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. 2018 May 9;9:1835. doi: 10.1038/s41467-018-03955-w

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© The Author(s) 2018

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. 1 — Identification of newly formed SecGFP-GPI endocytic vesicles using a pH pulsing assay. a Schematic (top panel) of the pH pulsing assay depicting the effect of pH on SecGFP-GPI fluorescence during endocytic vesicle budding. Note the fluorescence of SecGFP-GPI is retained at high pH (top and bottom left panel) or when exposed to low pH if sequestered in a newly formed endocytic vesicle (bottom right panel), and quenched only when exposed to low pH (top right panel). Sequential TIRF images of AGS cell co-expressing SecGFP-GPI and mCherry-ARF1 collected at pH 7, pH 5 and in the RFP channels (middle panel). Newly formed endocytic vesicles (inset) (identified as in Supplementary Figure 1c) are used to construct a single frame (yellow rectangle) of the montage depicted (bottom panel). b Average of the normalised fluorescence intensities of pH 5 and pH 7 traces at the site of newly formed SecGFP-GPI endocytic vesicles compared to their respective random traces constructed from 120 endocytic events (pH 5 and pH 7) and 3428 random spots, derived from 17 cells pooled from four independent experiments. c The graph shows the fold enrichment of fluorescence intensity over the local background of pH 5 vs. pH 7 at the time of formation of the endocytic vesicles (data from 1b). d–f Graphs show the average normalised fluorescence intensity vs. time traces for the recruitment of TagRFPt-CDC42 (d), mCherry-ARF1 (e) and mCherry-GBF1 (f) to the forming SecGFP-GPI endocytic sites, and its corresponding random intensity trace (n, Table 1). The random traces were derived from randomly assigned spots of the same radius as the endocytic regions, as detailed in S.I. Endocytic distribution at each time point was compared to the random distribution by Mann–Whitney U test, and the log₁₀ (p) [log₁₀ (0.05) is −1.3 and log₁₀ (0.001) is −2.5] is plotted below each trace (d–f). Representative montages from the respective data sets are depicted below the graphs (d–f). Arrowheads indicate the newly formed endocytic vesicle. Error bars, s.e.m. (b, d–f). Scale bar, 1.5 µm (a, d–f)