Figure 7. ChEL interaction improves recovery as well as secretion of Tg I-II-III.

(A) 293 cells were triply transfected with empty vector plus a plasmid encoding Tg region I-II-III (always 0.1 μg DNA per well) plus a plasmid encoding the secretory ChEL domain (at different DNA levels as shown). DNA in each transfection totaled 3.1 μg per well. Transfected cells were pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased for 6 hours, at which time the cells were lysed, and both lysates and media were immunoprecipitated with anti-Tg and analyzed by reducing 5.5% SDS-PAGE and fluorography, as shown. The position of a 76-kDa molecular mass marker is shown at left. The figure has been spliced at the position indicated by a black line (between lanes 2 and 3), but all data were derived from a single exposure of the same gel. (B) Cells were either untransfected or transfected with 0.5 μg plasmid DNA encoding I-II-III plus 2.5 μg of vector DNA or that encoding secretory ChEL. As a positive control, 2 μg of plasmid DNA encoding wild-type Tg was transfected in parallel. Cells were pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased for either 0 or 16 hours, at which time the cells were lysed and both lysates and media immunoprecipitated with anti-Tg and analyzed by reducing 5.5% SDS-PAGE and phosphorimaging, as shown. The intracellular band density at the 0 chase time was defined as 100%; based on this, the recovery of each band at 16 hours is shown. Total recovery of labeled I-II-III alone at 16 hours was approximately 35%, while total recovery of labeled I-II-III (cells plus media) in the presence of secretory ChEL was approximately 68%.