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: Y172 immunoreactivity colocalizes with S1R and Kv2.1 in C-boutons. (A1–D4) Representative confocal images of adult (P75) mouse spinal cord MNs double immunostained with antibodies against either S1R or Kv2.1 (red) and Y172 (green), as indicated in the panels; sections were counterstained with fluorescent Nissl stain (blue) for MN visualization. The areas delimited by dotted-line squares in (A4,C4) are shown at higher magnification in (B1–B4,D1–D4) , respectively. (E–H) Pixel profile analysis (F,H ) along the lines depicted in (E,G) , which were obtained from MNs immunolabeled with Y172 (green) and anti-S1R or Kv2.1 (red) antibodies; fluorescent Nissl staining (blue) for MN visualization can be seen in (E) . Note the overlap between the Y172 and S1R (F) or Kv2.1 (H) signals. (I,J) Squashed MNs immunolabeled with anti-NRG1 (I) and Y172 (J) antibodies; the circled profiles are shown at higher magnification in the insets; note that Y172-positive profiles were smaller and displayed more compact patterns than NRG1 clusters. Scale bars: A4,C4 = 10 μm (valid for A1–A3,C1–C3 ); B4,D4 = 1 μm (valid for B1–B3,D1–D3 ); G = 1 μm (valid for E ); and J = 20 μm (valid for I ).

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: Confocal, Staining, Analysis, Immunolabeling, Immunofluorescence

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: Changes in Y172 immunoreactivity in MNs from mutant mice (P60) overexpressing NRG1 type III. (A–F) The density (per 100 μm 2 MN soma, A,B ) and size (in μm 2 , C,D ) of total (A,C) and peripheral (periph., B,D ) Y172-positive profiles and the percentage of these profiles showing a spatial association with VAChT-positive C-boutons (E) and NRG1 type III-positive spots (F) in MNs from WT and NRG1 type III-overexpressing mice. Note that NRG1 type III overexpression was associated with a prominent decrease in the density of total Y172-positive profiles (A) and a significant increase in the number of those located peripherally (B) in MNs; the area of both total and peripheral Y172-positive profiles was dramatically increased in MNs from NRG1 type III-overexpressing animals (C,D) . Additionally, the percentage of Y172-positive profiles showing a close association with VAChT- (E) or NRG1 type III-positive (F) spots significantly increased or decreased, respectively, in MNs from NRG1 type III-overexpressing animals; 10–15 randomly selected MNs from 3 to 4 mice per condition were analyzed; * p < 0.05 and *** p < 0.001 vs. WT; student’s t -test). ( G1–G4) Representative confocal micrographs of an NRG1 type III-overexpressing MN immunostained with Y172 (green) and anti-NRG1 type III (red) antibodies and counterstained with fluorescent Nissl stain (blue) for neuron visualization. Note that, compared to MNs of adult CD1 mice (see, for instance, or ), MNs overexpressing NRG1 type III exhibit an enlargement of Y172-positive profiles located in the periphery of the cell body, which correlated with the redundant and expanded SSCs previously described in Salvany et al. ; note also the expansion of NRG1 type III immunolabeling peripherally located in MN soma. (H1–H3) A higher magnification image of the area delimited in (G4) by the dotted-line rectangle corresponding to Y172, NRG1 type III and merged channels, as indicated, is shown. Scale bars: G4 = 10 μm (valid for G1-G3 ); H3 = 2.5 μm (valid for H1, H2 ).

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: Mutagenesis, Over Expression, Confocal, Staining, Immunolabeling, Immunofluorescence