3h labeled para aminohippuric acid

Parental COS-7 cells were purchased from ATCC (Manassas, VA). [³H]-labeled para-aminohippuric acid was purchased from PerkinElmer (Waltham, MA). Membrane-impermeable biotinylation reagent sulfo-NHS-SS-biotin, streptavidin agarose beads, protein G-agarose beads, and protease inhibitor cocktail were obtained from Pierce Biotechnology (Rockford, IL). Plasmid for wild type human Nedd4–2 was generously provided by Dr. Peter Snyder from University of Iowa (Iowa City, IA). Site-directed mutagenesis kit was purchased from Agilent Technologies (Santa Clara, CA). Mouse anti-myc and anti-β-actin antibodies were purchased from Roche (Indianapolis, IN). Mouse anti-E-cadherin antibody was purchased from Abcam (Cambridge, MA). Mouse anti-FLAG antibody and anti-FLAG M2 affinity beads were purchased from Sigma-Aldrich (St. Louis, MO). Rabbit anti-phospho-Ser/Thr antibody was obtained from Cell Signaling Technology (Danvers, MA). Phorbol 12-myristate 13-acetate (PMA), staurosporine (St), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO).