We prepared two complexes: (1) 50S–HflX–AMP–PNP–GMP–PNP and other was 50S–HflX–AMP–PNP. To make these complexes, proteins and 50S subunits were incubated at a ratio of 23:1. First, proteins were incubated with either 5 mM AMP-PNP (Sigma-Aldrich) only or both 5 mM AMP–PNP (Sigma-Aldrich) and 4 mM GMP–PNP for 20 min at 4°C (in respect to reaction volume). 50S subunits were mixed with analogue-bound proteins in binding buffer (20 mM Tris-Cl, pH 7.6, 50 mM NH4Cl, 10 mM Mg(OAc)2, and 1 mM DTT) for 45 min at 4°C and then loaded on the sucrose gradient (10% to 40%). Ultracentrifugation was done at 140,000 rcf for 6 h at 4°C, and the gradient was then analyzed at 260 nm. Subunits containing fractions were pooled together, and sucrose was removed by binding buffer exchanged through a 100-kD cutoff Amicon-Ultra filtration unit. The sample was then separated on SDS-PAGE gel to confirm HflX binding.
Amp pnp
Manufactured by Merck Group
Sourced in Germany
AMP-PNP is a laboratory reagent used in biochemical and molecular biology research. It is an analog of adenosine triphosphate (ATP) that can be used to study enzymatic processes involving ATP. AMP-PNP acts as a non-hydrolyzable ATP analog, allowing for the investigation of ATP-dependent reactions without the hydrolysis of the ATP molecule.
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Lab products found in correlation
Adenosine triphosphate (atp), by Merck Group (6 mentions)
Atpγs, by Merck Group (4 mentions)
Adenosine diphosphate (adp), by Merck Group (4 mentions)
Fm4 64, by Thermo Fisher Scientific (3 mentions)
Amp pnp, by Cytoskeleton (2 mentions)
Taxol, by Cytoskeleton (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Sypro orange dye, by Thermo Fisher Scientific (2 mentions)
Heat shock protein 70, by Merck Group (1 mentions)
Methotrexate, by Merck Group (1 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
Control shrna lentiviral particles sc 108080, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg hrp, by Southern Biotech (1 mentions)
Anti mouse igg hrp, by Southern Biotech (1 mentions)
Normal mouse igg sc 2025, by Santa Cruz Biotechnology (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Mobicol column, by MoBiTec (1 mentions)
Synergy h4 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions)
Pilot one, by Huber (1 mentions)
Aquaspec cell, by Bruker (1 mentions)
55 protocols using amp pnp
1
Structural Analysis of 50S-HflX Complexes
2
Biochemical Compound Procurement Protocol
3
Dissecting Molecular Mechanisms of Neuronal Signaling
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The AAVrg-cre retrograde viruses were from Addgene (catalog 24593-AAVrg and 55632-AAVrg) (54 (link), 55 (link)). AAV2/5-EGFP and AAV-Cre were from the Viral Vector Core, University of Iowa. Kv3.1 shRNA lentiviral particles (sc-42720-V), WNK1 shRNA lentiviral particles (sc-39257-V), and control shRNA lentiviral particles (sc-108080) were from Santa Cruz. The following primary and secondary antibodies were used: anti-WNK1 (NB600-225 and AF2849, Novus Biologicals); anti-β3 tubulin (MAB1195, R&D Systems); p-WNK1 (pS382) antibody (SPC-1097, StressMarq); anti-Kv3.1 (NBP2-12903, Novus Biologicals), anti–GAPDH-HRP (sc-47724 HRP, Santa Cruz). Alexa Fluor secondary antibodies (A-11005, A-11001, A-11012) were from Thermo Fisher Scientific. Anti-rabbit IgG-HRP (4030-05, Southernbiotech), anti-mouse IgG-HRP (1030-05, Southernbiotech), anti-goat IgG-HRP (6425-05, Southernbiotech), normal Rabbit IgG (catalog 2729, Cell Signaling), normal mouse IgG (sc-2025, Santa Cruz) were used. Angiotensin II (A9525), anti-phospho-SPAK/OSR1 antibody (07-2273), TEA, ATP, AMP-PNP, and ATPγS were obtained from Sigma-Aldrich.
4
Isolation and Characterization of Dynein-Dynactin-BicD2 Complexes
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Five g of fresh mouse brains were washed with ice cold PMEE buffer (35 mM Pipes, pH 7.2, 5 mM MgSO4, 1 mM EGTA, 0.5 mM EDTA, 6.8% (w/v) glycerol), supplemented with 1X protease inhibitor and 1mM TCEP, then homogenized in an ice cold Dounce homogenizer. The homogenate was centrifuged at 36,000 × g for 15mins at 2°C and the supernatant centrifuged at 90,000 × g for 30mins at 2°C. BicD2N was added to the final supernatant at 500 nM and the mixture was incubated on ice for 2 hrs with gentle swirling every 20 mins to mix. 4mM MgSO4, 1mM GTP (Sigma Aldrich) and 4mM AMP-PNP (Sigma Aldrich) was added and the mixture incubated for 10 min at 37°C. AMP-PNP was used to promote tight binding of the dynein motor to the MT surface (Supplementary Fig. 5a ) The mixture was supplemented with 20μM taxol (Cytoskeleton Inc.) and further incubated for 15 mins at 37°C. The polymerized MTs, along with bound DDB particles, were pelleted by centrifuging at 21,000× g for 30 mins at 30°C. The pellet was washed by resuspension in PMEE buffer containing 4mM MgSO4, 1mM GTP, 4mM AMP-PNP and 20μM taxol and the MTs were pelleted as above. The final pellet was re-suspended in ten times its volume of PMEE buffer containing 4mM MgSO4, 1mM GTP, 4mM AMP-PNP and 20μM taxol at room temperature. The presence of DDB-MT complexes in the suspension was confirmed by SDS-PAGE and negative stain EM (Supplementary Fig. 5b,c ).
5
In vitro Protein-Protein Binding Assay
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To assess in vitro binding, the GST-tagged bait protein (15 µg GST-CtRsa4-UBL, 15 µg GST-CtYtm1-UBL or 30 µg GST-CtKap104) was incubated with 15 µg wild-type CtMIDAS or respective MIDAS loop mutants (as indicated in Fig. 5c, d and Fig. 7a ; Supplementary Fig. 6a, b ) and/or 140 µg CtRea1-NAAA+ ring (amino acid residues 1–2390) and incubated in binding buffer (50 mM Tris, pH 7.5, containing 80 mM NaCl, 5 mM MgCl2, 5% Glycerol, 2% DMSO, 2 mM Na2SO4, 0.01% NP-40, and 1 mM DTT) at 4 °C for 45 min. Nucleotides (ATP or AMPPNP, Sigma-Aldrich) or Rbin-1 (Axon Medchem) where added to a final concentration of 2 and 0.1 mM, respectively. Next, GST-tagged bait proteins and bound material were pulled-down by incubation with 70 µl GSH–agarose (Prontino, Macherey-Nagel) in Mobicol columns (MoBiTec), at 4 °C for 45 min. The flow-through was discarded and beads were washed five times with 500 µl binding buffer. Subsequently, elution was performed with the addition of 50 µl elution buffer (binding buffer containing 30 mM reduced GSH). Eluates were analyzed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis on 4–12% polyacrylamide gels (NuPAGE, Invitrogen) and Coomassie staining.
6
Fluorescence Polarization-based Assay for CasDinG
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Nucleic acid-binding activities of strep-tagged WT CasDinG and CasDinG mutants were monitored using a fluorescence polarization-based assay. Anisotropy data were collected using a BioTek Synergy H4 Hybrid Multi-Mode Microplate Reader with polarizers and bandpass filters. The polarizers and bandpass filters provided 485 ± 20 nm excitation and detection of fluorescence emission at 528 ± 20 nm. Each reaction (80 μl) contained a limiting concentration (10 nM) of 3′ FAM-labeled ssDNA (40 nt). CasDinG and 3′-end FAM-labeled nucleic acid were assayed at room temperature with increasing concentrations of CasDinG (0–2.5 μM) in a binding buffer (100 mM Tris pH 8.0, 1 mM TCEP and 5 mM MgCl2) with or without 1 mM AMP-PNP (Sigma Aldrich). Change in anisotropy relative to FAM-nucleic acid was plotted as a function of CasDinG concentration. The apparent dissociation constant (Kd) for the nucleic acid substrate was determined by fitting the raw data to a single site saturation binding model in GraphPad Prism for Windows version 9.3.0.
7
FTIR Analysis of InvCΔ79 Protein Interactions
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FTIR spectra were recorded in transmission mode using a Bruker VERTEX 70 FTIR spectrometer (Bruker, Ettlingen, Germany) equipped with an AquaSpec cell (Bruker) and a liquid nitrogen cooled MCT detector. InvCΔ79 (0.2 mM) in buffer B3 was mixed with magnesium chloride and either ADP, AMP‐PNP (Sigma‐Aldrich), or ATPγS (Jena Bioscience, Jena, Germany) in a ratio of 1:10:10, respectively. All samples were degassed, equilibrated at room temperature, and measured against buffer complemented with the respective ligand and magnesium chloride at the same concentration. Spectra were recorded at 25°C and the sample temperature was controlled with a Huber Ministat 125 with Pilot ONE (Huber, Offenburg, Germany). The spectral resolution was set to 2 cm−1 and a total of 64 scans were averaged before Fourier transformation. Analysis of the spectra was performed using OPUS 7.8 (Bruker). Atmospheric compensation (H2O, CO2, and aqueous solution) was applied to the background‐corrected sample spectra. Vector normalization was performed between 1,710 and 1,500 cm−1 to correct for concentration differences. Difference spectra were obtained by subtraction of the InvCΔ79 apo‐form spectrum from the spectra of the ligand‐containing samples.
8
Cell Cycle Synchronization and Stretch
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Cells were prepared as above and FBS withdrawal for 24 h was applied to bring cells to the same phase of the cell cycle. The same set of equi-biaxially stretch protocols was applied as for the control cells for 4 hours in the presence of the ATP analogue and ATPase inhibitor AMP-PNP (200 µM, Sigma-Aldrich) or hydrogen peroxide (H2O2, 50 µM, Sigma-Aldrich).
9
Protein Kinase A: Purification and Assay
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Rp-cAMPS (>99% purity) was purchased from Biolog, while cAMP (>98.5% purity) was purchased from Sigma-Aldrich and S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate (MTSL) was purchased from Toronto Research Chemicals. PKA R1a (91 to 379, 33 kDa), (119 to 379, 29 kDa), and (91 to 244, 17 kDa) constructs were expressed and purified according to previously published protocols (38 (link), 39 (link)). PKA C-subunit (40 kDa) was expressed (38 (link)) or purchased (P2645, Sigma-Aldrich). AMP-PNP was purchased from Sigma-Aldrich. The kinase substrates, PKS (GRTGRRNSI) and PKS2 (GRTGRANSI), were synthesized and purchased from GenScript. The Kinase-Glo reagents were purchased from Promega.
10
Topoisomerase II-Mediated DNA Cleavage Assay
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DNA cleavage assays were performed by incubating 5 ng pUC18 with FLAG-tagged topo IIβ (50, 100, and 200 fmol) in 10 μL cleavage buffer (50 mM Tris-HCl pH 8.0, 120 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 200 μM etoposide, and 30 μg/mL bovine serum albumin) at 37°C for 15 min. In the post-strand passage DNA cleavage reaction, 0.5 mM AMP-PNP (Sigma) was added to the reaction mixture. The cleavage reaction was terminated by adding 1% SDS and 0.2 μg/μL proteinase K (Roche). After incubation at 55°C for 1 h, samples were separated on 1% agarose gels, and DNA bands were detected by staining with GelRed Nucleic Acid Gel Stain.
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