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. Author manuscript; available in PMC: 2009 Oct 19.
Published in final edited form as: Traffic. 2008 Aug 9;9(11):1972–1983. doi: 10.1111/j.1600-0854.2008.00817.x

Figure 9. The Alix V domain binds ubiquitin-agarose beads.

Figure 9

A) Cells were transfected with the indicated HA-tagged expression plasmids. Twenty-four hour posttransfection, cell lysates were incubated with ubiquitin-agarose (Ub agarose) or protein A-agarose (PrA agarose) beads. Eluted complexes were resolved by SDS-PAGE and immunoblotted with anti-HA antibody. In the top ‘lysates’ panels, cell lysates were subjected directly to SDS-PAGE and immunoblotted with anti-HA antibody. Alix-F and TSG-F denote full-length Alix and Tsg101, respectively. Lanes C, mock-transfected controls. B) Binding of EIAV/ΔYPDL-Ub Gag to Alix requires an intact sorting motif. Cells were transfected with vectors expressing WT EIAV, EIAV/ΔYPDL, ΔYPDL-Ub or ΔYPDL-Ub L8A/I44A along with Alix full length (Alix-F) or Alix V (Alix-V) domain expression vector. Cell lysates were immunoprecipitated with anti-HA beads followed by western blot using anti-EIAV antibody (top). Western blot of cell lysates using anti-EIAV serum (middle) or anti-HA antibody (bottom).