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. 2011 Aug 18;7(8):e1002184. doi: 10.1371/journal.ppat.1002184

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Sakane et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Synthetic K51-mono-methylated Tat proteins were incubated with increasing amounts of recombinant LSD1/KDM1 (0, 0.5, 1, 2 µg) for 1 h at 37°C. Reaction products were analyzed by western blotting using α-Tat, α-Me1K51 Tat antibodies. (B) Purified histone proteins were subjected to the same procedure as in A and analyzed by α-Me2H3K4 or α-histone H3 antibodies. (C) Synthetic K51-mono-methylated Tat or K50-acetylated/K51-monomethylated Tat proteins were incubated with 1 µg of recombinant LSD1 as described in A. (D) Increase in K51-monomethylation of Tat in LSD1 shRNA-infected J-Lat A2 cells. Whole cell lysates isolated from J-Lat A2 cells infected with shRNAs directed against LSD1 or control shRNAs and stimulated with TNFα were analyzed by western blotting with indicated antibodies. Band intensities were quantified using ImageJ Software (NIH). R.I.: Relative intensities of bands as compared to the control-vector transduced cells (100%).