P < 0.0001.