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. 2008 Nov 3;183(3):513–526. doi: 10.1083/jcb.200804048

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Figure 1. — Binding of the retromer Vps subcomplex to active Rab7. (A) A Triton X-100 extract of microsomes was incubated with equivalent amounts of the indicated GST-Rab proteins preloaded with GMP-PNP or GDP. Bound proteins were eluted in Laemmli sample buffer and analyzed by SDS-PAGE and immunoblotting (IB) with antibodies to the Vps26, Vps29, or Vps35 subunits of retromer (top). The first lane represents 20% of the input. The approximate molecular masses of the different proteins are indicated in kilodaltons. Asterisks indicate contaminating proteins found in all of the GST preparations. (B) COS-7 cells were transfected with plasmids encoding myc-Vps26, FLAG-Vps29, and (HA)₃-Vps35, either individually or in the combinations indicated in the figure. Lysates from these cells were incubated with GST-Rab7–GMP-PNP–bound beads. Input and bound proteins were analyzed by SDS-PAGE and immunoblotting with antibodies to the myc, FLAG, and HA tags (top). (C) Immobilized GST, GST-Rab7, or GST-Rab9a preloaded with GDP or GTPγS was incubated with recombinant Vps26/29/35 trimer. Bound proteins were eluted and analyzed by SDS-PAGE and immunoblotting with antibodies to Vps29, Vps26, and Vps35 (top). The input represents 1.8% of the total recombinant Vps26/29/35 trimer used per reaction. The bottom panels show SDS-PAGE/Coomassie blue staining of the GST proteins used in these experiments.