Parkin (1.3 μM) was incubated with ubiquitylation assay components in a final volume of 30 μl (50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.12 μM UBA1, 2 μM UBE2L3, 0.83 μM His-SUMO-Miro1, 2 mM ATP, 88 μM Flag-ubiquitin and 0.31 μM TcPINK1 (WT/KD). Ubiquitylation reactions were incubated at 30 °C and 1050 rpm for 60 min. Ubiquitylation reactions were stopped by the addition of 5 U mL-1 Apyrase (New England BioLabs) and incubated for 10 min at 30 °C and agitated at 1050 rpm. Probe profiling was carried out by the addition of 7 or 9 (8 μM) and incubated for 10 h at 30 °C and 650 rpm. The reactions were terminated by the addition of LDS sample buffer containing 4 % 2-mercaptoethanol. Reaction mixtures were resolved by SDS-PAGE and immunoblotted with the following antibodies: anti-FLAG (Sigma, 1:10000), anti-Parkin (Santa-Cruz, 1:500) or anti-His (1:5000). For Fluorescence detection, 20 μl of sample were loaded on a 4-12 % gel and subjected to SDS-PAGE. Gels were washed with water for 30 minutes and then visualized by scanning with a FLA-5100 imaging system (Filter: LPG, Laser: 532 nm, Voltage: 400, FUJIFILM Life Science). The gel was subsequently coomassie stained.
Anti parkin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-Parkin is a high-quality antibody that targets the Parkin protein, a key regulator of mitochondrial function and quality control. This antibody can be used for various applications, including Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by Merck Group (6 mentions)
Anti tom20, by Santa Cruz Biotechnology (4 mentions)
Anti pink1, by Santa Cruz Biotechnology (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Ml 1 apyrase, by New England Biolabs (2 mentions)
Fla 5100 imaging system, by Fujifilm (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti pink1, by Cell Signaling Technology (2 mentions)
Anti gfp, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail, by Nacalai Tesque (1 mentions)
Anti brca1, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Anti γh2ax, by Merck Group (1 mentions)
Anti p62, by Cell Signaling Technology (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
Anti nrf2, by Bioss Antibodies (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti phospho p38, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Ecl system, by Cytiva (1 mentions)
15 protocols using anti parkin
1
Parkin-Miro1 Ubiquitylation Assay
2
Whole-cell Protein Extraction and Western Blotting
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To obtain whole-cell lysates, cells were harvested and lysed with 1 × RIPA buffer (Nacalai Tesque) containing protease inhibitor cocktail (Nacalai Tesque) and phosphatase inhibitor cocktail (Nacalai Tesque). Cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on a polyvinylidene fluoride membrane. Western blotting was performed with the following antibodies: anti-BRCA1 (Cell Signaling Technology [CST], Danvers, MA, USA #9010), anti-β-actin (Santa Cruz Biotechnology #47778), anti-TOM20 (Santa Cruz Biotechnology #11415), anti-COX IV (CST #11967), anti-FLAG (SIGMA #F1804), anti-γH2AX (Millipore Merck #05-636), anti-PINK1 (CST #6946), anti-Myc (CST #2276), and anti-Parkin (Santa Cruz Biotechnology #32282). Intensity of each protein band was quantified using ImageJ software (https://imagej.nih.gov/ij/ ). Each band intensity was standardized by β-actin. Uncropped original images merged with corresponding marker images are shown in Fig. S8 -17.
3
Comprehensive Protein Expression Analysis
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The method has been described previously [18 (link)]. Protein lysate from pancreatic tissue or isolated pancreatic acinar cells was prepared by homogenizing in RIPA containing protease and phosphatase inhibitor. Lysate samples (20 μg protein) were loaded on 15% polyacrylamide gel, then the gel was transferred to polyvinylidene fluoride (PVDF) membrane, and finally, blocked and incubated with primary antibody and secondary antibody. The images were detected by enhanced chemiluminescence detection system (California, USA). Anti-CBS (1 : 300) and anti-Nrf2 (1 : 1000) were purchased from Bioss (Beijing, China); anti-SIRT1 (1 : 1000) and anti-PGC1-α (1 : 1000) were purchased from Wanleibio (Shenyang, China); anti-phospho-P38 (1 : 1000), anti-P38 (1 : 1000), anti-LC3 (1 : 1000), anti-β-actin (1 : 1000), and anti-P62 (1 : 1000) were purchased from Cell Signaling Technology (Danvers, USA); anti-Parkin (1 : 1000) was from Santa Cruz Biotechnology (California, USA).
4
Acrylamide Induced Autophagy Signaling
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After treatment for different time points of acrylamide, THP-1 cells were lysed with equal volumes of ice-cold 150-µL lysis buffer 1 h later. After centrifugation at 13,000× g for 15 min, equal amounts of cell lysates were blotted and analyzed by western blot with anti-LC3 (MBL, Nagoya, Japan), anti-PINK1 (Cell Signaling Technology, Danvers, MA, USA), anti-parkin and GADPH antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactive bands were visualized by using horseradish peroxidase-conjugated secondary antibody and the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). The assay was performed by using the protocols recommended by the manufacturer.
5
Protein Expression Analysis in Mouse Tissues
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Mouse tissue samples or cells were homogenized in lysis buffer containing 1% protease inhibitors. Protein assay kit was used to determine the protein concentration. The antibodies used in this study were anti-HSF2(Santa Cruz, 1:1000), anti-PARL (1:1000, Santa Cruz), anti-PINK1 (1:1000, Santa Cruz), anti-Parkin (1:1000, Santa Cruz), anti-β-actin (1:5000, Abcam), and anti-GAPDH (1:5000, Abcam). The results were analyzed by ImageJ.
6
Mitochondrial Dynamics Regulate TRAIL-Induced Apoptosis in NSCLC Cells
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Human NSCLC cell lines (Calu-1, A549, and H460) and BEAS-2B normal bronchial epithelial cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and grown in RPMI-1640 medium (Gibco, Grand Island, NY, USA), supplemented with 10% heat-inactivated fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Recombinant human TRAIL was purchased from PeproTech (Rocky Hill, NJ, USA). Mouse monoclonal anti-Drp1, anti-phosphorylated (p)-Drp1(Ser616), anti-caspase-3, anti-PARP, anti-LC3, anti-p62, anti-COX IV, anti-PINK1, and anti-Parkin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal anti-Fis1, anti-Mfn1, anti-Mfn2, anti-Opa1, anti-β-actin, N-acetyl-l -cysteine (NAC), and mitochondrial division inhibitor 1 (Mdivi-1) were purchased from Sigma (St Louis, MO, USA). Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrinpentachloride (MnTMPyP) was obtained from Cayman Chemical (Ann Arbor, MI, USA).
7
Antibody Validation for Western Blot and Immunostaining
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The following primary antibodies were used: anti-β-actin (A5316; Sigma, 1:5000 dilution), anti-Syt11 (270,003; Synaptic System, 1:1000 dilution), anti-c-Myc (sc-789; Santa Cruz, 1:200 dilution, 1 μg mg−1 for IP), anti-c-Myc (M 4439; Sigma, 1 μg mg−1 for IP), anti-GFP (A11122; Invitrogen, 1:1000 dilution), anti-parkin (sc-32,282; Santa Cruz, 1:400 dilution), anti-Flag (F1804; Sigma, 1:1000 dilution), and anti-tyrosine hydroxylase (AB152; Millipore, 1:1000 dilution). IRDye 680CW goat anti-mouse IgG (LIC-926-32,220; LI-COR Biosciences, 1:5000 dilution) and IRDye 800CW goat anti-rabbit IgG (LIC-926-32,211; LI-COR Biosciences, 1:5000 dilution) were used for immunoblotting. Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (A11034, 1:5000 dilution), Alexa Fluor® 594 goat anti-rabbit IgG (H+L) (A11037, 1:5000 dilution), and Alexa Fluor® 633 goat anti-rabbit IgG (H+L) (A-21,071, 1:5000 dilution) were used for immunostaining.
8
Parkin-Mediated Ubiquitylation Assay
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Wild-type ParkinPhospho-Ser65 or indicated mutant of Parkin (2 μg or 0.2 μg) was incubated with ubiquitylation assay components in a final volume of 50 μl (50 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 0.12 μM UbE1, 1 μM UbcH7 and 2 μg 6xHis-Sumo-Miro, 2 mM ATP). About 5 μg of ubiquitin or ubiquitinPhospho-Ser65 was added as indicated. Ubiquitylation reactions were incubated at 30°C for 60 min and terminated by the addition of LDS sample buffer. For all assays, reaction mixtures were resolved by SDS–PAGE. Ubiquitylation reactions were subjected to immunoblotting with anti-FLAG antibody (Sigma, 1:10,000), anti-Parkin (Santa Cruz 1:5,000) or anti-SUMO1 (1:2,000).
9
Antibody Characterization for Cell Biology Research
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Antibodies used in this study include antiactin (MAB1501; EMD Millipore), anti–α-adaptin (sc-17771; Santa Cruz Biotechnology, Inc.), anti-CD63 (556019; BD), anti-DNA (61014; Progen), anti-Drp1 (611113; BD), anti-Eps15 (sc-534; Santa Cruz Biotechnology, Inc.), anti-Flag (anti-DDDDK, ab1257; Abcam), anti-Flag (F1804; Sigma-Aldrich), anti-GFP (ab6673; Abcam), anti-GFP (A6455; Invitrogen), anti-LAMP2 (sc-18822; Santa Cruz Biotechnology, Inc.), anti-LBPA (Z-SLBPA; Eschelon), anti-parkin (sc-32282; Santa Cruz Biotechnology, Inc.), anti-PDH E2/E3bp (ab110333; Abcam), anti-PINK1 (6946; Cell Signaling Technology), anti-PMP70 (SAB4200181; Sigma-Aldrich), anti-SDHA (ab14715; Abcam), anti-SNAP29 (ab138500; Abcam), anti-Stx17 (17815–1-AP; ProteinTech), anti-Stx17 (HPA001204; Sigma-Aldrich), anti-TIM23 (611222; BD), anti-TIP47 (Novus Biologicals), anti-TOM20 (sc-11414; Santa Cruz Biotechnology, Inc.), anti-UQCRFS1 (referred to herein as CIII-Rieske, ab14746; Abcam), anti-VAMP7 (sc-166394; Santa Cruz Biotechnology, Inc.), anti-VAMP8 (ab76021; Abcam), anti-VDAC1 (ab14734; Abcam), and anti-Vps41 (ab181078; Abcam). Unless otherwise specified, all reagents were purchased from Sigma-Aldrich. The TIP47 antibody was a gift from P. McPherson (McGill University, Montreal, Quebec, Canada).
10
Mitochondria-Endoplasmic Reticulum Interactions
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Oligomycin (O4876), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (C2920), antimycin A (A8674), rotenone (R8875), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (C2759), bafilomycin A1 (B1793), sodium pyruvate (P5280), and glucose (G7021) were purchased from Sigma-Aldrich (St. Quentin Fallavier, France). MitoTracker Deep Red (M22426) and l -glutamine (25030-024) were purchased from ThermoFisher Scientific (Illkirch, France). Cyto-ID (ENZ-51031) was purchased from Enzo Life Sciences (Villeurbanne, France). Anti-Mitofusin 2 (rabbit, 9482), anti-Grp75 (rabbit, 3593), anti-LC3B (rabbit, 2775), and anti-Myc (mouse, 2276) were from Cell Signaling Technology (Saint Quentin Yvelines, France). Anti-Tom20 (rabbit, sc-11415), Anti-Tom20-AF488 (rabbit, sc-17764), anti-Parkin (mouse, sc-32282), and anti-Ero1-Lα (mouse, sc-365526) were from Santa Cruz Biotechnology (Heidelberg, Germany). Anti-VDAC1 (mouse, ab14734), anti-Parkin (rabbit, ab15954), and anti-Beclin-1 (mouse, ab114071) were from Abcam (Paris, France). Anti-KDEL (mouse, ADI-SPA-827-D) was from Enzo Life Sciences. Anti-PACS-2 (rabbit, 19508-1-AP) was from Proteintech (Manchester, UK), and Anti-PACS-2 (rabbit, clone 18143) was previously reported [9 (link)]. Anti-IP3R1 (rabbit, 07-1213) was from Merck Millipore (Molsheim, France). Anti-β-actin (mouse, A2228) and anti-P62 (rabbit, P0067) were from Sigma-Aldrich.
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