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. 2018 Sep 26;293(45):17454–17463. doi: 10.1074/jbc.RA118.004028

Figure 2.

Figure 2.

Methylation of PRMT5 at Arg-505 is essential for its methyltransferase activity. A, Western blot analysis of extracts from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using FLAG and PRMT5 antibodies. GAPDH was used as a loading control. Blots are representative of three independent experiments. B, quantitative real-time PCR analysis of PRMT5 mRNA normalized to β-actin in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. C, Western blot analysis of extracted histones from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using H4R3me2s antibody. Histone H4 was used as a loading control. Blots are representative of three independent experiments. D, ChIP analysis of H4R3me2s enrichment at the γ-promoter in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t-tests were used to compare means. *, p < 0.05, **, p < 0.01 compared with the vector control. E, quantitative real-time PCR analysis of γ-globin mRNA normalized to β-actin in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t-tests were used to compare means. **, p < 0.01 compared with the vector control.