Ribbon determinants are localized to the Golgi. (A–L) Rescue of the ribbon by coinjection of Golgi extract with tubulin. Tubulin alone (A–D) or Golgi extract without (E–H) or with tubulin (I–L) were microinjected into the cytoplasts (asterisks). After 2 h of incubation, cells were stained for GFP (B, F, and J), tubulin (C, G, and K), and DNA (D, H, and L). Injection of tubulin or Golgi extract alone had no effect (B and F, asterisks), whereas Golgi extract together with tubulin reconstituted a Golgi ribbon (J, asterisk). Karyoplasts are marked by arrows. Bar, 10 µm. (M) Distribution of Golgi elements in the injected cytoplasts. A ribbon is formed if the Golgi is in no more than three continuous structures. (N) Equal amounts of protein (4 µg) of rat liver homogenate (H), cytosol (C), intermediate fraction (I), and Golgi membranes (RLG) were immunoblotted for the Golgi proteins GM130 and GRASP65, nuclear pore complex protein Nup153, ER enzyme PDI, motor protein dynein, and centrosome marker pericentrin. GM130 and GRASP65 were highly enriched on RLG membranes, whereas Nup153, PDI, dynein, and pericentrin were not detectable. (O) Medial/trans-Golgi proteins are enriched in the detergent extract. RLG membranes were extracted with n-octylglucoside, cleared by centrifugation (pellet [P]), and the supernatant was dialyzed to remove the detergent (S). Equal volumes of each fraction were immunoblotted for the indicated proteins. Cis-Golgi proteins were de-enriched (e.g., Golgin84, GM130, and GRASP65), whereas markers of the medial/trans-Golgi and the TGN were enriched in the extract (e.g., Golgin45, GRASP55, Vti1a, and TGN38). MT, microtubule; RSA, rat serum albumin.