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. Author manuscript; available in PMC: 2016 Sep 1.
Published in final edited form as: Virology. 2015 May 15;483:209–217. doi: 10.1016/j.virol.2015.04.014

Fig. 4.

Fig. 4

Signal peptide cleavage and glycosylation of CPXV219. (A) Western blots. HeLa cells were infected with 10 PFU per cell of viruses expressing CPXV219 proteins that contain a single FLAG epitope tag following the putative CPXV219 SP (SP-219), the VSV-G SP (VG-SP) replacing the CPXV 219 SP, or non-cleavable VSV G SP (VG*-SP) replacing the CPX V219 SP. After 18 h, whole cell lysates were treated with β–mercaptoethanol and SDS and analyzed by SDS-PAGE and Western blotting. Separate membranes were blotted with anti-FLAG clone M2 or anti-FLAG antibody clone M1 and visualized by infrared fluorescence (upper) and chemiluminescence (lower). (B) Glycosylation. HeLa cells were infected with 3 PFU per cell of CPXV V5-219-HA or parental CPXV-BR. After 18 h, the cells were harvested, lysed with 50 mM Tris pH 7.0, 150 mM NaCl, and 0.5% NP-40 detergent and nuclei removed by centrifugation. The clarified lysates were incubated with PNGaseF, EndoH, or left untreated and analyzed by SDS-PAGE and Western blotting. Antibodies to V5 (green) and HA (red) were visualized by infrared fluorescence. Yellow results from coincidence of red and green. The positions of marker proteins are shown on the left of each panel.