Journal: Cellular and Molecular Life Sciences

Article Title: Comparative study of PRPH2 D2 loop mutants reveals divergent disease mechanism in rods and cones

doi: 10.1007/s00018-023-04851-3

Figure Lengend Snippet: List of antibodies used in this study

Article Snippet: Syntaxin 3 , Rabbit , , 1:250 (IF), 1:500 (WB) , Novus Biologicals NBP1-86984 RRID:AB_11055708.

Techniques:

Journal: BMC Neuroscience

Article Title: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

doi: 10.1186/1471-2202-7-54

Figure Lengend Snippet: Syntaxins 1 through 4 are differentially distributed in the synaptic layers of the retina. A : Syntaxin 1 is localized to amacrine cell bodies (A) and processes in the inner plexiform layer (IPL). B : Syntaxin 2 also is localized to amacrine cell bodies (A), but shows a more restricted, punctate distribution in the IPL. C : Syntaxin 3 is localized to the glutamatergic ribbon synapses of photoreceptor and bipolar cell terminals in the outer plexiform layer (OPL) and IPL, respectively. Labeling is also present in the cell bodies of the photoreceptors and their inner segments (IS) and in bipolar cell bodies (B) and axons. D : Syntaxin 4 shows a very restricted, punctate distribution in the OPL and IPL. In the OPL, clusters of syntaxin 4 processes are associated with cone terminals (arrows). In the IPL, a distinct lamina of syntaxin 4 positive puncta is present at the INL/IPL border (arrowheads), with other puncta sparsely distributed deeper in the IPL (circle). E–H: Immunoblots for syntaxins 1 through 4 reveal a band at the appropriate molecular mass in membrane homogenates prepared from mouse retina and brain, confirming the expression of all four syntaxin isoforms in the mouse retina and the specificity of the antibodies and antisera. ONL, outer nuclear layer; INL, inner nuclear layer, GCL, ganglion cell layer. Scale bars = 20 μm for A–D.

Article Snippet: Anti-syntaxin 3 was a rabbit polyclonal antibody directed against the cytoplasmic domain of syntaxin 3 (Novus Biologicals, Littleton, CO; diluted 1:750–1:1000) [ , ].

Techniques: Labeling, Western Blot, Membrane, Expressing

Journal: BMC Neuroscience

Article Title: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

doi: 10.1186/1471-2202-7-54

Figure Lengend Snippet: Syntaxin 3 does not colocalize with syntaxin 1. A–C: Syntaxin 3 is localized to the glutamatergic photoreceptor terminals in the outer plexiform layer (OPL) and numerous bipolar cell terminals in the inner plexiform layer (IPL). Labeling is also present in the cell bodies and inner segments of the photoreceptors and in bipolar cell bodies and axons. Syntaxin 1 labeling is restricted to amacrine cells and amacrine cell processes. Images shown from a projection of 10 optical sections with total thickness of 1.93 μm. Box indicates area shown in panels D–F. D–F: Examination of syntaxin 3 and syntaxin 1 immunolabeling at higher magnification confirms that these syntaxin isoforms are present in different sets of synaptic terminals. Arrows indicate bipolar cell terminals labeled for syntaxin 3. Images shown from a projection of 15 optical sections with total thickness of 2.04 μm. IS, inner segments; ONL, Outer nuclear layer; INL, inner nuclear layer, GCL, ganglion cell layer. Scale bars = 20 μm for A–C; 10 μm for D–F.

Article Snippet: Anti-syntaxin 3 was a rabbit polyclonal antibody directed against the cytoplasmic domain of syntaxin 3 (Novus Biologicals, Littleton, CO; diluted 1:750–1:1000) [ , ].

Techniques: Labeling, Immunolabeling

Journal: BMC Neuroscience

Article Title: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

doi: 10.1186/1471-2202-7-54

Figure Lengend Snippet: Syntaxin 3 is associated with the glutamatergic ribbon synapses of photoreceptors and bipolar cells . A–C: Labeling for syntaxin 3 and VGLUT1, a marker for ribbon synapses, colocalize extensively in the photoreceptor terminals in the OPL and in bipolar cell terminals in the IPL. Box indicates area shown in panels D–F. D–F: Examination of syntaxin 3 and VGLUT1 immunolabeling in the IPL at higher magnification confirms that all bipolar cell terminals contain syntaxin 3. Images shown are from a single optical section. IS, inner segments; ONL, Outer nuclear layer; INL, inner nuclear layer, GCL, ganglion cell layer. Scale bars = 20 μm for A–C; 10 μm for D–F.

Article Snippet: Anti-syntaxin 3 was a rabbit polyclonal antibody directed against the cytoplasmic domain of syntaxin 3 (Novus Biologicals, Littleton, CO; diluted 1:750–1:1000) [ , ].

Techniques: Labeling, Marker, Immunolabeling

Journal: BMC Neuroscience

Article Title: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

doi: 10.1186/1471-2202-7-54

Figure Lengend Snippet: Syntaxin 3 is not expressed at putative conventional glutamatergic synapses . A . Syntaxin 3 labeling is present in numerous terminals in the inner plexiform layer (IPL), corresponding to the ribbon synapse-containing terminals of bipolar cells (see text for details). B. Vesicular glutamate transporter 3 (VGLUT3) is expressed in a putative glutamatergic amacrine cell type and its terminals (arrows) in the mid-IPL. C . There is no colocalization of syntaxin 3- and VGLUT3-positive terminals, although there is some contact between these terminals. Images shown from a projection of 14 optical sections with total thickness of 1.89 μm. INL, inner nuclear layer, GCL, ganglion cell layer. Scale bars = 10 μm.

Article Snippet: Anti-syntaxin 3 was a rabbit polyclonal antibody directed against the cytoplasmic domain of syntaxin 3 (Novus Biologicals, Littleton, CO; diluted 1:750–1:1000) [ , ].

Techniques: Labeling

Journal: BMC Neuroscience

Article Title: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

doi: 10.1186/1471-2202-7-54

Figure Lengend Snippet: Syntaxin 4 is highly restricted within both plexiform layers. A . Low magnification image of syntaxin 4 labeling. Labeling in the outer plexiform layer (OPL) is present in two sets of puncta. One set of small individual puncta in the distal OPL and a second set of puncta found in larger clusters in the proximal OPL. In the inner plexiform layer (IPL), syntaxin 4 puncta form a prominent, narrow stratum (brackets) at the border with the inner nuclear layer (INL) and in sparsely distributed individual puncta deeper in the IPL. Labeling for syntaxin 4 also is present in blood vessels (BV). Box indicates area shown in panel B. B: High magnification image showing syntaxin 4 puncta in the OPL. Small puncta appear as singlets or doublets in the distal OPL (arrows). In the proximal OPL, syntaxin 4 labeling is found in large clusters of puncta (arrowheads). C: Conventional fluorescence image showing a series of syntaxin 4 puncta ascending through the INL (arrowheads). The stratum of syntaxin-4 positive puncta at the INL/IPL border is also visible (brackets). Panels shown in A and B are from a projection of 46 optical sections of 10.7 μm total thickness. ONL, outer nuclear layer; GCL, ganglion cell layer. Scale bars = 20 μm for A; 10 μm for B and C.

Article Snippet: Anti-syntaxin 3 was a rabbit polyclonal antibody directed against the cytoplasmic domain of syntaxin 3 (Novus Biologicals, Littleton, CO; diluted 1:750–1:1000) [ , ].

Techniques: Labeling, Fluorescence

Journal: BMC Neuroscience

Article Title: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

doi: 10.1186/1471-2202-7-54

Figure Lengend Snippet: Syntaxin 4 labeling in the outer plexiform layer (OPL) is associated with processes post-synaptic to the photoreceptor terminals, not the terminals themselves. A–C: Double labeling for syntaxin 4 and the photoreceptor terminal marker PSD-95 show that syntaxin 4-positive puncta (arrows) are spatially associated with rod terminals (RT) in the distal OPL and with cone terminals (CT) in the proximal OPL. Images shown from a projection of 27 optical sections with total thickness of 3.79 μm. D–F: At high magnification, syntaxin 4-positive puncta in the distal OPL appear as singlets or doublets (arrows) enveloped by the terminals of rods (RT) labeled for PSD-95. Image shown is a projection of 22 optical sections with total thickness of 3.2 μm. G–I: In the proximal OPL, syntaxin 4 labeled structures (arrows) are associated with cone terminals (CT). Several syntaxin-4 positive puncta are enveloped by the cone terminal. In addition, syntaxin 4 labeling is localized to a cluster of processes in the OPL just proximal to the cone terminal (*) that can be seen to connect to the puncta within the cone terminal. Numerous rod terminals (RT) and associated syntaxin 4 puncta are visible distal to the cone terminal. Image shown is a projection of 34 optical sections with total thickness of 7.09 μm. J–L: Syntaxin 4-labeled structures (arrowheads) in the OPL do not colocalize with synaptic vesicles labeled for VGLUT1 in photoreceptor terminals (PT). Images shown from a projection of 13 optical sections with total thickness of 1.75 μm. M–O: Synaptic ribbons in rod and cone terminals do not label for syntaxin 4. However, syntaxin 4-positive puncta in the OPL are spatially associated with synaptic ribbons and often can be seen to follow the contours of the ribbons (arrowheads) labeled for kinesin II in rod and cone terminals. Circle indicates location of a cone terminal. Images shown from a projection of 12 optical sections with total thickness of 1.60 μm. ONL, outer nuclear layer. Scale bars = 5 μm for A–C, and J–O; 2 μm for D–I.

Article Snippet: Anti-syntaxin 3 was a rabbit polyclonal antibody directed against the cytoplasmic domain of syntaxin 3 (Novus Biologicals, Littleton, CO; diluted 1:750–1:1000) [ , ].

Techniques: Labeling, Marker

Journal: BMC Neuroscience

Article Title: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

doi: 10.1186/1471-2202-7-54

Figure Lengend Snippet: Syntaxin 4 is localized to the tips of horizontal cell processes at ribbon synapses, but not to dendrites of ON- or OFF-bipolar cells. A–C: Syntaxin 4 is present in the tips of horizontal cell processes in the ribbon synaptic complexes of rods and cones. Syntaxin 4 and calbindin, a horizontal cell marker, show double labeling in the individual puncta associated with rod terminals (arrowheads) and in the clusters of puncta associated with cone terminals (arrows) in the OPL, indicating that the syntaxin 4 is present at the tips of horizontal cell processes invading rod and cone terminals. Images shown from a projection of 37 optical sections with total thickness of 5.24 μm. D–F: Syntaxin 4 is not present in the dendrites of rod or ON-cone bipolar cells. Double labeling for syntaxin 4 and G o -alpha, a marker for rod and ON-cone bipolar cells, shows no colocalization. ON-bipolar cell dendrites reach up to the level of the syntaxin 4-positive puncta and terminate. In favorably oriented complexes in rod terminals (arrowheads), rod bipolar cell dendrites extending into the distal OPL terminate between the syntaxin 4-positive doublets corresponding to horizontal cell processes. No colocalization of syntaxin 4 and G o -alpha is present in ON-cone bipolar cell dendrites in cone terminals (ellipse). Image shown is a single optical section. G–I: Syntaxin 4 is not present at flat contacts between OFF-cone bipolar cells and cone terminals. Double labeling for syntaxin 4 and peanut agglutinin (PNA), a marker for flat contacts (arrowheads) between OFF-cone bipolar cells and cone terminals (CT) shows no colocalization. Syntaxin 4 labeling in processes directly proximal to the cone terminals is also visible (*). Images shown from a projection of 26 optical sections with total thickness of 6.71 μm. INL, inner nuclear layer. Scale bars = 10 μm for A–C; 5 μm for D–F; 2 μm for G–I.

Article Snippet: Anti-syntaxin 3 was a rabbit polyclonal antibody directed against the cytoplasmic domain of syntaxin 3 (Novus Biologicals, Littleton, CO; diluted 1:750–1:1000) [ , ].

Techniques: Marker, Labeling

Journal: BMC Neuroscience

Article Title: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

doi: 10.1186/1471-2202-7-54

Figure Lengend Snippet: Syntaxin 4 does not colocalize with syntaxin 1 or other presynaptic markers for conventional or ribbon synapses in the inner plexiform layer (IPL). A–C: Syntaxin 4-positive puncta (arrows) found in the inner nuclear layer (INL) and IPL do not show labeling for syntaxin 1, which is expressed in the processes and conventional synapses of amacrine cells. Image shown is a projection of 15 optical sections with total thickness of 2.19 μm. D–F: Syntaxin 4 positive puncta (arrows) only very rarely show any colocalization with synapsin I (circle), a marker for conventional presynaptic terminals. Image shown is a projection of 10 optical sections with total thickness of 1.31 μm. G–I: Syntaxin 4-labeling (arrows) is only rarely associated with presynaptic terminals identified by labeling for the ubiquitous presynaptic marker synaptic vesicle protein 2 (SV2; arrowheads). Terminals showing weak colocalization are indicated by the ellipse. Image shown is a projection of 9 optical sections with total thickness of 1.17 μm. J–L: Syntaxin 4-positive puncta in the IPL (arrows) do not label for VGLUT1, indicating that syntaxin 4 is not present in bipolar cell terminals. Occasionally syntaxin 4-positive puncta make contact with VGLUT1-positive bipolar cell terminals (arrowhead). Image shown is a projection of 13 optical sections with total thickness of 1.78 μm. GCL, ganglion cell layer. Scale bars = 5 μm for A–C and G–I; 10 μm for D–F and J–L.

Article Snippet: Anti-syntaxin 3 was a rabbit polyclonal antibody directed against the cytoplasmic domain of syntaxin 3 (Novus Biologicals, Littleton, CO; diluted 1:750–1:1000) [ , ].

Techniques: Labeling, Marker

Journal: BMC Neuroscience

Article Title: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

doi: 10.1186/1471-2202-7-54

Figure Lengend Snippet: Syntaxin 4 does not colocalize with the presynaptic active zone protein, bassoon, in the inner plexiform layer (IPL) . A–C: Syntaxin 4-positive puncta in the IPL (arrowheads) do not show colocalization with presynaptic active zones containing bassoon (arrows). However, close apposition of syntaxin 4-positive puncta and bassoon-containing active zones is present (circles). Single confocal plane shown. Scale bars = 10 μm.

Article Snippet: Anti-syntaxin 3 was a rabbit polyclonal antibody directed against the cytoplasmic domain of syntaxin 3 (Novus Biologicals, Littleton, CO; diluted 1:750–1:1000) [ , ].

Techniques:

Journal: BMC Neuroscience

Article Title: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

doi: 10.1186/1471-2202-7-54

Figure Lengend Snippet: Syntaxin 4-positive puncta in the distal inner plexiform layer (IPL) do not arise from dopaminergic or GABAergic CD15-positive amacrine cells . A–C: Syntaxin 4-positive puncta form a plexus along the border of the inner nuclear layer (INL) and IPL that co-stratifies with the processes of dopaminergic amacrine cells (A) labeled for tyrosine hydroxylase (TH). Syntaxin 4 and TH labeling do not colocalize (see D–F), but syntaxin 4-positive puncta contact the processes and cell bodies of the dopaminergic cells (arrowheads). Images shown from a projection of 73 optical sections with total thickness of 10.5 μm. D–F: Higher magnification image of syntaxin 4-positive puncta (arrows and arrowheads) and TH-positive dopaminergic amacrine cell processes in the IPL in a single optical plane. There is no colocalization of labels indicating that the syntaxin 4-positive puncta do not arise from the dopaminergic amacrine cells, but there is often contact between the syntaxin 4-positive puncta and the TH-positive processes (arrowheads). G–I: Syntaxin 4-positive puncta (arrows) form a distinctive plexus that co-stratifies with processes from GABAergic CD15-positive amacrine cells (A). The processes of the CD15-positive amacrine cells do not label for syntaxin 4, but there is contact between syntaxin 4-positive puncta and the processes and cell body of CD15-positive amacrine cells (arrowhead). Images shown from a projection of 57 optical sections with total thickness of 8.16 μm. J–L: Higher magnification image of syntaxin 4 and CD15 double labeling visualized in a single optical plane. Syntaxin 4-positive puncta (arrows) co-stratify with the CD15-positive plexus in the IPL and often contact CD15-positive amacrine cells at the cell body or on processes (arrowhead). Images shown from a projection of 14 optical sections with total thickness of 1.89 μm. GCL, ganglion cell layer. Scale bars = 20 μm for A–C; 10 μm for D–I; 5 μm for J–L.

Article Snippet: Anti-syntaxin 3 was a rabbit polyclonal antibody directed against the cytoplasmic domain of syntaxin 3 (Novus Biologicals, Littleton, CO; diluted 1:750–1:1000) [ , ].

Techniques: Labeling

Journal: BMC Neuroscience

Article Title: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

doi: 10.1186/1471-2202-7-54

Figure Lengend Snippet: Syntaxin 4 in the inner plexiform layer (IPL) is not associated with ganglion cell dendrites or with Müller glial cell processes. A–C: Double labeling for syntaxin 4 and the ganglion cell dendrite marker, MAP 1. Syntaxin 4-positive puncta (arrows) form a plexus along the inner nuclear layer (INL)/IPL border with a few puncta present more proximally in the IPL. Ganglion cell dendrites labeled for MAP-1 are present throughout the thickness of the IPL, but do not show syntaxin 4 labeling. Contact between syntaxin 4-positive puncta and ganglion cell dendrites is seen only rarely (arrowheads). Images shown from a projection of 24 optical sections with total thickness of 3.35 μm. D–F: Double labeling for syntaxin 4 and glutamine synthetase (GS), a marker for Müller glial cells, shows no colocalization of syntaxin 4-positive puncta in the IPL (arrowheads) or in the outer plexiform layer (OPL; arrows). Images shown from a projection of 14 optical sections with total thickness of 3.35 μm. OLM, outer limiting membrane; ONL, outer nuclear layer; GCL, ganglion cell layer. Scale bars = 10 μm for A–C; 20 μm for D–F.

Article Snippet: Anti-syntaxin 3 was a rabbit polyclonal antibody directed against the cytoplasmic domain of syntaxin 3 (Novus Biologicals, Littleton, CO; diluted 1:750–1:1000) [ , ].

Techniques: Labeling, Marker, Membrane

Journal: BMC Neuroscience

Article Title: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

doi: 10.1186/1471-2202-7-54

Figure Lengend Snippet: Primary antibodies and lectins

Article Snippet: Anti-syntaxin 3 was a rabbit polyclonal antibody directed against the cytoplasmic domain of syntaxin 3 (Novus Biologicals, Littleton, CO; diluted 1:750–1:1000) [ , ].

Techniques: