Assessment by western blot of the expression of recombinant porcine reproductive and respiratory syndrome virus-1 (PRRSV-1) soluble Glycosylated Protein 2 (sGP2), 3 (sGP3) or 4 (sGP4) in cell lysate and culture supernatants of transiently transfected HEK293T/Expi293F

Whole-cell lysates (A) and culture supernatants (B) were collected three days post-transfection, and proteins were detected using a horseradish peroxidase-labelled antibody specific for the histidine tag. Protein extracted from cell lysates was treated with or without peptide-N-glycosidase F (PNGase F) prior to assessment by western blot to investigate potential N-glycosylation heterogeneity (C). Purification of recombinant PRRSV-1 sGPs from cell culture supernatants was performed using affinity chromatography. Eluted samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then evaluated by western blot (D) and Coomassie protein staining (E) to assess purity. Non-transfected cells (non-Tx) were included as a negative control.