Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Schematic diagram showing domain organization and variant fragments of Doc2B and Munc13-1. ( B ) Binding of MUN to GST-Doc2B FL or its variant fragments measured by GST pull-down experiments and quantification of the binding. ( C ) ITC-based measurements of MUN binding to GST-Doc2B 13 to 37 (Mid, left) and to GST-Doc2B 13 to 80 (Mid-L, right). ( D ) ITC-based measurements of the binding affinities between GST-Doc2B FL or its variant fragments and MUN. ( E ) 2D 1 H- 15 N HSQC spectra of 13 C/ 15 N-labeled Doc2B 1 to 80 before (black) and after (red) addition of MUN. Cross-peaks of residues that are chosen for mutation to detect MUN binding are labeled along with their corresponding residue number (cyan). ( F ) Peak intensity alteration of 13 C/ 15 N-labeled Doc2B 1 to 80 protein expressed as ratio between integrated peak volumes after ( V ) and before ( V 0 ) addition of unlabeled MUN. ( G ) Binding of Doc2B FL or its variant mutations to GST-MUN measured by GST pull-down experiments and quantification of the binding. ( H ) Binding of MUN or its variant fragments to GST-Doc2B FL measured by GST pull-down experiments and quantification of the binding. Red asterisk shows the band of bound MUN-BC. ( I ) Binding of MUN or its variant mutations to GST-Doc2B FL measured by GST pull-down experiments and quantification of the binding. Data of GST pull-down experiments are processed by ImageJ (National Institutes of Health) and presented as means ± SEM ( n = 3). Statistical significance and P values were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. * P < 0.05; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: For detecting Doc2B binding to EphB2 or Munc13-1 from brain samples, adult mouse brain was initially ground in buffer B supplied with 1% Triton X-100 and proteinase inhibitor cocktail (TargetMol) and then incubated with GST-Doc2B FL at 4°C overnight.

Techniques: Variant Assay, Binding Assay, Labeling, Mutagenesis, Residue

Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Illustration of the FRET assay for detecting MUN-catalyzed SNARE complex assembly starting from the Munc18-1/Syx1 (1 to 261) complex in the presence of Syb2 (29 to 96), SN25, and MUN. FRET signal between BDPY-labeled Syb2 S61C (donor) and TMR-labeled SN25 S187C (acceptor) was monitored. ( B to D ) MUN-catalyzed SNARE complex assembly by addition of Doc2B Mid, Doc2B Mid-L, or Doc2B FL, respectively (B), addition of different concentrations of Doc2B Mid (C), and addition of MUN NFAA or Doc2B I20A, which disrupts Doc2B-MUN interaction (D). Decrease of donor fluorescence at 1500 s is shown in the column at the right of the chart. ( E ) Illustration of Munc13-catalyzed lipid mixing between liposomes bearing Syb2 (1 to 116) and liposomes bearing the Munc18-1/Syx1 (1 to 288) complex in the presence of SN25, Syt1 C2AB, Ca 2+ , and Munc13-1 (C 1 C 2 BMUN). Donor (NBD) fluorescence was monitored at 538 nm. ( F to H ) Munc13-catalyzed lipid mixing by addition of Doc2B Mid, Doc2B Mid-L, or Doc2B FL, respectively (F); addition of C 1 C 2 BMUN NFAA or Doc2B I20A (G). Munc13-catalyzed lipid mixing at 1000 s is shown in (H). F 1 , fluorescence intensity observed as a function time; F 0 , initial fluorescence intensity. Data are presented as means ± SEM ( n = 3). Statistical significance and P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: For detecting Doc2B binding to EphB2 or Munc13-1 from brain samples, adult mouse brain was initially ground in buffer B supplied with 1% Triton X-100 and proteinase inhibitor cocktail (TargetMol) and then incubated with GST-Doc2B FL at 4°C overnight.

Techniques: Labeling, Fluorescence, Liposomes

Journal: Frontiers in Molecular Neuroscience

Article Title: Localization of the Priming Factors CAPS1 and CAPS2 in Mouse Sensory Neurons Is Determined by Their N-Termini

doi: 10.3389/fnmol.2022.674243

Figure Lengend Snippet: Details of antibodies used in this study.

Article Snippet: Munc13-1 , Rabbit , Recombinant protein (aa 3–317 from rat Munc13-1) , Synaptic Systems 126103 , 1:500 (ICC).

Techniques: Recombinant, Purification, Sequencing