trpv4  (Alomone Labs)

Journal: Journal of Neuroinflammation

Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

doi: 10.1186/s12974-021-02315-8

Figure Lengend Snippet: TRPV4 activation led to enhanced expression of TNF receptor 1 (TNFR1). A Immunoblotting analysis showing that pre-injection of GSK101 enhanced the expression of TNFR1 in the retina, compared with the control group. B Bar chart summarizing mean expression levels of TNFR1 under different conditions. n = 5, p

Article Snippet: As negative controls, TRPV4 and GFAP antibodies were pre-adsorbed with TRPV4 blocking peptide (Alomone Labs) and GFAP blocking peptide (Bioss), respectively.

Techniques: Activation Assay, Expressing, Injection

Journal: Journal of Neuroinflammation

Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

doi: 10.1186/s12974-021-02315-8

Figure Lengend Snippet: TRPV4 activation led to increased phosphorylation of JAK2 and STAT3, thereby inducing NF-κB p65 translocation from the cytoplasm into the nucleus. A , C Immunoblotting analysis showing that GSK101 treatment increased the phosphorylation of STAT3 and JAK2, compared with the control group, but had no effect on the protein levels of JAK2 and STAT3 ( C ). B , D Bar charts summarizing mean expression levels of phosphorylated STAT3/STAT ( B ) and phosphorylated JAK2/JAK2 ( D ) under different conditions. n = 3 for all groups, * p

Article Snippet: As negative controls, TRPV4 and GFAP antibodies were pre-adsorbed with TRPV4 blocking peptide (Alomone Labs) and GFAP blocking peptide (Bioss), respectively.

Techniques: Activation Assay, Translocation Assay, Expressing

Journal: Journal of Neuroinflammation

Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

doi: 10.1186/s12974-021-02315-8

Figure Lengend Snippet: Müller cells express functional TRPV4. A Immunofluorescence images show that cells with red autofluorescence are Müller cells. A1 Immunofluorescence images show cells with red autofluorescence in rat retinal vertical slices acquired from TdTomato transgenic mouse retina (Tomato). A2 Immunofluorescence images show glutamine synthetase (GS) protein staining (green) in the slices depicted in A1 . A3 Immunofluorescence images show DAPI (blue) staining in the slices depicted in A1 . A4 shows merged images. Scale bar: 50 µm. B Representative trace recorded from a Müller cell identified by spontaneous red fluorescence, showing that perfusion with GSK101 (10 μM) caused significant depolarization of Müller membrane potential. C Bar chart showing GSK101-induced depolarization of Müller cell membrane potential. n = 4. * p

Article Snippet: As negative controls, TRPV4 and GFAP antibodies were pre-adsorbed with TRPV4 blocking peptide (Alomone Labs) and GFAP blocking peptide (Bioss), respectively.

Techniques: Functional Assay, Immunofluorescence, Transgenic Assay, Staining, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

doi: 10.1186/s12974-021-02315-8

Figure Lengend Snippet: TRPV4 activation enhances TNF-α production in retinal tissues. A Cumulative changes in TNF-α mRNA levels in saline-injected retinas (control) and retinas with GSK101 injection at 1 week. n = 5, * p

Article Snippet: As negative controls, TRPV4 and GFAP antibodies were pre-adsorbed with TRPV4 blocking peptide (Alomone Labs) and GFAP blocking peptide (Bioss), respectively.

Techniques: Activation Assay, Injection

Journal: Journal of Neuroinflammation

Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

doi: 10.1186/s12974-021-02315-8

Figure Lengend Snippet: TRPV4 activation enhances the expression of GFAP. A Immunofluorescence images show GFAP protein expression profiles in rat retinal vertical slices acquired from sham-operated retinas (saline-injected; control) ( A1 ), 1 µM GSK101-injected retinas ( A2 ), and 10 µM GSK101-injected retinas ( A3 ). Retinas that received no GFAP antibody served as negative controls ( A4 ). Double immunofluorescence staining showing GFAP expression when the GFAP antibody was pre-adsorbed with its blocking peptide (BP) ( A5 ). Scale bar: 20 µm. B Representative immunoblots showing changes in GFAP protein levels in control and 10 µM GSK101-injected retinas. C Bar chart summarizing mean expression levels of GFAP under different conditions. n = 6. * p

Article Snippet: As negative controls, TRPV4 and GFAP antibodies were pre-adsorbed with TRPV4 blocking peptide (Alomone Labs) and GFAP blocking peptide (Bioss), respectively.

Techniques: Activation Assay, Expressing, Immunofluorescence, Injection, Double Immunofluorescence Staining, Blocking Assay, Western Blot

Journal: Journal of Neuroinflammation

Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

doi: 10.1186/s12974-021-02315-8

Figure Lengend Snippet: TNF-α inhibition reduces TRPV4-mediated retinal cell apoptosis. A1 – A2 DAPI staining in GSK101-injected ( A1 ) and R7050 with GSK101-injected (R7050 + GSK101) ( A2 ) whole flat-mounted retinas at 7 days after injections in the regions at angle 0°. A3 – A4 Counterstained images with TUNEL staining detection of cell apoptosis (green). A5 – A6 Merged images of corresponding TUNEL and DAPI images. Scale bar, 50 µm (for all images). B Bar chart summarizing mean numbers of TUNEL-positive cells in each retina under different conditions. R7050 (1 µM, 2 μl) was pre-injected 1 day before the GSK101 injection. n = 5. ** p

Article Snippet: As negative controls, TRPV4 and GFAP antibodies were pre-adsorbed with TRPV4 blocking peptide (Alomone Labs) and GFAP blocking peptide (Bioss), respectively.

Techniques: Inhibition, Staining, Injection, TUNEL Assay

Journal: Journal of Neuroinflammation

Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

doi: 10.1186/s12974-021-02315-8

Figure Lengend Snippet: TRPV4 activation enhances TNF-α production in cultured Müller cells. A Morphology of cultured Müller cells. Scale bar: 50 µm. B GSK101 treatment enhanced GFAP protein levels in cultured Müller cells. n = 4. ** p

Article Snippet: As negative controls, TRPV4 and GFAP antibodies were pre-adsorbed with TRPV4 blocking peptide (Alomone Labs) and GFAP blocking peptide (Bioss), respectively.

Techniques: Activation Assay, Cell Culture

Journal: Journal of Neuroinflammation

Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

doi: 10.1186/s12974-021-02315-8

Figure Lengend Snippet: Changes in TRPV4 protein levels in retinas of rats with COH. A Representative immunoblots showing changes in TRPV4 protein levels in control and COH retinal extracts at different postoperative times (G1w, G2w, and G3w). B Bar chart summarizing mean expression levels of TRPV4 at different postoperative times. Data are presented as means ± standard errors of the mean. n = 7, 4, 6, and 5, respectively. ** p

Article Snippet: As negative controls, TRPV4 and GFAP antibodies were pre-adsorbed with TRPV4 blocking peptide (Alomone Labs) and GFAP blocking peptide (Bioss), respectively.

Techniques: Western Blot, Expressing

Journal: Journal of Neuroinflammation

Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

doi: 10.1186/s12974-021-02315-8

Figure Lengend Snippet: Schematic diagram showing the signaling pathway involved in TRPV4 activation-mediated TNF-α production in Müller cells and RGC apoptosis in COH retinas. NF-κB nuclear factor-kappa B, TNF-α tumor necrosis factor-α, TNFR1 TNF receptor 1

Article Snippet: As negative controls, TRPV4 and GFAP antibodies were pre-adsorbed with TRPV4 blocking peptide (Alomone Labs) and GFAP blocking peptide (Bioss), respectively.

Techniques: Activation Assay

Journal: Journal of Neuroinflammation

Article Title: TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway

doi: 10.1186/s12974-021-02315-8

Figure Lengend Snippet: Effects of GSK101 on retinal ganglion cell (RGC) apoptosis and survival. A TRPV4 activation leads to enhanced RGC apoptosis. A1 – A3 4′,6-diamidino-2-phenylindole (DAPI) staining in saline-injected (control) ( A1 ), GSK101-injected (GSK101) ( A2 ), and HC-067 with GSK101 (HC) ( A3 ) retinas at 1 week after injection in the regions at angle 0°. Images were acquired from whole flat-mounted retinal preparations. A4 – A6 , Counterstained images with TUNEL staining detection of RGC apoptosis (green). A7 – A9 , Merged images of corresponding TUNEL and DAPI images. Scale bar: 50 µm. B Bar chart summarizing mean numbers of TUNEL-positive cells in each retina under different conditions. n = 5 for all groups. C Pre-application of HC-067 reduces RGC apoptosis in COH retinas. C1 – C3 DAPI staining in sham-operated (control) ( C1 ), COH ( C2 ), and HC-067 with COH (HC + COH) ( C3 ) retinas at G2w. C4 – C6 Counterstained images with TUNEL staining detection of RGC apoptosis (green). C7 – C9 Merged images of corresponding TUNEL and DAPI images. Scale bar, 50 μm. D Bar chart summarizing mean numbers of TUNEL-positive cells in each retina under different conditions. n = 6 for all groups. * p

Article Snippet: As negative controls, TRPV4 and GFAP antibodies were pre-adsorbed with TRPV4 blocking peptide (Alomone Labs) and GFAP blocking peptide (Bioss), respectively.

Techniques: Activation Assay, Staining, Injection, TUNEL Assay

Journal: BioMed Research International

Article Title: Role of TRPV4 in the Diagnosis and Treatment of Helicobacter pylori Infection in Children with Duodenal Ulcers

doi: 10.1155/2022/2777882

Figure Lengend Snippet: FD20 content in serum. In Ctrl and DU groups, after treatment with TRPV4 agonist GSK1016790 A or inhibitor HC067047, the content of FD20 in serum was detected. Ctrl: nonduodenal ulcer; DU: duodenal ulcer. n = 3, ∗ P

Article Snippet: Subsequently, the sections were incubated with primary antibody to TRPV4 (Alomone labs, America) overnight at 4°C prior to incubation with an anti-rabbit secondary antibody (Biosharp, Hefei, China).

Techniques:

Journal: BioMed Research International

Article Title: Role of TRPV4 in the Diagnosis and Treatment of Helicobacter pylori Infection in Children with Duodenal Ulcers

doi: 10.1155/2022/2777882

Figure Lengend Snippet: The content of inflammatory factor TNF- α in tissues and serum. (a) In the Ctrl and DU groups, serum TNF- α content changes after treatment with TRPV4 agonists GSK1016790 A or inhibitor HC067047. (b) In the Ctrl and DU groups, duodenal tissue TNF- α content changes after treatment with TRPV4 agonists GSK1016790 A or inhibitor HC067047. Ctrl: nonduodenal ulcer; DU: duodenal ulcer. n = 3, ∗ P

Article Snippet: Subsequently, the sections were incubated with primary antibody to TRPV4 (Alomone labs, America) overnight at 4°C prior to incubation with an anti-rabbit secondary antibody (Biosharp, Hefei, China).

Techniques:

Journal: BioMed Research International

Article Title: Role of TRPV4 in the Diagnosis and Treatment of Helicobacter pylori Infection in Children with Duodenal Ulcers

doi: 10.1155/2022/2777882

Figure Lengend Snippet: TRPV4 is highly expressed in children with duodenal ulcer and has good diagnostic value. (a) and (b) Immunohistochemistry representative images (a) and summary (b) data showing the expression of TRPV4 in children with duodenal ulcer; Ctrl: nonduodenal ulcer; DU: duodenal ulcer. (c) ROC curve analyses of TRPV4. n = 11, ∗ P

Article Snippet: Subsequently, the sections were incubated with primary antibody to TRPV4 (Alomone labs, America) overnight at 4°C prior to incubation with an anti-rabbit secondary antibody (Biosharp, Hefei, China).

Techniques: Diagnostic Assay, Immunohistochemistry, Expressing

Journal: BioMed Research International

Article Title: Role of TRPV4 in the Diagnosis and Treatment of Helicobacter pylori Infection in Children with Duodenal Ulcers

doi: 10.1155/2022/2777882

Figure Lengend Snippet: Increased TRPV4 expression and enhanced calcium influx in duodenal ulcer mice. (a) Representative images showing hematoxylin-eosin staining of duodenum tissue. (b) and (c) Representative images (b) and summary (c) data showing the expression of TRPV4 in duodenal ulcer mice. (d) and (e) Representative traces (d) and summarized data (e) showing the changes in the intracellular Ca 2+ concentration of duodenal tissue between DU and Ctrl group. Ctrl: nonduodenal ulcer; DU: duodenal ulcer. n = 3, ∗ P

Article Snippet: Subsequently, the sections were incubated with primary antibody to TRPV4 (Alomone labs, America) overnight at 4°C prior to incubation with an anti-rabbit secondary antibody (Biosharp, Hefei, China).

Techniques: Expressing, Mouse Assay, Staining, Concentration Assay

Journal: bioRxiv

Article Title: TRPV4 interacts with mitochondrial proteins and acts as a mitochondrial structure-function regulator

doi: 10.1101/330993

Figure Lengend Snippet: Endogenous TRPV4 regulates mitochondrial morphology in primary cell. a. Representative image showing the presence of endogenous TRPV4 in HUVEC cell. Western blot analysis of HUVEC cells for TRPV4 is shown below. b. HUVEC cells were treated with TRPV4 agonist (4αPDD) and antagonist (RN1734) and subsequently immunostained for Hsp60. Representative images depict that in presence of 4αPDD (5 µM) mitochondria become aggregated in the perinuclear region (arrows). Mitochondrial morphology was virtually normal as compared to DMSO control in the presence of inhibitor RN1734 and control. Scale bar: 5 μm.

Article Snippet: The TRPV4-specific peptide (CDGHQQGYAPKWRAEDAPL, used as a blocking peptide of the TRPV4 antibody) was purchased from Alomone (Jerusalem, Israel).

Techniques: Western Blot

Journal: bioRxiv

Article Title: TRPV4 interacts with mitochondrial proteins and acts as a mitochondrial structure-function regulator

doi: 10.1101/330993

Figure Lengend Snippet: TRPV4 is endogenously present in mitochondria. a. Shown are the super resolution images of mitochondria isolated from rat brain and immunostained for TRPV4 (green). Isolated mitochondria were labelled with MitoTracker Red (upper and middle panel) or immunostained for Hsp60 (green, lower panel). TRPV4 is present in a subset of mitochondria, but not in all. b. Western blot analysis of different mitochondrial fractions (S1, S2 and Mitochondrial fraction) isolated from rat brain are shown. Endogenous TRPV4 is present in isolated mitochondrial fraction as well as in both inner and outer membrane fractions. c. Western blot analysis of mitochondrial fraction isolated from adipocytes, ChoKI-TRPV4 cells and CHO-KI-Mock cells probed with anti TRPV4 antibody in absence or presence of specific blocking peptide is shown. Immunoreactivity at the specific size (100 kDa) or at higher molecular weight is indicated by arrows. d-e. Quantification of relative abundance of TRPV4 in outer and inner mitochondrial membrane is shown (total mitochondrial TRPV4 is considered as 100%). Majority of the endogenous TRPV4 is present in the inner membrane rather than in outer membrane of mitochondria isolated from brain (ns = non-significant, n = 4, P value = 0.1181). In contrast, majority of the TRPV4 is present in the outer membrane rather than in the inner membrane of the mitochondria isolated from CHOKI-TRPV4 cell (n = 6, P value = 0.0141, n = 6).

Article Snippet: The TRPV4-specific peptide (CDGHQQGYAPKWRAEDAPL, used as a blocking peptide of the TRPV4 antibody) was purchased from Alomone (Jerusalem, Israel).

Techniques: Isolation, Western Blot, Blocking Assay, Molecular Weight

Journal: bioRxiv

Article Title: TRPV4 interacts with mitochondrial proteins and acts as a mitochondrial structure-function regulator

doi: 10.1101/330993

Figure Lengend Snippet: TRPV4 localizes in sperm mitochondria of different vertebrates and activation of TRPV4 disrupts mitochondrial coiling. a. Confocal images demonstrating the colocalization (indicated by arrows) of TRPV4 (green) with MitoTracker-Red labelled structures in fish, duck and human sperm. Some but not all sperm mitochondria revel the presence of TRPV4. In case of duck sperm, apart from mid piece region, colocalization is also observed at the tip of the head. b. Confocal images depicting the mitochondrial abnormality observed in bull sperm when TRPV4 is activated by 4αPDD (1 μM, 2 hours at 37°C). Activation induces blabbing or node-like structure (indicated by arrow) in the mid-piece regions. In some cases, sperm mitochondrial structure becomes fragmented into two halves. c. Shown are the super resolution (SIM) images demonstrating the mitochondrial coiling pattern in control and TRPV4-activated conditions. In control condition, mitochondrial coiling is intact and form regular helix-like structure. In TRPV4-activated condition, the regular helical organization is disrupted (kinks or cervices in the mitochondrial coiling region are indicated by arrows). For better visualization enlarged images are presented in each panel. Models showing the Ca 2+ -influx and abnormalities in the mitochondrial coiling in presence of TRPV4 activator. Scale bar: 5 μm (for left side panel) and 1 μm (for enlarged image).

Article Snippet: The TRPV4-specific peptide (CDGHQQGYAPKWRAEDAPL, used as a blocking peptide of the TRPV4 antibody) was purchased from Alomone (Jerusalem, Israel).

Techniques: Activation Assay, Fluorescence In Situ Hybridization

Journal: bioRxiv

Article Title: TRPV4 interacts with mitochondrial proteins and acts as a mitochondrial structure-function regulator

doi: 10.1101/330993

Figure Lengend Snippet: Modulation of TRPV4 alters mitochondrial morphology. a-c. CHOK1-V4 ( a ) or in CHOK1-Mock cells ( b ) expressing MitoDsRed was used and TRPV4 was activated with 4αPDD (5µM) or inhibited by RN1734 (10µM) for 8 hours. Mitochondria in CHOK1-V4 cells become spherical or round ball-like in shape after activation with 4αPDD (shown as arrow head) as compared to cylindrical or rod-like normal mitochondria observed in CHOK1-Mock cell (lower panel). TRPV4 activation leads to mitochondrial aggregation in TRPV4-positive mitochondria as compared to other conditions. The digitalized image of mitoDsRed intensity and an enlarged view filed of the same are represented on the right hand side. Arrowheads and arrows are indicating the round/spherical mitochondria and aggregated mitochondria respectively. Scale bar is 5 μm. c. The schematic model represents the abnormalities in the mitochondrial number and morphology. In case of TRPV4 activation, several mitochondria fuse together and form aggregated mitochondria as compared to other conditions. d-h. Quantitative changes in mitochondrial morphology in response to TRPV4 activation or inhibition is represented. For quantitative analysis, images of mitoDsRed expressing CHOK1-V4 or CHOK1-Mock cells were processed and all mitochondrial parameters were calculated. Represented graphs depict mitochondrial area (d), perimeter (e), area/perimeter (f), which increases significantly after TRPV4 activation in CHOK1-V4 cells. Aspect Ratio (AR) of mitochondria remain unchanged in presence of TRPV4 agonist in CHOK1-V4 cells (g). However, in CHOK1-Mock cells mitochondrial AR is higher (indicating elongated mitochondria) as compared to TRPV4-positive cell. The Form Factor (h) of TRPV4-postive mitochondria is 1000 fold higher in presence of 4αPDD as compared to control indicating that in 4αPDD treated condition mitochondria aggregates and gets interconnected to each other.

Article Snippet: The TRPV4-specific peptide (CDGHQQGYAPKWRAEDAPL, used as a blocking peptide of the TRPV4 antibody) was purchased from Alomone (Jerusalem, Israel).

Techniques: Expressing, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: TRPV4 interacts with mitochondrial proteins and acts as a mitochondrial structure-function regulator

doi: 10.1101/330993

Figure Lengend Snippet: TRPV4 does not colocalizes with other subcellular organelles. Confocal images of HaCaT cells transiently expressing TRPV4-GFP and sub-cellular marker proteins such as ER-CFP, Golgi-CFP and Peroxisome-CFP are shown. Cells were also labelled with Lysotracker Red and ER-tracker are shown. Similarly, HaCaT cells expressing TRPV4-GFP are immuno-labelled for sub-cellular organelles specific markers, such as with anti GM130 ab, anti-Calnexin ab and anti-KDEL ab, Enlarged images (indicated by white dotted line) represent the perinuclear region in details. Scale bar: 5 µm (for enlarged image).

Article Snippet: The TRPV4-specific peptide (CDGHQQGYAPKWRAEDAPL, used as a blocking peptide of the TRPV4 antibody) was purchased from Alomone (Jerusalem, Israel).

Techniques: Expressing, Marker

Journal: bioRxiv

Article Title: TRPV4 interacts with mitochondrial proteins and acts as a mitochondrial structure-function regulator

doi: 10.1101/330993

Figure Lengend Snippet: TRPV4 regulates mitochondrial membrane potential and Electron Transport Chain functions. a. CHOK1-V4 and CHOK1-Mock cells were treated with TRPV4 activator 4αPDD (5 μM) and inhibitor RN1734 (10 μM) for 8 hours. Subsequently JC-1 (5 μM) was added and confocal images were acquired by dual excitation wavelength 488nm (Green, for mitochondria with formation of JC-1 monomers at low mitochondrial potential) and 535nm (Red, for mitochondria with formation of J-aggregates at high membrane potentials). Scale bar is 10 μm. b. Red/Green intensity (in arbitrary unit) of more than 15 view fields for each condition was calculated by ImageJ. TRPV4 activation significantly decreases the mitochondrial membrane potential as compared to the control. Basal level mitochondrial potential is higher in CHOK1-Mock cell as compared to CHOK1-V4 cells. Statistical paired two test was performed and P-values are significant (n = 3). Bar graph representing the ±SEM. c-f. TRPV4 activation alters mitochondrial (ETC). Enzymatic activity of mitochondrial Electron Transport Chain complex I, II, III and IV was determined in isolated mitochondria purified from brain. Mitochondria were pre incubated with TRPV4 activators or inhibitors and the samples were analysed for enzymatic activity. Results indicate that enzymatic activities of Complex I and III are not altered significantly in presence of TRPV4 activator or inhibitor (c e). However enzymatic activities of complex II and IV is significantly altered due to TRPV4 activation or inhibition (d f). In each case, Ionomycin (Ca 2+ ionophore) and complex chain inhibitor shows significant decrease in complex activity. g. TRPV4 regulates Membrane Permeability Transition (MPT) in isolated mitochondria. TRPV4 activation by 4αPDD induces more MPT as compared to control. Similarly, TRPV4 inhibition by RN1734 reduces the formation of MPT inside the mitochondria. CaCl2 is used as a positive control for MPT.

Article Snippet: The TRPV4-specific peptide (CDGHQQGYAPKWRAEDAPL, used as a blocking peptide of the TRPV4 antibody) was purchased from Alomone (Jerusalem, Israel).

Techniques: Activation Assay, Activity Assay, Isolation, Purification, Incubation, Inhibition, Permeability, Positive Control

Journal: bioRxiv

Article Title: TRPV4 interacts with mitochondrial proteins and acts as a mitochondrial structure-function regulator

doi: 10.1101/330993

Figure Lengend Snippet: The C-terminus of TRPV4 interacts with different mitochondrial proteins. a. Schematic diagram representing the deletion constructs of TRPV4 used for the co-localization study. b. Confocal images representing the localization of TRPV4-Nt-RFP, TRPV4-TM-GFP and TRPV4-Ct-RFP with respect to mitochondrial markers. c. Equal amounts of purified MBP-TRPV4-Ct and MBP-LacZ was used for binding assay with intact mitochondria in presence or absence of Ca 2+ and GTP/ATP. Eluted protein samples were subsequently probed with anti-MBP and Hsp60 antibodies. d. Purified MBP-TRPV4-Ct or MBP-LacZ was used for pull down experiments with mitochondrial lysate in presence of Ca 2+ and GTP/ATP independently. Western blot analysis indicates that MBP-TRPV4-Ct, but not MBP-LacZ interacts with Hsp60, Mfn2, Mfn1 independently of the presence of Ca 2+ and GTP/ATP. Other mitochondrial proteins such as Opa1, DRP1 and Cyt C are not detectable among the eluted proteins. Presence of MBP-TRPV4-Ct or MBP-LacZ at equal amounts in the final eluted fractions is demonstrated by Coomassie staining. e-f. His-Mfn1 and His-Mfn2 interact directly with MBP-TRPV4-Ct but not with MBP-lacZ. Purified His-Mfn1 or His-Mfn2 immobilized on Ni-NTA beads and purified MBP-TRPV4-Ct or MBP-LacZ were incubated under various conditions as indicated and analysed by immunoblot analyses. The values mentioned in the right side represents the percentage [with respect to the input] of protein eluted. (In: Input amount of MBP-TRPV4-Ct/MBP-LacZ, FT: Supernatant collected after incubation, W2: 2nd wash fraction, Mito: Mitochondrial pellet fraction).

Article Snippet: The TRPV4-specific peptide (CDGHQQGYAPKWRAEDAPL, used as a blocking peptide of the TRPV4 antibody) was purchased from Alomone (Jerusalem, Israel).

Techniques: Construct, Purification, Binding Assay, Western Blot, Staining, Incubation

Journal: bioRxiv

Article Title: TRPV4 interacts with mitochondrial proteins and acts as a mitochondrial structure-function regulator

doi: 10.1101/330993

Figure Lengend Snippet: TRPV4 colocalizes with mitochondrial markers. a. Confocal images of HaCaT cells transiently expressing TRPV4-GFP and labelled with MitoTracker-Red. b-c. HaCaT cells transiently expressing TRPV4 immunostained with specific antibodies detecting Cytochrome C (b) and Hsp60 (c) are shown. TRPV4 colocalizes with these markers in a subset (indicated by arrows) but not in all mitochondria. d. Cells co-expressing Mito-RFP and TRPV4-GFP shows colocalization of TRPV4 with MitoDsRed. e. Confocal images of live HaCaT cells transiently expressing TRPV4-GFP and mitoDsRed. Enlarged confocal images depicting that in case of low expressing TRPV4-GFP positive cells, TRPV4 perfectly colocalizes with MitoDsRed within the mitochondria (indicated by arrows).

Article Snippet: The TRPV4-specific peptide (CDGHQQGYAPKWRAEDAPL, used as a blocking peptide of the TRPV4 antibody) was purchased from Alomone (Jerusalem, Israel).

Techniques: Expressing

Journal: bioRxiv

Article Title: TRPV4 interacts with mitochondrial proteins and acts as a mitochondrial structure-function regulator

doi: 10.1101/330993

Figure Lengend Snippet: TRPV4 regulates mitochondrial Ca 2+ -influx. Mito-pericam was transiently expressed in CHOK1-V4 or CHOK1-Mock cells and live cell imaging was performed to monitor the effect of TRPV4 activation (4αPDD, 5 μM) (a-b) or inhibition (RN1734, 10 μM) (c-d). Addition of 4αPDD causes massive mitochondrial Ca 2+ -influx in CHOK1-V4 cell (a). However addition of RN1734, reduces the mitochondrial Ca 2+ -level with time (c) . In CHOK1-Mock cell, Ca 2+ -levels remain mostly unchanged in the presence of TRPV4 activator (b) or inhibitor (d). Ca 2+ intensity graphs of multiple alive cells in each condition was quantified by ImageJ are represented on the right side). Thick dark black line represents the average value of mitochondrial Ca 2+ -level (Arrow indicates the time of addition of drug, dotted line represents initial value as 100%). Scale bar: 20 μm.

Article Snippet: The TRPV4-specific peptide (CDGHQQGYAPKWRAEDAPL, used as a blocking peptide of the TRPV4 antibody) was purchased from Alomone (Jerusalem, Israel).

Techniques: Live Cell Imaging, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: TRPV4 interacts with mitochondrial proteins and acts as a mitochondrial structure-function regulator

doi: 10.1101/330993

Figure Lengend Snippet: TRPV4 is present in the synaptosomal fraction of brain tissue. Tissue lysate was prepared from Rat brain and density gradient separation was done for different fractions and subsequently probed for the endogenous TRPV4. Lane 1. Microsome fraction, Lane 2: Light membrane fraction, Lane 3: Crude membrane fraction, Lane 4: Myelin fraction, Lane 5: Synaptosomal fraction, Lane 6: Synaptic junction fraction. TRPV4-specific faint band (∼ 98kDa, indicated by blue star) is present in light membrane fraction (lane 2) and in synaptosomal fraction (lane 5). In addition, another strong band for TRPV4 is also observed in these same fractions (indicated by red star). The presence of a specific peptide (corresponding to the C-terminus of TRPV4), the TRPV4-specifc band is abolished in the synaptosomal fraction (right side).

Article Snippet: The TRPV4-specific peptide (CDGHQQGYAPKWRAEDAPL, used as a blocking peptide of the TRPV4 antibody) was purchased from Alomone (Jerusalem, Israel).

Techniques:

Journal: bioRxiv

Article Title: TRPV4 interacts with mitochondrial proteins and acts as a mitochondrial structure-function regulator

doi: 10.1101/330993

Figure Lengend Snippet: Perinuclear localization of TRPV4 in different cell lines. (a) TRPV4-GFP was transiently expressed in neuronal (F11) and non-neuronal (HaCaT, Cos7, HeLa) cell lines where TRPV4-specific clusters are present in the perinuclear regions. Enlarged confocal images of each are shown in the right side. (b) Confocal images showing that localization of TRPV4 in cells either stably expressing TRPV4 (named CHOK1-V4) or an empty plasmid (CHOK1-Mock). Endogenous expression of TRPV4 is shown in HUVEC cells (lowermost panel). TRPV4 is detected by immunostaining with TRPV4-specific antibody (green) and the nucleus is stained by DAPI (blue). Scale bar: 10 µm (a), 5 µm (b).

Article Snippet: The TRPV4-specific peptide (CDGHQQGYAPKWRAEDAPL, used as a blocking peptide of the TRPV4 antibody) was purchased from Alomone (Jerusalem, Israel).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Immunostaining, Staining