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. 2014 Jul 1;25(13):1958–1968. doi: 10.1091/mbc.E13-10-0564

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© 2014 Lisaingo et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Generating cell populations expressing defined TRF1 protein levels. (A) Schematic of constructs encoding YFP-TRF1 (top) and TRF1 translated separately from YFP (bottom), driven by a CAG promoter. (B) FACS plots showing wild-type control, vector control (IRES-YFP), TRF1_IRES-YFP, and YFP-TRF1 transfected cells at 24 h posttransfection and gates used to sort negative, low, high, and high++ YFP populations. Numbers in gates indicate fold difference in median YFP levels compared with the low population. (C) Western blot analysis of wild-type control, vector control (IRES-YFP), and TRF1_IRES-YFP low and high sorted cell populations (lanes 1–4, respectively) and (D) YFP-TRF1 low (lane 1) and high (lane 2) sorted cell populations using anti-TRF1 antibody or anti-YFP antibody (D, lane 3). Bottom, GAPDH loading control. The molecular weights of TRF1 (solid arrowhead) and YFP-TRF1 (open arrowheads) are indicated. (E) Localization of low YFP-TRF1 (green) over the cell cycle with DAPI DNA stain (blue). Foci doublets in metaphase (insets, arrows) and singlets in anaphase (insets, arrowheads). Images are maximum-intensity projections.