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. 2017 Oct 20;8:1075. doi: 10.1038/s41467-017-01313-w

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — Incorporation of RAP1 into shelterin_core complex does not change TRF2:TIN2 complex stoichiometry, and there are two each of RAP1 and TRF2. Telomerase stimulation by shelterin complexes is also not affected by RAP1 inclusion. a Superose 6 gel-filtration trace of co-expressed shelterin_core (+RAP1) complex (RAP1–TRF2–TIN2–TPP1–POT1) showed a mono-disperse peak (left peak) with SDS-PAGE showing five distinct Coomassie-stained bands representing the five shelterin proteins. SEC-MALS analysis determined the protein complex to be 348.7 ± 1.7 kDa, with the bottom number in parentheses being the theoretical mass for a 2:2:1:1:1 (RAP1:TRF2:TIN2:TPP1:POT1) complex. The right peak is composed of excess un-incorporated RAP1, POT1 and TIN2. b Purified singly ybbR-tagged shelterin_core (+RAP1) complex labeled with Cy3-CoA dye and compared with Coomassie-stained bands as reference. *Molecular weight is an apparent value of SeeBlue Plus2 protein marker (Thermo Fisher) on a tris-glycine gel. c Quantification of fluorescence-intensity showed TRF2:TIN2 relative stoichiometry to be 2:1 (P < 0.05) while TRF2:RAP1 is 1:1. Error bars indicate s.d. for three independent experiments performed with three different protein preparations. d Direct telomerase assay to determine the effects of RAP1 inclusion on processivity stimulation by shelterin_core(−POT1) or shelterin_core complexes. e Total activity of telomerase primer elongation products (relative to no-protein lane) with bound shelterin protein complexes at various concentrations (nM). f Telomerase processivity (relative to no-protein lane) with each shelterin protein/complex. (For panels e and f, error bars indicate the range for two independent experiments.)