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. 2020 May 12;11:2355. doi: 10.1038/s41467-020-16146-3

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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 Yeast cells expressing either C-terminally (upper panel) or internally (lower panel) 3xMyc tagged Ptc5 (magenta) together with Ant1-YFP (cyan) were analyzed by immunofluorescence microscopy. Antibodies against Myc and GFP were used for detection. White color indicates colocalization in merged pictures. Scale bar represents 5 µm. b The fraction of peroxisomal Ptc5 signal to total Ptc5 signal was quantified using ImageJ⁵⁹. ‘OE’ indicates Ptc5-RFP-PTS under control of the tef1 promoter (Fig. 1c). ‘Endogenous’ refers to Ptc5-3xMyc-PTS (a). Data of three independent experiments (n = 3; each color represents one experiment) were plotted. Error bars represent standard error of the mean. c Organelles prepared from strains co-expressing either Ptc5-3xMyc (upper three western blots) or Ptc5-3xMyc-PTS (lower three western blots) together with the peroxisomal matrix protein GFP-Sps19 were analyzed by density gradient centrifugation and western blot. Por1 is a protein localized in the outer mitochondrial membrane. Twelve fractions were collected, starting from the top of the gradient, and analyzed. d Western blots from three independent experiments (n = 3; each color represents one experiment) were quantified using ImageJ to determine the ratio of peroxisomal to total Ptc5 signal of Ptc5-RFP-PTS (OE) and Ptc5-3xMyc-PTS (endogenous), respectively. Error bars represent standard error of the mean. Source data are provided in the Source data file.