Five microliters of lysate and 25 µL of bead eluent were resolved on 4 to 20% gradient Bis-Tris gels (ThermoFisher) and blotted onto activated polyvinylidene difluoride using wet transfer with settings at 120 V for 1 h. Following transfer, membranes were rinsed with PBS and then fixed with 4% paraformaldehyde as previously described (41 (link)). Membranes were then dried completely and reactivated with methanol just prior to use. Membranes were blocked in TBST containing 5% bovine serum albumin for 2 h at room temperature. Membranes were then incubated overnight at 4 °C with blocking buffer containing one of the following antibodies: 1) anti–alpha-synuclein “SYN1” (BD Lifesciences) and 2) “MJFR1” (Abcam), 3) anti-ubiquitin conjugates “UBCJ2” (Enzo Life Sciences), and 4) anti-PSER129 “EP1536Y” (Abcam). To determine enrichment of biotinylated proteins, one membrane was incubated with prepared ABC reagent, diluted 1:10 in blocking buffer for 1 h at room temperature (Vector Laboratories). The next day, membranes were washed three times for 10 min each time with TBST and incubated for 1 h with either anti-rabbit (1:5,000) or anti-mouse (1:10,000) HRP-conjugated secondary antibodies (Cell Signaling Technologies) diluted in blocking buffer. Membranes were then washed three times for 10 min each time with TBST and detected using chemiluminescence substrate (Bio-Rad).