Fig 2.
In vivo measurement of i6A modification of different tRNAs by the PHA6 assay. (A) Ethidium bromide-stained gel loaded with two different concentrations of total RNA. 1× and 2×, 5 μg and 10 μg of total RNA per lane, respectively. (B to G) Hybridization with ACL and body (TψC) probes for three different tRNASer tRNAs. The ACL probe hybridizes to the anticodon stem-loop region of tRNA. The body probe, also called the TψC probe, was used as a loading control probe and covers the entire TψC loop of tRNA. (H to K) Measurement of i6A modification of tRNALeuCAA and tRNALeuUAA in vivo by the PHA6 assay. The same blot was probed, stripped, and reprobed sequentially with the probes indicated to the right. (L and M) In vitro isopentylation assay using purified recombinant human TRIT1, [14C]DMAPP, and synthetic minihelices representing the anticodon stem-loops (ASLs) of the tRNAs indicated above the lanes. (L) Ethidium bromide-stained gel; (M) autoradiograph. Lane M, size markers, with sizes (in nucleotides) indicated to the left.