Fig. 3. The WW domain 1 Nedd4 mutant interacts with membrane-bound Spt23. (A) Cells transformed with Spt23 and the indicated Nedd4 expression constructs or vector alone control (V) were grown as described in the legend of Fig. 2C and lysed in RIPA buffer. The extract (600 µg per immunoprecipitation reaction) was pre-cleared for 1 hour with protein-G sepharose. After a brief centrifugation, the supernatant was transferred to a new tube containing 1 µg of the anti-FLAG antibody M5 or an isotype control (C). After a 4 hour incubation, protein-G sepharose was added and the mixtures were incubated for an additional 2 hours. Protein complexes were washed three times with cold lysis buffer, eluted from beads by resuspending in 1x SDS-PAGE loading buffer, and Nedd4 westerns were performed as described above. Ponceau S staining of the blot after transfer revealed equivalent amounts of immunoprecipitating antibody. (B) Immunoprecipiations were performed as described in (A), except that the Nedd4 polyclonal was used in the immunoprecipitations and the M5 antibody was used for the western blot.

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