aqp4  (Alomone Labs)

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Down-regulation of ASICs current and the calcium transients by disrupting PICK1 protects primary cultured mouse cortical neurons from OGD-Rep insults

doi:

Figure Lengend Snippet: ASIC1 and ASIC2a protein expression during OGD-Rep in three types of neurons. Cells were treated with OGD-Rep and prepared for immunofluorescence and western blot analysis. A, C. Representative pictures and summary data showing expressions of ASIC1 and ASIC2a using immunofluorescence. B, D. Representative bands and summary data of ASIC1 and ASIC2a expression in western blotting. Data are the mean ± SD in each group. *P

Article Snippet: Cerebral cortical neurons were collected and fixed with 4% paraformaldehyde in PBS for 30 min. Then permeabilized with PBS/0.3% Triton X-100 for 30 min, and all cells were blocked with 2% goat serum and 1% bovine serum albumin (BSA) in PBS for 1 h, and then incubated with anti-ASIC1 (Alomone labs, Israel) or anti-ASIC2a (Alomone labs, Israel) overnight at 4°C.

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: Frontiers in Pharmacology

Article Title: A Synthesized Glucocorticoid- Induced Leucine Zipper Peptide Inhibits Retinal Müller Cell Gliosis

doi: 10.3389/fphar.2018.00331

Figure Lengend Snippet: The synthesized GILZ-p inhibited LPS induced Müller cell gliosis. Western blot analysis was performed to determine the protein expression levels of glial fibrillary acidic protein (GFAP) (A,B) , and AQP4 (C,D) in Müller cells treated with 1000 ng/ml LPS in combination with different concentrations of GILZ-p (0.01, 0.1, 1, and 10 μM) for 24 h. β-actin was used as the loading control. The results of quantitative analysis, as determined by densitometric analysis, were expressed as relative to β-actin. Data represent the mean ± SE; the Mann–Whitney U -test was used for comparisons between two groups. n = 3 for each group. ∗ P

Article Snippet: The membranes were blocked in 5% non-fat milk at room temperature for 1 h and incubated with the following antibodies: anti-monocyte chemoattractant protein (MCP)-1 (ab25124; Abcam, Cambridge, United Kingdom), anti-intercellular adhesion molecule (ICAM)-1 (ab171123; Proteintech, Chicago, IL, United States), anti-IL1β (ab9787; Abcam), Anti-tumor necrosis factor (TNF)α (PB0270, Boster Biological Technology, Wuhan, China), rabbit anti-p65 polyclonal antibody (ab16502; Abcam), anti-phospho-NF-κB p65 (Ser536) rabbit monoclonal antibody (3033P; Cell Signaling Technology, Beverly, MA, United States), anti-AQP4 (300-314, Alomone Labs, Jerusalem, Israel), anti-glial fibrillary acidic protein (GFAP) (ab10062, Abcam), or rabbit anti β-actin antibody (ab69512; Abcam) overnight.

Techniques: Synthesized, Western Blot, Expressing, MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

doi: 10.1186/2051-5960-1-70

Figure Lengend Snippet: Alteration of GFAP and AQP4 in the cortex of EAE mice. A , Alterations of astrocytic proteins in the cortex at different stages of rrEAE. B , Summary of the alterations of intensities. C , Summary of increased GFAP + cells. (B, C) One-Way ANOVA followed by Fisher’s test. *, significant difference from Control for each antibody, p

Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-Kv1.4, anti-AQP4 (Alomone Labs, Jerusalem, Israel), and anti-PKCγ (Santa Cruz Biotechnology, Dallas, TX); goat polyclonal anti-GFAP (AbCAM, Cambridge, MA, USA); chicken polyclonal anti-Vimentin (Millipore, Temecula, CA); and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA).

Techniques: Mouse Assay

Journal: Acta Neuropathologica Communications

Article Title: Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

doi: 10.1186/2051-5960-1-70

Figure Lengend Snippet: Altered astrocytes in the hippocampus and cortex in Kv3.1 KO mice. The confocal image stacks of hippocampus (A-C) and cortex (D-F) were costained for GFAP (green) and AQP4 (red) from WT (A,D) , AnkG KO (B,E) and Kv3.1 KO (C,F) mice. The collapsed 2D image is on the top and 3 cross sections are at the bottom. The crossbars are centered on astrocyte endfeet with colocalizing AQP4 and GFAP. Scale bars, 100 μm.

Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-Kv1.4, anti-AQP4 (Alomone Labs, Jerusalem, Israel), and anti-PKCγ (Santa Cruz Biotechnology, Dallas, TX); goat polyclonal anti-GFAP (AbCAM, Cambridge, MA, USA); chicken polyclonal anti-Vimentin (Millipore, Temecula, CA); and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA).

Techniques: Mouse Assay

Journal: Acta Neuropathologica Communications

Article Title: Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

doi: 10.1186/2051-5960-1-70

Figure Lengend Snippet: Differentially altered expression of astrocytic proteins in the cerebellum of EAE mice. A , The confocal image stack of cerebellar molecular layer that was stained for GFAP (green), AQP4 (red) and Vim (blue) from a control mouse. Collapsed 2D image is on the left and 3 cross sections of 3D are on the right. B , The images at the peak stage of an rrEAE mouse. C , The images at the remitting stage of an rrEAE mouse. D , The images at the relapsing stage of an rrEAE mouse. E , The confocal images of cerebellar WM from a Thy1-YFP transgenic mouse. YFP (green), AQP4 (red) and GFAP (blue). In (A-E) , the crossbars are centered on astrocytic endfeet with colocalizing AQP4 and GFAP. F , The confocal images at the peak stage of chEAE. The crossbars show the lesion edge with upregulated AQP4 and GFAP. G , Structural diagram of cerebellar cortex. H , Summary of changes of protein levels during rrEAE in cerebellar molecular layer. One-Way ANOVA followed by Fisher’s test. *, significant difference from Control for each antibody, p

Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-Kv1.4, anti-AQP4 (Alomone Labs, Jerusalem, Israel), and anti-PKCγ (Santa Cruz Biotechnology, Dallas, TX); goat polyclonal anti-GFAP (AbCAM, Cambridge, MA, USA); chicken polyclonal anti-Vimentin (Millipore, Temecula, CA); and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA).

Techniques: Expressing, Mouse Assay, Staining, Transgenic Assay

Journal: Acta Neuropathologica Communications

Article Title: Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

doi: 10.1186/2051-5960-1-70

Figure Lengend Snippet: Activation of astrocytes in the hippocampus of EAE mice. A , Increased GFAP but not AQP4 staining in the hippocampus of EAE mice. B , Increased Vim staining in the hippocampus during EAE progression. C , Enlarged images of individual astrocytes in the hippocampus clearly show the increase of Vim staining. High magnification confocal image stacks were obtained from control (D) and EAE (E) Thy1-YFP transgenic mice. Images contain YFP (green), GFAP (blue) and AQP4 (red). The collapsed 2D image is on the left, and 3 cross sections are on the right. The crossbars reveal astrocytic endfeet with colocalizing AQP4 and GFAP. F , Summary of the levels of astrocytic proteins at different stages during EAE progression. One-Way ANOVA followed by Fisher’s test, *, significant difference from Control for each antibody, p

Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-Kv1.4, anti-AQP4 (Alomone Labs, Jerusalem, Israel), and anti-PKCγ (Santa Cruz Biotechnology, Dallas, TX); goat polyclonal anti-GFAP (AbCAM, Cambridge, MA, USA); chicken polyclonal anti-Vimentin (Millipore, Temecula, CA); and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA).

Techniques: Activation Assay, Mouse Assay, Staining, Transgenic Assay

Journal: Acta Neuropathologica Communications

Article Title: Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

doi: 10.1186/2051-5960-1-70

Figure Lengend Snippet: Upregulation of GFAP and AQP4 in the cerebellum in AnkG and Kv3.1 KO mice. A , High magnification image stacks of cerebellar molecular layer stained with anti-PKCγ (green) and anti-GFAP (red) antibodies. The collapsed 2D image is on the left, and 3 cross sections are on the right. B , Confocal image stacks from the AnkG KO mice. C , Confocal image stacks from the Kv3.1 KO mice. In (A,C) , the crossbars reveal radially oriented GFAP + Bergmann glial processes in (A) WT and (C) Kv3.1 KO mice. In (B) , the crossbars are centered on highly upregulated GFAP + astrocyte processes in the absence of Purkinje neurons in an AnkG KO mouse. D , A single confocal image of the granule cell layer in a WT mouse. E , An image from the AnkG KO mice. F , An image from the Kv3.1 KO mice. G , Normalized fluorescence intensity in the molecular layer. H , Normalized fluorescence intensity in the granule cell layer. (G, H) One-Way ANOVA followed by Fisher’s test. *, significant difference from Wildtype for each antibody, p

Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-Kv1.4, anti-AQP4 (Alomone Labs, Jerusalem, Israel), and anti-PKCγ (Santa Cruz Biotechnology, Dallas, TX); goat polyclonal anti-GFAP (AbCAM, Cambridge, MA, USA); chicken polyclonal anti-Vimentin (Millipore, Temecula, CA); and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA).

Techniques: Mouse Assay, Staining, Fluorescence

Journal: Acta Neuropathologica Communications

Article Title: Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

doi: 10.1186/2051-5960-1-70

Figure Lengend Snippet: Activation of astrocytes in spinal cord white matter. A , Clinical scores (top) and body weight (bottom) of mice with chEAE. B , White matter (WM) and gray matter (GM) in spinal cord longitudinal section was stained with FMG (green) and nuclear dye (blue). Box 1 shows both GM and WM and box 2 shows only WM. Spinal cord sections, control (C) and EAE peak (D) , were co-stained for GFAP (green, top), AQP4 (red), Hoechst (blue), and FMG (green, bottom). Co-staining of Kv1.4 (green, top), Vim (red), Hoechst (blue) and FMG (green, bottom) were also performed on control (E) and EAE (F) spinal cord sections. High magnification confocal image stacks were obtained from control (G) and EAE (H) Thy1-YFP transgenic mice. Images contain YFP (green), GFAP (blue) and AQP4 (red). The collapsed 2D image is on the left, and 3 cross sections are on the right. In (G) , the crossbars are centered on a putative node of Ranvier. In (H) , the crossbars are centered on the AQP4+/GFAP + lesion edge. Scale bars, 500 μm in C-F , 50 μm in G , H .

Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-Kv1.4, anti-AQP4 (Alomone Labs, Jerusalem, Israel), and anti-PKCγ (Santa Cruz Biotechnology, Dallas, TX); goat polyclonal anti-GFAP (AbCAM, Cambridge, MA, USA); chicken polyclonal anti-Vimentin (Millipore, Temecula, CA); and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA).

Techniques: Activation Assay, Mouse Assay, Staining, Transgenic Assay

Journal: PLoS ONE

Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

doi: 10.1371/journal.pone.0204041

Figure Lengend Snippet: Effects of AMD3100 on prazosin and cyclazosin induced ERK1/2 phosphorylation. ERK1/2 phosphorylation was measured and analyzed as in Fig 3 . Cells were pre- incubated with AMD3100 (10 μM, 15 min), followed by stimulation with vehicle (control, ctrl), prazosin (Praz) or cyclazosin (Cycl, 100 μM each) for 20 min. A. Representative images from Western blot experiments with cells transfected with CXCR4. The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in A. Data are mean ± SE. *: p

Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

Techniques: Incubation, Western Blot, Transfection, Migration

Journal: PLoS ONE

Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

doi: 10.1371/journal.pone.0204041

Figure Lengend Snippet: Screening of adrenergic receptor antagonists for CXCR4 and ACKR3 agonist activity in PRESTO-Tango β-arrestin recruitment assays. Data are mean ± SE from n = 4 independent experiments (in triplicates). Cells were stimulated with vehicle, 100 μM of individual AR antagonists or with 200 nM of CXCL12. Luminescence signals are expressed as fold of vehicle-treated cells (control, = 1). *: p

Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

doi: 10.1371/journal.pone.0204041

Figure Lengend Snippet: Prazosin and cyclazosin induce ERK1/2 phosphorylation. HEK293 cells transfected with CXCR4 ( A/B ) or ACKR3 ( C/D ) were stimulated with CXCL12 (100 nM), prazosin (100 μM) or cyclazosin (100 μM) for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2 (ERK1/2). A. Representative images from Western blot experiments with cells transfected with CXCR4. The migration position of molecular mass standards is indicated. B. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 3 independent experiments as in A. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min. C. Representative images from Western blot experiments with cells transfected with ACKR3. The migration position of molecular mass standards is indicated. D. Densitometric quantification of the band intensities, expressed as pERK1/2/ERK1/2, from n = 5 independent experiments as in B. Data are mean ± SE. Symbols (*: CXCL12; #: cyclazosin; $: prazosin) indicate significant differences vs. t = 0 min.

Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

Techniques: Transfection, Western Blot, Migration

Journal: PLoS ONE

Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

doi: 10.1371/journal.pone.0204041

Figure Lengend Snippet: Prazosin and cyclazosin induce internalization of CXCR4 and ACKR3 in hVSMCs. A. hVSMC were treated with 100 μM cyclazosin or prazosin at 37°C for 15 or 30 min, stained with anti-CXCR4/Alexa 647-conjugated anti-rabbit and anti-ACKR3/Alexa 488-conjugated anti-mouse and analyzed for receptor expression via flow cytometry. RFU: relative fluorescence units. The horizontal and vertical lines show the gating thresholds for CXCR4 (Alexa 647) and ACKR3 (Alexa 488). B-E . Quantification of CXCR4 ( B/C ) and ACKR3 ( D/E ) cell surface expression by flow cytometry. Cells were stimulated with 100 μM cyclazosin ( B/D ) or prazosin ( C/E ) as indicated. Data are mean ± SE from 4 independent experiments. *: p

Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

Techniques: Staining, Expressing, Flow Cytometry, Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

doi: 10.1371/journal.pone.0204041

Figure Lengend Snippet: Prazosin and cyclazosin induce chemical shift changes in the NMR spectra of CXCR4 and ACKR3 in membranes. 1 H- 13 C HSQC spectra of reductively methylated CXCR4 ( A-C ) and ACKR3 ( D-F ) membrane preparations were recorded without (black) and with (red) 200 μM prazosin ( A/D ), cyclazosin ( B/E ) or atipamezol ( C/F ). Black arrows indicate significant differences in chemical shifts or broadening (loss) of the signal.

Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

Techniques: Nuclear Magnetic Resonance, Methylation

Journal: PLoS ONE

Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

doi: 10.1371/journal.pone.0204041

Figure Lengend Snippet: CXCL12-inudced ERK1/2 phosphorylation in HEK293 cells is augmented after transfection with CXCR4 and ACKR3. HEK293 cells were transfected with empty vector (left), CXCR4 (center) or ACKR3 (right) and stimulated with 100 nM of CXCL12 for various time periods as indicated. ERK1/2 phosphorylation was monitored by Western blotting of cell lysates with anti-phophoERK1/2 (pERK) and anti-ERK1/2. The migration position of molecular mass standards is indicated.

Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

Techniques: Transfection, Plasmid Preparation, Western Blot, Migration

Journal: PLoS ONE

Article Title: Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

doi: 10.1371/journal.pone.0204041

Figure Lengend Snippet: Dose-dependent effects of prazosin and cyclazosin in PRESTO-Tango β-arrestin recruitment assays for CXCR4 and ACKR3. Data are mean ± SE from n = 3 independent experiments (in triplicates). RLU: relative luminescence units. A-C: CXCR4 PRESTO-Tango assays. A. Grey circles: cells treated with cyclazosin. Black circles: cells treated with prazosin. B. Cells were pre-incubated with vehicle (control, grey circles) or AMD3100 (10 μM; black circles) for 15 min, followed by stimulation with cyclazosin. **: p

Article Snippet: Cells were then blocked with 2% FBS in cold PBS for 30 min, followed by incubation with anti-CXCR4 (ACR-014, Alomone Labs) and anti-ACKR3 (MAB42273, R & D Systems) antibodies for 1h on ice.

Techniques: Incubation