Recombinant:
Article Title: Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons
Article Snippet: DOI: https://doi.org/10.7554/eLife.85561 16 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti- SNAP25 (mouse monoclonal) Covance SMI- 81R 1:1000 Antibody anti- VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti- tomosyn (rabbit polyclonal) Synaptic Systems 183103 1:1000 (WB) 1:500 (ICC) Antibody anti- tomosyn- 2 (rabbit polyclonal) Synaptic Systems 183203 1:1000 Antibody anti- alpha- tubulin (mouse monoclonal) Synaptic Systems 302211 1:1000 Antibody anti- actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti- synaptophysin (guinea pig polyclonal) Synaptic Systems 101004 1:1000 Antibody anti- MAP2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti- IA- 2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti- CHGA (rabbit polyclonal) Synaptic Systems 259003 1:500 Antibody anti- CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti- BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti- BDNF (mouse monoclonal) Biosensis BSENM- 1736–50 (clone 3C11) 1:500 Antibody anti- LAMP1 (rat monoclonal) DSHB hybridoma product 1D4B (supernatant)2 1:20 (ICC) 1:200 (WB) Antibody anti- LIMP2 (rabbit polyclonal) Novus Biologicals NB400- 129 1:1000 Antibody anti- GAPDH (rabbit polyclonal) Elabscience E- AB- 40337 1:2000 Antibody anti- TMEM87A (rabbit polyclonal) Novus Biologicals NBP1- 90532 1:100 Antibody anti- TGN38 (sheep polyclonal) Bio- Rad AHP499G 1:100 Recombinant DNA reagent pLenti- Syn(pr)- Cre- mCherry Wolzak et al., 2022 - Recombinant DNA reagent pLenti- Syn(pr)-ΔCre- mCherry Wolzak et al., 2022 - Cre lacking catalytic domain Recombinant DNA reagent pLenti- Syn(pr)- pre- NPYpHluorin Nassal et al., 2022 - Recombinant DNA reagent pLenti- mScarlet- T2A- tomosyn This paper - mouse isoform m (NM_001081344).
Article Title: Neuronal network inactivity potentiates neuropeptide release from mouse cortical neurons
Article Snippet: 102 103 eN eu r Ac ce pte d M an us cri pt Material and Methods 104 Key resources table 105 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus musculus) C57BL/6 J Charles River Strain code 631 Genetic reagent (Rattus norvegicus) Wistar (Crl:WI) Charles River Strain code: 003 Used for preparation of glia feeder layer Antibody anti-NPY (rabbit polyclonal) Cell Signaling 11976 1:400 Antibody anti-VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti-actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti-MAP-2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti-IA-2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti-CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti-BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti-GAPDH (rabbit polyclonal) Elabscience E-AB-40337 1:2000 Recombinant DNA reagent pLenti-Syn(pr)TeNT-IRESmCherry Hoogstraaten et al., 2020 - Recombinant DNA reagent pLenti-Syn(pr)pre-NPYpHluorin Nassal et al., 2022 - eN eu ro Ac ce pte d an us cri pt Reagent type (species) or resource Designation Source or reference Identifiers Additional information Sequence-based reagent qPCR primers are listed in Table 1 Peptide, recombinant protein 2.5% trypsin gibco 15090046 Peptide, recombinant protein papain Worthington Biochemical Corporation LS003127 Peptide, recombinant protein poly-L-ornithine Sigma P4957 Peptide, recombinant protein laminin Sigma L2020 Peptide, recombinant protein rat tail collagen BD Biosciences 354236 Peptide, recombinant protein poly-D-lysine Sigma P6407 Commercial assay or kit SensiFast cDNA Synthesis Kit Meridian Bioscience BIO-65054 Commercial assay or kit SensiFast SYBR Lo-ROX Kit Meridian Bioscience BIO-94020 Software, algorithm MATLAB MathWorks - Software, algorithm Prism GraphPad - Software, algorithm Fiji/ImageJ NIH - 106 eN eu ro Ac ce pt d M an us cri pt Table 1 107 List of qRT-PCR primers used in this study.
Sequencing:
Article Title: Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons
Article Snippet: DOI: https://doi.org/10.7554/eLife.85561 16 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti- SNAP25 (mouse monoclonal) Covance SMI- 81R 1:1000 Antibody anti- VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti- tomosyn (rabbit polyclonal) Synaptic Systems 183103 1:1000 (WB) 1:500 (ICC) Antibody anti- tomosyn- 2 (rabbit polyclonal) Synaptic Systems 183203 1:1000 Antibody anti- alpha- tubulin (mouse monoclonal) Synaptic Systems 302211 1:1000 Antibody anti- actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti- synaptophysin (guinea pig polyclonal) Synaptic Systems 101004 1:1000 Antibody anti- MAP2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti- IA- 2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti- CHGA (rabbit polyclonal) Synaptic Systems 259003 1:500 Antibody anti- CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti- BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti- BDNF (mouse monoclonal) Biosensis BSENM- 1736–50 (clone 3C11) 1:500 Antibody anti- LAMP1 (rat monoclonal) DSHB hybridoma product 1D4B (supernatant)2 1:20 (ICC) 1:200 (WB) Antibody anti- LIMP2 (rabbit polyclonal) Novus Biologicals NB400- 129 1:1000 Antibody anti- GAPDH (rabbit polyclonal) Elabscience E- AB- 40337 1:2000 Antibody anti- TMEM87A (rabbit polyclonal) Novus Biologicals NBP1- 90532 1:100 Antibody anti- TGN38 (sheep polyclonal) Bio- Rad AHP499G 1:100 Recombinant DNA reagent pLenti- Syn(pr)- Cre- mCherry Wolzak et al., 2022 - Recombinant DNA reagent pLenti- Syn(pr)-ΔCre- mCherry Wolzak et al., 2022 - Cre lacking catalytic domain Recombinant DNA reagent pLenti- Syn(pr)- pre- NPYpHluorin Nassal et al., 2022 - Recombinant DNA reagent pLenti- mScarlet- T2A- tomosyn This paper - mouse isoform m (NM_001081344).
Article Title: Neuronal network inactivity potentiates neuropeptide release from mouse cortical neurons
Article Snippet: 102 103 eN eu r Ac ce pte d M an us cri pt Material and Methods 104 Key resources table 105 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus musculus) C57BL/6 J Charles River Strain code 631 Genetic reagent (Rattus norvegicus) Wistar (Crl:WI) Charles River Strain code: 003 Used for preparation of glia feeder layer Antibody anti-NPY (rabbit polyclonal) Cell Signaling 11976 1:400 Antibody anti-VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti-actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti-MAP-2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti-IA-2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti-CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti-BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti-GAPDH (rabbit polyclonal) Elabscience E-AB-40337 1:2000 Recombinant DNA reagent pLenti-Syn(pr)TeNT-IRESmCherry Hoogstraaten et al., 2020 - Recombinant DNA reagent pLenti-Syn(pr)pre-NPYpHluorin Nassal et al., 2022 - eN eu ro Ac ce pte d an us cri pt Reagent type (species) or resource Designation Source or reference Identifiers Additional information Sequence-based reagent qPCR primers are listed in Table 1 Peptide, recombinant protein 2.5% trypsin gibco 15090046 Peptide, recombinant protein papain Worthington Biochemical Corporation LS003127 Peptide, recombinant protein poly-L-ornithine Sigma P4957 Peptide, recombinant protein laminin Sigma L2020 Peptide, recombinant protein rat tail collagen BD Biosciences 354236 Peptide, recombinant protein poly-D-lysine Sigma P6407 Commercial assay or kit SensiFast cDNA Synthesis Kit Meridian Bioscience BIO-65054 Commercial assay or kit SensiFast SYBR Lo-ROX Kit Meridian Bioscience BIO-94020 Software, algorithm MATLAB MathWorks - Software, algorithm Prism GraphPad - Software, algorithm Fiji/ImageJ NIH - 106 eN eu ro Ac ce pt d M an us cri pt Table 1 107 List of qRT-PCR primers used in this study.
Real-time Polymerase Chain Reaction:
Article Title: Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons
Article Snippet: DOI: https://doi.org/10.7554/eLife.85561 16 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti- SNAP25 (mouse monoclonal) Covance SMI- 81R 1:1000 Antibody anti- VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti- tomosyn (rabbit polyclonal) Synaptic Systems 183103 1:1000 (WB) 1:500 (ICC) Antibody anti- tomosyn- 2 (rabbit polyclonal) Synaptic Systems 183203 1:1000 Antibody anti- alpha- tubulin (mouse monoclonal) Synaptic Systems 302211 1:1000 Antibody anti- actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti- synaptophysin (guinea pig polyclonal) Synaptic Systems 101004 1:1000 Antibody anti- MAP2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti- IA- 2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti- CHGA (rabbit polyclonal) Synaptic Systems 259003 1:500 Antibody anti- CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti- BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti- BDNF (mouse monoclonal) Biosensis BSENM- 1736–50 (clone 3C11) 1:500 Antibody anti- LAMP1 (rat monoclonal) DSHB hybridoma product 1D4B (supernatant)2 1:20 (ICC) 1:200 (WB) Antibody anti- LIMP2 (rabbit polyclonal) Novus Biologicals NB400- 129 1:1000 Antibody anti- GAPDH (rabbit polyclonal) Elabscience E- AB- 40337 1:2000 Antibody anti- TMEM87A (rabbit polyclonal) Novus Biologicals NBP1- 90532 1:100 Antibody anti- TGN38 (sheep polyclonal) Bio- Rad AHP499G 1:100 Recombinant DNA reagent pLenti- Syn(pr)- Cre- mCherry Wolzak et al., 2022 - Recombinant DNA reagent pLenti- Syn(pr)-ΔCre- mCherry Wolzak et al., 2022 - Cre lacking catalytic domain Recombinant DNA reagent pLenti- Syn(pr)- pre- NPYpHluorin Nassal et al., 2022 - Recombinant DNA reagent pLenti- mScarlet- T2A- tomosyn This paper - mouse isoform m (NM_001081344).
Article Title: Neuronal network inactivity potentiates neuropeptide release from mouse cortical neurons
Article Snippet: 102 103 eN eu r Ac ce pte d M an us cri pt Material and Methods 104 Key resources table 105 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus musculus) C57BL/6 J Charles River Strain code 631 Genetic reagent (Rattus norvegicus) Wistar (Crl:WI) Charles River Strain code: 003 Used for preparation of glia feeder layer Antibody anti-NPY (rabbit polyclonal) Cell Signaling 11976 1:400 Antibody anti-VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti-actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti-MAP-2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti-IA-2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti-CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti-BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti-GAPDH (rabbit polyclonal) Elabscience E-AB-40337 1:2000 Recombinant DNA reagent pLenti-Syn(pr)TeNT-IRESmCherry Hoogstraaten et al., 2020 - Recombinant DNA reagent pLenti-Syn(pr)pre-NPYpHluorin Nassal et al., 2022 - eN eu ro Ac ce pte d an us cri pt Reagent type (species) or resource Designation Source or reference Identifiers Additional information Sequence-based reagent qPCR primers are listed in Table 1 Peptide, recombinant protein 2.5% trypsin gibco 15090046 Peptide, recombinant protein papain Worthington Biochemical Corporation LS003127 Peptide, recombinant protein poly-L-ornithine Sigma P4957 Peptide, recombinant protein laminin Sigma L2020 Peptide, recombinant protein rat tail collagen BD Biosciences 354236 Peptide, recombinant protein poly-D-lysine Sigma P6407 Commercial assay or kit SensiFast cDNA Synthesis Kit Meridian Bioscience BIO-65054 Commercial assay or kit SensiFast SYBR Lo-ROX Kit Meridian Bioscience BIO-94020 Software, algorithm MATLAB MathWorks - Software, algorithm Prism GraphPad - Software, algorithm Fiji/ImageJ NIH - 106 eN eu ro Ac ce pt d M an us cri pt Table 1 107 List of qRT-PCR primers used in this study.
cDNA Synthesis:
Article Title: Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons
Article Snippet: DOI: https://doi.org/10.7554/eLife.85561 16 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti- SNAP25 (mouse monoclonal) Covance SMI- 81R 1:1000 Antibody anti- VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti- tomosyn (rabbit polyclonal) Synaptic Systems 183103 1:1000 (WB) 1:500 (ICC) Antibody anti- tomosyn- 2 (rabbit polyclonal) Synaptic Systems 183203 1:1000 Antibody anti- alpha- tubulin (mouse monoclonal) Synaptic Systems 302211 1:1000 Antibody anti- actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti- synaptophysin (guinea pig polyclonal) Synaptic Systems 101004 1:1000 Antibody anti- MAP2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti- IA- 2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti- CHGA (rabbit polyclonal) Synaptic Systems 259003 1:500 Antibody anti- CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti- BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti- BDNF (mouse monoclonal) Biosensis BSENM- 1736–50 (clone 3C11) 1:500 Antibody anti- LAMP1 (rat monoclonal) DSHB hybridoma product 1D4B (supernatant)2 1:20 (ICC) 1:200 (WB) Antibody anti- LIMP2 (rabbit polyclonal) Novus Biologicals NB400- 129 1:1000 Antibody anti- GAPDH (rabbit polyclonal) Elabscience E- AB- 40337 1:2000 Antibody anti- TMEM87A (rabbit polyclonal) Novus Biologicals NBP1- 90532 1:100 Antibody anti- TGN38 (sheep polyclonal) Bio- Rad AHP499G 1:100 Recombinant DNA reagent pLenti- Syn(pr)- Cre- mCherry Wolzak et al., 2022 - Recombinant DNA reagent pLenti- Syn(pr)-ΔCre- mCherry Wolzak et al., 2022 - Cre lacking catalytic domain Recombinant DNA reagent pLenti- Syn(pr)- pre- NPYpHluorin Nassal et al., 2022 - Recombinant DNA reagent pLenti- mScarlet- T2A- tomosyn This paper - mouse isoform m (NM_001081344).
Article Title: Neuronal network inactivity potentiates neuropeptide release from mouse cortical neurons
Article Snippet: 102 103 eN eu r Ac ce pte d M an us cri pt Material and Methods 104 Key resources table 105 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus musculus) C57BL/6 J Charles River Strain code 631 Genetic reagent (Rattus norvegicus) Wistar (Crl:WI) Charles River Strain code: 003 Used for preparation of glia feeder layer Antibody anti-NPY (rabbit polyclonal) Cell Signaling 11976 1:400 Antibody anti-VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti-actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti-MAP-2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti-IA-2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti-CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti-BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti-GAPDH (rabbit polyclonal) Elabscience E-AB-40337 1:2000 Recombinant DNA reagent pLenti-Syn(pr)TeNT-IRESmCherry Hoogstraaten et al., 2020 - Recombinant DNA reagent pLenti-Syn(pr)pre-NPYpHluorin Nassal et al., 2022 - eN eu ro Ac ce pte d an us cri pt Reagent type (species) or resource Designation Source or reference Identifiers Additional information Sequence-based reagent qPCR primers are listed in Table 1 Peptide, recombinant protein 2.5% trypsin gibco 15090046 Peptide, recombinant protein papain Worthington Biochemical Corporation LS003127 Peptide, recombinant protein poly-L-ornithine Sigma P4957 Peptide, recombinant protein laminin Sigma L2020 Peptide, recombinant protein rat tail collagen BD Biosciences 354236 Peptide, recombinant protein poly-D-lysine Sigma P6407 Commercial assay or kit SensiFast cDNA Synthesis Kit Meridian Bioscience BIO-65054 Commercial assay or kit SensiFast SYBR Lo-ROX Kit Meridian Bioscience BIO-94020 Software, algorithm MATLAB MathWorks - Software, algorithm Prism GraphPad - Software, algorithm Fiji/ImageJ NIH - 106 eN eu ro Ac ce pt d M an us cri pt Table 1 107 List of qRT-PCR primers used in this study.
Software:
Article Title: Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons
Article Snippet: DOI: https://doi.org/10.7554/eLife.85561 16 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti- SNAP25 (mouse monoclonal) Covance SMI- 81R 1:1000 Antibody anti- VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti- tomosyn (rabbit polyclonal) Synaptic Systems 183103 1:1000 (WB) 1:500 (ICC) Antibody anti- tomosyn- 2 (rabbit polyclonal) Synaptic Systems 183203 1:1000 Antibody anti- alpha- tubulin (mouse monoclonal) Synaptic Systems 302211 1:1000 Antibody anti- actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti- synaptophysin (guinea pig polyclonal) Synaptic Systems 101004 1:1000 Antibody anti- MAP2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti- IA- 2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti- CHGA (rabbit polyclonal) Synaptic Systems 259003 1:500 Antibody anti- CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti- BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti- BDNF (mouse monoclonal) Biosensis BSENM- 1736–50 (clone 3C11) 1:500 Antibody anti- LAMP1 (rat monoclonal) DSHB hybridoma product 1D4B (supernatant)2 1:20 (ICC) 1:200 (WB) Antibody anti- LIMP2 (rabbit polyclonal) Novus Biologicals NB400- 129 1:1000 Antibody anti- GAPDH (rabbit polyclonal) Elabscience E- AB- 40337 1:2000 Antibody anti- TMEM87A (rabbit polyclonal) Novus Biologicals NBP1- 90532 1:100 Antibody anti- TGN38 (sheep polyclonal) Bio- Rad AHP499G 1:100 Recombinant DNA reagent pLenti- Syn(pr)- Cre- mCherry Wolzak et al., 2022 - Recombinant DNA reagent pLenti- Syn(pr)-ΔCre- mCherry Wolzak et al., 2022 - Cre lacking catalytic domain Recombinant DNA reagent pLenti- Syn(pr)- pre- NPYpHluorin Nassal et al., 2022 - Recombinant DNA reagent pLenti- mScarlet- T2A- tomosyn This paper - mouse isoform m (NM_001081344).
Article Title: Neuronal network inactivity potentiates neuropeptide release from mouse cortical neurons
Article Snippet: 102 103 eN eu r Ac ce pte d M an us cri pt Material and Methods 104 Key resources table 105 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus musculus) C57BL/6 J Charles River Strain code 631 Genetic reagent (Rattus norvegicus) Wistar (Crl:WI) Charles River Strain code: 003 Used for preparation of glia feeder layer Antibody anti-NPY (rabbit polyclonal) Cell Signaling 11976 1:400 Antibody anti-VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti-actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti-MAP-2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti-IA-2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti-CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti-BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti-GAPDH (rabbit polyclonal) Elabscience E-AB-40337 1:2000 Recombinant DNA reagent pLenti-Syn(pr)TeNT-IRESmCherry Hoogstraaten et al., 2020 - Recombinant DNA reagent pLenti-Syn(pr)pre-NPYpHluorin Nassal et al., 2022 - eN eu ro Ac ce pte d an us cri pt Reagent type (species) or resource Designation Source or reference Identifiers Additional information Sequence-based reagent qPCR primers are listed in Table 1 Peptide, recombinant protein 2.5% trypsin gibco 15090046 Peptide, recombinant protein papain Worthington Biochemical Corporation LS003127 Peptide, recombinant protein poly-L-ornithine Sigma P4957 Peptide, recombinant protein laminin Sigma L2020 Peptide, recombinant protein rat tail collagen BD Biosciences 354236 Peptide, recombinant protein poly-D-lysine Sigma P6407 Commercial assay or kit SensiFast cDNA Synthesis Kit Meridian Bioscience BIO-65054 Commercial assay or kit SensiFast SYBR Lo-ROX Kit Meridian Bioscience BIO-94020 Software, algorithm MATLAB MathWorks - Software, algorithm Prism GraphPad - Software, algorithm Fiji/ImageJ NIH - 106 eN eu ro Ac ce pt d M an us cri pt Table 1 107 List of qRT-PCR primers used in this study.
Quantitative RT-PCR:
Article Title: Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons
Article Snippet: DOI: https://doi.org/10.7554/eLife.85561 16 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti- SNAP25 (mouse monoclonal) Covance SMI- 81R 1:1000 Antibody anti- VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti- tomosyn (rabbit polyclonal) Synaptic Systems 183103 1:1000 (WB) 1:500 (ICC) Antibody anti- tomosyn- 2 (rabbit polyclonal) Synaptic Systems 183203 1:1000 Antibody anti- alpha- tubulin (mouse monoclonal) Synaptic Systems 302211 1:1000 Antibody anti- actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti- synaptophysin (guinea pig polyclonal) Synaptic Systems 101004 1:1000 Antibody anti- MAP2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti- IA- 2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti- CHGA (rabbit polyclonal) Synaptic Systems 259003 1:500 Antibody anti- CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti- BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti- BDNF (mouse monoclonal) Biosensis BSENM- 1736–50 (clone 3C11) 1:500 Antibody anti- LAMP1 (rat monoclonal) DSHB hybridoma product 1D4B (supernatant)2 1:20 (ICC) 1:200 (WB) Antibody anti- LIMP2 (rabbit polyclonal) Novus Biologicals NB400- 129 1:1000 Antibody anti- GAPDH (rabbit polyclonal) Elabscience E- AB- 40337 1:2000 Antibody anti- TMEM87A (rabbit polyclonal) Novus Biologicals NBP1- 90532 1:100 Antibody anti- TGN38 (sheep polyclonal) Bio- Rad AHP499G 1:100 Recombinant DNA reagent pLenti- Syn(pr)- Cre- mCherry Wolzak et al., 2022 - Recombinant DNA reagent pLenti- Syn(pr)-ΔCre- mCherry Wolzak et al., 2022 - Cre lacking catalytic domain Recombinant DNA reagent pLenti- Syn(pr)- pre- NPYpHluorin Nassal et al., 2022 - Recombinant DNA reagent pLenti- mScarlet- T2A- tomosyn This paper - mouse isoform m (NM_001081344).
Article Title: Neuronal network inactivity potentiates neuropeptide release from mouse cortical neurons
Article Snippet: 102 103 eN eu r Ac ce pte d M an us cri pt Material and Methods 104 Key resources table 105 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus musculus) C57BL/6 J Charles River Strain code 631 Genetic reagent (Rattus norvegicus) Wistar (Crl:WI) Charles River Strain code: 003 Used for preparation of glia feeder layer Antibody anti-NPY (rabbit polyclonal) Cell Signaling 11976 1:400 Antibody anti-VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti-actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti-MAP-2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti-IA-2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti-CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti-BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti-GAPDH (rabbit polyclonal) Elabscience E-AB-40337 1:2000 Recombinant DNA reagent pLenti-Syn(pr)TeNT-IRESmCherry Hoogstraaten et al., 2020 - Recombinant DNA reagent pLenti-Syn(pr)pre-NPYpHluorin Nassal et al., 2022 - eN eu ro Ac ce pte d an us cri pt Reagent type (species) or resource Designation Source or reference Identifiers Additional information Sequence-based reagent qPCR primers are listed in Table 1 Peptide, recombinant protein 2.5% trypsin gibco 15090046 Peptide, recombinant protein papain Worthington Biochemical Corporation LS003127 Peptide, recombinant protein poly-L-ornithine Sigma P4957 Peptide, recombinant protein laminin Sigma L2020 Peptide, recombinant protein rat tail collagen BD Biosciences 354236 Peptide, recombinant protein poly-D-lysine Sigma P6407 Commercial assay or kit SensiFast cDNA Synthesis Kit Meridian Bioscience BIO-65054 Commercial assay or kit SensiFast SYBR Lo-ROX Kit Meridian Bioscience BIO-94020 Software, algorithm MATLAB MathWorks - Software, algorithm Prism GraphPad - Software, algorithm Fiji/ImageJ NIH - 106 eN eu ro Ac ce pt d M an us cri pt Table 1 107 List of qRT-PCR primers used in this study.
Western Blot:
Article Title: Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons
Article Snippet: DOI: https://doi.org/10.7554/eLife.85561 16 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti- SNAP25 (mouse monoclonal) Covance SMI- 81R 1:1000 Antibody anti- VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti- tomosyn (rabbit polyclonal) Synaptic Systems 183103 1:1000 (WB) 1:500 (ICC) Antibody anti- tomosyn- 2 (rabbit polyclonal) Synaptic Systems 183203 1:1000 Antibody anti- alpha- tubulin (mouse monoclonal) Synaptic Systems 302211 1:1000 Antibody anti- actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti- synaptophysin (guinea pig polyclonal) Synaptic Systems 101004 1:1000 Antibody anti- MAP2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti- IA- 2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti- CHGA (rabbit polyclonal) Synaptic Systems 259003 1:500 Antibody anti- CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti- BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti- BDNF (mouse monoclonal) Biosensis BSENM- 1736–50 (clone 3C11) 1:500 Antibody anti- LAMP1 (rat monoclonal) DSHB hybridoma product 1D4B (supernatant)2 1:20 (ICC) 1:200 (WB) Antibody anti- LIMP2 (rabbit polyclonal) Novus Biologicals NB400- 129 1:1000 Antibody anti- GAPDH (rabbit polyclonal) Elabscience E- AB- 40337 1:2000 Antibody anti- TMEM87A (rabbit polyclonal) Novus Biologicals NBP1- 90532 1:100 Antibody anti- TGN38 (sheep polyclonal) Bio- Rad AHP499G 1:100 Recombinant DNA reagent pLenti- Syn(pr)- Cre- mCherry Wolzak et al., 2022 - Recombinant DNA reagent pLenti- Syn(pr)-ΔCre- mCherry Wolzak et al., 2022 - Cre lacking catalytic domain Recombinant DNA reagent pLenti- Syn(pr)- pre- NPYpHluorin Nassal et al., 2022 - Recombinant DNA reagent pLenti- mScarlet- T2A- tomosyn This paper - mouse isoform m (NM_001081344).
Article Title: Neuronal network inactivity potentiates neuropeptide release from mouse cortical neurons
Article Snippet: 102 103 eN eu r Ac ce pte d M an us cri pt Material and Methods 104 Key resources table 105 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus musculus) C57BL/6 J Charles River Strain code 631 Genetic reagent (Rattus norvegicus) Wistar (Crl:WI) Charles River Strain code: 003 Used for preparation of glia feeder layer Antibody anti-NPY (rabbit polyclonal) Cell Signaling 11976 1:400 Antibody anti-VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti-actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti-MAP-2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti-IA-2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti-CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti-BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti-GAPDH (rabbit polyclonal) Elabscience E-AB-40337 1:2000 Recombinant DNA reagent pLenti-Syn(pr)TeNT-IRESmCherry Hoogstraaten et al., 2020 - Recombinant DNA reagent pLenti-Syn(pr)pre-NPYpHluorin Nassal et al., 2022 - eN eu ro Ac ce pte d an us cri pt Reagent type (species) or resource Designation Source or reference Identifiers Additional information Sequence-based reagent qPCR primers are listed in Table 1 Peptide, recombinant protein 2.5% trypsin gibco 15090046 Peptide, recombinant protein papain Worthington Biochemical Corporation LS003127 Peptide, recombinant protein poly-L-ornithine Sigma P4957 Peptide, recombinant protein laminin Sigma L2020 Peptide, recombinant protein rat tail collagen BD Biosciences 354236 Peptide, recombinant protein poly-D-lysine Sigma P6407 Commercial assay or kit SensiFast cDNA Synthesis Kit Meridian Bioscience BIO-65054 Commercial assay or kit SensiFast SYBR Lo-ROX Kit Meridian Bioscience BIO-94020 Software, algorithm MATLAB MathWorks - Software, algorithm Prism GraphPad - Software, algorithm Fiji/ImageJ NIH - 106 eN eu ro Ac ce pt d M an us cri pt Table 1 107 List of qRT-PCR primers used in this study.
Immunocytochemistry:
Article Title: Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons
Article Snippet: DOI: https://doi.org/10.7554/eLife.85561 16 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti- SNAP25 (mouse monoclonal) Covance SMI- 81R 1:1000 Antibody anti- VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti- tomosyn (rabbit polyclonal) Synaptic Systems 183103 1:1000 (WB) 1:500 (ICC) Antibody anti- tomosyn- 2 (rabbit polyclonal) Synaptic Systems 183203 1:1000 Antibody anti- alpha- tubulin (mouse monoclonal) Synaptic Systems 302211 1:1000 Antibody anti- actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti- synaptophysin (guinea pig polyclonal) Synaptic Systems 101004 1:1000 Antibody anti- MAP2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti- IA- 2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti- CHGA (rabbit polyclonal) Synaptic Systems 259003 1:500 Antibody anti- CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti- BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti- BDNF (mouse monoclonal) Biosensis BSENM- 1736–50 (clone 3C11) 1:500 Antibody anti- LAMP1 (rat monoclonal) DSHB hybridoma product 1D4B (supernatant)2 1:20 (ICC) 1:200 (WB) Antibody anti- LIMP2 (rabbit polyclonal) Novus Biologicals NB400- 129 1:1000 Antibody anti- GAPDH (rabbit polyclonal) Elabscience E- AB- 40337 1:2000 Antibody anti- TMEM87A (rabbit polyclonal) Novus Biologicals NBP1- 90532 1:100 Antibody anti- TGN38 (sheep polyclonal) Bio- Rad AHP499G 1:100 Recombinant DNA reagent pLenti- Syn(pr)- Cre- mCherry Wolzak et al., 2022 - Recombinant DNA reagent pLenti- Syn(pr)-ΔCre- mCherry Wolzak et al., 2022 - Cre lacking catalytic domain Recombinant DNA reagent pLenti- Syn(pr)- pre- NPYpHluorin Nassal et al., 2022 - Recombinant DNA reagent pLenti- mScarlet- T2A- tomosyn This paper - mouse isoform m (NM_001081344).
Article Title: Neuronal network inactivity potentiates neuropeptide release from mouse cortical neurons
Article Snippet: 102 103 eN eu r Ac ce pte d M an us cri pt Material and Methods 104 Key resources table 105 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus musculus) C57BL/6 J Charles River Strain code 631 Genetic reagent (Rattus norvegicus) Wistar (Crl:WI) Charles River Strain code: 003 Used for preparation of glia feeder layer Antibody anti-NPY (rabbit polyclonal) Cell Signaling 11976 1:400 Antibody anti-VAMP2 (mouse monoclonal) Synaptic Systems 104211 1:2000 Antibody anti-actin (mouse monoclonal) Merck MAB1501 1:1000 Antibody anti-MAP-2 (chicken polyclonal) Abcam ab5392 1:5000 Antibody anti-IA-2 (PTPRN) (mouse monoclonal) Merck MABS469 1:100 Antibody anti-CHGB (rabbit polyclonal) Synaptic Systems 259103 1:500 Antibody anti-BDNF (mouse monoclonal) DSHB hybridoma product BDNF #9 (supernatant)1 1:4 Antibody anti-GAPDH (rabbit polyclonal) Elabscience E-AB-40337 1:2000 Recombinant DNA reagent pLenti-Syn(pr)TeNT-IRESmCherry Hoogstraaten et al., 2020 - Recombinant DNA reagent pLenti-Syn(pr)pre-NPYpHluorin Nassal et al., 2022 - eN eu ro Ac ce pte d an us cri pt Reagent type (species) or resource Designation Source or reference Identifiers Additional information Sequence-based reagent qPCR primers are listed in Table 1 Peptide, recombinant protein 2.5% trypsin gibco 15090046 Peptide, recombinant protein papain Worthington Biochemical Corporation LS003127 Peptide, recombinant protein poly-L-ornithine Sigma P4957 Peptide, recombinant protein laminin Sigma L2020 Peptide, recombinant protein rat tail collagen BD Biosciences 354236 Peptide, recombinant protein poly-D-lysine Sigma P6407 Commercial assay or kit SensiFast cDNA Synthesis Kit Meridian Bioscience BIO-65054 Commercial assay or kit SensiFast SYBR Lo-ROX Kit Meridian Bioscience BIO-94020 Software, algorithm MATLAB MathWorks - Software, algorithm Prism GraphPad - Software, algorithm Fiji/ImageJ NIH - 106 eN eu ro Ac ce pt d M an us cri pt Table 1 107 List of qRT-PCR primers used in this study.
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