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. 2021 Aug 19;13(8):1650. doi: 10.3390/v13081650

Figure 2.

Figure 2

L243, F245 and L249 are critical for VP24 nuclear export. (a,b) COS7 cells transfected to express the indicated proteins were treated 24 h post-transfection with or without LMB before live-cell CLSM (a) to determine the Fn/c for GFP (b); mean ± SEM; n ≥ 50 cells; data from single assays, representative of three independent assays. (c) COS7 cells transfected to express the indicated proteins were lysed 24 h post-transfection before immunoprecipitation for GFP. Lysates (input) and immunoprecipitates (IP) were analyzed by immunoblotting (IB) using antibodies against the indicated proteins. Results are representative of two independent assays. (d,e) COS7 cells co-transfected to express the indicated proteins were treated with or without LMB before fixation, immunofluorescent staining for NP (red) and CLSM (d) to determine the Fn/c for GFP (e); mean ± SEM, n ≥ 31 cells. Arrowheads indicate cells with detectable NP expression. (f) Alignment of the C-terminal 11 residues of VP24. Red and blue font indicate conserved and non-conserved residues, respectively; EBOV VP24 residues implicated in CRM1-dependent nuclear export highlighted in yellow; Q245 of MARV VP24 highlighted in blue. ****, p < 0.0001; NS, not significant; No add., no addition; WT, wildtype; NM, NES mutant. Scale bars, 30 μm.