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. 2017 Sep 5;127(10):3657–3674. doi: 10.1172/JCI93041

Figure 2. DNMT3A truncation mutants do not heterodimerize with WT DNMT3A and fail to form mutant DNMT3A homodimers.

Figure 2

(A) AlphaLISA assay schematic of assay for B–E, in which the interaction between DNMT3A-FLAG and DNMT3A-V5 molecules is measured. Anti-FLAG “donor” beads excited by light at 680 nm release singlet oxygen, which can excite anti-V5 “acceptor” beads within 200 nm, leading to emission light at 620 nm. (B) AlphaLISA measurement of dimerization of WT DNMT3A-V5 with DNMT3A-FLAG (WT, Q515*, E616fs, and L723fs), with crosstitration (4:1, 2:1, 1:1, 1:2, 1:4). (C) Quantification of DNMT3A-FLAG:DNMT3A-V5 homodimerization (WT:WT, Q515*:Q515*, E616fs:E616fs, and L723fs:L723fs) when mixed at 1:1 stoichiometric ratio. (D) Oligomerization of WT DNMT3A-V5 and DNMT3A-FLAG (WT, Q515*, E616fs, or L723fs), mixed at a 1:1 stoichiometric ratio, in response to NaCl. (E) WT DNMT3A-V5 and DNMT3A-FLAG (WT, Q515*, E616fs, or L723fs) oligomerization, mixed at a 1:1 stoichiometric ratio, in response to KCl. All data are shown as mean ± SD of 3 independent experiments, each performed in triplicate. *P < 0.05, 2-way ANOVA relative to WT:WT.