Fig. 6. The WW domain 1 is required for proteasome-dependent degradation of Spt23 in mammalian cells. H1299 cells were transfected with pCEP-FLAG-Spt23 and pCEP (V), pCEP-wt Nedd4 (wt) or pCEP containing the indicated Nedd4 mutants by Lipofectamine. After transfection, cells were re-fed with complete media (-) or media containing the proteasome inhibitor MG115 (+). Cells were incubated for 9 hours prior to harvesting. Extracts were prepared as described in the Materials and Methods and western blots were performed with the Nedd4 polyclonal or the anti-FLAG antibody M5. Ponceau S staining of the blot after transfer revealed equivalent loading of total protein.