Figure 5. ER exit of Tg region I-II-III in the absence and presence of ChEL domain.

(A) 293 cells were either untransfected (control) or transiently transfected with 2 μg plasmid DNA encoding either full-length wild-type mouse Tg or Tg region I-II-III. Cells were pulse labeled and chased and samples prepared and immunoprecipitated with anti-Tg as in Figure 4, with analysis by reducing 4% SDS-PAGE and fluorography. Arrows highlight secretion (or lack thereof) from cells to media. (B) The first lane represents untransfected control cells. The remaining cells transfected to express Tg region I-II-III were pulse labeled as in A and then lysed without or with 4 hours of chasing. Tg immunoprecipitates from cell lysates and media were either mock digested or digested with endoglycosidase H for 1 hour at 37°C, before reducing 4% SDS-PAGE and fluorography. Lanes were run contiguously but have been separated for clarity. The positions of molecular mass standards are shown at left. (C) Lower panels: 293 cells were transiently transfected with 0.5 μg plasmid DNA encoding I-II-III cotransfected with 2.5 μg of plasmid DNA encoding the secretory ChEL domain or untransfected controls. Cells were pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased for the times indicated. At each chase time, the cells were lysed and both lysates and media immunoprecipitated with polyclonal anti-Tg to recover both I-II-III and secretory ChEL proteins, as revealed by reducing 5.5% SDS-PAGE and fluorography. The upper panel represents a negative control with identical transfection of I-II-III but without cotransfection of the secretory ChEL plasmid.