A protein extraction kit (Beyotime, Jiangsu, PR China) was used to extract proteins from hepatic cells and liver tissue. The bicinchoninic acid method (Beyotime) was used to estimate the total protein concentration. Western blotting was then carried out according to a previously published method [17 (link)]. In this study, the antibodies used were: anti-PINK1 (catalog no. 23274-1-AP, 1:1000, Proteintech, Rosemont, IL, USA), anti-Parkin (catalog no. 14060-1-AP, 1:1000, Proteintech), anti-protein kinase B beta (AKT2) (catalog no. 17609-1-AP, 1:1000, Proteintech), anti-phospho-AKT2 (pAKT2) (Ser473) (catalog no. 28731-1-AP, 1:1000, Proteintech), anti-insulin receptor (INSR) (catalog no. 20433-1-AP, 1:1000, Proteintech), anti-glucose transporter type 2 (GLUT2) (catalog no. 20436-1-AP, 1:1000, Proteintech), anti-insulin receptor substrate 2 (IRS2) (catalog no. 20702-1-1AP, 1:1000, Proteintech), and anti-β-actin (catalog no. 66009-1-Ig, 1:1000, Proteintech). The secondary antibodies comprised goat anti-rabbit IgG (catalog no. SA00001-2, 1:1000, Proteintech) or goat anti-rat IgG (catalog no. GB23302, 1:1000, Servicebio, Wuhan, PR China) conjugated to horseradish peroxidase (HRP). Immunoreactive bands were captured and analyzed using Image J software (NIH, Bethesda, MD, USA).
Anti parkin
Manufactured by Proteintech
Sourced in United States
The Anti-Parkin is a primary antibody that targets the Parkin protein. Parkin is an E3 ubiquitin-protein ligase enzyme that plays a role in the control of mitochondrial function and dynamics. The Anti-Parkin antibody can be used in various laboratory techniques, such as Western blotting, immunohistochemistry, or immunocytochemistry, to detect and study the Parkin protein.
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Lab products found in correlation
Anti pink1, by Proteintech (5 mentions)
Anti β actin, by Proteintech (4 mentions)
Anti caspase 3, by Proteintech (2 mentions)
Anti bax, by Proteintech (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid method, by Beyotime (1 mentions)
Goat anti rabbit igg, by Proteintech (1 mentions)
Goat anti rat igg, by Wuhan Servicebio Technology (1 mentions)
Anti mfn2, by Proteintech (1 mentions)
Mdivi 1, by MedChemExpress (1 mentions)
Aicar, by MedChemExpress (1 mentions)
Anti vdac1, by Proteintech (1 mentions)
Compound c, by MedChemExpress (1 mentions)
Anti caspase 9, by Proteintech (1 mentions)
Anti mfn1, by Proteintech (1 mentions)
Anti drp1, by Proteintech (1 mentions)
Anti opa1, by Proteintech (1 mentions)
Anti lamin b, by Proteintech (1 mentions)
Anti γ h2a x ab81299, by Abcam (1 mentions)
Anti ampkα 2532, by Cell Signaling Technology (1 mentions)
7 protocols using anti parkin
1
Protein Extraction and Western Blotting Protocol
2
Mitochondrial Dysfunction and Cell Death
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TMZ, Compound C, AICAR, Mdivi1, MG132 and WY14643, were purchased from MedChemExpress company. MTT and N‐Acetyl‐L‐cysteine (NAC) were purchased from Sigma‐Aldrich. MitoTracker™ Red FM (M22425), Hoechst 33342 (H1399) and anti‐Ubiquitin WB Antibody (13–1600) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.) (1:1,000). Anti‐phospho‐Ubiquitin (Ser65) (ABS1513‐I) was purchased from Merck KGaA company (1:1000). Anti‐P53 (21891–1‐AP), anti‐PINK1 (23274–1‐AP), anti‐Parkin (14060–1‐AP), anti‐Drp1 (12957–1‐AP), anti‐Opa1 (27733–1‐AP), anti‐Mfn1 (13798–1‐AP), anti‐Mfn2 (12186–1‐AP), anti‐Caspase‐9 (10380–1‐AP), anti‐BAX (50599–2‐Ig), anti‐Caspase‐3 (19677–1‐AP), anti‐VDAC1 (10866–1‐AP), anti‐Lamin B (12987–1‐AP), anti‐Cytochrome c (10993–1‐AP), and anti‐β‐actin (60008–1‐Ig) were purchased from ProteinTech Group, Inc., (Chicago, IL, USA) (1:1,000). Anti‐γ‐H2A.X (ab81299) was purchased from Abcam (1:1000). Anti‐AMPKα (2532) and p‐AMPKα (50081) were purchased from Cell Signaling Technology, Inc. (Massachusetts, USA) (1:1000). Anti‐phospho‐DRP1(Ser616) (DF2972) was purchased from Affinity Biosciences (1:1000). Anti‐phospho‐DRP1(Ser637) (6319S) was purchased from Cell Signaling Technology, Inc. (1:1,000).
3
Mitochondrial Dynamics in Acupuncture
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Instruments needed are disposable acupuncture needles (0.2x13mm, Huanqiu, Suzhou, China), and HANS acupoint nerve stimulator (LH202H, Huawei Industry Development, Beijing, China). ATP assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The following primary antibodies were used in this study: anti-TOM20, anti-TIM23, anti-Pink1, anti-Parkin (Proteintech, Chicago, IL, USA), anti-LC3 (MBL, Tokyo, Japan), anti-GAPDH (Abcam, Boston, Mass, USA), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Servicebio, Wuhan, China), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Servicebio, Wuhan, China), Alexa Fluor 594 labeled goat anti-rabbit IgG (ZSGB-BIO, Beijing, China), Alexa Fluor 488 labeled goat anti-mouse IgG (ZSGB-BIO, Beijing, China), Prestained Protein Marker II (10–200kDa) (Servicebio, Wuhan, China).
4
Immunostaining for Autophagy and Lysosomal Markers
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Immunostaining was performed as described elsewhere, particularly in [19 (link)]. The following primary antibodies:anti-Lamp2 (1:100, Proteintech, Beijing, China), anti-LC3 (1:100, CST, Beverly, MA, USA), anti-p62 (1:100, CST, Beverly, MA, USA), anti-Cathepsin B (CTSB) (1:100, CST, Beverly, MA, USA) and anti-Cathepsin D (CTSD) (1:100, Proteintech, Beijing , China) and anti-Parkin (1:100, Proteintech, Beijing, China) and anti-COX IV (1:100, Proteintech, Beijing, China).
5
Western Blot Analysis of Autophagy and Apoptosis Markers in MAC-T Cells
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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).
6
Immunohistochemical Analysis of Mitochondrial Proteins
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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.
7
Western Blot Analysis of Synovial Proteins
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Western blotting was performed as previously described (28 (link)). The protein concentrations of the synovial tissues or cells were determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Proteins (30 µg) were separated by SDS-PAGE on a 10% gel and transferred to PVDF membranes (MilliporeSigma). The PVDF membranes were blocked with 5% non-fat dry milk for 2 h, followed by incubation with one of the following primary antibodies at 4°C overnight: Anti-MAGL (1:1,000, Abcam, cat. no. ab246902), anti-iNOS (1:1,000, ProteinTech Group, Inc., cat. no. 22226-1), anti-Arg1 (1:5,000, ProteinTech Group, Inc., cat. no. 16001-1), anti-TNF-α (1:1,000, ABclonal, cat. no. A20851), anti-IL-1β (1:1,000, ABclonal, cat. no. A16288), anti-IL-6 (1:1,000, ABclonal, cat. no. A11115), anti-PTEN-induced kinase 1 (PINK1) (1:1,000, ProteinTech Group, Inc., cat. no. 23274-1), anti-Parkin (1:1,000, ProteinTech Group, Inc., cat. no. 14060-1), or anti-β-actin (1:5,000, ProteinTech Group, Inc., cat. no. 81115-1). The following day, the membranes were washed and incubated with the secondary antibody (1:5,000, Abcam, cat. no. ab205718) for 2 h at room temperature. The proteins were detected using Pierce ECL Western Blotting Substrate (Thermo Fisher).
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