Figure 3.

Northern blotting analysis of mitochondrial tRNA. (A) APM gel electrophoresis. Five μg total RNAs of mutant and WT zebrafish were separated by polyacrylamide gel electrophoresis that contains 0.05 mg/ml APM, electroblotted onto a positively charged membrane, and hybridized with DIG-labeled oligonucleotide probes specific for tRNA^Lys, tRNA^Glu and tRNA^Ala, respectively. H₂O₂-treated tRNA was used as a control of complete oxidation of thiolation, eliminating the mobility shift. The retarded bands of 2-thiolated tRNAs and non-retarded bands of tRNA without thiolation are marked by arrows. (B) Northern blot analysis of tRNA under a denaturing condition. Five micrograms of total RNAs of mutant and WT zebrafish were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with the DIG-labeled oligonucleotide probes specific for tRNA^Lys, tRNA^Glu, tRNA^Gln, tRNA^Trp, tRNA^Leu(UUR), tRNA^Met, tRNA^Ala, and 5S rRNA as a loading control, respectively. (C) Five micrograms of total RNAs from mutant and WT zebrafish under acid conditions were electrophoresed at 4°C through an acid (pH 5.2) 10% polyacrylamide with 8 M urea gel, electroblotted, and hybridized with a DIG-labeled oligonucleotide probes specific for the tRNA^Lys, tRNA^Gln, tRNA^Leu(UUR), tRNA^Trp, tRNA^Ala and tRNA^Met, respectively. (D) Quantification of aminoacylated proportions of tRNAs in mutant and WT zebrafish. The calculations were based on three independent experiments. Graph details and symbols are explained in the legend to the Figure 2.