rabbit sera specific syntaxin 7 syntaxin 8  (Synaptic Systems)

Journal:

Article Title: Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity

doi: 10.1073/pnas.0511138103

Figure Lengend Snippet: Localization of SNAREs on early endosomes by using immunocytochemistry. (a) Fluorescence micrographs showing endosomes labeled with dextran-Alexa 488 (green channel) immunostained for syntaxin 13 (Left) and use 1 (Right) (red channel). To determine colocalization, linescans were performed through the intensity centers of green endosomes (examples indicated by white lines). (b) Representative linescan analysis, obtained from the images shown in a, showing intensity profiles of green (endosomes) and red (antibody-staining) signals. (c) Colocalization between early endosomes and SNARE proteins as determined by linescan analysis and correlation (see Materials and Methods). A correlation coefficient of 1 represents complete colocalization, whereas a correlation coefficient of ≈0 represent independent distribution. Coint, simultaneous labeling with dextran-Alexa 488 and dextran-Alexa 594 (positive control); 2nd AB, omission of the primary antibody (negative control); syphy, synaptophysin; syx, syntaxin; eb, endobrevin/VAMP8; β-tub, β-tubulin; tfr, transferrin receptor. Values are means of two independent experiments with 60 analyzed endosomes each. Error bars indicate the range of values.

Article Snippet: The following antibodies were described previously: rabbit sera specific for syntaxin 7 and syntaxin 8 ( 37 ), endobrevin ( 18 ), and vti1b ( 43 ), mouse monoclonal antibodies specific for synaptophysin Cl 7.2 ( 44 ), synaptobrevin Cl 69.1 ( 45 ), and SNAP-25 Cl 71.1 ( 46 ) (all available from Synaptic Systems, Göttingen, Germany), and syntaxin 1 HPC-1 ( 47 ).

Techniques: Immunocytochemistry, Fluorescence, Labeling, Staining, Positive Control, Negative Control

Journal:

Article Title: Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity

doi: 10.1073/pnas.0511138103

Figure Lengend Snippet: Effects of adding recombinant SNAREs on fusion of early endosomes. (a) Effects of Q-SNAREs considered to be involved in the fusion of early endosomes, including syntaxin 16 (syx 16), syntaxin 13 (syx 13), vti1a, and syntaxin 6 (syx 6). Values are means ± SEM of 7–10 independent experiments. (b) Effects of late endosomal and neuronal Q-SNAREs, including syntaxin 7 (syx 7), vti1b, syntaxin 8 (syx 8), syntaxin 1 (syx 1), and SNAP-25. Values are means ± SEM of 7–13 independent experiments. (c) Effects of R-SNAREs, including VAMP4, synaptobrevin/VAMP2 (syb), endobrevin/VAMP8 (eb), cellubrevin/VAMP3, Ti-VAMP/VAMP7, VAMP5, ykt6, and sec22. Values are means ± SEM of three to seven independent experiments. Individual Q-SNAREs were used at 12 μM, with R-SNAREs tested at 25 μM.

Article Snippet: The following antibodies were described previously: rabbit sera specific for syntaxin 7 and syntaxin 8 ( 37 ), endobrevin ( 18 ), and vti1b ( 43 ), mouse monoclonal antibodies specific for synaptophysin Cl 7.2 ( 44 ), synaptobrevin Cl 69.1 ( 45 ), and SNAP-25 Cl 71.1 ( 46 ) (all available from Synaptic Systems, Göttingen, Germany), and syntaxin 1 HPC-1 ( 47 ).

Techniques: Recombinant

Journal:

Article Title: Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity

doi: 10.1073/pnas.0511138103

Figure Lengend Snippet: Proteoliposomes containing the Q-SNAREs syntaxin 13, syntaxin 6, and vti1a show no specificity for R-SNAREs in fusion. (a) Fusion with liposomes containing VAMP4, endobrevin, or synaptobrevin. Fusion was monitored by fluorescence dequenching due to dilution of labeled phospholipids with unlabeled phospholipids during fusion (see Materials and Methods) and normalized to maximal fluorescence measured after adding detergent at the end of the reaction. As control, Q-SNARE liposomes were preincubated for 1 h at room temperature with purified endobrevin or synaptobrevin lacking the transmembrane domain (soluble fragment, final concentration of 30 μM) before starting the fusion reaction. (b–d) Dose-dependent inhibition by soluble R-SNAREs of fusion between liposomes containing the Q-SNAREs syntaxin13, vti1a, and syntaxin 6 and liposomes containing the R-SNARE VAMP4. Measurements were performed at 37°C with an overall protein concentration of 3 μM in the liposomes. The soluble R-SNAREs were added simultaneously with the acceptor liposomes at the start of the reaction (concentrations represent final assay concentrations).

Article Snippet: The following antibodies were described previously: rabbit sera specific for syntaxin 7 and syntaxin 8 ( 37 ), endobrevin ( 18 ), and vti1b ( 43 ), mouse monoclonal antibodies specific for synaptophysin Cl 7.2 ( 44 ), synaptobrevin Cl 69.1 ( 45 ), and SNAP-25 Cl 71.1 ( 46 ) (all available from Synaptic Systems, Göttingen, Germany), and syntaxin 1 HPC-1 ( 47 ).

Techniques: Fluorescence, Labeling, Purification, Concentration Assay, Inhibition, Protein Concentration