Effects of SARS-CoV-2 ORF8 mutations at glycosylation and cysteine residues on secretion.A, wild-type (WT) and mutant ORF8-mycFLAG were expressed in 293T cells and separated by SDS-PAGE. Total fractions (input) treated with 2-ME were analyzed by immunoblotting using anti-FLAG mAb and anti-β-tub antibody. B and C, the immunoprecipitated (IP) fractions were prepared using anti-FLAG mAb beads and treated with 2-ME (B) or without 2-ME (C). The IP fractions were analyzed by Western blot using an anti-FLAG mAb. The approximate sizes (kDa) of the molecular weight standards for SDS-PAGE are indicated on the left. 2-ME, 2-mercaptoethanol; ORF8, open reading frame 8; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.