Extended Data Fig. 3: Flow cytometry gating scheme, cell-cell fusion assays, GP2 antibody staining, and effects of GPC mutations on fusion activity.
From: Molecular organization of the New World arenavirus spike glycoprotein complex
a, Flow cytometry gating strategy for immunostaining experiments of GPC-transfected cells. PE: R-phycoerythrin. b, Flow cytometry gating strategy for immunostaining experiments evaluating cell surface expression of WT or mutant GPC constructs used in cell-cell fusion assays. c, Schematic describing the protocol used for split-GFP cell-cell fusion assays. Created with BioRender.com Mann, C. (2025) https://BioRender.com/imne67a. d,g, Representative images from split-GFP cell-cell fusion assays of HEK 293 T cells transiently transfected with WT or mutant JUNV GPC (d) and WT or mutant MACV GPC (g) with exposure of cells to pulse media at the indicated pHs. Images were obtained with a live-cell imager. Scale bar, 100 µm. For (d), data quantification is provided in Fig. 3j. For (g), quantification is provided in panel h. e,f, Results of immunostaining experiments of WT or mutant JUNV GPC (e) or WT or mutant MACV GPC (f). Staining was measured by flow cytometry using TfR1-sAD-Fc (a GP1-reactive reagent) or KL-AV-2A1 (a GP2-reactive antibody)30. Data are mean ± s.d. for experiments each performed in technical triplicate with the following numbers of independent experiments: for all samples in (e) (n = 4); for samples in (f), WT MACV (n = 6), all other samples (n = 4). One-way ANOVA with Dunnett’s multiple comparison test. ****P < 0.0001; ***P = 0.0004; **P = 0.0035 (e). ****P < 0.0001 (f). h, Quantification of data shown in g. Data are provided as GFP-positive area divided by total cell-covered area. Data are mean ± s.d. for experiments each performed in technical triplicate with the following numbers of independent experiments: WT (n = 5), E10A (n = 4), H67A (n = 4).
