Fig 1: Validation of i-tRAP by using biological samples. (A) Effects of weak alkali treatment on the charging ratios of tRNAGln(CUG) (left) and tRNAGly(GCC) (right). tRNA charging levels were quantified by using i-tRAP. Small RNA was extracted from HEK293T cells and deacylated the charged tRNA by weak alkali treatment with 200 mM Tris-HCl, pH 8.5, at 37°C for 10 min. (B) Northern blot quantification of tRNAGln(CUG) and tRNAGly(GCC) aminoacylation. Aminoacylation of tRNAGln(CUG) and tRNAGly(GCC) were analyzed by acid-urea gel electrophoresis and subsequent northern blot hybridization using probes for tRNAGln(CUG) and tRNAGly(GCC), respectively. tRNA charging levels were quantified as a ratio of charged-to-total tRNA. (C) Experimental scheme for panels D–F. TIG-1 cells cultured in an amino acid–rich medium were transfected with the indicated siRNAs and cultured for an additional 2 d. (D) QARS gene expression, (E) quantification of QARS by western blot, and (F) charging ratio of tRNAGln(CUG). Data, mean ± SEM; n = 3 (B) and 6 (A, D, E, and F); statistics, (A, D, E, and F) unpaired two-tailed Student's t-test. (*) P < 0.05.