Dopal

Human recombinant AS was purchased from Calbiochem (La Jolla, CA, USA), mutant A53T-AS from rPeptide (Bogart, GA, USA). DA, L-DOPA, Dithiothreitol, benomyl, and Dulbecco’s Modified Eagle’s medium (DMEM) were from Sigma (St. Louis, MO, USA). DOPAL was from Santa Cruz Biotech (Dallas, TX, USA). All these reagents were dissolved in Type 1 water. Mouse anti-AS antibody and cell culture medium were from Invitrogen (Camarillo, CA, USA). Human glioblastoma (U118MG (ATTC^R HTB-15^Trade;), non-adherent PC12 cells, and F-12K medium were from ATTC (Manassas, VA, USA) and human oligodendrocytes (MO3.13) from Cellutions Biosystems Ins (Burlington, Ontario, Canada). The aldehyde reductase inhibitor AL-1756 was a gift from Alcon Laboratories, Fort Worth, TX. Tolcapone was from Orion Pharma (Espoo, Finland). benomyl, AL-1576, and Tolcapone were dissolved in DMSO and stored at −20 °C. Co-incubation of PC12 cells with glial cells was done using ThinCert membranes from Greiner Bio-One North America Inc. (Monroe, NC, USA). For Western blotting NuPage LDS Sample Buffer (4X) (Invitrogen, ThermoFisher Scientific, Waltham, MA) containing sodium dodecyl sulfate (SDS) was used.

PC12 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA; PC12 cells catalog no. CRL-1721); F12K cell culture medium from Gibco Life Technologies (Grand Islands, NY); tolcapone (to block catechol-O-methyltransferase) from Orion Pharma (Espoo, Finland); DOPAL from Santa Cruz Biotechnology, Inc. (Dallas, TX); and Cys-DA from the NIMH Chemical Synthesis and Drug Supply Program (No. C-805).
Non-adherent, non-differentiated cells PC12 cells were stored in liquid nitrogen until passaged for experiments. The cells were grown in F12K medium with 15% horse serum and 2.5% fetal bovine serum and incubated at 37 °C in a 5% carbon dioxide atmosphere. The medium was replaced several times per week; the cells were passaged once per week.
At 24 hours prior to plating for experiments, the cells were centrifuged. The medium was replaced with medium containing 10 μM tolcapone. The experiments began after 24 hours of incubation in tolcapone-containing medium. The cells were collected, suspended in the same medium, and counted (Cellometer, Nexcelom Bioscience, Lawrence, MA). About 500,000 cells/well were plated in 12-well plates.