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. 2017 Feb 13;7:42271. doi: 10.1038/srep42271

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Copyright © 2017, The Author(s)

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Total RNA purified from PANC-1 cells was incubated in the absence or presence of silkworm recombinant ALKBH3 and then enzymatically digested to nucleosides by treatment with nuclease P1, phosphodiesterase II, and alkaline phosphatase. The nucleosides were subjected to LC-ESI-MS/MS and the peak areas of methylated nucleosides were normalized for that of cytidine. Relative abundance levels of 1-meA (A), 3-meC (B), N6-meA (C), and 5-meC (D) are shown. ALKBH3 demethylated 3-meC, 1-meA, and N6-meA in the presence of 2-OG and Fe(II) but not in their absence. Heat-denatured ALKBH3 showed no enzymatic activity for the methylated bases. ALKBH3 did not demethylate 5-meC, even in the presence of 2-OG and Fe(II). Data show means ± S.D. (n = 3). **p < 0.01.