Fig. 4. Inhibition of Spt23 processing by the WW domain 1 mutants requires WW domains 2 and 3, and both of these C-terminal WW domains are required for Spt23 binding. (A) Cells were first transformed using the pESC-FLAG-Spt23 expression construct and then with pYes (v), pYes-wt Nedd4 (wt), pYes-WW1 (-) and pYes-WW domain 1 mutant Nedd4 expression vectors harboring substitutions in the indicated WW domains. Cells were processed as described in the legend of Fig. 2C. Measurement of Nedd4 protein in the extract was performed by a western blot using the anti-Nedd4 polyclonal while the amount of membrane-bound (MB) and processed Spt23 was measured by the anti-FLAG tag antibody M5. Ponceau S staining of the blot after transfer revealed equivalent loading of total protein. (B) An equal amount of extract prepared from cells producing FLAG-tagged Spt23 and the indicated Nedd4 proteins were subjected to immunoprecipitation with the anti-FLAG antibody M5 or an isotype control antibody (C). The amount of Nedd4 protein immunoprecipitated with Spt23 was then determined by a western blot. Ponceau S staining of the blot after transfer revealed equivalent amounts of immunoprecipitating antibody.