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. Author manuscript; available in PMC: 2008 Feb 9.
Published in final edited form as: Mol Cell. 2007 Feb 9;25(3):385–397. doi: 10.1016/j.molcel.2007.01.024

Fig 1. Cdc48pGSTNpl4p/Ufd1p interacts with poly-ubiquitinated Mga2p120 and Mga2p90.

Fig 1

(A) (Left panel) Recombinant GST-tagged Cdc48p, Npl4p and Ufd1p proteins were produced in bacteria and purified with glutathione beads. GST-tagged Cdc48p and Ufd1p were liberated from beads using Procession Protease. Proteins were then mixed with bead-bound GST-Npl4p or GST alone control and protein complexes were allowed to form. Beads were pelleted, resuspended in SDS-PAGE loading buffer, boiled, resolved by SDS-PAGE, and the formation of Cdc48pGSTNpl4p/Ufd1p complex was verified by Coomassie blue staining. (Right Panel) Microsomes were isolated from yeast expressing FLAG-tagged Mga2p or empty vector control and solublized. Proteins were immunoprecipitated with anti-FLAG antibody conjugate agarose. Proteins were eluted with FLAG peptide, separated by SDS-PAGE, and visualized by Coomassie blue staining. (B) Agarose captured Mga2p120-Mga2p90 complexes were placed in in vitro ubiquitination reactions containing indicated E1, the E2 Ubc1p, and varying amounts of Rsp5p (600 ng, 200 ng, 66 ng). After termination, agarose beads were washed to remove enzymes and complexes were eluted with FLAG peptide. Western blotting was performed on duplicate gels with anti-FLAG and anti-Ub antibodies. * Denotes an alternatively process Mga2p product, see text for details. (C) Products from the in vitro ubiquitination reactions were incubated with glutathione beads containing GST or Cdc48pGSTNpl4p/Ufd1p. Beads were washed, resuspended in SDS-PAGE loading buffer and samples were boiled. Equal amounts of eluted material were run on duplicate 8.0% SDS-polyacrylamide gels and western blotting was carried-out separately with anti-FLAG and anti-Ub antibodies. Ponceau S staining of both membranes (only the one for the anti-FLAG blot is presented) was performed to show equivalent amount of recombinant proteins in each binding reaction. (D) Binding assays were carried-out using the indicated GST-fused proteins/complexes and ubiquitin modified or unmodified Mga2p120-Mga2p90 complexes.