Western blotting of primary neuronal lysate

For in vitro lysates, all cell lysis and harvesting steps took place on ice. Cells were washed twice with ice-cold 1× PBS and after the second wash, 150–400 μl of TNEB lysis buffer was added.

Approximately 100–200 μl of lysate was resuspended in an equal volume of Laemmli buffer with β-mercaptoethanol (1:20). Protein samples were heated to 95°C for 10 min and stored at −20°C. Equal protein concentrations were determined by DC Protein Assay Kit II and samples were separated on 4–12% Criterion Bis-Tris gels. Proteins were transferred to PVDF membranes and blocked in methanol for 5 min. After 10 min of drying, the membranes were incubated overnight with antibodies detecting CHIP, HSP70, HSC70, TOM20, p62, PINK1, or LC3 in 5% nonfat dry milk in TBS-Tween (0.1%) or in membrane blocking solution at 4°C. Membranes were washed three times with TBS-Tween and incubated with horseradish peroxidase conjugated secondary antibodies for 1 h at room temperature (RT). After three washes in TBS-Tween, protein bands were visualized using Western Lightning chemiluminescence plus enhanced luminol reagent. Western blots were analyzed using ImageJ (RRID:SCR_002285) to determine the mean relative densities of each protein band and fold changes were calculated using untreated cultures as controls. Data represent results from at least three independent experiments as indicated in the figure legends.