Ab2788

The mice were anesthetized using 3.6% chloral hydrate and then decapitated. Striatal tissues from these bilaterally injected mice were dissected out and processed for Western blot analysis. Briefly, after measurement of protein concentration, samples were diluted with five volumes of SDS-PAGE buffer. These diluted samples were separated by SDS-PAGE (12% with a 5% concentrating gel) in reducing conditions and electro-transferred to PVDF membranes (Millipore, US). After blocking for 2 h at room temperature with 5% milk in Tris-buffered saline (pH = 7.6) containing 0.05% Tween-20 (TBS-T), the membranes were incubated overnight at 4°C with anti-LC3B antibody (Abcam, 1:1,000, ab48394), anti-Beclin1 antibody (Abcam, 1:500, ab62557), anti-SQSTM1 antibody (Abcam,1:1,000, ab56416), anti-LAMP2A antibody (Abcam, ab18528,1:1,000), anti-Hsc70 antibody (Abcam,1:1,000, ab2788), anti-PSMC3 (Abcam, ab171974, 1:1,000), anti-Proteasome 20S beta 6 (abcam, ab150392, 1:1,000) and β-actin (Abcam, ab8226, 1:1,000). After three washes, the membranes were incubated with goat anti-Rabbit secondary antibody (Li-COR, P/N 925-32211, 1:5,000) or goat anti-Mouse secondary antibody (Li-COR, P/N 925-32210, 1:5,000) for 2 h at room temperature. The images of Western blot were taken by LI-COR Odyssey.

The cell precipitates and uEVs pellets were dissolved in RIPA buffer (Bioss, China), phenylmethanesulfonyl fluoride (PMSF) (Solarbio, China), and phosphatase inhibitor cocktail II (MedChemExpress, USA) were added, and the protein supernatant was collected after centrifugation. The extraction protein concentration was determined with a bicinchoninic acid protein assay kit (Servicebio, China). Equal amounts of proteins were separated via 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Vazyme, China). The proteins were subsequently transferred to 0.2 µm polyvinylidene difluoride membranes (Millipore, USA), which were blocked with 5% bovine serum albumin for 1 h and then incubated with primary antibodies overnight at 4 °C. The primary antibodies used were rabbit anti-CD63 (Abcam/ab92726, USA), rabbit anti-TSG101 (Abcam/ab2788, USA), rabbit anti-calnexin (Abcam/ab22595, USA), rabbit anti-NDUFS1 (ABclonal/A21192, China), and rabbit anti-GAPDH (Proteintech/10494–1-AP, China). Afterward, the membranes were incubated with a secondary antibody conjugated to horseradish peroxidase (Proteintech, China) for 1 h at room temperature and then visualized with a chemiluminescence imaging system (General Electric, USA).

Immunoprecipitation of Hsp70 was performed with an Immunoprecipitation Kit Dynabeads^® Protein G (Thermo Fisher Scientific, Schwerte, Germany) as described in the manufacturer's protocol. HT22 cells were lysed in PBS due to repeated disruption steps and 10 min centrifugation at 20,000 × g. Hsp70 was eluted using the mouse monoclonal Hsp70/Hsp72 antibody [C92F3A] (ADI-SPA-810, Enzo Life Sciences, Loerrach, Germany). Unbound fractions and immunoprecipitate were analyzed using protein carbonyl immunoblot. Hsp70 was detected using polyclonal rabbit Hsp701A antibody (PA5-28003, Thermo Fisher Scientific, Schwerte, Germany), which detects also Hsc70. To exclude that also Hsc70 is immunoprecipitated we detected also Hsc70 using the monoclonal Hsc70 antibody (ab2788, Abcam, Cambridge, UK).
To detect the interaction of Hsp70 with proteasomal subunits the Pierce Co-Immunoprecipitation kit (Thermo Fisher Scientific, Schwerte, Germany) was used, which allows no co-elution of antibody heavy and small chains, since the latter interferes in the analysis of proteasomal subunits in immunoblot detection. Hsp70 was detected using the mouse monoclonal Hsp70/Hsp72 antibody [C92F3A].