Fig. 3: p300BRPHZ phase separation requires both AIL and BD.
a HAT activity of untreated p300BRPHZ (blue) and SIRT2-treated p300BRPHZ (orange) measured by a fluorometric assay. Reactions were started by the addition of acetyl-CoA and quenched by flash-freeze at indicated time points. Data are presented as mean values ± SD; error bars represent SD from triplicate measurements. b Phase separation of SIRT2-treated p300BRPHZ. The reaction mixture contained 10 μM p300BRPHZ and 12% PEG. Addition of Acetyl-CoA led to the disassembly of the p300BRPHZ droplets. c Representative images of p300BRPHZ samples from (k) after incubation for 2 min under a microscope. d Quantification of droplets counted in images acquired for the sample in (c). The number of droplets was counted in five non-overlapping 50 µm × 50 µm square regions and the mean value was plotted. Error bar represents SD. e Schematics of the p300BRPHZ phase separation mechanisms that can occur simultaneously. Multivalent ‘in trans’ interactions between HAT and deacetylated AIL and/or BD and acetylated lysines outside AIL can promote the formation of condensates. f, g Representative images of WT p300BRPHZ (f) and N1132A mutant (g) samples (from at least three replicates) containing 13 µM protein and 12% PEG in a 50 µm × 50 µm square region on cover slides under a microscope. h LC–MS analysis of N1132A p300BRPHZ shown in (g). i, j Representative images of WT p300BRPHZ (i) and ΔAIL mutant (j) samples (from at least three replicates) containing 13 µM protein and 12% PEG in a 50 µm × 50 µm square region on cover slides under a microscope. k LC–MS analysis of ΔAIL p300BRPHZ shown in (j). Source data are provided in the Source Data file.
