Fig. 4
Analysis of the tRNA-binding activity of Ar-Larp. a Electrophoretic mobility shift assay (EMSA) analysis of the affinity between Ar-Larp and tRNAs. All tRNAs (25 μg per lane) were labeled with digoxigenin (DIG) using the DIG Oligonucleotide 3'-End Labeling Kit. Lanes 2–5 contain increasing amounts (0–1 μg) of GST-fused Ar-Larp. Ar-Larp-tRNA complexes were detected by Northern blotting (1 μg of GST was used as a control in lane 1). In lanes 7–8, excess tRNAs (250 and 500 μg, respectively) were added to the reaction system, which prohibited the binding of Ar-Larp to labeled tRNAs. In lanes 9–10, an anti-Ar-Larp antibody (0.5 and 2 μg, respectively) was added to the reaction system, and the super-shifted band of the Ar-Larp-anti-tRNA antibody complex was detected. b Competition EMSA analysis of the affinity between Ar-Larp and tRNAs and other RNAs. All tRNAs (25 μg per lane) were labeled with DIG using the DIG Oligonucleotide 3'-End Labeling Kit. Lanes 1–4 contain increasing amounts (0–1 μg) of GST-fused Ar-Larp. Ar-Larp-tRNA complexes were detected by Northern blotting. In lanes 5–8, excess RNAs (tRNA, 5.8 s rRNA, 5 s rRNA, and mRNAs, 500 μg each) were added to the reaction system as the competitors to prohibit the binding of Ar-Larp to labeled tRNAs. c EMSA analysis of the affinity between Ar-Larp and pre-tRNA and mature tRNA. Unlabeled tRNAs (25 μg per lane) were incubated with GST-Ar-Larp (0–1 μg). Ar-Larp-tRNA complexes were detected using probes specific to pre-tRNA (tRNALeu, c-left panel) and mature tRNA (tRNALeu, c-right panel) (1 μg of GST was used as a control in lane 1). d Detection of Ar-Larp-tRNA complexes by co-IP using an anti-Ar-Larp antibody and probes specific to tRNAs. Northern blot analysis of tRNAs from the pellets (co-immunoprecipitation) or total extract fractions (Input) of Artemia cells at each developmental stage (indicated in Fig. 1a, isotype antibody was used as a control)