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. 2009 Nov 15;20(22):4720–4729. doi: 10.1091/mbc.E09-06-0453

Figure 2.

Figure 2.

Tripartite complex of Rabex-5, Rab22 and Rab5. (A) GST-Rabex-5(81-399) pull-down of various forms of Rab5. Myc-Rab5:WT, Myc-Rab5:Q79L, and Myc-Rab5:S34N were expressed in BHK cells, respectively, and cell lysates were clarified by centrifugation at 10,000 × g. The postnuclear supernatants were either directly analyzed (10 μl) by immunoblot assays with the anti-Myc antibody to confirm protein expression or incubated (180 μl) with GST-Rabex-5(81-399) on glutathione-Sepharose 4B resin in the pull-down assays to determine protein interactions, as indicated. The results were reproducible in two independent experiments. Molecular mass standards (in kilodaltons) are indicated on the left of the panel. (B) GST-Rab22 pull-down of Rabex-5(81-399) and Rab5:S34N. Myc-Rabex-5(81-399) was either expressed alone or coexpressed with Myc-Rab5:S34N in BHK cells and cell lysates were clarified by centrifugation at 10,000 × g. The postnuclear supernatants were either directly analyzed (10 μl) by immunoblot assays with the anti-Myc antibody to confirm protein expression or incubated (180 μl) with GST-Rab22-GTPγS or GST-Rab22-GDPβS on glutathione-Sepharose 4B resin in the pull-down assays to determine protein interactions, as indicated. The bound proteins on the resin were identified by immunoblot assays with the anti-Myc antibody. The results were reproducible in two independent experiments. Molecular mass standards (in kilodaltons) are indicated on the left of the panels.