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