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. 2014 Apr 29;42(11):6901–6920. doi: 10.1093/nar/gku312

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — Recruitment of H3K4-specific methyltransferase activity by MYC is ASH2L dependent. (A) ASH2L was immunoprecipitated from Jurkat T cell F-buffer lysates with the indicated pre-immune and immune serum (548) and mAbs (4B5 and 4C5) and an isotype-specific control (5F4). The ASH2L-associated MTase activity was measured on core histones with the use of radiolabeled ³H-S-adenosylmethionine. The CB-stained gel shows the input of core histones. (B) ASH2L was immunoprecipitated from lysates of different cells using the ASH2L-specific 548 polyclonal antiserum (i) or the corresponding pre-immune serum (pi) or a combination of ASH2L-specific mAbs 4B5 and 4C5, and 5F4 for control. The ASH2L-associated MTase activity was measured on core histones with the use of radiolabeled ³H-S-adenosylmethionine. (C) The specificity of ASH2L-associated MTase activity from HEK293 was analyzed on recombinant histone H3 by using K4 and K9 methylation-specific antibodies as indicated. (D) ASH2L was immunoprecipitated from Jurkat T cell lysates. The indicated recombinant GST- or MBP-fusion proteins or GST and MBP alone as control were tested as substrates for ASH2L-associated MTase activity using ³H-S-adenosylmethionine. The arrowheads in the CB-stained gel indicate the input of the respective fusion protein. (E) and (F) Control vector, Flag-tagged MYC wt and mutants were expressed transiently in HEK293 cells, immunoprecipitated from F-buffer lysates using a Flag-specific antibody and the MYC-associated MTase activity was measured on recombinant GST-H3 N-terminal tails using ³H-S-adenosylmethionine. (G) HEK293T cells were transiently transfected with Flag-tagged MYC and pSuper constructs expressing an ASH2L-specific shRNA or a control shRNA. After immunoprecipitation of MYC from F-buffer lysates using a Flag-specific antibody, MYC-associated MTase activity was measured on recombinant Histone H3 using an H3K4me3-specific antibody. The expression of relevant proteins are shown for control. (H) HeLa cells were transiently transfected with pSuper constructs expressing either an ASH2L-specific shRNA or a control shRNA. The presence of ASH2L (mAb 4C5) and H3K4me3 (Abcam 8580) was visualized by immunofluorescence. Arrowheads and arrows indicate transfected and untransfected cells, respectively.