antibody anti trpv4 extracellular  (Alomone Labs)


Bioz Verified Symbol Alomone Labs is a verified supplier
Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    Alomone Labs antibody anti trpv4 extracellular
    PAR2 and <t>TRPV4</t> expression in the hippocampus. Immunohistochemistry discloses the expression of PAR2 and TRPV4 in the hippocampus. A comparable expression pattern is observed: high levels of PAR2 and TRPV4 are detected in CA1 stratum pyramidale (pcl, pyramidal cell layer; oriens, stratum oriens; rad, stratum radiatum; la-mol, stratum lacunosum-moleculare). No pronounced colocalization between PAR2 and GFAP was detected. Scale bars: 100 and 10 μm, n = 9 slices out of three animals.
    Antibody Anti Trpv4 Extracellular, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibody anti trpv4 extracellular/product/Alomone Labs
    Average 93 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    antibody anti trpv4 extracellular - by Bioz Stars, 2022-08
    93/100 stars

    Images

    1) Product Images from "Protease Activated Receptor 2 (PAR2) Induces Long-Term Depression in the Hippocampus through Transient Receptor Potential Vanilloid 4 (TRPV4)"

    Article Title: Protease Activated Receptor 2 (PAR2) Induces Long-Term Depression in the Hippocampus through Transient Receptor Potential Vanilloid 4 (TRPV4)

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00042

    PAR2 and TRPV4 expression in the hippocampus. Immunohistochemistry discloses the expression of PAR2 and TRPV4 in the hippocampus. A comparable expression pattern is observed: high levels of PAR2 and TRPV4 are detected in CA1 stratum pyramidale (pcl, pyramidal cell layer; oriens, stratum oriens; rad, stratum radiatum; la-mol, stratum lacunosum-moleculare). No pronounced colocalization between PAR2 and GFAP was detected. Scale bars: 100 and 10 μm, n = 9 slices out of three animals.
    Figure Legend Snippet: PAR2 and TRPV4 expression in the hippocampus. Immunohistochemistry discloses the expression of PAR2 and TRPV4 in the hippocampus. A comparable expression pattern is observed: high levels of PAR2 and TRPV4 are detected in CA1 stratum pyramidale (pcl, pyramidal cell layer; oriens, stratum oriens; rad, stratum radiatum; la-mol, stratum lacunosum-moleculare). No pronounced colocalization between PAR2 and GFAP was detected. Scale bars: 100 and 10 μm, n = 9 slices out of three animals.

    Techniques Used: Expressing, Immunohistochemistry

    PAR2 induces LTD through the activation of TRPV4. (A) Application of TRPV4-agonist (2 μM RN1747) causes LTD. (B) Removal of the TRPV4-agonist (2 μM RN1747) following induction of LTD does not affect the stability of synaptic depression. (C) In presence of the TRPV4-antagonist (10 μM RN1734) the TRPV4-agonist is not able to induce synaptic depression. (D) In a two pathways experimental setting, low frequency stimulation (LFS, 1 Hz, 900 pulses) and TRPV4-agonist application induce similar levels of LTD. (E) LFS-induced LTD is not blocked by the TRPV4-antagonist. (F) Application of PAR2-agonist (10 μM AC55541) in presence of a TRPV4-antagonist (10 μM RN1734) blocks PAR2-induced LTD. (G) Application of TRPV4-agonist (2 μM RN1747) in presence of PAR2-antagonist (50 μM FSLLRY-NH 2 ) does not affect TRPV4-induced LTD. (H) Once PAR2-agonist mediated LTD is established, the TRPV4-agonist (2 μM RN1747) does not further de-potentiate a second pathway at adjusted response level (upward arrow). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section, n = 12 slices for each experiments, refer to text for statistics.
    Figure Legend Snippet: PAR2 induces LTD through the activation of TRPV4. (A) Application of TRPV4-agonist (2 μM RN1747) causes LTD. (B) Removal of the TRPV4-agonist (2 μM RN1747) following induction of LTD does not affect the stability of synaptic depression. (C) In presence of the TRPV4-antagonist (10 μM RN1734) the TRPV4-agonist is not able to induce synaptic depression. (D) In a two pathways experimental setting, low frequency stimulation (LFS, 1 Hz, 900 pulses) and TRPV4-agonist application induce similar levels of LTD. (E) LFS-induced LTD is not blocked by the TRPV4-antagonist. (F) Application of PAR2-agonist (10 μM AC55541) in presence of a TRPV4-antagonist (10 μM RN1734) blocks PAR2-induced LTD. (G) Application of TRPV4-agonist (2 μM RN1747) in presence of PAR2-antagonist (50 μM FSLLRY-NH 2 ) does not affect TRPV4-induced LTD. (H) Once PAR2-agonist mediated LTD is established, the TRPV4-agonist (2 μM RN1747) does not further de-potentiate a second pathway at adjusted response level (upward arrow). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section, n = 12 slices for each experiments, refer to text for statistics.

    Techniques Used: Activation Assay

    TRPV4-mediated LTD depends on NMDAR-activity. (A) Similar to PAR2-induced LTD (c.f., Figures 1G,H ), the NMDAR-antagonist (50 μM APV) blocks TRPV4 (2 μM RN1747)-induced LTD, while (B) application of a TRPV4-agonist (2 μM RN1747) induces LTD in presence of the mGluR-antagonist (200 μM MCGP). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section.
    Figure Legend Snippet: TRPV4-mediated LTD depends on NMDAR-activity. (A) Similar to PAR2-induced LTD (c.f., Figures 1G,H ), the NMDAR-antagonist (50 μM APV) blocks TRPV4 (2 μM RN1747)-induced LTD, while (B) application of a TRPV4-agonist (2 μM RN1747) induces LTD in presence of the mGluR-antagonist (200 μM MCGP). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section.

    Techniques Used: Activity Assay

    2) Product Images from "Expression and Functional Role of TRPV4 in Bone Marrow-Derived CD11c+ Cells"

    Article Title: Expression and Functional Role of TRPV4 in Bone Marrow-Derived CD11c+ Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20143378

    TRPV4 was downregulated in mature CD11c + BMDCs. ( A ) Concentration dependence of immature (black bars) and mature (dark cyan bars) CD11c + BMDC responding fraction. ***, p
    Figure Legend Snippet: TRPV4 was downregulated in mature CD11c + BMDCs. ( A ) Concentration dependence of immature (black bars) and mature (dark cyan bars) CD11c + BMDC responding fraction. ***, p

    Techniques Used: Concentration Assay

    TRPV4-deficient BMDCs exhibited impaired FcR-dependent phagocytosis. ( A ) Representative confocal images of wild-type and Trpv4 KO BMDCs after treatment with uncoated or IgG-coated fluorescent microspheres. Scale bar, 20 µm. ( B ) Percentage of cells with internalized beads. Data were collected from 10 randomly selected fields per condition from three independent experiments. ***, p
    Figure Legend Snippet: TRPV4-deficient BMDCs exhibited impaired FcR-dependent phagocytosis. ( A ) Representative confocal images of wild-type and Trpv4 KO BMDCs after treatment with uncoated or IgG-coated fluorescent microspheres. Scale bar, 20 µm. ( B ) Percentage of cells with internalized beads. Data were collected from 10 randomly selected fields per condition from three independent experiments. ***, p

    Techniques Used:

    TRPV4 was dispensable in the differentiation of CD11c + BMDCs. ( A ) Color-coded two-dimensional t-distributed stochastic neighbor embedding (tSNE) representations of the total bone marrow-derived cell population (20,000 cells) defined by the surface markers CD11b, CD11c, and F4/80. ( B ) Histograms showing surface expression of the indicated markers in bone marrow-derived cells from wild-type (WT, black traces) and Trpv4 knockout (KO, red traces) mice. The shaded histograms represent specificity (fluorescence minus one) controls. The bar graph shows the percentage of different cell populations present in total bone marrow-derived cell cultures defined by the surface expression of CD11b, CD11c, and F4/80. The data are represented as mean ± SEM of nine independent experiments.
    Figure Legend Snippet: TRPV4 was dispensable in the differentiation of CD11c + BMDCs. ( A ) Color-coded two-dimensional t-distributed stochastic neighbor embedding (tSNE) representations of the total bone marrow-derived cell population (20,000 cells) defined by the surface markers CD11b, CD11c, and F4/80. ( B ) Histograms showing surface expression of the indicated markers in bone marrow-derived cells from wild-type (WT, black traces) and Trpv4 knockout (KO, red traces) mice. The shaded histograms represent specificity (fluorescence minus one) controls. The bar graph shows the percentage of different cell populations present in total bone marrow-derived cell cultures defined by the surface expression of CD11b, CD11c, and F4/80. The data are represented as mean ± SEM of nine independent experiments.

    Techniques Used: Derivative Assay, Expressing, Knock-Out, Mouse Assay, Fluorescence

    TRPV4 was functionally expressed in CD11c + bone marrow-derived cells (BMDCs). ( A ) Expression profile of selected Trp genes in the total granulocyte-macrophage colony-stimulating (GM-CSF)-differentiated bone marrow-derived cell population (black bars) and in CD11c + -purified BMDCs (light gray). Values are relative to GAPDH expression. ( B ) Confocal image of CD11c + BMDCs stained with an anti-TRPV4 antibody (red). The blue color corresponds to nuclear staining with DAPI. ( C–E ) Representative traces of intracellular Ca 2+ concentration in CD11c + BMDCs showing the effects of 300 nM of GSK1016790A (GSK). ATP (100 μM) was used as a positive control for intracellular Ca 2+ increase. The TRPV4 antagonist HC067047 was used at 10 μM. ( F ) Percentage of CD11c + BMDCs responding to the indicated stimulus. GSK, GSK1016790A (300 nM); HC, HC067047 (1 µM); Ca 2+ -free, Krebs with nominal [Ca 2+ ] supplemented with 2.5 mM EDTA; Caps, Capsaicin (1 nM); THC, trans-Δ 9 -tetrahydrocannabinol (10 µM). The responding fraction is indicated within each bar. ***, p
    Figure Legend Snippet: TRPV4 was functionally expressed in CD11c + bone marrow-derived cells (BMDCs). ( A ) Expression profile of selected Trp genes in the total granulocyte-macrophage colony-stimulating (GM-CSF)-differentiated bone marrow-derived cell population (black bars) and in CD11c + -purified BMDCs (light gray). Values are relative to GAPDH expression. ( B ) Confocal image of CD11c + BMDCs stained with an anti-TRPV4 antibody (red). The blue color corresponds to nuclear staining with DAPI. ( C–E ) Representative traces of intracellular Ca 2+ concentration in CD11c + BMDCs showing the effects of 300 nM of GSK1016790A (GSK). ATP (100 μM) was used as a positive control for intracellular Ca 2+ increase. The TRPV4 antagonist HC067047 was used at 10 μM. ( F ) Percentage of CD11c + BMDCs responding to the indicated stimulus. GSK, GSK1016790A (300 nM); HC, HC067047 (1 µM); Ca 2+ -free, Krebs with nominal [Ca 2+ ] supplemented with 2.5 mM EDTA; Caps, Capsaicin (1 nM); THC, trans-Δ 9 -tetrahydrocannabinol (10 µM). The responding fraction is indicated within each bar. ***, p

    Techniques Used: Derivative Assay, Expressing, Purification, Staining, Concentration Assay, Positive Control

    LPS-induced cytokine production occurred independently of TRPV4. ( A ) Representative confocal immunofluorescence microscopy images of fixed BMDCs untreated or treated with LPS (100 ng/mL). Cell stainings correspond to NF-κB p65 (red) and DAPI (nuclear, blue). Scale bar, 10 µm. The average linear intensity along the gray rectangle is represented next to the corresponding image. ( B ) Percentage of the total nuclear area stained by NF-κB p65 staining. The horizontal bar represents the mean. ***, p
    Figure Legend Snippet: LPS-induced cytokine production occurred independently of TRPV4. ( A ) Representative confocal immunofluorescence microscopy images of fixed BMDCs untreated or treated with LPS (100 ng/mL). Cell stainings correspond to NF-κB p65 (red) and DAPI (nuclear, blue). Scale bar, 10 µm. The average linear intensity along the gray rectangle is represented next to the corresponding image. ( B ) Percentage of the total nuclear area stained by NF-κB p65 staining. The horizontal bar represents the mean. ***, p

    Techniques Used: Immunofluorescence, Microscopy, Staining

    3) Product Images from "Extracellular Matrix Stiffness Alters TRPV4 Regulation in Chondrocytes"

    Article Title: Extracellular Matrix Stiffness Alters TRPV4 Regulation in Chondrocytes

    Journal: bioRxiv

    doi: 10.1101/2021.09.14.460172

    A) Immunofluorescent images of ATDC5 cells on normal gels (upper panel) treated with 30nM GSK101 for 28 hr and severe OA gels with control treatment (lower panel) stained for the nucleus (Hoechst, blue), f-actin (purple), and TRPV4 (white). The left panel shows the composite images of all stains (scale bar =10 μm). The middle panel shows nuclear and f-actin staining, and the right panel shows TRPV4 and nuclear staining. B) TRPV4 fluorescence of ATDC5 cells on normal, OA, and severe OA gels during control (dark blue) and 28 hr of 30 nM GSK101 (light blue) treatments. f-actin was used to draw ROI around the cells to measure TRPV4 fluorescence from max projections of z-stack images. Error Bars are ± SEM, * indicates p-value
    Figure Legend Snippet: A) Immunofluorescent images of ATDC5 cells on normal gels (upper panel) treated with 30nM GSK101 for 28 hr and severe OA gels with control treatment (lower panel) stained for the nucleus (Hoechst, blue), f-actin (purple), and TRPV4 (white). The left panel shows the composite images of all stains (scale bar =10 μm). The middle panel shows nuclear and f-actin staining, and the right panel shows TRPV4 and nuclear staining. B) TRPV4 fluorescence of ATDC5 cells on normal, OA, and severe OA gels during control (dark blue) and 28 hr of 30 nM GSK101 (light blue) treatments. f-actin was used to draw ROI around the cells to measure TRPV4 fluorescence from max projections of z-stack images. Error Bars are ± SEM, * indicates p-value

    Techniques Used: Staining, Fluorescence

    A) mRNA expression of anabolic factors (Sox-9, aggrecan, col2 and TRPV4) in ATDC5 cells on normal, OA and severe OA gels during control (dark blue) or 28 hr of 30 nM GSK101 (light blue) treatment. B) mRNA expression of catabolic chondrocyte factors, collagen I and MMP-13 in ATDC5 cells on normal, OA and severe OA gels during control (dark blue) or 28 hr of 30 nM GSK101 (light blue) treatment. Fold change was normalized to rplp0 expression for each sample and then normalized to the normal control condition. C) Western blotting shows TRPV4 protein levels for ATDC5 cells on normal, OA, and severe OA gels during control treatment. The bar graph represents the normalized TRPV4 protein level to GAPDH for each sample. Error bars are ± SEM, * indicates significance with a p-value
    Figure Legend Snippet: A) mRNA expression of anabolic factors (Sox-9, aggrecan, col2 and TRPV4) in ATDC5 cells on normal, OA and severe OA gels during control (dark blue) or 28 hr of 30 nM GSK101 (light blue) treatment. B) mRNA expression of catabolic chondrocyte factors, collagen I and MMP-13 in ATDC5 cells on normal, OA and severe OA gels during control (dark blue) or 28 hr of 30 nM GSK101 (light blue) treatment. Fold change was normalized to rplp0 expression for each sample and then normalized to the normal control condition. C) Western blotting shows TRPV4 protein levels for ATDC5 cells on normal, OA, and severe OA gels during control treatment. The bar graph represents the normalized TRPV4 protein level to GAPDH for each sample. Error bars are ± SEM, * indicates significance with a p-value

    Techniques Used: Expressing, Western Blot

    4) Product Images from "TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching"

    Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1901033

    p38 activation and macrophage phagocytosis in response to LPS is dependent on matrix stiffness. Phosphorylated and total A. p38 and B. JNK on various matrix stiffnesses in the physiologic range (1kPa, 8kPa, and 25kPa) from WT vs TRPV4 KO BMDMs quantified for LPS 15 minutes ( *p = 0.031). C. Macrophage phagocytosis of E. coli particles ± p38 inhibition (SB, BIRB) on various matrix stiffnesses ( *p
    Figure Legend Snippet: p38 activation and macrophage phagocytosis in response to LPS is dependent on matrix stiffness. Phosphorylated and total A. p38 and B. JNK on various matrix stiffnesses in the physiologic range (1kPa, 8kPa, and 25kPa) from WT vs TRPV4 KO BMDMs quantified for LPS 15 minutes ( *p = 0.031). C. Macrophage phagocytosis of E. coli particles ± p38 inhibition (SB, BIRB) on various matrix stiffnesses ( *p

    Techniques Used: Activation Assay, Inhibition

    TRPV4-mediated p38 MAPK and JNK molecular switch is regulated by dual-specificity phosphatase 1 (DUSP1). WT and TRPV4 KO BMDMs were incubated ± LPS as above for indicated time, and cells were lysed and analyzed by A. immunoblot for DUSP1 and B. band density quantified as DUSP1/GAPDH from immunoblot (n = 4) ( *p
    Figure Legend Snippet: TRPV4-mediated p38 MAPK and JNK molecular switch is regulated by dual-specificity phosphatase 1 (DUSP1). WT and TRPV4 KO BMDMs were incubated ± LPS as above for indicated time, and cells were lysed and analyzed by A. immunoblot for DUSP1 and B. band density quantified as DUSP1/GAPDH from immunoblot (n = 4) ( *p

    Techniques Used: Incubation

    TRPV4 function protects against lung injury in a murine Pseudomonas aeruginosa pneumonia model. Sterile beads or P. aeruginosa (PA, PAM57–15) were instilled intratracheally in TRPV4 KO and age-matched female congenic WT mice with BAL and tissue harvest performed at Day 3 (injury phase). TRPV4 deleted mice (TRPV4 KO) have greater A. inflammatory cell infiltration and B. BAL total protein compared to WT (* p
    Figure Legend Snippet: TRPV4 function protects against lung injury in a murine Pseudomonas aeruginosa pneumonia model. Sterile beads or P. aeruginosa (PA, PAM57–15) were instilled intratracheally in TRPV4 KO and age-matched female congenic WT mice with BAL and tissue harvest performed at Day 3 (injury phase). TRPV4 deleted mice (TRPV4 KO) have greater A. inflammatory cell infiltration and B. BAL total protein compared to WT (* p

    Techniques Used: Mouse Assay

    p38 MAPK and JNK are differentially regulated by TRPV4 after LPS or Pseudomonas aeruginosa . BMDMs were incubated ± LPS as above for indicated time cultured on tissue culture-treated plastic, and cells were lysed and analyzed by immunoblot for A. phosphorylated and total p38, ERK, and JNK compared to WT BMDMs (whole cell lysate). Band density quantified from immunoblot (n = 3–6) for B. p-p38/total p38 ( *p
    Figure Legend Snippet: p38 MAPK and JNK are differentially regulated by TRPV4 after LPS or Pseudomonas aeruginosa . BMDMs were incubated ± LPS as above for indicated time cultured on tissue culture-treated plastic, and cells were lysed and analyzed by immunoblot for A. phosphorylated and total p38, ERK, and JNK compared to WT BMDMs (whole cell lysate). Band density quantified from immunoblot (n = 3–6) for B. p-p38/total p38 ( *p

    Techniques Used: Incubation, Cell Culture

    TRPV4 mediates phagocytosis after LPS in healthy human through p38 MAPK. Monocyte derived and alveolar macrophages from healthy (n = 6) control subjects were incubated ± LPS ± TRPV4 inhibitor, HC, and phagocytosis of E. coli particles was measured in A. monocyte-derived and B. alveolar macrophages in healthy controls. HC alone had no effect. Representative immunoblot for phosphorylated and total p38 in C. healthy monocyte derived macrophages ± LPS 15 minutes and D. band density quantified ( *p
    Figure Legend Snippet: TRPV4 mediates phagocytosis after LPS in healthy human through p38 MAPK. Monocyte derived and alveolar macrophages from healthy (n = 6) control subjects were incubated ± LPS ± TRPV4 inhibitor, HC, and phagocytosis of E. coli particles was measured in A. monocyte-derived and B. alveolar macrophages in healthy controls. HC alone had no effect. Representative immunoblot for phosphorylated and total p38 in C. healthy monocyte derived macrophages ± LPS 15 minutes and D. band density quantified ( *p

    Techniques Used: Derivative Assay, Incubation

    TRPV4 mediates clearance of P. aeruginosa by macrophages. WT and TRPV4 KO mice were intratracheally administered ±GFP P. aeruginosa for 3 days. Representative confocal images of whole lung lavage cytospins of macrophages (open arrowhead) and neutrophils (filled arrowhead) in WT mice given IT sterile beads or GFP- P. aeruginosa after immunofluorescence with A. TRPV4 extracellular antibody (green, TRPV4) and C. anti-GFP (green, GFP P. aeruginosa ; anti-CD45, red; dapi, blue). B, D. Quantification of A, C . * , # p
    Figure Legend Snippet: TRPV4 mediates clearance of P. aeruginosa by macrophages. WT and TRPV4 KO mice were intratracheally administered ±GFP P. aeruginosa for 3 days. Representative confocal images of whole lung lavage cytospins of macrophages (open arrowhead) and neutrophils (filled arrowhead) in WT mice given IT sterile beads or GFP- P. aeruginosa after immunofluorescence with A. TRPV4 extracellular antibody (green, TRPV4) and C. anti-GFP (green, GFP P. aeruginosa ; anti-CD45, red; dapi, blue). B, D. Quantification of A, C . * , # p

    Techniques Used: Mouse Assay, Immunofluorescence

    TRPV4 modulates pro-inflammatory cytokine production through JNK. BMDMs were incubated ± LPS (100ng/mL, 24h) ± JNK inhibitor, SP600125 (20μM, 25h) ± p38 inhibitor, SB203580 (10μM, 25h), cultured on cell culture-treated plastic, and cytokines measured via ELISA. IL-6, CXCL2, and CXCL1 secretion ± LPS in A. WT and TRPV4 KO BMDMs and B. WT BMDMs ± SP600125 ± SB203580 ( *, # p
    Figure Legend Snippet: TRPV4 modulates pro-inflammatory cytokine production through JNK. BMDMs were incubated ± LPS (100ng/mL, 24h) ± JNK inhibitor, SP600125 (20μM, 25h) ± p38 inhibitor, SB203580 (10μM, 25h), cultured on cell culture-treated plastic, and cytokines measured via ELISA. IL-6, CXCL2, and CXCL1 secretion ± LPS in A. WT and TRPV4 KO BMDMs and B. WT BMDMs ± SP600125 ± SB203580 ( *, # p

    Techniques Used: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

    5) Product Images from "TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching"

    Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1901033

    p38 activation and macrophage phagocytosis in response to LPS is dependent on matrix stiffness. Phosphorylated and total A. p38 and B. JNK on various matrix stiffnesses in the physiologic range (1kPa, 8kPa, and 25kPa) from WT vs TRPV4 KO BMDMs quantified for LPS 15 minutes ( *p = 0.031). C. Macrophage phagocytosis of E. coli particles ± p38 inhibition (SB, BIRB) on various matrix stiffnesses ( *p
    Figure Legend Snippet: p38 activation and macrophage phagocytosis in response to LPS is dependent on matrix stiffness. Phosphorylated and total A. p38 and B. JNK on various matrix stiffnesses in the physiologic range (1kPa, 8kPa, and 25kPa) from WT vs TRPV4 KO BMDMs quantified for LPS 15 minutes ( *p = 0.031). C. Macrophage phagocytosis of E. coli particles ± p38 inhibition (SB, BIRB) on various matrix stiffnesses ( *p

    Techniques Used: Activation Assay, Inhibition

    TRPV4-mediated p38 MAPK and JNK molecular switch is regulated by dual-specificity phosphatase 1 (DUSP1). WT and TRPV4 KO BMDMs were incubated ± LPS as above for indicated time, and cells were lysed and analyzed by A. immunoblot for DUSP1 and B. band density quantified as DUSP1/GAPDH from immunoblot (n = 4) ( *p
    Figure Legend Snippet: TRPV4-mediated p38 MAPK and JNK molecular switch is regulated by dual-specificity phosphatase 1 (DUSP1). WT and TRPV4 KO BMDMs were incubated ± LPS as above for indicated time, and cells were lysed and analyzed by A. immunoblot for DUSP1 and B. band density quantified as DUSP1/GAPDH from immunoblot (n = 4) ( *p

    Techniques Used: Incubation

    TRPV4 function protects against lung injury in a murine Pseudomonas aeruginosa pneumonia model. Sterile beads or P. aeruginosa (PA, PAM57–15) were instilled intratracheally in TRPV4 KO and age-matched female congenic WT mice with BAL and tissue harvest performed at Day 3 (injury phase). TRPV4 deleted mice (TRPV4 KO) have greater A. inflammatory cell infiltration and B. BAL total protein compared to WT (* p
    Figure Legend Snippet: TRPV4 function protects against lung injury in a murine Pseudomonas aeruginosa pneumonia model. Sterile beads or P. aeruginosa (PA, PAM57–15) were instilled intratracheally in TRPV4 KO and age-matched female congenic WT mice with BAL and tissue harvest performed at Day 3 (injury phase). TRPV4 deleted mice (TRPV4 KO) have greater A. inflammatory cell infiltration and B. BAL total protein compared to WT (* p

    Techniques Used: Mouse Assay

    p38 MAPK and JNK are differentially regulated by TRPV4 after LPS or Pseudomonas aeruginosa . BMDMs were incubated ± LPS as above for indicated time cultured on tissue culture-treated plastic, and cells were lysed and analyzed by immunoblot for A. phosphorylated and total p38, ERK, and JNK compared to WT BMDMs (whole cell lysate). Band density quantified from immunoblot (n = 3–6) for B. p-p38/total p38 ( *p
    Figure Legend Snippet: p38 MAPK and JNK are differentially regulated by TRPV4 after LPS or Pseudomonas aeruginosa . BMDMs were incubated ± LPS as above for indicated time cultured on tissue culture-treated plastic, and cells were lysed and analyzed by immunoblot for A. phosphorylated and total p38, ERK, and JNK compared to WT BMDMs (whole cell lysate). Band density quantified from immunoblot (n = 3–6) for B. p-p38/total p38 ( *p

    Techniques Used: Incubation, Cell Culture

    TRPV4 mediates phagocytosis after LPS in healthy human through p38 MAPK. Monocyte derived and alveolar macrophages from healthy (n = 6) control subjects were incubated ± LPS ± TRPV4 inhibitor, HC, and phagocytosis of E. coli particles was measured in A. monocyte-derived and B. alveolar macrophages in healthy controls. HC alone had no effect. Representative immunoblot for phosphorylated and total p38 in C. healthy monocyte derived macrophages ± LPS 15 minutes and D. band density quantified ( *p
    Figure Legend Snippet: TRPV4 mediates phagocytosis after LPS in healthy human through p38 MAPK. Monocyte derived and alveolar macrophages from healthy (n = 6) control subjects were incubated ± LPS ± TRPV4 inhibitor, HC, and phagocytosis of E. coli particles was measured in A. monocyte-derived and B. alveolar macrophages in healthy controls. HC alone had no effect. Representative immunoblot for phosphorylated and total p38 in C. healthy monocyte derived macrophages ± LPS 15 minutes and D. band density quantified ( *p

    Techniques Used: Derivative Assay, Incubation

    TRPV4 mediates clearance of P. aeruginosa by macrophages. WT and TRPV4 KO mice were intratracheally administered ±GFP P. aeruginosa for 3 days. Representative confocal images of whole lung lavage cytospins of macrophages (open arrowhead) and neutrophils (filled arrowhead) in WT mice given IT sterile beads or GFP- P. aeruginosa after immunofluorescence with A. TRPV4 extracellular antibody (green, TRPV4) and C. anti-GFP (green, GFP P. aeruginosa ; anti-CD45, red; dapi, blue). B, D. Quantification of A, C . * , # p
    Figure Legend Snippet: TRPV4 mediates clearance of P. aeruginosa by macrophages. WT and TRPV4 KO mice were intratracheally administered ±GFP P. aeruginosa for 3 days. Representative confocal images of whole lung lavage cytospins of macrophages (open arrowhead) and neutrophils (filled arrowhead) in WT mice given IT sterile beads or GFP- P. aeruginosa after immunofluorescence with A. TRPV4 extracellular antibody (green, TRPV4) and C. anti-GFP (green, GFP P. aeruginosa ; anti-CD45, red; dapi, blue). B, D. Quantification of A, C . * , # p

    Techniques Used: Mouse Assay, Immunofluorescence

    TRPV4 modulates pro-inflammatory cytokine production through JNK. BMDMs were incubated ± LPS (100ng/mL, 24h) ± JNK inhibitor, SP600125 (20μM, 25h) ± p38 inhibitor, SB203580 (10μM, 25h), cultured on cell culture-treated plastic, and cytokines measured via ELISA. IL-6, CXCL2, and CXCL1 secretion ± LPS in A. WT and TRPV4 KO BMDMs and B. WT BMDMs ± SP600125 ± SB203580 ( *, # p
    Figure Legend Snippet: TRPV4 modulates pro-inflammatory cytokine production through JNK. BMDMs were incubated ± LPS (100ng/mL, 24h) ± JNK inhibitor, SP600125 (20μM, 25h) ± p38 inhibitor, SB203580 (10μM, 25h), cultured on cell culture-treated plastic, and cytokines measured via ELISA. IL-6, CXCL2, and CXCL1 secretion ± LPS in A. WT and TRPV4 KO BMDMs and B. WT BMDMs ± SP600125 ± SB203580 ( *, # p

    Techniques Used: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Omega-3 Fatty Acids Modulate TRPV4 Function Through Plasma Membrane Remodeling"

    Article Title: Omega-3 Fatty Acids Modulate TRPV4 Function Through Plasma Membrane Remodeling

    Journal: Cell reports

    doi: 10.1016/j.celrep.2017.09.029

    GSK101 elicits withdrawal responses in rat TRPV4-expressing worms. (A) Schematic representation of the withdrawal responses after addition of GSK101 drop in front of freely moving worms. (B) GSK101 dose-response profile for wild-type (WT [N2]) and TRPV4-expressing worms. (C) Inhibition of GSK101-mediated withdrawal responses in TRPV4 worms by HC067047 (2 µM). (D) Withdrawal responses elicited by 4α-Phorbol in WT and TRPV4 worms. (E) Withdrawal responses elicited by 1 M glycerol and nose touch in WT, osm9 , and TRPV4; osm9 strains. Bars are mean ± SEM, the number of worms tested during 3 assays sessions is indicated inside the bars. The asterisks indicate values significantly different from control. *** p
    Figure Legend Snippet: GSK101 elicits withdrawal responses in rat TRPV4-expressing worms. (A) Schematic representation of the withdrawal responses after addition of GSK101 drop in front of freely moving worms. (B) GSK101 dose-response profile for wild-type (WT [N2]) and TRPV4-expressing worms. (C) Inhibition of GSK101-mediated withdrawal responses in TRPV4 worms by HC067047 (2 µM). (D) Withdrawal responses elicited by 4α-Phorbol in WT and TRPV4 worms. (E) Withdrawal responses elicited by 1 M glycerol and nose touch in WT, osm9 , and TRPV4; osm9 strains. Bars are mean ± SEM, the number of worms tested during 3 assays sessions is indicated inside the bars. The asterisks indicate values significantly different from control. *** p

    Techniques Used: Expressing, Inhibition

    EPA supplementation enhances TRPV4 activity in HMVEC. (A) Representative whole-cell patch-clamp recordings (+80 mV) of control and EPA (100 µM)-treated HMVEC challenged with GSK101 (100 nM) and HC067047 (10 µM). (B) Box plots show the mean, median, standard deviation, and standard error of the mean from TRPV4 currents (I GSK101 - I HC / pF) obtained by whole-cell patch-clamp recordings (+80 mV) of control, EPA-, and ω -6 AA-treated HMVEC. (C) Left, representative current-voltage relationships determined by whole-cell patch-clamp recording of control and EPA (100 µM)-treated HMVEC challenged with GSK101 (100 nM) in the presence of 5 mM Ca 2+ . Right, bar graph of peak currents (at +80 mV) relative to the currents after 5 min of exposure to GSK101 (I max /I 5 min ). Bars are mean ± SEM. (D) HMVEC were challenged with isosmotic (IB, 320 mOsm), hyposmotic (HB, 240 mOsm), and GSK101 (100 nM) solutions and analyzed for their responses using Ca 2+ imaging (Fluo-4 AM); color bar indicates relative change in fluorescence intensity. Control and EPA (100–300 µM)-treated HMVEC were analyzed from 5 independent preparations. (E) Representative traces corresponding to normalized (ΔF/F) intensity changes of individual cells shown in (D). (F) Area under the curve of control and EPA-treated HMVEC challenged with hyposmotic buffer. Bars are mean ± SEM. The number of endothelial cells measured is indicated below the boxes and inside the bars. The asterisks indicate values significantly different from control. *** p
    Figure Legend Snippet: EPA supplementation enhances TRPV4 activity in HMVEC. (A) Representative whole-cell patch-clamp recordings (+80 mV) of control and EPA (100 µM)-treated HMVEC challenged with GSK101 (100 nM) and HC067047 (10 µM). (B) Box plots show the mean, median, standard deviation, and standard error of the mean from TRPV4 currents (I GSK101 - I HC / pF) obtained by whole-cell patch-clamp recordings (+80 mV) of control, EPA-, and ω -6 AA-treated HMVEC. (C) Left, representative current-voltage relationships determined by whole-cell patch-clamp recording of control and EPA (100 µM)-treated HMVEC challenged with GSK101 (100 nM) in the presence of 5 mM Ca 2+ . Right, bar graph of peak currents (at +80 mV) relative to the currents after 5 min of exposure to GSK101 (I max /I 5 min ). Bars are mean ± SEM. (D) HMVEC were challenged with isosmotic (IB, 320 mOsm), hyposmotic (HB, 240 mOsm), and GSK101 (100 nM) solutions and analyzed for their responses using Ca 2+ imaging (Fluo-4 AM); color bar indicates relative change in fluorescence intensity. Control and EPA (100–300 µM)-treated HMVEC were analyzed from 5 independent preparations. (E) Representative traces corresponding to normalized (ΔF/F) intensity changes of individual cells shown in (D). (F) Area under the curve of control and EPA-treated HMVEC challenged with hyposmotic buffer. Bars are mean ± SEM. The number of endothelial cells measured is indicated below the boxes and inside the bars. The asterisks indicate values significantly different from control. *** p

    Techniques Used: Activity Assay, Patch Clamp, Standard Deviation, Imaging, Fluorescence

    EPA supplementation increases ω -3 fatty acid eicosanoid derivatives in HMVEC and does not affect TRPV4 expression and trafficking. (A) EPA and ω -6 AA content in control and EPA (100 µM)-treated HMVEC, as determined by LC-MS. (B) ω .
    Figure Legend Snippet: EPA supplementation increases ω -3 fatty acid eicosanoid derivatives in HMVEC and does not affect TRPV4 expression and trafficking. (A) EPA and ω -6 AA content in control and EPA (100 µM)-treated HMVEC, as determined by LC-MS. (B) ω .

    Techniques Used: Expressing, Liquid Chromatography with Mass Spectroscopy

    EPA and 17,18-EEQ fully restore TRPV4 function in C. elegans . (A) GSK101-mediated withdrawal responses of TRPV4 and TRPV4; fat 3 mutants after worms were fed with specified PUFAs and eicosanoid derivatives (200 µM). Dotted red and blue lines represent the 20% and 45% thresholds for positive and intermediate responses, respectively. (B) Schematic representation of the effect of ETYA (non-metabolizable analogue of ω -6 AA) in worms. (C) Top inset, ω -6 PUFAs present in fat-1 and fat-1fat-4 . Bottom, withdrawal responses elicited by GSK101 in TRPV4; fat-3 , TRPV4; fat-3 supplemented with ETYA, TRPV4; fat-1, TRPV4; fat-1fat-4, and TRPV4; fat-1fat-4 supplemented with ω -6 AA. (D) Withdrawal responses elicited by 1 M glycerol and nose touch in TRPV4; osm9fat-3 mutants after being fed with EPA (200 µM). Bars are mean ± SEM, the number of worms tested during 3 assays sessions is indicated inside the bars. The asterisks indicate values significantly different from control. *** p
    Figure Legend Snippet: EPA and 17,18-EEQ fully restore TRPV4 function in C. elegans . (A) GSK101-mediated withdrawal responses of TRPV4 and TRPV4; fat 3 mutants after worms were fed with specified PUFAs and eicosanoid derivatives (200 µM). Dotted red and blue lines represent the 20% and 45% thresholds for positive and intermediate responses, respectively. (B) Schematic representation of the effect of ETYA (non-metabolizable analogue of ω -6 AA) in worms. (C) Top inset, ω -6 PUFAs present in fat-1 and fat-1fat-4 . Bottom, withdrawal responses elicited by GSK101 in TRPV4; fat-3 , TRPV4; fat-3 supplemented with ETYA, TRPV4; fat-1, TRPV4; fat-1fat-4, and TRPV4; fat-1fat-4 supplemented with ω -6 AA. (D) Withdrawal responses elicited by 1 M glycerol and nose touch in TRPV4; osm9fat-3 mutants after being fed with EPA (200 µM). Bars are mean ± SEM, the number of worms tested during 3 assays sessions is indicated inside the bars. The asterisks indicate values significantly different from control. *** p

    Techniques Used:

    PUFAs are required for TRPV4 function in C. elegans ). LA, linolenic acid; γLA, γ-linolenic acid; DγLA, dihomo-linolenic acid; ω -6 AA, arachidonic acid; EET, epoxy-eicosatrienoic acid; ALA, α-linolenic acid; STA, stearidonic acid; ω -3 AA; EPA, eicosapentaenoic acid; EEQ, 17’18’-epoxy eicosatetraenoic acid. (B) Withdrawal responses elicited by GSK101 in WT, TRPV4, TRPV4; fat-3 , and TRPV4; fat-3 worms supplemented with PUFAs. (C) Withdrawal responses elicited by 4α-Phorbol in WT, TRPV4, and TRPV4; fat-3 worms. (D) Withdrawal responses elicited by GSK101 in TRPV4 and TRPV4; fat-4 worms. (E) Withdrawal responses elicited by 1 M glycerol and nose touch in TRPV4; osm9 and TRPV4; osm9fat-3 strains. (F) Representative micrographs of TRPV4::GFP and TRPV4::GFP; fat-3 ASH neurons. (G) Box plots show the mean, median, and the 75 th to 25 th percentiles of the fluorescence intensity analysis from images in (F). The number of neurons imaged during 2 sessions is indicated below the boxes. (H) Schematic representation of the phospholipid synthesis. (I) GSK101 withdrawal responses after knocking down the expression of mboa-6 in TRPV4 worms. Bars are mean ± SEM, the number of worms tested during 3 assays sessions is indicated inside the bars. The asterisks indicate values significantly different from control. *** p .
    Figure Legend Snippet: PUFAs are required for TRPV4 function in C. elegans ). LA, linolenic acid; γLA, γ-linolenic acid; DγLA, dihomo-linolenic acid; ω -6 AA, arachidonic acid; EET, epoxy-eicosatrienoic acid; ALA, α-linolenic acid; STA, stearidonic acid; ω -3 AA; EPA, eicosapentaenoic acid; EEQ, 17’18’-epoxy eicosatetraenoic acid. (B) Withdrawal responses elicited by GSK101 in WT, TRPV4, TRPV4; fat-3 , and TRPV4; fat-3 worms supplemented with PUFAs. (C) Withdrawal responses elicited by 4α-Phorbol in WT, TRPV4, and TRPV4; fat-3 worms. (D) Withdrawal responses elicited by GSK101 in TRPV4 and TRPV4; fat-4 worms. (E) Withdrawal responses elicited by 1 M glycerol and nose touch in TRPV4; osm9 and TRPV4; osm9fat-3 strains. (F) Representative micrographs of TRPV4::GFP and TRPV4::GFP; fat-3 ASH neurons. (G) Box plots show the mean, median, and the 75 th to 25 th percentiles of the fluorescence intensity analysis from images in (F). The number of neurons imaged during 2 sessions is indicated below the boxes. (H) Schematic representation of the phospholipid synthesis. (I) GSK101 withdrawal responses after knocking down the expression of mboa-6 in TRPV4 worms. Bars are mean ± SEM, the number of worms tested during 3 assays sessions is indicated inside the bars. The asterisks indicate values significantly different from control. *** p .

    Techniques Used: Fluorescence, Expressing

    7) Product Images from "Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: Relevance to MLC disease pathogenesis"

    Article Title: Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: Relevance to MLC disease pathogenesis

    Journal: Neurobiology of Disease

    doi: 10.1016/j.nbd.2014.02.003

    TRPV4 recycling in WT and S280L MLC1 astrocytoma cell lines after hyposmotic stress. Astrocytoma cell lines expressing WT or mutated S280L MLC1 proteins were incubated with Alexa Fluor 488-conjugated Tf at 4 °C to allow binding to surface TfR. After washing out labeled Tf (T0), cells were incubated at 37 °C for 20 min and overnight (ON) in the presence of excess unlabeled Tf and then stained with anti-TRPV4 pAb. a,c. TRPV4 (red) and Alexa Fluor 488-conjugated Tf (green) partially colocalize in intracellular vesicles in WT MLC1 and S280L mutant astrocytoma cell plasma membranes at T0. b,d. After ON incubation with unlabeled Tf, TRPV4-Tf colocalization disappears in WT MLC1 astrocytoma cells (b) but not in S280L astrocytoma cells where TRPV4 still colocalizes with Tf in clustered intracytoplasmic vesicles (d, arrows). Scale Bars:10 μm. e. WB of total cell proteins (input lanes) and of enriched surface proteins after biotinylation (eluates) reveals that 30-min incubation in hyposmotic solution induces an increase in surface expression of TRPV4 in WT MLC1 astrocytoma cells but not in cells expressing S280L mutation. Molecular weight markers are indicated on the left (kDa).
    Figure Legend Snippet: TRPV4 recycling in WT and S280L MLC1 astrocytoma cell lines after hyposmotic stress. Astrocytoma cell lines expressing WT or mutated S280L MLC1 proteins were incubated with Alexa Fluor 488-conjugated Tf at 4 °C to allow binding to surface TfR. After washing out labeled Tf (T0), cells were incubated at 37 °C for 20 min and overnight (ON) in the presence of excess unlabeled Tf and then stained with anti-TRPV4 pAb. a,c. TRPV4 (red) and Alexa Fluor 488-conjugated Tf (green) partially colocalize in intracellular vesicles in WT MLC1 and S280L mutant astrocytoma cell plasma membranes at T0. b,d. After ON incubation with unlabeled Tf, TRPV4-Tf colocalization disappears in WT MLC1 astrocytoma cells (b) but not in S280L astrocytoma cells where TRPV4 still colocalizes with Tf in clustered intracytoplasmic vesicles (d, arrows). Scale Bars:10 μm. e. WB of total cell proteins (input lanes) and of enriched surface proteins after biotinylation (eluates) reveals that 30-min incubation in hyposmotic solution induces an increase in surface expression of TRPV4 in WT MLC1 astrocytoma cells but not in cells expressing S280L mutation. Molecular weight markers are indicated on the left (kDa).

    Techniques Used: Expressing, Incubation, Binding Assay, Labeling, Staining, Mutagenesis, Western Blot, Molecular Weight

    8) Product Images from "Regulation of TGFβ Signalling by TRPV4 in Chondrocytes"

    Article Title: Regulation of TGFβ Signalling by TRPV4 in Chondrocytes

    Journal: Cells

    doi: 10.3390/cells10040726

    Activation of TRPV4 modulates TGFβ signalling in a time-dependent manor. TC28a2 cells with SBE-nLUCp reporter were used to monitor TGFβ signalling. ( A ) Cells were stimulated with 10 ng/mL TGFβ3 or medium control, incubated for 15 min then stimulated with 100 nM GSK101 (activator) or DMSO control (vehicle) and then incubated for a further 3 h 45 min before SBE-nLUCp activity was determined. TRPV4 inhibitor (500 nM GSK219) was added to cells along with TGFβ3. ( B ) and ( C ) Cells were either not transfected (NT), mock transfected (TR), transfected with siRNA to TGFB1 (siTGFB1) or transfected with siRNA to TRPV4 (siTRPV4) for 24 h and then serum starved and incubated for a further 48 h. Following incubation, cells were stimulated with TGFβ3 ( B ) or media control ( C ) and then TRPV4 activated using GSK101, SBE-nLUCp activity determined as described in ( A ). ( D ) Schematic illustrating the order of stimulation/activation for A–C. ( E ) Cells were stimulated with 10 ng/mL TGFβ3. TRPV4 was activated (100 nM GSK101/DMSO control) either before (-ve mins), with (0 min) or after (+ve mins) TGFβ3 stimulation. ( F ) Cells were stimulated with 10 ng/mL TGFβ3 or medium control, incubated for 15 min then TRPV4 activated using 100 nM GSK101 or DMSO control. SBE-nLUCp activity was determined after the indicated amount of time post TGFβ3 stimulation. ( G ) Schematic representation of conditions shown in E. ( H ) Schematic representation of conditions shown in F. FC SBE RLU; fold change in SMAD-binding element relative light units NT; no treatment. Data in A combined from four independent experiments, and data in B–F combined from three independent experiments. Raw data are shown in Figures S3 and S5 . GSK101 treatment was normalised to the DMSO control for each siRNA/timepoint. Statistical differences were calculated by two-way ANOVA followed by Sidak’s multiple comparisons test; p
    Figure Legend Snippet: Activation of TRPV4 modulates TGFβ signalling in a time-dependent manor. TC28a2 cells with SBE-nLUCp reporter were used to monitor TGFβ signalling. ( A ) Cells were stimulated with 10 ng/mL TGFβ3 or medium control, incubated for 15 min then stimulated with 100 nM GSK101 (activator) or DMSO control (vehicle) and then incubated for a further 3 h 45 min before SBE-nLUCp activity was determined. TRPV4 inhibitor (500 nM GSK219) was added to cells along with TGFβ3. ( B ) and ( C ) Cells were either not transfected (NT), mock transfected (TR), transfected with siRNA to TGFB1 (siTGFB1) or transfected with siRNA to TRPV4 (siTRPV4) for 24 h and then serum starved and incubated for a further 48 h. Following incubation, cells were stimulated with TGFβ3 ( B ) or media control ( C ) and then TRPV4 activated using GSK101, SBE-nLUCp activity determined as described in ( A ). ( D ) Schematic illustrating the order of stimulation/activation for A–C. ( E ) Cells were stimulated with 10 ng/mL TGFβ3. TRPV4 was activated (100 nM GSK101/DMSO control) either before (-ve mins), with (0 min) or after (+ve mins) TGFβ3 stimulation. ( F ) Cells were stimulated with 10 ng/mL TGFβ3 or medium control, incubated for 15 min then TRPV4 activated using 100 nM GSK101 or DMSO control. SBE-nLUCp activity was determined after the indicated amount of time post TGFβ3 stimulation. ( G ) Schematic representation of conditions shown in E. ( H ) Schematic representation of conditions shown in F. FC SBE RLU; fold change in SMAD-binding element relative light units NT; no treatment. Data in A combined from four independent experiments, and data in B–F combined from three independent experiments. Raw data are shown in Figures S3 and S5 . GSK101 treatment was normalised to the DMSO control for each siRNA/timepoint. Statistical differences were calculated by two-way ANOVA followed by Sidak’s multiple comparisons test; p

    Techniques Used: Activation Assay, Incubation, Activity Assay, Transfection, Binding Assay

    TRPV4 is expressed and can be activated in TC28a2 chondrocytes. ( A ) Immunostaining for TRPV4 in TC28a2 chondrocytes, using anti-TRPV4 antibody and DAPI nuclear stain. Scale bar represents 100 µm. ( B ) Dose response of GSK101 on Fluo8 fluorescence 15 min post stimulation. ( C ) Fluorescence imaging of Fluo8-loaded TC28a2 cells 15 min post stimulation with 100 nM GSK101 or DMSO control. Scale bar represents 200 µm. ( D ) Representative traces of Fluo8 fluorescence following DMSO (upper), 100 nM GSK101 stimulation (middle) or 100 nM GSK101 stimulation in cells pre-incubated with 500 nM GSK219 (lower) for 15 min. Data representative of three independent experiments.
    Figure Legend Snippet: TRPV4 is expressed and can be activated in TC28a2 chondrocytes. ( A ) Immunostaining for TRPV4 in TC28a2 chondrocytes, using anti-TRPV4 antibody and DAPI nuclear stain. Scale bar represents 100 µm. ( B ) Dose response of GSK101 on Fluo8 fluorescence 15 min post stimulation. ( C ) Fluorescence imaging of Fluo8-loaded TC28a2 cells 15 min post stimulation with 100 nM GSK101 or DMSO control. Scale bar represents 200 µm. ( D ) Representative traces of Fluo8 fluorescence following DMSO (upper), 100 nM GSK101 stimulation (middle) or 100 nM GSK101 stimulation in cells pre-incubated with 500 nM GSK219 (lower) for 15 min. Data representative of three independent experiments.

    Techniques Used: Immunostaining, Staining, Fluorescence, Imaging, Incubation

    TRPV4 activation enhances TGFβ signalling through the JUN and SP1 transcription factors. ( A , B ) TRRUST analysis of genes significantly increased in RNAseq for each of the indicated experimental comparisons. ( C ) siRNA knockdown of JUN and SP1 prevents TRPV4 enhancement of TGFβ signalling. Data were combined from three independent experiments. GSK101 treatment was normalised to DMSO for each siRNA. Statistical differences were calculated using two-way ANOVA followed by Sidak’s multiple comparisons test; p
    Figure Legend Snippet: TRPV4 activation enhances TGFβ signalling through the JUN and SP1 transcription factors. ( A , B ) TRRUST analysis of genes significantly increased in RNAseq for each of the indicated experimental comparisons. ( C ) siRNA knockdown of JUN and SP1 prevents TRPV4 enhancement of TGFβ signalling. Data were combined from three independent experiments. GSK101 treatment was normalised to DMSO for each siRNA. Statistical differences were calculated using two-way ANOVA followed by Sidak’s multiple comparisons test; p

    Techniques Used: Activation Assay

    Reduction in extracellular calcium or calmodulin inhibition prevents GSK101 enhancement of TGFβ signalling. TC28a2 cells grown with indicated concentration of calcium ( A ) or KN93 ( B ) for ~16 h and then stimulated with TGFβ3 followed by DMSO (black) or GSK101 (red) after 15 min, and luciferase activity was determined 4 h after TGFβ3. ( A ) TRPV4 activation (using 100 nM GSK101) does not enhance TGFβ signalling at low calcium concentrations in medium. ( B ) Pre-treatment with calmodulin inhibitor (KN93) prevents TRPV4 activation (using 100 nM GSK101) of enhanced TGFβ signalling. ( C , D ) Schematics showing timing for calcium removal or calmodulin inhibition (KN93) in relation to stimulation/activation. Data were combined from three independent experiments. CFM; calcium-free medium, FC SBE RLU; fold change in SMAD-binding element relative light units. Statistical differences were calculated using two-way ANOVA followed by Sidak’s multiple comparisons test; p
    Figure Legend Snippet: Reduction in extracellular calcium or calmodulin inhibition prevents GSK101 enhancement of TGFβ signalling. TC28a2 cells grown with indicated concentration of calcium ( A ) or KN93 ( B ) for ~16 h and then stimulated with TGFβ3 followed by DMSO (black) or GSK101 (red) after 15 min, and luciferase activity was determined 4 h after TGFβ3. ( A ) TRPV4 activation (using 100 nM GSK101) does not enhance TGFβ signalling at low calcium concentrations in medium. ( B ) Pre-treatment with calmodulin inhibitor (KN93) prevents TRPV4 activation (using 100 nM GSK101) of enhanced TGFβ signalling. ( C , D ) Schematics showing timing for calcium removal or calmodulin inhibition (KN93) in relation to stimulation/activation. Data were combined from three independent experiments. CFM; calcium-free medium, FC SBE RLU; fold change in SMAD-binding element relative light units. Statistical differences were calculated using two-way ANOVA followed by Sidak’s multiple comparisons test; p

    Techniques Used: Inhibition, Concentration Assay, Luciferase, Activity Assay, Activation Assay, Binding Assay

    RNA-seq identification of TGFβ3 response genes that are enhanced by TRPV4 activation. ( A ) Experimental design for RNA-seq (triplicate). ( B ) Hierarchical clustering and ( C ) PCA analysis shows separation of DMSO, GSK101, TGFβ3+DMSO and TGFβ3+GSK101 treatment groups, the TGFβ3+GSK219 and TGFβ3+GSK219+GSK101 treatment groups both clustered with TGFβ3+DMSO. ( D ) Histogram indicating number of differentially expressed genes (DEGs) between experimental conditions according to DESeq2. ( E ) Venn diagram indicating commonality between genes significantly up regulated in GSK101 vs. DMSO, TGFβ3+DMSO vs. DMSO and TGFβ3+GSK101 vs. DMSO. ( F ) Scatter plot of significant genes comparing fold change in gene expression in GSK101 vs. DMSO and TGFβ3+DMSO vs. DMSO. ( G ) Venn diagram of genes significantly up regulated in TGFβ3+GSK101 vs. TGFβ3+DMSO or TGFβ3+DMSO vs. DMSO illustrating that GSK101 causes further enhancement of TGFβ response genes. ( H ) Scatter plot of significant genes comparing fold change in gene expression following TGFβ3+DMSO vs. DMSO and TGFβ3+GSK101 vs. TGFβ3+DMSO.
    Figure Legend Snippet: RNA-seq identification of TGFβ3 response genes that are enhanced by TRPV4 activation. ( A ) Experimental design for RNA-seq (triplicate). ( B ) Hierarchical clustering and ( C ) PCA analysis shows separation of DMSO, GSK101, TGFβ3+DMSO and TGFβ3+GSK101 treatment groups, the TGFβ3+GSK219 and TGFβ3+GSK219+GSK101 treatment groups both clustered with TGFβ3+DMSO. ( D ) Histogram indicating number of differentially expressed genes (DEGs) between experimental conditions according to DESeq2. ( E ) Venn diagram indicating commonality between genes significantly up regulated in GSK101 vs. DMSO, TGFβ3+DMSO vs. DMSO and TGFβ3+GSK101 vs. DMSO. ( F ) Scatter plot of significant genes comparing fold change in gene expression in GSK101 vs. DMSO and TGFβ3+DMSO vs. DMSO. ( G ) Venn diagram of genes significantly up regulated in TGFβ3+GSK101 vs. TGFβ3+DMSO or TGFβ3+DMSO vs. DMSO illustrating that GSK101 causes further enhancement of TGFβ response genes. ( H ) Scatter plot of significant genes comparing fold change in gene expression following TGFβ3+DMSO vs. DMSO and TGFβ3+GSK101 vs. TGFβ3+DMSO.

    Techniques Used: RNA Sequencing Assay, Activation Assay, Expressing

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Alomone Labs antibody anti trpv4 extracellular
    PAR2 and <t>TRPV4</t> expression in the hippocampus. Immunohistochemistry discloses the expression of PAR2 and TRPV4 in the hippocampus. A comparable expression pattern is observed: high levels of PAR2 and TRPV4 are detected in CA1 stratum pyramidale (pcl, pyramidal cell layer; oriens, stratum oriens; rad, stratum radiatum; la-mol, stratum lacunosum-moleculare). No pronounced colocalization between PAR2 and GFAP was detected. Scale bars: 100 and 10 μm, n = 9 slices out of three animals.
    Antibody Anti Trpv4 Extracellular, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibody anti trpv4 extracellular/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibody anti trpv4 extracellular - by Bioz Stars, 2022-08
    93/100 stars
      Buy from Supplier

    Image Search Results


    PAR2 and TRPV4 expression in the hippocampus. Immunohistochemistry discloses the expression of PAR2 and TRPV4 in the hippocampus. A comparable expression pattern is observed: high levels of PAR2 and TRPV4 are detected in CA1 stratum pyramidale (pcl, pyramidal cell layer; oriens, stratum oriens; rad, stratum radiatum; la-mol, stratum lacunosum-moleculare). No pronounced colocalization between PAR2 and GFAP was detected. Scale bars: 100 and 10 μm, n = 9 slices out of three animals.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Protease Activated Receptor 2 (PAR2) Induces Long-Term Depression in the Hippocampus through Transient Receptor Potential Vanilloid 4 (TRPV4)

    doi: 10.3389/fnmol.2017.00042

    Figure Lengend Snippet: PAR2 and TRPV4 expression in the hippocampus. Immunohistochemistry discloses the expression of PAR2 and TRPV4 in the hippocampus. A comparable expression pattern is observed: high levels of PAR2 and TRPV4 are detected in CA1 stratum pyramidale (pcl, pyramidal cell layer; oriens, stratum oriens; rad, stratum radiatum; la-mol, stratum lacunosum-moleculare). No pronounced colocalization between PAR2 and GFAP was detected. Scale bars: 100 and 10 μm, n = 9 slices out of three animals.

    Article Snippet: Immunohistochemistry The following primary antibodies were used for immunodetection: goat anti-PAR2 (sc-8205, Santa Cruz, 1:25), rabbit anti-TRPV4 (ACC-124, Alomone Labs, 1:50), rabbit anti-PAR2 (APR-032, Alomone Labs 1:500) and mouse anti-GFAP (G3893, Sigma-Aldrich, 1:2000).

    Techniques: Expressing, Immunohistochemistry

    PAR2 induces LTD through the activation of TRPV4. (A) Application of TRPV4-agonist (2 μM RN1747) causes LTD. (B) Removal of the TRPV4-agonist (2 μM RN1747) following induction of LTD does not affect the stability of synaptic depression. (C) In presence of the TRPV4-antagonist (10 μM RN1734) the TRPV4-agonist is not able to induce synaptic depression. (D) In a two pathways experimental setting, low frequency stimulation (LFS, 1 Hz, 900 pulses) and TRPV4-agonist application induce similar levels of LTD. (E) LFS-induced LTD is not blocked by the TRPV4-antagonist. (F) Application of PAR2-agonist (10 μM AC55541) in presence of a TRPV4-antagonist (10 μM RN1734) blocks PAR2-induced LTD. (G) Application of TRPV4-agonist (2 μM RN1747) in presence of PAR2-antagonist (50 μM FSLLRY-NH 2 ) does not affect TRPV4-induced LTD. (H) Once PAR2-agonist mediated LTD is established, the TRPV4-agonist (2 μM RN1747) does not further de-potentiate a second pathway at adjusted response level (upward arrow). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section, n = 12 slices for each experiments, refer to text for statistics.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Protease Activated Receptor 2 (PAR2) Induces Long-Term Depression in the Hippocampus through Transient Receptor Potential Vanilloid 4 (TRPV4)

    doi: 10.3389/fnmol.2017.00042

    Figure Lengend Snippet: PAR2 induces LTD through the activation of TRPV4. (A) Application of TRPV4-agonist (2 μM RN1747) causes LTD. (B) Removal of the TRPV4-agonist (2 μM RN1747) following induction of LTD does not affect the stability of synaptic depression. (C) In presence of the TRPV4-antagonist (10 μM RN1734) the TRPV4-agonist is not able to induce synaptic depression. (D) In a two pathways experimental setting, low frequency stimulation (LFS, 1 Hz, 900 pulses) and TRPV4-agonist application induce similar levels of LTD. (E) LFS-induced LTD is not blocked by the TRPV4-antagonist. (F) Application of PAR2-agonist (10 μM AC55541) in presence of a TRPV4-antagonist (10 μM RN1734) blocks PAR2-induced LTD. (G) Application of TRPV4-agonist (2 μM RN1747) in presence of PAR2-antagonist (50 μM FSLLRY-NH 2 ) does not affect TRPV4-induced LTD. (H) Once PAR2-agonist mediated LTD is established, the TRPV4-agonist (2 μM RN1747) does not further de-potentiate a second pathway at adjusted response level (upward arrow). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section, n = 12 slices for each experiments, refer to text for statistics.

    Article Snippet: Immunohistochemistry The following primary antibodies were used for immunodetection: goat anti-PAR2 (sc-8205, Santa Cruz, 1:25), rabbit anti-TRPV4 (ACC-124, Alomone Labs, 1:50), rabbit anti-PAR2 (APR-032, Alomone Labs 1:500) and mouse anti-GFAP (G3893, Sigma-Aldrich, 1:2000).

    Techniques: Activation Assay

    TRPV4-mediated LTD depends on NMDAR-activity. (A) Similar to PAR2-induced LTD (c.f., Figures 1G,H ), the NMDAR-antagonist (50 μM APV) blocks TRPV4 (2 μM RN1747)-induced LTD, while (B) application of a TRPV4-agonist (2 μM RN1747) induces LTD in presence of the mGluR-antagonist (200 μM MCGP). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Protease Activated Receptor 2 (PAR2) Induces Long-Term Depression in the Hippocampus through Transient Receptor Potential Vanilloid 4 (TRPV4)

    doi: 10.3389/fnmol.2017.00042

    Figure Lengend Snippet: TRPV4-mediated LTD depends on NMDAR-activity. (A) Similar to PAR2-induced LTD (c.f., Figures 1G,H ), the NMDAR-antagonist (50 μM APV) blocks TRPV4 (2 μM RN1747)-induced LTD, while (B) application of a TRPV4-agonist (2 μM RN1747) induces LTD in presence of the mGluR-antagonist (200 μM MCGP). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section.

    Article Snippet: Immunohistochemistry The following primary antibodies were used for immunodetection: goat anti-PAR2 (sc-8205, Santa Cruz, 1:25), rabbit anti-TRPV4 (ACC-124, Alomone Labs, 1:50), rabbit anti-PAR2 (APR-032, Alomone Labs 1:500) and mouse anti-GFAP (G3893, Sigma-Aldrich, 1:2000).

    Techniques: Activity Assay

    TRPV4 was downregulated in mature CD11c + BMDCs. ( A ) Concentration dependence of immature (black bars) and mature (dark cyan bars) CD11c + BMDC responding fraction. ***, p

    Journal: International Journal of Molecular Sciences

    Article Title: Expression and Functional Role of TRPV4 in Bone Marrow-Derived CD11c+ Cells

    doi: 10.3390/ijms20143378

    Figure Lengend Snippet: TRPV4 was downregulated in mature CD11c + BMDCs. ( A ) Concentration dependence of immature (black bars) and mature (dark cyan bars) CD11c + BMDC responding fraction. ***, p

    Article Snippet: Cells were blocked with antimouse CD16/32 polyclonal antibody (1 µg/mL, eBioscience) in 5% sheep serum (Sigma-Aldrich) for 3 h. After two rinsing steps with PBS, cells were incubated overnight at 4 °C with a rabbit anti-TRPV4 antibody (1:200, ACC-124, Alomone labs, Jerusalem, Israel).

    Techniques: Concentration Assay

    TRPV4-deficient BMDCs exhibited impaired FcR-dependent phagocytosis. ( A ) Representative confocal images of wild-type and Trpv4 KO BMDCs after treatment with uncoated or IgG-coated fluorescent microspheres. Scale bar, 20 µm. ( B ) Percentage of cells with internalized beads. Data were collected from 10 randomly selected fields per condition from three independent experiments. ***, p

    Journal: International Journal of Molecular Sciences

    Article Title: Expression and Functional Role of TRPV4 in Bone Marrow-Derived CD11c+ Cells

    doi: 10.3390/ijms20143378

    Figure Lengend Snippet: TRPV4-deficient BMDCs exhibited impaired FcR-dependent phagocytosis. ( A ) Representative confocal images of wild-type and Trpv4 KO BMDCs after treatment with uncoated or IgG-coated fluorescent microspheres. Scale bar, 20 µm. ( B ) Percentage of cells with internalized beads. Data were collected from 10 randomly selected fields per condition from three independent experiments. ***, p

    Article Snippet: Cells were blocked with antimouse CD16/32 polyclonal antibody (1 µg/mL, eBioscience) in 5% sheep serum (Sigma-Aldrich) for 3 h. After two rinsing steps with PBS, cells were incubated overnight at 4 °C with a rabbit anti-TRPV4 antibody (1:200, ACC-124, Alomone labs, Jerusalem, Israel).

    Techniques:

    TRPV4 was dispensable in the differentiation of CD11c + BMDCs. ( A ) Color-coded two-dimensional t-distributed stochastic neighbor embedding (tSNE) representations of the total bone marrow-derived cell population (20,000 cells) defined by the surface markers CD11b, CD11c, and F4/80. ( B ) Histograms showing surface expression of the indicated markers in bone marrow-derived cells from wild-type (WT, black traces) and Trpv4 knockout (KO, red traces) mice. The shaded histograms represent specificity (fluorescence minus one) controls. The bar graph shows the percentage of different cell populations present in total bone marrow-derived cell cultures defined by the surface expression of CD11b, CD11c, and F4/80. The data are represented as mean ± SEM of nine independent experiments.

    Journal: International Journal of Molecular Sciences

    Article Title: Expression and Functional Role of TRPV4 in Bone Marrow-Derived CD11c+ Cells

    doi: 10.3390/ijms20143378

    Figure Lengend Snippet: TRPV4 was dispensable in the differentiation of CD11c + BMDCs. ( A ) Color-coded two-dimensional t-distributed stochastic neighbor embedding (tSNE) representations of the total bone marrow-derived cell population (20,000 cells) defined by the surface markers CD11b, CD11c, and F4/80. ( B ) Histograms showing surface expression of the indicated markers in bone marrow-derived cells from wild-type (WT, black traces) and Trpv4 knockout (KO, red traces) mice. The shaded histograms represent specificity (fluorescence minus one) controls. The bar graph shows the percentage of different cell populations present in total bone marrow-derived cell cultures defined by the surface expression of CD11b, CD11c, and F4/80. The data are represented as mean ± SEM of nine independent experiments.

    Article Snippet: Cells were blocked with antimouse CD16/32 polyclonal antibody (1 µg/mL, eBioscience) in 5% sheep serum (Sigma-Aldrich) for 3 h. After two rinsing steps with PBS, cells were incubated overnight at 4 °C with a rabbit anti-TRPV4 antibody (1:200, ACC-124, Alomone labs, Jerusalem, Israel).

    Techniques: Derivative Assay, Expressing, Knock-Out, Mouse Assay, Fluorescence

    TRPV4 was functionally expressed in CD11c + bone marrow-derived cells (BMDCs). ( A ) Expression profile of selected Trp genes in the total granulocyte-macrophage colony-stimulating (GM-CSF)-differentiated bone marrow-derived cell population (black bars) and in CD11c + -purified BMDCs (light gray). Values are relative to GAPDH expression. ( B ) Confocal image of CD11c + BMDCs stained with an anti-TRPV4 antibody (red). The blue color corresponds to nuclear staining with DAPI. ( C–E ) Representative traces of intracellular Ca 2+ concentration in CD11c + BMDCs showing the effects of 300 nM of GSK1016790A (GSK). ATP (100 μM) was used as a positive control for intracellular Ca 2+ increase. The TRPV4 antagonist HC067047 was used at 10 μM. ( F ) Percentage of CD11c + BMDCs responding to the indicated stimulus. GSK, GSK1016790A (300 nM); HC, HC067047 (1 µM); Ca 2+ -free, Krebs with nominal [Ca 2+ ] supplemented with 2.5 mM EDTA; Caps, Capsaicin (1 nM); THC, trans-Δ 9 -tetrahydrocannabinol (10 µM). The responding fraction is indicated within each bar. ***, p

    Journal: International Journal of Molecular Sciences

    Article Title: Expression and Functional Role of TRPV4 in Bone Marrow-Derived CD11c+ Cells

    doi: 10.3390/ijms20143378

    Figure Lengend Snippet: TRPV4 was functionally expressed in CD11c + bone marrow-derived cells (BMDCs). ( A ) Expression profile of selected Trp genes in the total granulocyte-macrophage colony-stimulating (GM-CSF)-differentiated bone marrow-derived cell population (black bars) and in CD11c + -purified BMDCs (light gray). Values are relative to GAPDH expression. ( B ) Confocal image of CD11c + BMDCs stained with an anti-TRPV4 antibody (red). The blue color corresponds to nuclear staining with DAPI. ( C–E ) Representative traces of intracellular Ca 2+ concentration in CD11c + BMDCs showing the effects of 300 nM of GSK1016790A (GSK). ATP (100 μM) was used as a positive control for intracellular Ca 2+ increase. The TRPV4 antagonist HC067047 was used at 10 μM. ( F ) Percentage of CD11c + BMDCs responding to the indicated stimulus. GSK, GSK1016790A (300 nM); HC, HC067047 (1 µM); Ca 2+ -free, Krebs with nominal [Ca 2+ ] supplemented with 2.5 mM EDTA; Caps, Capsaicin (1 nM); THC, trans-Δ 9 -tetrahydrocannabinol (10 µM). The responding fraction is indicated within each bar. ***, p

    Article Snippet: Cells were blocked with antimouse CD16/32 polyclonal antibody (1 µg/mL, eBioscience) in 5% sheep serum (Sigma-Aldrich) for 3 h. After two rinsing steps with PBS, cells were incubated overnight at 4 °C with a rabbit anti-TRPV4 antibody (1:200, ACC-124, Alomone labs, Jerusalem, Israel).

    Techniques: Derivative Assay, Expressing, Purification, Staining, Concentration Assay, Positive Control

    LPS-induced cytokine production occurred independently of TRPV4. ( A ) Representative confocal immunofluorescence microscopy images of fixed BMDCs untreated or treated with LPS (100 ng/mL). Cell stainings correspond to NF-κB p65 (red) and DAPI (nuclear, blue). Scale bar, 10 µm. The average linear intensity along the gray rectangle is represented next to the corresponding image. ( B ) Percentage of the total nuclear area stained by NF-κB p65 staining. The horizontal bar represents the mean. ***, p

    Journal: International Journal of Molecular Sciences

    Article Title: Expression and Functional Role of TRPV4 in Bone Marrow-Derived CD11c+ Cells

    doi: 10.3390/ijms20143378

    Figure Lengend Snippet: LPS-induced cytokine production occurred independently of TRPV4. ( A ) Representative confocal immunofluorescence microscopy images of fixed BMDCs untreated or treated with LPS (100 ng/mL). Cell stainings correspond to NF-κB p65 (red) and DAPI (nuclear, blue). Scale bar, 10 µm. The average linear intensity along the gray rectangle is represented next to the corresponding image. ( B ) Percentage of the total nuclear area stained by NF-κB p65 staining. The horizontal bar represents the mean. ***, p

    Article Snippet: Cells were blocked with antimouse CD16/32 polyclonal antibody (1 µg/mL, eBioscience) in 5% sheep serum (Sigma-Aldrich) for 3 h. After two rinsing steps with PBS, cells were incubated overnight at 4 °C with a rabbit anti-TRPV4 antibody (1:200, ACC-124, Alomone labs, Jerusalem, Israel).

    Techniques: Immunofluorescence, Microscopy, Staining

    A) Immunofluorescent images of ATDC5 cells on normal gels (upper panel) treated with 30nM GSK101 for 28 hr and severe OA gels with control treatment (lower panel) stained for the nucleus (Hoechst, blue), f-actin (purple), and TRPV4 (white). The left panel shows the composite images of all stains (scale bar =10 μm). The middle panel shows nuclear and f-actin staining, and the right panel shows TRPV4 and nuclear staining. B) TRPV4 fluorescence of ATDC5 cells on normal, OA, and severe OA gels during control (dark blue) and 28 hr of 30 nM GSK101 (light blue) treatments. f-actin was used to draw ROI around the cells to measure TRPV4 fluorescence from max projections of z-stack images. Error Bars are ± SEM, * indicates p-value

    Journal: bioRxiv

    Article Title: Extracellular Matrix Stiffness Alters TRPV4 Regulation in Chondrocytes

    doi: 10.1101/2021.09.14.460172

    Figure Lengend Snippet: A) Immunofluorescent images of ATDC5 cells on normal gels (upper panel) treated with 30nM GSK101 for 28 hr and severe OA gels with control treatment (lower panel) stained for the nucleus (Hoechst, blue), f-actin (purple), and TRPV4 (white). The left panel shows the composite images of all stains (scale bar =10 μm). The middle panel shows nuclear and f-actin staining, and the right panel shows TRPV4 and nuclear staining. B) TRPV4 fluorescence of ATDC5 cells on normal, OA, and severe OA gels during control (dark blue) and 28 hr of 30 nM GSK101 (light blue) treatments. f-actin was used to draw ROI around the cells to measure TRPV4 fluorescence from max projections of z-stack images. Error Bars are ± SEM, * indicates p-value

    Article Snippet: A subset of cells received 30 nM GSK101 for 28 h. Cells were fixed in 4% paraformaldehyde, blocked for 1 hr in PBS containing 1% BSA/0.2% Fish Gelatin (Fisher Scientific), and probed for membrane TRPV4 using 1:200 rabbit anti-TRPV4 (Alomone Labs, ACC-124) overnight at 4°C.

    Techniques: Staining, Fluorescence

    A) mRNA expression of anabolic factors (Sox-9, aggrecan, col2 and TRPV4) in ATDC5 cells on normal, OA and severe OA gels during control (dark blue) or 28 hr of 30 nM GSK101 (light blue) treatment. B) mRNA expression of catabolic chondrocyte factors, collagen I and MMP-13 in ATDC5 cells on normal, OA and severe OA gels during control (dark blue) or 28 hr of 30 nM GSK101 (light blue) treatment. Fold change was normalized to rplp0 expression for each sample and then normalized to the normal control condition. C) Western blotting shows TRPV4 protein levels for ATDC5 cells on normal, OA, and severe OA gels during control treatment. The bar graph represents the normalized TRPV4 protein level to GAPDH for each sample. Error bars are ± SEM, * indicates significance with a p-value

    Journal: bioRxiv

    Article Title: Extracellular Matrix Stiffness Alters TRPV4 Regulation in Chondrocytes

    doi: 10.1101/2021.09.14.460172

    Figure Lengend Snippet: A) mRNA expression of anabolic factors (Sox-9, aggrecan, col2 and TRPV4) in ATDC5 cells on normal, OA and severe OA gels during control (dark blue) or 28 hr of 30 nM GSK101 (light blue) treatment. B) mRNA expression of catabolic chondrocyte factors, collagen I and MMP-13 in ATDC5 cells on normal, OA and severe OA gels during control (dark blue) or 28 hr of 30 nM GSK101 (light blue) treatment. Fold change was normalized to rplp0 expression for each sample and then normalized to the normal control condition. C) Western blotting shows TRPV4 protein levels for ATDC5 cells on normal, OA, and severe OA gels during control treatment. The bar graph represents the normalized TRPV4 protein level to GAPDH for each sample. Error bars are ± SEM, * indicates significance with a p-value

    Article Snippet: A subset of cells received 30 nM GSK101 for 28 h. Cells were fixed in 4% paraformaldehyde, blocked for 1 hr in PBS containing 1% BSA/0.2% Fish Gelatin (Fisher Scientific), and probed for membrane TRPV4 using 1:200 rabbit anti-TRPV4 (Alomone Labs, ACC-124) overnight at 4°C.

    Techniques: Expressing, Western Blot

    p38 activation and macrophage phagocytosis in response to LPS is dependent on matrix stiffness. Phosphorylated and total A. p38 and B. JNK on various matrix stiffnesses in the physiologic range (1kPa, 8kPa, and 25kPa) from WT vs TRPV4 KO BMDMs quantified for LPS 15 minutes ( *p = 0.031). C. Macrophage phagocytosis of E. coli particles ± p38 inhibition (SB, BIRB) on various matrix stiffnesses ( *p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

    doi: 10.4049/jimmunol.1901033

    Figure Lengend Snippet: p38 activation and macrophage phagocytosis in response to LPS is dependent on matrix stiffness. Phosphorylated and total A. p38 and B. JNK on various matrix stiffnesses in the physiologic range (1kPa, 8kPa, and 25kPa) from WT vs TRPV4 KO BMDMs quantified for LPS 15 minutes ( *p = 0.031). C. Macrophage phagocytosis of E. coli particles ± p38 inhibition (SB, BIRB) on various matrix stiffnesses ( *p

    Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

    Techniques: Activation Assay, Inhibition

    TRPV4-mediated p38 MAPK and JNK molecular switch is regulated by dual-specificity phosphatase 1 (DUSP1). WT and TRPV4 KO BMDMs were incubated ± LPS as above for indicated time, and cells were lysed and analyzed by A. immunoblot for DUSP1 and B. band density quantified as DUSP1/GAPDH from immunoblot (n = 4) ( *p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

    doi: 10.4049/jimmunol.1901033

    Figure Lengend Snippet: TRPV4-mediated p38 MAPK and JNK molecular switch is regulated by dual-specificity phosphatase 1 (DUSP1). WT and TRPV4 KO BMDMs were incubated ± LPS as above for indicated time, and cells were lysed and analyzed by A. immunoblot for DUSP1 and B. band density quantified as DUSP1/GAPDH from immunoblot (n = 4) ( *p

    Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

    Techniques: Incubation

    TRPV4 function protects against lung injury in a murine Pseudomonas aeruginosa pneumonia model. Sterile beads or P. aeruginosa (PA, PAM57–15) were instilled intratracheally in TRPV4 KO and age-matched female congenic WT mice with BAL and tissue harvest performed at Day 3 (injury phase). TRPV4 deleted mice (TRPV4 KO) have greater A. inflammatory cell infiltration and B. BAL total protein compared to WT (* p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

    doi: 10.4049/jimmunol.1901033

    Figure Lengend Snippet: TRPV4 function protects against lung injury in a murine Pseudomonas aeruginosa pneumonia model. Sterile beads or P. aeruginosa (PA, PAM57–15) were instilled intratracheally in TRPV4 KO and age-matched female congenic WT mice with BAL and tissue harvest performed at Day 3 (injury phase). TRPV4 deleted mice (TRPV4 KO) have greater A. inflammatory cell infiltration and B. BAL total protein compared to WT (* p

    Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

    Techniques: Mouse Assay

    p38 MAPK and JNK are differentially regulated by TRPV4 after LPS or Pseudomonas aeruginosa . BMDMs were incubated ± LPS as above for indicated time cultured on tissue culture-treated plastic, and cells were lysed and analyzed by immunoblot for A. phosphorylated and total p38, ERK, and JNK compared to WT BMDMs (whole cell lysate). Band density quantified from immunoblot (n = 3–6) for B. p-p38/total p38 ( *p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

    doi: 10.4049/jimmunol.1901033

    Figure Lengend Snippet: p38 MAPK and JNK are differentially regulated by TRPV4 after LPS or Pseudomonas aeruginosa . BMDMs were incubated ± LPS as above for indicated time cultured on tissue culture-treated plastic, and cells were lysed and analyzed by immunoblot for A. phosphorylated and total p38, ERK, and JNK compared to WT BMDMs (whole cell lysate). Band density quantified from immunoblot (n = 3–6) for B. p-p38/total p38 ( *p

    Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

    Techniques: Incubation, Cell Culture

    TRPV4 mediates phagocytosis after LPS in healthy human through p38 MAPK. Monocyte derived and alveolar macrophages from healthy (n = 6) control subjects were incubated ± LPS ± TRPV4 inhibitor, HC, and phagocytosis of E. coli particles was measured in A. monocyte-derived and B. alveolar macrophages in healthy controls. HC alone had no effect. Representative immunoblot for phosphorylated and total p38 in C. healthy monocyte derived macrophages ± LPS 15 minutes and D. band density quantified ( *p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

    doi: 10.4049/jimmunol.1901033

    Figure Lengend Snippet: TRPV4 mediates phagocytosis after LPS in healthy human through p38 MAPK. Monocyte derived and alveolar macrophages from healthy (n = 6) control subjects were incubated ± LPS ± TRPV4 inhibitor, HC, and phagocytosis of E. coli particles was measured in A. monocyte-derived and B. alveolar macrophages in healthy controls. HC alone had no effect. Representative immunoblot for phosphorylated and total p38 in C. healthy monocyte derived macrophages ± LPS 15 minutes and D. band density quantified ( *p

    Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

    Techniques: Derivative Assay, Incubation

    TRPV4 mediates clearance of P. aeruginosa by macrophages. WT and TRPV4 KO mice were intratracheally administered ±GFP P. aeruginosa for 3 days. Representative confocal images of whole lung lavage cytospins of macrophages (open arrowhead) and neutrophils (filled arrowhead) in WT mice given IT sterile beads or GFP- P. aeruginosa after immunofluorescence with A. TRPV4 extracellular antibody (green, TRPV4) and C. anti-GFP (green, GFP P. aeruginosa ; anti-CD45, red; dapi, blue). B, D. Quantification of A, C . * , # p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

    doi: 10.4049/jimmunol.1901033

    Figure Lengend Snippet: TRPV4 mediates clearance of P. aeruginosa by macrophages. WT and TRPV4 KO mice were intratracheally administered ±GFP P. aeruginosa for 3 days. Representative confocal images of whole lung lavage cytospins of macrophages (open arrowhead) and neutrophils (filled arrowhead) in WT mice given IT sterile beads or GFP- P. aeruginosa after immunofluorescence with A. TRPV4 extracellular antibody (green, TRPV4) and C. anti-GFP (green, GFP P. aeruginosa ; anti-CD45, red; dapi, blue). B, D. Quantification of A, C . * , # p

    Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

    Techniques: Mouse Assay, Immunofluorescence

    TRPV4 modulates pro-inflammatory cytokine production through JNK. BMDMs were incubated ± LPS (100ng/mL, 24h) ± JNK inhibitor, SP600125 (20μM, 25h) ± p38 inhibitor, SB203580 (10μM, 25h), cultured on cell culture-treated plastic, and cytokines measured via ELISA. IL-6, CXCL2, and CXCL1 secretion ± LPS in A. WT and TRPV4 KO BMDMs and B. WT BMDMs ± SP600125 ± SB203580 ( *, # p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: TRPV4 Protects the Lung from Bacterial Pneumonia via MAPK Molecular Pathway Switching

    doi: 10.4049/jimmunol.1901033

    Figure Lengend Snippet: TRPV4 modulates pro-inflammatory cytokine production through JNK. BMDMs were incubated ± LPS (100ng/mL, 24h) ± JNK inhibitor, SP600125 (20μM, 25h) ± p38 inhibitor, SB203580 (10μM, 25h), cultured on cell culture-treated plastic, and cytokines measured via ELISA. IL-6, CXCL2, and CXCL1 secretion ± LPS in A. WT and TRPV4 KO BMDMs and B. WT BMDMs ± SP600125 ± SB203580 ( *, # p

    Article Snippet: Antibodies and reagents The following primary antibodies were purchased: intracellular TRPV4 (Alomone Labs, Jerusalem, Israel), extracellular TRPV4 (Alomone Labs, Jerusalem, Israel), anti-phospho p38 (Thr180/Tyr182, Cell Signaling, MA), anti-p38 (Santa Cruz, CA), anti-phospho JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phospho ERK (Santa Cruz), anti-ERK (Cell Signaling), anti-phospho MK2 (Cell Signaling), anti-MK2 (Cell Signaling), anti-phospho MKK3/MKK6 (Cell Signaling), anti-MKK3 (Cell Signaling), anti-MKK6 (Cell Signaling), anti-GAPDH (Fitzgerald Industries International, Acton, MA), anti-DUSP1/MKP1 (Santa Cruz, CA), α-CD45 (BD Biosciences), and purified rabbit IgG from mouse serum (Sigma-Aldrich, St. Louis, MO).

    Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay