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. Author manuscript; available in PMC: 2016 Apr 16.
Published in final edited form as: Mol Cell. 2015 Mar 26;58(2):203–215. doi: 10.1016/j.molcel.2015.02.029

Figure 1. p300 Co-Purifies With HCT Activity From Nuclear Extract and Has Intrinsic HCT Activity In Vitro and In Cells.

Figure 1

(A) HCT partial purification scheme

(B–C) HCT activity co-elutes with HAT activity. HeLa S3 nuclear extracts were fractionated by the scheme detailed in (A). HCT and HAT activities were assayed by panKCr or panKAc immunoblot, respectively, at each round of fractionation and peak HCT activity was followed. The immublot readouts for HCT (B) and HAT activity (C) of the final Mono S fractions are shown here. S3 indicates crude unfractionated extract, L indicates the load/Mono S input, and FT indicates the column flow through. Equal volumes of each fraction were used in these assays. For MS/MS analysis of fractions with peak HCT activity (fractions 4, 5, and 6) see Figure S1C–E and Table S1.

(D) Immunoblot for p300 of the fractions assayed for activity in (B–C). Labels and loading are as in (B–C).

(E–F) HCT (E) and HAT (F) activities of purified recombinant p300, GCN5, and TIP60 were assayed using recombinant octamers as substrate. Reaction products were immunoblotted with the indicated antibodies.

(G) p300-mediated HCT and HAT reactions were performed as in (E–F) with the indicated reaction conditions. Reaction products were probed with the indicated site-specific antibodies.

(H) HeLa S3 cells were transfected with control or p300- and/or CBP-specific siRNA. 72 hours post-transfection, whole cell lysates and histones were prepared and immunoblotted with the indicated antibodies. Immunoblots of acid extracts are shown above the black line, while immunoblots of whole cell lysates are shown below the black line. See also Figure S1.