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. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: Arch Biochem Biophys. 2008 Aug 20;479(1):63–68. doi: 10.1016/j.abb.2008.08.009

Fig. 2. Cargo-sorting signals induce AP-1 polymerization on peptidoliposomes.

Fig. 2

(A) Bovine adrenal cytosol (5 mg/ml) was incubated with peptidoliposomes and GTPγS (100 µM) in a 500 µl reaction for 15 min at 37 °C. After pelleting the liposomes by centrifugation, the membranes were solubilized in 200 µl of 2 % taurocholate in assay buffer and loaded onto 10–25% linear sucrose gradient and centrifuged at 50,000 rpm for 3 hours. Samples were then analyzed as described in the experimental procedures. (B) Cargo-sorting signals promote recruitment of purified AP-1 to peptidoliposomes in an Arf-1/GTP-dependent manner. Affinity-purified AP-1 (15 nM) was incubated with the indicated peptidoliposomes, recombinant myristoylated Arf-1 (2 µM), and 100 µM GTPγS in a 100 µl reaction for 15 min at 37 C. The liposomes were then pelleted by centrifugation and subjected to SDS-PAGE and western blotting with either anti-γ-adaptin mAb 100/3 or anti-Arf mAb 1D9 antibodies, respectively; The doublelet detected by mAb 100/3 is the result of proteolytic cleavage of the appendage from a portion of the γ-subunits.