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. 2019 Mar;25(3):336–351. doi: 10.1261/rna.068114.118

FIGURE 4.

FIGURE 4.

Human Pus10 is the catalytic protein for tRNA Ψ54 synthesis. (A) tRNA Ψ54 synthase activity (percent Ψ54 in tRNATrp /percent Ψ27 in tRNAPhe by the same extract) (see Supplemental Fig. S2A) by extracts of wild-type (WT), vector control (Control), and Pus10-knockdown (KD1 and KD2) strains of PC3 cells (dark bars—based on 2D-TLC analyses in Supplemental Fig. S2A; light bars—another independent replicate) showing reduction of Ψ54 in knockdown cells. For an unknown reason, vector controls showed more activity than WT. (B) tRNA Ψ54 synthase activity (dark bars—based on analyses in Supplemental Fig. S2C; light bars—another independent replicate) presented as in A using WT, scrambled control (SC), and Pus10-knockdown (KD1) strains of HEK293T. (C) tRNA Ψ54 synthase activity (dark bars—based on analyses in Supplemental Fig. S2E; light bars—another independent replicate) as in A using HEK293T-KD1 cells transiently expressing codon-changed Pus10 and its D344A mutant. Transient transfection by empty vector (EV) was used to produce control cells.