Key resources table.
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (M. musculus) | C57BL/6 | Experimental Medicine, Panum Stable, University of Copenhagen. | ||
| Strain, strain background (M. musculus) | CD1 | Experimental Medicine, Panum Stable, University of Copenhagen. | ||
| Genetic reagent (M. musculus) | Synaptotagmin-7 (syt7) null allele | Maximov A, Lao Y, Li H, Chen X, Rizo J, Sørensen JB, Südhof TC. Genetic analysis of synaptotagmins-7 function in synaptic vesicle exocytosis. Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3986–3991. | PMID:18308933 | |
| Genetic reagent (M. musculus) | Synaptotagmin-1 (syt1) null allele | Geppert M, Goda Y, Hammer RE, LI C, Rosahl TW, Stevens CF, Südhof TC. 1994. Synaptotagmins I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79(4): 717–727. | PMID:7954835 | |
| Transfected construct (Rattus norwegicus) | p156rrl-pCMV- pH(ecliptic GFP)-TEV-rnSyt1 | This paper, Syt-1 WT | Local reference: Lenti #94 |
|
| Transfected construct (Rattus norwegicus) | p156rrl-pCMV- pH(ecliptic GFP)-TEV-rnSyt7S | This paper, Syt-7 WT | Local reference: Lenti #96 |
|
| Transfected construct (Rattus norwegicus) | p156rrl-pCMV- pH(ecliptic GFP)-TEV-rnSyt7S-C2AB/D225,227,233,357,359A | This paper, Syt-7 C2AB* | Local reference: Lenti #133 |
|
| Transfected construct (Rattus norwegicus) | p156rrl-pCMV- pH(ecliptic GFP)-TEV-rnSyt7S-C2B/D357,359A | This paper, Syt-7 C2B* | Local reference: Lenti #135 |
|
| Transfected construct (Rattus norwegicus) | p156rrl-pCMV- pH (ecliptic GFP)-TEV-rnSyt7S-C2A/D225,227,233A |
This paper, Syt-7 C2A* | Local reference: Lenti #136 |
|
| Transfected construct (Rattus norwegicus) | pSFV1-EGFP-ubMunc13-2 | Zikich D, Mezer A, Varoqueaux F, Sheinin A, Junge HJ, Nachiel E, Melamed R, Brose N, Gutman M, Ashery U. 2008. J. Neurosci. 28:1949–1960. | PMID:18287511 | Local reference: Semliki #486 |
| Antibody | Chicken anti-GFP | Abcam | Ab13970 RRID:AB_300798 |
1:500; 2 hr at room temperature |
| Antibody | Rabbit anti-Chromogranin A | Abcam | Ab15160 RRID:AB_301704 |
1:500; Overnight at four degrees |
| Antibody | Rabbit anti-synaptotagmin-1 | Gift from T. C. Südhof, Stanford, CA | W855 | 1:2000; 2 hr at room temperature |
| Antibody | Mouse anti-synaptotagmin-1 | Synaptic System | SySy: 105011 RRID:AB_887832 |
1:500; Overnight at 4 degrees or 2 hr at room temperature |
| Antibody | Rabbit anti-synaptotagmin-7 | Synaptic System | SySy: 105173 RRID:AB_887838 |
ICC: 1:500; Overnight at 4 degrees or 2 hr at room temperature WB: 1:500; Overnight at four degrees. |
| Antibody | Mouse anti-synaptotagmin-7 | Sigma-aldrich | MABN665 RRID:AB_2888943 |
1:200; Overnight/4 degrees |
| Antibody | anti-VCP | Abcam | Ab11433 RRID:AB_298039 |
1:10000; 1 hr at room temperature |
| Antibody | Goat anti-rabbit HRP | Agilent | Dako-P0448 RRID:AB_2617138 |
1:2000; 1 hr and30 min at room temperature |
| Antibody | Goat anti-mouse HRP | Agilent | Dako-P0447 RRID:AB_2617137 |
1:10000; 30 mins at room temperature |
| Antibody | Goat anti-mouse Alexa 546 | ThermoFisher Scientific | A11003 AB_2534071 |
1:500; 30 min at room temperature |
| Antibody | Goat anti-rabbit Alexa 647 | ThermoFisher Scientific | A21245 RRID:AB_2535813 |
1:500; 30 min at room temperature |
| Antibody | Goat anti-mouse Alexa 488 | Invitrogen | A11029 RRID:AB_2534088 |
1:500; 30 min at room temperature |
| Antibody | Goat anti-chicken Alexa 488 | Abcam |
Ab150169 RRID:AB_2636803 |
1:500; 30 min at room temperature |
| Commercial assay or kit | BCA Protein assay kit | Pierce | Pierce: 23227 | |
| Chemical compound, drug | NaCl | Sigma-aldrich | Sigma-aldrich: S9888 | |
| Chemical compound, drug | KCl | Sigma-aldrich | Sigma-aldrich: P5405 | |
| Chemical compound, drug | NaH2PO4 | Sigma-aldrich | Sigma-aldrich: S8282 | |
| Chemical compound, drug | Glucose | Sigma-aldrich | Sigma-aldrich: G8270 | |
| Chemical compound, drug | DMEM | Gibco | Gibco: 31966047 | |
| Chemical compound, drug | L-cysteine | Sigma-aldrich | Sigma-aldrich: C7352 | |
| Chemical compound, drug | CaCl2 | Sigma-aldrich | Sigma-aldrich: 499609 | |
| Chemical compound, drug | EDTA | Sigma-aldrich | Sigma-aldrich: E5134 | |
| Chemical compound, drug | Papain | Worthington Biochemical | Worthington Biochemical: LS003126 | |
| Chemical compound, drug | Albumin | Sigma-aldrich | Sigma-aldrich: A3095 | |
| Chemical compound, drug | Trypsin-inhibitor | Sigma-aldrich | Sigma-aldrich: T9253 | |
| Chemical compound, drug | Penicillin/streptomycin | Invitrogen | Invitrogen: 15140122 | |
| Chemical compound, drug | Insulin-transferrin-selenium-X | Invitrogen | Invitrogen: 51500056 | |
| Chemical compound, drug | Fetal calf serum | Invitrogen | Invitrogen: 10500064 | |
| Chemical compound, drug | MgCl2 | Sigma-aldrich | Sigma-aldrich: 449172 | |
| Chemical compound, drug | HEPES | Sigma-aldrich | Sigma-aldrich: H3375 | |
| Chemical compound, drug | Nitrophenyl-EGTA (NPE) | Synthesized at the Max-Planck-Institut for biophysical chemistry, Göttingen. | ||
| Chemical compound, drug | Fura-4F | Invitrogen | Invitrogen: F14174 | |
| Chemical compound, drug | Furaptra | Invitrogen | Invitrogen: M1290 | |
| Chemical compound, drug | Mg-ATP | Sigma-aldrich | Sigma-aldrich: A9187 | |
| Chemical compound, drug | GTP | Sigma-aldrich | Sigma-aldrich: G8877 | |
| Chemical compound, drug | Ascorbic acid | Sigma-aldrich | Sigma-aldrich: A5960 | |
| Chemical compound, drug | EGTA | Sigma-aldrich | Sigma-aldrich: E4378 | |
| Chemical compound, drug | Ethylmaleimide (NEM) | Sigma-aldrich | Sigma-aldrich: 04259 | |
| Chemical compound, drug | Phorbol 12-myristate 13-acetate (PMA) | Sigma-aldrich | Sigma-aldrich: P8139 |
|
| Chemical compound, drug | Paraformaldehyde | Sigma-aldrich | Sigma-aldrich: P6148 | |
| Chemical compound, drug | PIPES | Sigma-aldrich | Sigma-aldrich: 80635 | |
| Chemical compound, drug | Triton X-100 | Sigma-aldrich | Sigma-aldrich: T8787 | |
| Chemical compound, drug | BSA | Sigma-aldrich | Sigma-aldrich: A4503 | |
| Chemical compound, drug | Prolong Gold | Invitrogen | Invitrogen: P36934 | |
| Chemical compound, drug | Protease cocktail inhibitor | Invitrogen | Invitrogen: 87785 | |
| Chemical compound, drug | RIPA buffer | Invitrogen | Invitrogen: R0278 | |
| Chemical compound, drug | ECL plus western blotting substrate | Pierce | Pierce: 32132 | |
| Software, algorithm | Igor 8.0 | Wavemetrics | ||
| Software, algorithm | ImageJ | NIH software |