Figure 4
From: Studies on membrane topology, N-glycosylation and functionality of SARS-CoV membrane protein
Mapping the epitope of the human monoclonal α-M antibody S30. A, An exemplary schematic representation of M mutants used in this study. A FLAG-epitope was fused to the C-terminus or in addition to the N-terminus. B, M1–134 was in vitro translated in the presence of [35S] PROMIX and subsequently immunoprecipitated using human monoclonal α-M antibody S30. The precipitates were subjected to SDS-PAGE and radioactive signals were visualized by autoradiography. C and D, BHK-T7 cells were transfected with plasmids encoding various C-terminally truncated M mutants. At 24 h p.t., cells were lysed, sonicated and directly subjected to SDS-PAGE analysis followed by immunoblotting using polyclonal α-FLAG and human monoclonal α-M antibody S30 simultaneously. The proteins were visualized using an AF680-coupled α-rabbit and an IRDye800-coupled α-human antibody and the Odyssey™ Infrared Imaging System. Mc-g, complex glycosylated form of M. Arrowheads indicate the specific protein bands detected by α-M S30 antibody.