Fluorescence recovery after photobleaching assays detect mutation of the bromodomains and inhibition by small molecules. (A) Nuclei of U2OS cells transfected with plasmids encoding green fluorescent protein fused to wild-type BRD4, BRD4 bromodomain mutants or wild-type BRD4 treated with JQ1. The bleached area is indicated by a red circle. (B) Time dependence of fluorescence recovery in the bleached area for the BRD4 fluorescence recovery after photobleaching (FRAP) assay. Curves represent the means at each time point of at least ten cells in each group. Half-times of fluorescence recovery (t½) in FRAP assays for BRD4 (C) and BRD3 and TRIM24 (D). 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. wt, Wild type; N###F, Bromodomain mutants, indicating substitution made. *P < 0.05, significant difference from wild type.