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. 2022 Jan 19;12:1005. doi: 10.1038/s41598-022-04883-y

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© The Author(s) 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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(a–c) Effect of TSP extraction by protein extraction buffer. Comparison of solution states (a) and TSP concentrations (b) extracted from infiltrated leaves using three protein extraction buffers EB1 (PBS, EDTA, and 2-mercaptoethanol), EB2 (PBS and EDTA), and EB3 (only PBS). The TSP concentration was measured 2 h after extraction (2 h) or the sample thawed after freezing (F/T). Values are the means ± SE of three independent biological replicates. (c) Western blot analysis of M protein (25 kDa) and N protein (46 kDa) expression in TSP extracted by three extraction buffers. (d) The purification conditions of plant-derived VLPs were confirmed through 10–60% sucrose gradient sedimentation of ME and ME + N_FLAG TSP. M protein and N protein were observed in about 40% sucrose layer by western blot analysis with M and FLAG antibodies. (e) Identification of M and N proteins from VLPs purified by 40% sucrose cushion in TSP of ME and ME + N_FLAG. (f) Presence of N protein after purification of VLPs using 40% sucrose in N_FLAG single expression and ME + N_FLAG co-expression. All images were cropped. See Supplementary Figs. S4–S7 for full size of blot.