Figure 4

Fluorescence recovery after photobleaching assay utilising a multimerised CREBBP bromodomain construct to establish a robust assay window. (A) Domain organisation of CREBBP and representation of the regions of CREBBP incorporated into various green fluorescent protein (GFP) chimeric expression constructs. (B) Half-times of fluorescence recovery (t½) from fluorescence recovery after photobleaching (FRAP) assay using full-length CREBBP (EX1). (C) Nuclei of U2OS cells transfected with plasmids encoding GFP chimerised to wild-type (wt) or mutant multimerised CREBBP bromodomain (EX4), with or without 2.5 μM suberoylanilide hydroxamic acid (SAHA) and the inhibitor I-CBP112. The bleached area is indicated by a red circle. (D) Time dependence of fluorescence recovery in the bleached area of cells expressing wt or mutant EX4. (E) Half-times of fluorescence recovery (t½) of cells expressing wt or mutant EX4. Bars represent the mean t½ calculated from individual recovery curves of at least ten cells per group, and error bars depict the standard error of the mean. Where an inhibitor is used, concentration is 1 μM. N####F, Bromodomain mutants, indicating substitution made. ^†Addition of 2.5 μM SAHA. *P < 0.05, significant difference from wt^†.