Journal: Cells

Article Title: Sympathetic Stimulation Upregulates the Ca 2+ Channel Subunit, Ca V α2δ1, via the β1 and ERK 1/2 Pathway in Neonatal Ventricular Cardiomyocytes

doi: 10.3390/cells11020188

Figure Lengend Snippet: Expression of Ca V α2δ1 ( A ), Ca V α1C ( C ), Ca V β3 ( E ), and Ca V β2 ( G ) in neonatal rat cardiomyocytes. Total proteins were extracted from neonatal rat ventricular cardiomyocytes. Proteins were separated on an 8% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with anti-Ca V α2δ1, anti-Ca V α1C, anti-Ca V β3, anti-Ca V β2, and anti-GAPDH antibodies overnight and then incubated with HRP-conjugated goat anti-rabbit secondary antibody. Lanes were loaded with 30 μg of proteins. NE: cardiomyocytes treated with 1 µM norepinephrine. Graph showing the total protein expression of Ca V α2δ1 ( B ), Ca V α1C ( D ), Ca V β3 ( F ), and Ca V β2 ( H ) normalized to GAPDH. * p < 0.01 vs. Basal. Statistical analysis was performed using one-way ANOVA.

Article Snippet: Antibodies used were rabbit anti-Ca V α2δ1 (1:1000, Alomone ACC-015), rabbit Ca V α1C (1:1000, Alomone ACC-003), rabbit Ca V β2 (1:5000, Alomone ACC-105), rabbit Ca V β3 (1:500, Alomone ACC-008), rabbit Phospho-ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA, 9101S), Total ERK 1/2 (Cell Signaling Technology, 137F5) and rabbit GAPDH (Jackson, West Grove, PA, USA, 111-035-144).

Techniques: Expressing, Incubation

Journal: Cells

Article Title: Sympathetic Stimulation Upregulates the Ca 2+ Channel Subunit, Ca V α2δ1, via the β1 and ERK 1/2 Pathway in Neonatal Ventricular Cardiomyocytes

doi: 10.3390/cells11020188

Figure Lengend Snippet: Expression of Ca V α2δ1 ( A ) and Ca V β3 ( C ) in neonatal rat cardiomyocytes. Total proteins were extracted from neonatal rat ventricular cardiomyocytes. Cells were treated with 1 µM Norepinephrine (NE) for 24 h or pre-treated with metoprolol tartrate (β1-blocker) for 1 h followed by treatment with NE for 24 h. Proteins were separated on an 8% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with anti-Ca V α2δ1, anti-Ca V β3, and anti-GAPDH antibodies overnight and then incubated with HRP-conjugated goat anti-rabbit secondary antibody. Lanes were loaded with 30 μg of proteins. NE: cardiomyocytes treated with 1 µM norepinephrine. 24 h + metoprolol tartrate: Cardiomyocytes pre-treated with metoprolol tartrate (β1-blocker) for 1 h, followed by treatment with NE for 24 h. ( B ) Graph showing the total protein expression of Ca V α2δ1 normalized to GAPDH. * p < 0.01 vs. Basal untreated NRVMs; † p <0.01 vs. NE 24 h. (4 cardio-preparations). (D) Graph showing the total protein expression of Ca V β3 normalized to GAPDH. * p < 0.01 vs. Basal untreated NRVMs (4 cardio-preparations). Statistical analysis was performed using one-way ANOVA.

Article Snippet: Antibodies used were rabbit anti-Ca V α2δ1 (1:1000, Alomone ACC-015), rabbit Ca V α1C (1:1000, Alomone ACC-003), rabbit Ca V β2 (1:5000, Alomone ACC-105), rabbit Ca V β3 (1:500, Alomone ACC-008), rabbit Phospho-ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA, 9101S), Total ERK 1/2 (Cell Signaling Technology, 137F5) and rabbit GAPDH (Jackson, West Grove, PA, USA, 111-035-144).

Techniques: Expressing, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Transplantation of Neural Precursors Derived from Induced Pluripotent Cells Preserve Perineuronal Nets and Stimulate Neural Plasticity in ALS Rats

doi: 10.3390/ijms21249593

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: Anti- l -type Ca 2+ CP α1C , L-type Ca 2+ CP α1C/ICC , Rabbit polyclonal , 1:200 , Alomone Labs, Jerusalem, Israel.

Techniques: Marker

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 axotomized MNs. The sections of the spinal cord from adult (P60) mice subjected to unilateral sciatic nerve transection were immunostained with the Y172 antibody in combination with antibodies against VAChT (blue) and IBA1 (red), as indicated in the panels, to identify C-boutons and microglia, respectively; sections were also counterstained with fluorescent Nissl stain for MN visualization (shown in gray in F4,G4 ). (A1–B2) Ventral horn MNs in general view (A1–B2 ) and higher magnification (C1–E5) images from the contralateral (contra., nonaxotomized, A1, A2,C1–C5 ) and ipsilateral (ipsi., axotomized, B1, B2,D1–E5 ) sides of the spinal cord 1 (A1–D5) and 14 (E1–E5) days after nerve transection; note the nuclear Y172 immunostaining (B1,D1,E1) and the prominent IBA1-positive reactive microglia on the ipsilateral side (B2, D4,E4) in relation to those on the contralateral side (A1,A2,C1,C4) ; in (E1) , an MN with intense nuclear Y172 staining as well as numerous profiles immunoreactive with the monoclonal antibody can be seen. The areas delimited by dotted-line rectangles in (C4,D4,E4) are shown at higher magnifications in (C5,D5,E5) ; note, on the ipsilateral side (D4,E4) , the presence of abundant IBA1-positive microglial processes enwrapping MNs and contacting Y172-positive profiles, suggesting a role for microglia in the loss of peripheral cytoplasmic Y172 immunolabeling in axotomized MNs. (F1–G4) MNs on the ipsilateral side of spinal cord 30 (F1–F4) and 180 (G1–G4) days after axotomy; note in (F1–F4) the intense nuclear signal and the depletion of cytoplasmic peripheral profiles positive with the Y172 antibody. In (G1–G4) , an apparently healthy MN exhibiting both abundant cytoplasmic profiles and the absence of nuclear immunostaining with the Y172 antibody can be seen in the vicinity of an atrophic MN showing intense nuclear Y172 immunoreactivity. In all cases, MNs with nuclear Y172 signal displayed a reduced density of VAChT-positive C-boutons. (H,I ) The density (per 100 μm 2 of cell body) and size (in μm 2 ) of cytoplasmic Y172-positive profiles in MNs located on the contralateral and ipsilateral sides of the spinal cord on different days after unilateral sciatic nerve transection. (J) Changes (expressed as the % of the contralateral side) in the density of VAChT-positive puncta observed in ipsilateral side MNs on different days post-axotomy. The data in the graph are expressed as the mean ± SEM, * p < 0.05 and *** p < 0.001 vs. the contralateral side; n = 8–11 randomly selected MNs per side from three mice per day post-axotomy. Scale bars: B2 = 40 μm (valid for A1–B1 ); E4 = 20 μm (valid for C1–C4,D1–D4,E1–E3 ); E5 = 5 μm (valid for C5,D5,E5 ); G4 =10 μm (valid for F1–G3 ).

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: Staining, Immunostaining, Immunolabeling

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, Staining, Immunolabeling

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: 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: Western blot and immunocytochemical analyses with the Y172 antibody in NSC-34 cells (A–E4) and cultured MNs (F–K4) . (A) The time course of Y172 antigen expression levels in NSC-34 cells after a different number of days (0, 2, 4 and 8) (d) of differentiation, as determined by western blot analysis; the densitometric analysis was normalized to β-actin, which was used as a loading control. (B) Representative western blots of NSC-34 cells probed with the Y172 antibody used for quantification in (A) . (C1–E4) Representative confocal images of NSC-34 cells after 2 (C1–C4) , 4 (D1–D4) and 8 (E1–E4) days of differentiation stained with Y172 (green) and anti-VAChT (red) antibodies as well as DAPI (blue) for DNA, as indicated. Y172 immunostaining was found exclusively in the nuclei of NSC-34 cells following 2 days of differentiation (C1–4) ; however, granular immunostaining was observed in the cytoplasm of cell bodies and neurites after 4 days of differentiation (D1–4) ; after 8 days of differentiation, Y172 immunostaining decreased in the nucleus but markedly increased in the cytoplasm of cell bodies and proximal neurites (E1–4) . VAChT immunostaining displayed a diffuse and faintly positive pattern into the cell body cytoplasm, and although some positive puncta appear to be present, these were very scarce and did not have a defined location in NSC-34 cells. (F) The time course of Y172 antigen expression levels in MNs cultured for 3, 6 and 12 days, determined by western blot analysis; the densitometric analysis was normalized to β-actin, which was used as a loading control. (G) Representative western blots of cultured MNs probed with the Y172 antibody used for quantification in (F) . ( H1–K4) Representative confocal images of MNs cultured for 3 (H1–4) , 6 (I1–4) and 12 (J1–4) days stained with Y172 (green) and anti-VAChT (red) antibodies as well as DAPI (blue) for DNA, as indicated. Note the intense nuclear positivity but the faint and discrete cytoplasmic signal obtained with the Y172 antibody at 3 DIV (H1–4) ; however, at 6 DIV (I1–I4) , an increase in the cytoplasmic immunolabeling, mainly in the form of oval patches in the soma and proximal neurites, was observed. Following 12 days of culture (J1–4) , a reduction in Y172 immunolabeling was observed in the cytoplasm, although some positive profiles remained. The area delimited by the dotted rectangle in (J1–J4) is shown in (K1–K4) at higher magnification; note that some Y172-positive profiles were in close association with patches expressing VAChT (red, arrow). The data in (A,F) are shown as the mean ± SEM of 3–6 western blots from three independent cultures. Scale bars: E4,J4 = 20 μm (valid for C1-E3,H1–J3 , respectively); K4 = 4 μm (valid for K1–K3 ).

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: Western Blot, Cell Culture, Expressing, Staining, Immunostaining, Immunolabeling