Anti pink1

Total protein was extracted from rat embryonic spinal cords (E11 and E12) or C17.2 cells using radioimmunoprecipitation (RIPA) buffer (Solarbio Science & Technology, Beijing, China). Protein concentrations were determined by a bicinchoninic acid assay (Solarbio Science & Technology). Proteins were separated by 8%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat milk solution, the membranes were incubated with primary antibodies at 4°C overnight. Following primary antibodies were applied in the experiment: anti-LC3 (Abcam, Cambridge, MA, United States; 1:1000), anti-LAMP2 (ProteinTech, Chicago, IL, United States; 1:1000), anti-Sirt1 (Cell Signaling Technology, Boston, MA, United States; 1:1000), anti-Bhlhe40 (ProteinTech, 1:1000), anti-PINK1 (ProteinTech, 1:1000), anti-VDAC1 (Abcam, 1:2000) and anti-GAPDH (ProteinTech, 1:5000). On the following day, the membranes were washed, and incubated with corresponding secondary antibodies (ProteinTech; 1:5000) at room temperature for 1.5 h. Enhanced chemiluminescent (ECL) reagent (Millipore, Billerica, MA, United States) was applied to detect protein-antibody interactions, which were visualized using a chemiluminescence detection system (C300, Azure Biosystems, United States).

The western blot was performed as previously described^{53 (link)}. Anti-OXPHOS (Abcam Plc, Cambridge, United Kingdom; Cat# ab110413, RRID: AB_2629281), anti-HSP60 (Santa Cruz Biotechnology, CA, USA; Cat# sc-13115, RRID: AB_627758), anti-YME1L1 (Proteintech, Rosemont, Illinois, USA; Cat# 11510-1-AP, RRID: AB_2217459), anti-PINK1 (Proteintech, Rosemont, Illinois, USA; 23274-1-AP), anti-OPA1 (Cell Signaling Technology; Cat# 80471, RRID: AB_2734117), anti-VDAC (Cell Signaling Technology; Cat# 4866, RRID: AB_2272627), anti-DRP1 (Cell Signaling Technology; Cat# 8570, RRID: AB_10950498), anti-α-tubulin (Cell Signaling Technology; Cat# 2144, RRID: AB_2210548) and anti-GAPDH (Cell Signaling Technology; Cat# 2118, RRID: AB_561053), anti-LONP1 (Bioss Antibodies, Boston, Massachusetts, USA; Cat# bs-4245R, RRID: AB_11051909). All antibodies were used at dilution 1:1000. Ponceau was from Sigma-Aldrich (Saint Louis, MO, USA). The respective loading control normalized the western blotting results, and the final values were given in the percentage of the respective control group.

MAC-T cells were lysed in RIPA buffer containing a protease/phosphatase inhibitor cocktail on ice for 30 min.). Protein (equal amounts, 20 µg) were loaded on 10% or 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Roche). After blocking with 5% skim milk at 37°C for 1 h and the membranes were incubated with the following primary antibodies at 4°C overnight: anti-LC3 I/II (1:1000, #4108) and anti-cleaved-Caspase-3 (1:1000, #9664) from Cell Signaling Technology (Danvers, USA); anti-ATG5 (1:750, 10181-2-AP), anti-Beclin-1 (1:1000, 11036-1-AP), anti-p62/SQSTM1 (1:1000, 18420-1-AP), anti-ASC (1:1000, 10500-1-AP), anti-BAX (1:1000, 50599-2-Ig), anti-Bcl-2 (1:1000, 12789-1-AP), anti-Caspase-3 (1:1000), anti-PINK1 (1:1000, 23274-1-AP), anti-Parkin (1:1000, 14060-1-AP), anti-GAPDH (1:5000, 60004-1-AP) and anti-β-Actin (1:5000, 60008-1-AP) from Proteintech Group Inc (Rosemont, IL 60018, USA); anti-NLRP3 (1:500, AF2155) from Beyotime Biotechnology (Shanghai, China) and anti-Caspase-1 (1:1000, ab179515) from Abcam (Cambridge, UK). The immunoreactive bands were visualized with an ECL detection system (Tanon 6200 chemiluminescence imaging workstation, Tanon Science & Technology Co., Ltd. Shanghai, China). The protein bands were quantified by densitometry using ImageJ software (version 1.50).

Western blotting was performed as described previously. The primary antibodies included anti-USP8 (Proteintech, USA), anti-PINK1 (Proteintech, USA), anti-Parkin(CST, USA), anti-Ubiquitin (Proteintech, USA), anti-GAPDH (Proteintech, USA), anti-VDAC1 (Proteintech, USA), anti-Fis1 (Proteintech, USA), anti-Drp1 (CST, USA), anti-phospho-Drp1 (Ser616, CST, USA), anti-Bax, anti-Bcl2, anti-Mfn2, anti-SOD, anti-Mn-SOD, anti-ATG7, anti-Beclin1 (all were from Proteintech, USA), anti-P62 (CST, USA), anti-LC3II/I (Proteintech, USA), anti-LC3B (Proteintech, USA), anti-CAT (Proteintech, USA), anti- cytochrome C (Proteintech, USA), anti-Cleaved-caspase9 (Proteintech, USA). Densitometry was conducted with the image processing and analysis program AlphaView.SA, and the data were expressed as relative units.

The intestinal mucosal tissue specimens were embedded with paraffin, fixed with 4% paraformaldehyde, and sliced continuously with a slicer. They were then defatted and hydrated in a solution of xylene, then treated with a citrate buffer (0.01 M) under 800 W microwave, and incubated for 10 min in 3% hydrogen peroxide at room temperature. After that, 50 µL of primary antibody (PARL or PINK1 or Parkin) was added to the slices, followed by incubation at 4°C overnight. The antibodies used in this study were anti-HSF2(Santa Cruz, 1:200), anti-PARL (1:400, Proteintech), anti-PINK1 (1:300, Proteintech), anti-Parkin (1:250, Proteintech). Another 50 µl DAB was added for color development, and the dyeing time and degree were controlled under microscope observation. Double steam water was used to wash twice, 1 minute each time, and hematoxylin was used for restaining (1 min), followed by rinsing with 1% ammonia after removal. The slides were successively immersed in 95 and 100% ethanol, and were dehydrated twice in total. After blow drying, they were sealed with neutral resin and the results were observed under the microscope.