Figure 2: Dnmt3a is methylated in cells by overexpressing GLP or G9a. | Nature Communications

Figure 2: Dnmt3a is methylated in cells by overexpressing GLP or G9a.

From: MPP8 mediates the interactions between DNA methyltransferase Dnmt3a and H3K9 methyltransferase GLP/G9a

Figure 2

(a) hDNMT3A-WT or K47R mutant was coexpressed with GLP (WT or C1203A mutant) or G9a in GLP knockdown 293T cells for metabolic labelling with [methyl-3H]-Met (ref. 29). After Myc-immunoprecipitation, methylated-hDNMT3A is visualized by autoradiogram. (b) Myc-hDNMT3A was coexpressed with Flag-GLP in 293T cell, purified by Myc-IP and digested after SDS–PAGE for tandem mass spectrometry (LC-MS/MS) analysis. Mono- and di-methylation of K47 were detected. (c) Flag-tagged mDnmt3a was overexpressed together with mouse G9a (lane 2), or mouse GLP (lane 3), or a G9a catalytic dead mutant (lane 4) in 293T cells. Flag-tagged mDnmt3a was immunoprecipitated by anti-Flag antibody and subjected to western blot analysis by anti-mDnmt3aK44me2 antibody. (d) hDNMT3A-WT or K47R mutant was coexpressed with GLP or G9a in GLP knockdown 293T cells for IP-western analysis. Methylated-hDNMT3A is detected by anti-mDnmt3aK44me2 antibody (lanes 3 and 7). (e) Overexpressing GLP methylates endogenous Dnmt3a (indicated by the white arrow). Flag-GLP–WT or C1203A mutant (GLP dead) was expressed in control (lanes 1–3) or DNMT3A knockdown 293T cells (lanes 4–6). Nuclear pellet fraction was examined by western blot using indicated antibodies.

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