Journal: Scientific Reports

Article Title: A minimalist model to measure interactions between proteins and synaptic vesicles

doi: 10.1038/s41598-020-77887-1

Figure Lengend Snippet: Patterning strategy and SV immobilization. ( a ) Schematic representation of photopatterning of neutravidin on a glass coverslip. The glass coverslip was uniformly coated with an anti-fouling layer of PLL-g-PEG. The photoinitiator (PLPP) was added on top of the PLL-g-PEG layer and the substrate was exposed to UV light through a virtual photomask to activate the PLPP. Under UV light, the PLPP degraded the PLL-PEG layer, leaving accessible regions for neutravidin to attach. ( b ) Schematic of our strategy to attach the SVs to the substrate. The purified SVs (gray) were attached to the neutravidin (pink) functionalized glass coverslips via a biotinylated mouse anti-synaptotagmin antibody (orange). The attachment of antibodies was controlled by a secondary anti-mouse antibody labeled with Alexa Fluor 488 (green). To image the SVs, a single-domain antibody against vGLUT1, labeled with STAR635P (red) was employed. ( c ) Fluorescence micrographs of the individual steps of the vesicle immobilization strategy. The neutravidin functionalization step was assessed by fluorescently labeled neutravidin (neutravidin-FITC, left), the attachment of the biotinylated mouse anti-synaptotagmin antibody was tested with a secondary anti-mouse antibody labeled with Alexa Fluor 488 (center), and the SV attachment was tested with a single-domain antibody against vGLUT1 labeled with STAR635P (right). Note that the uniform fluorescence in the right image shows the bound SVs, whereas the small bright spots are aggregates. The images of neutravidin-FITC and anti-ms-Alexa488 were taken on two different glass coverslips. In the actual experiments, we used unlabeled neutravidin, so as to not interfere with the fluorescence of the mEGFP-tagged proteins. The scale bars are 25 μm.

Article Snippet: Biotinylated mouse anti-synaptotagmin monoclonal antibodies (Synaptic Systems GmbH, Göttingen, Germany), were added to neutravidin-functionalized coverslips (concentration of 0.01 mg/mL) and incubated for 1 hour.

Techniques: Purification, Labeling, Fluorescence

Journal: Cells

Article Title: Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions

doi: 10.3390/cells10030567

Figure Lengend Snippet: Evidence for Ca 2+ -dependent interaction between annexin A2 (ANXA2) and S100A13 under serum-free conditions. ( A ) Pull-down assay using Strep -tagII-S100A13. Strep -tagII-S100A13 and C6 glioma cell lysates were incubated in the absence (left lane) and presence (right lane) of 100 μM Ca 2+ . Results represent the immunoblot using anti-ANXA2 IgG (upper lanes) and anti-β-actin antibody (lower lanes). The pull-down assay in A showed that ANXA2 dimer interacts with Strep -tagII-S100A13 in C6 glioma cell lysates in a Ca 2+ -enhanced manner. In the previously published study , we have shown that p40 Syt-1 also interacts with Strep -tagII-S100A13 in C6 glioma cell lysates in a Ca 2+ -enhanced manner. As both pull-down assays using Strep -tagII-S100A13 have been done with the same C6 glioma cell lysates, it appears that S100A13 forms the same protein complex with both experiments p40 Syt-1 and ANXA2. The experiment was done using same C6 glioma cell lysates as previously reported to show the interaction between Strep- tagII-S100A13 and p40 Syt-1 . ( B ) Serum-free-induced release of S100A13, but not ANXA2 from C6 glioma cells. Results represent the time course of protein levels of S100A13, ANXA2 and β-actin in cells (upper panels) and conditioned medium (CM) (lower panel) by immunoblot analysis. Extracellular S100A13 was recovered from CM using immunoprecipitation. ( C ) Schematic model of interaction between His 6 -ANXA2 and Strep -tagII-S100A13 bound to Streptavidin plate in an ELISA-based protein binding assay. Inset: enhancement of the interaction in the presence of His 6 -p40 synaptotagmin-1 (Syt-1). ( D , E ) No effect of amlexanox (Amx) on the His 6 -ANXA2 binding to Strep -tagII-S100A13 in terms of analyses of His 6 -ANXA2 concentration-dependency ( D ) and its reciprocal plot ( E ). ( F ) His 6 -p40 Syt-1 concentration-dependent enhancement of His 6 -ANXA2 binding to Strep -tagII-S100A13 in the presence of Ca 2+ . Data are presented as the mean ± standard error of the mean (S.E.M.) from a Tukey–Kramer multiple comparison test. * p < 0.05 and ** p < 0.01. n = 4 experiments per group.

Article Snippet: For immunoblotting, immunoprecipitation, immunocytochemistry or intracellular antibody delivery experiments, the following antibodies were used: mouse anti-prothymosin alpha (ProT α ) monoclonal antibodies (Clones: 2F11 and 4F4, Alexis Biochemicals, Lausen, Switzerland), rat anti-ProT α monoclonal antibody (Clone: 1 – 21) (developed in our laboratory, Nagasaki University, Nagasaki, Japan) [ ], N-terminus recognition goat anti-annexin A2 (ANXA2) polyclonal antibody and horseradish peroxidase (HRP)-conjugated mouse anti-β-actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit anti-S100A13 antibody (kindly provided by Dr. T. Maciag, Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, ME, USA), mouse anti-synaptotagmin-1 (Syt-1) monoclonal antibody (Wako, Osaka, Japan), mouse monoclonal anti-Stx-1 antibody (Wako, Osaka, Japan), rabbit anti-ATP8A2 polyclonal antibody (Abnova, Taipei, Taiwan), and normal mouse or goat IgG (ICN/Cappel Inc., Durham, NC, USA).

Techniques: Pull Down Assay, Incubation, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Protein Binding, Binding Assay, Concentration Assay