Fig. 2

Analysis of amino acid substitutions at N-glycan consensus sequence 413–415 (g4) for expression. a Cells were transfected with either the wt F, g4, g4AST or the g4NSA gene. At 24 h p.t., cells were metabolically labeled for 15 min (pulse) and then incubated for 2 h in serum-free nonradioactive medium (chase). After immunoprecipitation of F proteins from cell lysates and separation on a 12% SDS-gel under reducing conditions, samples were analyzed by autoradiography. wt: wild-type; b Cell surface expression of CedV F proteins. Cells were transfected with either wt F, g4, g4AST or the g4NSA gene. At 24 h p.t., MDCK-2 cells expressing F proteins were surface-labeled with biotin on ice. After cell lysis, biotinylated proteins were immunoprecipitated using NeutrAvidin beads and subjected to SDS-PAGE under non-reducing conditions. Precipitated F proteins were visualized using an antibody against the HA-tag (H6908), HRP-labeled secondary antibodies and chemiluminescence. Representative blots are shown from four independent experiments. c Intracellular localization of wt and mutant CedV F proteins in MDCK-2 cells. F proteins are stained with anti-HA-tag specific primary antibodies and AlexaFluor488-conjugated secondary antibodies. The endoplasmic reticulum (ER) was visualized using a pDS Red2-ER plasmid-derived red fluorescent labeling. Representative images from two independent experiments are displayed. Inserts show magnifications of indicated areas. Magnification, × 63