Figure 4.

APC/C interacts directly with the nucleosome acidic patch. (A) APC/C composition with proteins identified to be patch 1- and 2-dependent by mass spectrometry and/or western blot indicated. (B) Pulldown of WT or acidic patch mutant (AP mut) nucleosomes using immobilized recombinant twin-Strep-tagged APC/C alone or with WT or nucleosome binding deficient mutant of LANA (LANA mut). Histones are labeled with carboxyrhodamine (*) to facilitate high sensitivity detection by fluorescence imaging. Coomassie stained (top) and fluorescent images (bottom) of the same gel are shown. For fluorescent images, two different photomultiplier tube (PMT) settings were used for imaging (700 and 900). A PMT setting of 700 results in minimal pixel saturation. Imaging at a PMT setting of 900, which results in significant pixel saturation in input samples, increases sensitivity of elution samples to demonstrate minimal nucleosome binding in NCP AP mut and LANA WT elutions.