To perform the in vitro sumoylation assay, one microgram of purified Stu2p‐TAP was incubated with 5 μg of His6‐Smt3p‐GG, His6‐Ubc9p, 2 μg Aos1/Uba2p, 4 mM ATP and 7 μL of an ATP regeneration system (3.5 U mL−1 creatine kinase, 10 mM creatine phosphate and 0.6 U mL−1 inorganic pyrophosphatase (Sigma Chemical Company, St. Louis, MO)). The mixture was incubated for 2 hr at 30 °C. To stop the reaction, 3x Laemmli sample buffer plus 5% beta‐mercaptoethanol was added and samples were boiled for 5 min. Reaction products were subjected to 6% SDS‐PAGE and visualized by western blot analysis. The presence of Stu2p‐TAP was confirmed using mouse anti‐HA (Santa Cruz Biotechnology, CA).
Creatine kinase
Manufactured by Merck Group
Sourced in United States
Creatine kinase is an enzyme found in various tissues, including the heart, brain, and skeletal muscles. It plays a crucial role in energy metabolism by catalyzing the reversible conversion of creatine and ATP to phosphocreatine and ADP. This enzymatic activity is essential for the storage and rapid release of energy in cells, supporting various physiological processes.
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Lab products found in correlation
Creatine phosphate, by Merck Group (6 mentions)
Fm4 64, by Thermo Fisher Scientific (4 mentions)
Inorganic pyrophosphatase, by Merck Group (3 mentions)
Adenosine triphosphate (atp), by Merck Group (3 mentions)
Anti ubiquitin antibody, by Santa Cruz Biotechnology (3 mentions)
Phosphocreatine, by Merck Group (2 mentions)
Digitonin, by Merck Group (2 mentions)
Creatine phosphate, by Roche (2 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (2 mentions)
Untagged luciferase, by Promega (2 mentions)
Quinacrine, by Merck Group (2 mentions)
Superdex 75 10 300 gl size exclusion chromatography column, by GE Healthcare (2 mentions)
Recombinant human h5b, by Enzo Life Sciences (2 mentions)
Anti mbp antibody, by New England Biolabs (2 mentions)
Bovine ubiquitin, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (2 mentions)
Mouse anti ha, by Santa Cruz Biotechnology (1 mentions)
Cyclooctatetraene, by Merck Group (1 mentions)
Ix83 celltirf, by Olympus (1 mentions)
28 protocols using creatine kinase
1
In vitro Stu2p sumoylation assay
2
Single-Molecule Imaging of Multidrug Resistance Protein
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Single-molecule experiments were performed at room temperature (23 ± 1°C) on an objective-type total-internal-reflection fluorescence microscope (Olympus IX83 cellTIRF). Microfluidic imaging chambers were passivated with a mixture of PEG and biotin-PEG (Laysan Bio), and incubated with 0.8 µM streptavidin (Invitrogen) followed by 2 nM fluorescently labeled, His-tagged bMRP1 that had been preincubated with biotinylated anti-His6 antibodies (Invitrogen) for 1 hr on ice in a buffer containing 50 mM Tris-HCl pH 8.0, 150 mM KCl, 2 mM MgCl2, 0.06% digitonin, 0.5 mg/mL BSA, 10 mM phosphocreatine (Sigma), and 0.1 mg/mL creatine kinase (Sigma). A triplet-state quenching cocktail (Dave et al., 2009 (link)) of 1 mM cyclooctatetraene (Sigma), 1 mM 4-nitrobenzyl alcohol (Sigma), and 1 mM Trolox (Sigma), as well as an oxygen scavenging system (Aitken et al., 2008 (link)) containing 10 nM protocatechuate-3,4-dioxygenase (Sigma) and 2.5 mM protocatechuic acid (Sigma) were supplemented to the imaging buffer. ATP and/or LTC4 were included in the imaging buffer at concentrations specified in the text. Fluorescence signals were split with a W-View Gemini-2C (Hamamatsu), directed to two CMOS cameras (Flash 4.0 v3, Hamamatsu), and acquired by MetaMorph software (Molecular Devices) at a frame rate specified in the text.
3
VCA Injury Assessment via ELISA and Histology
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Prior to implantation of VCAs, perfusates from cold stored VCAs were collected and levels of creatine kinase (Sigma, USA), and HMGB1 (LifeSpan BioSciences, USA) were measured by ELISA as markers of VCA injury. For histological examination, VCAs were isolated and subsequently fixed, processed to paraffin, and four μm sections stained with Hematoxylin and Eosin (HE) and scored for graft injury from 0-5 using a semiquantitative histology scoring system (26 (link)).
4
Nuclear Export Assay of NP Mutants
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HeLa cells (1×105) were cultured on coverslips in 12-well plates, transfected with pCAGGS harboring wild-type NP or mutant NP-NES3 Φ1, Φ2, Φ3, or Φ4, and then examined in a nuclear export assay [5] (link). Briefly, the cell membrane was selectively permeabilized by treatment with 50 µg/ml digitonin (Sigma) for 5 min on ice. The cells were then incubated with apyrase (10 units/ml; Sigma) on ice for 5 min to deplete intracellular ATP, washed, and then incubated in PBS to remove all cytoplasmic components. Fresh HeLa cell lysates (prepared by lysing cells in 10 mM Tris-Cl (pH 7.4), 2 mM MgCl2, 3 mM CaCl2, and 0.3 M sucrose) supplemented with protease inhibitor cocktail, 5 mg/ml bovine serum albumin, 1 mM ATP (Sigma), 5 mM creatine phosphate (Roche), and 32 units/ml creatine kinase (Sigma) were added to the cells and incubated at 30°C for 1 h to allow nuclear export to proceed. The cells were then stained with an anti-NP Ab followed by anti-mouse Alexa Fluor 488 and Hoechst 33342. Fluorescence images were taken under a confocal laser-scanning microscope (FV 1000, Olympus).
5
Oligonucleotide Purification and Dilution
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All oligonucleotides were purchased from Thermo Fisher Scientific (Germany) or from Integrated DNA Technologies (Belgium), and purified by the manufacturer using reverse phase HPLC (for the primers) and PAGE (for the IO). All oligonucleotides and genomic DNA were diluted and stored in Tris-EDTA (TE) buffer (10 mM Tris-HCL, 0.5 mM EDTA pH 8.0). Details of all the oligonucleotides used in this study are provided in Table S1 . All reagents and buffers, including creatine kinase and sucrose phosphorylase, were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A). T4 single-stranded DNA binding protein (gp32) and Bacillus subtilis DNA Polymerase I, Large Fragment (BSU) were purchased from New England Biolabs (Ipswich, MA).
6
Biochemical Reagents and Compounds Protocol
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Eugenol (purity=99%), ryanodine, BDM, procaine, MgATP, disodium phosphocreatine,
EGTA, imidazole, methanesulfonic acid, calcium chloride, and creatine kinase were
purchased from Sigma Chemical Co. (USA). All other reagents were analytical grade and
were purchased from Merck (Germany).
EGTA, imidazole, methanesulfonic acid, calcium chloride, and creatine kinase were
purchased from Sigma Chemical Co. (USA). All other reagents were analytical grade and
were purchased from Merck (Germany).
7
FMRP Sumoylation In Vitro Assay
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Immobilized GST-FMRP/His-FMRP dimers were incubated with 0.15 μg of E1-activating complex (Enzo Life science), 0.1 μg of E2 Ubc9 (Enzo Life science), 3 μg of SUMO1-GG in 20 μL of in vitro SUMO reaction mix (20 mM HEPES pH 7.3, 110 mM KOAc, 2 mM Mg(OAc)2, 0.5 mM EGTA, 1 mM DTT 0.05% Tween 20, 0.2 mg mL−1 ovalbumin) including the ATP regenerating system (20 mM ATP, 10 mM creatine phosphate, 3.5 U mL−1 of creatine kinase, and 0.6 U mL−1 of inorganic pyrophosphatase (Sigma-Aldrich) for 2 h at 30 °C). After centrifugation for 5 min at 3000×g at 4 °C, the supernatant containing the released His-FMRP (1–160) and the pellet containing the remaining immobilized GST-FMRP/His-FMRP dimers were denatured at 95 °C for 10 min in 5× Laemmli buffer containing 7.5% β-mercaptoethanol and analyzed by immunoblotting with FMRP #2F5-1 antibodies.
8
Purification of Fluorescent Proteins
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We used previously published protocols to purify Hsp104 (44 (link)), Ssa1 (66 (link)), Sis1 (67 (link)), Ydj1 (44 (link)), His-tagged Luciferase (43 (link)), and GFPuv (68 (link)). Creatine kinase was purchased from Sigma–Aldrich (10127566001). Untagged Luciferase was purchased from Promega (E1701). cfSGFP gene (45 ) was synthesized and cloned by Genescript to pET3a plasmid and expressed in the Escherichia coli BL21(DE3) codon+ strain. The soluble fraction of the lysate was mixed 1:1 with 96% ethanol and centrifuged. Supernatant was loaded on a Q-Sepharose fast flow (GE Healthcare Bio-Sciences AB) column in 40 mM Tris–HCl, pH 7.5, 10% glycerol, 50 mM NaCl, and eluted with 40 mM Tris–HCl, pH 7.5, 10% glycerol, 300 mM NaCl. Fractions with cfSGFP were dialyzed into 40 mM Tris–HCl, pH 7.5, 20% glycerol, 50 mM NaCl, heated for 15 min at 73.2 °C, and rapidly cooled down to precipitate protein impurities. The soluble fraction was stored at −80 °C. Protein concentrations are indicated in the figures. All the protein concentrations refer to monomer.
9
Ubiquitination Assay of ARI Proteins
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ubiquitination assays were carried out as described previously (Hardtke et al., 2002; Stone et al., 2005 ). Reactions (25 μL) containing 50 mM Tris–HCl, pH 7.5; 10 mM MgCl2; 0.05 mM ZnCl2; 1 mM ATP; 0.2 mM dithiothreitol; (DTT) 10 mM phosphocreatine; 0.1 unit of creatine kinase (Sigma–Aldrich); 50 ng of human recombinant E1 (Sigma–Aldrich); 6 μg of purified E2 AtUBC11; 2–8 μg of eluted GST-ARI12 or GST-ARI8 (positive control); and 2 μg ubiquitin (Sigma–Aldrich) were incubated at 30 °C for 3 h. Reactions were stopped by adding 5 μL of 6× loading buffer (0.3 M Tris–HCl pH 6.8, 0.6 M DTT, 12% [w/v] SDS, 60% [v/v] glycerol, 0.06–0.1% [w/v] Bromophenol blue), 3 min denaturation at 95 °C and loaded on a 10% SDS-PAGE followed by Western blotting using anti-ubiquitin antibodies (1:1000 in TBST; P4D1; Santa Cruz Biotechnology, Texas, USA). To confirm the presence of the recombinant GST-tagged ARI proteins and their monoubiquitination the blots were stripped and rehybridized with anti-GST antibodies (1:15,000) in 5% milk.
10
Reconstitution of cis-SNARE Complex
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