Figure 1.

Kalirin and Trio isoforms are expressed in secretory tissues and cell lines. (A) The indicated lysates (100 μg protein) were fractionated on 5% polyacrylamide gels and probed for Kalirin (top panel) or Trio (bottom panel). The mobility of recombinant Kalirin isoforms fractionated in parallel is indicated. The domain structures of the various Kalirin and Trio isoforms are depicted. Ctx, adult rat parietal cortex; AP, adult rat anterior pituitary; Adr, adult rat whole adrenal gland; AtT-20, mouse corticotrope tumor cells; GH3, rat somatomammotrope tumor cells; PC12, rat pheochromocytoma cells. The arrow indicates a nonspecific cross-reactive band. (B) AtT-20 postnuclear supernatant was subjected to velocity centrifugation (left panel); samples collected from the top were fractionated on 4–20% polyacrylamide gels and probed as indicated. Fractions enriched in unprocessed POMC and intermediate (1 and 2) were pooled as a source of immature secretory granules (ISGs). Fractions enriched in the 16-kDa fragment (4 and 5) were pooled as a source of mature secretory granules (MSGs). Pooled ISGs (middle) and MSGs (right) were subjected to equilibrium centrifugation. Fractions from these gradients were probed for Kalirin, Trio, TGN/immature granule/endosome marker VAMP4, POMC products, and the plasma membrane marker SNAP25.