Transient expression of GPCMV gM and gN homologs and analysis of tagged proteins by western blot.

<p><b>A</b>. Predicted amino acid sequence for GP73 (gN) with potential glycosylation sites shaded in green. A truncated version of gN was generated by deletion of the first 40 codons, which included the majority of the predicted signal peptide sequence (underlined, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135567#pone.0135567.s004" target="_blank">S4 Fig</a>). The truncated gN(s) initiated from the first internal methionine (shaded in red). <b>B</b>. Figure of gN and gM expression constructs. Full length gN tagged with C-terminal mCherry was designated gNmCherry, B(i). Full length gN with C-terminal FLAG epitope tag was designated gN(f), B(ii), and truncated gN designated gN(s), B(iii). Full length gM was C-terminal tagged with GFP and designated gMGFP, B(iv). Size of predicted MW of tagged proteins indicated (kDa). <b>C</b>. Western blots were performed on transient plasmid expression of gN and gM tagged proteins in GPL cells. C(i) gNmCherry expression detected using anti-mCherry antibody. Lanes: (1) kDa ladder; (2) and (3) gNmcherry; (4) mock untransfected GPL cell lysate. C(ii) gN(f)FLAG expression detected using anti-FLAG antibody. Lanes: (1) kDa ladder; (2) and (3) gN(f)FLAG. C(iii) gN(s)FLAG expression detected using anti-FLAG antibody. Lanes: (1) kDa ladder; (2) and (3) gN(s)FLAG (4) mock untransfected GPL cell lysate. C(iv) gMGFP expression detected using anti-GFP antibody. Lanes: (1) kDa ladder; (2) and (3) gM; (4) mock untransfected GPL cell lysate. <b>D</b>. gN and gM expression in the presence or absence of glycosylation inhibitor. D(i) Western blot of gN(f)FLAG in the presence of tunicamycin (lane 2) or absence (lane 1). D(ii) Western blot of gN(s)FLAG in the presence of tunicamycin (lane 2) or absence (lane 1). D(iii) Western blot of gMGFP in the presence of tunicamycin (lane 2) or absence (lane 1). Control mock untransfected GPL cell lysate lane 3 D(i)-(iii).