Downregulation of Nedd2 in PC12 cells by Penetratin 1-linked antisense oligonucleotide to Nedd2 (V-ANedd). Naive and neuronally differentiated PC12 cells (pretreated for at least 7 d with NGF) were plated on Matrigel-coated multichamber slides. V-ANedd (400 nm) was added to the indicated cultures after plating. Naive cultures were grown in RPMI 1640 medium with 5% FCS/10% horse serum; neuronal cultures were grown in RPMI 1640 medium with 1% horse serum and NGF (100 ng/ml). After overnight maintenance, cells were fixed in ice-cold methanol and then immunostained with anti-N-Nedd (see Materials and Methods). Cells were observed with a Nikon fluorescence microscope, 120× magnification.