2.3. Generation of Recombinant sGP

Expression plasmids for pCAGGS-EBOV (Mayinga), SUDV (Boniface), BDBV (Butalya), or RESTV (Pennsylvania 89) sGP were transfected in 293F cells according to the manufacturer’s instructions. After incubation for 4 days, the supernatant was cleared from cell debris by centrifugation twice at 3500 rpm for 10 min at 4 °C. The supernatants containing each sGP were collected and concentrated in Amicon Ultra 30 K filters (Millipore) to a final concentration of 1–2 mg/mL and stored at −80 °C. Supernatant of nontransfected 293F cells was also concentrated using the same method and used as a control protein. Expression of each sGP was confirmed by mixing the concentrated protein 1:1 with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing 20% β-mercaptoethanol and incubated at 99 °C for 10 min. SDS-PAGE with all samples was performed in parallel on Tris-Glycine eXtended (TGX) criterion pre-cast gels (Bio-Rad Laboratories). The gel was processed for silver staining, using the Pierce Silver Stain Kit (Thermo Fisher Scientific, USA).