Figure 7.

Synthesis and delivery of secretory proteins in hSec10 overexpressing cells. (a) Western blot by using antibody against gp80 demonstrates increased steady-state levels of gp80 in three hSec10 transfected clones (1, 2, 3) compared with control cells (C). (b–g) Synthesis and secretion assays were performed by pulse and pulse-chase with [³⁵S]methionine. (b) hSec10 overexpressing and control cells were metabolically pulse labeled for 20 min with [³⁵S]methionine and the amount of newly synthesized gp80 was measured. (c) Other secretory proteins were also increased in hSec10 overexpressing versus control cells. Cells were pulse labeled for 20 min with [³⁵S]methionine, and chased for 1 h. Then the apical (A) and basolateral (B) media were collected and aliquots run on SDS-PAGE and analyzed by phosphorimaging. The entire gel is shown and the position of gp80 indicated by the arrow. We show this experiment in triplicate to demonstrate that even though there is some variability, there is a general consistency with respect to the increase in apical and basolateral protein secretion across a broad spectrum of secretory proteins. (d and f) hSec10 overexpressing and control cells were metabolically pulse labeled for 20 min. The apical and basolateral media were collected for the indicated time intervals during the subsequent chase. Aliquots were analyzed by SDS-10% PAGE and radioactivity associated with gp80 was determined by using a phosphorimager. Total secretion of gp80 was increased both apically (d) and basolaterally (f). (e and g) Data in d and f were replotted in e and g, respectively, to emphasize the kinetics of secretion, i.e., the fraction of the total gp80 secreted as a function of time. Data for each condition were normalized, so that the total gp80 secreted into the apical (e) or basolateral (g) media by the end of the 120-min time course was taken as 100%. The cumulative secretion at each time point was then plotted. These results indicate that although the amounts of gp80 secreted apically and basolaterally were increased by hSec10 transfection, the kinetics of secretion, measured as cumulative percent of total over time, was unchanged.