anti mouse trpv4  (Alomone Labs)


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    Structured Review

    Alomone Labs anti mouse trpv4
    Transfection of large mouse cholangiocytes (MLC) with plasmid for transient receptor potential vanilloid member 4 <t>(TRPV4)</t> small interfering (si)RNA decreases protein and RNA expression. A : representative Western blot demonstrating change in TRPV4 protein levels in cells transfected with nontargeting siRNA (scramble) and cells transfected with TRPV4 siRNA. β-Actin was used as loading control. B : cumulative data demonstrating change in TRPV4 protein ( left ) and mRNA ( right ) levels in control cells, cells transfected with nontargeting siRNA (scramble), and cells transfected with TRPV4 siRNA (* P
    Anti Mouse Trpv4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse trpv4/product/Alomone Labs
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    anti mouse trpv4 - by Bioz Stars, 2022-10
    95/100 stars

    Images

    1) Product Images from "Mechanosensor transient receptor potential vanilloid member 4 (TRPV4) regulates mouse cholangiocyte secretion and bile formation"

    Article Title: Mechanosensor transient receptor potential vanilloid member 4 (TRPV4) regulates mouse cholangiocyte secretion and bile formation

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00176.2019

    Transfection of large mouse cholangiocytes (MLC) with plasmid for transient receptor potential vanilloid member 4 (TRPV4) small interfering (si)RNA decreases protein and RNA expression. A : representative Western blot demonstrating change in TRPV4 protein levels in cells transfected with nontargeting siRNA (scramble) and cells transfected with TRPV4 siRNA. β-Actin was used as loading control. B : cumulative data demonstrating change in TRPV4 protein ( left ) and mRNA ( right ) levels in control cells, cells transfected with nontargeting siRNA (scramble), and cells transfected with TRPV4 siRNA (* P
    Figure Legend Snippet: Transfection of large mouse cholangiocytes (MLC) with plasmid for transient receptor potential vanilloid member 4 (TRPV4) small interfering (si)RNA decreases protein and RNA expression. A : representative Western blot demonstrating change in TRPV4 protein levels in cells transfected with nontargeting siRNA (scramble) and cells transfected with TRPV4 siRNA. β-Actin was used as loading control. B : cumulative data demonstrating change in TRPV4 protein ( left ) and mRNA ( right ) levels in control cells, cells transfected with nontargeting siRNA (scramble), and cells transfected with TRPV4 siRNA (* P

    Techniques Used: Transfection, Plasmid Preparation, RNA Expression, Western Blot

    Flow-stimulated increases in intracellular calcium concentration ([Ca 2+ ] i ) in large mouse cholangiocytes (MLC). Representative Ca 2+ fluorescence in response to fluid flow. MLC on coverglass were loaded with fura-2 AM and exposed to flow (2 mL/min; shear = 0.64 dyne/cm 2 ); y -axis values represent the ratio of fluorescence at 340 and 380 nm; x -axis represents time (bar = 100 s). Control cells shown ( A ) vs. cells exposed to HC-067047 (HC, 10 µM; B ) and after transfection with transient receptor potential vanilloid member 4 (TRPV4) small interfering (si)RNA ( C ). Cells were exposed to the Ca 2+ ionophore ionomycin, as an internal control. D : cumulative data demonstrating the change in [Ca 2+ ] i in response to fluid flow (shear = 0.64 dyne/cm 2 ) in control cells ( n = 78) and in the presence of HC-067047 (HC, n = 61), or after transfection with TRPV4 siRNA ( n = 49). Values represent change in fluorescence ratio 340/380. Maximal increase vs. basal # P ≤ 0.01; inhibitor HC067047 vs. control ** P ≤ 0.01; * P
    Figure Legend Snippet: Flow-stimulated increases in intracellular calcium concentration ([Ca 2+ ] i ) in large mouse cholangiocytes (MLC). Representative Ca 2+ fluorescence in response to fluid flow. MLC on coverglass were loaded with fura-2 AM and exposed to flow (2 mL/min; shear = 0.64 dyne/cm 2 ); y -axis values represent the ratio of fluorescence at 340 and 380 nm; x -axis represents time (bar = 100 s). Control cells shown ( A ) vs. cells exposed to HC-067047 (HC, 10 µM; B ) and after transfection with transient receptor potential vanilloid member 4 (TRPV4) small interfering (si)RNA ( C ). Cells were exposed to the Ca 2+ ionophore ionomycin, as an internal control. D : cumulative data demonstrating the change in [Ca 2+ ] i in response to fluid flow (shear = 0.64 dyne/cm 2 ) in control cells ( n = 78) and in the presence of HC-067047 (HC, n = 61), or after transfection with TRPV4 siRNA ( n = 49). Values represent change in fluorescence ratio 340/380. Maximal increase vs. basal # P ≤ 0.01; inhibitor HC067047 vs. control ** P ≤ 0.01; * P

    Techniques Used: Concentration Assay, Fluorescence, Transfection

    Specific agonists of transient receptor potential vanilloid member 4 (TRPV4) activate currents and increase intracellular calcium concentration ([Ca 2+ ] i ) in large mouse cholangiocytes (MLC). A, top : representative whole cell recording. The specific TRPV4 agonist 4α -phorbol-12,13-didecanoate (4αPDD; 1 µM) activates whole cell currents. Currents measured at +100 mV (○) and at −100 mV (●) are shown. Voltage protocol −100 mV to +100 mV ramp every 5 s. Bottom : cells on coverglass were loaded with fura-2 AM, washed with PBS, and exposed to 4αPDD (1 µM); y -axis values represent the ratio of fluorescence at 340 and at 380 nm. B, top : the specific TRPV4 agonist GSK (100 nM) activates whole cell currents. Currents measured at +100 mv (○) and at −100 mV (●) are shown. Bottom : fura-2-loaded MLC cells were exposed to glycogen synthase kinase (GSK; 100 nM). The y -axis values represent the ratio of fluorescence at 340 and at 380 nm. C, top : cumulative data demonstrating maximal current density (pA/pF) measured at +100 mV in response to 4αPDD or GSK. Currents were significantly inhibited by ruthenium red (RR, 10 µM, n = 9), HC-067047 (HC, 10 µM, n = 16), and after transfection with TRPV4 small interfering (si)RNA ( n = 8). Bottom : cumulative data demonstrating the change in [Ca 2+ ] i in response to 4αPDD or GSK in control cells ( n = 78) and in the presence of RR ( n = 46), HC ( n = 54), or after transfection with TRPV4 siRNA ( n = 27). Values represent relative change in fluorescence ratio 340/380. Maximal increase vs. basal ** P ≤ 0.01, inhibitor vs. control, * P ≤ 0.05, vs. control ** P ≤ 0.01, * P
    Figure Legend Snippet: Specific agonists of transient receptor potential vanilloid member 4 (TRPV4) activate currents and increase intracellular calcium concentration ([Ca 2+ ] i ) in large mouse cholangiocytes (MLC). A, top : representative whole cell recording. The specific TRPV4 agonist 4α -phorbol-12,13-didecanoate (4αPDD; 1 µM) activates whole cell currents. Currents measured at +100 mV (○) and at −100 mV (●) are shown. Voltage protocol −100 mV to +100 mV ramp every 5 s. Bottom : cells on coverglass were loaded with fura-2 AM, washed with PBS, and exposed to 4αPDD (1 µM); y -axis values represent the ratio of fluorescence at 340 and at 380 nm. B, top : the specific TRPV4 agonist GSK (100 nM) activates whole cell currents. Currents measured at +100 mv (○) and at −100 mV (●) are shown. Bottom : fura-2-loaded MLC cells were exposed to glycogen synthase kinase (GSK; 100 nM). The y -axis values represent the ratio of fluorescence at 340 and at 380 nm. C, top : cumulative data demonstrating maximal current density (pA/pF) measured at +100 mV in response to 4αPDD or GSK. Currents were significantly inhibited by ruthenium red (RR, 10 µM, n = 9), HC-067047 (HC, 10 µM, n = 16), and after transfection with TRPV4 small interfering (si)RNA ( n = 8). Bottom : cumulative data demonstrating the change in [Ca 2+ ] i in response to 4αPDD or GSK in control cells ( n = 78) and in the presence of RR ( n = 46), HC ( n = 54), or after transfection with TRPV4 siRNA ( n = 27). Values represent relative change in fluorescence ratio 340/380. Maximal increase vs. basal ** P ≤ 0.01, inhibitor vs. control, * P ≤ 0.05, vs. control ** P ≤ 0.01, * P

    Techniques Used: Concentration Assay, Fluorescence, Transfection

    2) Product Images from "Mechanosensor transient receptor potential vanilloid member 4 (TRPV4) regulates mouse cholangiocyte secretion and bile formation"

    Article Title: Mechanosensor transient receptor potential vanilloid member 4 (TRPV4) regulates mouse cholangiocyte secretion and bile formation

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00176.2019

    Transfection of large mouse cholangiocytes (MLC) with plasmid for transient receptor potential vanilloid member 4 (TRPV4) small interfering (si)RNA decreases protein and RNA expression. A : representative Western blot demonstrating change in TRPV4 protein levels in cells transfected with nontargeting siRNA (scramble) and cells transfected with TRPV4 siRNA. β-Actin was used as loading control. B : cumulative data demonstrating change in TRPV4 protein ( left ) and mRNA ( right ) levels in control cells, cells transfected with nontargeting siRNA (scramble), and cells transfected with TRPV4 siRNA (* P
    Figure Legend Snippet: Transfection of large mouse cholangiocytes (MLC) with plasmid for transient receptor potential vanilloid member 4 (TRPV4) small interfering (si)RNA decreases protein and RNA expression. A : representative Western blot demonstrating change in TRPV4 protein levels in cells transfected with nontargeting siRNA (scramble) and cells transfected with TRPV4 siRNA. β-Actin was used as loading control. B : cumulative data demonstrating change in TRPV4 protein ( left ) and mRNA ( right ) levels in control cells, cells transfected with nontargeting siRNA (scramble), and cells transfected with TRPV4 siRNA (* P

    Techniques Used: Transfection, Plasmid Preparation, RNA Expression, Western Blot

    Flow-stimulated increases in intracellular calcium concentration ([Ca 2+ ] i ) in large mouse cholangiocytes (MLC). Representative Ca 2+ fluorescence in response to fluid flow. MLC on coverglass were loaded with fura-2 AM and exposed to flow (2 mL/min; shear = 0.64 dyne/cm 2 ); y -axis values represent the ratio of fluorescence at 340 and 380 nm; x -axis represents time (bar = 100 s). Control cells shown ( A ) vs. cells exposed to HC-067047 (HC, 10 µM; B ) and after transfection with transient receptor potential vanilloid member 4 (TRPV4) small interfering (si)RNA ( C ). Cells were exposed to the Ca 2+ ionophore ionomycin, as an internal control. D : cumulative data demonstrating the change in [Ca 2+ ] i in response to fluid flow (shear = 0.64 dyne/cm 2 ) in control cells ( n = 78) and in the presence of HC-067047 (HC, n = 61), or after transfection with TRPV4 siRNA ( n = 49). Values represent change in fluorescence ratio 340/380. Maximal increase vs. basal # P ≤ 0.01; inhibitor HC067047 vs. control ** P ≤ 0.01; * P
    Figure Legend Snippet: Flow-stimulated increases in intracellular calcium concentration ([Ca 2+ ] i ) in large mouse cholangiocytes (MLC). Representative Ca 2+ fluorescence in response to fluid flow. MLC on coverglass were loaded with fura-2 AM and exposed to flow (2 mL/min; shear = 0.64 dyne/cm 2 ); y -axis values represent the ratio of fluorescence at 340 and 380 nm; x -axis represents time (bar = 100 s). Control cells shown ( A ) vs. cells exposed to HC-067047 (HC, 10 µM; B ) and after transfection with transient receptor potential vanilloid member 4 (TRPV4) small interfering (si)RNA ( C ). Cells were exposed to the Ca 2+ ionophore ionomycin, as an internal control. D : cumulative data demonstrating the change in [Ca 2+ ] i in response to fluid flow (shear = 0.64 dyne/cm 2 ) in control cells ( n = 78) and in the presence of HC-067047 (HC, n = 61), or after transfection with TRPV4 siRNA ( n = 49). Values represent change in fluorescence ratio 340/380. Maximal increase vs. basal # P ≤ 0.01; inhibitor HC067047 vs. control ** P ≤ 0.01; * P

    Techniques Used: Concentration Assay, Fluorescence, Transfection

    Specific agonists of transient receptor potential vanilloid member 4 (TRPV4) activate currents and increase intracellular calcium concentration ([Ca 2+ ] i ) in large mouse cholangiocytes (MLC). A, top : representative whole cell recording. The specific TRPV4 agonist 4α -phorbol-12,13-didecanoate (4αPDD; 1 µM) activates whole cell currents. Currents measured at +100 mV (○) and at −100 mV (●) are shown. Voltage protocol −100 mV to +100 mV ramp every 5 s. Bottom : cells on coverglass were loaded with fura-2 AM, washed with PBS, and exposed to 4αPDD (1 µM); y -axis values represent the ratio of fluorescence at 340 and at 380 nm. B, top : the specific TRPV4 agonist GSK (100 nM) activates whole cell currents. Currents measured at +100 mv (○) and at −100 mV (●) are shown. Bottom : fura-2-loaded MLC cells were exposed to glycogen synthase kinase (GSK; 100 nM). The y -axis values represent the ratio of fluorescence at 340 and at 380 nm. C, top : cumulative data demonstrating maximal current density (pA/pF) measured at +100 mV in response to 4αPDD or GSK. Currents were significantly inhibited by ruthenium red (RR, 10 µM, n = 9), HC-067047 (HC, 10 µM, n = 16), and after transfection with TRPV4 small interfering (si)RNA ( n = 8). Bottom : cumulative data demonstrating the change in [Ca 2+ ] i in response to 4αPDD or GSK in control cells ( n = 78) and in the presence of RR ( n = 46), HC ( n = 54), or after transfection with TRPV4 siRNA ( n = 27). Values represent relative change in fluorescence ratio 340/380. Maximal increase vs. basal ** P ≤ 0.01, inhibitor vs. control, * P ≤ 0.05, vs. control ** P ≤ 0.01, * P
    Figure Legend Snippet: Specific agonists of transient receptor potential vanilloid member 4 (TRPV4) activate currents and increase intracellular calcium concentration ([Ca 2+ ] i ) in large mouse cholangiocytes (MLC). A, top : representative whole cell recording. The specific TRPV4 agonist 4α -phorbol-12,13-didecanoate (4αPDD; 1 µM) activates whole cell currents. Currents measured at +100 mV (○) and at −100 mV (●) are shown. Voltage protocol −100 mV to +100 mV ramp every 5 s. Bottom : cells on coverglass were loaded with fura-2 AM, washed with PBS, and exposed to 4αPDD (1 µM); y -axis values represent the ratio of fluorescence at 340 and at 380 nm. B, top : the specific TRPV4 agonist GSK (100 nM) activates whole cell currents. Currents measured at +100 mv (○) and at −100 mV (●) are shown. Bottom : fura-2-loaded MLC cells were exposed to glycogen synthase kinase (GSK; 100 nM). The y -axis values represent the ratio of fluorescence at 340 and at 380 nm. C, top : cumulative data demonstrating maximal current density (pA/pF) measured at +100 mV in response to 4αPDD or GSK. Currents were significantly inhibited by ruthenium red (RR, 10 µM, n = 9), HC-067047 (HC, 10 µM, n = 16), and after transfection with TRPV4 small interfering (si)RNA ( n = 8). Bottom : cumulative data demonstrating the change in [Ca 2+ ] i in response to 4αPDD or GSK in control cells ( n = 78) and in the presence of RR ( n = 46), HC ( n = 54), or after transfection with TRPV4 siRNA ( n = 27). Values represent relative change in fluorescence ratio 340/380. Maximal increase vs. basal ** P ≤ 0.01, inhibitor vs. control, * P ≤ 0.05, vs. control ** P ≤ 0.01, * P

    Techniques Used: Concentration Assay, Fluorescence, Transfection

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    Alomone Labs intracellular trpv4
    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 <t>TRPV4</t> 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
    Intracellular Trpv4, 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/intracellular trpv4/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    intracellular trpv4 - by Bioz Stars, 2022-10
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    94
    Alomone Labs antibody anti trpv4
    <t>TRPV4</t> deficiency does not alter cortical tubular apoptosis upon acute ischaemic kidney injury. ( A ) Representative ×200 images of TUNEL labelling on WT and Trpv4 KO kidney sections 24 hours after renal ischaemia. Thin white arrows indicate TUNEL–positive tubular cells. Thick white arrows indicate TUNEL–positive interstitial cells. Yellow bars represent 100 μm. ( B ) Number of TUNEL–positive cortical tubular cells per ×200 field. Each dot represents a single animal and is the mean of 5 randomly selected cortical fields. n = 6 for both WT and Trpv4 KO mice, two-tailed unpaired t-test. G, glomerulus; N, necrosis. ( C ) Serum lactate dehydrogenase (LDH) levels in WT and Trpv4 KO mice 24 hours after renal ischaemia. n = 5 for both WT and Trpv4 KO mice, two-tailed unpaired t-test, *P
    Antibody Anti Trpv4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibody anti trpv4/product/Alomone Labs
    Average 94 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    antibody anti trpv4 - by Bioz Stars, 2022-10
    94/100 stars
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    93
    Alomone Labs antibody anti trpv4 extracellular
    <t>TRPV4</t> was downregulated in mature CD11c + BMDCs. ( A ) Concentration dependence of immature (black bars) and mature (dark cyan bars) CD11c + BMDC responding fraction. ***, p
    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, 2022-10
    93/100 stars
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    Image Search Results


    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: 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: 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: 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: 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: 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: 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: 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

    TRPV4 deficiency does not alter cortical tubular apoptosis upon acute ischaemic kidney injury. ( A ) Representative ×200 images of TUNEL labelling on WT and Trpv4 KO kidney sections 24 hours after renal ischaemia. Thin white arrows indicate TUNEL–positive tubular cells. Thick white arrows indicate TUNEL–positive interstitial cells. Yellow bars represent 100 μm. ( B ) Number of TUNEL–positive cortical tubular cells per ×200 field. Each dot represents a single animal and is the mean of 5 randomly selected cortical fields. n = 6 for both WT and Trpv4 KO mice, two-tailed unpaired t-test. G, glomerulus; N, necrosis. ( C ) Serum lactate dehydrogenase (LDH) levels in WT and Trpv4 KO mice 24 hours after renal ischaemia. n = 5 for both WT and Trpv4 KO mice, two-tailed unpaired t-test, *P

    Journal: Scientific Reports

    Article Title: Transient Receptor Potential Vanilloid 4 Channel Deficiency Aggravates Tubular Damage after Acute Renal Ischaemia Reperfusion

    doi: 10.1038/s41598-018-23165-0

    Figure Lengend Snippet: TRPV4 deficiency does not alter cortical tubular apoptosis upon acute ischaemic kidney injury. ( A ) Representative ×200 images of TUNEL labelling on WT and Trpv4 KO kidney sections 24 hours after renal ischaemia. Thin white arrows indicate TUNEL–positive tubular cells. Thick white arrows indicate TUNEL–positive interstitial cells. Yellow bars represent 100 μm. ( B ) Number of TUNEL–positive cortical tubular cells per ×200 field. Each dot represents a single animal and is the mean of 5 randomly selected cortical fields. n = 6 for both WT and Trpv4 KO mice, two-tailed unpaired t-test. G, glomerulus; N, necrosis. ( C ) Serum lactate dehydrogenase (LDH) levels in WT and Trpv4 KO mice 24 hours after renal ischaemia. n = 5 for both WT and Trpv4 KO mice, two-tailed unpaired t-test, *P

    Article Snippet: ImmunofluorescenceFive μm thick cryosections were post-fixed in ice-cold acetone, air-dried, rehydrated and blocked with 10% normal donkey serum (Jackson ImmunoResearch) for 30 min. Next, sections were incubated overnight at 4 °C with the following primary antibodies: rat anti-Ly-6B.2 (Gr1) (1:300; MCA771G; AbD Serotec), rabbit anti-TRPV4 (1:50; acc-034, Alomond Labs), rabbit anti-aquaporin-1 (1:300; AB2219, Millipore), rabbit anti-sodium-chloride symporter (1:500; AB3553; Millipore), goat anti-aquaporin-2 (1:200, sc-9882; Santa Cruz Biotechnologies), rabbit anti-sodium-potassium-chloride cotransporter 2 (1:1000; a gift from Sebastian Bachmann, Charité Berlin).

    Techniques: TUNEL Assay, Mouse Assay, Two Tailed Test

    Expression of renal injury and pro-inflammatory markers. ( A ) Renal mRNA levels of lipocalin 2 ( Lcn2 ; also known as neutrophil gelatinase–associated lipocalin [ Ngal ]), ( B ) kidney injury molecule 1 ( Kim1 ) and pro–inflammatory cytokine ( C ) interleukin 1 beta ( Il1b ) and ( D ) Il6 and ( E ) tumor necrosis factor ( Tnf ) in sham and in Trpv4 KO and WT mice following IRI. Renal gene expression data are determined in n = 4 for sham-operated WT and Trpv4 KO mice and n = 6 for I/R–injured WT and Trpv4 KO mice, two-way ANOVA. In all cases P Treatment

    Journal: Scientific Reports

    Article Title: Transient Receptor Potential Vanilloid 4 Channel Deficiency Aggravates Tubular Damage after Acute Renal Ischaemia Reperfusion

    doi: 10.1038/s41598-018-23165-0

    Figure Lengend Snippet: Expression of renal injury and pro-inflammatory markers. ( A ) Renal mRNA levels of lipocalin 2 ( Lcn2 ; also known as neutrophil gelatinase–associated lipocalin [ Ngal ]), ( B ) kidney injury molecule 1 ( Kim1 ) and pro–inflammatory cytokine ( C ) interleukin 1 beta ( Il1b ) and ( D ) Il6 and ( E ) tumor necrosis factor ( Tnf ) in sham and in Trpv4 KO and WT mice following IRI. Renal gene expression data are determined in n = 4 for sham-operated WT and Trpv4 KO mice and n = 6 for I/R–injured WT and Trpv4 KO mice, two-way ANOVA. In all cases P Treatment

    Article Snippet: ImmunofluorescenceFive μm thick cryosections were post-fixed in ice-cold acetone, air-dried, rehydrated and blocked with 10% normal donkey serum (Jackson ImmunoResearch) for 30 min. Next, sections were incubated overnight at 4 °C with the following primary antibodies: rat anti-Ly-6B.2 (Gr1) (1:300; MCA771G; AbD Serotec), rabbit anti-TRPV4 (1:50; acc-034, Alomond Labs), rabbit anti-aquaporin-1 (1:300; AB2219, Millipore), rabbit anti-sodium-chloride symporter (1:500; AB3553; Millipore), goat anti-aquaporin-2 (1:200, sc-9882; Santa Cruz Biotechnologies), rabbit anti-sodium-potassium-chloride cotransporter 2 (1:1000; a gift from Sebastian Bachmann, Charité Berlin).

    Techniques: Expressing, Mouse Assay

    Analysis of renal tubular TRPV4 expression in the kidney of WT mice by immunostaining. ( A ) The proximal tubule marker aquaporin-1 (Aqp1), ( D ) the collecting duct marker aquaporin-2 (Aqp2) and ( C ) partly the distal convoluted tubule marker sodium-chloride symporter (Slc12a3) co-localize with TRPV4. ( B ) The thick ascending limb marker sodium-potassium-chloride cotransporter 2 (Slc12a2) does not co-localize with TRPV4. White squares mark equivalent tubular sections represented on adjacent sections. White arrows point to the same tubular structures seen on two serial cuts stained by different antibodies. Yellow bar represents 50 μm.

    Journal: Scientific Reports

    Article Title: Transient Receptor Potential Vanilloid 4 Channel Deficiency Aggravates Tubular Damage after Acute Renal Ischaemia Reperfusion

    doi: 10.1038/s41598-018-23165-0

    Figure Lengend Snippet: Analysis of renal tubular TRPV4 expression in the kidney of WT mice by immunostaining. ( A ) The proximal tubule marker aquaporin-1 (Aqp1), ( D ) the collecting duct marker aquaporin-2 (Aqp2) and ( C ) partly the distal convoluted tubule marker sodium-chloride symporter (Slc12a3) co-localize with TRPV4. ( B ) The thick ascending limb marker sodium-potassium-chloride cotransporter 2 (Slc12a2) does not co-localize with TRPV4. White squares mark equivalent tubular sections represented on adjacent sections. White arrows point to the same tubular structures seen on two serial cuts stained by different antibodies. Yellow bar represents 50 μm.

    Article Snippet: ImmunofluorescenceFive μm thick cryosections were post-fixed in ice-cold acetone, air-dried, rehydrated and blocked with 10% normal donkey serum (Jackson ImmunoResearch) for 30 min. Next, sections were incubated overnight at 4 °C with the following primary antibodies: rat anti-Ly-6B.2 (Gr1) (1:300; MCA771G; AbD Serotec), rabbit anti-TRPV4 (1:50; acc-034, Alomond Labs), rabbit anti-aquaporin-1 (1:300; AB2219, Millipore), rabbit anti-sodium-chloride symporter (1:500; AB3553; Millipore), goat anti-aquaporin-2 (1:200, sc-9882; Santa Cruz Biotechnologies), rabbit anti-sodium-potassium-chloride cotransporter 2 (1:1000; a gift from Sebastian Bachmann, Charité Berlin).

    Techniques: Expressing, Mouse Assay, Immunostaining, Marker, Staining

    Flow cytometric analysis of renal granulocyte infiltration. ( A ) Gating strategy: Pre–gating on live cells using Fixable Viability Dye eFluor 660 and further gating on single cells. ( B ) Representative flow cytometry data of infiltrating Ly6G–positive cells (granulocytes) and F4/80–positive cells (macrophages) in sham and I/R–injured kidneys of WT and Trpv4 KO mice. ( C ) Quantification of infiltrating Ly6G–positive cells and ( D ) F4/80-positive cells. n = 4 for sham-operated WT and Trpv4 KO mice and n = 6 for I/R–injured WT and Trpv4 KO mice, two-way ANOVA, Sidak’s multiple comparisons test. In both cases P Treatment

    Journal: Scientific Reports

    Article Title: Transient Receptor Potential Vanilloid 4 Channel Deficiency Aggravates Tubular Damage after Acute Renal Ischaemia Reperfusion

    doi: 10.1038/s41598-018-23165-0

    Figure Lengend Snippet: Flow cytometric analysis of renal granulocyte infiltration. ( A ) Gating strategy: Pre–gating on live cells using Fixable Viability Dye eFluor 660 and further gating on single cells. ( B ) Representative flow cytometry data of infiltrating Ly6G–positive cells (granulocytes) and F4/80–positive cells (macrophages) in sham and I/R–injured kidneys of WT and Trpv4 KO mice. ( C ) Quantification of infiltrating Ly6G–positive cells and ( D ) F4/80-positive cells. n = 4 for sham-operated WT and Trpv4 KO mice and n = 6 for I/R–injured WT and Trpv4 KO mice, two-way ANOVA, Sidak’s multiple comparisons test. In both cases P Treatment

    Article Snippet: ImmunofluorescenceFive μm thick cryosections were post-fixed in ice-cold acetone, air-dried, rehydrated and blocked with 10% normal donkey serum (Jackson ImmunoResearch) for 30 min. Next, sections were incubated overnight at 4 °C with the following primary antibodies: rat anti-Ly-6B.2 (Gr1) (1:300; MCA771G; AbD Serotec), rabbit anti-TRPV4 (1:50; acc-034, Alomond Labs), rabbit anti-aquaporin-1 (1:300; AB2219, Millipore), rabbit anti-sodium-chloride symporter (1:500; AB3553; Millipore), goat anti-aquaporin-2 (1:200, sc-9882; Santa Cruz Biotechnologies), rabbit anti-sodium-potassium-chloride cotransporter 2 (1:1000; a gift from Sebastian Bachmann, Charité Berlin).

    Techniques: Flow Cytometry, Mouse Assay

    Renal function and damage after renal ischaemia/reperfusion injury (IRI). ( A ) Serum creatinine levels of Trpv4 KO (▪) and WT (○) mice at baseline, 6 and 24 hours after renal IRI. Please note that at baseline all serum creatinine levels were below the measurement limit (18 µmol/L). n = 6 for both WT and Trpv4 KO mice, repeated measurement two-way ANOVA (P Time

    Journal: Scientific Reports

    Article Title: Transient Receptor Potential Vanilloid 4 Channel Deficiency Aggravates Tubular Damage after Acute Renal Ischaemia Reperfusion

    doi: 10.1038/s41598-018-23165-0

    Figure Lengend Snippet: Renal function and damage after renal ischaemia/reperfusion injury (IRI). ( A ) Serum creatinine levels of Trpv4 KO (▪) and WT (○) mice at baseline, 6 and 24 hours after renal IRI. Please note that at baseline all serum creatinine levels were below the measurement limit (18 µmol/L). n = 6 for both WT and Trpv4 KO mice, repeated measurement two-way ANOVA (P Time

    Article Snippet: ImmunofluorescenceFive μm thick cryosections were post-fixed in ice-cold acetone, air-dried, rehydrated and blocked with 10% normal donkey serum (Jackson ImmunoResearch) for 30 min. Next, sections were incubated overnight at 4 °C with the following primary antibodies: rat anti-Ly-6B.2 (Gr1) (1:300; MCA771G; AbD Serotec), rabbit anti-TRPV4 (1:50; acc-034, Alomond Labs), rabbit anti-aquaporin-1 (1:300; AB2219, Millipore), rabbit anti-sodium-chloride symporter (1:500; AB3553; Millipore), goat anti-aquaporin-2 (1:200, sc-9882; Santa Cruz Biotechnologies), rabbit anti-sodium-potassium-chloride cotransporter 2 (1:1000; a gift from Sebastian Bachmann, Charité Berlin).

    Techniques: Mouse Assay

    Treatment of primary proximal tubular cells (PTCs) of WT and Trpv4 KO mice with hypoxia-inducible factor 1-alpha (HIF1α)-stabilizing agent CoCl 2 . mRNA expression of ( A ) HIF1α target gene Vegfa, ( B ) stress cytokine interleukin 6 (Il6), bona fide NF-κB target gene nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IkBa), and ( D ) endoplasmatic reticulum stress marker CCAAT/enhancer binding protein homologous protein (Chop). Each dot is a mean of 2 technical repeats and represents a pool of PTCs isolated from 2–3 animals of the same genotype. Data of two independent experiments. n = 4 for untreated and n = 5 for CoCl 2 -treated WT and Trpv4 KO PTCs, two-way ANOVA. In all cases P Treatment

    Journal: Scientific Reports

    Article Title: Transient Receptor Potential Vanilloid 4 Channel Deficiency Aggravates Tubular Damage after Acute Renal Ischaemia Reperfusion

    doi: 10.1038/s41598-018-23165-0

    Figure Lengend Snippet: Treatment of primary proximal tubular cells (PTCs) of WT and Trpv4 KO mice with hypoxia-inducible factor 1-alpha (HIF1α)-stabilizing agent CoCl 2 . mRNA expression of ( A ) HIF1α target gene Vegfa, ( B ) stress cytokine interleukin 6 (Il6), bona fide NF-κB target gene nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IkBa), and ( D ) endoplasmatic reticulum stress marker CCAAT/enhancer binding protein homologous protein (Chop). Each dot is a mean of 2 technical repeats and represents a pool of PTCs isolated from 2–3 animals of the same genotype. Data of two independent experiments. n = 4 for untreated and n = 5 for CoCl 2 -treated WT and Trpv4 KO PTCs, two-way ANOVA. In all cases P Treatment

    Article Snippet: ImmunofluorescenceFive μm thick cryosections were post-fixed in ice-cold acetone, air-dried, rehydrated and blocked with 10% normal donkey serum (Jackson ImmunoResearch) for 30 min. Next, sections were incubated overnight at 4 °C with the following primary antibodies: rat anti-Ly-6B.2 (Gr1) (1:300; MCA771G; AbD Serotec), rabbit anti-TRPV4 (1:50; acc-034, Alomond Labs), rabbit anti-aquaporin-1 (1:300; AB2219, Millipore), rabbit anti-sodium-chloride symporter (1:500; AB3553; Millipore), goat anti-aquaporin-2 (1:200, sc-9882; Santa Cruz Biotechnologies), rabbit anti-sodium-potassium-chloride cotransporter 2 (1:1000; a gift from Sebastian Bachmann, Charité Berlin).

    Techniques: Mouse Assay, Expressing, Marker, Binding Assay, Isolation

    Immunolabeling of TRPV1 and TRPV4 in cochlear preparations of V3KO mice with normal hearing. (A,B) Representative images of TRPV1 (green; A ) and TRPV4 (green; B ) in HCs from the base turn of cochlear preparations from V3WT and V3KO mice with normal hearing. Cell bodies (myosin 7A, red) and cell nuclei (DAPI, blue) of OHCs in a three-row pattern and IHCs in a single-row pattern are shown. Scale bars = 10 μm. (C) IF intensity of TRPV1 and TRPV4 in cochlear OHCs of V3WT (solid bar) and V3KO (open bar) mice with normal hearing. Data are shown as mean ± SEM ( n = 5 per group). ** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Transient Receptor Potential Cation Channel Subfamily Vanilloid 4 and 3 in the Inner Ear Protect Hearing in Mice

    doi: 10.3389/fnmol.2019.00296

    Figure Lengend Snippet: Immunolabeling of TRPV1 and TRPV4 in cochlear preparations of V3KO mice with normal hearing. (A,B) Representative images of TRPV1 (green; A ) and TRPV4 (green; B ) in HCs from the base turn of cochlear preparations from V3WT and V3KO mice with normal hearing. Cell bodies (myosin 7A, red) and cell nuclei (DAPI, blue) of OHCs in a three-row pattern and IHCs in a single-row pattern are shown. Scale bars = 10 μm. (C) IF intensity of TRPV1 and TRPV4 in cochlear OHCs of V3WT (solid bar) and V3KO (open bar) mice with normal hearing. Data are shown as mean ± SEM ( n = 5 per group). ** P

    Article Snippet: The following primary antibodies were used: rabbit myosin-VIIa (Bioscience, Allentown, PA, USA), mouse myosin-VIIa (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rat phalloidin-iFluor 555 (Abcam, Cambridge, MA, USA), mouse or rabbit anti-TRPV3 antibody (Genentech, San Francisco, CA, USA), mouse anti-TRPV1 antibody and rabbit anti-TRPV4 antibody (Alomone Labs, Jerusalem, Israel).

    Techniques: Immunolabeling, Mouse Assay

    Model of the role of TRPV3 and TRPV4 in cochlear HCs in hearing. (A) Normal expression of TRPV3 and TRPV4 in cochlear HCs is critical for maintaining a normal number of HCs and normal hearing. (B) The loss of TRPV3 leads to compensatory upregulation of TRPV4 in cochlear HCs, which preserves the HC population and normal hearing. (C) Failure of compensatory upregulation or downregulation of TRPV4 in cochlear HCs under TRPV3 deficiency leads to cochlear impairment and hearing loss.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Transient Receptor Potential Cation Channel Subfamily Vanilloid 4 and 3 in the Inner Ear Protect Hearing in Mice

    doi: 10.3389/fnmol.2019.00296

    Figure Lengend Snippet: Model of the role of TRPV3 and TRPV4 in cochlear HCs in hearing. (A) Normal expression of TRPV3 and TRPV4 in cochlear HCs is critical for maintaining a normal number of HCs and normal hearing. (B) The loss of TRPV3 leads to compensatory upregulation of TRPV4 in cochlear HCs, which preserves the HC population and normal hearing. (C) Failure of compensatory upregulation or downregulation of TRPV4 in cochlear HCs under TRPV3 deficiency leads to cochlear impairment and hearing loss.

    Article Snippet: The following primary antibodies were used: rabbit myosin-VIIa (Bioscience, Allentown, PA, USA), mouse myosin-VIIa (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rat phalloidin-iFluor 555 (Abcam, Cambridge, MA, USA), mouse or rabbit anti-TRPV3 antibody (Genentech, San Francisco, CA, USA), mouse anti-TRPV1 antibody and rabbit anti-TRPV4 antibody (Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing

    The upregulation of TRPV4 protects V3KO mice against kanamycin challenge. (A) ABR thresholds in response to click and 3-ms pure tones of 8, 16, 24, and 32 kHz were measured in V3WT (solid bar) and V3KO (open bar) mice before (pre-KM) and after (post-KM) kanamycin administration ( n = 10 per group). (B,C) Immunolabeling of TRPV4 (green) in the apex, middle, and base of the cochlea from V3WT mice with impaired hearing and V3KO mice with normal hearing after kanamycin treatment. Cell bodies (myosin 7A, red) and nuclei (DAPI, blue) of cochlear HCs are shown. Scale bars = 10 μm. (D) IF intensity of TRPV4 in cochlear OHCs from V3WT mice with normal hearing after saline treatment (NS, solid bar), V3WT mice with impaired hearing after kanamycin (KM) treatment (gray bar), and V3KO mice with normal hearing after kanamycin treatment (open bar). (E) Quantitative analysis of the number of IHCs and OHCs per 100 μm in apex, middle, and base of cochlea from the above three groups. Data are shown as mean ± SEM ( n = 10 per group). * # P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Transient Receptor Potential Cation Channel Subfamily Vanilloid 4 and 3 in the Inner Ear Protect Hearing in Mice

    doi: 10.3389/fnmol.2019.00296

    Figure Lengend Snippet: The upregulation of TRPV4 protects V3KO mice against kanamycin challenge. (A) ABR thresholds in response to click and 3-ms pure tones of 8, 16, 24, and 32 kHz were measured in V3WT (solid bar) and V3KO (open bar) mice before (pre-KM) and after (post-KM) kanamycin administration ( n = 10 per group). (B,C) Immunolabeling of TRPV4 (green) in the apex, middle, and base of the cochlea from V3WT mice with impaired hearing and V3KO mice with normal hearing after kanamycin treatment. Cell bodies (myosin 7A, red) and nuclei (DAPI, blue) of cochlear HCs are shown. Scale bars = 10 μm. (D) IF intensity of TRPV4 in cochlear OHCs from V3WT mice with normal hearing after saline treatment (NS, solid bar), V3WT mice with impaired hearing after kanamycin (KM) treatment (gray bar), and V3KO mice with normal hearing after kanamycin treatment (open bar). (E) Quantitative analysis of the number of IHCs and OHCs per 100 μm in apex, middle, and base of cochlea from the above three groups. Data are shown as mean ± SEM ( n = 10 per group). * # P

    Article Snippet: The following primary antibodies were used: rabbit myosin-VIIa (Bioscience, Allentown, PA, USA), mouse myosin-VIIa (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rat phalloidin-iFluor 555 (Abcam, Cambridge, MA, USA), mouse or rabbit anti-TRPV3 antibody (Genentech, San Francisco, CA, USA), mouse anti-TRPV1 antibody and rabbit anti-TRPV4 antibody (Alomone Labs, Jerusalem, Israel).

    Techniques: Mouse Assay, Mass Spectrometry, Immunolabeling

    Immunolabeling of TRPV4 in HCs in cochlear preparations of V3KO mice with impaired hearing. (A) Representative images of TRPV4 (green) in HCs from the base turn of cochlear preparations in V3WT (top) and V3KO (middle) mice with normal hearing and V3KO mice with impaired hearing (bottom). Cell bodies (myosin 7A, red) and cell nuclei (DAPI, blue) are shown. Scale bars = 10 μm. (B) IF intensity of TRPV4 in the apex, middle, and base turn of cochlear OHCs from V3WT (solid bar) and V3KO (gray bar) mice with normal hearing and V3KO mice with impaired hearing (open bar). (C) Quantitative analysis of the number of cochlear HCs per 100 μm in the apex, middle, and base of cochlea samples from the above three groups. Data are shown as mean ± SEM ( n = 5 per group). * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Transient Receptor Potential Cation Channel Subfamily Vanilloid 4 and 3 in the Inner Ear Protect Hearing in Mice

    doi: 10.3389/fnmol.2019.00296

    Figure Lengend Snippet: Immunolabeling of TRPV4 in HCs in cochlear preparations of V3KO mice with impaired hearing. (A) Representative images of TRPV4 (green) in HCs from the base turn of cochlear preparations in V3WT (top) and V3KO (middle) mice with normal hearing and V3KO mice with impaired hearing (bottom). Cell bodies (myosin 7A, red) and cell nuclei (DAPI, blue) are shown. Scale bars = 10 μm. (B) IF intensity of TRPV4 in the apex, middle, and base turn of cochlear OHCs from V3WT (solid bar) and V3KO (gray bar) mice with normal hearing and V3KO mice with impaired hearing (open bar). (C) Quantitative analysis of the number of cochlear HCs per 100 μm in the apex, middle, and base of cochlea samples from the above three groups. Data are shown as mean ± SEM ( n = 5 per group). * P

    Article Snippet: The following primary antibodies were used: rabbit myosin-VIIa (Bioscience, Allentown, PA, USA), mouse myosin-VIIa (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rat phalloidin-iFluor 555 (Abcam, Cambridge, MA, USA), mouse or rabbit anti-TRPV3 antibody (Genentech, San Francisco, CA, USA), mouse anti-TRPV1 antibody and rabbit anti-TRPV4 antibody (Alomone Labs, Jerusalem, Israel).

    Techniques: Immunolabeling, Mouse 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