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. 2022 Jul 6;13:3880. doi: 10.1038/s41467-022-31564-1

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© The Author(s) 2022, corrected publication 2022

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. 5 — a Schematic diagram of wild-type Fsx1, four mutants and AtHAP2ΔTM → GPI. SP signal peptide, FL fusion loop. For colors and abbreviations see legend of Fig. 2. b Quantification of content-mixing (cell–cell fusion) in populations of cells expressing vectors (n = 7), Fsx1 (wt) (n = 7), its mutants (ΔFL → AG₄A (n = 6), ΔDIV → EFF-1 stem (n = 4), ΔTMs → EFF-1 TM (n = 4), ΔTMs → GPI (n = 3), or AtHAP2ΔTM → GPI (n = 3). Bar chart showing means ± SEM. Comparisons by one-way ANOVA followed by Bonferroni’s test against the vector (black) and against Fsx1 (red). ns = non-significant, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file. c Representative merged images from the experiments in (b): magenta (RFP); green (GFP) and blue (DAPI). Fused cells with RFP and GFP (arrows). Scale bars, 20 µm. See also Supplementary Fig. 7f. d Immunoblot of EFF-1-V5, control (untransfected cells) and Fsx1-V5 expressing cells. “Surface” indicates surface biotinylation followed by affinity purification using neutravidin agarose beads; “Total” indicates the expression in whole cell extracts. Actin is used as a loading control. The amount of initial cells for Fsx1 is 4 times higher than EFF-1. n = 3. e Surface biotinylation as explained in panel d for cells expressing Fsx1-V5 (WT), ΔFL → AG₄A-V5, ΔDIV → EFF-1 stem-V5 or ΔTMs→EFF-1 TM-V5. n = 3. f Immunofluorescence images on non-permeabilized cells expressing Fsx1-FLAG (WT), AFF-1-FLAG (negative control, cytotail), Fsx1-ΔFL → AG₄A-FLAG, Fsx1-ΔDIV → EFF-1 stem-FLAG, AFF-1-FLAG (permeabilized), Fsx1-ΔTMs → EFF-1 TM-FLAG, Fsx1-ΔTMs → GPI and AtHAP2-ΔTM → GPI. The FLAG tag was inserted before the first TM or the GPI signal of each construct except for C. elegans AFF-1 in which the FLAG is at C-terminal after the cytoplasmic tail. Transfected BHK cells were incubated with anti-FLAG antibody on ice before fixation. Non-permeabilized staining of FLAG antibody showed the surface expression of Fsx1 and the mutants. C. elegans AFF-1 tagged with a cytoplasmic FLAG is a negative control for non-permeabilized staining. Permeabilized staining of CeAFF-1-FLAG shows the localization on plasma membrane and internal compartments (see also Supplementary Fig. 7g). Scale bars, 10 µm.