rabbit polyclonal antibody against trpv4 channels  (Alomone Labs)

Journal: PLoS ONE

Article Title: Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells

doi: 10.1371/journal.pone.0011654

Figure Lengend Snippet: a. Co-immunoprecipitation of actin and tubulin with TRPV4. Cell extracts from CHO-KI cells stably expressing TRPV4 (lane 1) was immunoprecipitated by TRPV4 antibody (lane 2) or by a non-specific antibody (lane 3). Blots were probed for TRPV4 (left side), tubulin (middle) and actin (right side). b. Co-immunoprecipitation of tubulin with TRPV4. Extracts from DRG (lane 1) was immunoprecipitated by TRPV4 antibody (lane 2) or by a non-specific antibody (lane 3). Blots were probed for TRPV4 (upper panel) and tubulin (lower side). c. MBP-TRPV4-Ct (lane 2-3) but not MBP-LacZ (lane 4–5) forms specific complexes when incubated with mammalian brain extract (lane 1), both in presence (lane 2 and 4) or absence (lane 3 and 5) of Ca 2+ (1 mM). Presence of PKCε, actin and tubulin are observed only in lane 2 and 3. Neurofilament in the pull down samples is visible only after exposing for a prolonged time. Presence of CamKII is noted only in the presence of Ca 2+ (lane 2). Note that the amount of MBP-LacZ used, as a negative control for the pull down experiment is much more than MBP-TRPV4-Ct. d. Tubulin interacts with TRPV4-Ct directly. MBP-LacZ (lane 1–2) or MBP-TRPV4-Ct (lane 3–4) was incubated with buffer only (lane 1 and 3) or with purified tubulin (lane 2 and 4). Pulled down samples were probed for different isotype-specific and different post-translationally modified tubulins. e. Actin interacts directly with TRPV4-Ct. MBP-TRPV4-Ct (lane 1–2) or MBP-LacZ (lane 3–4) was incubated with purified actin (lane 1–4) either in the presence (lane 1-and 3) or absence (lane 2 and 4) of Ca 2+ (1 mM) and subsequently probed for bound actin. f. Soluble tubulin and actin competes for the C-terminal cytoplasmic fragment of TRPV4. MBP-TRPV4-Ct was incubated with only tubulin (lane 1), with only actin (lane 2), or both tubulin and actin in a sequential manner (lane 3–4). Prior incubation of tubulin inhibits further binding of actin (lane 3). Similarly, prior incubation of actin significantly reduces the further binding of tubulin (lane 4).

Article Snippet: Taxol, Nocodazole, 4αPDD, bovine actin, tetramethylrhodamine isothiocyanate-labelled IB4 from Griffonia simplicifolia , antibodies against α-tubulin (clone DM1A), β-tubulin (clone D66), tyrosinated tubulin (clone TUB1A2), polyglutamylated tubulin (clone B3), acetylated tubulin (clone 611-B-1), phospho-serine (Clone PSR-45), β-tubulin sub type III (clone SDL.3D10), neurofilament 116 kDa (clone NN18) and the affinity purified rabbit polyclonal antibody against C-terminal cytoplasmic domain of TRPV4 were purchased from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Immunoprecipitation, Stable Transfection, Expressing, Incubation, Negative Control, Purification, Modification, Binding Assay

Journal: PLoS ONE

Article Title: Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells

doi: 10.1371/journal.pone.0011654

Figure Lengend Snippet: MBP and MBP-TRPV4-Ct were centrifuged at 70000 g/30 min/4°C and only soluble proteins present in the supernatant were used for all co-sedimentation experiments. a. MBP-TRPV4-Ct co-sediments with polymerized actin filaments. Actin was polymerized either in presence of MBP-TRPV4-Ct (lane 1 and 4), in presence of MBP only (lane 2 and 5) or in buffer only (lane 3 and 6). Polymerized actin filaments and associated proteins were isolated from remaining soluble actin and unbound proteins by centrifugal separation of pellets ( P , lane 1–3) from corresponding supernatants ( S , lane 4–6). The entire amount of MBP remains in the supernatant (lane 5) while a significant amount of MBP-TRPV4-Ct appears in the pellet (lane 1). Arrows indicate the position of respective proteins. b. MBP-TRPV4-Ct co-sediments with microtubules. Taxol-stabilized microtubules (left panel) were incubated with MBP (lane 1–2), MBP-TRPV4-Ct (lane 3–4) or with buffer only (lane 5–6) followed by the centrifugal separation of pellet ( P ) consisting MT and bound proteins from supernatant ( S ) consisting of soluble tubulin and other unbound proteins (left side panel). In right side panel, soluble tubulin and GTP was incubated with MBP (lane 1–2), MBP-TRPV4-Ct (lane 3–4) or buffer only (lane 5–6) followed by separation of pellet ( P ) and supernatant ( S ). Note the specific presence of MBP-TRPV4-Ct in the pellet in both cases (in lane 4). c. MBP-TRPV4-Ct stabilizes microtubules against depolymerizing factors. Microtubules was formed form soluble tubulin in buffer (lane 1), along with MBP (lane 2) or along with MBP-TRPV4-Ct (lane 3) in control condition (left most panel), in presence of Nocodazole (middle left panel), in presence of Ca 2+ (middle right side) or in presence of both Nocodazole and Ca 2+ (right most). Microtubules and bound proteins present in the pellet fraction ( P ) were isolated from unpolymerized tubulin and unbound proteins remaining in the supernatant ( S ) by centrifugal separation. Note the enhancement of polymerized microtubules (represented by tubulin present in lane 3, P fraction in every conditions) due to the presence of MBP-TRPV4-Ct.

Article Snippet: Taxol, Nocodazole, 4αPDD, bovine actin, tetramethylrhodamine isothiocyanate-labelled IB4 from Griffonia simplicifolia , antibodies against α-tubulin (clone DM1A), β-tubulin (clone D66), tyrosinated tubulin (clone TUB1A2), polyglutamylated tubulin (clone B3), acetylated tubulin (clone 611-B-1), phospho-serine (Clone PSR-45), β-tubulin sub type III (clone SDL.3D10), neurofilament 116 kDa (clone NN18) and the affinity purified rabbit polyclonal antibody against C-terminal cytoplasmic domain of TRPV4 were purchased from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Sedimentation, Isolation, Incubation

Journal: PLoS ONE

Article Title: Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells

doi: 10.1371/journal.pone.0011654

Figure Lengend Snippet: a–c. Shown are the live-cell confocal images of F11 cells expressing TRV4-GFP (green) and RFP-actin (red). Presences of TRPV4-GFP specifically in actin-enriched structures are shown. a. Enlarged view of lamellipodia and at the tip of the actin filaments are shown. b–c. Enlarged view of focal adhesion point-like structures (b) and cell cortex with actin ribs (c) are shown. Arrows indicate the localization of TRPV4-GFP at filopodial tips. d–f. TRPV4 co-localizes with microtubule cytoskeleton. Shown are the confocal images of F11 cells immunostained for TRPV4 (green) and tyrosinated tubulin (red). Arrows indicate presence and acumulation of microtubules in thin filopodial structures (d, upper panel) and thin lamellipodial structures (e, middle panel). The status of the microtubules in the non-transfected cells are shown in below (f, lower panel).

Article Snippet: Taxol, Nocodazole, 4αPDD, bovine actin, tetramethylrhodamine isothiocyanate-labelled IB4 from Griffonia simplicifolia , antibodies against α-tubulin (clone DM1A), β-tubulin (clone D66), tyrosinated tubulin (clone TUB1A2), polyglutamylated tubulin (clone B3), acetylated tubulin (clone 611-B-1), phospho-serine (Clone PSR-45), β-tubulin sub type III (clone SDL.3D10), neurofilament 116 kDa (clone NN18) and the affinity purified rabbit polyclonal antibody against C-terminal cytoplasmic domain of TRPV4 were purchased from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Expressing, Transfection

Journal: PLoS ONE

Article Title: Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells

doi: 10.1371/journal.pone.0011654

Figure Lengend Snippet: a . Shown are the confocal time series images of live F11 cell expressing TRPV4-GFP (green) and RFP-Actin (red). Fluorescence images were superimposed on the DIC images. Addition of 4αPDD (1 µM) results in growth cone retraction of the transfected cell (T) but not from the non-transfected (NT) cells. b. Prolonged activation of endogenous TRPV4 reduces neurite outgrowth. Shown are the images of cultured DRG neurons stained for IB4 (red) and βIII tubulin (green). IB4-positive neurons extend their neurites in control condition (upper panel). Majority of the IB4-positive neurons do not produce any neurite when 4αPDD at low dose (0.1 µM) was applied for 36 hours (middle panel). An enlarged view of an IB4-positive and an IB4-negative neuron is shown in the lower panel. Note that the majority of the IB4-negative neurons remain unaffected even in the presence of 4αPDD. c. CHO-KI-TRPV4 cells that express low level of TRPV4 or CHO-KI-Mock cells that do not express TRPV4 were activated with 4αPDD (1 µM). After activation, cells were extracted by detergent in isotonic buffer and fixed subsequently by PFA. Cells were immunostained for actin (green) and tubulin (red). CHO-KI-TRPV4 cells loose all the peripheral microtubules but retain filamentous actin after activation and extraction. The stable MTOC regions are marked with arrows. In contrast, CHO-KI-Mock cells remain unaffected. Intensity of the microtubule is shown (red and blue indicate highest and lowest intensity respectively).

Article Snippet: Taxol, Nocodazole, 4αPDD, bovine actin, tetramethylrhodamine isothiocyanate-labelled IB4 from Griffonia simplicifolia , antibodies against α-tubulin (clone DM1A), β-tubulin (clone D66), tyrosinated tubulin (clone TUB1A2), polyglutamylated tubulin (clone B3), acetylated tubulin (clone 611-B-1), phospho-serine (Clone PSR-45), β-tubulin sub type III (clone SDL.3D10), neurofilament 116 kDa (clone NN18) and the affinity purified rabbit polyclonal antibody against C-terminal cytoplasmic domain of TRPV4 were purchased from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Expressing, Fluorescence, Transfection, Activation Assay, Cell Culture, Staining

Journal: PLoS ONE

Article Title: Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells

doi: 10.1371/journal.pone.0011654

Figure Lengend Snippet: Shown are the confocal images of live F11 cells expressing TRPV4-GFP (green) and Actin-RFP (red). a. Activation of TRPV4 results in merging of several actin-ribs at the tips and further transition of lamellipodial structures to filopodial structures. The arrow indicates the region and direction of the cell retraction at the same time. b. Activation of TRPV4 results in lateral initiation of filopodial structures from neurites and further elongation of them.

Article Snippet: Taxol, Nocodazole, 4αPDD, bovine actin, tetramethylrhodamine isothiocyanate-labelled IB4 from Griffonia simplicifolia , antibodies against α-tubulin (clone DM1A), β-tubulin (clone D66), tyrosinated tubulin (clone TUB1A2), polyglutamylated tubulin (clone B3), acetylated tubulin (clone 611-B-1), phospho-serine (Clone PSR-45), β-tubulin sub type III (clone SDL.3D10), neurofilament 116 kDa (clone NN18) and the affinity purified rabbit polyclonal antibody against C-terminal cytoplasmic domain of TRPV4 were purchased from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Expressing, Activation Assay

Journal: PLoS ONE

Article Title: Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells

doi: 10.1371/journal.pone.0011654

Figure Lengend Snippet: a. Taxol (1 µM, 30 minutes) reduces TRPV4-mediated Ca 2+ -influx in Cos7 cells. Shown are the normalized average ratiometric Ca 2+ -influx in arbitrary units (AU). TRPV4 was activated by a pulse (indicated by a black line) of 4αPDD for 10 sec. The dark blue line (C1) represents average response due to first pulse in control conditions (n = 32), the light blue line (C2) represents average response due to second pulse in control conditions (n = 32), the blackish green line (T1) represents average response due to first pulse under the influence of Taxol-stabilized microtubules (n = 42) and the light green line (T2) represents the average response due to second pulse under the Taxol-stabilized microtubules (n = 30). b. Cells with Taxol-stabilized microtubules reveal reductions in total Ca 2+ -influx due to TRPV4 activation. Total Ca 2+ -influx was calculated from the total area appeared by the ratiometric calcium-influx graph (as shown in figure a) for each cell and was calculated by Origin 7G software. At the <0.05 level, the difference of the population means between 2 nd pulse (untreated and taxol-treated, one sample t-test) is significant (*). The difference in case of 1 st pulse is non-significant. c. Taxol stabilized cells reveal a trend for time delay to reach in maximum response. The time (in seconds) each cell took to reach its maximum response (indicated by filled triangles) was plotted. Times needed during first pulse in control conditions (dark blue), second pulse in control conditions (light blue), first pulse with Taxol (blackish green) and second pulse with Taxol (light green) are indicated. The average values (indicated by filled squares) and the standard deviations are also shown for each condition. The average time differences remain non-significant.

Article Snippet: Taxol, Nocodazole, 4αPDD, bovine actin, tetramethylrhodamine isothiocyanate-labelled IB4 from Griffonia simplicifolia , antibodies against α-tubulin (clone DM1A), β-tubulin (clone D66), tyrosinated tubulin (clone TUB1A2), polyglutamylated tubulin (clone B3), acetylated tubulin (clone 611-B-1), phospho-serine (Clone PSR-45), β-tubulin sub type III (clone SDL.3D10), neurofilament 116 kDa (clone NN18) and the affinity purified rabbit polyclonal antibody against C-terminal cytoplasmic domain of TRPV4 were purchased from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Activation Assay, Software

Journal: Cardiology Journal

Article Title: Transient receptor potential channel TRPV4 mediates TGF- β 1-induced differentiation of human ventricular fibroblasts

doi: 10.5603/CJ.a2019.0050

Figure Lengend Snippet: Transforming growth factor beta (TGF-β) induces TRPV4 mRNA expression. Cardiac fibroblasts were treated with 2 ng/mL TGF-β and analyzed for the expression of transient receptor potential (TRP) channels by a polymerase chain reaction (PCR); A. Representative image of an agarose gel showing PCR amplification products; B. Semi-quantitative analysis of TRPV4 mRNA expression relative to α-actin (*p < 0.05 vs. untreated cells).

Article Snippet: Polyclonal antibodies against TRPV4 were obtained from Alomone Labs (Jerusalem, Israel), plasminogen activator inhibitor-1 (PAI-1)-specific antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), α -SMA antibody was from Abcam (Cambridge, MA, USA), and monoclonal antibodies against phosphorylated (p)-ERK and total ERK were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification

Journal: Cardiology Journal

Article Title: Transient receptor potential channel TRPV4 mediates TGF- β 1-induced differentiation of human ventricular fibroblasts

doi: 10.5603/CJ.a2019.0050

Figure Lengend Snippet: Transforming growth factor beta (TGF-β) induces TRPV4 protein expression and differentiation of ventricular fibroblasts. Cardiac fibroblasts were treated with 2 ng/mL TGF-β1 and analyzed for protein expression of TRPV4, smooth muscle actin alpha (α-SMA), and plasminogen activator inhibitor-1 (PAI-1) by Western blotting; A. Representative Western blotting image; B–D. Relative protein expression of TRPV (B), plasminogen activator inhibitor- 1 (PAI-1) (C), and smooth muscle actin alpha (α-SMA) (D) after TGF-β treatment (*p < 0.05, **p < 0.01, and ***p < 0.001, respectively, vs. untreated cells); con — control.

Article Snippet: Polyclonal antibodies against TRPV4 were obtained from Alomone Labs (Jerusalem, Israel), plasminogen activator inhibitor-1 (PAI-1)-specific antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), α -SMA antibody was from Abcam (Cambridge, MA, USA), and monoclonal antibodies against phosphorylated (p)-ERK and total ERK were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot

Journal: Cardiology Journal

Article Title: Transient receptor potential channel TRPV4 mediates TGF- β 1-induced differentiation of human ventricular fibroblasts

doi: 10.5603/CJ.a2019.0050

Figure Lengend Snippet: Transient receptor potential V4 (TRPV4) is involved in fibroblast differentiation. Cardiac fibroblasts (CFs) were pre-treated with the indicated concentrations of TRPV4 agonist GSK1016790A or antagonist RN-9893 and then treated with 2 ng/mL Transforming growth factor beta (TGF-β) for 24 h. CF conversion into myofibroblasts was assessed based on smooth muscle actin alpha (α-SMA) expression analyzed by Western blotting.

Article Snippet: Polyclonal antibodies against TRPV4 were obtained from Alomone Labs (Jerusalem, Israel), plasminogen activator inhibitor-1 (PAI-1)-specific antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), α -SMA antibody was from Abcam (Cambridge, MA, USA), and monoclonal antibodies against phosphorylated (p)-ERK and total ERK were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot

Journal: Cardiology Journal

Article Title: Transient receptor potential channel TRPV4 mediates TGF- β 1-induced differentiation of human ventricular fibroblasts

doi: 10.5603/CJ.a2019.0050

Figure Lengend Snippet: ERK activation is involved in TRPV4-mediated differentiation of human ventricular fibroblasts. Cardiac fibroblasts (CFs) were treated with 2 ng/mL of transforming growth factor beta (TGF-β) in the presence or absence of selective ERK inhibitor U0126 (10 μM), reactive oxygen species scavenger N-acetyl-L-cysteine (NAC 0.5 mM), or TRPV4 antagonist RN-9893 (50 μM) and analyzed for the expression of p-ERK (T202/Y204) and smooth muscle actin alpha (α-SMA).

Article Snippet: Polyclonal antibodies against TRPV4 were obtained from Alomone Labs (Jerusalem, Israel), plasminogen activator inhibitor-1 (PAI-1)-specific antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), α -SMA antibody was from Abcam (Cambridge, MA, USA), and monoclonal antibodies against phosphorylated (p)-ERK and total ERK were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Expressing

Journal: Cardiology Journal

Article Title: Transient receptor potential channel TRPV4 mediates TGF- β 1-induced differentiation of human ventricular fibroblasts

doi: 10.5603/CJ.a2019.0050

Figure Lengend Snippet: TRPV4 mediates Ca2+ influx required for cardiac fibroblasts (CFs) differentiation; A. Ca2+ influx was elicited by 2 mM Ca2+ and further augmented by the treatment with TRPV4 agonist GSK1016790A; B. Ca2+ influx was compared between control CFs (clear bar) and transforming growth factor beta (TGF-β)-treated CFs (gray bar); the treatment with TRPV4 agonist GSK1016790A further increased Ca2+ influx (*p < 0.05 vs. control).

Article Snippet: Polyclonal antibodies against TRPV4 were obtained from Alomone Labs (Jerusalem, Israel), plasminogen activator inhibitor-1 (PAI-1)-specific antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), α -SMA antibody was from Abcam (Cambridge, MA, USA), and monoclonal antibodies against phosphorylated (p)-ERK and total ERK were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques:

Journal: Cardiology Journal

Article Title: Transient receptor potential channel TRPV4 mediates TGF- β 1-induced differentiation of human ventricular fibroblasts

doi: 10.5603/CJ.a2019.0050

Figure Lengend Snippet: A signaling model for TRPV4-mediated differentiation of human ventricular fibroblasts. Transforming growth factor beta (TGF-β) signaling upregulates the expression of TRPV4, which induces Ca2+ influx and ERK1/2 activation, ultimately resulting in cardiac fibroblasts conversion into myofibroblasts; α-SMA — smooth muscle actin alpha.

Article Snippet: Polyclonal antibodies against TRPV4 were obtained from Alomone Labs (Jerusalem, Israel), plasminogen activator inhibitor-1 (PAI-1)-specific antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), α -SMA antibody was from Abcam (Cambridge, MA, USA), and monoclonal antibodies against phosphorylated (p)-ERK and total ERK were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Activation Assay