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. 2008 May;19(5):1942–1951. doi: 10.1091/mbc.E08-02-0110

Figure 2.

Figure 2.

AP-3 recruitment is ARF-1 dependent. (A) Liposomes with LAMP-1, LIMP-II, and no cytoplasmic domain (−cd) were incubated with cytosol and GTPγS. AP-1γ, AP-3σA/B, and ARF-1 bound to liposomes were detected after SDS-PAGE and Western blotting by using specific antibodies. (B) Liposomes with LIMP-II cytoplasmic domains were incubated with ARF-depleted cytosol (3 mg/ml) and supplemented or not with recombinant, myristoylated ARF-1 (30 μg/ml) with or without GTPγS and with or without brefeldin A (100 μg/ml). AP-1γ, AP-3σA/B, and ARF-1 bound to liposomes were detected after SDS-PAGE and Western blotting. (C) Top, liposomes with increasing amounts of LIMP-II wt (♦), LIMP-II L18A, I19A (■), or no cytoplasmic domain (−cd) (♦) were incubated with cytosol and GTPγS. Bottom, liposomes with increasing amounts of gpI (♦), gpI Y10, 23A, DAC (■), or no cytoplasmic domain (−cd) (♦) were incubated with cytosol in the presence of GTPγS. After incubation, AP-1γ, AP-2α, AP-3σA/B, clathrin, and COP-Iβ bound to liposomes were detected after SDS-PAGE and Western blotting. (D) Quantification of C. The clathrin signal was quantified and normalized to the values obtained with gpI-containing liposomes.