FIGURE 5.

Secretion rescue of mutant rdw-Tg by co-expressed wild-type Tg. Side-by-side, identical wells of 293 cells were co-transfected with plasmids encoding empty vector plus wild-type Tg-Myc (wt-Tg-Myc) and rdw-Tg-GFP such that total plasmid DNA and rdw-Tg-GFP DNA was held constant in all samples (wt:rdw plasmid ratio 6:1); Control lanes were transfected only with empty vector. At 48 h post-transfection, the medium was changed, and cells were cultured for an additional 24 h in complete growth medium. The cells were lysed, and 3.33% of each cell lysate and 1% of each collection of medium were resolved by SDS-PAGE and analyzed by immunoblotting with anti-GFP antibody. Left panels: direct immunoblotting of lysates and media. Middle panels: endoglycosidase H (Endo H) digestion of the samples indicated prior to immunoblotting. Note that the small mobility shift observed after endo H digestion of the medium is distinct from the larger shift seen for intracellular Tg, indicating Golgi sugar modification of Asn-linked glycans on all secreted rdw-Tg-GFP molecules (although some sugar chains on each molecule remain endo H-sensitive). Right panel: media from co-transfected or control cells were immunoprecipitated with α-Myc antibody, and co-precipitated rdw-Tg-GFP (co-IP) was analyzed by immunoblotting as in the other panels.