rabbit polyclonal anti trpv2 antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit polyclonal anti trpv2 antibody
    Light stimulation promotes cell migration mediated by <t>TRPV2</t> in C2C12 myoblasts expressing eNpHR. ( A ) The role of TRPV2 channels in cell migration was evaluated with a cell migration tracking assay on C2C12 myoblasts expressing eNpHR. Cells were seeded at low density and the migration velocity (µm/min) was assessed for 15 h with a JuliStage system. C2C12 myoblast migration was evaluated for five conditions: without treatment (n=432), co-transfected with TRPV2[E594K] (n=290), treated with DMSO 1/1000 (n=180), treated with 100 µM Tranilast (n=285), and co-transfected with TRPV2[E594K] plus treated with 100 µM Tranilast (n=89) ($ corresponds to the comparison with C2C12 myoblasts without treatment, * corresponds to the comparison with C2C12 myoblasts treated with DMSO 1/1000) ($$$$ and ****: p
    Rabbit Polyclonal Anti Trpv2 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal anti trpv2 antibody - by Bioz Stars, 2021-12
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    Images

    1) Product Images from "Fine tuning of calcium constitutive entry by optogenetically-controlled membrane polarization: impact on cell migration"

    Article Title: Fine tuning of calcium constitutive entry by optogenetically-controlled membrane polarization: impact on cell migration

    Journal: bioRxiv

    doi: 10.1101/2020.06.04.134205

    Light stimulation promotes cell migration mediated by TRPV2 in C2C12 myoblasts expressing eNpHR. ( A ) The role of TRPV2 channels in cell migration was evaluated with a cell migration tracking assay on C2C12 myoblasts expressing eNpHR. Cells were seeded at low density and the migration velocity (µm/min) was assessed for 15 h with a JuliStage system. C2C12 myoblast migration was evaluated for five conditions: without treatment (n=432), co-transfected with TRPV2[E594K] (n=290), treated with DMSO 1/1000 (n=180), treated with 100 µM Tranilast (n=285), and co-transfected with TRPV2[E594K] plus treated with 100 µM Tranilast (n=89) ($ corresponds to the comparison with C2C12 myoblasts without treatment, * corresponds to the comparison with C2C12 myoblasts treated with DMSO 1/1000) ($$$$ and ****: p
    Figure Legend Snippet: Light stimulation promotes cell migration mediated by TRPV2 in C2C12 myoblasts expressing eNpHR. ( A ) The role of TRPV2 channels in cell migration was evaluated with a cell migration tracking assay on C2C12 myoblasts expressing eNpHR. Cells were seeded at low density and the migration velocity (µm/min) was assessed for 15 h with a JuliStage system. C2C12 myoblast migration was evaluated for five conditions: without treatment (n=432), co-transfected with TRPV2[E594K] (n=290), treated with DMSO 1/1000 (n=180), treated with 100 µM Tranilast (n=285), and co-transfected with TRPV2[E594K] plus treated with 100 µM Tranilast (n=89) ($ corresponds to the comparison with C2C12 myoblasts without treatment, * corresponds to the comparison with C2C12 myoblasts treated with DMSO 1/1000) ($$$$ and ****: p

    Techniques Used: Migration, Expressing, Transfection

    Expression of TRPV2 channels in C2C12 myoblasts. ( A ) Assessment of TRPV2 mRNA expression in C2C12 myoblasts by RT-PCR. ( B ) Western blot analysis of TRPV2 and GAPDH expression in C2C12 myoblasts, HEK293 cells stably expressing TRPV2 (HEK TRPV2), and control HEK293 cells (HEK ctrl). TRPV2 and GAPDH were detected sequentially on the same blot (stripped twice). 5 µg of proteins were deposited for HEK cell lysates and 10 µg for C2C12 myoblasts (n=4). ( C ) Confocal images at two magnifications showing immuno-localization of TRPV2 (red) and nuclei staining (blue) in C2C12 myoblasts (scale bar: 20 µm).
    Figure Legend Snippet: Expression of TRPV2 channels in C2C12 myoblasts. ( A ) Assessment of TRPV2 mRNA expression in C2C12 myoblasts by RT-PCR. ( B ) Western blot analysis of TRPV2 and GAPDH expression in C2C12 myoblasts, HEK293 cells stably expressing TRPV2 (HEK TRPV2), and control HEK293 cells (HEK ctrl). TRPV2 and GAPDH were detected sequentially on the same blot (stripped twice). 5 µg of proteins were deposited for HEK cell lysates and 10 µg for C2C12 myoblasts (n=4). ( C ) Confocal images at two magnifications showing immuno-localization of TRPV2 (red) and nuclei staining (blue) in C2C12 myoblasts (scale bar: 20 µm).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Stable Transfection, Staining

    Involvement of TRPV2 channels in the mediation of constitutive calcium entry during light stimulation. ( A ) Representative traces of Fura-2 normalized ratio in response to a light stimulation of 30 s at 48 mW/cm 2 (orange rectangle) in C2C12 myoblasts expressing eNpHR-YFP treated with DMSO 1/1000 (vehicle) or with 100 µM Tranilast, a TRPV2 inhibitor. ( B ) Maximum amplitude (∆F/F0) of the Fura-2 fluorescence response to light stimulation in eNpHR-expressing C2C12 myoblasts treated with DMSO 1/1000 (n=40) or with 100 µM Tranilast (n=45). ( C ) Confocal images showing immuno-expression of the negative-dominant TRPV2[E594K] tagged with a flag sequence (red) co-transfected (TRPV2[E594K] right panels) or not (Ctrl, left panel) with eNpHR-YFP (green) in C2C12 myoblasts. ( D ) Representative traces of normalized Fura-2 ratio during a light stimulation of 30 s at 48 mW/cm 2 (orange rectangle) in C2C12 myoblasts expressing eNpHR-YFP co-transfected (TRPV2[E594K]) or not (Ctrl) with the negative-dominant TRPV2[E594K]. ( E ) Maximum amplitude (∆F/F0) of the Fura-2 fluorescence response to light stimulation in control C2C12 myoblasts expressing eNpHR-YFP (Ctrl, n=31) or in C2C12 myoblasts expressing eNpHR-YFP co-transfected with negative-dominant TRPV2[E594K] (TRPV2[E594K], n=102). Data are presented as mean ± SEM. *** and **** represent significant differences with p
    Figure Legend Snippet: Involvement of TRPV2 channels in the mediation of constitutive calcium entry during light stimulation. ( A ) Representative traces of Fura-2 normalized ratio in response to a light stimulation of 30 s at 48 mW/cm 2 (orange rectangle) in C2C12 myoblasts expressing eNpHR-YFP treated with DMSO 1/1000 (vehicle) or with 100 µM Tranilast, a TRPV2 inhibitor. ( B ) Maximum amplitude (∆F/F0) of the Fura-2 fluorescence response to light stimulation in eNpHR-expressing C2C12 myoblasts treated with DMSO 1/1000 (n=40) or with 100 µM Tranilast (n=45). ( C ) Confocal images showing immuno-expression of the negative-dominant TRPV2[E594K] tagged with a flag sequence (red) co-transfected (TRPV2[E594K] right panels) or not (Ctrl, left panel) with eNpHR-YFP (green) in C2C12 myoblasts. ( D ) Representative traces of normalized Fura-2 ratio during a light stimulation of 30 s at 48 mW/cm 2 (orange rectangle) in C2C12 myoblasts expressing eNpHR-YFP co-transfected (TRPV2[E594K]) or not (Ctrl) with the negative-dominant TRPV2[E594K]. ( E ) Maximum amplitude (∆F/F0) of the Fura-2 fluorescence response to light stimulation in control C2C12 myoblasts expressing eNpHR-YFP (Ctrl, n=31) or in C2C12 myoblasts expressing eNpHR-YFP co-transfected with negative-dominant TRPV2[E594K] (TRPV2[E594K], n=102). Data are presented as mean ± SEM. *** and **** represent significant differences with p

    Techniques Used: Expressing, Fluorescence, Sequencing, Transfection

    Schematic model of the effect of light-induced activation of halorhodopsin pump on the activation of calcium constitutive entry pathway through TRPV2 channels and on the modulation of cell migration. ( 1 ) Light stimulation of C2C12 myoblasts expressing eNpHR (orange) leads to membrane polarization by chloride ion entry (yellow) which gives rise to constitutive calcium entry (red) through TRPV2 channel (blue) by increasing the driving force for Ca 2+ across the plasma membrane. This light-induced calcium entry increases cell migration in a manner that can be abolished ( 2 ) by the TRPV2 inhibitor Tranilast (purple) and the negative-dominant TRPV2[E594K] transcript (green).
    Figure Legend Snippet: Schematic model of the effect of light-induced activation of halorhodopsin pump on the activation of calcium constitutive entry pathway through TRPV2 channels and on the modulation of cell migration. ( 1 ) Light stimulation of C2C12 myoblasts expressing eNpHR (orange) leads to membrane polarization by chloride ion entry (yellow) which gives rise to constitutive calcium entry (red) through TRPV2 channel (blue) by increasing the driving force for Ca 2+ across the plasma membrane. This light-induced calcium entry increases cell migration in a manner that can be abolished ( 2 ) by the TRPV2 inhibitor Tranilast (purple) and the negative-dominant TRPV2[E594K] transcript (green).

    Techniques Used: Activation Assay, Migration, Expressing

    2) Product Images from "Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels"

    Article Title: Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels

    Journal: Cell calcium

    doi: 10.1016/j.ceca.2016.01.001

    Influx in stretched cardiomyocytes in presence of TRPV2 channels antibodies
    Figure Legend Snippet: Influx in stretched cardiomyocytes in presence of TRPV2 channels antibodies

    Techniques Used:

    TRPV2 protein expression in isolated WT and mdx cardiomyocytes
    Figure Legend Snippet: TRPV2 protein expression in isolated WT and mdx cardiomyocytes

    Techniques Used: Expressing, Isolation

    TRPV2 expression in heart tissue from WT and mdx mice
    Figure Legend Snippet: TRPV2 expression in heart tissue from WT and mdx mice

    Techniques Used: Expressing, Mouse Assay

    3) Product Images from "Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels"

    Article Title: Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels

    Journal: Cell calcium

    doi: 10.1016/j.ceca.2016.01.001

    Influx in stretched cardiomyocytes in presence of TRPV2 channels antibodies
    Figure Legend Snippet: Influx in stretched cardiomyocytes in presence of TRPV2 channels antibodies

    Techniques Used:

    TRPV2 protein expression in isolated WT and mdx cardiomyocytes
    Figure Legend Snippet: TRPV2 protein expression in isolated WT and mdx cardiomyocytes

    Techniques Used: Expressing, Isolation

    TRPV2 expression in heart tissue from WT and mdx mice
    Figure Legend Snippet: TRPV2 expression in heart tissue from WT and mdx mice

    Techniques Used: Expressing, Mouse Assay

    4) Product Images from "Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels"

    Article Title: Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels

    Journal: Cell calcium

    doi: 10.1016/j.ceca.2016.01.001

    Influx in stretched cardiomyocytes in presence of TRPV2 channels antibodies
    Figure Legend Snippet: Influx in stretched cardiomyocytes in presence of TRPV2 channels antibodies

    Techniques Used:

    TRPV2 protein expression in isolated WT and mdx cardiomyocytes
    Figure Legend Snippet: TRPV2 protein expression in isolated WT and mdx cardiomyocytes

    Techniques Used: Expressing, Isolation

    TRPV2 expression in heart tissue from WT and mdx mice
    Figure Legend Snippet: TRPV2 expression in heart tissue from WT and mdx mice

    Techniques Used: Expressing, Mouse Assay

    5) Product Images from "Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels"

    Article Title: Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels

    Journal: Cell calcium

    doi: 10.1016/j.ceca.2016.01.001

    Influx in stretched cardiomyocytes in presence of TRPV2 channels antibodies
    Figure Legend Snippet: Influx in stretched cardiomyocytes in presence of TRPV2 channels antibodies

    Techniques Used:

    TRPV2 protein expression in isolated WT and mdx cardiomyocytes
    Figure Legend Snippet: TRPV2 protein expression in isolated WT and mdx cardiomyocytes

    Techniques Used: Expressing, Isolation

    TRPV2 expression in heart tissue from WT and mdx mice
    Figure Legend Snippet: TRPV2 expression in heart tissue from WT and mdx mice

    Techniques Used: Expressing, Mouse Assay

    6) Product Images from "TRPV2 activation reorganizes actin cytoskeleton, induces neurite initiation and branching by altering cAMP levels"

    Article Title: TRPV2 activation reorganizes actin cytoskeleton, induces neurite initiation and branching by altering cAMP levels

    Journal: bioRxiv

    doi: 10.1101/817684

    Endogenous TRPV2 activation increases cell size and enhances neuritogenesis. a. Activation of TRPV2 induces more neurites. The F-11 cell was grown in control condition, activated with Probenecid (10μM for 12 hours) or inhibited with Tranilast (75μM for 12 hours). Cells were quantified and percentage of cells without any neurite, with at-least one primary (1°) neurite, and with at-least one secondary (2°) neurite originated from primary neurite are plotted. b. Length of the 1° neurites (n =109 for control, 89 for Probenecid-treated conditions and 143 for Tranilast-treated conditions are plotted in ascending orders. For cells without any 1° neurite, the length of the 1° neurite is considered as zero. c. Length of the 2° neurites (n =109 for control, 89 for Probenecid-treated conditions and 143 for Tranilast-treated conditions) are plotted in ascending orders. For 1° neurite without any 2° neurite, the length of the 2° neurite is considered as zero. d-f. Length of the total cell (d), 1° neurite (e) and 2° neurite (f) from cells grown in control condition, activated with Probenecid or inhibited with Tranilast are plotted. The average length of the 2° neurites is non-significantly (ns) different when cells were grown in different conditions. P values ≤ 0.001, 0.01, 0.5, 0.1 are considered as ***, **, *, and ns respectively.
    Figure Legend Snippet: Endogenous TRPV2 activation increases cell size and enhances neuritogenesis. a. Activation of TRPV2 induces more neurites. The F-11 cell was grown in control condition, activated with Probenecid (10μM for 12 hours) or inhibited with Tranilast (75μM for 12 hours). Cells were quantified and percentage of cells without any neurite, with at-least one primary (1°) neurite, and with at-least one secondary (2°) neurite originated from primary neurite are plotted. b. Length of the 1° neurites (n =109 for control, 89 for Probenecid-treated conditions and 143 for Tranilast-treated conditions are plotted in ascending orders. For cells without any 1° neurite, the length of the 1° neurite is considered as zero. c. Length of the 2° neurites (n =109 for control, 89 for Probenecid-treated conditions and 143 for Tranilast-treated conditions) are plotted in ascending orders. For 1° neurite without any 2° neurite, the length of the 2° neurite is considered as zero. d-f. Length of the total cell (d), 1° neurite (e) and 2° neurite (f) from cells grown in control condition, activated with Probenecid or inhibited with Tranilast are plotted. The average length of the 2° neurites is non-significantly (ns) different when cells were grown in different conditions. P values ≤ 0.001, 0.01, 0.5, 0.1 are considered as ***, **, *, and ns respectively.

    Techniques Used: Activation Assay

    Activation of TRPV2 induce rapid translocation in leading edges but not on the filopodial tips. a. Live cell image of F-11 cell co transfected with TRPV2-GFP and actin-RFP shows that activation of TRPV2 by Probenecid alters actin cytoskeleton dynamics leading to changes in cell morphology, such as extension of cell membrane (upper panel), formation of massive lamellipodium (middle panel) and merging of lamellipodium (lower panel). b. Activation of TRPV2-GFP by 2APB (indicated by green arrow at 886 th frame) also causes changes in sub-membranous actin cytoskeleton and results in rapid translocation of TRPV2-GFP to the leading edges, merging of actin-ribs in the lamellipodium boundary. Intensity profile of the TRPV2-GFP and actin-RFP are provided in below. c. Shown are the enlarged portion of the leading edge of a F11 cell expressing TRPV2-GFP and actin-RFP before and after activation with 2APB. The filopodial tips are marked with white arrows. TRPV2-GFP is mainly present in the filopodial base but not in the filopodial tips.
    Figure Legend Snippet: Activation of TRPV2 induce rapid translocation in leading edges but not on the filopodial tips. a. Live cell image of F-11 cell co transfected with TRPV2-GFP and actin-RFP shows that activation of TRPV2 by Probenecid alters actin cytoskeleton dynamics leading to changes in cell morphology, such as extension of cell membrane (upper panel), formation of massive lamellipodium (middle panel) and merging of lamellipodium (lower panel). b. Activation of TRPV2-GFP by 2APB (indicated by green arrow at 886 th frame) also causes changes in sub-membranous actin cytoskeleton and results in rapid translocation of TRPV2-GFP to the leading edges, merging of actin-ribs in the lamellipodium boundary. Intensity profile of the TRPV2-GFP and actin-RFP are provided in below. c. Shown are the enlarged portion of the leading edge of a F11 cell expressing TRPV2-GFP and actin-RFP before and after activation with 2APB. The filopodial tips are marked with white arrows. TRPV2-GFP is mainly present in the filopodial base but not in the filopodial tips.

    Techniques Used: Activation Assay, Translocation Assay, Transfection, Expressing

    Activation of endogenous TRPV2 increases cAMP level in F-11 cells. a. Western blot analysis of F11 cell extracts for CREB and phospho-CREB levels. F11 cells were either grown on control conditions or treated with activators or inhibitors for 12 hours before the samples were prepared. b-c. F11 cells grown in control conditions or treated with activators or inhibitors for 12 hours. Cells were analyzed for CREB and phospho-CREB levels by FACS analysis. Activation of TRPV2 does not alter CREB levels much, but alters phosphor-CREB levels (n = 3). d. F11 cells were transfected with FRET-based cAMP-sensor molecule (Epac-SH188) and the respective confocal images acquired in different spectra are shown. e. Multiple F11 cells transfected with the cAMP sensor were quantified and the ratio of acceptor/donor values are shown. Each dot represents the “absolute values” obtained from a single cell. Live F11 cells were imaged for total 200 frames (≈ 5 Min in total, time interval between each frame is 0.5 sec) and cells were activated with Probenecid at 25 th Frame. Decreased values suggest increased cAMP formation after addition of TRPV2 activators. P values that are ≥ 0.1 and ≤ 0.01 are considered as non-significant (ns) and * respectively. f-g. Shown is the ratio of acceptor/donor in live F11 cell transfected with the cAMP sensor. Cells were activated with Probenecid at 25 th Frame. The initial value of the ratio is normalized and considered at 1. Gradual decline in values suggest increased cAMP formation after addition of TRPV2 activators, both in whole cell (f) and in specific ROI drawn on the neurites and branching points (g).
    Figure Legend Snippet: Activation of endogenous TRPV2 increases cAMP level in F-11 cells. a. Western blot analysis of F11 cell extracts for CREB and phospho-CREB levels. F11 cells were either grown on control conditions or treated with activators or inhibitors for 12 hours before the samples were prepared. b-c. F11 cells grown in control conditions or treated with activators or inhibitors for 12 hours. Cells were analyzed for CREB and phospho-CREB levels by FACS analysis. Activation of TRPV2 does not alter CREB levels much, but alters phosphor-CREB levels (n = 3). d. F11 cells were transfected with FRET-based cAMP-sensor molecule (Epac-SH188) and the respective confocal images acquired in different spectra are shown. e. Multiple F11 cells transfected with the cAMP sensor were quantified and the ratio of acceptor/donor values are shown. Each dot represents the “absolute values” obtained from a single cell. Live F11 cells were imaged for total 200 frames (≈ 5 Min in total, time interval between each frame is 0.5 sec) and cells were activated with Probenecid at 25 th Frame. Decreased values suggest increased cAMP formation after addition of TRPV2 activators. P values that are ≥ 0.1 and ≤ 0.01 are considered as non-significant (ns) and * respectively. f-g. Shown is the ratio of acceptor/donor in live F11 cell transfected with the cAMP sensor. Cells were activated with Probenecid at 25 th Frame. The initial value of the ratio is normalized and considered at 1. Gradual decline in values suggest increased cAMP formation after addition of TRPV2 activators, both in whole cell (f) and in specific ROI drawn on the neurites and branching points (g).

    Techniques Used: Activation Assay, Western Blot, FACS, Transfection

    Ectopic expression of TRPV2 alters cell morphology and polarity. Shown are the confocal images of different cells or their specialized areas expressing TRPV2-GFP. a. Different non-neuronal cells (CHO-K1, HaCaT, HEK293, and SaOS) become enlarged after expressing TRPV2-GFP while non-transfected cells remain normal in sizes. (T and NT represents transfected and non-transfected cells respectively). All the cells were stained for Tyrosinated tubulin (Red, YL1/2 Ab) and DNA (Blue, DAPI). b. TRPV2-GFP localizes in the specialized cellular structures such as at growth cone, neurite and filopodia when expressed in Neuro2A (neuronal) cells. c-d. TRPV2-GFP localizes in the similar specialized cellular structures such as at growth cone, neurite, filopodia in fixed (c) as well as in live (d) F11 (neuronal) cells. In each case, GFP fluorescence is superimposed with the DIC images.
    Figure Legend Snippet: Ectopic expression of TRPV2 alters cell morphology and polarity. Shown are the confocal images of different cells or their specialized areas expressing TRPV2-GFP. a. Different non-neuronal cells (CHO-K1, HaCaT, HEK293, and SaOS) become enlarged after expressing TRPV2-GFP while non-transfected cells remain normal in sizes. (T and NT represents transfected and non-transfected cells respectively). All the cells were stained for Tyrosinated tubulin (Red, YL1/2 Ab) and DNA (Blue, DAPI). b. TRPV2-GFP localizes in the specialized cellular structures such as at growth cone, neurite and filopodia when expressed in Neuro2A (neuronal) cells. c-d. TRPV2-GFP localizes in the similar specialized cellular structures such as at growth cone, neurite, filopodia in fixed (c) as well as in live (d) F11 (neuronal) cells. In each case, GFP fluorescence is superimposed with the DIC images.

    Techniques Used: Expressing, Transfection, Staining, Fluorescence

    TRPV2 colocalizes with sub-membranous actin cytoskeleton and interacts with actin. a. TRPV2 co-localizes with Phalloidin in filopodia and specific cellular areas. b. Live cell image of a F11 cell co-expressing TRPV2-GFP (green) and actin-RFP (red) demonstrating colocalization of TRPV2-GFP and actin-RFP in specific cellular regions such as in neurites and in filopodia. c. The C-terminus of TRPV2 interacts with actin. Soluble brain extract supernatant (lane 1) was pulled down with purified MBP-LacZ (lane 4-5), MBP-TRPV2-Ct (lane 6-7) and probed for bound actin by Western blot analysis.
    Figure Legend Snippet: TRPV2 colocalizes with sub-membranous actin cytoskeleton and interacts with actin. a. TRPV2 co-localizes with Phalloidin in filopodia and specific cellular areas. b. Live cell image of a F11 cell co-expressing TRPV2-GFP (green) and actin-RFP (red) demonstrating colocalization of TRPV2-GFP and actin-RFP in specific cellular regions such as in neurites and in filopodia. c. The C-terminus of TRPV2 interacts with actin. Soluble brain extract supernatant (lane 1) was pulled down with purified MBP-LacZ (lane 4-5), MBP-TRPV2-Ct (lane 6-7) and probed for bound actin by Western blot analysis.

    Techniques Used: Expressing, Purification, Western Blot

    TRPV2-mediated neuritogenesis is dependent on wild type TRPV2 but independent of intracellular Ca 2+ -levels. a. Only Wild-type TRPV2-GFP induces long neurites. Shown are the F11 cells expressing TRPV2-Wt-GFP, or TRPV2-mutants or GFP only. Cells were stained with DAPI and images were acquired by confocal microscopy. b. F11 cells were grown in absence or in presence of BAPTA-AM and were either treated with TRPV2 activator (Probenecid) or TRPV2 inhibitor (Tranilast). Cells were stained with Phalloidin (Green), Microtubules (Red) and DAPI (Blue).
    Figure Legend Snippet: TRPV2-mediated neuritogenesis is dependent on wild type TRPV2 but independent of intracellular Ca 2+ -levels. a. Only Wild-type TRPV2-GFP induces long neurites. Shown are the F11 cells expressing TRPV2-Wt-GFP, or TRPV2-mutants or GFP only. Cells were stained with DAPI and images were acquired by confocal microscopy. b. F11 cells were grown in absence or in presence of BAPTA-AM and were either treated with TRPV2 activator (Probenecid) or TRPV2 inhibitor (Tranilast). Cells were stained with Phalloidin (Green), Microtubules (Red) and DAPI (Blue).

    Techniques Used: Expressing, Staining, Confocal Microscopy

    Exogenous expression of TRPV2 induces neuritogenesis and enhances cell elongation. a-b. Representative fluorescence microscopic images demonstrating Neuro2A (a) and F11 (b) cells expressing TRPV2-GFP. Transfected cells (T) become much elongated and have a higher number of neurites with complex branches compared to non-transfected (NT) cells. c. Expression of TRPV2-GFP enhances neuritogenesis. Percentage of F-11 cells having at-least one primary neurite, two primary neurites, more than 2 primary neurites or no neurites were quantified (n =149 for non-transfected cells, 104 for GFP expressing cells and n =131 for TRPV2-GFP expressing cells). d. Quantification of the length of the entire cell (n = 149), primary (1°) neurite (n = 100) and secondary (2°) neurites (n = 80) originated from F11 cells over expressing TRPV2-GFP are shown. The mean cell length becomes significantly different (p-value) while the mean length of the 1° and 2° neurites are not-significantly different. e-f. Length of the 1° neurites and 2° neurites (n =131 TRPV2-GFP expressing cells, 104 GFP expressing cells, and n = 149 non-transfected cells) present in F11 cells are plotted in ascending orders. For cells without any 1° neurite, the length of the 1° neurite is considered as zero (e). Similarly, for 1° neurites without any 2° neurite, the length of the 2° neurite is considered as zero (f). g-i. Ratio (of length) of 1° neurites to total cell (g), 2° neurite to total cell (h) and 1° to 2° neurite (i) are plotted for each cell. Marginal differences in these ratios between TRPV2-GFP expressing cells and GFP only expressing cells or non-transfected cells are observed. P values ≤ 0.001, 0.01, 0.5, 0.1 are considered as ***, **, *, and ns respectively.
    Figure Legend Snippet: Exogenous expression of TRPV2 induces neuritogenesis and enhances cell elongation. a-b. Representative fluorescence microscopic images demonstrating Neuro2A (a) and F11 (b) cells expressing TRPV2-GFP. Transfected cells (T) become much elongated and have a higher number of neurites with complex branches compared to non-transfected (NT) cells. c. Expression of TRPV2-GFP enhances neuritogenesis. Percentage of F-11 cells having at-least one primary neurite, two primary neurites, more than 2 primary neurites or no neurites were quantified (n =149 for non-transfected cells, 104 for GFP expressing cells and n =131 for TRPV2-GFP expressing cells). d. Quantification of the length of the entire cell (n = 149), primary (1°) neurite (n = 100) and secondary (2°) neurites (n = 80) originated from F11 cells over expressing TRPV2-GFP are shown. The mean cell length becomes significantly different (p-value) while the mean length of the 1° and 2° neurites are not-significantly different. e-f. Length of the 1° neurites and 2° neurites (n =131 TRPV2-GFP expressing cells, 104 GFP expressing cells, and n = 149 non-transfected cells) present in F11 cells are plotted in ascending orders. For cells without any 1° neurite, the length of the 1° neurite is considered as zero (e). Similarly, for 1° neurites without any 2° neurite, the length of the 2° neurite is considered as zero (f). g-i. Ratio (of length) of 1° neurites to total cell (g), 2° neurite to total cell (h) and 1° to 2° neurite (i) are plotted for each cell. Marginal differences in these ratios between TRPV2-GFP expressing cells and GFP only expressing cells or non-transfected cells are observed. P values ≤ 0.001, 0.01, 0.5, 0.1 are considered as ***, **, *, and ns respectively.

    Techniques Used: Expressing, Fluorescence, Transfection, Significance Assay

    Activation of endogenous TRPV2 leads to immediate membrane ruffling and alters filopodial and growth cone dynamics. a-c. Representative live cell imaging of F11 cells expressing TRPV2-GFP (green) merged with DIC are shown. When cells were left un-treated (a), there is not much change in the morphology. Inhibition of TRPV2 activity by Tranilast (b) leads to a quick reduction of cell size suggesting that TRPV2 activity contributes to the maintenance of cell morphology. Activation of TRPV2 with Probenecid (c) results in rapid membrane-ruffling leading to changes in cell morphology. In each case, GFP fluorescence is superimposed with the DIC images. d. Shown are the time-series images of enlarged sections of a F11 cell expressing TRPV2-GFP that have been treated with Probenecid (indicated by a red arrow). Activation of TRPV2-GFP results in different events such as initiation of neurites, branching of neurites and growth cone dynamics that are controlled by sub-membranous actin cytoskeleton leading to changes in membrane ruffling. Intensity of the GFP fluorescence is represented in rainbow colors. The time gap between each image frame 0.04 Sec.
    Figure Legend Snippet: Activation of endogenous TRPV2 leads to immediate membrane ruffling and alters filopodial and growth cone dynamics. a-c. Representative live cell imaging of F11 cells expressing TRPV2-GFP (green) merged with DIC are shown. When cells were left un-treated (a), there is not much change in the morphology. Inhibition of TRPV2 activity by Tranilast (b) leads to a quick reduction of cell size suggesting that TRPV2 activity contributes to the maintenance of cell morphology. Activation of TRPV2 with Probenecid (c) results in rapid membrane-ruffling leading to changes in cell morphology. In each case, GFP fluorescence is superimposed with the DIC images. d. Shown are the time-series images of enlarged sections of a F11 cell expressing TRPV2-GFP that have been treated with Probenecid (indicated by a red arrow). Activation of TRPV2-GFP results in different events such as initiation of neurites, branching of neurites and growth cone dynamics that are controlled by sub-membranous actin cytoskeleton leading to changes in membrane ruffling. Intensity of the GFP fluorescence is represented in rainbow colors. The time gap between each image frame 0.04 Sec.

    Techniques Used: Activation Assay, Live Cell Imaging, Expressing, Inhibition, Activity Assay, Fluorescence

    Schematic representation of plausible cellular and molecular events regulated by TRPV2 leading to enhanced neuritogenesis. a. TRPV2 activation induces a cascade of cellular events involving both Ca 2+ -dependent and Ca 2+ -independent signaling leading to cytoskeletal remodeling. TRPV2 activation induces new 1° neurites, branching events and induce new 2° neurites leading to enhanced neuritogenesis. However, once initiated, further extension of the formed neurites seem to be independent of TRPV2 activation. b. Schematic representation of a neuronal cells with neurites and their relative intracellular Ca 2+ -levels. TRPV2 seem to contribute to the regulation of Ca 2+ -homeostasis in different parts of the cells and especially in the leading ends where Ca 2+ -levels are usually high. c. Shown is a simplified model demonstrating the changes in cell morphology and development of complex neurites due to activation of TRPV2. d. A simplified model represents the involvement of different molecular factors such as TRPV2, actin, microtubule and Ca 2+ in specialized subcellular structures (indicated by dotted box) such as in filopodia and in neuronal growth cone.
    Figure Legend Snippet: Schematic representation of plausible cellular and molecular events regulated by TRPV2 leading to enhanced neuritogenesis. a. TRPV2 activation induces a cascade of cellular events involving both Ca 2+ -dependent and Ca 2+ -independent signaling leading to cytoskeletal remodeling. TRPV2 activation induces new 1° neurites, branching events and induce new 2° neurites leading to enhanced neuritogenesis. However, once initiated, further extension of the formed neurites seem to be independent of TRPV2 activation. b. Schematic representation of a neuronal cells with neurites and their relative intracellular Ca 2+ -levels. TRPV2 seem to contribute to the regulation of Ca 2+ -homeostasis in different parts of the cells and especially in the leading ends where Ca 2+ -levels are usually high. c. Shown is a simplified model demonstrating the changes in cell morphology and development of complex neurites due to activation of TRPV2. d. A simplified model represents the involvement of different molecular factors such as TRPV2, actin, microtubule and Ca 2+ in specialized subcellular structures (indicated by dotted box) such as in filopodia and in neuronal growth cone.

    Techniques Used: Activation Assay

    Activation of endogenous TRPV2 affects primary neurites more than secondary neurites. a-c. The ratio of primary neurite length to total cell length (a), or secondary neurite length to total cell length (b) or secondary neurite to primary neurite length were quantified for cells grown in different conditions. This ratio is significantly higher when cells are grown in presence of TRPV2 activator Probenecid (10μM for 12 hours) and mean is unaltered when grown in presence of inhibitor Tranilast 75μM for 12 hours (n =109 for control, 89 for Probenecid-treated conditions and n = 143 for Tranilast-treated conditions). d-e. Cells were grown in different conditions and the distance between cell body to the origin of first secondary neurite developed was quantified. Activation of TRPV2 increases the distance (of primary neurite) between cell body to the first point where secondary neurite originates. The mean/average distance is unaltered when grown in presence of inhibitor Tranilast. The values were plotted in ascending order (e). The P values ≤ 0.001, 0.01, 0.5, 0.1 are considered as ***, **, *, and ns respectively.
    Figure Legend Snippet: Activation of endogenous TRPV2 affects primary neurites more than secondary neurites. a-c. The ratio of primary neurite length to total cell length (a), or secondary neurite length to total cell length (b) or secondary neurite to primary neurite length were quantified for cells grown in different conditions. This ratio is significantly higher when cells are grown in presence of TRPV2 activator Probenecid (10μM for 12 hours) and mean is unaltered when grown in presence of inhibitor Tranilast 75μM for 12 hours (n =109 for control, 89 for Probenecid-treated conditions and n = 143 for Tranilast-treated conditions). d-e. Cells were grown in different conditions and the distance between cell body to the origin of first secondary neurite developed was quantified. Activation of TRPV2 increases the distance (of primary neurite) between cell body to the first point where secondary neurite originates. The mean/average distance is unaltered when grown in presence of inhibitor Tranilast. The values were plotted in ascending order (e). The P values ≤ 0.001, 0.01, 0.5, 0.1 are considered as ***, **, *, and ns respectively.

    Techniques Used: Activation Assay

    F11 cells endogenously express functional TRPV2. a. Immunofluorescence images of F-11 cells stained with anti-TRPV2 antibody in the absence (lower panel) or presence (upper panel) of specific blocking peptide are shown. b. Western blot analysis of F11 cell extract probed with anti-TRPV2 antibody are shown. The presence of specific blocking peptide diminished the TRPV2-specific immunoreactivity completely. c. Live cell imaging of F11 cells loaded with Fluo-4 demonstrating the transient and sharp increase in the intracellular Ca 2+ -level immediately after treating the cells with a specific activator (Probenecid, 250μM). The interval between each time frame is 0.5 sec. d. Similar Ca 2+ -imaging of F-11 cells shows an immediate drop in intracellular Ca 2+ -level followed by application of specific inhibitor (Tranilast, 75μM). Further application of specific activator (Probenecid, 250μM) cause again sudden increase in Ca 2+ -level. g-h. Quantification of intracellular Ca 2+ -levels as shown above (c-d) are represented. In each case, fluorescence intensity (in arbitrary unit) from multiple cells (n =10) are shown. The average value is shown as a thick black line. The interval between each time frame is 0.5 sec.
    Figure Legend Snippet: F11 cells endogenously express functional TRPV2. a. Immunofluorescence images of F-11 cells stained with anti-TRPV2 antibody in the absence (lower panel) or presence (upper panel) of specific blocking peptide are shown. b. Western blot analysis of F11 cell extract probed with anti-TRPV2 antibody are shown. The presence of specific blocking peptide diminished the TRPV2-specific immunoreactivity completely. c. Live cell imaging of F11 cells loaded with Fluo-4 demonstrating the transient and sharp increase in the intracellular Ca 2+ -level immediately after treating the cells with a specific activator (Probenecid, 250μM). The interval between each time frame is 0.5 sec. d. Similar Ca 2+ -imaging of F-11 cells shows an immediate drop in intracellular Ca 2+ -level followed by application of specific inhibitor (Tranilast, 75μM). Further application of specific activator (Probenecid, 250μM) cause again sudden increase in Ca 2+ -level. g-h. Quantification of intracellular Ca 2+ -levels as shown above (c-d) are represented. In each case, fluorescence intensity (in arbitrary unit) from multiple cells (n =10) are shown. The average value is shown as a thick black line. The interval between each time frame is 0.5 sec.

    Techniques Used: Functional Assay, Immunofluorescence, Staining, Blocking Assay, Western Blot, Live Cell Imaging, Imaging, Fluorescence

    7) Product Images from "TRPV2 interacts with actin and reorganizes submembranous actin cytoskeleton"

    Article Title: TRPV2 interacts with actin and reorganizes submembranous actin cytoskeleton

    Journal: Bioscience Reports

    doi: 10.1042/BSR20200118

    Activation of TRPV2 induces rapid translocation in leading edges but not on the filopodial tips ( A ) Live cell imaging of F11 cell co-transfected with TRPV2-GFP and actin-RFP shows that activation of TRPV2 by Probenecid alters actin cytoskeleton dynamics leading to changes in cell morphology, such as extension of cell membrane (upper panel), formation of massive lamellipodia (middle panel) and merging of lamellipodia (lower panel). ( B ) Activation of TRPV2-GFP by 2APB (indicated by green arrow at 886th frame) also causes changes in submembranous actin cytoskeleton and results in rapid translocation of TRPV2-GFP to the leading edges, merging of actin-ribs in the lamellipodium boundary. Intensity profile of the TRPV2-GFP and actin-RFP are provided below. ( C ) Enlarged section of the leading edge of a F11 cell expressing TRPV2-GFP and actin-RFP before and after activation with 2APB. The filopodial tips are marked with white arrows. TRPV2-GFP is mainly present in the filopodial base but not in the filopodial tips.
    Figure Legend Snippet: Activation of TRPV2 induces rapid translocation in leading edges but not on the filopodial tips ( A ) Live cell imaging of F11 cell co-transfected with TRPV2-GFP and actin-RFP shows that activation of TRPV2 by Probenecid alters actin cytoskeleton dynamics leading to changes in cell morphology, such as extension of cell membrane (upper panel), formation of massive lamellipodia (middle panel) and merging of lamellipodia (lower panel). ( B ) Activation of TRPV2-GFP by 2APB (indicated by green arrow at 886th frame) also causes changes in submembranous actin cytoskeleton and results in rapid translocation of TRPV2-GFP to the leading edges, merging of actin-ribs in the lamellipodium boundary. Intensity profile of the TRPV2-GFP and actin-RFP are provided below. ( C ) Enlarged section of the leading edge of a F11 cell expressing TRPV2-GFP and actin-RFP before and after activation with 2APB. The filopodial tips are marked with white arrows. TRPV2-GFP is mainly present in the filopodial base but not in the filopodial tips.

    Techniques Used: Activation Assay, Translocation Assay, Live Cell Imaging, Transfection, Expressing

    Schematic representation of plausible cellular and molecular events regulated by TRPV2 leading to enhanced neuritogenesis ( A ) TRPV2 activation induces a cascade of cellular events involving both Ca 2+ -dependent and Ca 2+ -independent signaling leading to cytoskeletal remodeling. TRPV2 activation induces new 1° neurites, branching events and induce new 2° neurites leading to enhanced neuritogenesis. However, once initiated, further extension of the formed neurites seems to be independent of TRPV2 activation. ( B ) Schematic representation of a neuronal cell with neurites and their relative intracellular Ca 2+ -levels. TRPV2 seems to contribute to the regulation of Ca 2+ -homeostasis in different parts of the cells and especially in the leading ends where Ca 2+ -levels are usually high. ( C ) Simplified model demonstrating the changes in cell morphology and development of complex neurites due to activation of TRPV2. ( D ) A simplified model represents the involvement of different molecular factors such as TRPV2, actin, microtubule and Ca 2+ in specialized subcellular structures (indicated by dotted box) such as in filopodia and in neuronal growth cone.
    Figure Legend Snippet: Schematic representation of plausible cellular and molecular events regulated by TRPV2 leading to enhanced neuritogenesis ( A ) TRPV2 activation induces a cascade of cellular events involving both Ca 2+ -dependent and Ca 2+ -independent signaling leading to cytoskeletal remodeling. TRPV2 activation induces new 1° neurites, branching events and induce new 2° neurites leading to enhanced neuritogenesis. However, once initiated, further extension of the formed neurites seems to be independent of TRPV2 activation. ( B ) Schematic representation of a neuronal cell with neurites and their relative intracellular Ca 2+ -levels. TRPV2 seems to contribute to the regulation of Ca 2+ -homeostasis in different parts of the cells and especially in the leading ends where Ca 2+ -levels are usually high. ( C ) Simplified model demonstrating the changes in cell morphology and development of complex neurites due to activation of TRPV2. ( D ) A simplified model represents the involvement of different molecular factors such as TRPV2, actin, microtubule and Ca 2+ in specialized subcellular structures (indicated by dotted box) such as in filopodia and in neuronal growth cone.

    Techniques Used: Activation Assay

    Neuritogenesis is dependent on wildtype TRPV2 ( A ) Only wildtype TRPV2-GFP induces long neurites. The images represent F11 cells expressing TRPV2-Wt-GFP, or TRPV2-mutants in pcDNA3.1 or GFP-only. To find the status of neurites in TRPV2 mutant overexpressing cells were stained with anti-TRPV2 antibody and DAPI and images were acquired by confocal microscopy. ( B ) Quantification of the cells with neurite after expressing GFP only, TRPV2-GFP and TRPV2 mutant are shown. Cells were analyzed by quantifying the cell length without any neurites, with one primary neurite, with two primary neurites and cells with three or more primary neurites. Cells used for the quantification are as follows: GFP-only (104 cells), TRPV2-GFP (26 cells), TRPV2-N571T (49 cells), TRPV2-N572T (25 cells) and TRPV2-NN- 571/572-TT (16 cells) from three independent experiments.
    Figure Legend Snippet: Neuritogenesis is dependent on wildtype TRPV2 ( A ) Only wildtype TRPV2-GFP induces long neurites. The images represent F11 cells expressing TRPV2-Wt-GFP, or TRPV2-mutants in pcDNA3.1 or GFP-only. To find the status of neurites in TRPV2 mutant overexpressing cells were stained with anti-TRPV2 antibody and DAPI and images were acquired by confocal microscopy. ( B ) Quantification of the cells with neurite after expressing GFP only, TRPV2-GFP and TRPV2 mutant are shown. Cells were analyzed by quantifying the cell length without any neurites, with one primary neurite, with two primary neurites and cells with three or more primary neurites. Cells used for the quantification are as follows: GFP-only (104 cells), TRPV2-GFP (26 cells), TRPV2-N571T (49 cells), TRPV2-N572T (25 cells) and TRPV2-NN- 571/572-TT (16 cells) from three independent experiments.

    Techniques Used: Expressing, Mutagenesis, Staining, Confocal Microscopy

    Ectopic expression of TRPV2 alters cell morphology and polarity Confocal microscopy images of different cells and their specialized regions expressing TRPV2-GFP. ( A ) Different non-neuronal cells (CHO-K1, HaCaT, HEK293 and SaOS) become enlarged after expressing TRPV2-GFP, while non-transfected cells retain their normal size (T and NT represent transfected and non-transfected cells, respectively). All cells were stained for Tyrosinated tubulin (Red, YL1/2 Ab) and DNA (Blue, DAPI). ( B ) TRPV2-GFP localizes in specialized cellular structures such as at growth cone, neurite and filopodia when expressed in Neuro2A (neuronal) cells. ( C,D ) TRPV2-GFP localizes in similar specialized cellular structures such as at growth cone, neurite, filopodia in fixed (C) as well as in live (D) F11 (neuronal) cells. In each case, GFP fluorescence is superimposed with the DIC images. ( E ) F11 cells expressing only-GFP do not induce filopodia or neurites. Fluorescence image is superimposed with DIC image. Dotted lines show enlarged areas of the cell.
    Figure Legend Snippet: Ectopic expression of TRPV2 alters cell morphology and polarity Confocal microscopy images of different cells and their specialized regions expressing TRPV2-GFP. ( A ) Different non-neuronal cells (CHO-K1, HaCaT, HEK293 and SaOS) become enlarged after expressing TRPV2-GFP, while non-transfected cells retain their normal size (T and NT represent transfected and non-transfected cells, respectively). All cells were stained for Tyrosinated tubulin (Red, YL1/2 Ab) and DNA (Blue, DAPI). ( B ) TRPV2-GFP localizes in specialized cellular structures such as at growth cone, neurite and filopodia when expressed in Neuro2A (neuronal) cells. ( C,D ) TRPV2-GFP localizes in similar specialized cellular structures such as at growth cone, neurite, filopodia in fixed (C) as well as in live (D) F11 (neuronal) cells. In each case, GFP fluorescence is superimposed with the DIC images. ( E ) F11 cells expressing only-GFP do not induce filopodia or neurites. Fluorescence image is superimposed with DIC image. Dotted lines show enlarged areas of the cell.

    Techniques Used: Expressing, Confocal Microscopy, Transfection, Staining, Fluorescence

    Activation of TRPV2 leads to immediate membrane ruffling and alters filopodial and growth cone dynamics ( A–C ) Representative live cell imaging of F11 cells expressing TRPV2-GFP (green) merged with DIC are shown. When cells were left untreated (A), there is no significant change in the morphology. Inhibition of TRPV2 activity by Tranilast (B) leads to a quick reduction in cell size suggesting that TRPV2 activity contributes to the maintenance of cell morphology. Activation of TRPV2 with Probenecid (C) results in rapid membrane-ruffling leading to changes in cell morphology. In each case, GFP fluorescence is superimposed with DIC. ( D ) Time-series images of enlarged sections of a F11 cell expressing TRPV2-GFP that have been treated with Probenecid (indicated by a red arrow). Activation of TRPV2-GFP results in different events such as initiation of neurites that are controlled by submembranous actin cytoskeleton leading to changes in membrane ruffling. Intensity of the GFP fluorescence is represented in rainbow colors. The time gap between each image frame 0.04 s.
    Figure Legend Snippet: Activation of TRPV2 leads to immediate membrane ruffling and alters filopodial and growth cone dynamics ( A–C ) Representative live cell imaging of F11 cells expressing TRPV2-GFP (green) merged with DIC are shown. When cells were left untreated (A), there is no significant change in the morphology. Inhibition of TRPV2 activity by Tranilast (B) leads to a quick reduction in cell size suggesting that TRPV2 activity contributes to the maintenance of cell morphology. Activation of TRPV2 with Probenecid (C) results in rapid membrane-ruffling leading to changes in cell morphology. In each case, GFP fluorescence is superimposed with DIC. ( D ) Time-series images of enlarged sections of a F11 cell expressing TRPV2-GFP that have been treated with Probenecid (indicated by a red arrow). Activation of TRPV2-GFP results in different events such as initiation of neurites that are controlled by submembranous actin cytoskeleton leading to changes in membrane ruffling. Intensity of the GFP fluorescence is represented in rainbow colors. The time gap between each image frame 0.04 s.

    Techniques Used: Activation Assay, Live Cell Imaging, Expressing, Inhibition, Activity Assay, Fluorescence

    TRPV2 co-localizes with submembranous actin cytoskeleton and interacts with actin ( A ) TRPV2 co-localizes with Phalloidin in filopodia and specific cellular areas. ( B ) Live cell image of F11 cell co-expressing TRPV2-GFP (green) and actin-RFP (red) demonstrating co-localization of TRPV2-GFP and actin-RFP in specific cellular regions such as in neurites and in filopodia. ( C ) The C-terminus of TRPV2 interacts with actin. Soluble brain extract supernatant (lane 1) was pulled down with purified MBP-LacZ (lanes 4–5), MBP-TRPV2-Ct (lanes 6–7) and was probed for bound actin by Western blot analysis.
    Figure Legend Snippet: TRPV2 co-localizes with submembranous actin cytoskeleton and interacts with actin ( A ) TRPV2 co-localizes with Phalloidin in filopodia and specific cellular areas. ( B ) Live cell image of F11 cell co-expressing TRPV2-GFP (green) and actin-RFP (red) demonstrating co-localization of TRPV2-GFP and actin-RFP in specific cellular regions such as in neurites and in filopodia. ( C ) The C-terminus of TRPV2 interacts with actin. Soluble brain extract supernatant (lane 1) was pulled down with purified MBP-LacZ (lanes 4–5), MBP-TRPV2-Ct (lanes 6–7) and was probed for bound actin by Western blot analysis.

    Techniques Used: Expressing, Purification, Western Blot

    Exogenous expression of TRPV2 induces neuritogenesis and enhances cell elongation ( A,B ) Representative fluorescence microscopic images demonstrating Neuro2A (A) and F11 (B) cells expressing TRPV2-GFP. Transfected cells (T) become much elongated and have a higher number of neurites with complex branches compared with non-transfected (NT) cells. ( C ) Expression of TRPV2-GFP enhances neuritogenesis. Percentage of F11 cells having at least one primary neurite, two primary neurites, more than two primary neurites or no neurites were quantified ( n =149 for non-transfected cells, 104 for GFP-expressing cells and n =131 for TRPV2-GFP expressing cells). ( D ) Quantification of the length of the entire cell ( n =149), primary (1°) neurite ( n =100) and secondary (2°) neurites ( n =80) originating from F11 cells overexpressing TRPV2-GFP are shown. The 131 transfected cells show 301 primary neurites and 94 secondary neurites. Whereas 149 non-transfected cells show 21 primary neurites and 4 secondary neurites. The mean cell length becomes significantly different ( P -value), while the mean length of the 1° and 2° neurites are non-significantly different. ( E,F ) Length of the 1° and 2° neurites ( n =131 TRPV2-GFP expressing cells, 104 GFP-expressing cells, and n =149 non-transfected cells) present in F11 cells are plotted in ascending orders. For cells without any 1° neurite, the length of the 1° neurite is considered as zero (E). Similarly, for 1° neurite without any 2° neurite, the length of the 2° neurite is considered as zero (F). ( G – I ) Ratio (of length) of 1° neurites to total cell (G), 2° neurite to total cell (H) and 1° to 2° neurites (I) are plotted for each cell. Marginal differences in these ratios between TRPV2-GFP expressing cells and GFP-only expressing cells or non-transfected cells are observed. P -values ≤0.001, 0.5, 0.1 are considered as ***, * and ns, respectively.
    Figure Legend Snippet: Exogenous expression of TRPV2 induces neuritogenesis and enhances cell elongation ( A,B ) Representative fluorescence microscopic images demonstrating Neuro2A (A) and F11 (B) cells expressing TRPV2-GFP. Transfected cells (T) become much elongated and have a higher number of neurites with complex branches compared with non-transfected (NT) cells. ( C ) Expression of TRPV2-GFP enhances neuritogenesis. Percentage of F11 cells having at least one primary neurite, two primary neurites, more than two primary neurites or no neurites were quantified ( n =149 for non-transfected cells, 104 for GFP-expressing cells and n =131 for TRPV2-GFP expressing cells). ( D ) Quantification of the length of the entire cell ( n =149), primary (1°) neurite ( n =100) and secondary (2°) neurites ( n =80) originating from F11 cells overexpressing TRPV2-GFP are shown. The 131 transfected cells show 301 primary neurites and 94 secondary neurites. Whereas 149 non-transfected cells show 21 primary neurites and 4 secondary neurites. The mean cell length becomes significantly different ( P -value), while the mean length of the 1° and 2° neurites are non-significantly different. ( E,F ) Length of the 1° and 2° neurites ( n =131 TRPV2-GFP expressing cells, 104 GFP-expressing cells, and n =149 non-transfected cells) present in F11 cells are plotted in ascending orders. For cells without any 1° neurite, the length of the 1° neurite is considered as zero (E). Similarly, for 1° neurite without any 2° neurite, the length of the 2° neurite is considered as zero (F). ( G – I ) Ratio (of length) of 1° neurites to total cell (G), 2° neurite to total cell (H) and 1° to 2° neurites (I) are plotted for each cell. Marginal differences in these ratios between TRPV2-GFP expressing cells and GFP-only expressing cells or non-transfected cells are observed. P -values ≤0.001, 0.5, 0.1 are considered as ***, * and ns, respectively.

    Techniques Used: Expressing, Fluorescence, Transfection

    F11 cells endogenously express functional TRPV2 ( A ) Immunofluorescence images of F11 cells stained with anti-TRPV2 antibody in the absence (lower panel) or presence (upper panel) of specific blocking peptides are shown. ( B ) Western blot analysis of F11 cell extract probed with anti-TRPV2 antibody are shown. The presence of specific blocking peptide diminished the TRPV2-specific immunoreactivity completely. ( C ) Live cell imaging of F11 cells incubated with Fluo-4 demonstrating the transient and sharp increase in the intracellular Ca 2+ -level immediately after treating the cells with a specific activator (Probenecid, 250 µM). The interval between each time frame is 0.5 s. ( D ) Similar Ca 2+ -imaging of F11 cells shows an immediate drop in intracellular Ca 2+ - levels followed by application of specific inhibitor (Tranilast, 75 µM). Further application of specific activator (Probenecid, 250 µM) causes a sudden increase in Ca 2+ -level. ( E,F ) Quantification of intracellular Ca 2+ -levels as shown above (C,D) are represented. In each case, fluorescence intensity (in arbitrary units) from multiple cells ( n =10) are shown. The average value is shown as a thick black line. The interval between each time frame is 0.5 s.
    Figure Legend Snippet: F11 cells endogenously express functional TRPV2 ( A ) Immunofluorescence images of F11 cells stained with anti-TRPV2 antibody in the absence (lower panel) or presence (upper panel) of specific blocking peptides are shown. ( B ) Western blot analysis of F11 cell extract probed with anti-TRPV2 antibody are shown. The presence of specific blocking peptide diminished the TRPV2-specific immunoreactivity completely. ( C ) Live cell imaging of F11 cells incubated with Fluo-4 demonstrating the transient and sharp increase in the intracellular Ca 2+ -level immediately after treating the cells with a specific activator (Probenecid, 250 µM). The interval between each time frame is 0.5 s. ( D ) Similar Ca 2+ -imaging of F11 cells shows an immediate drop in intracellular Ca 2+ - levels followed by application of specific inhibitor (Tranilast, 75 µM). Further application of specific activator (Probenecid, 250 µM) causes a sudden increase in Ca 2+ -level. ( E,F ) Quantification of intracellular Ca 2+ -levels as shown above (C,D) are represented. In each case, fluorescence intensity (in arbitrary units) from multiple cells ( n =10) are shown. The average value is shown as a thick black line. The interval between each time frame is 0.5 s.

    Techniques Used: Functional Assay, Immunofluorescence, Staining, Blocking Assay, Western Blot, Live Cell Imaging, Incubation, Imaging, Fluorescence

    8) Product Images from "Fine Tuning of Calcium Constitutive Entry by Optogenetically-Controlled Membrane Polarization: Impact on Cell Migration"

    Article Title: Fine Tuning of Calcium Constitutive Entry by Optogenetically-Controlled Membrane Polarization: Impact on Cell Migration

    Journal: Cells

    doi: 10.3390/cells9071684

    Involvement of TRPV2 channels in the mediation of constitutive calcium entry during light stimulation. ( A ) Representative traces of Fura-2 normalized ratio in response to a light stimulation of 30 s at 48 mW/cm 2 (orange rectangle) in C2C12 myoblasts expressing eNpHR-YFP treated with DMSO 1/1000 (vehicle) or with 100 µM Tranilast, a TRPV2 inhibitor. ( B ) Maximum amplitude (ΔF/F0) of the Fura-2 fluorescence response to light stimulation in eNpHR-expressing C2C12 myoblasts treated with DMSO 1/1000 ( n =4 0) or with 100 µM Tranilast ( n = 45). ( C ) Confocal images showing immuno-expression of the negative-dominant TRPV2[E594K] tagged with a flag sequence (red) co-transfected (TRPV2[E594K] right panels) or not (Ctrl, left panel) with eNpHR-YFP (green) in C2C12 myoblasts. ( D ) Representative traces of normalized Fura-2 ratio during a light stimulation of 30 s at 48 mW/cm 2 (orange rectangle) in C2C12 myoblasts expressing eNpHR-YFP co-transfected (TRPV2[E594K]) or not (Ctrl) with the negative-dominant TRPV2[E594K]. ( E ) Maximum amplitude (ΔF/F0) of the Fura-2 fluorescence response to light stimulation in control C2C12 myoblasts expressing eNpHR-YFP (Ctrl, n = 31) or in C2C12 myoblasts expressing eNpHR-YFP co-transfected with negative-dominant TRPV2[E594K] (TRPV2[E594K], n = 102). Data are presented as mean ± SEM. *** and **** represent significant differences with p
    Figure Legend Snippet: Involvement of TRPV2 channels in the mediation of constitutive calcium entry during light stimulation. ( A ) Representative traces of Fura-2 normalized ratio in response to a light stimulation of 30 s at 48 mW/cm 2 (orange rectangle) in C2C12 myoblasts expressing eNpHR-YFP treated with DMSO 1/1000 (vehicle) or with 100 µM Tranilast, a TRPV2 inhibitor. ( B ) Maximum amplitude (ΔF/F0) of the Fura-2 fluorescence response to light stimulation in eNpHR-expressing C2C12 myoblasts treated with DMSO 1/1000 ( n =4 0) or with 100 µM Tranilast ( n = 45). ( C ) Confocal images showing immuno-expression of the negative-dominant TRPV2[E594K] tagged with a flag sequence (red) co-transfected (TRPV2[E594K] right panels) or not (Ctrl, left panel) with eNpHR-YFP (green) in C2C12 myoblasts. ( D ) Representative traces of normalized Fura-2 ratio during a light stimulation of 30 s at 48 mW/cm 2 (orange rectangle) in C2C12 myoblasts expressing eNpHR-YFP co-transfected (TRPV2[E594K]) or not (Ctrl) with the negative-dominant TRPV2[E594K]. ( E ) Maximum amplitude (ΔF/F0) of the Fura-2 fluorescence response to light stimulation in control C2C12 myoblasts expressing eNpHR-YFP (Ctrl, n = 31) or in C2C12 myoblasts expressing eNpHR-YFP co-transfected with negative-dominant TRPV2[E594K] (TRPV2[E594K], n = 102). Data are presented as mean ± SEM. *** and **** represent significant differences with p

    Techniques Used: Expressing, Fluorescence, Sequencing, Transfection

    Schematic model of the effect of light-induced activation of halorhodopsin pump on the activation of calcium constitutive entry pathway through TRPV2 channels and on the modulation of cell migration. ( 1 ) Light stimulation of C2C12 myoblasts expressing eNpHR (orange) leads to membrane polarization by chloride ion entry (yellow) which gives rise to constitutive calcium entry (red) through TRPV2 channel (blue) by increasing the driving force for Ca 2+ across the plasma membrane. This light-induced calcium entry increases cell migration in a manner that can be abolished ( 2 ) by the TRPV2 inhibitor Tranilast (purple) and the negative-dominant TRPV2[E594K] transcript (green).
    Figure Legend Snippet: Schematic model of the effect of light-induced activation of halorhodopsin pump on the activation of calcium constitutive entry pathway through TRPV2 channels and on the modulation of cell migration. ( 1 ) Light stimulation of C2C12 myoblasts expressing eNpHR (orange) leads to membrane polarization by chloride ion entry (yellow) which gives rise to constitutive calcium entry (red) through TRPV2 channel (blue) by increasing the driving force for Ca 2+ across the plasma membrane. This light-induced calcium entry increases cell migration in a manner that can be abolished ( 2 ) by the TRPV2 inhibitor Tranilast (purple) and the negative-dominant TRPV2[E594K] transcript (green).

    Techniques Used: Activation Assay, Migration, Expressing

    Expression of transient receptor potential vanilloid 2 (TRPV2) channels in C2C12 myoblasts. ( A ) Assessment of TRPV2 mRNA expression in C2C12 myoblasts by RT-PCR. ( B ) Western blot analysis of TRPV2 and GAPDH expression in C2C12 myoblasts, human embryonic kidney (HEK)293 cells stably expressing TRPV2 (HEK TRPV2), and control HEK293 cells (HEK ctrl). TRPV2 and GAPDH were detected sequentially on the same blot (stripped twice). 5 µg of proteins were deposited for HEK cell lysates and 10 µg for C2C12 myoblasts ( n = 4). ( C ) Confocal images at two magnifications showing immuno-localization of TRPV2 (red) and nuclei staining (blue) in C2C12 myoblasts (scale bar: 20 µm).
    Figure Legend Snippet: Expression of transient receptor potential vanilloid 2 (TRPV2) channels in C2C12 myoblasts. ( A ) Assessment of TRPV2 mRNA expression in C2C12 myoblasts by RT-PCR. ( B ) Western blot analysis of TRPV2 and GAPDH expression in C2C12 myoblasts, human embryonic kidney (HEK)293 cells stably expressing TRPV2 (HEK TRPV2), and control HEK293 cells (HEK ctrl). TRPV2 and GAPDH were detected sequentially on the same blot (stripped twice). 5 µg of proteins were deposited for HEK cell lysates and 10 µg for C2C12 myoblasts ( n = 4). ( C ) Confocal images at two magnifications showing immuno-localization of TRPV2 (red) and nuclei staining (blue) in C2C12 myoblasts (scale bar: 20 µm).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Stable Transfection, Staining

    Light stimulation promotes cell migration mediated by TRPV2 in C2C12 myoblasts expressing eNpHR. ( A ) The role of TRPV2 channels in cell migration was evaluated with a cell migration tracking assay on C2C12 myoblasts expressing eNpHR. Cells were seeded at low density and the migration velocity (µm/min) was assessed for 15 h with a JuliStage system. C2C12 myoblast migration was evaluated for five conditions: without treatment ( n = 432), co-transfected with TRPV2[E594K] ( n = 290), treated with DMSO 1/1000 ( n = 180), treated with 100 µM Tranilast ( n = 285), and co-transfected with TRPV2[E594K] plus treated with 100 µM Tranilast ( n = 89) ($ corresponds to the comparison with C2C12 myoblasts without treatment, * corresponds to the comparison with C2C12 myoblasts treated with DMSO 1/1000) ($$$$ and ****: p
    Figure Legend Snippet: Light stimulation promotes cell migration mediated by TRPV2 in C2C12 myoblasts expressing eNpHR. ( A ) The role of TRPV2 channels in cell migration was evaluated with a cell migration tracking assay on C2C12 myoblasts expressing eNpHR. Cells were seeded at low density and the migration velocity (µm/min) was assessed for 15 h with a JuliStage system. C2C12 myoblast migration was evaluated for five conditions: without treatment ( n = 432), co-transfected with TRPV2[E594K] ( n = 290), treated with DMSO 1/1000 ( n = 180), treated with 100 µM Tranilast ( n = 285), and co-transfected with TRPV2[E594K] plus treated with 100 µM Tranilast ( n = 89) ($ corresponds to the comparison with C2C12 myoblasts without treatment, * corresponds to the comparison with C2C12 myoblasts treated with DMSO 1/1000) ($$$$ and ****: p

    Techniques Used: Migration, Expressing, Transfection

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    Alomone Labs rabbit polyclonal anti trpv2 antibody
    Light stimulation promotes cell migration mediated by <t>TRPV2</t> in C2C12 myoblasts expressing eNpHR. ( A ) The role of TRPV2 channels in cell migration was evaluated with a cell migration tracking assay on C2C12 myoblasts expressing eNpHR. Cells were seeded at low density and the migration velocity (µm/min) was assessed for 15 h with a JuliStage system. C2C12 myoblast migration was evaluated for five conditions: without treatment (n=432), co-transfected with TRPV2[E594K] (n=290), treated with DMSO 1/1000 (n=180), treated with 100 µM Tranilast (n=285), and co-transfected with TRPV2[E594K] plus treated with 100 µM Tranilast (n=89) ($ corresponds to the comparison with C2C12 myoblasts without treatment, * corresponds to the comparison with C2C12 myoblasts treated with DMSO 1/1000) ($$$$ and ****: p
    Rabbit Polyclonal Anti Trpv2 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Light stimulation promotes cell migration mediated by TRPV2 in C2C12 myoblasts expressing eNpHR. ( A ) The role of TRPV2 channels in cell migration was evaluated with a cell migration tracking assay on C2C12 myoblasts expressing eNpHR. Cells were seeded at low density and the migration velocity (µm/min) was assessed for 15 h with a JuliStage system. C2C12 myoblast migration was evaluated for five conditions: without treatment (n=432), co-transfected with TRPV2[E594K] (n=290), treated with DMSO 1/1000 (n=180), treated with 100 µM Tranilast (n=285), and co-transfected with TRPV2[E594K] plus treated with 100 µM Tranilast (n=89) ($ corresponds to the comparison with C2C12 myoblasts without treatment, * corresponds to the comparison with C2C12 myoblasts treated with DMSO 1/1000) ($$$$ and ****: p

    Journal: bioRxiv

    Article Title: Fine tuning of calcium constitutive entry by optogenetically-controlled membrane polarization: impact on cell migration

    doi: 10.1101/2020.06.04.134205

    Figure Lengend Snippet: Light stimulation promotes cell migration mediated by TRPV2 in C2C12 myoblasts expressing eNpHR. ( A ) The role of TRPV2 channels in cell migration was evaluated with a cell migration tracking assay on C2C12 myoblasts expressing eNpHR. Cells were seeded at low density and the migration velocity (µm/min) was assessed for 15 h with a JuliStage system. C2C12 myoblast migration was evaluated for five conditions: without treatment (n=432), co-transfected with TRPV2[E594K] (n=290), treated with DMSO 1/1000 (n=180), treated with 100 µM Tranilast (n=285), and co-transfected with TRPV2[E594K] plus treated with 100 µM Tranilast (n=89) ($ corresponds to the comparison with C2C12 myoblasts without treatment, * corresponds to the comparison with C2C12 myoblasts treated with DMSO 1/1000) ($$$$ and ****: p

    Article Snippet: Cells were stained using the blocking solution containing rabbit polyclonal anti-TRPV2 antibody (1:200, ACC-039, Alomone labs) or rabbit polyclonal anti-FLAG antibody (1:100, F7425, Sigma-Aldrich) overnight at 4°C, followed by incubation with donkey anti-rabbit secondary antibody coupled with red fluorescent Alexa Fluor 555 (1:400, Molecular Probes, ThermoFisher Scientific) for 2 hours at room temperature.

    Techniques: Migration, Expressing, Transfection

    Expression of TRPV2 channels in C2C12 myoblasts. ( A ) Assessment of TRPV2 mRNA expression in C2C12 myoblasts by RT-PCR. ( B ) Western blot analysis of TRPV2 and GAPDH expression in C2C12 myoblasts, HEK293 cells stably expressing TRPV2 (HEK TRPV2), and control HEK293 cells (HEK ctrl). TRPV2 and GAPDH were detected sequentially on the same blot (stripped twice). 5 µg of proteins were deposited for HEK cell lysates and 10 µg for C2C12 myoblasts (n=4). ( C ) Confocal images at two magnifications showing immuno-localization of TRPV2 (red) and nuclei staining (blue) in C2C12 myoblasts (scale bar: 20 µm).

    Journal: bioRxiv

    Article Title: Fine tuning of calcium constitutive entry by optogenetically-controlled membrane polarization: impact on cell migration

    doi: 10.1101/2020.06.04.134205

    Figure Lengend Snippet: Expression of TRPV2 channels in C2C12 myoblasts. ( A ) Assessment of TRPV2 mRNA expression in C2C12 myoblasts by RT-PCR. ( B ) Western blot analysis of TRPV2 and GAPDH expression in C2C12 myoblasts, HEK293 cells stably expressing TRPV2 (HEK TRPV2), and control HEK293 cells (HEK ctrl). TRPV2 and GAPDH were detected sequentially on the same blot (stripped twice). 5 µg of proteins were deposited for HEK cell lysates and 10 µg for C2C12 myoblasts (n=4). ( C ) Confocal images at two magnifications showing immuno-localization of TRPV2 (red) and nuclei staining (blue) in C2C12 myoblasts (scale bar: 20 µm).

    Article Snippet: Cells were stained using the blocking solution containing rabbit polyclonal anti-TRPV2 antibody (1:200, ACC-039, Alomone labs) or rabbit polyclonal anti-FLAG antibody (1:100, F7425, Sigma-Aldrich) overnight at 4°C, followed by incubation with donkey anti-rabbit secondary antibody coupled with red fluorescent Alexa Fluor 555 (1:400, Molecular Probes, ThermoFisher Scientific) for 2 hours at room temperature.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Stable Transfection, Staining

    Involvement of TRPV2 channels in the mediation of constitutive calcium entry during light stimulation. ( A ) Representative traces of Fura-2 normalized ratio in response to a light stimulation of 30 s at 48 mW/cm 2 (orange rectangle) in C2C12 myoblasts expressing eNpHR-YFP treated with DMSO 1/1000 (vehicle) or with 100 µM Tranilast, a TRPV2 inhibitor. ( B ) Maximum amplitude (∆F/F0) of the Fura-2 fluorescence response to light stimulation in eNpHR-expressing C2C12 myoblasts treated with DMSO 1/1000 (n=40) or with 100 µM Tranilast (n=45). ( C ) Confocal images showing immuno-expression of the negative-dominant TRPV2[E594K] tagged with a flag sequence (red) co-transfected (TRPV2[E594K] right panels) or not (Ctrl, left panel) with eNpHR-YFP (green) in C2C12 myoblasts. ( D ) Representative traces of normalized Fura-2 ratio during a light stimulation of 30 s at 48 mW/cm 2 (orange rectangle) in C2C12 myoblasts expressing eNpHR-YFP co-transfected (TRPV2[E594K]) or not (Ctrl) with the negative-dominant TRPV2[E594K]. ( E ) Maximum amplitude (∆F/F0) of the Fura-2 fluorescence response to light stimulation in control C2C12 myoblasts expressing eNpHR-YFP (Ctrl, n=31) or in C2C12 myoblasts expressing eNpHR-YFP co-transfected with negative-dominant TRPV2[E594K] (TRPV2[E594K], n=102). Data are presented as mean ± SEM. *** and **** represent significant differences with p

    Journal: bioRxiv

    Article Title: Fine tuning of calcium constitutive entry by optogenetically-controlled membrane polarization: impact on cell migration

    doi: 10.1101/2020.06.04.134205

    Figure Lengend Snippet: Involvement of TRPV2 channels in the mediation of constitutive calcium entry during light stimulation. ( A ) Representative traces of Fura-2 normalized ratio in response to a light stimulation of 30 s at 48 mW/cm 2 (orange rectangle) in C2C12 myoblasts expressing eNpHR-YFP treated with DMSO 1/1000 (vehicle) or with 100 µM Tranilast, a TRPV2 inhibitor. ( B ) Maximum amplitude (∆F/F0) of the Fura-2 fluorescence response to light stimulation in eNpHR-expressing C2C12 myoblasts treated with DMSO 1/1000 (n=40) or with 100 µM Tranilast (n=45). ( C ) Confocal images showing immuno-expression of the negative-dominant TRPV2[E594K] tagged with a flag sequence (red) co-transfected (TRPV2[E594K] right panels) or not (Ctrl, left panel) with eNpHR-YFP (green) in C2C12 myoblasts. ( D ) Representative traces of normalized Fura-2 ratio during a light stimulation of 30 s at 48 mW/cm 2 (orange rectangle) in C2C12 myoblasts expressing eNpHR-YFP co-transfected (TRPV2[E594K]) or not (Ctrl) with the negative-dominant TRPV2[E594K]. ( E ) Maximum amplitude (∆F/F0) of the Fura-2 fluorescence response to light stimulation in control C2C12 myoblasts expressing eNpHR-YFP (Ctrl, n=31) or in C2C12 myoblasts expressing eNpHR-YFP co-transfected with negative-dominant TRPV2[E594K] (TRPV2[E594K], n=102). Data are presented as mean ± SEM. *** and **** represent significant differences with p

    Article Snippet: Cells were stained using the blocking solution containing rabbit polyclonal anti-TRPV2 antibody (1:200, ACC-039, Alomone labs) or rabbit polyclonal anti-FLAG antibody (1:100, F7425, Sigma-Aldrich) overnight at 4°C, followed by incubation with donkey anti-rabbit secondary antibody coupled with red fluorescent Alexa Fluor 555 (1:400, Molecular Probes, ThermoFisher Scientific) for 2 hours at room temperature.

    Techniques: Expressing, Fluorescence, Sequencing, Transfection

    Schematic model of the effect of light-induced activation of halorhodopsin pump on the activation of calcium constitutive entry pathway through TRPV2 channels and on the modulation of cell migration. ( 1 ) Light stimulation of C2C12 myoblasts expressing eNpHR (orange) leads to membrane polarization by chloride ion entry (yellow) which gives rise to constitutive calcium entry (red) through TRPV2 channel (blue) by increasing the driving force for Ca 2+ across the plasma membrane. This light-induced calcium entry increases cell migration in a manner that can be abolished ( 2 ) by the TRPV2 inhibitor Tranilast (purple) and the negative-dominant TRPV2[E594K] transcript (green).

    Journal: bioRxiv

    Article Title: Fine tuning of calcium constitutive entry by optogenetically-controlled membrane polarization: impact on cell migration

    doi: 10.1101/2020.06.04.134205

    Figure Lengend Snippet: Schematic model of the effect of light-induced activation of halorhodopsin pump on the activation of calcium constitutive entry pathway through TRPV2 channels and on the modulation of cell migration. ( 1 ) Light stimulation of C2C12 myoblasts expressing eNpHR (orange) leads to membrane polarization by chloride ion entry (yellow) which gives rise to constitutive calcium entry (red) through TRPV2 channel (blue) by increasing the driving force for Ca 2+ across the plasma membrane. This light-induced calcium entry increases cell migration in a manner that can be abolished ( 2 ) by the TRPV2 inhibitor Tranilast (purple) and the negative-dominant TRPV2[E594K] transcript (green).

    Article Snippet: Cells were stained using the blocking solution containing rabbit polyclonal anti-TRPV2 antibody (1:200, ACC-039, Alomone labs) or rabbit polyclonal anti-FLAG antibody (1:100, F7425, Sigma-Aldrich) overnight at 4°C, followed by incubation with donkey anti-rabbit secondary antibody coupled with red fluorescent Alexa Fluor 555 (1:400, Molecular Probes, ThermoFisher Scientific) for 2 hours at room temperature.

    Techniques: Activation Assay, Migration, Expressing

    Influx in stretched cardiomyocytes in presence of TRPV2 channels antibodies

    Journal: Cell calcium

    Article Title: Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels

    doi: 10.1016/j.ceca.2016.01.001

    Figure Lengend Snippet: Influx in stretched cardiomyocytes in presence of TRPV2 channels antibodies

    Article Snippet: Immunolabelling with a-TRPV2 (ACC-039, Alomone lab) was performed using standard protocol including a permeabilization step with 0.1% Triton X-100 in PBS for 10 min.

    Techniques:

    TRPV2 protein expression in isolated WT and mdx cardiomyocytes

    Journal: Cell calcium

    Article Title: Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels

    doi: 10.1016/j.ceca.2016.01.001

    Figure Lengend Snippet: TRPV2 protein expression in isolated WT and mdx cardiomyocytes

    Article Snippet: Immunolabelling with a-TRPV2 (ACC-039, Alomone lab) was performed using standard protocol including a permeabilization step with 0.1% Triton X-100 in PBS for 10 min.

    Techniques: Expressing, Isolation

    TRPV2 expression in heart tissue from WT and mdx mice

    Journal: Cell calcium

    Article Title: Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels

    doi: 10.1016/j.ceca.2016.01.001

    Figure Lengend Snippet: TRPV2 expression in heart tissue from WT and mdx mice

    Article Snippet: Immunolabelling with a-TRPV2 (ACC-039, Alomone lab) was performed using standard protocol including a permeabilization step with 0.1% Triton X-100 in PBS for 10 min.

    Techniques: Expressing, Mouse Assay