Figure 1.

ARF and coatomer binding in one- and two-stage reactions. The amount of membrane-bound ARF and coatomer (β-COP) determined by Western blot analysis of binding reactions. Lanes 1 and 2 show one-stage reactions in which ARF and coatomer were incubated together. Lanes 3–6 show the results from two-stage reactions in which the membranes were first incubated with ARF but not coatomer, reisolated, and then incubated in a second stage with coatomer but not ARF. As controls, membranes (lane 2) or ARF (lane 3) were excluded from stage 1 or coatomer (lane 4) was excluded from both stages. All incubations were carried out at 37°C except lane 5, which was carried out at 0°C.