trpv4  (Alomone Labs)

Journal: iScience

Article Title: The role of arachidonic acid metabolites in the subtype classification and pathogenesis of primary aldosteronism

doi: 10.1016/j.isci.2025.114598

Figure Lengend Snippet: Arachidonic acid promotes Ca 2+ uptake and increases CYP11B2 expression in adrenal cortical cells (A) Confirmation of the expression of CYP11B2 in primary adrenal cortical cells. Scale bars, 50 μm. (B) Cell viability of primary adrenal cortical cells treated with different doses of arachidonic acid (AA) ( n = 4). (C) Aldosterone levels in cellular supernatant of primary adrenal cortical cells treated with different doses of AA ( n = 6). (D) Aldosterone levels in cellular supernatant of primary adrenal cortical cells treated with different doses of Ang II (left) or endothelin-1 (right) along with AA ( n = 3). (E) Representative western blots showing levels of CYP11B2, CYP11B1, CYP17A1, and HSD3B2 in primary adrenal cortical cells treated with vehicle or AA. The quantitative results are shown on the right ( n = 3). (F) Changes of cytoplasmic Ca 2+ , labeled with Fura-2 AM, in primary adrenal cortical cells treated with 1 mM thapsigargin (TG) stimulation in a 1 mM extracellular Ca 2+ solution after preincubation with vehicle or AA ( n = 12). (G) Changes of mitochondrial Ca 2+ , labeled with Rhod-2 AM, in digitonin-permeabilized primary adrenal cortical cells treated with 200 μM ATP stimulation in a 300 μM extracellular Ca 2+ solution after preincubation with vehicle or AA ( n = 12). (H) Changes of endoplasmic reticulum (ER) Ca 2+ , labeled with Mag Fura-2 AM, in digitonin-permeabilized primary adrenal cortical cells treated with ATP stimulation in a Ca 2+ -free extracellular solution after preincubation with vehicle or AA ( n = 12). (I) Representative western blots showing levels of KCNJ5, Na + /K + ATPase alpha-1 subunit, NCX-1, Letm 1, MCU, VDAC, RyR2, and IP 3 R in primary adrenal cortical cells treated with vehicle or AA. (Left) Representative western blots showing levels of TRPC1, TRPC3, TRPC6, TRPV1, and TRPV4 in primary adrenal cortical cells treated with vehicle or AA. (Right) The quantitative results are shown on the left ( n = 3). The results are expressed as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 compared with vehicle group; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 compared with 1 μM AA group by one-way ANOVA (B and C) and by Student’s t test (D–I).

Article Snippet: TRPV4 , Alomone , Cat#ACC-034; RRID: AB_2040264.

Techniques: Expressing, Western Blot, Labeling

Journal: eLife

Article Title: TRPV3 channel activity helps cortical neurons stay active during fever

doi: 10.7554/eLife.102412

Figure Lengend Snippet: ( A ) TRPV3 immunostaining (green) in mouse primary somatosensory (S1) cortex, striatum, thalamus, and hippocampus, with DAPI counterstain (blue). Top row left: Postnatal day (P)7; bottom row left: P14; bottom row right: P21. Top row right: Sections incubated with TRPV3 antibody plus TRPV3 blocking peptide in S1 cortex, striatum, thalamus, and hippocampus (green) with DAPI counterstain (blue). Remaining TRPV3 staining is shown in green with DAPI in blue. ( B ) TRPV3 and TRPV4 immunostaining in S1 cortex, striatum, thalamus, and hippocampus at P14. Top row left: TRPV3 (green) and TRPV4 (brick red). Top row right: TRPV3 (green) and TRPV4 (brick red) with DAPI counterstain (blue). Bottom row left: TRPV4 immunostaining in S1BF (inset from top row left) at 10x. Bottom row right: TRPV3 immunostaining in S1BF (inset from top row left) at 10x.

Article Snippet: Slides were blocked in 10% normal goat serum for 1 hr at room temperature, then incubated overnight at 4°C with anti-TRPV3-Biotin antibody (#ACC-033-B), TRPV3 blocking peptide (#BLP-CC033), and/or anti-TRPV4 antibody (#ACC-034) (Alomone Labs, Israel).

Techniques: Immunostaining, Incubation, Blocking Assay, Staining

Journal: eLife

Article Title: TRPV3 channel activity helps cortical neurons stay active during fever

doi: 10.7554/eLife.102412

Figure Lengend Snippet: ( A ) Setup for recording L4-evoked post-synaptic potential and spiking in an excitatory cortical pyramidal neuron (PN) with an intracellular blocker of TRPV3 channels (Forsythoside B, 50 µM) (left) or TRPV4 channels (RN1734, 10 µM) (right) at just-subthreshold V m at 30°C, 36°C, and 39°C in mouse S1 cortex. ( B ) Percentages of cell types obtained from experiment in A . ( C ) Evoked spikes in L2/3 cortical PNs during temperature elevations to 30°C, 36°C, and 39°C under three conditions: no blockers, TRPV3 blocker (Forsythoside B, 50 µM), or TRPV4 blocker (RN1734, 10 µM). ( D ) Correlation between post-synaptic potential (PSP) peak and spike threshold (ST). r =Pearson correlation coefficient with Deming linear regression. ( E ) Same as ( C ) for the L4-evoked late PSP peak. ( F ) Same as ( C ) for input resistance (R in ). Each data point in C – F represents an individual cell. Data were collected from 26 cells in 7 animals for the TRPV3 blocker, 24 cells in 6 animals for the TRPV4 blocker, and 37 cells in 14 animals for the no-block condition. Mean ± SEM is shown in C , E , and F . Statistical significance was assessed using one- or two-way repeated-measures ANOVA with Tukey’s or Sidak post-hoc test ( α =0.05). In D , correlations were evaluated using Pearson’s r with Deming linear regression.

Article Snippet: Slides were blocked in 10% normal goat serum for 1 hr at room temperature, then incubated overnight at 4°C with anti-TRPV3-Biotin antibody (#ACC-033-B), TRPV3 blocking peptide (#BLP-CC033), and/or anti-TRPV4 antibody (#ACC-034) (Alomone Labs, Israel).

Techniques: Blocking Assay

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: A. Schematic of P. aeruginosa mouse model with collection of bronchoalveolar lavage (BAL) cells and lung tissue homogenate in alveolar macrophage-specific Trpv4 KO mice ( Cd11c Cre: Trpv4 fl/fl ) and parental controls ( Trpv4 fl/fl ). Created with biorender.com . B. Percent BAL neutrophil, macrophage, and lymphocyte cells counts. n=14–15 mice per group. * p ≤ 0.05, p denotes macrophages in Cd11c Cre: Trpv4 fl/fl mice vs Trpv4 fl/fl mice, ** p ≤0.01, p denotes neutrophils Cd11c Cre: Trpv4 fl/fl vs Trpv4 fl/fl by Student’s t-test. C. CCL3 and CXCL1 chemokine secretion in BAL fluid (BALF) in Trpv4 fl/fl and Cd11cCre: Trpv4 fl/fl by LEGENDplex (pg/mL). n=14–15 mice per group. * p ≤ 0.05, p denotes Cd11c Cre: Trpv4 fl/fl vs Trpv4 fl/fl by Student’s t-test. D. Lung tissue homogenate utilized for flow cytometry with gating on percent CD45 + , F4/80 + , and Siglec-F − population for IL-1β and CCL2 in Cd11c Cre: Trpv4 fl/fl and Trpv4 fl/fl mice as quantified in E. n=14–15 mice per group. * p ≤ 0.05, p denotes IL-1β in Cd11c Cre: Trpv4 fl/fl mice vs Trpv4 fl/fl mice, *** p ≤ 0.001, p denotes CCL2 in Cd11c Cre: Trpv4 fl/fl mice vs Trpv4 fl/fl mice by Student’s t-test.

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Derivative Assay, Expressing, In Vivo, Flow Cytometry

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: A. The mRNA expression of NF-κB genes, including IL - 1β and Cxcl1 , were measured in BMDMs from WT and Trpv4 KO mice ± TLR agonists: TLR1/2 (Pam3CSK4, 100 ng/ml), TLR4 (LPS 100 ng/ml), TLR3 (Poly I:C 10 μg/ml), and TLR9 (CpGDNA 5 μg/ml) at 1 hour. n=3–4 biological replicates per group, *p ≤ 0.05, ** p ≤ 0.01 denotes WT vs KO ± TLR agonist. Student’s t-test comparing Trpv4 KO vs WT BMDMs within a TLR group. B. The same NF-κB genes in response to the TLR agonists were measured at 6 hours. n=3–4 biological replicates per group, *p ≤ 0.05, ** p ≤ 0.01 denotes WT vs KO ± TLR agonist. Student’s t-test comparing Trpv4 KO vs WT BMDMs within a TLR group. C. UMAP cluster plot shows 9 clusters in WT and Trpv4 KO BMDMs ± LPS after 10X genomics. D. Within the Il1β+_Cxcl2+ cluster, the GO BP gene pathway analysis comparing either LPS-stimulated WT or KO represented significant pathways as measured by −Log10 transformed p -value (x-axis; light green: increased KO vs WT, dark green: decreased KO vs WT). n=3 biological replicates per group.

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Expressing, Transformation Assay

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: A. The transcriptional activity of NF-κB response elements was evaluated in NF-κB luciferase transfected HeLa cells, additionally transfected with either empty vector (EV) or TRPV4 plasmid (TRPV4 overexpression) ± LPS (100 ng/mL, 16 hours) and BMDMs from mVenus-RELA (p65) reporter mice transfected with either control (Scrambled) siRNA or TRPV4 siRNA ± LPS (1 μg/mL, 1 hour). HeLa TRPV4 overexpression n=3 biological replicates per group, ****p ≤ 0.0001 , p denotes EV vs TRPV4 expression ± LPS by Student’s t-test and mVenus p65 BMDM. n=5 biological replicates per group, * p ≤ 0.05, ****p ≤ 0.0001 downregulation p denotes Scrambled siRNA vs TRPV4 siRNA ± LPS by Student’s t-test. B. BMDMs from WT and Trpv4 KO mice transfected with control (Scrambled) or p65 siRNA. IL1β secretion was measured by ELISA in the conditional medium from BMDMs treated with LPS (100 ng/mL, 24 hours). n=6 biological replicates per group, Student’s t-test shows no statistical difference. C. BMDMs from WT and Trpv4 KO mice were treated with LPS in the indicated time course. The protein levels of IκBα with representative blot and quantification. n=3 biological replicates per group, * p ≤ 0.05, p denotes IκBα protein WT vs KO at 10 minutes by Student’s t-test. D. BMDMs from WT and Trpv4 KO mice were treated with LPS in the indicated time course. The protein levels of nuclear and cytoplasmic fractions of NF-κB/p65 with representative blot and quantification of nuclear fraction. n=3 biological replicates per group, * p ≤ 0.05, p denotes p65 protein WT vs KO at 30 minutes by Student’s t-test.

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Activity Assay, Translocation Assay, Luciferase, Transfection, Plasmid Preparation, Over Expression, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: A. Sequence alignment of published structure of IκBα (blue) and TRPV4 N-terminal domain shows sequence similarity (grey residues with green selected for structural mapping) within the interacting interface (yellow box and yellow helices) of p65/p50 (red helices) using CLC genomics work bench (Qiagen). Within these homologous regions conserved residues between IκBα and TRPV4 are seen (#1–4, PDB:1NFI). B. 293T cells overexpressing the indicated proteins, Myc-tagged TRPV4 full-length (FL) and Flag-tagged p65, were co-immunoprecipitated for Myc and immunoblotted for Flag and Myc. n=3 biological replicates per group. C. WT BMDMs ± LPS (100 ng/mL, 1 hour) were co-immunoprecipitated for p65 and immunoblotted for p65 and TRPV4, Trpv4 KO as a negative control. n=3 biological replicates per group. D. Trpv4 KO BMDMs were transduced with lentivirus encoding either EV or Myc-tagged TRPV4 full-length ± LPS (100 ng/mL, 1 hour) and stained for Myc (green) and p65 (red); Scale bar: 50 μm, 40X original magnification; magnified cells (inset) shown to see location of TRPV4 and p65 interaction indicated by the yellow color and p65 and nucleus (DAPI) co-localization indicated by the pink color. p65 colocalization with Myc (TRPV4) (at least 25 high power fields were analyzed) and DAPI (40–50 cells were analyzed) was quantified by Pearson’s coefficient. n=3 biological replicates per group. **** p ≤ 0.0001, *** p ≤ 0.001, p denotes Myc-TRPV4 ± LPS by Student’s t-test. E. Schematic demonstrating BiFC method used to determine protein-protein interaction. F. HeLa cells co-transduced with plasmids encoding either empty vector (EV) constructs, VN-tagged TRPV4, or VC-tagged p65. TRPV4 and p65 bind as indicated by VN and VC green fluorescence in the perinuclear region (white arrows); nucleus blue, Scale bar: 75 μm; 40X original magnification; magnified cells (inset) to see location of TRPV4-p65 interaction. Whole image Venus intensity was quantified. n=3 biological replicates per group (at least 25 high power fields were analyzed). ** p ≤ 0.01, p denotes EV vs VN-TRPV4 and VC-p65 by Student’s t-test.

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Sequencing, Immunoprecipitation, Negative Control, Transduction, Staining, Plasmid Preparation, Construct, Fluorescence

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: A. Schematic of NanoBiT constructs with LargeBiT (Lg-BiT) empty vector (green), TRPV4 full length (blue), TRPV4 ANKRD deleted (ANKRD del; burnt orange) and SmallBiT (Sm-BiT) EV (purple) and p65 full length (red). B. 293T cells transfected with plasmids as in A exhibit luminescence with TRPV4 and p65 full length (blue and red stripes) that is decreased with TRPV4 ANKRD del (burnt orange and red stripes) with immunoblot confirmation of transfection efficiency ( Fig. S6 ). n=5 biological replicates per group, **** p ≤ 0.0001, p denotes TRPV4 ± ANKRD deletion by Student’s t-test. C. Trpv4 KO BMDMs transduced with LV as in A exhibit the same luminescence pattern as in B. n=3 biological replicates per group, **** p ≤ 0.0001, p denotes TRPV4 ± ANKRD deletion by Student’s t-test.

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Binding Assay, Construct, Plasmid Preparation, Transfection, Western Blot, Transduction

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: A. WT and Trpv4 KO BMDMs were treated with LPS for the indicated times and cell fractionation of cytosol and endoplasmic reticulum (ER) performed. Quantification of TRPV4 was performed by immunoblot of biochemically isolated cell fractions. n=3 biological replicates per group, * p ≤ 0.05, *** p ≤ 0.001, p denotes ± LPS by One-way ANOVA with Sidak’s MCT. B. Trpv4 KO BMDMs were transduced with Myc-tagged TRPV4 full-length (FL) (stained green) and stimulated with LPS (1 hour). ER was stained with calnexin (red). Co-localization between TRPV4 and calnexin was quantified by Pearson’s coefficient. Data represents scores of Myc-positive individual cells from combined experiments. Scale bar: 50 μm; 40X original magnification; magnified cells (inset) shown to see location of TRPV4 and calnexin interaction. n=3 biological replicates per group (10–40 cells analyzed per experiment), **** p ≤ 0.0001, p denotes ± LPS by Student’s t-test. C. BMDMs from WT mice were treated with LPS for the indicated times, and plasma membrane TRPV4 was measured by immunoblot of biochemically isolated cell fractions. n=3 biological replicates per group, * p ≤ 0.05, p denotes ± LPS by One-way ANOVA with Fischer’s LSD.

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Clinical Proteomics, Membrane, Cell Fractionation, Western Blot, Isolation, Transduction, Staining

Journal: Science signaling

Article Title: The mechanosensitive channel TRPV4 inhibits pulmonary inflammation by limiting NF-KB signaling in alveolar macrophages

doi: 10.1126/scisignal.adt1539

Figure Lengend Snippet: 1. Under basal conditions, TRPV4 N-terminal tail ANKRD domain interacts with the NF-κB subunit p65, in the complex with IκBα on the endoplasmic reticulum (ER) membrane to limit translocation of p65 to the nucleus for cytokine transcription and secretion. 2. Upon TLR agonism, IκKβ phosphorylates IκBα (3) , which leads IκBα dissociation and proteasome degradation (4). 5. An increased fraction of p65 dissociates from the ER and free p65 translocates to the nucleus thereby limiting pro-inflammatory cytokine transcription and secretion. 6. The dissociated TRPV4 fraction translocates to the plasma membrane. Created with biorender.com .

Article Snippet: Primary antibodies to extracellular and intracellular TRPV4 (Alomone Labs, Jerusalem Israel; Cell Signaling), GAPDH (Fitzgerald Industries International, Acton, MA), total NF-κB/p65 (Cell Signaling), IκBα (Santa Cruz Biotechnology), Integrin β-1 (BD biosciences), β-actin (Abcam), and GSK1016790A (GSK, Sigma-Aldrich) were obtained from commercial vendors.

Techniques: Membrane, Translocation Assay, Clinical Proteomics