Stress-induced phospho-ubiquitin formation causes parkin degradation

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

To prepare crude mitochondrial and cytosolic fractions from cells, the following protocol was followed. Cells and lysates were kept on ice for the duration of the procedure, and all centrifugation steps were performed at 4°C. First, cells were sprayed from the dish in PBS and pelleted by centrifugation. Cells from four wells of a 24-well plate were combined in one tube. Analysis of mRNA by qPCR was performed as in [14] . Briefly, total cellular RNA was extracted using TRI reagent (Molecular Research Center). cDNA was synthesized using the firststrand cDNA synthesis kit (Origene). qPCR was performed using FastStart SYBR Green Master The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 Immunofluorescence (for quantification of viral titer) Cells were fixed for 15 min in 4% paraformaldehyde and then washed 3 times with 1X PBS. Cells were then blocked with Superblock blocking buffer supplemented with 0.3% Triton X-100 for 1-2 h at room temperature and incubated overnight at 4°C with primary antibody in Superblock/Triton X (chicken anti-GFP, 1:1000, #A10262, Thermo Fisher Scientific) with gentle shaking. The next day, primary antibody was washed off in 3x8-minute washes with gentle shaking using 1X PBS. Cells were then incubated with fluorescent secondary antibody in Superblock/Triton X for 1 hour with gentle shaking (AlexaFluor-488 anti-chicken, 1:1000, #A11039, Thermo Fisher Scientific). Cells were then washed 3 X 8 minutes in 1X PBS with gentle shaking. Hoechst 33328 was added in the first wash (1:2500). Cells were kept in 1X PBS before imaging using an inverted fluorescence microscope.