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. 2023 Jul 28;19:73. doi: 10.1186/s13007-023-01049-3

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 5 — FLIM analysis of AP1-mV NLS-mCh and AP1-mV Ap1-mCh in N. benthamiana leaf cells. FLIM experiments were performed in N. benthamiana leaf epidermis cells. Fusion proteins were expressed from the UBQ10 promoter and imaged 3–4 days after infiltration. A Average lifetime image of individual nuclei expressing AP1-mV, AP1-mV NLS-mCh and Ap1-mV AP1-mCh (A’–A’’ respectively). Nucleoli were excluded from FLIM analysis. (Scale bars: A–A’’: 6 µm) B BINDING [%] (grey) and FRET efficiencies [%] (white) for AP1-mV, AP1-mV NLS-mCh and AP1-mV AP1-mCh. For each analysed nucleus average BINDING and a corresponding average lifetime were fitted. Mean BINDING of the donor only sample AP1-mV was 1.23% and most values were below 10%. Therefore, nuclei with BINDING below 10% were excluded from FRET efficiency calculation. Co-expression of NLS-mCh with AP1-mV did not lead to significant higher BINDING (3.06%) compared to the donor only sample, while AP1-mV AP1-mCh showed increased BINDING (29.89%) with an average FRET efficiency of 39.98%. Statistical groups were assigned after multiple comparison with Kruskal–Wallis and a Post hoc test using the criterium Fisher’s least significant difference (alpha parameter is 0.05). (Dashed blue line marks the BINDING cut-off of 10%; Number of repetitions are indicated below BINDING values and number of images with BINDING above 10% are indicated below the FRET efficiency values in the bottom of the plot)