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. 2018 Dec 12;47(4):2056–2074. doi: 10.1093/nar/gky1241

Figure 4.

Figure 4.

Aminoacylation assays. (A) In vitro aminoacylation of transcribed human mitochondrial mutant and wild type tRNAThr. The unmodified human mitochondrial wild type (G42) and mutant (A42) tRNAThr were incubated with human TARS2 in the presence of [14C] threonine. Samples were withdrawn and stopped after 5, 10, 15 or 20 min, respectively. The relative aminoaylation efficiency was calculated from the initial phase of the reaction. The calculations were based on three independent determinations. Graph shows the results of a representative experiment. (B) In vivo aminoacylation assays. Two micrograms of total mitochondrial RNA purified from various cell lines under acid conditions were electrotrophoresed at 4°C through an acid (pH 5.0) 10% polycrylamide/8M urea gel, electroblotted and hybridized with a DIG-labeled oligonucleotide probe specific for the tRNAThr. The blots were then stripped and rehybridized with probes specific for the tRNAIle, tRNALeu(CUN) and tRNASer(AGY), respectively. (C) The proportion of aminoacylated tRNAThr in the mutant, control cybrid cell lines and HUVECs. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 3.