Figure 3

Fluorescence recovery after photobleaching assays performed with the addition of suberoylanilide hydroxamic acid to establish a robust assay window. (A) Nuclei of U2OS cells transfected with plasmids encoding green fluorescent protein chimerised to wild-type or mutant SMARCA2, with or without 2.5 μM suberoylanilide hydroxamic acid (SAHA) and the inhibitor PFI-3. The bleached area is indicated by a red circle. (B) Time dependence of fluorescence recovery in the bleached area for the SMARCA2 fluorescence recovery after photobleaching (FRAP) assay. Half-times of fluorescence recovery (t½) in the FRAP assays for SMARCA2 (C), BAZ2A, BRD7, ATAD2, GCN5L2, ZMYND11 and BRD1 (D) and BRD3 (E). 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, except GSK2801, which is 5 μM. wt, Wild type; X####F, Bromodomain mutants, indicating substitution made. ^†Addition of 2.5 μM SAHA. *P < 0.05, significant difference from wild type^†.