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. 2010 Aug 23;5(8):e12335. doi: 10.1371/journal.pone.0012335

Figure 2. Release of P-TEFb from the 7SK snRNP by the P-TEFb binding domain of Brd4.

Figure 2

A) A schematic of Brd4 constructs used. BD1, Bromodomain 1; BD2, Bromodomain 2; ET, Extraterminal domain; H1, H2 and H3, Helical domains 1, 2 and 3. Brd4 1209–1362 contains three helical regions, Brd4 1209–1362 Δ1329–1345 is missing helical region 3 which is required for P-TEFb binding. B) Recombinant protein expressed and purified from E. coli. A contaminating band can be seen in the Brd4 mutant preparation and this was confirmed to be an E. coli protein by Western blot (data not shown), not full length Brd4. M, Marker; 1, Brd4 1209–1362; 2, Brd4 1209–1362 Δ1329–1345. C) Indicated amounts of Brd4 were added into the release reaction for the indicated times. D) Same as in C except the Brd4 mutant missing helical domain 3 was used for the reactions. E) Brd4 release was quantified from three independent experiments. Two independent experiments were done to calculate the mean for the Brd4 helical domain 3 mutant. The y-axis is a measure of percent of Cdk9 left in the complex. All error bars represent standard error.