rabbit anti-syntaxin-16  (Synaptic Systems)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells

doi: 10.1016/j.mcpro.2023.100704

Figure Lengend Snippet: Summary of SV, SNARE, and other trafficking proteins enriched in VGluT3 immunoisolates at P23 and identified by MS analysis

Article Snippet: The following primary antibodies were used: rabbit anti-VAMP-7 (232003, Synaptic Systems), rabbit anti-syntaxin-6 (110062, Synaptic Systems), rabbit anti-syntaxin-7 (110072, Synaptic Systems), rabbit anti-syntaxin-8 (110083, Synaptic Systems), rabbit anti-syntaxin-12/13 (110133, Synaptic Systems), rabbit anti-syntaxin-16 (110162, Synaptic Systems), rabbit anti-SCAMP1 (PA1-739, Thermo Fisher Scientific), mouse anti-V-ATPase (149011, Synaptic Systems), mouse anti-SV2B (119111, Synaptic Systems), rabbit anti-Vti1A (165002, Synaptic Systems), rabbit anti-PKC alpha [Y124] (ab32376, Abcam), rabbit anti-YKT6 (ab236583, Abcam), rabbit anti-VAP-A (249002, Synaptic Systems), mouse anti-VCP (MA3-004, Thermo Fisher Scientific), mouse anti-otoferlin [13A9] (ab53233, Abcam), rabbit anti-otoferlin (178003, Synaptic Systems), rabbit anti-VGluT3 (135203, Synaptic Systems), guinea pig anti-VGluT3 (135204, Synaptic Systems), mouse anti-synapsin-1 (106001, Synaptic Systems), goat IgG anti-CtBP2 [E−16] (sc-5967, Santa Cruz Biotechnology), rabbit anti-Munc18-1 (116002, Synaptic Systems), rabbit anti-Munc18-2 (116102, Synaptic Systems), rabbit anti-Munc18-3 (116202, Synaptic Systems), and mouse anti-VAMP-2 (104211, Synaptic Systems).

Techniques: Membrane

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells

doi: 10.1016/j.mcpro.2023.100704

Figure Lengend Snippet: Immunolocalization analysis of trafficking proteins in the adult organ of Corti . A and B , Syntaxin-6 ( A ) and syntaxin-16 ( B ), enriched in our MS experiments, were previously shown to be expressed in IHCs. C–F , The Golgi and endosomal markers YKT6 ( C ) and VtiA ( D ), the ER and presynaptic plasma membrane protein VAP-A ( E ), the ER protein VCP ( F ) are expressed in IHCs. Note that all proteins localize to the basolateral region of the IHCs, although in some cases the protein is also expressed in afferent (postsynaptic) and efferent fibers of SGNs. Images correspond to high magnification views of representative P15–25 IHCs immunolabeled with antibodies against the candidate proteins ( red ), the ribbon marker CtBP2/RIBEYE ( green ), and the IHC marker otoferlin ( blue ). In ( C–F ), the upper panels show overviews of representative IHCs, displaying maximum intensity projections of 5 to 10 confocal optical sections through the longitudinal axis of the IHCs (scale bars: 5 μm). The bottom panels show a zoom into the synaptic area, displaying single confocal optical sections through the longitudinal axis of a single IHC at the basal region (scale bars: 2 μm). IHC, inner hair cell; SGN, spiral ganglion neuron.

Article Snippet: The following primary antibodies were used: rabbit anti-VAMP-7 (232003, Synaptic Systems), rabbit anti-syntaxin-6 (110062, Synaptic Systems), rabbit anti-syntaxin-7 (110072, Synaptic Systems), rabbit anti-syntaxin-8 (110083, Synaptic Systems), rabbit anti-syntaxin-12/13 (110133, Synaptic Systems), rabbit anti-syntaxin-16 (110162, Synaptic Systems), rabbit anti-SCAMP1 (PA1-739, Thermo Fisher Scientific), mouse anti-V-ATPase (149011, Synaptic Systems), mouse anti-SV2B (119111, Synaptic Systems), rabbit anti-Vti1A (165002, Synaptic Systems), rabbit anti-PKC alpha [Y124] (ab32376, Abcam), rabbit anti-YKT6 (ab236583, Abcam), rabbit anti-VAP-A (249002, Synaptic Systems), mouse anti-VCP (MA3-004, Thermo Fisher Scientific), mouse anti-otoferlin [13A9] (ab53233, Abcam), rabbit anti-otoferlin (178003, Synaptic Systems), rabbit anti-VGluT3 (135203, Synaptic Systems), guinea pig anti-VGluT3 (135204, Synaptic Systems), mouse anti-synapsin-1 (106001, Synaptic Systems), goat IgG anti-CtBP2 [E−16] (sc-5967, Santa Cruz Biotechnology), rabbit anti-Munc18-1 (116002, Synaptic Systems), rabbit anti-Munc18-2 (116102, Synaptic Systems), rabbit anti-Munc18-3 (116202, Synaptic Systems), and mouse anti-VAMP-2 (104211, Synaptic Systems).

Techniques: Membrane, Immunolabeling, Marker

Journal: The Journal of Biological Chemistry

Article Title: Interaction of Calcium-dependent Activator Protein for Secretion 1 (CAPS1) with the Class II ADP-ribosylation Factor Small GTPases Is Required for Dense-core Vesicle Trafficking in the trans -Golgi Network *

doi: 10.1074/jbc.M110.137414

Figure Lengend Snippet: Endogenous CAPS1 is associated with the Golgi membrane. A and B, electron micrograms of submicrosomal fractions affinity-purified using immune magnetic beads. Cerebellar microsomal fractions of P21 mice were incubated with magnetic beads coated with anti-CAPS1 antibody (A) or anti-VAMP2 antibody (B), and immunoaffinity-purified submicrosomal fractions were subjected to electron microscopic observation. Scale bars, 200 nm. C, Western blot analysis of submicrosomal fractions affinity-purified using normal rabbit IgG, anti-CAPS1 antibody, or anti-VAMP2 antibody immunomagnetic beads. Each immunopurified fraction was immunoblotted with anti-CAPS1, anti-VAMP2, anti-VAMP4, anti-SNAP25, anti-Bcl-2, anti-BiP/GRP78, anti-pan-ARF (1D9), anti-GM130, anti-GS28, anti-p115, anti-Stx5, anti-Stx6, anti-Stx16, and anti-Vti1a antibodies. Anti-pan-ARF (1D9) antibody recognizes all of the ARF family proteins (supplemental Fig. S3).

Article Snippet: The following primary antibodies were also used for Western blotting: rabbit polyclonal anti-CAPS2 (1:1,000 dilution) ( 5 ), mouse monoclonal anti-FLAG (1:1,000 dilution; catalog no. F1804, Sigma), rat monoclonal anti-hemagglutinin epitope (HA) (1:1,000 dilution; catalog no. 1867423, Roche Applied Science), mouse monoclonal anti-VAMP2 (1:10,000 dilution; catalog no. 104211, Synaptic Systems), rabbit polyclonal anti-VAMP4 (1:1,000 dilution; catalog no. PA1-768, Affinity BioReagents), mouse monoclonal SNAP25 (1:2,500 dilution; catalog no. SMI-81, Sternberger), mouse monoclonal anti-ARF (clone 1D9) (1:500 dilution; catalog no. ab2806, Abcam), mouse monoclonal anti-GM130 (1:250 dilution; catalog no. 610822, BD Biosciences), mouse monoclonal anti-GS28 (1:1,000 dilution; catalog no. 611184, BD Biosciences), mouse monoclonal anti-p115 (1:1,000 dilution; catalog no. 612260, BD Biosciences), mouse monoclonal anti-Bcl-2 (1:500 dilution; catalog no. 610538, BD Biosciences), mouse monoclonal anti-BiP/GRP78 (1:250 dilution; catalog no. 610978, BD Biosciences), rabbit polyclonal anti-syntaxin 5 (1:1,000 dilution; catalog no. 110053, Synaptic Systems), mouse monoclonal anti-syntaxin 6 (1:2,500 dilution; catalog no. 610635, BD Biosciences), rabbit polyclonal anti-syntaxin 16 (1:1,000 dilution; catalog no. 110162, Synaptic Systems), mouse monoclonal anti-Vti1a (1:2,500 dilution; catalog no. 611220, BD Biosciences), and goat polyclonal anti-GST (1:1,000 dilution; catalog no. 27-4577, Amersham Biosciences).

Techniques: Affinity Purification, Magnetic Beads, Incubation, Purification, Western Blot