Figure 4.

Pulse-chase analysis of PC processing. At the end of the pulse (0 min, lane 1), two precursors of ∼70 and ∼55 kDa (open circles) are seen that are sensitive to endo H digestion (lane 2), yielding an ∼50-kDa doublet (closed circle). At 10 min of chase (lane 4), two bands, a mature glycosylated ∼140-kDa band (asterisk) and an immature glycosylated ∼100-kDa band (arrowhead), are seen. Both are insensitive to endo H but sensitive to sialidase digestion, yielding ∼170- and ∼95-kDa bands (closed circles). After 20 min of chase (lanes 7–9), a 250-kDa band gradually accumulates that most likely represents a dimer of PC (diamond). At 60 min of chase (lanes 10–12), the two PC precursors (∼70 and ∼55 kDa) are no longer present, but the two sialidase-sensitive bands persist. Size markers indicate molecular mass in kilodaltons. MDCK-PC8 cells, stably expressing PC, were pulsed for 15 min with [³⁵S]EasyTag Express protein-labeling mix and chased in medium containing excess unlabeled methionine and cysteine as described in MATERIALS AND METHODS. Lysates were collected at 0–60 min after pulse, and PC was immunoprecipitated with affinity-purified anti-PC IgG (0601). Immunoprecipitates were incubated with or without endo H (Streptomyces plicatus) or sialidase (Arthrobacter ureafaciens), separated by SDS-PAGE, and detected by fluorography.