Figure 4.
Immunoprecipitation of membrane-bound coatomer and its individual subunits. In vitro-translated [35S]ARF-(Tmd)Phe-82 was bound to CHO Golgi membranes in the presence of GTP[γS] and coatomer as described in Fig. 2, lane 2. After irradiation of membrane-bound ARF, the membranes were solubilized in detergent-buffer and the coatomer complex was immunoprecipitated using anti-coatomer-antibody (lane 2), anti-γ-COP antibody (lanes 3 and 4), anti-β′-COP antibody (lanes 5 and 6), and anti-β-COP antibody (lanes 7 and 8). In some cases, coatomer was denatured prior to immunoprecipitation to allow immunoprecipitation of the individual subunits (lanes 4, 6, and 8). Immunoprecipitation with a pre-immune serum is shown in lane 1. (a) As a control, immunoprecipitated material from incubations shown in b was analyzed for the presence of individual coatomer subunits by SDS/PAGE (19) and Western blot analysis with the respective antibodies. (b) Immunoprecipitated material was analyzed by SDS/PAGE (19) and autoradiography.