Regulation of Nedd2 by V-ANedd. Naive PC12 cells were grown for 24 hr in serum-free RPMI 1640 medium in the presence or absence of NGF (100 ng/ml) and with or withoutV-ANedd (400 nm) or V-SNedd(400 nm), as indicated. The cells were extracted in sample buffer, the extracts were boiled, and equal amounts of protein were resolved by 10% SDS-PAGE and transferred to nitrocellulose. Blots were probed with (A) anti-N-Nedd at 1:500 or (B) anti-C-Nedd at 1:330, and staining was visualized with ECL. Bands were quantified with Scion Image software and normalized against peripherin levels. The level of downregulation is representative of that obtained in five independent experiments.