Fig. 5.

Sialic acid is a terminal modification occurring immediately before secretion. A, HEK cell lines stably expressing HA-tagged WT human adiponectin or the K1–5R mutant (lacking the five known glycosylated lysines in the variable and collagenous domains) were metabolically labeled for 2 h before replacement of the radiolabeled amino acids with an excess of unlabeled amino acids and continued culture for increasing periods of ‘chase’. Lysates and media were harvested and immunoprecipitated with HA antibody before analysis by SDS-PAGE and autoradiography. B, 2DE and Western blotting of lysates, with and without Brefeldin A (BFA) treatment, and medium from CHO cells stably expressing human adiponectin. Data are representative of two independent experiments.