Figure 7. Analysis of Brd4 recruitment to the transcription site.

(A) YFP-Brd4 is not enriched at the inactive transcription site marked by Cherry-lac repressor (panels a–c). YFP-Brd4 and endogenous Brd4, immunostained with antibody Brd4CA, are strongly recruited to the active site marked by Cherry-tTA-ER (panels d–f and g–i, respectively). Transcription was induced for 3 hr by the addition of tamoxifen. Scale bar represents 5 µm. Scale bar in enlarged inset represents 1 µm. (B) Quantification of Cherry-tTA-ER (top panel, solid red circles) and YFP-Brd4 (lower panel, solid green circles) accumulations at the transcription site during activation. Tamoxifen was added to the media immediately after the first time point (∼0 min). Images were collected every 1.5 min for ∼40 min. Measured intensities were normalized to the high and low plateau values and fitted to a logistic fit (solid black line). The initial accumulation (blue Xs and arrows) is when the fitted curve reaches 5% of the total accumulation. The set of graphs represents the average of 6 cells imaged from 4 different coverslips on 4 different days. Error bars represent the standard deviation.