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. 2012 Oct 1;287(46):38755–38766. doi: 10.1074/jbc.M112.412015

FIGURE 4.

FIGURE 4.

TAF7 binds and is phosphorylated by BRD4. A, TAF7 associates with BRD4 in vivo. BRD4 was immunoprecipitated (IP) from HeLa nuclear extract using α-BRD4 and immunoblotted with α-TAF7. B, TAF7 interacts directly with BRD4. BRD4 (0.5, 1.0, and 1.5 μg) was pulled down with 0.5 μg of GST-TAF7 immobilized on GST beads and immunoblotted with α-BRD4. BRD4 (1.5 μg) with GST beads alone, or insect cell control extract (CE) made in parallel with recombinant BRD4 were used as negative controls. C, TAF7 binds BRD4 kinase subdomains. Upper, map of BRD4 proteins used. Middle, α-TAF7 immunoblot of 0.3 μg of GST-TAF7 (input) recovered by pull-down with 1.0 μg of wild type FLAG-BRD4 or equimolar amounts of FLAG-BRD4 mutants. Lower, quantitation of recovered GST-TAF7. D, BRD4 interacts with the central serine-rich region of TAF7. Upper, α-BRD4 immunoblot of 0.6 μg of BRD4 recovered by pull-down with 1 μg of full-length GST-TAF7 or equimolar amounts of GST-TAF7 sections. Middle, quantitation of recovered BRD4. Lower, map of TAF7 proteins used showing interaction sites of kinases. E, TAF7 is phosphorylated by BRD4. Autoradiograph of a kinase assay with TAF7 (0.15 μg) alone (first lane) or with recombinant murine BRD4 (0.3 μg) or human BRD4 immunoprecipitated from HeLa nuclear extract, with or without 1 μm flavopiridol and 100 μm roscovitine. F, TAF7 phosphorylation is dependent on BRD4 kinase domains. Autoradiograph showing 0.1 μg of TAF7 phosphorylated by 0.3 μg of BRD4 WT or equimolar amounts of BRD4 mutants. G, BRD4 phosphorylates the C-terminal end of TAF7. Autoradiograph showing 0.75 μg of full-length GST-TAF7 or equimolar amounts of GST-TAF7 sections phosphorylated by 0.6 μg of BRD4 (additional band migrating at ∼98 kDa is most likely hyper-phosphorylated TAF7 accentuated in the 15% gel used here). H, phosphorylation of BRD4 modulates its ability to phosphorylate TAF7. BRD4 (0.3 μg) that was unphosphorylated, autophosphorylated, or pre-phosphorylated with CAK (0.1 μg), PTEFb (0.1 μg), or both was used to phosphorylate 0.1 μg of TAF7 in the presence of 100 μm roscovitine and 1 μm flavopiridol.