eIF2A knockdown does not alter RAN translation in transfected cells. (A) Western blot showing efficiency of eIF2A KD in HEK293 cells following transfection with increasing concentrations of eIF2A siRNA. The starred lane indicates siRNA concentration used in subsequent experiments. GAPDH was used as a loading control. (B and C) NLuc expression of indicated reporters expressed from DNA plasmids in HEK293 cells 24 h post transfection with non-targeting or eIF2A siRNAs. NLuc levels are expressed relative to levels in cells transfected with the non-targeting siRNA, n = 12. (D) Schematic of CUG and ACG-NLuc reporter mRNAs, and NLuc expression of indicated reporters expressed from DNA plasmids transfected into HEK293 cells 24 h post transfection with non-targeting or eIF2A siRNAs. NLuc levels are expressed relative to levels in cells transfected with the non-targeting siRNA, n = 9. (E) NLuc expression of indicated reporters expressed from ARCA-capped mRNAs transfected into HEK293 cells 24 h post transfection with non-targeting or eIF2A siRNAs. NLuc levels are expressed relative to levels in cells transfected with the non-targeting siRNA, n = 6. (F) NLuc expression of indicated reporters expressed from A-capped RNAs transfected into HEK293 cells 24 h post transfection with non-targeting or eIF2A siRNAs. NLuc levels are expressed relative to levels in cells transfected with the non-targeting siRNA, n = 6. All graphs represent mean with error bars ± standard deviation. *P < 0.05, **P < 0.01, Two-way ANOVA with Sidak’s multiple comparison test.