Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2012 Sep 18;7(9):e44717. doi: 10.1371/journal.pone.0044717

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2012 Eskat et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

PMC Copyright notice

Typical HEp-2 cell nuclei are displayed in (A) and (D) showing centromere location of all four CEN proteins. Two of these locations, spot 1 and spot 2 in each of the two graphs, were selected for fluorescence intensity analysis before and after acceptor bleaching (see enlargements below). Spot 1 served as control and showed no detectable intensity change. At spot 2, the acceptor fluorophore mCherry was bleached (compare pre-bleach and post-bleach in (A) and (D)). In (B) and (E), the time course of the fluorescence intensity of the donor and the acceptor of both FRET pairs are shown. The acceptor intensities in the ROI (“region of interest”; open squares) were averaged and normalized to the mean intensity measured at the three time points before bleaching. The donor intensities in the ROI were averaged and normalized to the intensity measured at the time point after bleaching. Bleaching of the acceptor resulted in a fluorescence intensity increase of the donor (black dots) for EGFP-CENP-U (B) indicating the presence of FRET (see arrow), but no fluorescence intensity increase for EGFP-CENP-Q (E) indicating the absence of FRET. (C) and (F): Donor fluorescence intensity variation observed during acceptor bleaching normalized to the intensity measured at the first time point after bleaching. Control: spot 1 (acceptor not bleached) yielding E_var (grey bars), FRET measurement: spot 2 (acceptor bleached) yielding E_FRET (black bars). For protein pairs indicated, number of observed single cases (grouped into E_var or E_FRET value ranges of 4%) displayed versus values of E_var or E_FRET. (C) EGFP-CENP-U (donor) and CENP-R-mCherry (acceptor): distribution of E_FRET (40 bleached kinetochores) is clearly distinct from the distribution of E_var (39 non-bleached kinetochores) indicating FRET. (F): EGFP-CENP-Q (donor) and CENP-P-mCherry (acceptor): distribution of E_FRET (52 bleached kinetochores) superimposes the distribution of E_var (51 non-bleached kinetochores) indicating no FRET.