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. 2018 Aug 10;8:11951. doi: 10.1038/s41598-018-30356-2

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© The Author(s) 2018

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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Western blot analysis of rNDVs expressing S or S2 protein of IBV. The expression of codon optimized S, and S2 proteins and non-codon optimized S protein of IBV were detected by Western blot analysis in infected DF-1 cell lysates, using a chicken polyclonal anti IBV serum (A-upper panel & B). The panel B was added to show the expression of non-codon optimized S protein clearly, since the expression of non-codon optimized S protein was not clear in panel A. For the codon optimized S protein of IBV expressed from rNDV (A-lane 3 and B-lane 2) two bands on top (~170–220 kDa) represent uncleaved S protein (S0) or polymeric forms of S1 or S2 protein. The ~130 kDa band, ~95 kDa band and ~60 kDa band represent S2 or S1 subunit of cleaved S protein. In the case of non-codon optimized S protein expressed from rNDV (2A-lane 2 and B-lane 1), two bands on top (~170–220 kDa) represent uncleaved S protein (S0) or polymeric forms of S2 or S1 protein and ~95 kDa band represents S2 or S1 subunit of cleaved S protein. In the case of rNDV/IBV- S2 (A-lane 1), there are two bands (~170–220 kDa) on top, representing polymeric forms of S2 protein, the ~105 kDa and the ~95 kDa band representing S2 subunit. Lane 4 of panel A and lane 3 of panel B represent rNDV as control. Lane 5 of panel A represents non-infected DF-1 cells. The incorporation of codon optimized S2 and S proteins and non-codon optimized S protein of IBV in NDV particles were detected by Western blot (C-upper panel). Two bands (~170–220 kDa) on top represent uncleaved S protein (S0) or polymeric forms of S2 or S1 protein (C-lane 2). The ~95 kDa and the ~60 kDa band represent S2 or S1 subunit of cleaved S protein (C-lane 2). The two bands (~170–220 kDa) on top represent polymeric forms of S2 protein, the ~105 kDa band and the ~95 kDa band represent S2 subunit(C-lane 4). The lane 1 and 3 of panel C represent purified rNDV control and purified rNDV expressing non-codon optimized S protein, respectively. A monoclonal anti-NDV/HN antibody was used to detect the 70 kDa of HN protein of NDV in DF-1 cell lysates (A-lower panel); rNDV/S2 (lane 1), rNDV/IBV-non-cod.opt.S (lane 2), rNDV/IBV- cod.opt.S (lane 3), rNDV (lane 4) and incorporated in NDV particles (C-lower panel); rNDV (lane 1), rNDV/IBV-cod.opt.S (lane 2), rNDV/IBV-non-cod.opt.S (lane 3) and rNDV/S2 (lane 4). The full-length gels are presented in supplementary Figure S1.