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
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
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, Mouse Assay, Over Expression