Stress-induced phospho-ubiquitin formation causes parkin degradation

Lyudmila Kovalchuke; Eugene V. Mosharov; Oren A. Levy; Lloyd A. Greene

Inhibitors and antioxidants were dissolved in water when possible and in DMSO when not water-soluble. In most cases, stock solutions of the drugs were diluted in fresh medium before addition to cells (with the exception of cycloheximide, in which case fresh medium was added to cells the day before treatment and cycloheximide stock solution was added directly to cells). In such cases, drugs and cellular stressors (L-DOPA, H2O2, CCCP) were added to cells at the same time. In the cases of carbidopa and cycloheximide, the latter were added to cells before treatment with L-DOPA. Cells were pretreated with carbidopa for 1.5 hours and cycloheximide for ~15 minutes before L-DOPA addition. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/484857 doi: bioRxiv preprint precast Bis-Tris gels, MOPS SDS running buffer (or MES SDS running buffer for low molecular weight proteins), and NuPAGE transfer buffer. Generally, 4-12% gels were used and ~20 μg of protein was added per well. For one sample per gel, half the sample was added in a well adjacent to the full volume of sample to serve as a standard (see below). Proteins were transferred onto a nitrocellulose membrane for ~2 hours at 35V at 4°C. Following transfer, membranes were blocked for 1 hour at room temperature with gentle shaking in 5% milk in TBST (TBS + 1% Tween 20).