After the designated treatments were performed, liver tissues and cell pellets were lysed with RIPA buffer supplemented with protease inhibitors. Total proteins were separated via 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated overnight with rabbit antibodies against NLRP3 (Zymed, South San Francisco, CA, USA), cleaved caspase 1, cleaved caspase 3 (BD PharMingen, San Diego, California, USA), IL-1β (R&D Systems, USA), IL-18 (Santa Cruz Biotechnology Inc., Santa Cruz,California, USA), LC3-I, LC3-II (Sigma, USA), NF-κB, Beclin-1, PINK1, Parkin and β-actin primary antibodies (Cell Signaling Technology, Danvers, MA, USA) at 4°C. Then, the membranes were treated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Cell Signaling, CA, USA) and developed with a chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL, USA). Densitometry analysis was performed using ImageJ software, and the relative levels of protein in each group were normalized to the loading control.
Parkin
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China
Parkin is a protein that plays a role in regulating the ubiquitin-proteasome system, a cellular process involved in the degradation of damaged or unnecessary proteins. Parkin functions as an E3 ubiquitin ligase, which attaches ubiquitin molecules to target proteins, marking them for destruction by the proteasome.
Automatically generated - may contain errors
Lab products found in correlation
Pink1, by Cell Signaling Technology (14 mentions)
Cox 4, by Cell Signaling Technology (10 mentions)
Beclin 1, by Cell Signaling Technology (8 mentions)
Bnip3, by Cell Signaling Technology (8 mentions)
β actin, by Cell Signaling Technology (8 mentions)
Gapdh, by Cell Signaling Technology (7 mentions)
Cleaved caspase 3, by Cell Signaling Technology (6 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (5 mentions)
Lc3a b, by Cell Signaling Technology (5 mentions)
Tom20, by Cell Signaling Technology (5 mentions)
Caspase 3, by Cell Signaling Technology (4 mentions)
β actin, by Merck Group (4 mentions)
Sirt1, by Cell Signaling Technology (4 mentions)
Pgc 1α, by Cell Signaling Technology (4 mentions)
Lc3 2, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Phospho drp1, by Cell Signaling Technology (3 mentions)
Pink1, by Novus Biologicals (3 mentions)
Caspase 9, by Cell Signaling Technology (3 mentions)
Cleaved caspase 9, by Cell Signaling Technology (3 mentions)
58 protocols using parkin
1
Western Blot Analysis of Liver Proteins
2
Protein Expression Analysis in Renal Cortex
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The renal cortex was homogenized with RIPA Lysis Buffer. After centrifugation, protein concentration was determined by bicinchoninic acid (BCA) protein assay kit. The protein samples were then denatured in boiling water for 10 min. Equal amounts of proteins were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin in tris-buffered saline (TBS) for 1 h and incubated overnight at 4°C with following antibodies: Parkin (Cell Signaling Technology, cat. no. 2132), DRP1 (Abcam, cat. no. ab184247), LC3B (Cell Signaling Technology, cat. no. 2775), BNIP3L/Nix (Cell Signaling Technology, cat. no. 12396), caspase3 (Cell Signaling Technology, cat. no. 9662), cleaved caspase3 (Cell Signaling Technology, cat. no. 9661), and β-actin (Sigma, cat. no. A1978). Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, followed by enhanced chemiluminescence reaction. Quantification was performed by the Gel and Graph Digitizing System. The full western blot bands of LC3B, Parkin, BNIP3L, DRP1, caspase3, cleaved caspase3 were shown in Supplementary Figures S1 –S6 .
3
Immunofluorescence Assay for Parkin and Sirt1
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A total of 1 × 106 cells were seeded into the culture dish, washed with PBS, and permeabilized with PBS containing 0.1% Triton X-100 and 0.1% sodium citrate at 4°C. Next, the samples were blocked with 10% goat serum albumin for 1 h and cultured at 4°C overnight with the primary antibodies Parkin (1/1,000, Cell Signaling Technology, Beverly, MA, USA) and Sirt1 (1/1,000, Cell Signaling Technology). Following PBS washing three times, the samples were incubated with Alexa Fluor 488 donkey anti-rabbit antibody (1/1,000, Invitrogen) for 1 h. Cells were observed under an inverted fluorescence microscope at 40× magnification (BX51, Olympus).
4
Western Blot Analysis of Cellular Proteins
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Lysates from cells and tissues were subjected to 10 % SDS-PAGE (Bio-Rad, Pre-gel, MA); separated proteins were transferred onto Polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Seoul, Korea). Membranes were blocked with TBS-based blocking buffer (Li-COR Biosciences, Lincoln, NE) for 1h at room temperature, and incubated overnight at 4 °C with primary antibodies p16, SOD1,SOD2, ERK, p-ERK, CREB, p-CREB, p-elF2α, Beclin1, Bip, ATG12, LC3B, p62, BNIP3, PINK1, Parkin, PPARα, FATP4, Tau, P-tau, Beta-amyloid, GFAP, iba-1, beta-actin (1:1000, Cell Signaling Technology, Danvers, MA). Membranes were then washed with TBST three times for 10 min each and incubated with horseradish peroxidase-conjugated secondary antibodies (1:3000, Cell Signaling Technology, Danvers, MA) for 1 h at room temperature and washed again. Signals were detected by Odyssey-LC chemiluminsescent imaging system (LI-COR, Lincoln, NE). Signals were averaged and expressed as described in figure legend.
5
Quantitative Protein Expression Analysis
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Total proteins were isolated from liver tissue as described previously. Specific antibodies against Nrf2, HO-1, NQO-1, Bcl-2, Bax, Cyto C, PARP, Caspase-3, p62, Beclin-1, Parkin, LC3, PGC-1α, OPA-1, Mfn-2, Drp-1, NF-κB, β-actin and Lamin B1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Specific antibodies against PINK1 were purchased from Abcam (Cambridge, MA, USA). β-actin, and Lamin B1 were used to quantify protein expression. Densitometric quantifications of indicated proteins were analyzed by Image J (NIH, Bethesda, MD, USA). Quantifications of liver sections were analyzed by Image Pro plus (Media Cybernetics, Rockville, MD, USA).
6
Multimodal Mitochondrial Function Analysis
7
Comprehensive Mitophagy Regulation Assay
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The primary antibodies used were RhoA (#2117, for WB), Parkin (#2132), COX-IV (#11967), VDAC (#4661), lamin A/C (#2032), Rho-GDI (#2564), HA (#3724), PKD (#90039), P-PKD S916 (#2051), GAPDH (#2118), α-actinin (#3134) and LC3B (#3868) from Cell Signaling Technology; PINK1 from Novus Biologicals (#NB600-973); RhoA (SC-418, for IP) and ubiquitin (SC-8017) from Santa Cruz Biotechnology; miniSOG from Kerafast (#EFH004). Horseradish peroxidase (HRP)-conjugated secondary antibodies for WB, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), cycloheximide (CHX), MG-132, CID755673, Bafilomycin A1, Evans blue and triphenyltetrazolium chloride (TTC) were purchased from MilliporeSigma. Y-27632 was purchased from Cell Signaling Technology. DharmaFECT-1 and LysoTracker Blue were purchased from Thermo Fisher Scientific. C3 exoenzyme was purchased from Cytoskeletton, Inc.
8
Mitochondrial Dynamics and Signaling Pathways
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Primary antibodies used: Akt1 (2938), Akt2 (5239), Akt1/2 (9272), GSK‐3α/β (5676), LC3 (4108S), Lamin A/C (2032), NFR1 (12381), PGC‐1α (2178), phospho‐p38 MAPK (9215), phospho‐Akt1 (9018), phospho‐Akt2 (8599), phospho‐DRP1 (Ser616) (4494), phospho‐GSK‐3α/β (9331), Parkin (2132S), Tfam (7495S), VDAC (4866) (Cell Signaling); β‐actin (A5441) (Sigma); PINK1 (ab23707), Mfn‐2 (ab124773) (Abcam); Drp1 (611113) (BD Biosciences); phospho‐PGC‐1α (S571) (AF6650) (R&D Systems); p38 (sc‐7972) and Tom20 (sc‐11415) (Santa Cruz); SPC (AB3786) (Millipore).
9
Immunohistochemical Analysis of Ubiquitin-Specific Protease 22, Parkin, and Ki-67
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Sections were deparaffinized in xylene and hydrated in a series of graded ethanol. Antigen retrieval was performed using DAKO PT-link pH 6 or pH 9 for 20 min a 98 °C; endogenous peroxidase was inhibited by 0.3% hydrogen peroxide for 10 min. Sections were then incubated 1 h at RT with anti-USP22 (1:200, retrieval pH6, Abcam, Cambridge, UK), -Parkin (1:100 retrieval pH9, Cell Signaling Technology, Danvers, MA, USA) and Ki-67 (1:200 retrieval pH6, Dako, Camarillo, CA, USA), washed in 1 × PBS pH 7.4 and re-incubated for 30 min at RT with Dako EnVision® (Dako, Camarillo, CA, USA) + Dual Link System-HRP (DAB+) and finally counterstained with Hematoxylin.
Percentages of positive tumor cells scored 0 (0% to 25%), 1 (26% to 50%), 2 (51% to 75%), and 3 (76% to 100%), staining intensity was graded as follows: 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The arithmetic sum of the two scores are considered for statistical purposes.
Percentages of positive tumor cells scored 0 (0% to 25%), 1 (26% to 50%), 2 (51% to 75%), and 3 (76% to 100%), staining intensity was graded as follows: 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The arithmetic sum of the two scores are considered for statistical purposes.
10
Mitochondrial Quality Control and Arterial Stiffening
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