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. 2020 Mar 9;11:1283. doi: 10.1038/s41467-020-14932-7

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© The Author(s) 2020

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. 2 — a Diagram of the mCherry-M2x24 construct + TO1-Biotin controlled by a doxycycline inducible promoter. Followed by representative fixed maximum projections (left & centre) and live cell images of observed cellular foci (right). b Diagram of the mCherry-MS2v5x24 construct + tdMCP-EGFP under the control of a doxycycline inducible promoter. Followed by representative fixed maximum projections (left & centre) and live cell images of observed cellular foci (right). c Diagram of the mCherry-MS2v5x24 construct + TO1-Biotin under the control of a doxycycline inducible promoter. Followed by representative fixed maximum projections (left & centre) and live cell images of observed cellular foci (right). For (a–c) TO1-Biotin signal is in yellow, EGFP signal is in green, mCherry is in red, Hoechst 33258 is in blue and scale bars = 10 µm and 1 µm inset. Fixed cell images depict expression in Cos-7 cells and live cell images depict expression in HEK 293 T cells. d Representative bleaching curve for an individual foci from a fixed Cos-7 cell, (yellow) fitted with a maximum likelihood step finding algorithm⁵⁷ (black). e Distribution of 378 calculated bleaching step sizes from 70 individual mCherry-M2x24 foci from eight fixed cells. f Fixed cell maximum foci intensity distributions of mCherry-MS2v5x24 (black) and mCherry-M2x24 (yellow) in the presence of 200 nM TO1-Biotin. The M2x24 distribution quantifies~7000 foci from 13 cells. g Maximum live-cell foci intensities for cells expressing mCherry-M2x24 (yellow) and mCherry-MS2v5x24 (black) in the presence of 200 nM TO1-Biotin. n = 183 and 187 foci, respectively.