Figure 2.
Quantitative measurement confirms that BD245 work together to create high affinity to H3K14ac. (A) The indicated GST‐BD constructs of PBRM1 were expressed and purified from Escherichia coli, pulled down by the indicated peptides, and immunoblotted with anti‐GST antibody. (B) The bands in (A) were quantified with NIH ImageJ, and the ratios of bands associated with H3K14ac/H3 were calculated from three experiments. The P‐values were calculated using the two‐tailed Student's t‐test. *: P < 0.05; **: P < 0.01; ***: P < 0.001. N.S.: nonsignificant. (C) Top: The indicated GST‐BD constructs of PBRM1 were expressed and purified from Escherichia coli. Similar amount of GST‐fusion protein was incubated with 3ug of nucleosome purified from HeLa cells, washed then analyzed with indicated antibodies. Arrow: the full‐length GST‐BD12 protein. Bottom: The intensity of the pulled down H3 or H3K14ac bands was determined with ImageJ from three different experiments, and the ratios of H3/input and H3K14ac/input were calculated and plotted. The error bars represent standard error of the mean. (D) GST‐BD fusion proteins (left) or cleaved and purified BD proteins (right) were pulled down with indicated peptides and analyzed with western blots. (E) BLI analysis of the interaction between PBRM1 BD proteins and the H3K14ac peptide. Binding activity was normalized to maximum response and is reported as relative binding. The association phase takes place from 0 to 900 s; the dissociation phase takes place from 901 to 2200 s. (F) Binding parameters of PBRM1 BD constructs and H3 peptide interactions. Apparent K d values () were obtained for the interaction between PBRM1 BD proteins and H3 peptides by fitting BLI data to a double exponential function. The H3:H3K14ac reports the preference of PBRM1 BD proteins to bind the H3K14ac peptide over the nonacetylated H3 peptide.