Gtx110089

NP-40 Lysis Buffer (Beyotime, Shanghai, China) was used to extract total proteins as in our previous study19 (link). After extraction, the proteins were stored at −80 °C for subsequent western blot analysis. The analysis was performed as described previously57 (link). Briefly, proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane, and then incubated with primary rabbit antibodies specific to PINK1 (1:1000, ab23707, polyclonal, Abcam, Cambridge, MA, UK), Parkin (1:1000, A0968, polyclonal, ABclonal, College Park, MD, USA), Hsp60 (1:5000, GTX110089, polyclonal, Genetex Inc., Irvine, CA, USA) and GAPDH (1:2000, Zhongshan Golden Bridge Biotechnology, Beijing, China) or mouse antibodies specific to COX IV (1:2000, GTX101499, polyclonal, Genetex Inc., Irvine, CA, USA) and β-actin (1:2000, Zhongshan Golden Bridge Biotechnology, Beijing, China). The samples were incubated with horseradish peroxidase (HRP) -conjugated secondary antibodies (1:20000; ZB-2305 and ZB-2301, Zhongshan Golden Bridge Biotechnology, Beijing, China), and developed with Immobilon Western Chemiluminescent reagents (Millipore, Billerica, MA, USA).

The protocol for detection of reference protein markers by WB, ELISA and enzymatic assays is available at protocols.io (dx.doi.org/10.17504/protocols.io.bgc4jsyw), and antibody information at S1 Table. The enrichment in ER, cytosol, nucleus, mitochondria, PM, cis-Golgi and trans-Golgi was assessed by detection of Grp78, Gapdh, histone H3, Hsp60, flotillin 1, golgin A5 and golgin-97 proteins, respectively. Membranes were revealed with SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, MA, USA) and images acquired in a LI-COR C-DiGit Chemiluminescence Western Blot Scanner (LI-COR Biosciences, NE, USA).
Grp78, Gapdh and histone H3 were identified with 250X Endoplasmic Reticulum Fraction Western Blot Cocktail and 2500X horseradish peroxidase (HRP) Conjugated Secondary Antibody Cocktail (ab139415, Abcam, Cambridge, MA, USA), diluted 2000 and 2500 times, respectively.
For golgin-97, primary antibody (GTX114445, GeneTex, CA, USA) and an anti-rabbit IgG conjugated to HRP (ab205718, Abcam, Cambridge, MA, USA) were both diluted 2000 times. Anti flotillin 1 (GTX104769, GeneTex, CA, USA), anti Hsp60 (GTX110089, GeneTex, CA, USA) and anti golgin A5 (GTX104255, GeneTex, CA, USA) were diluted 2000, 10000 and 2000 times, respectively.