Pulse-chase labeling of full-length polycystin-2 and polycystin-2 (1–703). Stably transfected HeLa and LLC-PK1 cells inducibly producing HA epitope–tagged full-length polycystin-2 (a–c) and polycystin-2 (1–703; d–f) proteins were metabolically labeled with 35S-methionine/cysteine. The HA-tagged proteins were immunoprecipitated with an anti-HA epitope antibody, digested with PNGase F and endoglycosidase H (Endo H) when indicated, run on a polyacrylamide gel, and visualized by autoradiography. (a–c) The full-length polycystin-2 can be digested with both glycosidases at any time point after the pulse. Because the top band (filled arrowhead) disappeared after a digest with calf intestinal phosphatase (not depicted), it represents a phosphorylated form of polycystin-2. (d–f) The truncated polycystin-2 protein migrated as a smear (large arrow) ∼2 h after the pulse. The smear was resistant to endoglycosidase H and therefore probably represents protein that has progressed beyond the cis-Golgi apparatus. Because both bottom bands (filled and open arrowhead) collapsed into one faster-migrating band (small arrow) after endoglycosidase H treatment, they represent a pool of truncated polycystin-2 that did not reach the mid-Golgi apparatus. Molecular masses (in kD) are indicated on the left of each panel.
