Journal: PLoS ONE

Article Title: Effects of Combinatorial Treatment with Pituitary Adenylate Cyclase Activating Peptide and Human Mesenchymal Stem Cells on Spinal Cord Tissue Repair

doi: 10.1371/journal.pone.0015299

Figure Lengend Snippet: (A). Astrocytes were treated with PACAP (20 ng/ml), or indirectly co-cultured with hMSCs in the absence and presence of PACAP (20 ng/ml) for 48 hours. The cultures were subjected to Q-PCR for measurement of the mRNA levels of GLT-1 and GLAST. Data are presented as mean ± SEM and expressed as the ratio of GLT-1 (GLAST) mRNA levels compared to control. * p <0.05 versus control. (B). Astrocytes were indirectly co-cultured with hMSCs in the absence or presence of PACAP for 48 hours. Membrane proteins were then extracted for western blotting using anti-GLT-1 antibody. The same blot was reprobed with anti-P2X 7 R antibody. P2X 7 R levels are presented as a loading control. Relative intensity of GLT-1 levels normalized to P2X 7 R was measured. Data are presented as mean ± SEM and expressed as a percentage of GLT-1 levels in the group with combinatorial treatment compared to that detected in the group co-cultured only with hMSCs. * p <0.05 versus the group only co-cultured with hMSCs. (C). Astrocytes were infected by lentivirus carrying lenti-GLT-1-GFP, and then co-cultured with hMSCs in the absence or presence of PACAP. Strong punctate fluorescence spots (arrowheads) were observed in the processes of astrocytes co-cultured with hMSCs in the presence of PACAP, when compared to that seen in astrocytes co-cultured only with hMSCs (arrows). Scale bar, 50 µm. (D). Astrocytes were treated with PACAP, or co-cultured with hMSCs in the absence or presence of PACAP. After 48 or 72 hours, the cultures were subjected to [ 3 H]-L-glutamate uptake analysis as described in . Data are presented as mean ± SEM and expressed as a ratio of the glutamate uptake in each treated group compared to that detected in the control group. * p <0.05 versus control.

Article Snippet: The protein was identified by incubating the membrane with anti-GLT-1 antibody (1∶1000; Millipore) or anti- purinergic P2X 7 R antibody (1∶1000; Alomone Labs, Israel) overnight at 4°C, followed by HRP-conjugated secondary antibody (1∶2000) and ECL solution.

Techniques: Cell Culture, Western Blot, Infection, Fluorescence

Journal: BMC Neuroscience

Article Title: Is TrpM5 a reliable marker for chemosensory cells? Multiple types of microvillous cells in the main olfactory epithelium of mice

doi: 10.1186/1471-2202-9-115

Figure Lengend Snippet: Antisera used to characterize TrpM5 cells

Article Snippet: P2X2 , purinergic receptor – transduction component , Alomone Labs APR003 , AN-06.

Techniques: Transduction

Journal: Frontiers in Physiology

Article Title: Myosin 5a in the Urinary Bladder: Localization, Splice Variant Expression, and Functional Role in Neurotransmission

doi: 10.3389/fphys.2022.890102

Figure Lengend Snippet: Purinergic responses of WT and DBA bladders. (A) Comparison of the contractile response of WT (black bar) and DBA (gray bar) bladder smooth muscle to the purinergic receptor agonist, αβmeATP (10 µM). Contractions of WT and DBA bladders were not significantly different under this condition ( N = 11 WT, N = 13 DBA; p = 0.21). (B) Western blot comparing expression of the P2X 1 purinergic receptor in WT and DBA extracts from bladder tissue devoid of mucosa. β-actin, shown in bottom panel, served as loading control. Molecular weights of mass standards (in kDa) are indicated at tick marks. (C) Quantitative data for WT (black circles) and DBA (gray squares) are graphed at right. The intensity of P2X 1 receptor immunoreactivity, normalized by intensity of β-actin, was not different between groups ( N = 3, p = 0.2).

Article Snippet: The P2X 1 purinergic receptor (P2X 1 R) antibody (APR-001, RRID:AB_2040052) was from Alomone Labs (Jerusalem, Israel).

Techniques: Western Blot, Expressing

Journal: The Journal of Neuroscience

Article Title: Kv4.1, a Key Ion Channel For Low Frequency Firing of Dentate Granule Cells, Is Crucial for Pattern Separation

doi: 10.1523/JNEUROSCI.1541-19.2020

Figure Lengend Snippet: Comparison between Kv4.1 and Kv4.2 expression in the hippocampus. A, Both Kv4.1 and Kv4.2 antibodies selectively recognize the channel in HEK293 cells. HEK293 cells were transfected with either AcGFP-tagged Kv4.1 cDNA (Kv4.1-AcGFP Tf) or Kv4.2 (Kv4.2-AcGFP Tf). Forty-eight hours after transfection, the cells were stained using Kv4.1 or Kv4.2 antibodies. Scale bar, 20 μm. B, Representative fluorescence immunostaining images show that Kv4.1 (top) is highly expressed in DG while Kv4.2 (bottom) is broadly expressed in the hippocampus of 8-week-old mice. Scale bars, 500 μm (left) and 50 μm (middle and right). C, Summary bar graphs of the relative fluorescence intensity (F − F0) shown in the DG and CA1 for Kv4.1 (n = 10, left) and Kv4.2 (n = 5, right). (Kv4.1; GCL, 20.3 ± 2.0; ML, 11.9 ± 1.3; Pyr, 12.0 ± 2.3; Rad, 11.9 ± 2.2; GCL vs ML, p = 0.0037; GCL vs Pyr, p = 0.013; Pyr vs Rad, p = 0.97; Kv4.2; GCL, 24.2 ± 2.3; ML, 39.1 ± 5.8; Pyr, 25.1 ± 2.3; Rad, 33.5 ± 3.3; GCL vs ML, p = 0.037; Pyr vs Rad, p = 0.016). D, Western blot analysis for Kv4.1 and Kv4.2 in DG and CA1 of 8-week-old mice. Right, DG exhibits higher Kv4.1 expression level compared with CA1; 1.00 ± 0.02 in DG, 0.53 ± 0.05 in CA1, n = 8; p < 0.0001. Paired t test. Prospero-related homeobox 1 (Prox) and α-tubulin used as a marker for DG and loading control, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, N.S. (not significant) p > 0.05 by Student's t-test.

Article Snippet: The following antibodies were purchased from commercial sources: anti-Kv4.3, APC-017, Alomone; anti-Prox1 antibody, PRB-238C-200, BioLegend; anti-α-tubulin, T5168, Sigma-Aldrich.

Techniques: Expressing, Transfection, Staining, Fluorescence, Immunostaining, Western Blot, Marker

Journal: Journal of Neuroinflammation

Article Title: Morphine immunomodulation prolongs inflammatory and postoperative pain while the novel analgesic ZH853 accelerates recovery and protects against latent sensitization

doi: 10.1186/s12974-019-1480-x

Figure Lengend Snippet: Immunohistochemistry following CFA and drug. Tissue was taken 25 days after CFA in the CFA-then-drug paradigm and 21 days after CFA in the drug-then-CFA paradigm. a CFA then drug. Staining at this time point did not show higher microglial activation in any group, but pp38 was decreased by ZH853, and IL-1β and CGRP were increased by morphine relative to vehicle and ZH853. Astrocyte activation and P2X7Rs were increased in morphine-treated animals compared to ZH853-treated animals. b Drug then CFA. Microglial activation was greater in animals pretreated with morphine- versus vehicle- and ZH853-treated animals. pp38 was increased by morphine and decreased by ZH853, producing a significant difference between drugs. IL-1β was significantly decreased by ZH853 relative to morphine and vehicle. CGRPR staining was increased by morphine relative to vehicle- or ZH853-treated animals. Astrocyte activation was decreased by ZH853 relative to vehicle and morphine, and P2X7R was increased by morphine relative to vehicle and ZH853. n indicated in bar graphs. Error bars indicate SEM. One-way ANOVAs were performed for each marker. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: After two washes in PBS and blocking with 5% normal horse serum/0.3% Triton X-100, sections were incubated in the following primary antibodies: calcitonin gene-related peptide (CGRP) (rabbit, T-4032, Peninsula Labs, San Carlos, CA), glial fibrillary acidic protein monoclonal (GFAP) (mouse, Astro6 MA5-12023, ThermoFisher, Carlsbad, CA), Anti-CD11b/c (OX42) (rabbit, CBL1512-100UG, Millipore Sigma, St. Louis, MO), purinergic receptor 7 (P2X7R) (rabbit, #APR-008, Alomone Labs, Jerusalem, Israel), phosphorylated-p38 MAP kinase (pp38) (rabbit, #4511, Cell Signaling Technology, Danvers, MA), or interleukin-1beta (IL-1β) (rabbit, ab9787, Abcam, Cambridge, MA) and were incubated for 24 h at 4 °C on a slow rocker.

Techniques: Immunohistochemistry, Staining, Activation Assay, Marker

Journal: Journal of Neuroinflammation

Article Title: Morphine immunomodulation prolongs inflammatory and postoperative pain while the novel analgesic ZH853 accelerates recovery and protects against latent sensitization

doi: 10.1186/s12974-019-1480-x

Figure Lengend Snippet: Representative images of IHC from the Drug-then-CFA paradigm. Each analysis was done on images acquired with the same exposure, gain, and aperture across treatment groups, but exposure and gain were adjusted for each antibody. All images were taken on a × 10 objective and microglia were imaged at × 20 for the inset of OX42. OX42 labels microglia, pp38 labels phosphorylated p38 Map Kinase, IL-1β labels interleukin-1beta, CGRP labels α-calcitonin gene-related peptide, Astro6 labels glial fibrillary acidic protein (GFAP), and P2X7R labels the P2X purinoceptor 7

Article Snippet: After two washes in PBS and blocking with 5% normal horse serum/0.3% Triton X-100, sections were incubated in the following primary antibodies: calcitonin gene-related peptide (CGRP) (rabbit, T-4032, Peninsula Labs, San Carlos, CA), glial fibrillary acidic protein monoclonal (GFAP) (mouse, Astro6 MA5-12023, ThermoFisher, Carlsbad, CA), Anti-CD11b/c (OX42) (rabbit, CBL1512-100UG, Millipore Sigma, St. Louis, MO), purinergic receptor 7 (P2X7R) (rabbit, #APR-008, Alomone Labs, Jerusalem, Israel), phosphorylated-p38 MAP kinase (pp38) (rabbit, #4511, Cell Signaling Technology, Danvers, MA), or interleukin-1beta (IL-1β) (rabbit, ab9787, Abcam, Cambridge, MA) and were incubated for 24 h at 4 °C on a slow rocker.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Morphine immunomodulation prolongs inflammatory and postoperative pain while the novel analgesic ZH853 accelerates recovery and protects against latent sensitization

doi: 10.1186/s12974-019-1480-x

Figure Lengend Snippet: Regression analyses of IHC markers versus von Frey scores. Individual scores for both von Frey at the time point just prior to perfusion and integrated density (or cell count for pp38) from both paradigms of CFA and drug treatment were plotted for all drug groups. pp38 and P2X7R are consistently correlated with pain. P values are listed on each graph

Article Snippet: After two washes in PBS and blocking with 5% normal horse serum/0.3% Triton X-100, sections were incubated in the following primary antibodies: calcitonin gene-related peptide (CGRP) (rabbit, T-4032, Peninsula Labs, San Carlos, CA), glial fibrillary acidic protein monoclonal (GFAP) (mouse, Astro6 MA5-12023, ThermoFisher, Carlsbad, CA), Anti-CD11b/c (OX42) (rabbit, CBL1512-100UG, Millipore Sigma, St. Louis, MO), purinergic receptor 7 (P2X7R) (rabbit, #APR-008, Alomone Labs, Jerusalem, Israel), phosphorylated-p38 MAP kinase (pp38) (rabbit, #4511, Cell Signaling Technology, Danvers, MA), or interleukin-1beta (IL-1β) (rabbit, ab9787, Abcam, Cambridge, MA) and were incubated for 24 h at 4 °C on a slow rocker.

Techniques: Cell Counting

Journal: Journal of Neuroinflammation

Article Title: Morphine immunomodulation prolongs inflammatory and postoperative pain while the novel analgesic ZH853 accelerates recovery and protects against latent sensitization

doi: 10.1186/s12974-019-1480-x

Figure Lengend Snippet: Schematic of postulated mechanisms of microglial activation by morphine but not by ZH853. Proposed mechanisms by which morphine activates glia or enhances trauma-induced activation include (1) induction of neuronal CGRP that activates microglial CGRP receptors , (2) binding at toll-like receptors (TLRs) alone or in concert with damage- or pathogen-associated molecular patterns (DAMPs, PAMPS) , and (3) activation of purinergic P2X receptors including by upregulation through MOR (but see ). Each of these mechanisms causes phosphorylation of the mitogen-activated protein kinase (MAPK) p38 to increase transcription and translation of cytokines, including interleukin-1β (IL-1β , example shown), P2X7R activation also induces NOD-like receptor family, pyrin domain containing 3 (NLRP3) to become a complex (inflammasome) that contains and releases active caspase-1 which cleaves pro-IL-1β into the active cytokine IL-1β . IL-1β activates IL-1 receptors on astrocytes and neurons, further increasing inflammation. Ultimately, this proinflammatory pathway causes dorsal horn plasticity that leads to central sensitization and increased pain

Article Snippet: After two washes in PBS and blocking with 5% normal horse serum/0.3% Triton X-100, sections were incubated in the following primary antibodies: calcitonin gene-related peptide (CGRP) (rabbit, T-4032, Peninsula Labs, San Carlos, CA), glial fibrillary acidic protein monoclonal (GFAP) (mouse, Astro6 MA5-12023, ThermoFisher, Carlsbad, CA), Anti-CD11b/c (OX42) (rabbit, CBL1512-100UG, Millipore Sigma, St. Louis, MO), purinergic receptor 7 (P2X7R) (rabbit, #APR-008, Alomone Labs, Jerusalem, Israel), phosphorylated-p38 MAP kinase (pp38) (rabbit, #4511, Cell Signaling Technology, Danvers, MA), or interleukin-1beta (IL-1β) (rabbit, ab9787, Abcam, Cambridge, MA) and were incubated for 24 h at 4 °C on a slow rocker.

Techniques: Activation Assay, Binding Assay

Journal: Scientific Reports

Article Title: Potential Orphan Drug Therapy of Intravesical Liposomal Onabotulinumtoxin-A for Ketamine-Induced Cystitis by Mucosal Protection and Anti-inflammation in a Rat Model

doi: 10.1038/s41598-018-24239-9

Figure Lengend Snippet: Alterations of neuroreceptors protein expression in the mucosa layer or smooth muscle layer of the bladder for all groups (n = 8). Western blot analyses with specific antibodies to the TRPV1 receptor and P2X 3 receptor of the rat mucosal layer as well as M 2 -and M 3 - mAChRs and the purinergic P2X 1 receptor of the rat detrusor layer were performed in 3 groups. ( A ) TRPV1 receptor: The TRPV1 antibody produced a clear single band at 95 kDa. ( B ) Purinergic P2X 3 mature receptor: the predominant P2X 3 form (65 kDa). ( C ) M 2 –mAChR of bladder detrusor layer. The M 2 –mAChR antibody produced a clear single band between 50kD and 75kD. ( D ) M 3 –mAChR of bladder detrusor layer. ( E ) Purinergic P2X 1 receptor. Experiments were repeated two times and representative blots are shown. Data of proteins expression (ratios of signal intensities of investigated receptors relative to GAPDH) were calculated with 8 samples in each group. These data of Mean ± SE were standardized and expressed in percentage in which the value of the control group is treated as 100%. Theses values were shown in the bar graph. An asterisk indicates a significant difference between controls and other groups (One-way ANOVA with Dunnett’s test, p < 0.05). The grouping of blots was cropped from the same gel for each protein. The full-length gels and blots are included in the Supplementary Figure .

Article Snippet: Antibodies raised against SNAP25 (1: 1000 dilution; Cell signal), E-cadherin (1:1000 dilution; Cell Signal), nerve growth factor (1:200 dilution; Cell Signal), IL-1β (1:500 dilution; Abcam) IL-6 (1:1000 dilution; Abcam), TNF-α (1:250 dilution, Santa Cruz), NF-κB (1: 2500 dilution; Cell SiZgnal), COX-2 (1:200 dilution; Abcam), TRPV1 receptor (1:1000 dilution; Alomone), purinergic receptor P2X 1 (1:10000 dilution; Alomone), purinergic receptor P2X 3 (1: 1000 dilution; Alomone), M 2 -mAChR (1:1000 dilution; Alomone), M 3 -mAChR (1:1000 dilution; Alomone), and GAPDH (1: 10,000 dilution; Millipore) were used.

Techniques: Expressing, Western Blot, Produced

Journal: Scientific Reports

Article Title: Potential Orphan Drug Therapy of Intravesical Liposomal Onabotulinumtoxin-A for Ketamine-Induced Cystitis by Mucosal Protection and Anti-inflammation in a Rat Model

doi: 10.1038/s41598-018-24239-9

Figure Lengend Snippet: Alterations of neuroreceptors protein expression in the mucosa layer or smooth muscle layer of the bladder for all groups (n = 8). Western blot analyses with specific antibodies to the TRPV1 receptor and P2X 3 receptor of the rat mucosal layer as well as M 2 -and M 3 - mAChRs and the purinergic P2X 1 receptor of the rat detrusor layer were performed in 3 groups. ( A ) TRPV1 receptor: The TRPV1 antibody produced a clear single band at 95 kDa. ( B ) Purinergic P2X 3 mature receptor: the predominant P2X 3 form (65 kDa). ( C ) M 2 –mAChR of bladder detrusor layer. The M 2 –mAChR antibody produced a clear single band between 50kD and 75kD. ( D ) M 3 –mAChR of bladder detrusor layer. ( E ) Purinergic P2X 1 receptor. Experiments were repeated two times and representative blots are shown. Data of proteins expression (ratios of signal intensities of investigated receptors relative to GAPDH) were calculated with 8 samples in each group. These data of Mean ± SE were standardized and expressed in percentage in which the value of the control group is treated as 100%. Theses values were shown in the bar graph. An asterisk indicates a significant difference between controls and other groups (One-way ANOVA with Dunnett’s test, p < 0.05). The grouping of blots was cropped from the same gel for each protein. The full-length gels and blots are included in the Supplementary Figure .

Article Snippet: Antibodies raised against SNAP25 (1: 1000 dilution; Cell signal), E-cadherin (1:1000 dilution; Cell Signal), nerve growth factor (1:200 dilution; Cell Signal), IL-1β (1:500 dilution; Abcam) IL-6 (1:1000 dilution; Abcam), TNF-α (1:250 dilution, Santa Cruz), NF-κB (1: 2500 dilution; Cell SiZgnal), COX-2 (1:200 dilution; Abcam), TRPV1 receptor (1:1000 dilution; Alomone), purinergic receptor P2X 1 (1:10000 dilution; Alomone), purinergic receptor P2X 3 (1: 1000 dilution; Alomone), M 2 -mAChR (1:1000 dilution; Alomone), M 3 -mAChR (1:1000 dilution; Alomone), and GAPDH (1: 10,000 dilution; Millipore) were used.

Techniques: Expressing, Western Blot, Produced