A radio-immunoprecipitation assay lysis buffer, phenylmethanesulfonyl fluoride, and phosphatase inhibitor cocktail (Beyotime) were utilized to isolate proteins from cells and exosomes. The protein concentrations were evaluated by the BCA Protein Assay Kit (Beyotime) and adjusted. Proteins were separated and transferred onto polyvinylidene difluoride membranes (Millipore). Five percent non-fat powdered milk (Beyotime) was then used to block membranes for 1 h. After that, the membranes were incubated with different primary antibodies against LC3 (1:1000, Abmart), beclin1 (1:2000; Proteintech), Parkin (1:1000; ABclonal), PINK1 (1:1000; Abcam), CD206 (1:1000; Boster), Arginase-1 (Arg-1, 1:1000; Proteintech), CD63 (1:1000; UMIBIO), TSG101 (1:1000; UMIBIO), Aβ oligomer (1:1000, Abcam),Aβ1-42(1:1000,CST) separately overnight at 4°C. Afterward, members were incubated with corresponding secondary antibodies (Beyotime) for 1 hour at room temperature. The enhanced chemiluminescence (Tanon) was used to observe the blots. ImageJ software was used to quantify each band density.
Parkin
Manufactured by ABclonal
Parkin is a lab equipment product designed for protein purification. It plays a core function in the isolation and purification of Parkin, a protein involved in cellular processes. The detailed specifications and intended uses are not available in this concise and unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
Pink1, by Abcam (2 mentions)
Pink1, by ABclonal (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Cd206, by Boster Bio (1 mentions)
Arginase 1 arg 1, by Proteintech (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Beclin 1, by Proteintech (1 mentions)
Vdac1, by Proteintech (1 mentions)
Flag tag (flag), by ABclonal (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Gapdh, by ABclonal (1 mentions)
Cd206, by R&D Systems (1 mentions)
Tcs sp8, by Leica camera (1 mentions)
Occludin, by Bio-Synthesis (1 mentions)
Beclin1, by ABclonal (1 mentions)
β actin, by ABclonal (1 mentions)
Dimethyl sulfoxide (dmso), by Maclin Biochemical Technology (1 mentions)
5 protocols using parkin
1
Exosomal Protein Analysis by Western Blot
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted from collected cells and lysed in ice-cold RIPA buffer (Beyotime) for 15 min on ice with a protease inhibitor. Protein lysates were separated via SDS-PAGE and transferred onto PVDF membranes (Millipore) incubated with indicated primary antibodies and then secondary antibodies conjugated HRP. The ECL kit (Millipore) was used for detection of targeted signals. Antibodies used in western blot were as follows: GAPDH (ABclonal), DYNLT1 (Abcam), VDAC1 (Proteintech), Parkin (ABclonal), Flag (ABclonal), HA (ABclonal).
3
Immunofluorescence Imaging of Cellular Pathways
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Cells were resuspended on 35-mm confocal dishes and subjected to different treatments. Then, cells or brain tissue section were washed, fixed, permeabilized, and blocked. Next, they were incubated with different primary antibodies overnight at 4°C (LC3, 1:100; Abmart), Parkin (1:100; ABclonal), PINK1 (1:100; Abcam), CD206 (1:100; R&D Systems), and Arg-1 (1:200; Proteintech), and incubated with corresponding secondary antibodies (1:600; Invitrogen) for 1 h at room temperature. Finally, Cell nuclei were counterstained with the 4′,6-diamidino-2-phenylindole solution. The confocal microscope (TCS-SP8; Leica) was utilized to acquire images.
4
Protein Expression Analysis of Tight Junctions and Mitophagy
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Protein isolated from IPEC-J2 cells was separated by SDS gel electrophoresis and transferred to PVDF membranes. Then, membranes were incubated with tight junction primary antibodies (Claudin-1, Claudin-3, ZO-1, N-cad and Occludin) (Biosynthesis Biotechnology Inc., Beijing, China), mitophagy primary antibodies (LC3, P62, Beclin1, Parkin and PINK1) (ABclonal, Wuhan, China), a housekeeping antibody (β-actin) and a secondary antibody (ABclonal, Wuhan, China), respectively. Finally, the bands were quantified using a Tanon-5200 (Tanon, Shanghai, China) [29 (link),30 (link)].
5
Cardioprotective Effects of Natural Compounds
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The following reagents were used: DMEM, FBS, penicillin/streptomycin (Meilunbio, Dalian, China), cTnI Elisa kits (Sangon Biotech, Shanghai, China), TNF-α, IL-1β, and IL-6 Elisa kits (MyBioSource, San Diego, United States), MDA kits, SOD kits, and NADH kits (Solarbio, Beijing, China), CCK-8 kits (Beyotime Biotechnology, Shanghai, China), ROS fluorimetric kits, JC-1 fluorescent dye, protein extraction kits, and BCA kits (Meilunbio, Dalian, China). IL-1 (1:100), PINK1(1:1,000), Parkin (1:1,000), AMPK(1:1,000), Akt (1:1,000), P13K(1:1,000), Tom20 (1:1,000), β-tubulin (1:1,000), mTOR (1:1,000), and an HRP-conjugated GAPDH (1:1,000) antibody were obtained from Abclonal Biotechnology (Wuhan, China). The RIPA lysis buffer and loading buffer were purchased from Meilunbio (Dalian, China). PVDF membranes were obtained from Merck (New Jersey, United States). DMSO was purchased from Macklin (Shanghai, China), and doxorubicin, breviscapine, and dexrazoxane were purchased from Widely (Wuhan, China). The Mdivi-1 inhibitor was purchased from Selleck Chemicals (Houston, United States).
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