Molecular characterization and sub-cellular localization of MPXV197.

<p><b>A)</b> Schematic representation of MPXV 197 with predicted signal peptide (SP, blue, SignalP), transmembrane domains (TM, green, TMPred), and N-linked glycosylation sites (red, NetNGlyc 1.0). <b>B)</b> CHO cells were transduced with Ad-197/Ad-tTA (‘Ad-197’) or Ad-tTA only (‘Ad-control’) for 24 hours and lysed in sample buffer prior to electrophoretic separation and immunoblotting with αFLAG. Right panel: Overexposure reveals a >250 kDa band (asterisk). <b>C)</b> CHO cells were transduced as in B). After 24 h, cell surface proteins were biotinylated followed by immunoprecipitation with NeutrAvidin, electrophoretic separation and immunoblotting with αFLAG. <b>D)</b> 24 h after transduction with the indicated expression vectors, CHO cells were metabolically labeled for 45 min followed by chase for 0.5, 1, and 3 h. Cell lysates were immunoprecipitated with αFLAG. In the right panel, samples were treated with EndoH or left untreated prior to electrophoretic separation. EndoH sensitive proteins are indicated by asterisks. <b>E)</b> Sub-cellular localization of C- and N-terminal FLAG fusions of MPXV197 was determined by IFA using αFLAG. CHO cells were either permeabilized (‘Intracellular’) or non-permeabilized (‘Cell-surface’) prior to IFA. Scale bar is 20 µm. Arrows indicate the plasma membrane.