Fig. 5.
A, Pulse/chase analysis of proSAAS processing in AtT-20 cells overexpressing rat proSAAS. Cells were labeled with [3H]Leu for 20 min and then chased for 0, 15, 30, or 90 min in unlabeled Leu, and then the media and cell extracts were immunoprecipitated with antisera to the C-terminal region (245–260) or the N-terminal region (42–59) of pro- SAAS. The immunoprecipitates were fractionated on a denaturing polyacrylamide gel that was treated with Fluro-hance (Research Products International) and exposed to film for 3 d. The position and molecular mass (in kilodaltons) of prestained molecular weight markers (Life Technologies) are indicated. B,Analysis of media from wild-type AtT-20 cells (bottom) and rat proSAAS overexpressing cells (top) using matrix-assisted laser desorption ionization time of flight mass spectrometry. Predicted POMC- and proSAAS-derived peptides that are within 1 mass unit of the observed peaks are indicated.LPH, lipotropin; ACTH, adrenocorticotropic hormone; CLIP, corticotropin-like immunoreactive peptide.