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. 2021 Oct 26;11:21048. doi: 10.1038/s41598-021-99887-5

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© The Author(s) 2021

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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Immunoblotting studies on arenaviral NPs in transfected mammalian cells. (a–c) Immunoblotting analyses of whole-cell lysates obtained from monkey Vero E6 cells transfected with a construct expressing either wt or mutUGV1-NP-FLAG, or UHV1-NP-FLAG (a), wt or mutJUNV-NP-HA, or LCMV-NP-HA (b), HISV1-NP-FLAG (c), at three dpt after incubation at either 37 °C or 30 °C. Non-transfected (Mock) samples are provided as negative controls. 40 µg of protein per sample were loaded on standard SDS-PAGE gels, followed by immunoblotting. The nitrocellulose membranes were incubated sequentially with the following antibodies in the presented order: (1) mouse anti-FLAG tag 1:500 (a,c) or mouse anti-HA tag 1:500 (b); (2) rabbit anti-MTS-NP 1:200 (a,b) or rabbit anti-Hartmani-NP 1:500 (c); (3) mouse anti-tubulin 1:500 (a–c). Anti-tubulin specific signal at known molecular weight did not require membrane stripping. Tubulin (in red, secondary antibody: IRDye 680RD Donkey anti-mouse) was used as a reference for loading. Reptarenavirus and hartmanivirus (65–68 kDa), and mammarenavirus (63–65 kDa) NPs are indicated (black arrows). (a) Left panel: FLAG tag in red (secondary antibody: IRDye 680RD Donkey anti-mouse); middle panel: MTS-NP in green (secondary antibody: IRDye 800CW Donkey anti-rabbit); right panel: merged image. (b) Left panel: HA tag in red (secondary antibody: IRDye 680RD Donkey anti-mouse); middle panel: MTS-NP in green (secondary antibody: IRDye 800CW Donkey anti-rabbit); right panel: merged image. (c) Left panel: FLAG tag in red (secondary antibody: IRDye 680RD Donkey anti-mouse); middle panel: Hartmani-NP in green (secondary antibody: IRDye 800CW Donkey anti-rabbit); right panel: merged image. Immunodetection was performed using the Odyssey Infrared Imaging System (LICOR, Biosciences) providing also the molecular marker (Precision Plus Protein Dual Color Standards, Bio-Rad) used. Full-length blots are presented in Supplementary Fig. S8.