Inactive DNMT3B Splice Variants Modulate De Novo DNA Methylation (original) (raw)
Figure 4
DNMT3B3 weakens, while DNMT3B4 strongly inhibits, DNA binding by DNMT3B2.
(A) Binding to a 420 base pair DNA fragment was measured by EMSAs for increasing concentrations of C-terminal DNMT3B2, DNMT3B3 and DNMT3B4 proteins. Bound and unbound DNA species are indicated. (B) DNA binding was also measured for C-terminal DNMT3B2:DNMT3B2, DNMT3B2:DNMT3B3, and DNMT3B2:DNMT3B4 co-complexes at increasing protein concentrations. (C and D) Graphical representation of EMSAs from A and B, respectively, performed at least in triplicate. The percent of protein bound to DNA was calculated by band quantification using ImageQuant. Points: mean, bars: standard error. Results were fit using nonlinear regression, saturation-binding (one-site-specific binding with Hill slope). Kd values: 3B2ct = 0.22 µM, 3B3ct = 0.16 µM, 3B4ct = 2.42 µM, 3B2ct+3B2ct = 0.4 µM, 3B2ct+3B3ct = 1.5 µM, 3B2ct+3B4ct = N/A.