Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| strain, strain background (M. musculus) |
C57BL/6 | |||
| strain, strain background (M. musculus) |
CD1 | |||
| genetic reagent (M. musculus) |
Doc2a null allele | Sakaguchi G, Manabe T, Kobayashi K, Orita S, Sasaki T, Naito A, Maeda M, Igarashi H, Katsuura G, Nishioka H, Mizoguchi A, Itohara S, Takahashi T, Takai Y. Doc2alpha is an activity- dependent modulator of excitatory synaptic transmission. Eur J Neurosci. 1999 Dec;11(12):4262–8. |
PMID: 10594652 | |
| genetic reagent (M. musculus) |
Doc2b null allele | Groffen AJ, Martens S, Díez Arazola R, Cornelisse LN, Lozovaya N, de Jong AP, Goriounova NA, Habets RL, Takai Y, Borst JG, Brose N, McMahon HT, Verhage M. Doc2b is a high-affinity Ca2+ sensor for spontaneous neurotransmitter release. Science. 2010 Mar 26;327 (5973):1614–8. |
PMID: 20150444 | |
| 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 synaptotagmin-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. Synaptotagmin I: a major Ca2 + sensor for transmitter release at a central synapse. Cell 79(4): 717–727. |
PMID: 7954835 | |
| genetic reagent (M. musculus) |
Unc13b null allele | Varoqueaux F, Sigler A, Rhee J-S, Brose N, Enk C, Reim K, Rosenmund C. Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-medicated vesicle priming. Proc Natl Acad Sci U S A. 2002. Jun 25; 99(13):9037–9042. |
PMID: 12070347 | |
| transfected construct (R. norvegicus) |
pSFV-Doc2b-WT- IRES-EGFP |
Friedrich R, Groffen AJ, Connell E, van Weering JR, Gutman O, Henis YI, Davletov B, Ashery U. DOC2B acts as a calcium switch and enhances vesicle fusion. J Neurosci. 2008 Jul 2;28(27):6794–806. |
Genbank Accession: EU635444.1 |
local reference: SG#334 |
| transfected construct (R. norvegicus) |
pSFV-Doc2b-DN- IRES-EGFP |
Friedrich R, Groffen AJ, Connell E, van Weering JR, Gutman O, Henis YI, Davletov B, Ashery U. DOC2B acts as a calcium switch and enhances vesicle fusion. J Neurosci. 2008 Jul 2;28(27):6794–806. |
Genbank Accession: EU635445.1 |
derived from EU635444.1 with indicated mutations |
| transfected construct (R. norvegicus) |
pSFV-Doc2b-6A- IRES-EGFP |
this paper | derived from EU635444.1 with indicated mutations |
|
| transfected construct (R. norvegicus) |
pSFV-Doc2b-KE- IRES-EGFP |
this paper | derived from EU635444.1 with indicated mutations |
|
| transfected construct (R. norvegicus) |
pSFV-Doc2b-TCT- IRES-EGFP |
this paper | derived from EU635444.1 with indicated mutations |
|
| transfected construct (R. norvegicus) |
pSFV-Doc2b-MID (14-41)-IRES-EGFP |
this paper | derived from EU635444.1 with indicated mutations |
|
| transfected construct (R. norvegicus) |
pSFV-Doc2b-MID (Scrambled)- IRES-EGFP |
this paper | derived from EU635444.1 with indicated mutations |
|
| transfected construct (R. norvegicus) |
pLenti- Doc2bEGFP-WT |
this paper | derived from EU635444.1; local reference: J274#2 |
|
| transfected construct (R. norvegicus) |
pLenti- Doc2bEGFP-DN |
this paper | local reference: J274#7 |
|
| transfected construct (R. norvegicus) |
pLenti- Doc2bEGFP-6A |
this paper | local reference: K102#33 |
|
| transfected construct (R. norvegicus) |
pSFV-EGFP- Doc2B-DeltaMID |
this paper | local reference: #480 | |
| transfected construct () | ||||
| transfected construct () | ||||
| transfected construct () | ||||
| biological sample () | ||||
| antibody | anti-syntaxin | Synaptic System | SySy: 110011 | dil. 1:1000; Overnight/room temperature |
| antibody | anti-GFP | Abcam | Ab13970 | dil. 1:1000; Overnight/room temperature |
| antibody | secondary Goat anti-mouse Alexa 488 |
Invitrogen | Invitrogen: A11029 | dil. 1:1000; Overnight/4 deg. |
| antibody | secondary Goat anti-chicken Alexa 488 |
Abcam | Ab150169 | dil. 1:1000; Overnight/4 deg. |
| antibody | anti- synaptotagmin-1 |
Synaptic System | SySy: 105011 | dil. 1:1000; Overnight/4 deg. |
| antibody | anti- synaptotagmin-7 |
Synaptic System | SySy: 105173 | dil. 1:500; Overnight/4 deg. |
| antibody | anti-VCP | Abcam | Ab11433 | dil. 1:2000; 1h/room temperature |
| antibody | mouse mAb against Syb2, clone 69.1 |
Synaptic Systems GmbH | cat#104211 | 1:2500 diluted in PBS+ 2% BSA, incubated 2 hr at RT |
| antibody | rabbit pAb against Doc2b |
Groffen AJ, Brian EC, Dudok JJ, Kampmeijer J, Toonen RF, Verhage M. Ca(2+)-induced recruitment of the secretory vesicle protein DOC2B to the target membrane. J Biol Chem. 2004 May 28;279(22):23740–7. |
PMID: 15033971 | 1:133 diluted in PBS+ 2% BSA, incubated 2 hr at RT (note: Doc2b and Syb2 stainings were performed separately on different coverslips to rule out potential spectral overlap)/For western blotting: dil. 1:500; Overnight at 4 deg |
| antibody | Goat anti-rabbit HRP | Agilent | Dako-P0448 | dil. 1:10000; 1h30/room temperature |
| antibody | Goat anti-mouse HRP | Agilent | Dako-P0447 | dil. 1:10000; 1h30/room temperature |
| antibody | Goat-anti-Rabbit Alexa546 |
ThermoFisher Scientific | cat#A-11010 | 1:1000 diluted in PBS+ 2% BSA |
| antibody | Goat-anti-Mouse Alexa647 |
ThermoFisher Scientific | cat#10739374 | 1:1000 diluted in PBS+ 2% BSA |
| antibody | ||||
| recombinant DNA reagent | ||||
| sequence-based reagent | ||||
| peptide, recombinant protein |
||||
| commercial assay or kit | BCA Protein assay kit |
Pierce | Pierce: 23227 | |
| commercial assay or kit | ||||
| commercial assay or kit | ||||
| 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 biophycial 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 | Vitamin C | Sigma-aldrich | Sigma-aldrich: A5960 | |
| chemical compound, drug | EGTA | Sigma-aldrich | Sigma-aldrich: E4378 | |
| 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 | |
| chemical compound, drug | ||||
| chemical compound, drug | ||||
| chemical compound, drug | ||||
| chemical compound, drug | ||||
| software, algorithm | Igor | wavemetrics | ||
| software, algorithm | ImageJ | NIH software |