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. 1998 Mar 9;140(5):1013–1021. doi: 10.1083/jcb.140.5.1013

Figure 6.

Figure 6

p115 binds to giantin on COPI vesicles and GM130 on Golgi remnants. Golgi membranes were incubated at 37°C with interphase (30 min) or mitotic cytosol (20 min) in the presence of GTPγS, salt was added, and samples were fractionated to separate COPI vesicles from Golgi remnants (see Materials and Methods). Samples (8–10 μg) were incubated for 15 min on ice with 100 ng p115. Antibodies to GM130 and giantin were used to pretreat the membranes for 15 min on ice before addition of p115. The GM130 peptide was added together with the p115 at a 70-fold molar excess. Membranes were reisolated, fractionated by SDS-PAGE, the amount of bound p115 quantitated by Western blotting and normalized for β-COP (COPI vesicles) or GM130 (Golgi remnants). Membrane recovery was 80–90% for COPI vesicles and 70–80% for Golgi remnants. Results are expressed as a percentage of the interphase vesicles or remnants ± SEM (n = 3–5, depending on the experiment). The 100% values were 88 ± 5.8 ng p115/10 μg interphase vesicles and 112 ± 18.3 ng/10 μg interphase remnants. (A) Binding to COPI vesicles. (B) Binding to Golgi remnants.