Fig. 5. Spt23 processing is inhibited by WW-domain1- or ligase-defective Nedd4 and Rsp5 mutants. (A) Cells were transformed first with the pESC-FLAG-Spt23 expression construct and then with the indicated Nedd4 and Rsp5 expression constructs. Cells were processed as described in the legend of Fig. 2C. 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) Equal amount of extract prepared from cells producing FLAG-tagged Spt23 and the indicated Rsp5 proteins were subjected to immunoprecipitation with the anti-HA antibody 12CA5 (12) 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.