Figure 8.

γ-Tubulin is involved in the nucleation of Golgi-based MTs. (A) As indicated, purified Golgi was preincubated either with anti–γ-tubulin or with preimmune mouse IgGs for 1 h at 37°C and then used in the in vitro MT assembly assay. Samples were processed for the immunofluorescence labeling of α-tubulin. (B) Purified rat liver Golgi was washed as indicated with 2 M KCl and then rinsed three times in PEM buffer before being assayed for the presence of tubulins or to be used in the in vitro MT assembly assay. Samples were analyzed for the presence of tubulins by Western blotting, as described in Figure 7. After the in vitro MT assembly assays, samples were subjected to the immunofluorescence labeling of α-tubulin. (C) Salt-washed Golgi membranes were incubated (1 h, room temperature) with crude WIF-B cytosol or cytosol that was immunodepleted of γ-tubulin and then rinsed twice in PEM buffer and either analyzed by Western blot for the presence of peripherally bound γ-tubulin or used in the in vitro MT assembly assay, followed by the immunofluorescence labeling of α-tubulin. Scale bar, 10 μm.