Figure 2.

Photoaffinity labeling of recombinant HDAC enzymes. (a) 5 μg of each of the indicated recombinant HDAC enzymes were incubated with 1-BP (at 4 μM) for 5 min, followed by UV irradiation for 1 h, and rhodamine azide was added by click chemistry. A fluorescent image of an SDS-PAGE is shown and fluorescent dye markers are shown at the left of the gel. Note that HDAC3 consists of the enzyme plus its required cofactor NcoR2, which is a recombinant fragment that is also crosslinked by 1-BP. Minor bands at ~80 kDa and above represent multimers of HDAC3/NcoR2. Reactions for the class I HDACs 1, 2, 3 and 8 were analyzed on a separate gel from the reactions with the class II HDACs 4 and 5. (b) Competition with 106. HDAC3/NcoR2 was incubated with or without 106 at 10 μM, for 2 h at RT, prior to the addition of 1-BP (10 μM), followed by photocrosslinking and click chemistry as in a. (c) Determination of the half-life of the 1-BP/HDAC3 complex. 1-BP and recombinant HDAC3/NcoR2 were pre-incubated for 2 h prior to the addition of a 20-fold molar excess of 106, and samples were withdrawn at the indicated times, UV crosslinked and a rhodamine-azide was added by click chemistry. The inset shows a fluorescence image of an SDS-PAGE analysis of these samples, and the graph is a plot of the natural log of the fraction 1-BP/HDAC3 remaining at each time point, relative to the zero time point, versus time. ImageQuant software was used to quantify the data, which were normalized for HDAC3 protein concentration in each sample (determined by western blotting, not shown). A least-squares fit of the data (solid line, R² = 0.934) yields a t_1/2 of ~4 h.