Fig. 7

Granule sorting is mediated by both vasopressin and glycopeptide. a Stable AtT20 cells expressing the indicated provasopressin constructs were labeled with [³⁵S]methionine for 30 min and chased for an additional 2 h. From cell lysates and media, products were immunoprecipitated and analyzed by gel electrophoresis and autoradiography. The positions of full-size glycosylated (FLg) and unglycosylated forms (FLu) and of constructs without glycopeptide (∆gp) are indicated. b–e AtT20 cells stably expressing wild-type provasopressin (wt) or the mutants without the glycopeptide (∆gp) or with the V5xA mutant hormone sequence (5xA) or both (5xA∆gp) were stained for CgA as a granule marker, NPII, and nuclei (blue). Granules concentrate in terminal patches. Bar: 10 μm. f Quantitation of NPII staining in granule patches normalized for content of CgA. NPII/CgA ratios of ~70 images per construct (45 images for untransfected AtT20 cells, −) containing 4–5 cells each are summarized as a box plot showing the median and the center 50% of values in the box, with the whiskers including the 10th to the 90th percentiles. The central dot represents the mean. The original quantitation is shown on the left in gray and white. In black and gray, the data are shown upon correction for the relative amounts of pulse-labeled protein recovered from both media and cell lysates after a 2-h chase (three independent experiments including that shown in panel a; wt, 1.0; V5xA, 0.77 ± 0.20; ∆gp, 0.78 ± 0.20; V5xA∆gp, 0.68 ± 0.19). All pairwise comparisons, except wild-type vs. V∆gp, are significantly different according to an unpaired two-tailed t test (p < 0.001). The most important ones are indicated above the graph