Figure 4.

Reconciling discrepant predictions of TBP fast components. (A) We were able to roughly match two published FRAP curves by approximating the different imaging conditions used in the two studies (15,20). Our two FRAP curves were obtained on the same cells expressing similar levels of TBP. The difference in fast component between conditions 1 and 2 is ∼30%. (See Fig. 1 legend for the procedure to estimate fast components). (B) We renormalized the curve from condition 2 with the true bleach depth by measuring the intensity in the bleach region as quickly as possible after the condition 2 photobleach. This reduced the difference in fast component between conditions 1 and 2 to ∼20%. (C) We applied the photobleaches of conditions 1 and 2 to H2B-GFP in live cells and found an ∼20% difference in reversible fractions, roughly accounting for the remaining difference from B. (D) We performed FRAP using a circular photobleach on cells expressing low levels of TBP and then applied our correction procedure for photoswitching to the TBP curve, yielding an estimated fast component of 26%. The uncorrected H2B curve shows the level of photoswitching present under these conditions.