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. 2011 Nov 14;287(2):1090–1099. doi: 10.1074/jbc.M111.282855

FIGURE 2.

FIGURE 2.

Acetylated cyclin T1 binds bromodomains in BRD4. A, coimmunoprecipitation of HA-tagged wild type or acetylation-deficient (4KR) cyclin T1 with FLAG-tagged short BRD4 722 or BRD4 722ΔBD proteins. Immunoprecipitations (IP) were performed with FLAG-agarose, and associated cyclin T1 proteins (indicated by arrow) were examined by Western blotting (WB) with antibodies against the HA tag. Asterisk indicates a protein nonspecifically recognized by the HA antibodies. The relative intensities shown in this and all following figures were quantified using the ImageJ software available at rsb.info.nih.gov. ND, not determined. B, coimmunoprecipitation of HA-tagged wild type or acetylation-deficient (4KR) cyclin T1 with FLAG-tagged short BRD4 722, BRD4 722ΔBD1, or BRD4 722ΔBD2 proteins. Immunoprecipitations were performed with FLAG-agarose, and associated cyclin T1 proteins were examined by Western blotting with antibodies against the HA tag. Expression of overexpressed proteins was analyzed using indicated antibodies by Western blotting. C, GST pulldowns of GST, GST-cyclin T1, or acetylated GST-cyclin T1 proteins with truncated in vitro transcribed/translated FLAG-tagged BRD4 lacking the PID (BRD4 722 aa) or a mutant in which bromodomains have been deleted (BRD4 722ΔBD). Coimmunoprecipitated BRD4 proteins were detected by Western blotting with FLAG antibodies. Loading of 10% input is shown.