The in vitro autocleavage assay was performed as described previously [6 (link),27 (link)]. Each toxin protein was diluted in 20 mM Tris buffer (pH 7.4) in a final volume of 100 μL. Cleavage was initiated by addition of 10 μM inositol hexakisphosphate (InsP6) (Sigma, St. Louis, MO, USA) and the mixture was incubated at 37 °C for 12 h. To investigate the dependence of the effect on the time course and the concentration of InsP6, the toxins were incubated with 5, 10, and 20 μM InsP6 for 4, 8, and 12 h, respectively. The reaction was stopped by SDS-PAGE sample loading buffer, and analyzed by western blot using anti-TcdBGTD (the GTD of TcdB, amino acids 1–543) antiserum prepared by our laboratory.
Insp6
Manufactured by Merck Group
Sourced in United States
InsP6 is a laboratory product manufactured by Merck Group. It is an inositol hexakisphosphate compound that serves as a research tool for scientific investigations. The core function of InsP6 is to provide a chemical compound for analytical and experimental purposes in various fields of study.
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Lab products found in correlation
Glu plasminogen, by Enzyme Research (6 mentions)
Human fibrinogen, by Merck Group (2 mentions)
Apyrase from potato, by Merck Group (2 mentions)
Chemicals for all buffers described below, by Merck Group (2 mentions)
Human collagen type 3, by Southern Biotech (2 mentions)
May gr nwald solution, by Carl Roth (2 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions)
85srcl2, by PerkinElmer (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Synapt g2 si hdms, by Waters Corporation (1 mentions)
Ultrapure water, by Fujifilm (1 mentions)
Ammonium bicarbonate, by Avantor (1 mentions)
Lc ms grade acetonitrile, by Avantor (1 mentions)
Hexadeutero myo inositol trispyrophosphate itpp d6, by LGC (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Ammonium formate, by Merck Group (1 mentions)
Fibrinogen, by Thermo Fisher Scientific (1 mentions)
Human α thrombin, by Prolytix (1 mentions)
13 protocols using insp6
1
In vitro autocleavage assay of toxins
2
Synthesis and Purification of Inositol Phosphates
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Adenophostin A34 , Ins(1,3,4,5)P4 (ref. 63 ) scyllo-InsP5 (ref. 31 (link)), Bz(1,2,3,4)P4 (ref. 32 (link)), 1-PA-InsP5 (ref. 36 (link)), 2-OH-5-PA-InsP4 (ref. 37 ), 2-FAM-Ins(1,3,4,5,6)P5 (ref. 38 (link)) were synthesized as previously reported. The identities and purities of these compounds were confirmed by 1H and 31P NMR spectroscopy. Ins(1,3,5,6)P4, Ins(3,4,5,6)P4, Ins(1,3,4,5,6)P5, Ins(1,4,5)P3, Ins(1,4,6)P3, Ins(1,5,6)P3, were purchased from Cayman Chemical Company. InsP6 was purchased from Sigma. Details of the new syntheses of Ins(1,4,5,6)P4, Ins(4,5,6)P3, Ins(1,3,4,6)P4, 2-O-Bn-Ins(1,4,5,6)P4 and 2,3-di-O-Bn-Ins(1,4,5,6)P4 are given in the Supplementary Methods .
3
Preparing Inositol Hexakisphosphate Solution
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4
Radiolabeling Protocol with InsP6
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85SrCl2 (> 111 GBq/g) was purchased from PerkinElmer (Waltham, MA, USA). InsP6 was purchased from Sigma-Aldrich (St. Louis, MO, USA) as phytic acid sodium salt hydrate, InsP6•6Na+•6H2O. Other reagents were of reagent grade and used as received. All sections of this report adhere to the ARRIVE Guidelines for reporting animal research [14 (link)]. A completed ARRIVE guidelines checklist is included in supporting information (S1 File ).
5
Radium-223 Chloride Bioavailability
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[223Ra]RaCl2 was obtained from Bayer Yakuhin, Ltd (Osaka, Japan). Chlorella powder was supplied by Daesang Corp. (Seoul, Korea). InsP6 was purchased from Sigma-Aldrich (St. Louis, MO, USA) as phytic acid sodium salt hydrate, InsP6·6Na+·6H2O. Other reagents were of reagent grade and were used as received.
6
Recombinant Protein Expression Vectors
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Restriction enzymes and Gibson Assembly® cloning reagents were obtained from New England Biolabs and polymerases were obtained from Life Technologies. TOP10 competent cells and InsP6 (also known as phytic acid) were obtained from Sigma-Aldrich. Other common chemicals, antibiotics, and growth media components were from Sigma-Aldrich or Thermo Fisher.
Bacterial expression vectors for production of recombinant proteins pMCSG7, pMCSG53, and pMCSG58 were obtained from Dr. Andrzej Joachimiak of the Midwest Center for Structural Genomics (MCSG). The vector pMCSG7 consists of a LIC site and encodes for an N-terminal TEV cleavable 6xHis-tagged protein [15 (link)]. The vector pMCSG53 is an upgraded version of pMCSG7 that also carries the rare codon tRNA genes argU and ileX [16 (link)]. The vector pMCSG58 is similar to pMCSG53 except it encodes for a C-terminal 6xHis-tagged protein [16 (link)]. Proteins were expressed in kanamycin-resistant BL21(DE3)MAGIC E. coli strain. The pMagic plasmid also provides a second copy of the rare codon tRNA gene argU ([17 (link)] and A. Joachimiak, personal communication).
Bacterial expression vectors for production of recombinant proteins pMCSG7, pMCSG53, and pMCSG58 were obtained from Dr. Andrzej Joachimiak of the Midwest Center for Structural Genomics (MCSG). The vector pMCSG7 consists of a LIC site and encodes for an N-terminal TEV cleavable 6xHis-tagged protein [15 (link)]. The vector pMCSG53 is an upgraded version of pMCSG7 that also carries the rare codon tRNA genes argU and ileX [16 (link)]. The vector pMCSG58 is similar to pMCSG53 except it encodes for a C-terminal 6xHis-tagged protein [16 (link)]. Proteins were expressed in kanamycin-resistant BL21(DE3)MAGIC E. coli strain. The pMagic plasmid also provides a second copy of the rare codon tRNA gene argU ([17 (link)] and A. Joachimiak, personal communication).
7
In vitro autoprocessing of TcdB toxin
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The in vitro autoprocessing activity of TcdBs was performed in a 20 uL reaction with 200 nM of toxin, cleavage buffer (150 mM NaCl, 20 mM Tris pH 7.4), and 10 mM InsP6 (Sigma-Aldrich, St. Louis, MO, USA) [23 (link)]. The reaction was incubated for 0, 2.5, 5, 7.5, 10, 20, 30, 60, and 90 min at 37 °C. The reaction was stopped by addition of loading buffer and boiling at 95 °C for 5 min. The samples were separated by 7.5% SDS-PAGE and then analyzed by Coomassie staining. The activity of each toxin was quantified by comparing the densities of the bands of full-length and processed protein using Image J software (NIH, Bethesda, MD, USA). The data were plotted using nonlinear regression and in GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA [29 (link)].
8
mTORC2 Interactome Profiling by MS
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InsP6 (Sigma-Aldrich) was directly dissolved in 10 mM ammonium acetate (pH 8.5) and diluted to 50 μM. mTORC2 in cryo-EM buffer was buffer-exchanged and concentrated in 10 mM ammonium acetate (pH 8.5) using an Amicon Ultra-0.5 mL—MWCO 100kDa. The concentrated complex was mixed with an equal volume of Phenol at pH 8, thoroughly vortexed for 30 s, and incubated at room temperature for 30 min. The tube was then centrifuged for 5 min at 15,000g. The aqueous phase was then used for MS. A sample containing only buffer and no protein was subjected to the same treatment for reference. The samples were then mixed with four volumes of injection buffer [90% acetonitrile, 9% methanol, 50 mM ammonium acetate (pH 7)] and directly injected using a Hamilton syringe in Synapt G2-SI HDMS (Waters) in negative mode and using the T-Wave IMS.
9
Inositol Phosphate Analysis by LC-MS
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LC-MS grade acetonitrile and ammonium bicarbonate were purchased from Honeywell Burdick & Jackson (Morristown, NJ, USA). Ultrapure water was obtained from Wako Pure Chemical Industries (Osaka, Japan). Ultrapure-grade ammonium hydroxide (28% w/v) was obtained from Kanto Chemical (Tokyo, Japan). InsP 6 , ammonium formate, and ammonium acetate were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Hexadeutero-myo-inositol trispyrophosphate (ITPP-d 6 ) was purchased from Toronto Research Chemicals (North York, Canada). InsP 7 was synthesized from myo-inositol using fluorenylmethyl phosphoramidite chemistry as previously described [22] .
Louis, MO, USA). Hexadeutero-myo-inositol trispyrophosphate (ITPP-d 6 ) was purchased from Toronto Research Chemicals (North York, Canada). InsP 7 was synthesized from myo-inositol using fluorenylmethyl phosphoramidite chemistry as previously described [22] .
10
Synthesis and Characterization of Inositol Phosphates
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