Figure 5.
ARF interacts with β-COP in COPI-coated vesicles. In the presence of [35S]ARF-(Tmd)Phe-82, isolated CHO Golgi membranes were incubated with bovine brain cytosol and GTP[γS]. The COPI-coated vesicles were separated from their donor membranes by sucrose density gradient centrifugation and the gradients were fractionated. (a) The amount of total 35S radioactivity present in each gradient fraction. (b) Analysis of the gradient fractions for the presence of β-COP and ARF by SDS/PAGE and subsequent immunoblotting using their respective antibodies. (c) Irradiation of the gradient fractions and subsequent analysis of crosslink products by SDS/PAGE and autoradiography. (d) Analysis of gradient fractions by SDS/PAGE and autoradiography without prior irradiation.