anti trpc6  (Alomone Labs)


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    Structured Review

    Alomone Labs anti trpc6
    The mechanism by which <t>TRPC6</t> contributes to VSMC differentiation. A ) Immunofluorescence imaging of SM22α in TGF-β1-stimulated C3H10T1/2 cells in the presence of additional extracellular KCl. Cells were treated with TGF-β1 and indicated concentrations of KCl for 48 h. Representative images (left) and quantification of SM22α-positive cells (right) are shown ( n = 5). Scale bars, 50 μm. Nuclei were visualized with DAPI. B ) Membrane association of PTEN in TGF-β1-stimulated C3H10T1/2 cells in the presence or absence of additional extracellular KCl (30 mM). Cells were treated with TGF-β1 (5 ng/ml) and KCl for 48 h. Densitometric analysis of membrane-associated PTEN protein normalized to total PTEN protein (right, n = 5); increases are shown relative to untreated control cells. C ) PTEN membrane association in siRNA-transfected C3H10T1/2 cells. Cells were stimulated with TGF-β1 (5 ng/ml) for 12 h. Increases are shown relative to untreated siControl-transfected cells. D ) TRPC6 permeates Na + and Ca 2+ in proliferating VSMCs, which causes a slight depolarization of membrane potential that affects anionic phospholipid distribution in the inner leaflet of the plasma membrane. The C2 domain of PTEN binds to Ca 2+ in the proximal region of the mouth of the TRPC6 channel and brings PTEN close to the substrate PI(3,4,5)P 3 in the plasma membrane via interaction with clustered anionic phospholipids such as PS. TGF-β1 stimulation suppressed TRPC6 channel activity, which hyperpolarized membrane potential and inhibited Ca 2+ influx, resulting in dissociation of PTEN from the plasma membrane and accumulation of PI(3,4,5)P 3 that induces Akt activation. * P
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    Images

    1) Product Images from "TRPC6 regulates phenotypic switching of vascular smooth muscle cells through plasma membrane potential-dependent coupling with PTEN"

    Article Title: TRPC6 regulates phenotypic switching of vascular smooth muscle cells through plasma membrane potential-dependent coupling with PTEN

    Journal: The FASEB Journal

    doi: 10.1096/fj.201802811R

    The mechanism by which TRPC6 contributes to VSMC differentiation. A ) Immunofluorescence imaging of SM22α in TGF-β1-stimulated C3H10T1/2 cells in the presence of additional extracellular KCl. Cells were treated with TGF-β1 and indicated concentrations of KCl for 48 h. Representative images (left) and quantification of SM22α-positive cells (right) are shown ( n = 5). Scale bars, 50 μm. Nuclei were visualized with DAPI. B ) Membrane association of PTEN in TGF-β1-stimulated C3H10T1/2 cells in the presence or absence of additional extracellular KCl (30 mM). Cells were treated with TGF-β1 (5 ng/ml) and KCl for 48 h. Densitometric analysis of membrane-associated PTEN protein normalized to total PTEN protein (right, n = 5); increases are shown relative to untreated control cells. C ) PTEN membrane association in siRNA-transfected C3H10T1/2 cells. Cells were stimulated with TGF-β1 (5 ng/ml) for 12 h. Increases are shown relative to untreated siControl-transfected cells. D ) TRPC6 permeates Na + and Ca 2+ in proliferating VSMCs, which causes a slight depolarization of membrane potential that affects anionic phospholipid distribution in the inner leaflet of the plasma membrane. The C2 domain of PTEN binds to Ca 2+ in the proximal region of the mouth of the TRPC6 channel and brings PTEN close to the substrate PI(3,4,5)P 3 in the plasma membrane via interaction with clustered anionic phospholipids such as PS. TGF-β1 stimulation suppressed TRPC6 channel activity, which hyperpolarized membrane potential and inhibited Ca 2+ influx, resulting in dissociation of PTEN from the plasma membrane and accumulation of PI(3,4,5)P 3 that induces Akt activation. * P
    Figure Legend Snippet: The mechanism by which TRPC6 contributes to VSMC differentiation. A ) Immunofluorescence imaging of SM22α in TGF-β1-stimulated C3H10T1/2 cells in the presence of additional extracellular KCl. Cells were treated with TGF-β1 and indicated concentrations of KCl for 48 h. Representative images (left) and quantification of SM22α-positive cells (right) are shown ( n = 5). Scale bars, 50 μm. Nuclei were visualized with DAPI. B ) Membrane association of PTEN in TGF-β1-stimulated C3H10T1/2 cells in the presence or absence of additional extracellular KCl (30 mM). Cells were treated with TGF-β1 (5 ng/ml) and KCl for 48 h. Densitometric analysis of membrane-associated PTEN protein normalized to total PTEN protein (right, n = 5); increases are shown relative to untreated control cells. C ) PTEN membrane association in siRNA-transfected C3H10T1/2 cells. Cells were stimulated with TGF-β1 (5 ng/ml) for 12 h. Increases are shown relative to untreated siControl-transfected cells. D ) TRPC6 permeates Na + and Ca 2+ in proliferating VSMCs, which causes a slight depolarization of membrane potential that affects anionic phospholipid distribution in the inner leaflet of the plasma membrane. The C2 domain of PTEN binds to Ca 2+ in the proximal region of the mouth of the TRPC6 channel and brings PTEN close to the substrate PI(3,4,5)P 3 in the plasma membrane via interaction with clustered anionic phospholipids such as PS. TGF-β1 stimulation suppressed TRPC6 channel activity, which hyperpolarized membrane potential and inhibited Ca 2+ influx, resulting in dissociation of PTEN from the plasma membrane and accumulation of PI(3,4,5)P 3 that induces Akt activation. * P

    Techniques Used: Immunofluorescence, Imaging, Transfection, Activity Assay, Activation Assay

    2) Product Images from "Cell-cell Interaction Underlies Formation of Fluid in the Male Reproductive Tract of the Rat"

    Article Title: Cell-cell Interaction Underlies Formation of Fluid in the Male Reproductive Tract of the Rat

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200409205

    Identification and localization of TRPC and COX-1 in the rat epididymis. (A) RT-PCR and Western blot analysis of TRPC and COX-1 transcripts and proteins. RT-PCR revealed only TRPC1, 3, and 6 transcripts but not TRPC2, 4, and 5 transcripts (not depicted). The PCR products for TRPC1, 3, and 6 were more intense in the caudal than in the proximal region of the epididymis, whereas that of COX-1 was more uniformly distributed along the epididymis. The PCR product for S16 serves as the internal standard. In parallel with the RNA expression, Western blots also show the expression of TRPC1, 3, and 6 proteins in the rat epididymis. (B) Immunohistochemical localization of TRPC1, TRPC3, and TRPC6 proteins in the rat epididymis using polyclonal rabbit anti-TRPC1, anti-TRPC3, and anti-TRPC6 antibodies (1:100 dilution). Higher magnifications are shown in insets. Negative controls were obtained by incubation with antigen-preabsorbed antibodies. Phase contrast images of the corresponding stained sections are shown below. (C) Consecutive sections of the rat cauda epididymidis showing positive immunoreactivity (fluorescence) for COX-1 and TRPC3, respectively. Both proteins were restricted to basal cells in the epithelium but not to the other cell types. The two sections were overlaid to demonstrate coexistence of TRPC3 and COX-1 in the basal cells. Phase contrast image is shown to reveal the relationship of the cells. Basal cells are indicated by arrows.
    Figure Legend Snippet: Identification and localization of TRPC and COX-1 in the rat epididymis. (A) RT-PCR and Western blot analysis of TRPC and COX-1 transcripts and proteins. RT-PCR revealed only TRPC1, 3, and 6 transcripts but not TRPC2, 4, and 5 transcripts (not depicted). The PCR products for TRPC1, 3, and 6 were more intense in the caudal than in the proximal region of the epididymis, whereas that of COX-1 was more uniformly distributed along the epididymis. The PCR product for S16 serves as the internal standard. In parallel with the RNA expression, Western blots also show the expression of TRPC1, 3, and 6 proteins in the rat epididymis. (B) Immunohistochemical localization of TRPC1, TRPC3, and TRPC6 proteins in the rat epididymis using polyclonal rabbit anti-TRPC1, anti-TRPC3, and anti-TRPC6 antibodies (1:100 dilution). Higher magnifications are shown in insets. Negative controls were obtained by incubation with antigen-preabsorbed antibodies. Phase contrast images of the corresponding stained sections are shown below. (C) Consecutive sections of the rat cauda epididymidis showing positive immunoreactivity (fluorescence) for COX-1 and TRPC3, respectively. Both proteins were restricted to basal cells in the epithelium but not to the other cell types. The two sections were overlaid to demonstrate coexistence of TRPC3 and COX-1 in the basal cells. Phase contrast image is shown to reveal the relationship of the cells. Basal cells are indicated by arrows.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction, RNA Expression, Expressing, Immunohistochemistry, Incubation, Staining, Fluorescence

    3) Product Images from "Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes"

    Article Title: Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2008.153676

    DiC8-PIP 2 inhibits Ang II-evoked whole-cell cation currents and single TRPC6 channel activity in rabbit mesenteric artery myocytes Aa and b , inclusion of 100 μ m diC8-PIP 2 in the patch pipette solution inhibits whole-cell cation currents evoked by 1 n m Ang II. Ac , mean I–V relationship showing diC8-PIP 2 inhibits Ang II-evoked cation currents at all potentials tested ( n = at least 4 per point). Ba , 100 μ m diC8-PIP 2 inhibited TRPC activity in an inside-out patch which was initially activated by 1 n m Ang II in a cell-attached patch (i/o, inside-out patch). Bb , 100 μ m diC8-PIP 2 inhibited TRPC activity induced by 10 μ m OAG in an inside-out patch. C , inhibitory action of diC8-PIP 2 on OAG-evoked TRPC6 activity in inside-out patches had an IC 50 value of 7.6 μ m ( n = at least 4 per point).
    Figure Legend Snippet: DiC8-PIP 2 inhibits Ang II-evoked whole-cell cation currents and single TRPC6 channel activity in rabbit mesenteric artery myocytes Aa and b , inclusion of 100 μ m diC8-PIP 2 in the patch pipette solution inhibits whole-cell cation currents evoked by 1 n m Ang II. Ac , mean I–V relationship showing diC8-PIP 2 inhibits Ang II-evoked cation currents at all potentials tested ( n = at least 4 per point). Ba , 100 μ m diC8-PIP 2 inhibited TRPC activity in an inside-out patch which was initially activated by 1 n m Ang II in a cell-attached patch (i/o, inside-out patch). Bb , 100 μ m diC8-PIP 2 inhibited TRPC activity induced by 10 μ m OAG in an inside-out patch. C , inhibitory action of diC8-PIP 2 on OAG-evoked TRPC6 activity in inside-out patches had an IC 50 value of 7.6 μ m ( n = at least 4 per point).

    Techniques Used: Activity Assay, Transferring

    Agents that deplete PIP 2 evoke TRPC6 channel activity A and B , respectively, 20 μ m wortmannin and 100 μ m LY294002 transiently activated channel activity in cell-attached patches ( a ) which had similar amplitude histograms ( b ) and unitary conductances and E r values ( c ). C , 50 μg ml −1 poly- l -lysine activated channel currents in an inside-out patch ( a ) which had a similar properties to those evoked by wortmannin and LY294002 ( b and c ).
    Figure Legend Snippet: Agents that deplete PIP 2 evoke TRPC6 channel activity A and B , respectively, 20 μ m wortmannin and 100 μ m LY294002 transiently activated channel activity in cell-attached patches ( a ) which had similar amplitude histograms ( b ) and unitary conductances and E r values ( c ). C , 50 μg ml −1 poly- l -lysine activated channel currents in an inside-out patch ( a ) which had a similar properties to those evoked by wortmannin and LY294002 ( b and c ).

    Techniques Used: Activity Assay

    Anti-PIP 2 antibody potentiates TRPC6 channel activity Aa , 1: 200 dilution of anti-PIP 2 antibodies transiently increased TRPC6 activity in an inside-out patch which was induced by 1 n m Ang II in cell-attached mode. Subsequently the Ang II-evoked response was inhibited in the presence of the anti-PIP 2 antibody. Ab , 1: 200 dilution of anti-PIP 2 antibodies produced a sustained increase of OAG-evoked TRPC6 activity in an inside-out patch. B , mean data of effect of anti-PIP 2 antibodies on Ang II- and OAG-induced TRPC6 activity ( n = 6 for all conditions, ** P
    Figure Legend Snippet: Anti-PIP 2 antibody potentiates TRPC6 channel activity Aa , 1: 200 dilution of anti-PIP 2 antibodies transiently increased TRPC6 activity in an inside-out patch which was induced by 1 n m Ang II in cell-attached mode. Subsequently the Ang II-evoked response was inhibited in the presence of the anti-PIP 2 antibody. Ab , 1: 200 dilution of anti-PIP 2 antibodies produced a sustained increase of OAG-evoked TRPC6 activity in an inside-out patch. B , mean data of effect of anti-PIP 2 antibodies on Ang II- and OAG-induced TRPC6 activity ( n = 6 for all conditions, ** P

    Techniques Used: Activity Assay, Produced

    Wortmannin-induced depletion of PIP 2 decreases Ang II-evoked and increases OAG-induced TRPC6 activity in cell-attached patches A shows that Ang II-evoked TRPC6 activity ( a ) is decreased after pre-treatment with wortmannin ( b ). B illustrates that OAG-induced TRPC6 activity ( a ) is markedly increased following pre-treatment with wortmannin ( b ). C , mean data showing effect of wortmannin on Ang II- and OAG-induced TRPC6 activity (*** P
    Figure Legend Snippet: Wortmannin-induced depletion of PIP 2 decreases Ang II-evoked and increases OAG-induced TRPC6 activity in cell-attached patches A shows that Ang II-evoked TRPC6 activity ( a ) is decreased after pre-treatment with wortmannin ( b ). B illustrates that OAG-induced TRPC6 activity ( a ) is markedly increased following pre-treatment with wortmannin ( b ). C , mean data showing effect of wortmannin on Ang II- and OAG-induced TRPC6 activity (*** P

    Techniques Used: Activity Assay

    4) Product Images from "TRPC3 Activation by Erythropoietin Is Modulated by TRPC6"

    Article Title: TRPC3 Activation by Erythropoietin Is Modulated by TRPC6

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M804734200

    Western blot ( WB ) of HEK 293T cells transfected ( Tx'd ) with Epo-R, BFP-TRPC3, and BFP-TRPC6. HEK 293T cells were cotransfected with Epo-R, and BFP-TRPC3, BFP-TRPC6, or both. Equivalent amounts of protein lysates were loaded in each lane, and Western blotting was performed with anti-TRC3, anti-TRPC6, or anti-Epo-R antibodies, followed by ECL.
    Figure Legend Snippet: Western blot ( WB ) of HEK 293T cells transfected ( Tx'd ) with Epo-R, BFP-TRPC3, and BFP-TRPC6. HEK 293T cells were cotransfected with Epo-R, and BFP-TRPC3, BFP-TRPC6, or both. Equivalent amounts of protein lysates were loaded in each lane, and Western blotting was performed with anti-TRC3, anti-TRPC6, or anti-Epo-R antibodies, followed by ECL.

    Techniques Used: Western Blot, Transfection

    Interaction of transfected and endogenous TRPC3 and TRPC6. A, V5-TRPC3 and/or FLAG-TRPC6 were expressed in HEK 293T cells with Epo-R. Immunoprecipitation ( IP ) was performed on lysates with anti-V5 antibody or anti-FLAG-agarose. Western blotting ( WB ) was performed after immunoprecipitation with anti-V5 or anti-FLAG antibodies. Representative results of five experiments are shown. B, immunoprecipitation was performed on lysates from TF-1 erythroid cells with anti-TRPC3 or anti-TRPC6 antibody to detect endogenous interactions. Western blotting was performed after immunoprecipitation with anti-TRPC3 or anti-TRPC3 antibodies. Representative results of two experiments are shown.
    Figure Legend Snippet: Interaction of transfected and endogenous TRPC3 and TRPC6. A, V5-TRPC3 and/or FLAG-TRPC6 were expressed in HEK 293T cells with Epo-R. Immunoprecipitation ( IP ) was performed on lysates with anti-V5 antibody or anti-FLAG-agarose. Western blotting ( WB ) was performed after immunoprecipitation with anti-V5 or anti-FLAG antibodies. Representative results of five experiments are shown. B, immunoprecipitation was performed on lysates from TF-1 erythroid cells with anti-TRPC3 or anti-TRPC6 antibody to detect endogenous interactions. Western blotting was performed after immunoprecipitation with anti-TRPC3 or anti-TRPC3 antibodies. Representative results of two experiments are shown.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot

    Schema of TRPC3/TRPC6 chimeras. A, schematic model of TRPC3-C6C, TRPC6-C3N, and TRPC6-C3C chimeras. B, Western blot ( WB ) of lysates from HEK 293T cells transfected with BFP-TRPC3, BFP-TRPC6, BFP-TRPC6-C3C, BFP-TRPC6-C3N, or BFP-TRPC3-C6C. Blots were probed with antibodies that recognize the C terminus of human TRPC3 (anti-TRPC3 (C)), the N terminus of murine and human TRPC3 (anti-TRPC3 (N)), or the N terminus of TRPC6 (anti-TRPC6 (N)). Representative results of two experiments are shown.
    Figure Legend Snippet: Schema of TRPC3/TRPC6 chimeras. A, schematic model of TRPC3-C6C, TRPC6-C3N, and TRPC6-C3C chimeras. B, Western blot ( WB ) of lysates from HEK 293T cells transfected with BFP-TRPC3, BFP-TRPC6, BFP-TRPC6-C3C, BFP-TRPC6-C3N, or BFP-TRPC3-C6C. Blots were probed with antibodies that recognize the C terminus of human TRPC3 (anti-TRPC3 (C)), the N terminus of murine and human TRPC3 (anti-TRPC3 (N)), or the N terminus of TRPC6 (anti-TRPC6 (N)). Representative results of two experiments are shown.

    Techniques Used: Western Blot, Transfection

    Interaction of TRPC3/TRPC6 chimeras with Epo-R. A, HEK 293T cells were transfected ( Tx'd ) with Epo-R and V5-TRPC3 or V5-TRPC3-C6C. Lysates were immunoprecipitated ( IP ) with anti-V5, anti-Epo-R antibodies, or normal rabbit serum ( NRS ). Western blots ( WB ) of lysates and immunoprecipitates were probed with anti-V5-HRP or anti-Epo-R and appropriate secondary antibody. Representative results of three similar experiments are shown. B, HEK 293T cells were transfected with Epo-R and FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N. Lysates were immunoprecipitated with anti-FLAG-agarose, anti-Epo-R, or normal rabbit serum. Western blots of lysates and immunoprecipitates were probed with anti-FLAG or anti-Epo-R antibodies. Representative results of three experiments are shown. C, densitometry was used to quantitate Epo-R, V5-TRPC3, FLAG-TRPC6, and chimeric channel bands from three experiments using transfected HEK 293T cells. The Epo-R to V5-TRPC3 or V5-TRPC3-C6C ratio or Epo-R to FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N ratio was calculated and normalized to V5-TRPC3 or FLAG-TRPC6 to allow comparison between experiments. The mean normalized ratio ± S.E. was determined for three separate experiments. The Epo-R/V5-TRPC3-C6C ratio was significantly less than the Epo-R/V5-TRPC3 ratio ( * , p ≤ 0.01), and the Epo-R/FLAG-TRPC6-C3C ratio was significantly greater than the Epo-R/FLAG-TRPC6 or FLAG-TRPC6-C3N ratio ( ** , *** , p ≤ 0.001).
    Figure Legend Snippet: Interaction of TRPC3/TRPC6 chimeras with Epo-R. A, HEK 293T cells were transfected ( Tx'd ) with Epo-R and V5-TRPC3 or V5-TRPC3-C6C. Lysates were immunoprecipitated ( IP ) with anti-V5, anti-Epo-R antibodies, or normal rabbit serum ( NRS ). Western blots ( WB ) of lysates and immunoprecipitates were probed with anti-V5-HRP or anti-Epo-R and appropriate secondary antibody. Representative results of three similar experiments are shown. B, HEK 293T cells were transfected with Epo-R and FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N. Lysates were immunoprecipitated with anti-FLAG-agarose, anti-Epo-R, or normal rabbit serum. Western blots of lysates and immunoprecipitates were probed with anti-FLAG or anti-Epo-R antibodies. Representative results of three experiments are shown. C, densitometry was used to quantitate Epo-R, V5-TRPC3, FLAG-TRPC6, and chimeric channel bands from three experiments using transfected HEK 293T cells. The Epo-R to V5-TRPC3 or V5-TRPC3-C6C ratio or Epo-R to FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N ratio was calculated and normalized to V5-TRPC3 or FLAG-TRPC6 to allow comparison between experiments. The mean normalized ratio ± S.E. was determined for three separate experiments. The Epo-R/V5-TRPC3-C6C ratio was significantly less than the Epo-R/V5-TRPC3 ratio ( * , p ≤ 0.01), and the Epo-R/FLAG-TRPC6-C3C ratio was significantly greater than the Epo-R/FLAG-TRPC6 or FLAG-TRPC6-C3N ratio ( ** , *** , p ≤ 0.001).

    Techniques Used: Transfection, Immunoprecipitation, Western Blot

    Schema of chimeras of subdivided TRPC3 and TRPC3 C termini. Amino acid ( AA ) composition of TRPC3 and TRPC6 chimeras and localization of TRP, CRIB, and coiled-coil domains are shown.
    Figure Legend Snippet: Schema of chimeras of subdivided TRPC3 and TRPC3 C termini. Amino acid ( AA ) composition of TRPC3 and TRPC6 chimeras and localization of TRP, CRIB, and coiled-coil domains are shown.

    Techniques Used:

    Immunolocalization of TRPC3 and Epo-R. HEK 293T cells were transfected ( Tx'd ) with Ext-V5-TRPC3, Epo-R, with or without TRPC6. Cells were permeabilized or not. Cells were stained with anti-V5, anti-Epo-R, or anti-TRPC6 antibodies, and then with Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit antibodies. Nuclei were identified by 4′,6-diamidino-2-phenylindole staining. Representative results of images obtained in the midplane of the cell with confocal microscopy are shown.
    Figure Legend Snippet: Immunolocalization of TRPC3 and Epo-R. HEK 293T cells were transfected ( Tx'd ) with Ext-V5-TRPC3, Epo-R, with or without TRPC6. Cells were permeabilized or not. Cells were stained with anti-V5, anti-Epo-R, or anti-TRPC6 antibodies, and then with Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit antibodies. Nuclei were identified by 4′,6-diamidino-2-phenylindole staining. Representative results of images obtained in the midplane of the cell with confocal microscopy are shown.

    Techniques Used: Transfection, Staining, Confocal Microscopy

    Modulation of membrane insertion of TRPC3 by Epo detected by cell surface biotinylation. HEK 293T cells transfected ( Tx'd ) with Epo-R and V5-TRPC3 without ( A and B ) or with FLAG-TRPC6 ( C and D ) were stimulated with 40 units/ml Epo. Biotinylation of cell surface proteins was performed, and V5-TRPC3 immunoprecipitated ( IP ) from lysates with anti-V5 antibody. Western blots ( WB ) were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-V5-HRP to detect total V5-TRPC3. Representative results of Western blots from four experiments are shown in A and C . Biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0, 1, 5, 10, and 20 min from four experiments are shown ( B and D ). * indicates a significant difference in the ratio compared with time 0 ( p ≤ 0.02). E, Epo-stimulated cell surface expression of endogenous TRPC3 was examined using BFU-E-derived erythroblasts at day 10 of methylcellulose culture (two experiments) or erythroblasts from phase II day 8 of liquid culture (one experiment). Cells were stimulated with 40 units/ml Epo for 0 or 5 min and biotinylated, and TRPC3 was immunoprecipitated from lysates with anti-TRPC3 antibody. Western blots were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-TRPC3 to detect total TRPC3. A representative result of three Western blots is shown. F, biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0 and 5 min from the three experiments are shown. No significant difference in the ratio at 5 min compared with time 0 was detected.
    Figure Legend Snippet: Modulation of membrane insertion of TRPC3 by Epo detected by cell surface biotinylation. HEK 293T cells transfected ( Tx'd ) with Epo-R and V5-TRPC3 without ( A and B ) or with FLAG-TRPC6 ( C and D ) were stimulated with 40 units/ml Epo. Biotinylation of cell surface proteins was performed, and V5-TRPC3 immunoprecipitated ( IP ) from lysates with anti-V5 antibody. Western blots ( WB ) were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-V5-HRP to detect total V5-TRPC3. Representative results of Western blots from four experiments are shown in A and C . Biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0, 1, 5, 10, and 20 min from four experiments are shown ( B and D ). * indicates a significant difference in the ratio compared with time 0 ( p ≤ 0.02). E, Epo-stimulated cell surface expression of endogenous TRPC3 was examined using BFU-E-derived erythroblasts at day 10 of methylcellulose culture (two experiments) or erythroblasts from phase II day 8 of liquid culture (one experiment). Cells were stimulated with 40 units/ml Epo for 0 or 5 min and biotinylated, and TRPC3 was immunoprecipitated from lysates with anti-TRPC3 antibody. Western blots were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-TRPC3 to detect total TRPC3. A representative result of three Western blots is shown. F, biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0 and 5 min from the three experiments are shown. No significant difference in the ratio at 5 min compared with time 0 was detected.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Expressing, Derivative Assay

    Plasma membrane insertion of TRPC3/TRPC3 chimeras detected with cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-C6C, V5-TRPC3-C6C1, V5-TRPC3-C6C2, FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N and Epo-R. Lysates were prepared, and immunoprecipitation ( IP ) performed with anti-V5 antibody or anti-FLAG-agarose. Western blotting ( WB ) was performed on immunoprecipitation pellets with streptavidin-HRP to detect biotinylation and either anti-V5-HRP to detect total TRPC3 chimeras or anti-TRPC6 or anti-TRPC3-N antibodies to detect total TRPC6 chimeras. Representative results of two experiments are shown. Tx'd , transfected.
    Figure Legend Snippet: Plasma membrane insertion of TRPC3/TRPC3 chimeras detected with cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-C6C, V5-TRPC3-C6C1, V5-TRPC3-C6C2, FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N and Epo-R. Lysates were prepared, and immunoprecipitation ( IP ) performed with anti-V5 antibody or anti-FLAG-agarose. Western blotting ( WB ) was performed on immunoprecipitation pellets with streptavidin-HRP to detect biotinylation and either anti-V5-HRP to detect total TRPC3 chimeras or anti-TRPC6 or anti-TRPC3-N antibodies to detect total TRPC6 chimeras. Representative results of two experiments are shown. Tx'd , transfected.

    Techniques Used: Expressing, Immunoprecipitation, Western Blot, Transfection

    5) Product Images from "The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *"

    Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.238360

    Schema of TRPC3/TRPC6 chimera. A , domains and motifs in the TRPC3 C terminus. B , representation of the proximal (C1) and distal (C2) C termini of TRPC3 and TRPC6. C , schematic models of TRPC3 chimeras: TRPC3-C6C1, TRPC3-C6C2, and TRPC3-C6TRP. D , schematic models of TRPC6 chimeras: TRPC6-C3C1, TRPC6-C3C2, TRPC6-C3TRP, and TRPC6-C3TRP-C3C2. For B–D , the origin of the TRP domain is shown by the color of the oval . The amino acid ( AA ) number at the beginning and at the end of the C1 and C2 domains contributed by TRPC3 ( top ) or TRPC6 ( bottom ) is indicated. FLAG-tagged chimeras expressed FLAG at the N terminus, and V5-tagged chimeras expressed V5 at the C terminus of the channel.
    Figure Legend Snippet: Schema of TRPC3/TRPC6 chimera. A , domains and motifs in the TRPC3 C terminus. B , representation of the proximal (C1) and distal (C2) C termini of TRPC3 and TRPC6. C , schematic models of TRPC3 chimeras: TRPC3-C6C1, TRPC3-C6C2, and TRPC3-C6TRP. D , schematic models of TRPC6 chimeras: TRPC6-C3C1, TRPC6-C3C2, TRPC6-C3TRP, and TRPC6-C3TRP-C3C2. For B–D , the origin of the TRP domain is shown by the color of the oval . The amino acid ( AA ) number at the beginning and at the end of the C1 and C2 domains contributed by TRPC3 ( top ) or TRPC6 ( bottom ) is indicated. FLAG-tagged chimeras expressed FLAG at the N terminus, and V5-tagged chimeras expressed V5 at the C terminus of the channel.

    Techniques Used:

    Role of the distal C terminus of TRPC3 and TRPC6 in regulation by Epo-R. HEK 293T cells were transfected with BFP-TRPC3, BFP-TRPC6, BFP-TRPC6-C3TRP, BFP-TRPC6-C3C2, BFP-TRPC6-C3TRP-C3C2, BFP-TRPC3-C6 802–809, or BFP-TRPC6-C3TRP-C3 741–748 chimeras and Epo-R. Fura Red-loaded cells were treated with 40 units/ml Epo. To quantitate [Ca 2+ ] i , F 440 / F 490 was measured at base line and by monitoring over 20 min after Epo stimulation. Shown is the percentage increase in F 440 / F 490 above base line (mean ± S.E. (error bars) percentage increase) = peak F 440 / F 490 divided by base line F 440 / F 490 × 100% − 100% (base line). The numbers of individual cells studied were as follows: BFP-TRPC3 (PBS 56, Epo 102), BFP-TRPC6 (PBS 57, Epo 103), BFP-TRPC6-C3TRP (PBS 20, Epo 34), BFP-TRPC6-C3C2 (PBS 20, Epo 35), BFP-TRPC6-C3TRP-C3C2 (PBS 30, Epo 64), BFP-TRPC3-C6 802–809 (PBS 26, Epo 49), or BFP-TRPC6-C3TRP-C3 741–748 (PBS 12, Epo 30). *, significantly greater percentage increase in F 440 / F 490 compared with Epo-stimulated cells expressing wild type TRPC6 ( p
    Figure Legend Snippet: Role of the distal C terminus of TRPC3 and TRPC6 in regulation by Epo-R. HEK 293T cells were transfected with BFP-TRPC3, BFP-TRPC6, BFP-TRPC6-C3TRP, BFP-TRPC6-C3C2, BFP-TRPC6-C3TRP-C3C2, BFP-TRPC3-C6 802–809, or BFP-TRPC6-C3TRP-C3 741–748 chimeras and Epo-R. Fura Red-loaded cells were treated with 40 units/ml Epo. To quantitate [Ca 2+ ] i , F 440 / F 490 was measured at base line and by monitoring over 20 min after Epo stimulation. Shown is the percentage increase in F 440 / F 490 above base line (mean ± S.E. (error bars) percentage increase) = peak F 440 / F 490 divided by base line F 440 / F 490 × 100% − 100% (base line). The numbers of individual cells studied were as follows: BFP-TRPC3 (PBS 56, Epo 102), BFP-TRPC6 (PBS 57, Epo 103), BFP-TRPC6-C3TRP (PBS 20, Epo 34), BFP-TRPC6-C3C2 (PBS 20, Epo 35), BFP-TRPC6-C3TRP-C3C2 (PBS 30, Epo 64), BFP-TRPC3-C6 802–809 (PBS 26, Epo 49), or BFP-TRPC6-C3TRP-C3 741–748 (PBS 12, Epo 30). *, significantly greater percentage increase in F 440 / F 490 compared with Epo-stimulated cells expressing wild type TRPC6 ( p

    Techniques Used: Transfection, Expressing

    Membrane and cytoskeletal association of TRPC3, TRPC6, and TRPC3/TRPC6 chimeras. Proteins from transfected HEK 293T cells ( A–C ) or UT-7/Epo cells ( C ) were fractionated, purified, and analyzed by Western blotting ( WB ). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Quality of fractionation was confirmed by probing Western blots with anti-Na + K + -ATPase (membrane), anti-vimentin (cytoskeletal fraction), and anti-GAPDH (cytosol marker) antibodies. Equivalent input from lysates was confirmed by probing with anti-actin. A , fractionation with the Qiagen cell fractionation kit. Membrane and cytoskeletal association of TRPC3/6 channels and chimeras. Three to nine experiments (depending on the FLAG-tagged construct) were performed, and representative results are shown. B , fractionation using the 0.5% Triton X-100 extraction method. Representative results of three experiments are shown. C , subcellular fractionation of endogenous TRPC3 and TRPC6 in UT-7/Epo cells with the Qiagen fractionation kit. For all preparations, 100 μg/lane was loaded except for the cytoskeletal fraction of transfected cells, where 50 μg was loaded per lane. Endogenous TRPC3 was detected using anti-TRPC3-C antibody. Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody. Transfected HEK 293T cells were used as controls. Representative results of four experiments are shown. lys , whole cell lysate; M , membrane fraction; Csk , cytoskeletal fraction. *, bands that did not disappear with peptide blocking, indicating that they are nonspecific.
    Figure Legend Snippet: Membrane and cytoskeletal association of TRPC3, TRPC6, and TRPC3/TRPC6 chimeras. Proteins from transfected HEK 293T cells ( A–C ) or UT-7/Epo cells ( C ) were fractionated, purified, and analyzed by Western blotting ( WB ). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Quality of fractionation was confirmed by probing Western blots with anti-Na + K + -ATPase (membrane), anti-vimentin (cytoskeletal fraction), and anti-GAPDH (cytosol marker) antibodies. Equivalent input from lysates was confirmed by probing with anti-actin. A , fractionation with the Qiagen cell fractionation kit. Membrane and cytoskeletal association of TRPC3/6 channels and chimeras. Three to nine experiments (depending on the FLAG-tagged construct) were performed, and representative results are shown. B , fractionation using the 0.5% Triton X-100 extraction method. Representative results of three experiments are shown. C , subcellular fractionation of endogenous TRPC3 and TRPC6 in UT-7/Epo cells with the Qiagen fractionation kit. For all preparations, 100 μg/lane was loaded except for the cytoskeletal fraction of transfected cells, where 50 μg was loaded per lane. Endogenous TRPC3 was detected using anti-TRPC3-C antibody. Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody. Transfected HEK 293T cells were used as controls. Representative results of four experiments are shown. lys , whole cell lysate; M , membrane fraction; Csk , cytoskeletal fraction. *, bands that did not disappear with peptide blocking, indicating that they are nonspecific.

    Techniques Used: Transfection, Purification, Western Blot, Construct, Fractionation, Marker, Cell Fractionation, Blocking Assay

    TRP domains, leucine zipper motifs, and AMPK binding site in TRPC3 and TRPC6. A , amino acid compositions of TRP domains and leucine zipper motifs of TRPC3 and TRPC6 are shown. Letters in boldface type indicate exchanged amino acids in the chimeras. For TRP domain exchange, the boldface sequences in the TRP domain of TRPC6 were exchanged with those of TRPC3 to create TRPC3-C6TRP. The boldface amino acids of TRPC3 TRP were exchanged with those of TRPC6 to create TRPC6-C3TRP. For leucine zipper exchange, the boldface sequences in the leucine zipper of TRPC6 were exchanged with those of TRPC3 to create TRPC3-C6LZ. The boldface amino acids of TRPC3 were exchanged with those of TRPC6 leucine zipper to create TRPC6-C3LZ. B , exchange of TRPC3 741–748 and TRPC6 802–809 is shown. Amino acids contributed by TRPC3 ( top ) or TRPC6 ( bottom ) are indicated, and localization to the C1 or C2 part of the C terminus is shown.
    Figure Legend Snippet: TRP domains, leucine zipper motifs, and AMPK binding site in TRPC3 and TRPC6. A , amino acid compositions of TRP domains and leucine zipper motifs of TRPC3 and TRPC6 are shown. Letters in boldface type indicate exchanged amino acids in the chimeras. For TRP domain exchange, the boldface sequences in the TRP domain of TRPC6 were exchanged with those of TRPC3 to create TRPC3-C6TRP. The boldface amino acids of TRPC3 TRP were exchanged with those of TRPC6 to create TRPC6-C3TRP. For leucine zipper exchange, the boldface sequences in the leucine zipper of TRPC6 were exchanged with those of TRPC3 to create TRPC3-C6LZ. The boldface amino acids of TRPC3 were exchanged with those of TRPC6 leucine zipper to create TRPC6-C3LZ. B , exchange of TRPC3 741–748 and TRPC6 802–809 is shown. Amino acids contributed by TRPC3 ( top ) or TRPC6 ( bottom ) are indicated, and localization to the C1 or C2 part of the C terminus is shown.

    Techniques Used: Binding Assay

    Plasma membrane insertion of TRPC3/TRPC6 chimeras detected with cell surface biotinylation. HEK 293T cells transfected ( Tx'd ) with Epo-R and V5-TRPC3, V5-TRPC3-C6TRP, V5-TRPC6, V5-TRPC6-C3TRP, V5-TRPC6-C3C2, V5-TRPC6-C3TRP-C3C2, or V5-TRPC6-C3TRP-C3 741–748 were stimulated with 0–40 units/ml Epo for 0–5 min. Biotinylation of cell surface proteins was performed, and V5-tagged proteins were immunoprecipitated ( IP ) from lysates with anti-V5 antibody. Western blots ( WB ) of immunoprecipitates were probed with streptavidin-HRP to detect biotinylated protein and then stripped and reprobed with anti-V5-HRP to detect total protein. Representative results of Western blots from three experiments are shown. Biotinylated and total protein bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. ( error bars ) values of the biotinylated/total protein ratios from three experiments after 5 min of stimulation are shown. *, significant difference in the ratio compared with time 0 ( p
    Figure Legend Snippet: Plasma membrane insertion of TRPC3/TRPC6 chimeras detected with cell surface biotinylation. HEK 293T cells transfected ( Tx'd ) with Epo-R and V5-TRPC3, V5-TRPC3-C6TRP, V5-TRPC6, V5-TRPC6-C3TRP, V5-TRPC6-C3C2, V5-TRPC6-C3TRP-C3C2, or V5-TRPC6-C3TRP-C3 741–748 were stimulated with 0–40 units/ml Epo for 0–5 min. Biotinylation of cell surface proteins was performed, and V5-tagged proteins were immunoprecipitated ( IP ) from lysates with anti-V5 antibody. Western blots ( WB ) of immunoprecipitates were probed with streptavidin-HRP to detect biotinylated protein and then stripped and reprobed with anti-V5-HRP to detect total protein. Representative results of Western blots from three experiments are shown. Biotinylated and total protein bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. ( error bars ) values of the biotinylated/total protein ratios from three experiments after 5 min of stimulation are shown. *, significant difference in the ratio compared with time 0 ( p

    Techniques Used: Transfection, Immunoprecipitation, Western Blot

    Subcellular localization of TRPC3, TRPC6, TRPC3/TRPC6 chimeras, PLCγ, and Epo-R. A , proteins from HEK 293T cells transfected with FLAG-TRPC3, FLAG-TRPC3-C6TRP, FLAG-TRPC6, FLAG-TRPC6-C3TRP-C3C2, and Epo-R were fractionated, purified, and analyzed by Western blotting ( WB ). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Transfected Epo-R was detected with anti-Epo-R antibody, and endogenous PLCγ was detected with anti-PLCγ antibody. Results from four fractionation experiments using FLAG-tagged constructs and two experiments using V5-tagged constructs were similar, and representative results with FLAG constructs are shown. B , representative results showing quality of fractionation by probing Western blots with anti-GAPDH, anti-Na + K + -ATPase, anti-lamin, and anti-vimentin (markers for cytosol, membrane, nuclear, and cytoskeletal fractions, respectively).
    Figure Legend Snippet: Subcellular localization of TRPC3, TRPC6, TRPC3/TRPC6 chimeras, PLCγ, and Epo-R. A , proteins from HEK 293T cells transfected with FLAG-TRPC3, FLAG-TRPC3-C6TRP, FLAG-TRPC6, FLAG-TRPC6-C3TRP-C3C2, and Epo-R were fractionated, purified, and analyzed by Western blotting ( WB ). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Transfected Epo-R was detected with anti-Epo-R antibody, and endogenous PLCγ was detected with anti-PLCγ antibody. Results from four fractionation experiments using FLAG-tagged constructs and two experiments using V5-tagged constructs were similar, and representative results with FLAG constructs are shown. B , representative results showing quality of fractionation by probing Western blots with anti-GAPDH, anti-Na + K + -ATPase, anti-lamin, and anti-vimentin (markers for cytosol, membrane, nuclear, and cytoskeletal fractions, respectively).

    Techniques Used: Transfection, Purification, Western Blot, Construct, Fractionation

    Role of TRP domains in regulation of TRPC3 and TRPC6 by Epo-R. HEK 293T cells were transfected with BFP-TRPC3, BFP-TRPC6, BFP-TRPC3-C6TRP, or BFP-TRPC6-C3TRP chimeras and Epo-R. Fura Red-loaded cells were treated with 40 units/ml Epo. To quantitate [Ca 2+ ] i , F 440 / F 490 was measured at base line and by monitoring over 20 min after Epo stimulation. Shown is the percentage increase in F 440 / F 490 above base line (mean ± S.E. ( error bars ) percentage increase) = peak F 440 / F 490 divided by base line F 440 / F 490 ). *, significantly greater percentage increase in F 440 / F 490 compared with Epo-stimulated cells expressing wild type TRPC6 ( p
    Figure Legend Snippet: Role of TRP domains in regulation of TRPC3 and TRPC6 by Epo-R. HEK 293T cells were transfected with BFP-TRPC3, BFP-TRPC6, BFP-TRPC3-C6TRP, or BFP-TRPC6-C3TRP chimeras and Epo-R. Fura Red-loaded cells were treated with 40 units/ml Epo. To quantitate [Ca 2+ ] i , F 440 / F 490 was measured at base line and by monitoring over 20 min after Epo stimulation. Shown is the percentage increase in F 440 / F 490 above base line (mean ± S.E. ( error bars ) percentage increase) = peak F 440 / F 490 divided by base line F 440 / F 490 ). *, significantly greater percentage increase in F 440 / F 490 compared with Epo-stimulated cells expressing wild type TRPC6 ( p

    Techniques Used: Transfection, Expressing

    6) Product Images from "TRPC channels are necessary mediators of pathologic cardiac hypertrophy"

    Article Title: TRPC channels are necessary mediators of pathologic cardiac hypertrophy

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1001825107

    dnTRPC6 inhibits pathological cardiac hypertrophy and heart failure. ( A and B ) Ca 2+ influx tracings in adult ventricular myocytes isolated from dnTRPC6 TG mice subjected to TAC. ( C ) Western blots of TRPC6 and GAPDH protein in hearts from WT and dnTRPC6 TG mice. ( D ) HW/BW ratio in WT and dnTRPC6 TG mice after PE/AngII infusion for 2 weeks. *, P
    Figure Legend Snippet: dnTRPC6 inhibits pathological cardiac hypertrophy and heart failure. ( A and B ) Ca 2+ influx tracings in adult ventricular myocytes isolated from dnTRPC6 TG mice subjected to TAC. ( C ) Western blots of TRPC6 and GAPDH protein in hearts from WT and dnTRPC6 TG mice. ( D ) HW/BW ratio in WT and dnTRPC6 TG mice after PE/AngII infusion for 2 weeks. *, P

    Techniques Used: Isolation, Mouse Assay, Western Blot

    7) Product Images from "STIM2 regulates both intracellular Ca2+ distribution and Ca2+ movement in skeletal myotubes"

    Article Title: STIM2 regulates both intracellular Ca2+ distribution and Ca2+ movement in skeletal myotubes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18256-3

    Decreased protein content level of TRPC6, JP1, or CaM1 by STIM2-knockdown. ( a ) Lysate of the STIM2-knockdown myotubes was subjected to an immunoblot assay with one of the antibodies against thirteen proteins that mediate or regulate skeletal muscle functions. α -Actin was used as a loading control. Three independent experiments per each protein were conducted. JP, junctophilin; CSQ, calsequestin. The immunoblot data were cropped from the immunoblot images of different gels and were grouped. The full-length blots are presented in Supplementary Figs 6 to 19 . Among them, the expression level of TRPC6, JP1, or CaM1 was significantly decreased (indicated by asterisks). ( b ) The protein content level in (a) is presented as bar graphs. Bar graphs are presented as the mean ± S.E. for three independent experiments. *Significant difference compared with the scrambled siRNA control ( P
    Figure Legend Snippet: Decreased protein content level of TRPC6, JP1, or CaM1 by STIM2-knockdown. ( a ) Lysate of the STIM2-knockdown myotubes was subjected to an immunoblot assay with one of the antibodies against thirteen proteins that mediate or regulate skeletal muscle functions. α -Actin was used as a loading control. Three independent experiments per each protein were conducted. JP, junctophilin; CSQ, calsequestin. The immunoblot data were cropped from the immunoblot images of different gels and were grouped. The full-length blots are presented in Supplementary Figs 6 to 19 . Among them, the expression level of TRPC6, JP1, or CaM1 was significantly decreased (indicated by asterisks). ( b ) The protein content level in (a) is presented as bar graphs. Bar graphs are presented as the mean ± S.E. for three independent experiments. *Significant difference compared with the scrambled siRNA control ( P

    Techniques Used: Expressing

    8) Product Images from "Characterization of pressure-mediated vascular tone in resistance arteries from bile duct-ligated rats"

    Article Title: Characterization of pressure-mediated vascular tone in resistance arteries from bile duct-ligated rats

    Journal: Oncotarget

    doi: 10.18632/oncotarget.15409

    Expression of ion channels in small mesenteric resistance arteries from SHAM- and BDL-rats A . Immunoblots for Ca v 1.2, TRPC3 and β-actin (loading control) from small mesenteric artery homogenates indicate no differences among both groups. The bar graphs show densitometry analyses for Ca v 1.2 and TRPC3. B . qPCR for Ca v 1.2. Representative IHC photographs and qPCR indicate that in the arteries from the BDL-rats, expressions of C ., D . BK Ca and E ., F . TRPC6 are reduced when compared to SHAM-rats ( n = 3-5/group). G . The immunoblots for both BK Ca and TRPC6 and their loading controls are shown. The bar graphs show densitometry analyses for BK Ca and TRPC6 ( n = 3/group). * P
    Figure Legend Snippet: Expression of ion channels in small mesenteric resistance arteries from SHAM- and BDL-rats A . Immunoblots for Ca v 1.2, TRPC3 and β-actin (loading control) from small mesenteric artery homogenates indicate no differences among both groups. The bar graphs show densitometry analyses for Ca v 1.2 and TRPC3. B . qPCR for Ca v 1.2. Representative IHC photographs and qPCR indicate that in the arteries from the BDL-rats, expressions of C ., D . BK Ca and E ., F . TRPC6 are reduced when compared to SHAM-rats ( n = 3-5/group). G . The immunoblots for both BK Ca and TRPC6 and their loading controls are shown. The bar graphs show densitometry analyses for BK Ca and TRPC6 ( n = 3/group). * P

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemistry

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    Alomone Labs anti trpc6
    The mechanism by which <t>TRPC6</t> contributes to VSMC differentiation. A ) Immunofluorescence imaging of SM22α in TGF-β1-stimulated C3H10T1/2 cells in the presence of additional extracellular KCl. Cells were treated with TGF-β1 and indicated concentrations of KCl for 48 h. Representative images (left) and quantification of SM22α-positive cells (right) are shown ( n = 5). Scale bars, 50 μm. Nuclei were visualized with DAPI. B ) Membrane association of PTEN in TGF-β1-stimulated C3H10T1/2 cells in the presence or absence of additional extracellular KCl (30 mM). Cells were treated with TGF-β1 (5 ng/ml) and KCl for 48 h. Densitometric analysis of membrane-associated PTEN protein normalized to total PTEN protein (right, n = 5); increases are shown relative to untreated control cells. C ) PTEN membrane association in siRNA-transfected C3H10T1/2 cells. Cells were stimulated with TGF-β1 (5 ng/ml) for 12 h. Increases are shown relative to untreated siControl-transfected cells. D ) TRPC6 permeates Na + and Ca 2+ in proliferating VSMCs, which causes a slight depolarization of membrane potential that affects anionic phospholipid distribution in the inner leaflet of the plasma membrane. The C2 domain of PTEN binds to Ca 2+ in the proximal region of the mouth of the TRPC6 channel and brings PTEN close to the substrate PI(3,4,5)P 3 in the plasma membrane via interaction with clustered anionic phospholipids such as PS. TGF-β1 stimulation suppressed TRPC6 channel activity, which hyperpolarized membrane potential and inhibited Ca 2+ influx, resulting in dissociation of PTEN from the plasma membrane and accumulation of PI(3,4,5)P 3 that induces Akt activation. * P
    Anti Trpc6, 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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    The mechanism by which TRPC6 contributes to VSMC differentiation. A ) Immunofluorescence imaging of SM22α in TGF-β1-stimulated C3H10T1/2 cells in the presence of additional extracellular KCl. Cells were treated with TGF-β1 and indicated concentrations of KCl for 48 h. Representative images (left) and quantification of SM22α-positive cells (right) are shown ( n = 5). Scale bars, 50 μm. Nuclei were visualized with DAPI. B ) Membrane association of PTEN in TGF-β1-stimulated C3H10T1/2 cells in the presence or absence of additional extracellular KCl (30 mM). Cells were treated with TGF-β1 (5 ng/ml) and KCl for 48 h. Densitometric analysis of membrane-associated PTEN protein normalized to total PTEN protein (right, n = 5); increases are shown relative to untreated control cells. C ) PTEN membrane association in siRNA-transfected C3H10T1/2 cells. Cells were stimulated with TGF-β1 (5 ng/ml) for 12 h. Increases are shown relative to untreated siControl-transfected cells. D ) TRPC6 permeates Na + and Ca 2+ in proliferating VSMCs, which causes a slight depolarization of membrane potential that affects anionic phospholipid distribution in the inner leaflet of the plasma membrane. The C2 domain of PTEN binds to Ca 2+ in the proximal region of the mouth of the TRPC6 channel and brings PTEN close to the substrate PI(3,4,5)P 3 in the plasma membrane via interaction with clustered anionic phospholipids such as PS. TGF-β1 stimulation suppressed TRPC6 channel activity, which hyperpolarized membrane potential and inhibited Ca 2+ influx, resulting in dissociation of PTEN from the plasma membrane and accumulation of PI(3,4,5)P 3 that induces Akt activation. * P

    Journal: The FASEB Journal

    Article Title: TRPC6 regulates phenotypic switching of vascular smooth muscle cells through plasma membrane potential-dependent coupling with PTEN

    doi: 10.1096/fj.201802811R

    Figure Lengend Snippet: The mechanism by which TRPC6 contributes to VSMC differentiation. A ) Immunofluorescence imaging of SM22α in TGF-β1-stimulated C3H10T1/2 cells in the presence of additional extracellular KCl. Cells were treated with TGF-β1 and indicated concentrations of KCl for 48 h. Representative images (left) and quantification of SM22α-positive cells (right) are shown ( n = 5). Scale bars, 50 μm. Nuclei were visualized with DAPI. B ) Membrane association of PTEN in TGF-β1-stimulated C3H10T1/2 cells in the presence or absence of additional extracellular KCl (30 mM). Cells were treated with TGF-β1 (5 ng/ml) and KCl for 48 h. Densitometric analysis of membrane-associated PTEN protein normalized to total PTEN protein (right, n = 5); increases are shown relative to untreated control cells. C ) PTEN membrane association in siRNA-transfected C3H10T1/2 cells. Cells were stimulated with TGF-β1 (5 ng/ml) for 12 h. Increases are shown relative to untreated siControl-transfected cells. D ) TRPC6 permeates Na + and Ca 2+ in proliferating VSMCs, which causes a slight depolarization of membrane potential that affects anionic phospholipid distribution in the inner leaflet of the plasma membrane. The C2 domain of PTEN binds to Ca 2+ in the proximal region of the mouth of the TRPC6 channel and brings PTEN close to the substrate PI(3,4,5)P 3 in the plasma membrane via interaction with clustered anionic phospholipids such as PS. TGF-β1 stimulation suppressed TRPC6 channel activity, which hyperpolarized membrane potential and inhibited Ca 2+ influx, resulting in dissociation of PTEN from the plasma membrane and accumulation of PI(3,4,5)P 3 that induces Akt activation. * P

    Article Snippet: Anti-TRPC6 (Alomone labs, Jerusalem, Israel), anti-glyceraldehyde 3-phosphate dehydrogenase (Santa Cruz Biotechnology, Dallas, TX, USA), anti-SM22α, anti-phospho-Akt (Ser473), anti-Akt, and anti-PTEN (Cell Signaling Technology, Danvers, MA, USA) antibodies were used at 1:2000 dilutions.

    Techniques: Immunofluorescence, Imaging, Transfection, Activity Assay, Activation Assay

    DiC8-PIP 2 inhibits Ang II-evoked whole-cell cation currents and single TRPC6 channel activity in rabbit mesenteric artery myocytes Aa and b , inclusion of 100 μ m diC8-PIP 2 in the patch pipette solution inhibits whole-cell cation currents evoked by 1 n m Ang II. Ac , mean I–V relationship showing diC8-PIP 2 inhibits Ang II-evoked cation currents at all potentials tested ( n = at least 4 per point). Ba , 100 μ m diC8-PIP 2 inhibited TRPC activity in an inside-out patch which was initially activated by 1 n m Ang II in a cell-attached patch (i/o, inside-out patch). Bb , 100 μ m diC8-PIP 2 inhibited TRPC activity induced by 10 μ m OAG in an inside-out patch. C , inhibitory action of diC8-PIP 2 on OAG-evoked TRPC6 activity in inside-out patches had an IC 50 value of 7.6 μ m ( n = at least 4 per point).

    Journal: The Journal of Physiology

    Article Title: Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes

    doi: 10.1113/jphysiol.2008.153676

    Figure Lengend Snippet: DiC8-PIP 2 inhibits Ang II-evoked whole-cell cation currents and single TRPC6 channel activity in rabbit mesenteric artery myocytes Aa and b , inclusion of 100 μ m diC8-PIP 2 in the patch pipette solution inhibits whole-cell cation currents evoked by 1 n m Ang II. Ac , mean I–V relationship showing diC8-PIP 2 inhibits Ang II-evoked cation currents at all potentials tested ( n = at least 4 per point). Ba , 100 μ m diC8-PIP 2 inhibited TRPC activity in an inside-out patch which was initially activated by 1 n m Ang II in a cell-attached patch (i/o, inside-out patch). Bb , 100 μ m diC8-PIP 2 inhibited TRPC activity induced by 10 μ m OAG in an inside-out patch. C , inhibitory action of diC8-PIP 2 on OAG-evoked TRPC6 activity in inside-out patches had an IC 50 value of 7.6 μ m ( n = at least 4 per point).

    Article Snippet: Anti-TRPC6 and anti-PIP2 antibodies Polyclonal TRPC6 antibody generated in rabbits against an intracellular epitope was purchased from Alomone Laboratories (Jerusalem, Israel) and the selectivity and negligible cross-reactivity of this antibody for its target protein has been previously confirmed ( ; ).

    Techniques: Activity Assay, Transferring

    Agents that deplete PIP 2 evoke TRPC6 channel activity A and B , respectively, 20 μ m wortmannin and 100 μ m LY294002 transiently activated channel activity in cell-attached patches ( a ) which had similar amplitude histograms ( b ) and unitary conductances and E r values ( c ). C , 50 μg ml −1 poly- l -lysine activated channel currents in an inside-out patch ( a ) which had a similar properties to those evoked by wortmannin and LY294002 ( b and c ).

    Journal: The Journal of Physiology

    Article Title: Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes

    doi: 10.1113/jphysiol.2008.153676

    Figure Lengend Snippet: Agents that deplete PIP 2 evoke TRPC6 channel activity A and B , respectively, 20 μ m wortmannin and 100 μ m LY294002 transiently activated channel activity in cell-attached patches ( a ) which had similar amplitude histograms ( b ) and unitary conductances and E r values ( c ). C , 50 μg ml −1 poly- l -lysine activated channel currents in an inside-out patch ( a ) which had a similar properties to those evoked by wortmannin and LY294002 ( b and c ).

    Article Snippet: Anti-TRPC6 and anti-PIP2 antibodies Polyclonal TRPC6 antibody generated in rabbits against an intracellular epitope was purchased from Alomone Laboratories (Jerusalem, Israel) and the selectivity and negligible cross-reactivity of this antibody for its target protein has been previously confirmed ( ; ).

    Techniques: Activity Assay

    Anti-PIP 2 antibody potentiates TRPC6 channel activity Aa , 1: 200 dilution of anti-PIP 2 antibodies transiently increased TRPC6 activity in an inside-out patch which was induced by 1 n m Ang II in cell-attached mode. Subsequently the Ang II-evoked response was inhibited in the presence of the anti-PIP 2 antibody. Ab , 1: 200 dilution of anti-PIP 2 antibodies produced a sustained increase of OAG-evoked TRPC6 activity in an inside-out patch. B , mean data of effect of anti-PIP 2 antibodies on Ang II- and OAG-induced TRPC6 activity ( n = 6 for all conditions, ** P

    Journal: The Journal of Physiology

    Article Title: Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes

    doi: 10.1113/jphysiol.2008.153676

    Figure Lengend Snippet: Anti-PIP 2 antibody potentiates TRPC6 channel activity Aa , 1: 200 dilution of anti-PIP 2 antibodies transiently increased TRPC6 activity in an inside-out patch which was induced by 1 n m Ang II in cell-attached mode. Subsequently the Ang II-evoked response was inhibited in the presence of the anti-PIP 2 antibody. Ab , 1: 200 dilution of anti-PIP 2 antibodies produced a sustained increase of OAG-evoked TRPC6 activity in an inside-out patch. B , mean data of effect of anti-PIP 2 antibodies on Ang II- and OAG-induced TRPC6 activity ( n = 6 for all conditions, ** P

    Article Snippet: Anti-TRPC6 and anti-PIP2 antibodies Polyclonal TRPC6 antibody generated in rabbits against an intracellular epitope was purchased from Alomone Laboratories (Jerusalem, Israel) and the selectivity and negligible cross-reactivity of this antibody for its target protein has been previously confirmed ( ; ).

    Techniques: Activity Assay, Produced

    Wortmannin-induced depletion of PIP 2 decreases Ang II-evoked and increases OAG-induced TRPC6 activity in cell-attached patches A shows that Ang II-evoked TRPC6 activity ( a ) is decreased after pre-treatment with wortmannin ( b ). B illustrates that OAG-induced TRPC6 activity ( a ) is markedly increased following pre-treatment with wortmannin ( b ). C , mean data showing effect of wortmannin on Ang II- and OAG-induced TRPC6 activity (*** P

    Journal: The Journal of Physiology

    Article Title: Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes

    doi: 10.1113/jphysiol.2008.153676

    Figure Lengend Snippet: Wortmannin-induced depletion of PIP 2 decreases Ang II-evoked and increases OAG-induced TRPC6 activity in cell-attached patches A shows that Ang II-evoked TRPC6 activity ( a ) is decreased after pre-treatment with wortmannin ( b ). B illustrates that OAG-induced TRPC6 activity ( a ) is markedly increased following pre-treatment with wortmannin ( b ). C , mean data showing effect of wortmannin on Ang II- and OAG-induced TRPC6 activity (*** P

    Article Snippet: Anti-TRPC6 and anti-PIP2 antibodies Polyclonal TRPC6 antibody generated in rabbits against an intracellular epitope was purchased from Alomone Laboratories (Jerusalem, Israel) and the selectivity and negligible cross-reactivity of this antibody for its target protein has been previously confirmed ( ; ).

    Techniques: Activity Assay

    Recombinant circulating factors increase stretch-evoked cationic current in mouse podocytes. Cells were cultured for 24 hr in either control medium, or medium containing suPAR or TNF as indicated, after which whole-cell recordings were made. (a) Representative examples of currents evoked shortly after making whole-cell contact (left), in the same cell 2–3 minutes after switching to a 70% hypoosmotic external medium (center) and after bath application of hypoosmotic medium containing 50 μm La 3+ (right), which inhibits TRPC6-mediated currents in podocytes. Currents shown were recorded during application of a ramp voltage command (−80 mV to + 80 mV over 2.5 sec). Note increase in amplitude of stretch-evoked currents in cells that had been cultured in suPAR or TNF compared to cell cultured in control medium. (b) Mean fold increases in current at +80 mV recorded in hypoosmotic stretch solution relative to baseline current in normal bath solution for cells cultured in control medium or suPAR. Data are mean ± SEM with 10 cells in each group. (c) Mean fold increase in stretch-evoked current ± SEM in cells that had been cultured in TNF for 24 hr compared to cells cultured in control media. Asterisks indicate P

    Journal: Biochimica et biophysica acta

    Article Title: Changes in podocyte TRPC channels evoked by plasma and sera from patients with recurrent FSGS and by putative glomerular permeability factors

    doi: 10.1016/j.bbadis.2017.06.010

    Figure Lengend Snippet: Recombinant circulating factors increase stretch-evoked cationic current in mouse podocytes. Cells were cultured for 24 hr in either control medium, or medium containing suPAR or TNF as indicated, after which whole-cell recordings were made. (a) Representative examples of currents evoked shortly after making whole-cell contact (left), in the same cell 2–3 minutes after switching to a 70% hypoosmotic external medium (center) and after bath application of hypoosmotic medium containing 50 μm La 3+ (right), which inhibits TRPC6-mediated currents in podocytes. Currents shown were recorded during application of a ramp voltage command (−80 mV to + 80 mV over 2.5 sec). Note increase in amplitude of stretch-evoked currents in cells that had been cultured in suPAR or TNF compared to cell cultured in control medium. (b) Mean fold increases in current at +80 mV recorded in hypoosmotic stretch solution relative to baseline current in normal bath solution for cells cultured in control medium or suPAR. Data are mean ± SEM with 10 cells in each group. (c) Mean fold increase in stretch-evoked current ± SEM in cells that had been cultured in TNF for 24 hr compared to cells cultured in control media. Asterisks indicate P

    Article Snippet: Rabbit antibodies against TRPC6 (ACC-017) and TRPC5 (ACC-020) were obtained from Alomone Labs (Jerusalem, Israel), antibodies against podocin (sc-21009) and β3-integrin (sc-14009) were obtained from Santa Cruz (Santa Cruz, CA).

    Techniques: Recombinant, Cell Culture, Size-exclusion Chromatography

    Effects of recombinant human suPAR and TNF on TRPC6 and podocin in immortalized podocytes. (a) Two isoforms of suPAR at 10 ng/ml for 24 hr caused increase steady-state surface expression of TRPC6 measured by cell surface biotinylation assays, and reduced podocin abundance measured by immunoblot of total cellular lysates. Summary bar graphs (mean ± SD) of three replications of these experiments are shown to the right of representative blots. We could not discern any consistent difference in the activities of these two recombinant preparations at this concentration at the limits of resolution of this in vitro assay. (b) Culturing podocytes with 10 ng/ml TNF for 24 hr causes an increase in the steady-state surface expression of TRPC6 measured by biotinyation assays but has no effect on overall abundance of podocin. (c) TNF and suPAR by themselves at a lower concentration (1 ng/ml) do not cause a notable increase in surface TRPC6 by themselves but a robust increase occurs when both factors are present at 1 ng/ml. Asterisks indicate P

    Journal: Biochimica et biophysica acta

    Article Title: Changes in podocyte TRPC channels evoked by plasma and sera from patients with recurrent FSGS and by putative glomerular permeability factors

    doi: 10.1016/j.bbadis.2017.06.010

    Figure Lengend Snippet: Effects of recombinant human suPAR and TNF on TRPC6 and podocin in immortalized podocytes. (a) Two isoforms of suPAR at 10 ng/ml for 24 hr caused increase steady-state surface expression of TRPC6 measured by cell surface biotinylation assays, and reduced podocin abundance measured by immunoblot of total cellular lysates. Summary bar graphs (mean ± SD) of three replications of these experiments are shown to the right of representative blots. We could not discern any consistent difference in the activities of these two recombinant preparations at this concentration at the limits of resolution of this in vitro assay. (b) Culturing podocytes with 10 ng/ml TNF for 24 hr causes an increase in the steady-state surface expression of TRPC6 measured by biotinyation assays but has no effect on overall abundance of podocin. (c) TNF and suPAR by themselves at a lower concentration (1 ng/ml) do not cause a notable increase in surface TRPC6 by themselves but a robust increase occurs when both factors are present at 1 ng/ml. Asterisks indicate P

    Article Snippet: Rabbit antibodies against TRPC6 (ACC-017) and TRPC5 (ACC-020) were obtained from Alomone Labs (Jerusalem, Israel), antibodies against podocin (sc-21009) and β3-integrin (sc-14009) were obtained from Santa Cruz (Santa Cruz, CA).

    Techniques: Recombinant, Expressing, Concentration Assay, In Vitro

    Effect of TRPC6 knockdown on BMSC cell cycle progression. Experiments were performed 4 days after transfection with siRNA. siCon, negative control siRNA; siTRPC6, siRNA specifically targeting TRPC6. (A) Cell cycle distribution of siCon- and siTRPC6-transfected BMSCs. Data are representative of six experiments. (B) Average percentages of cells residing in respective cell cycle stages for each siRNA treatment ( n = 6 respectively). * * Statistically significant ( P

    Journal: British Journal of Pharmacology

    Article Title: TRPC6 regulates cell cycle progression by modulating membrane potential in bone marrow stromal cells

    doi: 10.1111/bph.12840

    Figure Lengend Snippet: Effect of TRPC6 knockdown on BMSC cell cycle progression. Experiments were performed 4 days after transfection with siRNA. siCon, negative control siRNA; siTRPC6, siRNA specifically targeting TRPC6. (A) Cell cycle distribution of siCon- and siTRPC6-transfected BMSCs. Data are representative of six experiments. (B) Average percentages of cells residing in respective cell cycle stages for each siRNA treatment ( n = 6 respectively). * * Statistically significant ( P

    Article Snippet: Antibodies against TRPC1 and TRPC6 were purchased from Alomone labs (Jerusalem, Israel).

    Techniques: Transfection, Negative Control

    Schematic diagram of the role of the TRPC6 channel in cell cycle progression of BMSCs. In the G 1 phase, the highest expression level of TRPC6 induces membrane depolarization ( Depo ) and functionally suppresses Ca 2+ entry via SOC channels (indicated by ‘−’) in BMSCs. On the contrary, the lowest expression level of TRPC6 in the S phase maintains RMP at a more hyperpolarized level ( Hyper ) and enhances SOC-mediated Ca 2+ entry (indicated by ‘+’). The highest expression levels of STIM/Orai and TRPC1 molecules in the S phase also contribute to an enhanced SOCE. V m , membrane potential.

    Journal: British Journal of Pharmacology

    Article Title: TRPC6 regulates cell cycle progression by modulating membrane potential in bone marrow stromal cells

    doi: 10.1111/bph.12840

    Figure Lengend Snippet: Schematic diagram of the role of the TRPC6 channel in cell cycle progression of BMSCs. In the G 1 phase, the highest expression level of TRPC6 induces membrane depolarization ( Depo ) and functionally suppresses Ca 2+ entry via SOC channels (indicated by ‘−’) in BMSCs. On the contrary, the lowest expression level of TRPC6 in the S phase maintains RMP at a more hyperpolarized level ( Hyper ) and enhances SOC-mediated Ca 2+ entry (indicated by ‘+’). The highest expression levels of STIM/Orai and TRPC1 molecules in the S phase also contribute to an enhanced SOCE. V m , membrane potential.

    Article Snippet: Antibodies against TRPC1 and TRPC6 were purchased from Alomone labs (Jerusalem, Israel).

    Techniques: Expressing

    Cell cycle-dependent changes in the expression of TRPC1, TRPC6 and all subtypes of STIM and Orai in BMSCs. (A) mRNA expression profile of TRPC, STIM and Orai subtypes in non-synchronized BMSCs. Representative data of conventional PCR obtained from BMSC mRNAs extracted from seven rats. (B) phase-contrast images of cell cycle-synchronized BMSCs. Each cell cycle stage is labelled. The scale bar indicates 100 μm. All photographs were taken 4 days after drug application. Control (cont): non-synchronized cells. (C) representative histograms of cell cycle phase-synchronized BMSCs. All data were obtained 4 days after drug application. (D) Cell cycle distribution presented as % at different cell cycle stages evaluated by flow cytometry. Data are the averages of six experiments for each stage. * * Significantly different from the control (unsynchronized) for each cell cycle stage ( P

    Journal: British Journal of Pharmacology

    Article Title: TRPC6 regulates cell cycle progression by modulating membrane potential in bone marrow stromal cells

    doi: 10.1111/bph.12840

    Figure Lengend Snippet: Cell cycle-dependent changes in the expression of TRPC1, TRPC6 and all subtypes of STIM and Orai in BMSCs. (A) mRNA expression profile of TRPC, STIM and Orai subtypes in non-synchronized BMSCs. Representative data of conventional PCR obtained from BMSC mRNAs extracted from seven rats. (B) phase-contrast images of cell cycle-synchronized BMSCs. Each cell cycle stage is labelled. The scale bar indicates 100 μm. All photographs were taken 4 days after drug application. Control (cont): non-synchronized cells. (C) representative histograms of cell cycle phase-synchronized BMSCs. All data were obtained 4 days after drug application. (D) Cell cycle distribution presented as % at different cell cycle stages evaluated by flow cytometry. Data are the averages of six experiments for each stage. * * Significantly different from the control (unsynchronized) for each cell cycle stage ( P

    Article Snippet: Antibodies against TRPC1 and TRPC6 were purchased from Alomone labs (Jerusalem, Israel).

    Techniques: Expressing, Polymerase Chain Reaction, Flow Cytometry, Cytometry

    TRPC6 knockdown induces a negative shift in the membrane potential (RMP) and increases SOCE of BMSC. Experiments were performed 4 days after transfection with siRNA. siCon, control siRNA; siTRPC6, TRPC6-specific siRNA; siTRPC1, TRPC1-specific siRNA. (A) RMP was evaluated as a DiBAC 4 (3) fluorescence in siCon-, siTRPC6- and siTRPC1-treated BMSCs. Columns show the averaged RMP values from five independent experiments (each represents the mean of more than 60 cells). (B) Columns show the means of RMP recorded by the current clamp method, for siCon ( n = 10), siTRPC6 ( n = 15) and siTRPC1-treated ( n = 11) BMSCs. (C) Typical traces of SOCE in siCon- and siTRPC6-treated cells. Each trace shows the averaged time course of at least 50 cells evaluated on the same day. (D) Summary of the magnitude of SOCE in siCon- or siTRPC6-treated cells. Data represent the averages of five independent experiments (each represents the mean of at least 50–70 cells). In panels A, B and D, the symbol ‘ * * ’ means significantly different from the control ( P

    Journal: British Journal of Pharmacology

    Article Title: TRPC6 regulates cell cycle progression by modulating membrane potential in bone marrow stromal cells

    doi: 10.1111/bph.12840

    Figure Lengend Snippet: TRPC6 knockdown induces a negative shift in the membrane potential (RMP) and increases SOCE of BMSC. Experiments were performed 4 days after transfection with siRNA. siCon, control siRNA; siTRPC6, TRPC6-specific siRNA; siTRPC1, TRPC1-specific siRNA. (A) RMP was evaluated as a DiBAC 4 (3) fluorescence in siCon-, siTRPC6- and siTRPC1-treated BMSCs. Columns show the averaged RMP values from five independent experiments (each represents the mean of more than 60 cells). (B) Columns show the means of RMP recorded by the current clamp method, for siCon ( n = 10), siTRPC6 ( n = 15) and siTRPC1-treated ( n = 11) BMSCs. (C) Typical traces of SOCE in siCon- and siTRPC6-treated cells. Each trace shows the averaged time course of at least 50 cells evaluated on the same day. (D) Summary of the magnitude of SOCE in siCon- or siTRPC6-treated cells. Data represent the averages of five independent experiments (each represents the mean of at least 50–70 cells). In panels A, B and D, the symbol ‘ * * ’ means significantly different from the control ( P

    Article Snippet: Antibodies against TRPC1 and TRPC6 were purchased from Alomone labs (Jerusalem, Israel).

    Techniques: Transfection, Fluorescence