LPS, isoamyl alcohol, 2-methylbutane, picrotoxin, 3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid, sodium orthovanadate, and monoclonal anti-mouse β-actin antibodies were all purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse ASC and caspase-1 antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX), anti-phosphotyrosine antibodies from Cell Signaling (Danvers, MA), anti-mouse and human IL-1β antibodies from R&D Systems (Minneapolis, MN), and anti-human caspase-1 antibodies from BIOMOL International LP (Plymouth Meeting, PA). Recombinant mature mouse IL-1β was also purchased from R&D Systems. Biotin conjugated anti- mouse and rabbit secondary antibodies were from GE Healthcare UK Limited (Little Chalfont, Buckinghamshire, UK) and anti-goat IgG from Jackson ImmunoResearch (West Grove, PA). For inflammasome stimulation, ATP, nigericin, alum Imject, apoSAA, poly (dA:dT), and anthrax lethal factor and protective antigen were purchased from Amersham Biosciences (Piscatawy, NJ), Invivogen (San Diego, CA), Thermo Scientific (Waltham, MA), PeproTech Inc. (Rocky Hill, NJ), Invivogen, and BEI Resources (Manassas, VA), respectively. Muscimol was acquired from MP Biomedicals (Santa Ana, CA), and ethanol from Pharmco AAPER (Brookfield, CT). ASC (Tyr144) phospho-specific antibody was purchased from ECM Biosciences (Versailles, KY).
Adenosine triphosphate (atp)
Manufactured by Cytiva
Sourced in United States
ATP (Adenosine Triphosphate) is a fundamental molecule found in all living organisms. It serves as the primary energy currency of cells, providing the energy required for various cellular processes. ATP is involved in numerous biochemical reactions and plays a crucial role in energy production, storage, and transfer within biological systems.
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Lab products found in correlation
Coelenterazine, by Santa Cruz Biotechnology (2 mentions)
Envision plate reader, by PerkinElmer (2 mentions)
Amp pnp, by Roche (2 mentions)
Muscimol, by MP Biomedicals (1 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions)
Anti mouse asc and caspase 1 antibodies, by Santa Cruz Biotechnology (1 mentions)
Anti phosphotyrosine antibody, by Cell Signaling Technology (1 mentions)
Anti mouse and human il 1β antibodies, by R&D Systems (1 mentions)
Biotin conjugated anti mouse and rabbit secondary antibodies, by GE Healthcare (1 mentions)
γ 32p atp, by Cytiva (1 mentions)
Hepes, by Avantor (1 mentions)
Dithiothreitol (dtt), by Serva Electrophoresis (1 mentions)
Eb dye, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Chitin column, by New England Biolabs (1 mentions)
Dna modification enzymes, by New England Biolabs (1 mentions)
Dna oligonucleotide, by Integrated DNA Technologies (1 mentions)
Radioactive nucleotides, by PerkinElmer (1 mentions)
Bradford protein stain, by Bio-Rad (1 mentions)
Potassium chloride (kcl), by Avantor (1 mentions)
10 protocols using adenosine triphosphate (atp)
1
Inflammasome Activation Reagents and Antibodies
2
Kinase Activity Assay of S6K2 and PRMT6
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HEK293 cells were transfected with Myc-PRMT6 and EE-S6K2 expression plasmids. The cell extracts were subjected to immunoprecipitation with either anti-EE-tag or anti-Myc-tag antibodies following treatment for 30 min with the signal transduction inhibitors: 100 nM Rapamycin, 50 µM LY294002, or DMSO (vehicle). The immune complexes were washed three times with lysis buffer followed by a single wash with kinase assay buffer (50 mM HEPES [pH 7.5], 10 mM MgCl2, 1 mM dithiothreitol, 10 mM β-glycerophosphate). The kinase reaction was initiated in 25 μL of kinase assay buffer supplemented with 1 μM protein kinase A inhibitor (Calbiochem; San Diego, CA, USA), 50 μM ATP, 5 μCi of [γ-32P] ATP (Amersham Biosciences; Piscataway, NJ, USA), and 20 μg of 80S ribosomes isolated from a rat liver as a substrate (source of S6 protein (rpS6)) [5 (link),46 (link)]. The reaction was carried out at 30 °C for 30 min and terminated by the addition of the 5X SDS-PAGE sample buffer and boiling for 10 min. The proteins were resolved by SDS-PAGE. The amount of [γ-32P] incorporated into the proteins was assessed by a Fujifilm FLA-2000 phosphoimager apparatus (Bio-Rad; Hercules, CA, USA). Control IP was achieved by incubating lysates with protein A-Sepharose beads.
3
Nucleotide Synthesis Reaction Optimization
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The following reagents were used: [3H]-CDP ([5-3H]cytidine 5′-diphosphate) (Amersham Biosciences, TRK338); CDP (cytidine-5′-diphosphate) (MP Biochemical, 100529); ATP (adenosine 5′-triphosphate) (Amersham Biosciences, 27-1006-03); THP (tris(hydroxypropyl)phosphine) (Novagen, 71194); DTT (dithiothreitol) (Serva, 39759.02); BMS (bis(2-thioethyl)sulphone) (Apollo Scientific Ltd., OR8251T); TCEP (tris(carboxyethyl)phosphine) (Sigma, 646547-10X1ML); HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid) (Amresco, 0511-250G); dATP-Agarose, γ-aminohexyl-dATP-Agarose (Jena Bioscience, AC-122L); DEAE Sepharose FF (Pharmacia, 17-0709-01); and DTBS ((S)-(1,4-dithiobutyl)-2-amine or (S)-2-aminobutane-1,4-dithiol) (Sigma-Aldrich, 774405-1G). The dithiols DTT, BMS, and DTBA were always dissolved immediately before use.
4
Measuring Mitochondrial Calcium Uptake
5
Flow Cytometry Analysis of CD4+ Cells
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Blood and spleen cells (1 × 106) were stained with APC-labeled anti-CD4 mAb (BD Biosciences). In some experiments, stained cells were pre-warmed (37°C) in phosphate-buffered saline with 3% bovine serum albumin (Sigma-Aldrich) and then incubated with 25–500 μM ATP (Amersham Bioscience, USA), lysed iRBC supernatant or medium alone for 15 min. The non-infected RBCs (nRBCs) and iRBCs (2 × 108) were lysed with 200 μl of lysis buffer (40 mM NH4Cl, 4.2 mM Tris [pH 7.4]) for 5 min at 4°C. Spleen cells (1 × 106) were incubated with 200 μl of RBC supernatants diluted 1:5 in cold RPMI with 1% heat-inactivated fetal calf serum (FCS). The fluorescent 2.5 μM EB dye (Sigma-Aldrich) was added, and the samples were immediately analyzed by flow cytometry.
6
Purification and Phosphorylation of Aurora-A
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Aurora-A was expressed as intein–Aurora-A fusion protein in the BL21 (DE3) Escherichia coli strain. The soluble fraction from bacterial extracts was loaded on to a 10 ml chitin column (New England Biolabs). The bound fraction was treated with 100 mM DTT (dithiothreitol) to induce intein self-splicing33 (link). A non-radioactive reaction was performed using 500 μg purified Aurora-A incubated with 50 ng Src (Enzo Life Sciences) in 50 μl kinase buffer (100 mM Tris-HCl, pH 7.2, 3 mM MgCl2, 1 mM NaF, 1 mM DTT) for 1 h at 37 °C in the presence of 1 mM ATP (Amersham Biosciences). Then, 1 μg recombinant His-tagged histone-H3 (Enzo Life Sciences) was added to the kinase reaction mix for 15 min at 37 °C. For the pre-incubation, 50 μM PP2 was added to Src for 10 min at 37 °C, before the addition of Aurora-A. Then the samples were analysed by SDS–PAGE.
7
Measuring Mitochondrial Calcium Dynamics
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The recombinant Ca 2+ -sensitive photoprotein aequorin specifically targeted to the mitochondrial matrix (52) was employed to measure Ca 2+ uptake by the mitochondria which reflects the Ca 2+ microdomains generated at the mouth of the InsP3 channels upon their opening (53) . For cytosolic and mitochondrial calcium measurements using the aequorin method, OBs were plated into a 96-well plate at 50-60% of confluence and transduced for 48h in their culture medium with adenovirus expressing cytosolic or mitochondrial aequorin (cytAEQ or mtAEQ). 48h post adenoviral transduction cells were incubated (1 hr, 37°C) with the prosthetic group coelenterazine (5 μM, 1 h at 37°C, Santa Cruz Biotechnology) in CaCl 2 -containing Krebs-Ringer buffer (KRB -CaCl 2 , 125 mM NaCl, 1 mM Na 3 PO 4 , 1 mM MgSO 4 , 1 mM CaCl 2 , 5.5 mM glucose, 5 mM KCl, 20 mM HEPES, pH 7.4). After aequorin reconstitution, cells were placed in 70 μl of KRB-CaCl 2 and cytosolic or mitochondrial Ca 2+ transients were evoked by applying 100 μM ATP (Amersham Biosciences) in KRB.
Cytosolic and mitochondrial calcium measurements were carried out using a PerkinElmer EnVision plate reader equipped with two injector units. Output data were analyzed and calibrated with a custom-made macro-enabled Excel workbook.
Cytosolic and mitochondrial calcium measurements were carried out using a PerkinElmer EnVision plate reader equipped with two injector units. Output data were analyzed and calibrated with a custom-made macro-enabled Excel workbook.
8
Biochemical Characterization of T-Antigen
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Radioactive nucleotides were from Perkin Elmer. ATP was from Cytiva and adenylyl imidodiphosphate (AMP-PNP) was from Roche. DNA modification enzymes were from New England Biolabs. DNA oligonucleotides were from Integrated DNA Technologies except for those with MeP linkages, which were from Gene Link (Elmsford, NY). Full-length T-Antigen was from Millipore Sigma (SRP2093), and the N-terminal truncation, T-Ag131–627, of T-Ag was expressed in E. coli as detailed in the SI Appendix . Protein concentrations were determined using the Bio-Rad Bradford Protein stain using BSA as a standard.
9
BrxL Oligomerization Assays
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BrxL constructs (WT and E280Q) were purified via metal affinity chromatography and SEC, and fractions from the monomer peak were flash frozen and stored at -80°C in the presence of 20% glycerol. To assess if ATP or AMP-PNP induced multimer assembly in the absence of DNA, a sample was thawed on ice, diluted with 20 mM Tris (pH 7.5), 150 mM NaCl, 5 mM MgCl2 and concentrated to ∼2.8 mg/ml (∼39 μM); the final concentration of glycerol was ∼5%. The sample was split into three aliquots and AMP-PNP (Roche) or ATP (Cytiva) were added to a final concentration of 1.25 mM; a third sample had no added nucleotide. Reactions were incubated at room temperature for 60 min, then loaded onto a HiLoad 16/60 Supderdex 200 (Cytiva) equilibrated in 25 mM Tris (pH 7.5), 150 mM NaCl and eluted over 130 ml at 1.5 ml min−1.
To assess multimer assembly in the presence of DNA, 20 μM BrxL (WT or E280Q) was incubated with 0.25 μM dsDNA (the same 207 bp fragment used for EMSA assays) in a final volume of 200 μl under the same conditions described above. Samples were then separated on an SEC650 analytical column (BioRad). Where indicated, ATP and AMP-PNP were used at 1.25 mM final concentrations.
To assess multimer assembly in the presence of DNA, 20 μM BrxL (WT or E280Q) was incubated with 0.25 μM dsDNA (the same 207 bp fragment used for EMSA assays) in a final volume of 200 μl under the same conditions described above. Samples were then separated on an SEC650 analytical column (BioRad). Where indicated, ATP and AMP-PNP were used at 1.25 mM final concentrations.
10
Glycolysis Pathway Characterization
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ADP, creatine, disodium hydrogen phosphate, NAD+, potassium sodium tartrate, sodium acetate (Sigma Aldrich, Ireland), ATP (Cytiva), carnosine (Apollo Scientific, United Kingdom), 3,5-dinitrosalicyclic acid, glycogen, hydrochloric acid, sodium hydroxide (Acrós Organic-ThermoFisher, UK), L-Lactic Acid assay kit (Megazyme, Ireland), magnesium chloride, potassium chloride (VWR Chemicals, Ireland), perchloric acid (Alfa Aesar, United Kingdom), potassium hydroxide (Merck Emsure, Ireland), liquid nitrogen (AirProducts, Ireland). All chemicals were at least of reagent grade.
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