antibody guinea pig polyclonal anti- vesicular gaba transporter (vgat)  (Synaptic Systems)

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet: ( A ) Diagram depicting the treatment of hippocampal brain slices obtained from 3-month-old adult WT mice with vehicle (Ctrl) or recombinant DKK3 protein. Synapses were evaluated by confocal microscopy and electrophysiological recordings. ( B ) Confocal images of the CA3 SR region labeled with the presynaptic excitatory marker vGLUT1 (green) and the postsynaptic marker PSD-95 (red). Arrows indicate excitatory synapses as colocalized pre- and postsynaptic puncta. Scale bar = 5 μm and 2.5 μm in zoomed-in pictures. Quantification is shown on the right-hand side (Mann-Whitney test, n=5 animals per condition). ( C ) Representative mEPSC traces recorded at –60 mV from CA3 cells. Stars indicate mEPSC events. Quantification of mEPSC frequency and amplitude is shown on the right-hand side (Mann-Whitney test, n=10–13 cells from five animals). ( D ) Confocal images of the CA3 SR region labeled with the presynaptic inhibitory marker vGAT (green) and the postsynaptic marker gephyrin (red). Arrows indicate inhibitory synapses as colocalized pre- and postsynaptic puncta. Scale bar = 5 μm and 2.5 μm in zoomed-in pictures. Quantification is shown on the right-hand side (Mann-Whitney test, n=4 animals per condition). ( E ) Representative mIPSC traces recorded at 0 mV from CA3 cells. Stars indicate mIPSC events. Quantification of mIPSC frequency and amplitude is shown on the right-hand side (Student’s T-test for mIPSC frequency and Mann-Whitney test for mIPSC amplitude, n=11–12 cells from five to seven animals).

Article Snippet: Antibody , Guinea pig polyclonal anti- vesicular GABA Transporter (vGAT) , Synaptic Systems , Cat# 131 004, RRID: AB_887873 , 1:500 for IF.

Techniques: Recombinant, Confocal Microscopy, Labeling, Marker, MANN-WHITNEY

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet: ( A ) Confocal images of the TUNEL assay (red) in the hippocampus CA3 area (DAPI in blue). Graph shows the number of cells positive for TUNEL. Scale bar = 20 μm (n=2 animals). ( B ) Confocal images show the impact of different DKK3 concentrations on cell number (NeuN in red) and vGLUT1 (green) in the hippocampus CA3 area. Graph shows the number of NeuN + cells per 100 μm 3 . Scale bar = 150 μm (One-way ANOVA followed by Tukey’s post-hoc test, ns, n=2 animals, 2–3 brain slices per animal). ( C ) Confocal images from hippocampal CA1 SR show the effect of DKK3 on excitatory synapses (colocalized vGLUT1 puncta in green and PSD-95 puncta in red). Arrows indicate excitatory synapses. Scale bar = 5 μm and 2.5 μm in zoomed-in pictures. Quantification is shown on the right-hand side (Mann-Whitney test, n=3 animals). ( D ) Confocal images from hippocampal CA1 SR show the effect of DKK3 on inhibitory synapses (colocalized vGAT puncta in green and gephyrin puncta in red). Arrows point to inhibitory synapses. Scale bar = 5 μm and 2.5 μm in zoomed-in pictures. Quantification is shown on the right-hand side (Mann-Whitney test, n=4 animals).

Article Snippet: Antibody , Guinea pig polyclonal anti- vesicular GABA Transporter (vGAT) , Synaptic Systems , Cat# 131 004, RRID: AB_887873 , 1:500 for IF.

Techniques: TUNEL Assay, MANN-WHITNEY

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet: ( A ) Diagram of the canonical Wnt pathway through inhibition of GSK3β (Wnt/GSK3β pathway), resulting in elevation of β-catenin and transcriptional activation via TCF/LEF. ( B ) Confocal images show excitatory synapses, visualized by colocalization of vGLUT1 (green) and Homer1 (red), as well as β-catenin puncta (grey) in the CA3 SR after treatment with vehicle (Ctrl) or DKK3 in the absence or presence of BIO. Arrows indicate extra-synaptic β-catenin puncta. Scale bar = 5 μm. Quantification of extrasynaptic β-catenin puncta density as a percentage of control is shown on the right-hand side (Two-Way ANOVA followed by Tukey’s multiple comparisons, n=2–3 brain slices/animal from five animals). ( C ) Confocal images show excitatory synapses (co-localized vGLUT1 puncta in green and PSD-95 puncta in red) in the CA3 SR after treatment with vehicle (Ctrl) or DKK3 in the absence or presence of BIO. Scale bar = 5 μm and 2.5 μm. Graph shows the quantification of puncta density of pre- and postsynaptic markers and excitatory synapses as a percentage of control (Kruskal-Wallis followed by Dunn’s multiple comparisons, n=5 animals). ( D ) Diagram of the Wnt pathway through activation of JNK (Wnt/JNK pathway), resulting in increased levels of phospho-JNK and transcriptional changes. ( E ) Representative immunoblots of phospho-JNK Thr183/Tyr185 (P-JNK) and total JNK of brain slices treated with DKK3 and/or the JNK inhibitor CC-930. Actin was used as a loading control. Graph shows densitometric quantification of P-JNK vs. total JNK relative to the control condition (Kruskal-Wallis followed by Dunn’s multiple comparisons, n=2 brain slices/animal from four to five animals). ( F ) Confocal images showing inhibitory synapses defined by the colocalization of vGAT (green) and gephyrin (red) puncta in the CA3 SR after treatment with vehicle (Ctrl) or DKK3 in the absence or presence of CC-930. Scale bar = 5 μm and 2.5 μm. Graph shows the quantification of puncta density of pre and postsynaptic markers and inhibitory synapses as a percentage of control (Kruskal-Wallis followed by Dunn’s multiple comparisons, n=5 animals). Figure 4—source data 1. Uncropped western blot gels. Raw and annotated WB images. The representative western blot images for are indicated within a blue square.

Article Snippet: Antibody , Guinea pig polyclonal anti- vesicular GABA Transporter (vGAT) , Synaptic Systems , Cat# 131 004, RRID: AB_887873 , 1:500 for IF.

Techniques: Inhibition, Activation Assay, Control, Western Blot

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet: ( A ) Confocal images show excitatory synapses (co-localized vGLUT1 in green and PSD-95 in red) in the CA3 SR after treatment with vehicle (Ctrl) or DKK3 in the absence or presence of CHIR99021 (CHIR) for 4 hr. Scale bar = 5 μm and 2.5 μm Graph shows the quantification of puncta density for pre and postsynaptic markers as well as excitatory synapse number as a percentage of control (Kruskal-Wallis followed by Dunn’s multiple comparisons, n=2–3 brain slices from three animals). ( B ) Confocal images showing inhibitory synapses defined by the colocalization of vGAT (green) and Gephyrin (red) in the CA3 SR after treatment with vehicle (Ctrl) or DKK3 in the absence or presence of BIO. Scale bar = 5 μm and 2.5 μm. Graph shows the quantification of puncta density of pre and postsynaptic markers as well as inhibitory synapse number as a percentage of control (Kruskal-Wallis followed by Dunn’s multiple comparisons, n=3–4 animals). ( C ) Confocal images showing excitatory synapses (colocalized Bassoon in green and Homer1 in red) in the CA3 SR after vehicle (Ctrl) or DKK3 treatment in the absence or presence of CC-930. Scale bar = 5 μm and 2.5 μm. Graph shows the quantification of pre and postsynaptic markers as well as excitatory synapse number as a percentage of control (Two-Way ANOVA followed by Tukey’s multiple comparisons, n=2–3 brain slices from three animals).

Article Snippet: Antibody , Guinea pig polyclonal anti- vesicular GABA Transporter (vGAT) , Synaptic Systems , Cat# 131 004, RRID: AB_887873 , 1:500 for IF.

Techniques: Control

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet: ( A ) Diagram showing the experimental design. Three-month-old WT mice were injected with AAV9 scrambled (Scr) or Dkk3 shRNA in the CA3 region. Confocal images showing GFP (green) and DKK3 (red) in Scr- and Dkk3 -shRNA injected hippocampus. Scale bar = 145 μm. Graph shows quantification of DKK3 intensity in the area injected with the viruses. ( B ) Confocal images from CA3 SR show excitatory synapses (colocalized vGLUT1 puncta in green and PSD-95 puncta in red). Arrows indicate excitatory synapses. Scale bar = 5 μm and 2.5 μm in zoomed-in images. Quantification is shown on the right-hand side (Student’s T-test, n=5 animals per condition). ( C ) Representative mEPSC traces recorded at –60 mV from CA3 cells. Stars indicate mEPSC events. Quantification of mEPSC frequency and amplitude is shown on the right-hand side (Student’s T-test, n=8–9 cells from four animals). ( D ) Confocal images from CA3 SR show inhibitory synapses (colocalized vGAT in green and gephyrin in red). Arrows point to inhibitory synapses. Scale bar = 5 μm and 2.5 μm in zoomed-in pictures. Quantification is shown on the right-hand side (Student’s T-test, n=5 animals). ( E ) Representative mIPSC traces recorded at 0 mV from CA3 cells. Stars indicate mIPSC events. Quantification of mIPSC frequency and amplitude is shown on the right-hand side (Mann-Whitney test, n=10–12 cells from six animals).

Article Snippet: Antibody , Guinea pig polyclonal anti- vesicular GABA Transporter (vGAT) , Synaptic Systems , Cat# 131 004, RRID: AB_887873 , 1:500 for IF.

Techniques: Injection, shRNA, MANN-WHITNEY

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet: ( A ) Diagram depicting the experimental design. In green, 3-month-old WT and J20 mice were injected bilaterally with AAV9-Scr shRNA or AAV9- Dkk3 shRNA in the CA3 region. The density of synapses was evaluated at 4-month-old before plaque deposition starts. In blue, 7-month-old J20 mice were injected bilaterally with AAV9-Scr shRNA or AAV9- Dkk3 shRNA in the CA3 region. The density of synapses around plaques was evaluated at 9-month-old. ( B, C ) Representative confocal images from the CA3 SR region of 4-month-old WT and J20 mice. Images show ( B ) excitatory synapses (Bassoon in green and Homer1 in red) and ( C ) inhibitory synapses (vGAT in green and Gephyrin in red). Arrows point to synapses. Scale bar = 2.5 μm. Quantification of synapse number as a percentage relative to WT-Scr shRNA animals is shown on the right-hand side (Two-Way ANOVA followed by Tukey’s post-hoc test, n=9–11 animals per condition and 2–3 brain slices per animal). ( D, E ) Representative confocal images from the CA3 SR region of 9-month-old J20 mice. Images show an Aβ plaque (6E10; blue) and ( D ) excitatory synapses or ( C ) inhibitory synapses at different distances relative to the core of the plaque. Scale bar = 2.5 μm. Graphs show synapse number per 200 μm 3 at each distance (Two-Way ANOVA followed by Tukey’s post-hoc test, n=6–7 animals per condition and 2–3 brain slices per animal).

Article Snippet: Antibody , Guinea pig polyclonal anti- vesicular GABA Transporter (vGAT) , Synaptic Systems , Cat# 131 004, RRID: AB_887873 , 1:500 for IF.

Techniques: Injection, shRNA

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet: ( A ) Confocal images of the CA3 area of the hippocampus show that endogenous DKK3 is downregulated in the hippocampus of wildtype and J20 injected with Dkk3 shRNA AAV9 virus. Scale bar = 38 μm. ( B, C ) Graphs show ( B ) excitatory (Bassoon and Homer1) and ( C ) inhibitory (vGAT and Gephyrin) puncta density as a percentage relative to WT Scr shRNA group in 4 months old WT and J20 mice (Two-Way ANOVA followed by Tukey’s post-hoc test, n=9–11 animals per condition and 3 brain slices per animal). ( D, E ) Graphs show ( D ) excitatory (Bassoon and Homer1) and ( E ) inhibitory (vGAT and Gephyrin) puncta density at each distance from the core plaque as density per 200 μm 3 in 9 months old J20 mice (Two-Way ANOVA followed by Tukey’s post-hoc test. n=7–8 animals per condition and 3 brain slices per animal). ( F ) DKK3 downregulation does not affect plaque load in the hippocampus. Confocal images of Aβ (6E10 in green) and DKK3 (red) in the CA3 SR of 9 months old J20 mice. Scale bar = 10 µm. Graphs show the quantification of Aβ coverage, plaque number and plaque area in the CA3 (Student’s T-test, ns, n=6 per condition). ( G ) DKK3 downregulation does not affect Dkk1 expression in the hippocampus. Dkk1 expression was evaluated in the hippocampus of 4-month-old WT and J20 mice injected with Scr or Dkk3 shRNA. Graph shows Dkk1 mRNA levels normalized to the control group (Kruskal-Wallis followed by Dunn’s multiple comparisons, ns, n=4 animals per condition). ( H ) Increased expression of Dkk1 does not affect Dkk3 mRNA levels. Dkk3 expression was examined in the hippocampus of iDkk1 mice compared to control mice after induction of Dkk1 for 14 days. Graph shows Dkk1 and Dkk3 mRNA levels normalized to the control group (Student’s T-test, n=5–6 animals per condition).

Article Snippet: Antibody , Guinea pig polyclonal anti- vesicular GABA Transporter (vGAT) , Synaptic Systems , Cat# 131 004, RRID: AB_887873 , 1:500 for IF.

Techniques: Injection, shRNA, Virus, Expressing, Control

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet:

Article Snippet: Antibody , Guinea pig polyclonal anti- vesicular GABA Transporter (vGAT) , Synaptic Systems , Cat# 131 004, RRID: AB_887873 , 1:500 for IF.

Techniques: Isolation, Knock-Out, Control, Sequencing, shRNA, Recombinant, TUNEL Assay, cDNA Synthesis, Fluorsave, Software, Microscopy, Immunofluorescence, Patch Clamp

Journal: Frontiers in Cellular Neuroscience

Article Title: The Y172 Monoclonal Antibody Against p-c-Jun (Ser63) Is a Marker of the Postsynaptic Compartment of C-Type Cholinergic Afferent Synapses on Motoneurons

doi: 10.3389/fncel.2019.00582

Figure Lengend Snippet: Immunocytochemical analysis of the spatial relationships between peripheral cytoplasmic Y172 immunolabeling and excitatory and inhibitory afferent inputs to MNs of CD1 mice. Spinal cord sections were double immunostained with Y172 (green) and either anti-VAChT, VGluT1 or VGAT (for cholinergic, glutamatergic or GABAergic synapses, respectively, all red) and processed for fluorescent Nissl staining for MN visualization (blue). (A1–A5) Representative maximum intensity projections from confocal Z-stacked images showing Y172 and VAChT immunoreactivity in an MN of an adult (P75) mouse. Note the distribution of Y172-positive spots in the cytoplasm of the cell body; while some immunoreactive patches were located around the nucleus, others were peripherally distributed and exhibited a close association with VAChT-positive puncta. The area delimited by the dotted-lined rectangle in (A2) is shown at higher magnification in (A3–A5) ; note that, while the peripherally located Y172-positive spot was in contact with a VAChT-positive punctum, the spot that was more internally located did not exhibit any association with VAChT immunolabeling. (B,C) Pixel profile analysis (C) along a line crossing a multifluorescent-labeled VAChT/Y172 synapse (shown in B ) demonstrating the dissociation of presynaptic VAChT immunostaining and postsynaptic Y172-positive staining; the blue channel, corresponding to fluorescent Nissl staining, is not included in the graph. (D) Volume rendering of a high magnification confocal image of a C-bouton double immunolabeled with anti-VAChT (red) and Y172 antibodies (green) demonstrating the nonoverlapping and separate distribution of both signals; the blue channel corresponds to fluorescent Nissl stain for MN visualization. (E1–F5) Representative Z-staked images showing MNs immunostained with Y172 and either anti-VGluT1 (E1–E5) or anti-VGAT (F1–F5) antibodies. Note that neither VGluT1- nor VGAT-containing puncta were associated with Y172-positive profiles; the occasional degree of pixel overlapping observed in some cases was due to the random close proximity between Y172 and VGluT1 or VGAT immunoreactivity. (G–J) Pixel profile analysis (H,J) along the lines depicted in (G,I) ; in (G) , the yellow line delimits the periphery of an MN by passing through different Y172- and VGluT1-positive spots, whereas in (I) , the lines cross two spots with VGluT1 immunoreactivity (1, red) and one spot with Y172 immunoreactivity (2, green); note the absence of colocalization between the two signals in (H,J) . (K) The time course of changes in the number of Y172- and VAChT-positive profiles per 100 μm of soma perimeter in spinal cord MNs from mice at different ages. (L–N) The percentage of peripheral Y172-positive profiles closely associated with puncta positive for VAChT, VGluT1, or VGAT, and vice versa, in adult (P75) MNs is shown in (L–N) , respectively. The data are expressed as the mean ± SEM; 200–300 profiles from 10 to 15 randomly selected MNs (3–4 animals) per condition were analyzed; * p < 0.05 vs. Y172+ immunoreactivity; student’s t -test. Scale bars: A1 = 10 μm (valid for A2 ); D = 1 μm; E1,F1 = 10 μm (valid for E2,F2 ); F5 = 1.5 μm (valid for A3–A5,E3–E5,F3,F4 ); G = 10 μm.

Article Snippet: The primary antibodies used were rabbit monoclonal anti-phospho-c-Jun (serine [Ser]63) clone Y172 (diluted 1:300, hereafter referred to as the Y172 antibody; Abcam, Cambridge, UK; cat. ab32385 or Millipore, Burlington, MA, USA; cat.# 04-212); rabbit polyclonal anti-phospho-c-Jun (Ser63; 1:100; Cell Signaling, Danvers, MA, USA; cat.# 9261); rabbit polyclonal anti-phospho-c-Jun (Ser73; 1:100; Cell Signaling; cat.# 9164); guinea pig polyclonal anti-synaptophysin 1 (1:500; Synaptic Systems, Goettingen, Germany; cat.# 101004); guinea pig polyclonal anti-vesicular acetylcholine transporter (VAChT; 1:500; Synaptic Systems, Goettingen, Germany; cat.# 139105); guinea pig polyclonal anti-vesicular glutamate transporter 1 (VGluT1, 1:500; Synaptic Systems, Goettingen, Germany; cat.# 135304); guinea pig polyclonal anti-vesicular GABA transporter (VGAT, 1:200; Synaptic Systems, Goettingen, Germany; cat.# 131004); mouse monoclonal anti-synaptic vesicle glycoprotein 2A (SV2, 1:1,000; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# AB_2315386); mouse monoclonal anti-sigma-1 receptor (S1R, 1:50; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-137075); mouse monoclonal anti-Kv2.1 voltage-gated potassium channel (Kv2.1, 1:100; NeuroMab, Davis, CA, USA; cat.# 73-014); sheep polyclonal anti-choline acetyltransferase (ChAT, 1:1,000; Abcam cat.# Ab18736); rabbit polyclonal anti-ChAT (1:200; Millipore, Burlington, MA, USA; cat.# AB143); rabbit polyclonal anti-neuregulin-1 (NRG1) type III (extracellular, 1:250; Alomone labs, Jerusalem, Israel, cat.# ANR 113); mouse monoclonal anti-NRG-CRD, type III, clone N126B/31 (1:250; Millipore; cat.# MABN534); rabbit polyclonal anti-NRG1 1 α/β 1/2 (1:300; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-348); mouse monoclonal anti-Golgi matrix protein of 130 kDa (GM130, 1:200; BD Biosciences, San Jose, CA, USA; cat.# 610822); mouse monoclonal anti-lysosomal membrane glycoprotein (LAMP-1), clone ID4B (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# ID4B); mouse monoclonal anti-KDEL (Lys-Asp-Glu-Leu motif) receptor (KDELR), clone KR-10 (1:50; Stressgen Biotechnologies, San Diego, CA, USA; cat.# VAA-PT048); mouse monoclonal anti-protein disulfide-isomerase (PDI), clone 1D3 (1:200; Enzo Life Sciences, Farmingdale, NY, USA; cat.# ADI-SPA-891); and mouse monoclonal anti-calcitonin gene-related peptide (CGRP; 1:100; Abcam, Cambridge, UK; cat.# ab81887).

Techniques: Immunolabeling, Staining, Labeling, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: The Y172 Monoclonal Antibody Against p-c-Jun (Ser63) Is a Marker of the Postsynaptic Compartment of C-Type Cholinergic Afferent Synapses on Motoneurons

doi: 10.3389/fncel.2019.00582

Figure Lengend Snippet: p-c-Jun-like immunodetection with antibodies other than Y172 under basal conditions and in axotomized MNs (30 days after sciatic nerve transection at P60). (A1–E4) Representative images of spinal cord MNs double immunostained with different polyclonal antibodies (pAbs) against c-Jun (green) phosphorylated at either Ser63 (A1–B4) or Ser73 (C1–E4) and VAChT (red); fluorescent Nissl stain (blue in A4,B4,C4,D4 , and E4 ) was used to visualize MNs. The images in (A1,B1) , which show the p-c-Jun (Ser63) channel, were obtained following the modification of scanning parameters to achieve a higher sensitivity of detection than that used for the Y172 antibody. Note the absence of p-c-Jun (Ser63) nuclear immunostaining and the presence of immunolabeled cytoplasmic profiles mainly located in the periphery of the cell body (arrows in A3,A4 ) under basal conditions (A1–A4) . Thirty days after unilateral sciatic nerve transection (B1–B4) , the same antibody revealed prominent nuclear immunostaining in MNs located on the ipsilateral (operated) side of the spinal cord (white arrow in B4 ) but an almost complete absence of cytoplasmic positive profiles. The intense immunoreactivity in the nucleus of the presumably axotomized MN was in contrast with the faint nuclear immunostaining in the neighboring neuron, likely corresponding to a nonlesioned MN (blue arrow in B4). (C1–C4) Negative immunostaining with the polyclonal antibody against p-c-Jun (Ser73) was observed in both the nucleus and cytoplasm of an MN under basal conditions. (D1–E4) The same antibody showed intense positive nuclear immunostaining and the absence of immunostained cytoplasmic profiles in axotomized MNs, both in the ipsilateral and contralateral side of the spinal cord. (F) Representative western blots of mouse spinal cord extracts probed with the antibodies against p-c-Jun used in our study; a monoclonal antibody against c-Jun (c-Jun mAb) was also included, and β-actin was used as a loading control. Note that a band corresponding to ~43–48 kDa, as expected for p-c-Jun, was observed; the same band was also seen in western blots performed with the anti-c-Jun antibody. (G) Densitometric analysis of p-c-Jun bands obtained in western blots by probing with the antibodies used for analysis. The data were normalized to β-actin; the bars represent the values (mean ± SEM) of extracts from three mice. Scale bar: E4 = 20 μm (valid for A1–E3 ).

Article Snippet: The primary antibodies used were rabbit monoclonal anti-phospho-c-Jun (serine [Ser]63) clone Y172 (diluted 1:300, hereafter referred to as the Y172 antibody; Abcam, Cambridge, UK; cat. ab32385 or Millipore, Burlington, MA, USA; cat.# 04-212); rabbit polyclonal anti-phospho-c-Jun (Ser63; 1:100; Cell Signaling, Danvers, MA, USA; cat.# 9261); rabbit polyclonal anti-phospho-c-Jun (Ser73; 1:100; Cell Signaling; cat.# 9164); guinea pig polyclonal anti-synaptophysin 1 (1:500; Synaptic Systems, Goettingen, Germany; cat.# 101004); guinea pig polyclonal anti-vesicular acetylcholine transporter (VAChT; 1:500; Synaptic Systems, Goettingen, Germany; cat.# 139105); guinea pig polyclonal anti-vesicular glutamate transporter 1 (VGluT1, 1:500; Synaptic Systems, Goettingen, Germany; cat.# 135304); guinea pig polyclonal anti-vesicular GABA transporter (VGAT, 1:200; Synaptic Systems, Goettingen, Germany; cat.# 131004); mouse monoclonal anti-synaptic vesicle glycoprotein 2A (SV2, 1:1,000; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# AB_2315386); mouse monoclonal anti-sigma-1 receptor (S1R, 1:50; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-137075); mouse monoclonal anti-Kv2.1 voltage-gated potassium channel (Kv2.1, 1:100; NeuroMab, Davis, CA, USA; cat.# 73-014); sheep polyclonal anti-choline acetyltransferase (ChAT, 1:1,000; Abcam cat.# Ab18736); rabbit polyclonal anti-ChAT (1:200; Millipore, Burlington, MA, USA; cat.# AB143); rabbit polyclonal anti-neuregulin-1 (NRG1) type III (extracellular, 1:250; Alomone labs, Jerusalem, Israel, cat.# ANR 113); mouse monoclonal anti-NRG-CRD, type III, clone N126B/31 (1:250; Millipore; cat.# MABN534); rabbit polyclonal anti-NRG1 1 α/β 1/2 (1:300; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-348); mouse monoclonal anti-Golgi matrix protein of 130 kDa (GM130, 1:200; BD Biosciences, San Jose, CA, USA; cat.# 610822); mouse monoclonal anti-lysosomal membrane glycoprotein (LAMP-1), clone ID4B (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# ID4B); mouse monoclonal anti-KDEL (Lys-Asp-Glu-Leu motif) receptor (KDELR), clone KR-10 (1:50; Stressgen Biotechnologies, San Diego, CA, USA; cat.# VAA-PT048); mouse monoclonal anti-protein disulfide-isomerase (PDI), clone 1D3 (1:200; Enzo Life Sciences, Farmingdale, NY, USA; cat.# ADI-SPA-891); and mouse monoclonal anti-calcitonin gene-related peptide (CGRP; 1:100; Abcam, Cambridge, UK; cat.# ab81887).

Techniques: Immunodetection, Staining, Modification, Immunostaining, Immunolabeling, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Direct Reprogramming of Adult Human Somatic Stem Cells Into Functional Neurons Using Sox2, Ascl1 , and Neurog2

doi: 10.3389/fncel.2018.00155

Figure Lengend Snippet: Phenotypic specification of lineage reprogrammed hUCMSC-derived iNs. (A) Principal component analysis (PCA) of gene expression among cells reprogrammed with Sox2/Neurog2 or Sox2/Ascl1 . Genes used in the PCA are involved in neurotransmitter identity. Note the significant overlap between the two cell populations, suggesting that expression of either Sox2/Neurog2 or Sox2/Ascl1 / may elicit similar neuronal phenotypes. (B) Heat map showing the relative expression of 9 genes involved in the specification of different neuronal phenotypes. Observe the variable expression of genes essential for the specification of distinct neurotransmitter identities in iNs derived from hUCMSCs lineage-converted through the expression of either Sox2/Neurog2 or Sox2/Ascl1 . Choline O-acetyltransferase (CHAT), Tyrosine hydroxylase (TH), Tryptophan hydroxylase 2 (TPH2), Vesicular Glutamate Transporter 1 (VGLUT1 or SLC17A7), GABA Vesicular Transporter (VGAT or SLC32A1), FEZ family zinc finger 2 (FEZF2), T-box brain 1 (TBR1), SATB homeobox 2 (SATB2), COUP-TF-Interacting Protein 2 (CTIP2 or BCL11B).

Article Snippet: The following primary antibodies and dilutions were used: chicken anti-Green Fluorescent Protein (GFP, Aves Labs, 1:1,000), rabbit anti-Red Fluorescent Protein (RFP, Rockland, 1:1,000), mouse anti-major microtubule associated protein (MAP2; Sigma, 1:500), guinea pig polyclonal anti-vesicular GABA transporter (vGAT, Synaptic Systems, 1:200), and polyclonal anti-vesicular glutamate transporter 1 (vGLUT11, Synaptic Systems, 1:1,000).

Techniques: Derivative Assay, Gene Expression, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Direct Reprogramming of Adult Human Somatic Stem Cells Into Functional Neurons Using Sox2, Ascl1 , and Neurog2

doi: 10.3389/fncel.2018.00155

Figure Lengend Snippet: Protein expression of vesicular transporters of lineage reprogrammed hUCMSC-derived iNs. Immunostaining for DSRED (red), GFP (green), vGAT (white; left panel) or vGLUT1 (white; right panel) and DAPI (blue), 15 days post transfection (dpt). Scale bar represents 20μm. (A–H) hUCMSC transfected with control plasmids encoding only reporter proteins GFP and DSRED. Note that cells displayed classical mesenchymal cell morphologies and did not express neither vGAT or vGLUT1. (I–P) hUCMSC transfected with Sox2 and Ascl1 . (Q–Y) hUCMSC transfected with Sox2 and Neurog2 . (I,J) Observe the expression of the vesicular GABA transporter (VGAT) in Sox2/Ascl1 -derived iN (yellow arrows). The inset shows a high magnification view of the boxed area.

Article Snippet: The following primary antibodies and dilutions were used: chicken anti-Green Fluorescent Protein (GFP, Aves Labs, 1:1,000), rabbit anti-Red Fluorescent Protein (RFP, Rockland, 1:1,000), mouse anti-major microtubule associated protein (MAP2; Sigma, 1:500), guinea pig polyclonal anti-vesicular GABA transporter (vGAT, Synaptic Systems, 1:200), and polyclonal anti-vesicular glutamate transporter 1 (vGLUT11, Synaptic Systems, 1:1,000).

Techniques: Expressing, Derivative Assay, Immunostaining, Transfection, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Disruption of Slc4a10 augments neuronal excitability and modulates synaptic short-term plasticity

doi: 10.3389/fncel.2015.00223

Figure Lengend Snippet: Synaptic expression of Slc4a10 in the cortex. (A′–A′′′) Overlap of Slc4a10 and VGAT, a marker for GABAergic presynapses (green: Slc4a10, red: VGAT). (B′–B′′′) Slc4a10 co-localizes also with the postsynaptic GABA A -receptor in cortical neurons (green: Slc4a10, red: GABA A - α5).

Article Snippet: For co-stainings, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:500, Synaptic Systems) and polyclonal guinea pig anti-GABA A receptor subunit α5 (1:4000; Redecker et al., ).

Techniques: Expressing, Marker