Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination. Valencia-Sanchez et al.
Description
Original gels and membranes images shown in Figures 3, 4 and 7, which were used in EMSA gels images and Western Blots images, respectively. The images were used to calculate app Kd constants of Dot1L to Nucleosomes, and to monitor H3K79 histone methylation by Dot1L.
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Nucleosome binding assay Increasing amounts of catalytic domains of Dot1L or yeast Dot1 proteins (50 nM to 1000 nM) were incubated with 12 nM recombinant nucleosomes in EMSA buffer (10 mM Tris pH 7.5, 100 mM NaCl, 2.5% Glycerol and 1 mM DTT) at room temperature for 30 min. The binding reactions were resolved on native polyacrylamide gels (6% PAGE, 0.2 X TBE), stained with SYBR™ Gold (Thermo Fisher), visualized on a Typhoon Trio+ scanner (Molecular Dynamics), and quantified using the program ImageQuant 5.2v (Molecular Dynamics). The amount of Dot1L or yeast Dot1 bound to nucleosomes was determined by measuring the decrease in free nucleosome in each reaction. The background was subtracted from Dot1-free samples. The free DNA was taken under in consideration for the calculation of free nucleosome. The apparent KD and the Hill coefficient for each binding curve were calculated by fitting the specific binding with Hill slope equation using the program Prism 7 (GraphPad). The final parameters were calculated from at least 3 independent experiments. (n=3/data point). Histone Methyl Transferase assay (HMT) The reactions were performed by mixing the indicated amounts of recombinant human Dot1L (1 pmol at the highest concentration) and nucleosomes (10 pmol) in the HMT assay buffer (50 mM Tris-HCl pH 8.5, 50 mM NaCl, 5 mM MgCl2 and 1 mM DTT) with 10 uM SAM at 30 C for 1 hour or 3 hours, in 25 μL final volume. The reactions were stopped by the addition of 5.0 μL 5X SDS buffer. The products were resolved in a 15% SDS-PAGE gel and transferred in to 0.2-μm polyvinylidene difluoride PDVF membrane (Bio-Rad). The histone methylation level was determined by incubating the membranes with anti-H3K79Me3 (Abcam ab2621, 1:1000) or anti-H3K79Me2 (Abcam ab3594, 1:1000) for 12 hours at 4C. The western blots were developed using ECL reagent (Thermo Fisher), and imaged in the ChemiDoc system (Bio-Rad). The loading control was determined with FastBlue staining, or blotting with anti-H4 antibody (Abcam ab7311, 1:1000).