IRES activity is inhibited under ribosomal protein deficiency. (A) Schematic representation of the transcript produced by the bicistronic reporter plasmid with cap-dependent βgal and IRES-dependent CAT expression. (B) Hek293T cells were pretreated with 20 and 100nM rapamycin (gray and white bars, respectively) or with DMSO (black bars) for 12 hours. Cells were transfected with a βgal-CAT bicistronic reporter plasmid lacking IRES sequence (ev) or harboring the 5′UTR of LamB1, Bag1, and Csde1 between the βgal and CAT open reading frame. Cells were harvested 48 hours after transfection, and βgal and CAT were measured using ELISA assays. All βgal-CAT ratios were normalized to the ratio in lysate containing the bicistronic construct without IRES, which was set to 1 in each condition. Error bars indicate SEM. (n = 4). (C) Hek293T cells were treated with Scrambled (Sc), Rps19, and Rpl11 siRNA and transfected after 12 hours with the same bicistronic plasmids. IRES activity is represented by the CAT/βgal ratio. Data shown are means ± SEM (n = 4). *P < .05; **P < .01. (D) Western blot showing knockdown of RPS19 and RPL11 by siRNA in Hek293T cells 3 days after transfection.