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. Author manuscript; available in PMC: 2014 Dec 12.
Published in final edited form as: Nature. 2014 May 21;510(7504):283–287. doi: 10.1038/nature13320

Extended Data Figure 6. SMYD3 specifically methylates MAP3K2 at lysine 260 in vitro.

Extended Data Figure 6

a, SMYD3 methylates MAP3K2 on protein arrays. Representative image (n=3 independent experiments) showing a SMYD3 methylation assay on a ProtoArray. The close-up shows the two independent MAP3K2 spots on the array being methylated. b, SMYD3 is detected in the cytoplasm and not the nucleus in LKR10 cells. Immunoblot analysis with the indicated antibodies of LKR10 cell lysates biochemically separated into cytoplasmic, nuclear, and chromatin fractions (see Methods). c, SMYD3 catalytic efficiency is two orders of magnitude greater on MAP3K2 than on H4. kcat, KM, and kcat/KM values of SMYD3 activity on recombinant H4 and MAP3K2 as substrates are shown. d, Schematic of the H3K4* mutant form used in (d). Note that the only lysine available to be methylated in H3 is present at K4. e, In vitro methylation assay on full-length recombinant MAP3K2, H3, or H3K4* with recombinant SMYD3 and PRDM9. Top panels: short and long exposure autoradiograms of the methylation assay. No signal was detected for SMYD3 on H3 and H3K4* after long exposures. The asterisk and line indicate breakdown products of MAP3K2 that contain K260 and can be detected in this methylation assay upon long exposure. Bottom panel: Coomassie stain of proteins in the reaction. f, Positive control of activity for enzymes used in Fig. 3f-g on their known respective substrates (MAP3K2, histone H3, nucleosome or RelA as indicated). g, In vitro methylation assays using MAP3K2 K260meo, me1, me2 or me3 peptides as SMYD3 substrates. Dot blot is shown as control of peptide's comparable concentration used for the methylation assay. h, Mass spectrometry analysis of SMYD3 methylation activity on unmodified MAP3K2 K260 peptide. i, Specificity of the indicated MAP3K2-K260me antibodies in dot blot assays using MAP3K2 K260meo, me1, me2 or me3 peptides. j, MAP3K2 is methylated in cells upon SMYD3 overexpression. Immunoblot analysis with the indicated antibodies from 293T cells lysates after Flag immunoprecipitation in cells overexpressing Flag-MAP3K2 and/or HA-SMYD3.