Evaluating SARS-CoV-2 E Protein Expression

E protein expression in the C-KCE-E was evaluated by immunofluorescence (IFA) and western blot. The monoclonal antibodies (mAbs) against E and the polyclonal antibodies (pAbs) against gC were produced as described previously [13 (link),14 (link)]. For IFA, the CEFs were infected at an MOI of 1 with C-KCE or C-KCE-E. The mAbs against E (made by us) was used as the primary antibodies. The secondary antibodies were fluorescein isothiocyanate-conjugated goat anti-mouse IgGs (for E detection) (Santa Cruz). The CEFs nuclei were stained with 4’-6-diamidino-2-phenylindole (DAPI). Additionally, the cells were observed with a laser-scanning confocal microscope (Carl Zeiss, Heidenheim, Germany). The results were analyzed using the software, Image J (NIH, Bethesda, MD, USA). For western blot analysis, E expression was carried out in CEFs infected with C-KCE-E and C-KCE at an MOI of 1. mAbs against E, pAbs against gC (made by us) and mAbs against GAPDH (Santa Cruz, CA, USA) for the control were used as primary antibodies; goat HRP-conjugated anti-rabbit or anti-mouse IgGs were used as secondary antibodies. The bands were visualized using ECL detection reagents (Thermo, Waltham, MA, USA), according to the manufacturer’s instructions.