anti trpc3  (Alomone Labs)


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    Structured Review

    Alomone Labs anti trpc3
    Schematic model of <t>TRPC3</t> transmembrane domains and protein binding sites. Predicted transmembrane domains, calcium entry pore, IP 3 R, and PLCγ SH2 binding sites and deleted and substituted sites in TRPC3-DEL and TRPC3-SUB are shown. aa , amino
    Anti Trpc3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 36 article reviews
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    anti trpc3 - by Bioz Stars, 2022-09
    95/100 stars

    Images

    1) Product Images from "TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *"

    Article Title: TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M710231200

    Schematic model of TRPC3 transmembrane domains and protein binding sites. Predicted transmembrane domains, calcium entry pore, IP 3 R, and PLCγ SH2 binding sites and deleted and substituted sites in TRPC3-DEL and TRPC3-SUB are shown. aa , amino
    Figure Legend Snippet: Schematic model of TRPC3 transmembrane domains and protein binding sites. Predicted transmembrane domains, calcium entry pore, IP 3 R, and PLCγ SH2 binding sites and deleted and substituted sites in TRPC3-DEL and TRPC3-SUB are shown. aa , amino

    Techniques Used: Protein Binding, Binding Assay

    Association of TRPC3 IP 3 R binding mutants with IP 3 R. IP 3 R type II and V5-TRPC3, V5-TRPC3-DEL, or V5-TRPC3-SUB were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-V5 or anti-IP 3 R antibodies or normal rabbit
    Figure Legend Snippet: Association of TRPC3 IP 3 R binding mutants with IP 3 R. IP 3 R type II and V5-TRPC3, V5-TRPC3-DEL, or V5-TRPC3-SUB were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-V5 or anti-IP 3 R antibodies or normal rabbit

    Techniques Used: Binding Assay, Immunoprecipitation

    Dose response and time course of [Ca 2+ ] i after Epo stimulation of HEK 293T cells transfected with TRPC3 and Epo-R. A , Epo dose response. HEK 293T cells transfected with TRPC3 and Epo-R were stimulated with 0–40 units/ml Epo. [Ca 2+ ] i was measured
    Figure Legend Snippet: Dose response and time course of [Ca 2+ ] i after Epo stimulation of HEK 293T cells transfected with TRPC3 and Epo-R. A , Epo dose response. HEK 293T cells transfected with TRPC3 and Epo-R were stimulated with 0–40 units/ml Epo. [Ca 2+ ] i was measured

    Techniques Used: Transfection

    Requirement for external calcium in the Epo-stimulated calcium increase in HEK 293T cells. Fura Red-loaded HEK 293T cells were transfected with BFP-TRPC3 and Epo-R. A , cells were treated with 40 units/ml Epo in the presence (0.68 m m ) or absence (2
    Figure Legend Snippet: Requirement for external calcium in the Epo-stimulated calcium increase in HEK 293T cells. Fura Red-loaded HEK 293T cells were transfected with BFP-TRPC3 and Epo-R. A , cells were treated with 40 units/ml Epo in the presence (0.68 m m ) or absence (2

    Techniques Used: Transfection

    Interaction of TRPC3 with PLCγ SH2 binding site substitutions with PLCγ. PLCγ and V5-TRPC3, V5-TRPC3-F4, or V5-TRPC3-Y226F were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-PLCγ
    Figure Legend Snippet: Interaction of TRPC3 with PLCγ SH2 binding site substitutions with PLCγ. PLCγ and V5-TRPC3, V5-TRPC3-F4, or V5-TRPC3-Y226F were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-PLCγ

    Techniques Used: Binding Assay, Immunoprecipitation

    Western blot of HEK 293T cells transfected with siRNA targeted to PLC γ. Lysates were prepared from HEK 293T cells transfected ( Tx ' d ) with or without BFP-TRPC3 and Epo-R, and siRNA was targeted to PLCγ or control siRNA. Blots were probed
    Figure Legend Snippet: Western blot of HEK 293T cells transfected with siRNA targeted to PLC γ. Lysates were prepared from HEK 293T cells transfected ( Tx ' d ) with or without BFP-TRPC3 and Epo-R, and siRNA was targeted to PLCγ or control siRNA. Blots were probed

    Techniques Used: Western Blot, Transfection, Planar Chromatography

    Endogenous expression of TRPC3 in human hematopoietic cells. Western blotting was performed on lysates from UT-7 and TF-1 Epo-responsive cell lines, from CD34 + cells and from day 10 and 14 BFU-E-derived erythroblasts. Equivalent amounts of protein
    Figure Legend Snippet: Endogenous expression of TRPC3 in human hematopoietic cells. Western blotting was performed on lysates from UT-7 and TF-1 Epo-responsive cell lines, from CD34 + cells and from day 10 and 14 BFU-E-derived erythroblasts. Equivalent amounts of protein

    Techniques Used: Expressing, Western Blot, Derivative Assay

    Plasma membrane externalization of TRPC3 detected by cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-DEL, V5-TRPC3-SUB, or V5-TRPC3-F4 and Epo-R. Lysates were prepared, and immunoprecipitation
    Figure Legend Snippet: Plasma membrane externalization of TRPC3 detected by cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-DEL, V5-TRPC3-SUB, or V5-TRPC3-F4 and Epo-R. Lysates were prepared, and immunoprecipitation

    Techniques Used: Expressing, Immunoprecipitation

    Association of TRPC3 and TRPC3-F4 with PLCγ or Epo-R. A , PLCγ and V5-TRPC3 or V5-TRPC3-F4 were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-PLCγ or anti-V5 antibodies or normal rabbit
    Figure Legend Snippet: Association of TRPC3 and TRPC3-F4 with PLCγ or Epo-R. A , PLCγ and V5-TRPC3 or V5-TRPC3-F4 were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-PLCγ or anti-V5 antibodies or normal rabbit

    Techniques Used: Immunoprecipitation

    2) Product Images from "Interaction between mitsugumin 29 and TRPC3 participates in regulating Ca2+ transients in skeletal muscle"

    Article Title: Interaction between mitsugumin 29 and TRPC3 participates in regulating Ca2+ transients in skeletal muscle

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2015.06.096

    A reduction in Ca 2+ transients in response to membrane depolarization, and the disruption of the binding between endogenous MG29 and TRPC3 in mouse primary skeletal myotubes expressing Δ116-MG29
    Figure Legend Snippet: A reduction in Ca 2+ transients in response to membrane depolarization, and the disruption of the binding between endogenous MG29 and TRPC3 in mouse primary skeletal myotubes expressing Δ116-MG29

    Techniques Used: Binding Assay, Expressing

    Co-immunoprecipitation of TRPC3 with each MG29 portion
    Figure Legend Snippet: Co-immunoprecipitation of TRPC3 with each MG29 portion

    Techniques Used: Immunoprecipitation

    3) Product Images from "Transient Receptor Potential Channel Activation Causes a Novel Form of [Ca 2+]i Oscillations and Is Not Involved in Capacitative Ca 2+ Entry in Glial Cells"

    Article Title: Transient Receptor Potential Channel Activation Causes a Novel Form of [Ca 2+]i Oscillations and Is Not Involved in Capacitative Ca 2+ Entry in Glial Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.23-11-04737.2003

    Effect of anti-TRPC3 antisense oligonucleotides on OAG-induced [Ca 2 + ] i oscillations. Astrocytes were treated for 36 hr with vehicle, and 100 μg/ml TRPC3 sense or antisense oligonucleotides were loaded with fura-2 and then exposed to 100 μ
    Figure Legend Snippet: Effect of anti-TRPC3 antisense oligonucleotides on OAG-induced [Ca 2 + ] i oscillations. Astrocytes were treated for 36 hr with vehicle, and 100 μg/ml TRPC3 sense or antisense oligonucleotides were loaded with fura-2 and then exposed to 100 μ

    Techniques Used:

    4) Product Images from "Modeling Suggests TRPC3 Hydrogen Bonding and Not Phosphorylation Contributes to the Ataxia Phenotype of the Moonwalker Mouse"

    Article Title: Modeling Suggests TRPC3 Hydrogen Bonding and Not Phosphorylation Contributes to the Ataxia Phenotype of the Moonwalker Mouse

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.5b00235

    Homology modeling reveals potential for hydrogen bonding. (A) Sequence alignment of mouse TRPC3 to rat TRPV1 and to the S4–S5 linker of the Kv 1.2/2.1 chimera used to create the homology models. A cut of 21 amino acids (indicated by double lines) was made at the end of S3 to facilitate modeling. Transmembrane helices are indicated by black boxes with the corresponding label for each S1–S6 transmembrane helix above. Coloring is according to the Clustal coloring scheme. The TRPC3 Mwk mutation in the S4–S5 linker region is indicated by a double-lined box. Arrows indicate residues highlighted in other panels of the figure. Image made with JalView. (B) Top-ranked (according to MODELLER score) mouse TRPC3 homology model on the TRPV1 apo structure (PDB entry 3J5P) with the Kv 1.2/2.1 channel chimera structure for the S4–S5 linker shown from a top view and a side view. Coloring indicates separate chains of the tetrameric channel. The S1–S4 helices of the purple chain have been removed for the sake of clarity in the side view figure. (C) Homology models of TRPC3 indicate that the threonine 635 (in green) mutated in the Mwk mouse has the potential for hydrogen bonding with the end of helix S6. The first quadrant shows an overlap of four models and the resulting T635 orientation. The remaining quadrants show three possible hydrogen bonding partners: S735 (cyan), Y736 (magenta), and the adjacent R634 (yellow). (D) Overexpression of the TRPC3 mutants Mwk (T635A) and T635V significantly induced cell death in mouse Neuro-2a cells (mean ± SEM; n = 3; ANOVA followed by the Newman–Keuls post hoc test; p
    Figure Legend Snippet: Homology modeling reveals potential for hydrogen bonding. (A) Sequence alignment of mouse TRPC3 to rat TRPV1 and to the S4–S5 linker of the Kv 1.2/2.1 chimera used to create the homology models. A cut of 21 amino acids (indicated by double lines) was made at the end of S3 to facilitate modeling. Transmembrane helices are indicated by black boxes with the corresponding label for each S1–S6 transmembrane helix above. Coloring is according to the Clustal coloring scheme. The TRPC3 Mwk mutation in the S4–S5 linker region is indicated by a double-lined box. Arrows indicate residues highlighted in other panels of the figure. Image made with JalView. (B) Top-ranked (according to MODELLER score) mouse TRPC3 homology model on the TRPV1 apo structure (PDB entry 3J5P) with the Kv 1.2/2.1 channel chimera structure for the S4–S5 linker shown from a top view and a side view. Coloring indicates separate chains of the tetrameric channel. The S1–S4 helices of the purple chain have been removed for the sake of clarity in the side view figure. (C) Homology models of TRPC3 indicate that the threonine 635 (in green) mutated in the Mwk mouse has the potential for hydrogen bonding with the end of helix S6. The first quadrant shows an overlap of four models and the resulting T635 orientation. The remaining quadrants show three possible hydrogen bonding partners: S735 (cyan), Y736 (magenta), and the adjacent R634 (yellow). (D) Overexpression of the TRPC3 mutants Mwk (T635A) and T635V significantly induced cell death in mouse Neuro-2a cells (mean ± SEM; n = 3; ANOVA followed by the Newman–Keuls post hoc test; p

    Techniques Used: Sequencing, Mutagenesis, Over Expression

    The gain-of-function Mwk mutation in TRPC3 causes increased cell death and calcium signaling in neuronal cells. (A) Equal amounts of TRPC3 are expressed at the cell surface in the mouse cerebellum. Biotinylated cerebellar slice cultures from wild-type (WT) and Mwk mice were lysed and subjected to pull-down experiments using streptavidin beads, followed by immunoblotting for TRPC3 and actin. Abbreviations: I, input; S, supernatant; B, pellet (biotinylated fraction). (B) Overexpression of Mwk (T635A) but not wild-type (WT) TRPC3 significantly induced cell death in mouse Neuro-2a cells (mean ± SEM; n = 3; ANOVA followed by Bonferroni’s post hoc test; p
    Figure Legend Snippet: The gain-of-function Mwk mutation in TRPC3 causes increased cell death and calcium signaling in neuronal cells. (A) Equal amounts of TRPC3 are expressed at the cell surface in the mouse cerebellum. Biotinylated cerebellar slice cultures from wild-type (WT) and Mwk mice were lysed and subjected to pull-down experiments using streptavidin beads, followed by immunoblotting for TRPC3 and actin. Abbreviations: I, input; S, supernatant; B, pellet (biotinylated fraction). (B) Overexpression of Mwk (T635A) but not wild-type (WT) TRPC3 significantly induced cell death in mouse Neuro-2a cells (mean ± SEM; n = 3; ANOVA followed by Bonferroni’s post hoc test; p

    Techniques Used: Mutagenesis, Mouse Assay, Over Expression

    5) Product Images from "Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels"

    Article Title: Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2009.00890.x

    Effect of candesartan or telmisartan but not amlodipine on TRPC expression in mesenteric arterioles. Long-term administration of angiotensin AT1 receptor antagonist telmisartan or candesartan, but not of calcium channel blocker amlodipine reduces TRPC1, TRPC3 and TRPC5 channel protein expression in vivo. The angiotensin AT1 receptor antagonist telmisartan (5 mg/kg per day) or candesartan (4 mg/kg per day), calcium channel blocker amlodipine (10 mg/kg per day), or placebo were administered to SHR by gavage for 16 weeks. Representative immunoblottings of TRPC channel protein expressions in mesenteric arterioles from treated SHR (Fig. 7A) and summary data are shown (Fig. 7B). * P
    Figure Legend Snippet: Effect of candesartan or telmisartan but not amlodipine on TRPC expression in mesenteric arterioles. Long-term administration of angiotensin AT1 receptor antagonist telmisartan or candesartan, but not of calcium channel blocker amlodipine reduces TRPC1, TRPC3 and TRPC5 channel protein expression in vivo. The angiotensin AT1 receptor antagonist telmisartan (5 mg/kg per day) or candesartan (4 mg/kg per day), calcium channel blocker amlodipine (10 mg/kg per day), or placebo were administered to SHR by gavage for 16 weeks. Representative immunoblottings of TRPC channel protein expressions in mesenteric arterioles from treated SHR (Fig. 7A) and summary data are shown (Fig. 7B). * P

    Techniques Used: Expressing, In Vivo

    Inhibition of norepinephrine-induced vasomotion in mesenteric arterioles from SHR by specific anti-TRPC antibodies Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions (A), in the presence of anti-TRPC1 antibodies (B), anti-TRPC3 antibodies (C), anti-TRPC3 antibodies with antigenic peptide (D), anti-TRPC5 antibodies (E), anti-TRPC1 plus anti-TRPC3 antibodies (F) or in the presence of anti-β-actin antibodies (G). Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions after short-term exposure to hypotonic 0.45% NaCl without antibody (H), or after short-term exposure to hypotonic 0.45% NaCl plus TRPC3 antibody (I). Summary data (J) show that inhibition of TRPC channels by specific anti-TRPC antibodies significantly attenuates norepinephrine-induced vasomotion in SHR. Data are mean ± S.E.M. of n = 6 independent experiments. * P
    Figure Legend Snippet: Inhibition of norepinephrine-induced vasomotion in mesenteric arterioles from SHR by specific anti-TRPC antibodies Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions (A), in the presence of anti-TRPC1 antibodies (B), anti-TRPC3 antibodies (C), anti-TRPC3 antibodies with antigenic peptide (D), anti-TRPC5 antibodies (E), anti-TRPC1 plus anti-TRPC3 antibodies (F) or in the presence of anti-β-actin antibodies (G). Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions after short-term exposure to hypotonic 0.45% NaCl without antibody (H), or after short-term exposure to hypotonic 0.45% NaCl plus TRPC3 antibody (I). Summary data (J) show that inhibition of TRPC channels by specific anti-TRPC antibodies significantly attenuates norepinephrine-induced vasomotion in SHR. Data are mean ± S.E.M. of n = 6 independent experiments. * P

    Techniques Used: Inhibition

    Expression of TRPC1, TRPC3 and TRPC5 channels in mesenteric arterioles from SHR. Representative immunoblottings (A) and summary data (B) of TRPC1, TRPC3, TRPC4, TRPC5,TRPC6 channels and TRPC3 antibody with its respective antigenic peptide the protein expression in mesenteric arterioles from WKY (open bars) and SHR (filled bars). Data are mean ± S.E.M. of n = 4 independent experiments. * P
    Figure Legend Snippet: Expression of TRPC1, TRPC3 and TRPC5 channels in mesenteric arterioles from SHR. Representative immunoblottings (A) and summary data (B) of TRPC1, TRPC3, TRPC4, TRPC5,TRPC6 channels and TRPC3 antibody with its respective antigenic peptide the protein expression in mesenteric arterioles from WKY (open bars) and SHR (filled bars). Data are mean ± S.E.M. of n = 4 independent experiments. * P

    Techniques Used: Expressing

    Inhibition of norepinephrine-induced calcium increase in mesenteric arterioles from SHR by specific anti-TRPC antibodies. Representative tracings of norepinephrine-induced calcium increase in mesenteric arterioles from SHR under control conditions and in the presence of anti-TRPC1 antibodies (A), anti-TRPC3 antibodies (B), anti-TRPC5 antibodies (C), anti-TRPC1 plus anti-TRPC3 plus anti-TRPC5 antibodies (D), anti-TRPC3 antibodies plus TRPC3 antigenic peptide (E) or anti-β-actin antibodies (F). Summary data (G) shows the effects of specific anti-TRPC antibodies on norepinephrine-induced calcium influx in mesenteric arterioles from SHR. Data are mean ± S.E.M. of n = 8 independent experiments. * P
    Figure Legend Snippet: Inhibition of norepinephrine-induced calcium increase in mesenteric arterioles from SHR by specific anti-TRPC antibodies. Representative tracings of norepinephrine-induced calcium increase in mesenteric arterioles from SHR under control conditions and in the presence of anti-TRPC1 antibodies (A), anti-TRPC3 antibodies (B), anti-TRPC5 antibodies (C), anti-TRPC1 plus anti-TRPC3 plus anti-TRPC5 antibodies (D), anti-TRPC3 antibodies plus TRPC3 antigenic peptide (E) or anti-β-actin antibodies (F). Summary data (G) shows the effects of specific anti-TRPC antibodies on norepinephrine-induced calcium influx in mesenteric arterioles from SHR. Data are mean ± S.E.M. of n = 8 independent experiments. * P

    Techniques Used: Inhibition

    6) Product Images from "Tadalafil Treatment Delays the Onset of Cardiomyopathy in Dystrophin‐Deficient Hearts"

    Article Title: Tadalafil Treatment Delays the Onset of Cardiomyopathy in Dystrophin‐Deficient Hearts

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.116.003911

    Tadalafil (Tad) modulates TRPC 6 and protease levels and activity in golden retriever muscular dystrophy ( GRMD) left ventricle (LV). A, Gene expression of Pde5 , Trpc3 , and Trpc6 in control and Tad‐treated GRMD LV (normalized to Gapdh and displayed as arbitrary units (AU) relative to untreated GRMD values). All values and individual values are presented, and group values are represented as mean± SD . Representative blots of Tad‐induced changes in TRPC 6, μ‐calpain, m‐calpain, cardiac troponin I ( cTnI) , spectrin 150‐kDa cleavage product, α‐actinin 80‐kDa cleavage product, integrin β1 75‐kDa cleavage product, dysferlin C72 fragment, full‐length utrophin, and fibronectin protein content (B), along with percentage difference between untreated and treated values (Cohen's d shown to display effect size). Ponceau Red staining is used as a loading control. C, TRPC 6 and TRPC 3 immunoprecipitation (IP) results for phosphorylated threonine (p‐Thr) with blot density quantified as phopho‐ to total (p/t) protein ratio.
    Figure Legend Snippet: Tadalafil (Tad) modulates TRPC 6 and protease levels and activity in golden retriever muscular dystrophy ( GRMD) left ventricle (LV). A, Gene expression of Pde5 , Trpc3 , and Trpc6 in control and Tad‐treated GRMD LV (normalized to Gapdh and displayed as arbitrary units (AU) relative to untreated GRMD values). All values and individual values are presented, and group values are represented as mean± SD . Representative blots of Tad‐induced changes in TRPC 6, μ‐calpain, m‐calpain, cardiac troponin I ( cTnI) , spectrin 150‐kDa cleavage product, α‐actinin 80‐kDa cleavage product, integrin β1 75‐kDa cleavage product, dysferlin C72 fragment, full‐length utrophin, and fibronectin protein content (B), along with percentage difference between untreated and treated values (Cohen's d shown to display effect size). Ponceau Red staining is used as a loading control. C, TRPC 6 and TRPC 3 immunoprecipitation (IP) results for phosphorylated threonine (p‐Thr) with blot density quantified as phopho‐ to total (p/t) protein ratio.

    Techniques Used: Activity Assay, Expressing, Staining, Immunoprecipitation

    7) Product Images from "Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein kinase G"

    Article Title: Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein kinase G

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

    doi: 10.1073/pnas.0304471101

    Effect of TRPC3 overexpression on store-operated Ca 2+ influx in PKG-HEK cells. ( A ) Immunoblots with an anti-PGK antibody showed that PKG-HEK cells, a HEK293 cell line stably transfected with PKG construct, expressed a much higher level of PKG compared
    Figure Legend Snippet: Effect of TRPC3 overexpression on store-operated Ca 2+ influx in PKG-HEK cells. ( A ) Immunoblots with an anti-PGK antibody showed that PKG-HEK cells, a HEK293 cell line stably transfected with PKG construct, expressed a much higher level of PKG compared

    Techniques Used: Over Expression, Western Blot, Stable Transfection, Transfection, Construct

    Effect of cGMP, KT5823 on TRPC3-mediated store-operated Ca 2+ influx in HEK293 cells without PKG transfection. 8-BrcGMP (2 mM) and KT5823 (1 μM) had no effect on the store-operated Ca 2+ influx in the HEK293 cells that were transiently transfected
    Figure Legend Snippet: Effect of cGMP, KT5823 on TRPC3-mediated store-operated Ca 2+ influx in HEK293 cells without PKG transfection. 8-BrcGMP (2 mM) and KT5823 (1 μM) had no effect on the store-operated Ca 2+ influx in the HEK293 cells that were transiently transfected

    Techniques Used: Transfection

    Effect of cGMP, KT5823, and H8 on TRPC3-mediated store-operated Ca 2+ influx in PKG-HEK cells. ( A ) Store-operated Ca 2+ influx in TRPC3-transfected PKG-HEK cells. Shown is the mean ± SE ( n = 14 experiments). ( B ) Store-operated Ca 2+ influx in PKG-HEK
    Figure Legend Snippet: Effect of cGMP, KT5823, and H8 on TRPC3-mediated store-operated Ca 2+ influx in PKG-HEK cells. ( A ) Store-operated Ca 2+ influx in TRPC3-transfected PKG-HEK cells. Shown is the mean ± SE ( n = 14 experiments). ( B ) Store-operated Ca 2+ influx in PKG-HEK

    Techniques Used: Transfection

    Direct PKG phosphorylation on TRPC3 proteins. ( A ) Immunoblot of TRPC3 proteins immunoprecipitated by an anti-TRPC3 (Alomone Laboratories). The antibody used for immunoblot was a second anti-TRPC3 antibody (A-15, Santa Cruz Biotechnology). ( B ) In vitro
    Figure Legend Snippet: Direct PKG phosphorylation on TRPC3 proteins. ( A ) Immunoblot of TRPC3 proteins immunoprecipitated by an anti-TRPC3 (Alomone Laboratories). The antibody used for immunoblot was a second anti-TRPC3 antibody (A-15, Santa Cruz Biotechnology). ( B ) In vitro

    Techniques Used: Immunoprecipitation, In Vitro

    8) Product Images from "Mild hypoxia-induced cardiomyocyte hypertrophy via up-regulation of HIF-1α-mediated TRPC signalling"

    Article Title: Mild hypoxia-induced cardiomyocyte hypertrophy via up-regulation of HIF-1α-mediated TRPC signalling

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2011.01497.x

    Enlarged OAG-induced TRPC3/TRPC6 current in HIF-1α overexpressing cardiomyocytes. (A) Representative I–V curves obtained after application of OAG (30 μM) in NC (blue line), HIF-1α overexpressing cardiomyocytes (red line) and SC205346 treatment 1 hr before cardiomyocytes transfected with HIF-1α (green line). The stimulation protocol is displayed in the left panel. (B) Mean data (±S.E.M.; n = 10 of NC, n = 9 of HIF-1α overexpressing cardiomyocytes, and n = 5 of SC205346 + HIF-1α) of the OAG-induced TRPC density at +50 mV and the effect of pharmacological agents ( n = 5 in each condition). # P
    Figure Legend Snippet: Enlarged OAG-induced TRPC3/TRPC6 current in HIF-1α overexpressing cardiomyocytes. (A) Representative I–V curves obtained after application of OAG (30 μM) in NC (blue line), HIF-1α overexpressing cardiomyocytes (red line) and SC205346 treatment 1 hr before cardiomyocytes transfected with HIF-1α (green line). The stimulation protocol is displayed in the left panel. (B) Mean data (±S.E.M.; n = 10 of NC, n = 9 of HIF-1α overexpressing cardiomyocytes, and n = 5 of SC205346 + HIF-1α) of the OAG-induced TRPC density at +50 mV and the effect of pharmacological agents ( n = 5 in each condition). # P

    Techniques Used: Transfection

    HIF-1α overexpression enhances TRPC3, TRPC6 expression and Ca 2+ -calcineurin signals under MH. Cardiac myocytes were transiently transfected with vectors of HIF-1α or pcDNA3.1 (NC), then exposed to MH for 6 hrs. HIF-1α overexpression promoted the up-regulation of TRPC3 and TRPC6 mRNA (A), protein (B and C) expression, elevation of [Ca 2+ ] i (E) and enhanced calcineurin (D) expression induced by MH. * P
    Figure Legend Snippet: HIF-1α overexpression enhances TRPC3, TRPC6 expression and Ca 2+ -calcineurin signals under MH. Cardiac myocytes were transiently transfected with vectors of HIF-1α or pcDNA3.1 (NC), then exposed to MH for 6 hrs. HIF-1α overexpression promoted the up-regulation of TRPC3 and TRPC6 mRNA (A), protein (B and C) expression, elevation of [Ca 2+ ] i (E) and enhanced calcineurin (D) expression induced by MH. * P

    Techniques Used: Over Expression, Expressing, Transfection

    MH triggers elevation of TRPC-mediated Ca 2+ signals. Bar graphs of TRPC1, TRPC3 and TRPC6 mRNA by real-time PCR (A) and protein expression by Western blotting (B) in cultured neonatal cardiac myocytes under MH condition at the indicated time points. (C) Elevation of [Ca 2+ ] i in cardiomyocytes under MH conditions was inhibited by two TRPC channel blockers, 2-APB and SK F3586. 2-APB (30 μM) and SK F3586 (10 μM) were given to cardiomyocytes 1 hr before MH exposure. (D) Enhanced calcineurin in cardiac myocytes cultured under MH conditions was inhibited by SK F3586. (E) TRPC channel blocker SK F96365 blocked p-NFAT decrease induced by MH. Total NFAT was not changed under the indicated condition. (F) SK F96365 blocked MCIP1 mRNA increase induced by MH. (G) SK F96365 prevented MH-induced cardiomyocytes hypertrophy by α-actinin immunostaining (H) SK F96365 completely inhibited ANP, BNP and β-MHC mRNA up-regulation induced by MH. Western blot band is a representative result from three independent experiments. * P
    Figure Legend Snippet: MH triggers elevation of TRPC-mediated Ca 2+ signals. Bar graphs of TRPC1, TRPC3 and TRPC6 mRNA by real-time PCR (A) and protein expression by Western blotting (B) in cultured neonatal cardiac myocytes under MH condition at the indicated time points. (C) Elevation of [Ca 2+ ] i in cardiomyocytes under MH conditions was inhibited by two TRPC channel blockers, 2-APB and SK F3586. 2-APB (30 μM) and SK F3586 (10 μM) were given to cardiomyocytes 1 hr before MH exposure. (D) Enhanced calcineurin in cardiac myocytes cultured under MH conditions was inhibited by SK F3586. (E) TRPC channel blocker SK F96365 blocked p-NFAT decrease induced by MH. Total NFAT was not changed under the indicated condition. (F) SK F96365 blocked MCIP1 mRNA increase induced by MH. (G) SK F96365 prevented MH-induced cardiomyocytes hypertrophy by α-actinin immunostaining (H) SK F96365 completely inhibited ANP, BNP and β-MHC mRNA up-regulation induced by MH. Western blot band is a representative result from three independent experiments. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Cell Culture, Immunostaining, Aqueous Normal-phase Chromatography

    SC205346 attenuates TRPC3, TRPC6 expression and Ca 2+ -calcineurin signals in MH cardiomyocytes. Cardiac myocytes were treated with SC205346 (30 μM) 1 hr before MH. SC205346 attenuated the up-regulation of TRPC3 and TRPC6 mRNA (A), protein (B and C) expression, elevation of [Ca 2+ ] i (E) and enhanced calcineurin (D) expression induced by MH. * P
    Figure Legend Snippet: SC205346 attenuates TRPC3, TRPC6 expression and Ca 2+ -calcineurin signals in MH cardiomyocytes. Cardiac myocytes were treated with SC205346 (30 μM) 1 hr before MH. SC205346 attenuated the up-regulation of TRPC3 and TRPC6 mRNA (A), protein (B and C) expression, elevation of [Ca 2+ ] i (E) and enhanced calcineurin (D) expression induced by MH. * P

    Techniques Used: Expressing

    9) Product Images from "Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels"

    Article Title: Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2009.00890.x

    Effect of candesartan or telmisartan but not amlodipine on TRPC expression in mesenteric arterioles. Long-term administration of angiotensin AT1 receptor antagonist telmisartan or candesartan, but not of calcium channel blocker amlodipine reduces TRPC1, TRPC3 and TRPC5 channel protein expression in vivo. The angiotensin AT1 receptor antagonist telmisartan (5 mg/kg per day) or candesartan (4 mg/kg per day), calcium channel blocker amlodipine (10 mg/kg per day), or placebo were administered to SHR by gavage for 16 weeks. Representative immunoblottings of TRPC channel protein expressions in mesenteric arterioles from treated SHR (Fig. 7A) and summary data are shown (Fig. 7B). * P
    Figure Legend Snippet: Effect of candesartan or telmisartan but not amlodipine on TRPC expression in mesenteric arterioles. Long-term administration of angiotensin AT1 receptor antagonist telmisartan or candesartan, but not of calcium channel blocker amlodipine reduces TRPC1, TRPC3 and TRPC5 channel protein expression in vivo. The angiotensin AT1 receptor antagonist telmisartan (5 mg/kg per day) or candesartan (4 mg/kg per day), calcium channel blocker amlodipine (10 mg/kg per day), or placebo were administered to SHR by gavage for 16 weeks. Representative immunoblottings of TRPC channel protein expressions in mesenteric arterioles from treated SHR (Fig. 7A) and summary data are shown (Fig. 7B). * P

    Techniques Used: Expressing, In Vivo

    Inhibition of norepinephrine-induced vasomotion in mesenteric arterioles from SHR by specific anti-TRPC antibodies Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions (A), in the presence of anti-TRPC1 antibodies (B), anti-TRPC3 antibodies (C), anti-TRPC3 antibodies with antigenic peptide (D), anti-TRPC5 antibodies (E), anti-TRPC1 plus anti-TRPC3 antibodies (F) or in the presence of anti-β-actin antibodies (G). Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions after short-term exposure to hypotonic 0.45% NaCl without antibody (H), or after short-term exposure to hypotonic 0.45% NaCl plus TRPC3 antibody (I). Summary data (J) show that inhibition of TRPC channels by specific anti-TRPC antibodies significantly attenuates norepinephrine-induced vasomotion in SHR. Data are mean ± S.E.M. of n = 6 independent experiments. * P
    Figure Legend Snippet: Inhibition of norepinephrine-induced vasomotion in mesenteric arterioles from SHR by specific anti-TRPC antibodies Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions (A), in the presence of anti-TRPC1 antibodies (B), anti-TRPC3 antibodies (C), anti-TRPC3 antibodies with antigenic peptide (D), anti-TRPC5 antibodies (E), anti-TRPC1 plus anti-TRPC3 antibodies (F) or in the presence of anti-β-actin antibodies (G). Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions after short-term exposure to hypotonic 0.45% NaCl without antibody (H), or after short-term exposure to hypotonic 0.45% NaCl plus TRPC3 antibody (I). Summary data (J) show that inhibition of TRPC channels by specific anti-TRPC antibodies significantly attenuates norepinephrine-induced vasomotion in SHR. Data are mean ± S.E.M. of n = 6 independent experiments. * P

    Techniques Used: Inhibition

    Expression of TRPC1, TRPC3 and TRPC5 channels in mesenteric arterioles from SHR. Representative immunoblottings (A) and summary data (B) of TRPC1, TRPC3, TRPC4, TRPC5,TRPC6 channels and TRPC3 antibody with its respective antigenic peptide the protein expression in mesenteric arterioles from WKY (open bars) and SHR (filled bars). Data are mean ± S.E.M. of n = 4 independent experiments. * P
    Figure Legend Snippet: Expression of TRPC1, TRPC3 and TRPC5 channels in mesenteric arterioles from SHR. Representative immunoblottings (A) and summary data (B) of TRPC1, TRPC3, TRPC4, TRPC5,TRPC6 channels and TRPC3 antibody with its respective antigenic peptide the protein expression in mesenteric arterioles from WKY (open bars) and SHR (filled bars). Data are mean ± S.E.M. of n = 4 independent experiments. * P

    Techniques Used: Expressing

    Inhibition of norepinephrine-induced calcium increase in mesenteric arterioles from SHR by specific anti-TRPC antibodies. Representative tracings of norepinephrine-induced calcium increase in mesenteric arterioles from SHR under control conditions and in the presence of anti-TRPC1 antibodies (A), anti-TRPC3 antibodies (B), anti-TRPC5 antibodies (C), anti-TRPC1 plus anti-TRPC3 plus anti-TRPC5 antibodies (D), anti-TRPC3 antibodies plus TRPC3 antigenic peptide (E) or anti-β-actin antibodies (F). Summary data (G) shows the effects of specific anti-TRPC antibodies on norepinephrine-induced calcium influx in mesenteric arterioles from SHR. Data are mean ± S.E.M. of n = 8 independent experiments. * P
    Figure Legend Snippet: Inhibition of norepinephrine-induced calcium increase in mesenteric arterioles from SHR by specific anti-TRPC antibodies. Representative tracings of norepinephrine-induced calcium increase in mesenteric arterioles from SHR under control conditions and in the presence of anti-TRPC1 antibodies (A), anti-TRPC3 antibodies (B), anti-TRPC5 antibodies (C), anti-TRPC1 plus anti-TRPC3 plus anti-TRPC5 antibodies (D), anti-TRPC3 antibodies plus TRPC3 antigenic peptide (E) or anti-β-actin antibodies (F). Summary data (G) shows the effects of specific anti-TRPC antibodies on norepinephrine-induced calcium influx in mesenteric arterioles from SHR. Data are mean ± S.E.M. of n = 8 independent experiments. * P

    Techniques Used: Inhibition

    10) 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:

    FRAP. A, localization of GFP-TRPC3 in HEK 293T cells transfected with GFP-TRPC3 and Epo-R examined with confocal microscopy and differential interference contrast ( DIC ). B, representative FRAP experiment in HEK 293T expressing Epo-R and GFP-TRPC3. Upper panels show untreated cells before photobleaching and then at 10, 40, and 80 s after photobleaching. Lower panels show recovery of cells photobleached in the presence of Epo (40 units/ml). Area of membrane bleached is shown in prominent white lines. C, schema showing calculation of half-time ( t ½ ) and the mobile fraction. When the region is bleached, fluorescence decreases from F i , the initial fluorescence value, to F o at time t o immediately after photobleaching. Fluorescence recovers over time until it reaches plateau value F p . t ½ is the time to recovery of half of plateau fluorescence ( F p ). a = the immobile fraction, b = the mobile fraction, calculated from F i i , F p , and F o . D, graph from a representative FRAP experiment showing relative fluorescence recovery after photobleaching (IgorPro analysis). E , t ½ (in seconds) of fluorescence recovery with or without Epo stimulation ( n = number of cells). F, mobile fraction with and without Epo stimulation ( n = number of cells). Mean ± S.E. calculated using one-way analysis of variance ( * , p ≤ 0.001).
    Figure Legend Snippet: FRAP. A, localization of GFP-TRPC3 in HEK 293T cells transfected with GFP-TRPC3 and Epo-R examined with confocal microscopy and differential interference contrast ( DIC ). B, representative FRAP experiment in HEK 293T expressing Epo-R and GFP-TRPC3. Upper panels show untreated cells before photobleaching and then at 10, 40, and 80 s after photobleaching. Lower panels show recovery of cells photobleached in the presence of Epo (40 units/ml). Area of membrane bleached is shown in prominent white lines. C, schema showing calculation of half-time ( t ½ ) and the mobile fraction. When the region is bleached, fluorescence decreases from F i , the initial fluorescence value, to F o at time t o immediately after photobleaching. Fluorescence recovers over time until it reaches plateau value F p . t ½ is the time to recovery of half of plateau fluorescence ( F p ). a = the immobile fraction, b = the mobile fraction, calculated from F i i , F p , and F o . D, graph from a representative FRAP experiment showing relative fluorescence recovery after photobleaching (IgorPro analysis). E , t ½ (in seconds) of fluorescence recovery with or without Epo stimulation ( n = number of cells). F, mobile fraction with and without Epo stimulation ( n = number of cells). Mean ± S.E. calculated using one-way analysis of variance ( * , p ≤ 0.001).

    Techniques Used: Transfection, Confocal Microscopy, Expressing, Fluorescence

    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

    11) Product Images from "Interaction between mitsugumin 29 and TRPC3 participates in regulating Ca2+ transients in skeletal muscle"

    Article Title: Interaction between mitsugumin 29 and TRPC3 participates in regulating Ca2+ transients in skeletal muscle

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2015.06.096

    A reduction in Ca 2+ transients in response to membrane depolarization, and the disruption of the binding between endogenous MG29 and TRPC3 in mouse primary skeletal myotubes expressing Δ116-MG29
    Figure Legend Snippet: A reduction in Ca 2+ transients in response to membrane depolarization, and the disruption of the binding between endogenous MG29 and TRPC3 in mouse primary skeletal myotubes expressing Δ116-MG29

    Techniques Used: Binding Assay, Expressing

    Co-immunoprecipitation of TRPC3 with each MG29 portion
    Figure Legend Snippet: Co-immunoprecipitation of TRPC3 with each MG29 portion

    Techniques Used: Immunoprecipitation

    12) Product Images from "Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels"

    Article Title: Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2009.00890.x

    Effect of candesartan or telmisartan but not amlodipine on TRPC expression in mesenteric arterioles. Long-term administration of angiotensin AT1 receptor antagonist telmisartan or candesartan, but not of calcium channel blocker amlodipine reduces TRPC1, TRPC3 and TRPC5 channel protein expression in vivo. The angiotensin AT1 receptor antagonist telmisartan (5 mg/kg per day) or candesartan (4 mg/kg per day), calcium channel blocker amlodipine (10 mg/kg per day), or placebo were administered to SHR by gavage for 16 weeks. Representative immunoblottings of TRPC channel protein expressions in mesenteric arterioles from treated SHR (Fig. 7A) and summary data are shown (Fig. 7B). * P
    Figure Legend Snippet: Effect of candesartan or telmisartan but not amlodipine on TRPC expression in mesenteric arterioles. Long-term administration of angiotensin AT1 receptor antagonist telmisartan or candesartan, but not of calcium channel blocker amlodipine reduces TRPC1, TRPC3 and TRPC5 channel protein expression in vivo. The angiotensin AT1 receptor antagonist telmisartan (5 mg/kg per day) or candesartan (4 mg/kg per day), calcium channel blocker amlodipine (10 mg/kg per day), or placebo were administered to SHR by gavage for 16 weeks. Representative immunoblottings of TRPC channel protein expressions in mesenteric arterioles from treated SHR (Fig. 7A) and summary data are shown (Fig. 7B). * P

    Techniques Used: Expressing, In Vivo

    Inhibition of norepinephrine-induced vasomotion in mesenteric arterioles from SHR by specific anti-TRPC antibodies Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions (A), in the presence of anti-TRPC1 antibodies (B), anti-TRPC3 antibodies (C), anti-TRPC3 antibodies with antigenic peptide (D), anti-TRPC5 antibodies (E), anti-TRPC1 plus anti-TRPC3 antibodies (F) or in the presence of anti-β-actin antibodies (G). Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions after short-term exposure to hypotonic 0.45% NaCl without antibody (H), or after short-term exposure to hypotonic 0.45% NaCl plus TRPC3 antibody (I). Summary data (J) show that inhibition of TRPC channels by specific anti-TRPC antibodies significantly attenuates norepinephrine-induced vasomotion in SHR. Data are mean ± S.E.M. of n = 6 independent experiments. * P
    Figure Legend Snippet: Inhibition of norepinephrine-induced vasomotion in mesenteric arterioles from SHR by specific anti-TRPC antibodies Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions (A), in the presence of anti-TRPC1 antibodies (B), anti-TRPC3 antibodies (C), anti-TRPC3 antibodies with antigenic peptide (D), anti-TRPC5 antibodies (E), anti-TRPC1 plus anti-TRPC3 antibodies (F) or in the presence of anti-β-actin antibodies (G). Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions after short-term exposure to hypotonic 0.45% NaCl without antibody (H), or after short-term exposure to hypotonic 0.45% NaCl plus TRPC3 antibody (I). Summary data (J) show that inhibition of TRPC channels by specific anti-TRPC antibodies significantly attenuates norepinephrine-induced vasomotion in SHR. Data are mean ± S.E.M. of n = 6 independent experiments. * P

    Techniques Used: Inhibition

    Expression of TRPC1, TRPC3 and TRPC5 channels in mesenteric arterioles from SHR. Representative immunoblottings (A) and summary data (B) of TRPC1, TRPC3, TRPC4, TRPC5,TRPC6 channels and TRPC3 antibody with its respective antigenic peptide the protein expression in mesenteric arterioles from WKY (open bars) and SHR (filled bars). Data are mean ± S.E.M. of n = 4 independent experiments. * P
    Figure Legend Snippet: Expression of TRPC1, TRPC3 and TRPC5 channels in mesenteric arterioles from SHR. Representative immunoblottings (A) and summary data (B) of TRPC1, TRPC3, TRPC4, TRPC5,TRPC6 channels and TRPC3 antibody with its respective antigenic peptide the protein expression in mesenteric arterioles from WKY (open bars) and SHR (filled bars). Data are mean ± S.E.M. of n = 4 independent experiments. * P

    Techniques Used: Expressing

    Inhibition of norepinephrine-induced calcium increase in mesenteric arterioles from SHR by specific anti-TRPC antibodies. Representative tracings of norepinephrine-induced calcium increase in mesenteric arterioles from SHR under control conditions and in the presence of anti-TRPC1 antibodies (A), anti-TRPC3 antibodies (B), anti-TRPC5 antibodies (C), anti-TRPC1 plus anti-TRPC3 plus anti-TRPC5 antibodies (D), anti-TRPC3 antibodies plus TRPC3 antigenic peptide (E) or anti-β-actin antibodies (F). Summary data (G) shows the effects of specific anti-TRPC antibodies on norepinephrine-induced calcium influx in mesenteric arterioles from SHR. Data are mean ± S.E.M. of n = 8 independent experiments. * P
    Figure Legend Snippet: Inhibition of norepinephrine-induced calcium increase in mesenteric arterioles from SHR by specific anti-TRPC antibodies. Representative tracings of norepinephrine-induced calcium increase in mesenteric arterioles from SHR under control conditions and in the presence of anti-TRPC1 antibodies (A), anti-TRPC3 antibodies (B), anti-TRPC5 antibodies (C), anti-TRPC1 plus anti-TRPC3 plus anti-TRPC5 antibodies (D), anti-TRPC3 antibodies plus TRPC3 antigenic peptide (E) or anti-β-actin antibodies (F). Summary data (G) shows the effects of specific anti-TRPC antibodies on norepinephrine-induced calcium influx in mesenteric arterioles from SHR. Data are mean ± S.E.M. of n = 8 independent experiments. * P

    Techniques Used: Inhibition

    13) Product Images from "Transient Receptor Potential Channel Canonical Type 3 Deficiency Antagonizes Myofibroblast Transdifferentiation In Vivo"

    Article Title: Transient Receptor Potential Channel Canonical Type 3 Deficiency Antagonizes Myofibroblast Transdifferentiation In Vivo

    Journal: BioMed Research International

    doi: 10.1155/2020/1202189

    Effects of TRPC3 on ROS production and mitochondrial function in human fibroblasts. Mitochondrial oxygen consumption was measured in human fibroblasts treated with TGF β 1 (10 ng/mL) for 24 h via Oxygraph-2k high-resolution respirometry. (a) The test protocol is shown. (b) The values are expressed in pmol/s per 10 6 cells. Summarized data for oxygen consumption capacity. N = 6, ∗ p
    Figure Legend Snippet: Effects of TRPC3 on ROS production and mitochondrial function in human fibroblasts. Mitochondrial oxygen consumption was measured in human fibroblasts treated with TGF β 1 (10 ng/mL) for 24 h via Oxygraph-2k high-resolution respirometry. (a) The test protocol is shown. (b) The values are expressed in pmol/s per 10 6 cells. Summarized data for oxygen consumption capacity. N = 6, ∗ p

    Techniques Used:

    Trpc3 −/− attenuated myofibroblast transdifferentiation through inhibition of NOX4/pSmad in vivo. Wound granulation tissues were harvested 6 days after wounding, and immunochemistry with a primary antibody against α SMA was performed. (a) Representative immunohistochemical images showing α SMA expression in wound granulation tissues from Trpc3 +/+ mice and Trpc3 −/− mice. Vascular smooth muscle cells were stained with an anti- α SMA antibody in granulation tissues from Trpc3 +/+ mice as an internal positive control. The scale bar represents 100 μ m. (b, c) Western blot analysis of TGF β 1, α SMA, fibronectin (Fibro), and Col1a1 levels in wound granulation tissues from Trpc3 +/+ and Trpc3 −/− mice. n = 6. ∗ p
    Figure Legend Snippet: Trpc3 −/− attenuated myofibroblast transdifferentiation through inhibition of NOX4/pSmad in vivo. Wound granulation tissues were harvested 6 days after wounding, and immunochemistry with a primary antibody against α SMA was performed. (a) Representative immunohistochemical images showing α SMA expression in wound granulation tissues from Trpc3 +/+ mice and Trpc3 −/− mice. Vascular smooth muscle cells were stained with an anti- α SMA antibody in granulation tissues from Trpc3 +/+ mice as an internal positive control. The scale bar represents 100 μ m. (b, c) Western blot analysis of TGF β 1, α SMA, fibronectin (Fibro), and Col1a1 levels in wound granulation tissues from Trpc3 +/+ and Trpc3 −/− mice. n = 6. ∗ p

    Techniques Used: Inhibition, In Vivo, Immunohistochemistry, Expressing, Mouse Assay, Staining, Positive Control, Western Blot

    Schematic illustration depicting the mechanism by which enhanced TRPC3-mediated mitochondrial Ca 2+ homeostasis and ROS generation contribute to myofibroblast transdifferentiation via the NOX4/pSmad pathway in the hypertrophic scars.
    Figure Legend Snippet: Schematic illustration depicting the mechanism by which enhanced TRPC3-mediated mitochondrial Ca 2+ homeostasis and ROS generation contribute to myofibroblast transdifferentiation via the NOX4/pSmad pathway in the hypertrophic scars.

    Techniques Used:

    Effects of TRPC3 on myofibroblast transdifferentiation of human fibroblasts. Human fibroblasts were treated with TGF β 1 alone (10 ng/mL) or with TGF β 1 and Pyr3 (10 μ mol/L) for 12 h (upper panel) or 24 h (lower panel). (a) Immunofluorescence staining and (b) myofibroblasts ( α SMA + , green) and nucleic acid staining (blue). This experiment was repeated three times, and the percentage of α SMA + myofibroblasts was quantified over the three experiments. The bars are 100 μ m. ∗ p
    Figure Legend Snippet: Effects of TRPC3 on myofibroblast transdifferentiation of human fibroblasts. Human fibroblasts were treated with TGF β 1 alone (10 ng/mL) or with TGF β 1 and Pyr3 (10 μ mol/L) for 12 h (upper panel) or 24 h (lower panel). (a) Immunofluorescence staining and (b) myofibroblasts ( α SMA + , green) and nucleic acid staining (blue). This experiment was repeated three times, and the percentage of α SMA + myofibroblasts was quantified over the three experiments. The bars are 100 μ m. ∗ p

    Techniques Used: Immunofluorescence, Staining

    Effect of TRPC3 inhibition on cytoplasmic and mitochondrial Ca 2+ homeostasis in human fibroblasts. SOCE was measured with thapsigargin (TG), and (a–d) [Ca 2+ ] cyt and (e–h) [Ca 2+ ] mito were quantified in human primary-cultured fibroblasts under control conditions or under TGF β 1 (10 ng/mL) treatment for 12 h (a, b, e, f) or 24 h (c, d, g, h). TG (1 μ mol/L) was added after preincubation with Pyr3 (10 μ mol/L). The ATP concentration was 200 μ mol/L in a 0.3 mmol/L extracellular Ca 2+ solution after preincubation with Pyr3 (10 μ mol/L). n = 6 per group. ∗∗ p
    Figure Legend Snippet: Effect of TRPC3 inhibition on cytoplasmic and mitochondrial Ca 2+ homeostasis in human fibroblasts. SOCE was measured with thapsigargin (TG), and (a–d) [Ca 2+ ] cyt and (e–h) [Ca 2+ ] mito were quantified in human primary-cultured fibroblasts under control conditions or under TGF β 1 (10 ng/mL) treatment for 12 h (a, b, e, f) or 24 h (c, d, g, h). TG (1 μ mol/L) was added after preincubation with Pyr3 (10 μ mol/L). The ATP concentration was 200 μ mol/L in a 0.3 mmol/L extracellular Ca 2+ solution after preincubation with Pyr3 (10 μ mol/L). n = 6 per group. ∗∗ p

    Techniques Used: Inhibition, Cell Culture, Concentration Assay

    14) 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:

    FRAP. A, localization of GFP-TRPC3 in HEK 293T cells transfected with GFP-TRPC3 and Epo-R examined with confocal microscopy and differential interference contrast ( DIC ). B, representative FRAP experiment in HEK 293T expressing Epo-R and GFP-TRPC3. Upper panels show untreated cells before photobleaching and then at 10, 40, and 80 s after photobleaching. Lower panels show recovery of cells photobleached in the presence of Epo (40 units/ml). Area of membrane bleached is shown in prominent white lines. C, schema showing calculation of half-time ( t ½ ) and the mobile fraction. When the region is bleached, fluorescence decreases from F i , the initial fluorescence value, to F o at time t o immediately after photobleaching. Fluorescence recovers over time until it reaches plateau value F p . t ½ is the time to recovery of half of plateau fluorescence ( F p ). a = the immobile fraction, b = the mobile fraction, calculated from F i i , F p , and F o . D, graph from a representative FRAP experiment showing relative fluorescence recovery after photobleaching (IgorPro analysis). E , t ½ (in seconds) of fluorescence recovery with or without Epo stimulation ( n = number of cells). F, mobile fraction with and without Epo stimulation ( n = number of cells). Mean ± S.E. calculated using one-way analysis of variance ( * , p ≤ 0.001).
    Figure Legend Snippet: FRAP. A, localization of GFP-TRPC3 in HEK 293T cells transfected with GFP-TRPC3 and Epo-R examined with confocal microscopy and differential interference contrast ( DIC ). B, representative FRAP experiment in HEK 293T expressing Epo-R and GFP-TRPC3. Upper panels show untreated cells before photobleaching and then at 10, 40, and 80 s after photobleaching. Lower panels show recovery of cells photobleached in the presence of Epo (40 units/ml). Area of membrane bleached is shown in prominent white lines. C, schema showing calculation of half-time ( t ½ ) and the mobile fraction. When the region is bleached, fluorescence decreases from F i , the initial fluorescence value, to F o at time t o immediately after photobleaching. Fluorescence recovers over time until it reaches plateau value F p . t ½ is the time to recovery of half of plateau fluorescence ( F p ). a = the immobile fraction, b = the mobile fraction, calculated from F i i , F p , and F o . D, graph from a representative FRAP experiment showing relative fluorescence recovery after photobleaching (IgorPro analysis). E , t ½ (in seconds) of fluorescence recovery with or without Epo stimulation ( n = number of cells). F, mobile fraction with and without Epo stimulation ( n = number of cells). Mean ± S.E. calculated using one-way analysis of variance ( * , p ≤ 0.001).

    Techniques Used: Transfection, Confocal Microscopy, Expressing, Fluorescence

    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

    15) Product Images from "Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels"

    Article Title: Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2009.00890.x

    Effect of candesartan or telmisartan but not amlodipine on TRPC expression in mesenteric arterioles. Long-term administration of angiotensin AT1 receptor antagonist telmisartan or candesartan, but not of calcium channel blocker amlodipine reduces TRPC1, TRPC3 and TRPC5 channel protein expression in vivo. The angiotensin AT1 receptor antagonist telmisartan (5 mg/kg per day) or candesartan (4 mg/kg per day), calcium channel blocker amlodipine (10 mg/kg per day), or placebo were administered to SHR by gavage for 16 weeks. Representative immunoblottings of TRPC channel protein expressions in mesenteric arterioles from treated SHR (Fig. 7A) and summary data are shown (Fig. 7B). * P
    Figure Legend Snippet: Effect of candesartan or telmisartan but not amlodipine on TRPC expression in mesenteric arterioles. Long-term administration of angiotensin AT1 receptor antagonist telmisartan or candesartan, but not of calcium channel blocker amlodipine reduces TRPC1, TRPC3 and TRPC5 channel protein expression in vivo. The angiotensin AT1 receptor antagonist telmisartan (5 mg/kg per day) or candesartan (4 mg/kg per day), calcium channel blocker amlodipine (10 mg/kg per day), or placebo were administered to SHR by gavage for 16 weeks. Representative immunoblottings of TRPC channel protein expressions in mesenteric arterioles from treated SHR (Fig. 7A) and summary data are shown (Fig. 7B). * P

    Techniques Used: Expressing, In Vivo

    Inhibition of norepinephrine-induced vasomotion in mesenteric arterioles from SHR by specific anti-TRPC antibodies Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions (A), in the presence of anti-TRPC1 antibodies (B), anti-TRPC3 antibodies (C), anti-TRPC3 antibodies with antigenic peptide (D), anti-TRPC5 antibodies (E), anti-TRPC1 plus anti-TRPC3 antibodies (F) or in the presence of anti-β-actin antibodies (G). Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions after short-term exposure to hypotonic 0.45% NaCl without antibody (H), or after short-term exposure to hypotonic 0.45% NaCl plus TRPC3 antibody (I). Summary data (J) show that inhibition of TRPC channels by specific anti-TRPC antibodies significantly attenuates norepinephrine-induced vasomotion in SHR. Data are mean ± S.E.M. of n = 6 independent experiments. * P
    Figure Legend Snippet: Inhibition of norepinephrine-induced vasomotion in mesenteric arterioles from SHR by specific anti-TRPC antibodies Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions (A), in the presence of anti-TRPC1 antibodies (B), anti-TRPC3 antibodies (C), anti-TRPC3 antibodies with antigenic peptide (D), anti-TRPC5 antibodies (E), anti-TRPC1 plus anti-TRPC3 antibodies (F) or in the presence of anti-β-actin antibodies (G). Representative tracings of norepinephrine-induced vasomotion in mesenteric arterioles from SHR under control conditions after short-term exposure to hypotonic 0.45% NaCl without antibody (H), or after short-term exposure to hypotonic 0.45% NaCl plus TRPC3 antibody (I). Summary data (J) show that inhibition of TRPC channels by specific anti-TRPC antibodies significantly attenuates norepinephrine-induced vasomotion in SHR. Data are mean ± S.E.M. of n = 6 independent experiments. * P

    Techniques Used: Inhibition

    Expression of TRPC1, TRPC3 and TRPC5 channels in mesenteric arterioles from SHR. Representative immunoblottings (A) and summary data (B) of TRPC1, TRPC3, TRPC4, TRPC5,TRPC6 channels and TRPC3 antibody with its respective antigenic peptide the protein expression in mesenteric arterioles from WKY (open bars) and SHR (filled bars). Data are mean ± S.E.M. of n = 4 independent experiments. * P
    Figure Legend Snippet: Expression of TRPC1, TRPC3 and TRPC5 channels in mesenteric arterioles from SHR. Representative immunoblottings (A) and summary data (B) of TRPC1, TRPC3, TRPC4, TRPC5,TRPC6 channels and TRPC3 antibody with its respective antigenic peptide the protein expression in mesenteric arterioles from WKY (open bars) and SHR (filled bars). Data are mean ± S.E.M. of n = 4 independent experiments. * P

    Techniques Used: Expressing

    Inhibition of norepinephrine-induced calcium increase in mesenteric arterioles from SHR by specific anti-TRPC antibodies. Representative tracings of norepinephrine-induced calcium increase in mesenteric arterioles from SHR under control conditions and in the presence of anti-TRPC1 antibodies (A), anti-TRPC3 antibodies (B), anti-TRPC5 antibodies (C), anti-TRPC1 plus anti-TRPC3 plus anti-TRPC5 antibodies (D), anti-TRPC3 antibodies plus TRPC3 antigenic peptide (E) or anti-β-actin antibodies (F). Summary data (G) shows the effects of specific anti-TRPC antibodies on norepinephrine-induced calcium influx in mesenteric arterioles from SHR. Data are mean ± S.E.M. of n = 8 independent experiments. * P
    Figure Legend Snippet: Inhibition of norepinephrine-induced calcium increase in mesenteric arterioles from SHR by specific anti-TRPC antibodies. Representative tracings of norepinephrine-induced calcium increase in mesenteric arterioles from SHR under control conditions and in the presence of anti-TRPC1 antibodies (A), anti-TRPC3 antibodies (B), anti-TRPC5 antibodies (C), anti-TRPC1 plus anti-TRPC3 plus anti-TRPC5 antibodies (D), anti-TRPC3 antibodies plus TRPC3 antigenic peptide (E) or anti-β-actin antibodies (F). Summary data (G) shows the effects of specific anti-TRPC antibodies on norepinephrine-induced calcium influx in mesenteric arterioles from SHR. Data are mean ± S.E.M. of n = 8 independent experiments. * P

    Techniques Used: Inhibition

    16) Product Images from "TRPC6 inactivation does not affect loss of renal function in nephrotoxic serum glomerulonephritis in rats, but reduces severity of glomerular lesions"

    Article Title: TRPC6 inactivation does not affect loss of renal function in nephrotoxic serum glomerulonephritis in rats, but reduces severity of glomerular lesions

    Journal: Biochemistry and Biophysics Reports

    doi: 10.1016/j.bbrep.2018.12.006

    Effects of NTS treatment on abundance of TRPC3, TRPC6 and TRPC5 in renal cortex. This was quantified by immunoblot from renal cortex. As described previously (Kim et al. [15] , [19] ), TRPC3 is increased in renal cortex of Trpc6 del/del rats but this does not increase further following NTS treatment. NTS increases TRPC3 and TRPC6 in Trpc6 wt/wt rats. There were not changes in TRPC5 associated with genotype or NTS treatment.
    Figure Legend Snippet: Effects of NTS treatment on abundance of TRPC3, TRPC6 and TRPC5 in renal cortex. This was quantified by immunoblot from renal cortex. As described previously (Kim et al. [15] , [19] ), TRPC3 is increased in renal cortex of Trpc6 del/del rats but this does not increase further following NTS treatment. NTS increases TRPC3 and TRPC6 in Trpc6 wt/wt rats. There were not changes in TRPC5 associated with genotype or NTS treatment.

    Techniques Used:

    17) Product Images from "Mitsugumin 53 regulates extracellular Ca2+ entry and intracellular Ca2+ release via Orai1 and RyR1 in skeletal muscle"

    Article Title: Mitsugumin 53 regulates extracellular Ca2+ entry and intracellular Ca2+ release via Orai1 and RyR1 in skeletal muscle

    Journal: Scientific Reports

    doi: 10.1038/srep36909

    Increased expression levels of TRPC3 and TRPC4 by full-length MG53 or PRY-SPRY, and the binding of TRPC3 to MG53. ( a ) Lysate from myotubes over-expressing full-length MG53 or PRY-SPRY was subjected to an immunoblot assay with one of the antibodies against six proteins that are known to be expressed and/or to mediate extracellular Ca 2+ entry into skeletal muscle. α -Actin was used as a loading control. Three independent experiments per each protein were conducted. ( b ) The expression levels of TRPC3 and TRPC4 (indicated by asterisks in a) were presented as bar graphs. Bar graphs were presented as the mean ± S.E. for three independent experiments. *Significant difference versus corresponding Vector control ( p
    Figure Legend Snippet: Increased expression levels of TRPC3 and TRPC4 by full-length MG53 or PRY-SPRY, and the binding of TRPC3 to MG53. ( a ) Lysate from myotubes over-expressing full-length MG53 or PRY-SPRY was subjected to an immunoblot assay with one of the antibodies against six proteins that are known to be expressed and/or to mediate extracellular Ca 2+ entry into skeletal muscle. α -Actin was used as a loading control. Three independent experiments per each protein were conducted. ( b ) The expression levels of TRPC3 and TRPC4 (indicated by asterisks in a) were presented as bar graphs. Bar graphs were presented as the mean ± S.E. for three independent experiments. *Significant difference versus corresponding Vector control ( p

    Techniques Used: Expressing, Binding Assay, Plasmid Preparation

    18) Product Images from "TRPC6 inactivation does not affect loss of renal function in nephrotoxic serum glomerulonephritis in rats, but reduces severity of glomerular lesions"

    Article Title: TRPC6 inactivation does not affect loss of renal function in nephrotoxic serum glomerulonephritis in rats, but reduces severity of glomerular lesions

    Journal: Biochemistry and Biophysics Reports

    doi: 10.1016/j.bbrep.2018.12.006

    ), TRPC3 is increased in renal cortex of Trpc6 del/del rats but this does not increase further following NTS treatment. NTS increases TRPC3 and TRPC6 in Trpc6 wt/wt rats. There were not changes in TRPC5 associated with genotype or NTS treatment.
    Figure Legend Snippet: ), TRPC3 is increased in renal cortex of Trpc6 del/del rats but this does not increase further following NTS treatment. NTS increases TRPC3 and TRPC6 in Trpc6 wt/wt rats. There were not changes in TRPC5 associated with genotype or NTS treatment.

    Techniques Used:

    19) Product Images from "Effects of TRPC6 Inactivation on Glomerulosclerosis and Renal Fibrosis in Aging Rats"

    Article Title: Effects of TRPC6 Inactivation on Glomerulosclerosis and Renal Fibrosis in Aging Rats

    Journal: Cells

    doi: 10.3390/cells10040856

    Immunoblot analysis of TRPC6, TRPC3 and TRPC5 abundance from renal cortex of Trpc6 wt/wt and Trpc6 del/del rats at 2 and 12 months of age as indicated. Representative blots are shown to the left. Bar graphs to the right denote mean ± SD of densitometric analyses with three animals in each group.
    Figure Legend Snippet: Immunoblot analysis of TRPC6, TRPC3 and TRPC5 abundance from renal cortex of Trpc6 wt/wt and Trpc6 del/del rats at 2 and 12 months of age as indicated. Representative blots are shown to the left. Bar graphs to the right denote mean ± SD of densitometric analyses with three animals in each group.

    Techniques Used:

    20) Product Images from "lncRNA DLX6-AS1 Promotes Proliferation of Laryngeal Cancer Cells by Targeting the miR-26a/TRPC3 Pathway"

    Article Title: lncRNA DLX6-AS1 Promotes Proliferation of Laryngeal Cancer Cells by Targeting the miR-26a/TRPC3 Pathway

    Journal: Cancer Management and Research

    doi: 10.2147/CMAR.S237181

    The effects of the DLX6-AS1/miR-26a/TRPC3 axis on mitochondrial functions and cell proliferation. ( A, B ) The effects of DLX6-AS1 siRNA and miR-26a on the expression levels of TRPC3 using Western blotting in laryngeal cancer cells. *** P
    Figure Legend Snippet: The effects of the DLX6-AS1/miR-26a/TRPC3 axis on mitochondrial functions and cell proliferation. ( A, B ) The effects of DLX6-AS1 siRNA and miR-26a on the expression levels of TRPC3 using Western blotting in laryngeal cancer cells. *** P

    Techniques Used: Expressing, Western Blot

    MiR-26a functioned as a link between DLX6-AS1 and TRPC3. ( A ) The detailed binding sequence of miR-26a and wild-type TRPC3 3ʹ-UTR was presented, with the mutation sequence also presented. ( B ) The effects of miR-26a mimic and inhibitor on the expression levels of TRPC3 was demonstrated using Western blotting. * P
    Figure Legend Snippet: MiR-26a functioned as a link between DLX6-AS1 and TRPC3. ( A ) The detailed binding sequence of miR-26a and wild-type TRPC3 3ʹ-UTR was presented, with the mutation sequence also presented. ( B ) The effects of miR-26a mimic and inhibitor on the expression levels of TRPC3 was demonstrated using Western blotting. * P

    Techniques Used: Binding Assay, Sequencing, Mutagenesis, Expressing, Western Blot

    The effects of lncRNA DLX6-AS1-mediated TRPC3 upregulation on cell proliferation and calcium homeostasis. ( A ) The effects of DLX6-AS1 specific siRNA on the expression levels of TRPC3 using Western blotting. ** P
    Figure Legend Snippet: The effects of lncRNA DLX6-AS1-mediated TRPC3 upregulation on cell proliferation and calcium homeostasis. ( A ) The effects of DLX6-AS1 specific siRNA on the expression levels of TRPC3 using Western blotting. ** P

    Techniques Used: Expressing, Western Blot

    21) Product Images from "TRPC3 Regulates the Proliferation and Apoptosis Resistance of Triple Negative Breast Cancer Cells through the TRPC3/RASA4/MAPK Pathway"

    Article Title: TRPC3 Regulates the Proliferation and Apoptosis Resistance of Triple Negative Breast Cancer Cells through the TRPC3/RASA4/MAPK Pathway

    Journal: Cancers

    doi: 10.3390/cancers11040558

    TRPC3 blockade induced apoptosis in MDA-MB-231 cells through activation of ERK 1/2. ( A ) decrease in the percentage of cell proliferation in response to Pyr3 (1.0 μM for 72 h) was attenuated by pre-treatment with ERK1/2 inhibitor PD98059 (5.0 μM for 24 h) as measured by MTT assay. Pre-treatment of MDA-MB-231 cells with p38 MAPK inhibitor SB202190 (1.0 μM for 24 h) and JNK inhibitor SP600125 (1.0 μM for 24 h) did not reverse the effect of Pyr3. Values are mean ± SEM ( n = 3). ** p
    Figure Legend Snippet: TRPC3 blockade induced apoptosis in MDA-MB-231 cells through activation of ERK 1/2. ( A ) decrease in the percentage of cell proliferation in response to Pyr3 (1.0 μM for 72 h) was attenuated by pre-treatment with ERK1/2 inhibitor PD98059 (5.0 μM for 24 h) as measured by MTT assay. Pre-treatment of MDA-MB-231 cells with p38 MAPK inhibitor SB202190 (1.0 μM for 24 h) and JNK inhibitor SP600125 (1.0 μM for 24 h) did not reverse the effect of Pyr3. Values are mean ± SEM ( n = 3). ** p

    Techniques Used: Multiple Displacement Amplification, Activation Assay, MTT Assay

    TRPC3 regulated calcium influx, proliferation and apoptosis of MDA-MB-231. ( A ) representative Ca 2+ imaging traces reflected changes in the level of cytosolic free calcium over time in MDA-MB-231. Average fluo-4 fluorescence intensity was transiently increased in response to 100 μM ATP when external Ca 2+ was absent. Addition of external calcium (1.8 mM) led to an increase in fluorescence intensity; a marked decrease of the fluorescence intensity was observed when 0.5/1.0 μM Pyr3 was applied. Our results showed that TRPC3 blocker Pyr3 abolished ATP-induced Ca 2+ influx in MDA-MB-231. F/F0: fluorescence (F) normalized to baseline fluorescence (F0). Traces of fluorescence intensity are average of at least three independent experiments, with 75–100 cells measured in total; ( B ) blocking TRPC3 by Pyr3 (0.5/1.0 μM for 72 h) decreased the percentage of viable MDA-MB-231 cells in a concentration-dependent manner when compared to DMSO control as measured by an MTT assay. OD570 values of 0.1% DMSO ( v / v ) solvent control group was set as 100% of cell viability. Values are mean ± SEM ( n = 5). *** p
    Figure Legend Snippet: TRPC3 regulated calcium influx, proliferation and apoptosis of MDA-MB-231. ( A ) representative Ca 2+ imaging traces reflected changes in the level of cytosolic free calcium over time in MDA-MB-231. Average fluo-4 fluorescence intensity was transiently increased in response to 100 μM ATP when external Ca 2+ was absent. Addition of external calcium (1.8 mM) led to an increase in fluorescence intensity; a marked decrease of the fluorescence intensity was observed when 0.5/1.0 μM Pyr3 was applied. Our results showed that TRPC3 blocker Pyr3 abolished ATP-induced Ca 2+ influx in MDA-MB-231. F/F0: fluorescence (F) normalized to baseline fluorescence (F0). Traces of fluorescence intensity are average of at least three independent experiments, with 75–100 cells measured in total; ( B ) blocking TRPC3 by Pyr3 (0.5/1.0 μM for 72 h) decreased the percentage of viable MDA-MB-231 cells in a concentration-dependent manner when compared to DMSO control as measured by an MTT assay. OD570 values of 0.1% DMSO ( v / v ) solvent control group was set as 100% of cell viability. Values are mean ± SEM ( n = 5). *** p

    Techniques Used: Multiple Displacement Amplification, Imaging, Fluorescence, Blocking Assay, Concentration Assay, MTT Assay

    Dominant negative (DN) of TRPC3 attenuated proliferation, induced apoptosis and sensitized cell death to chemotherapeutic agents in MDA-MB-231. ( A ) recombinant adenoviruses (Ad) harboring GFP (Ad-GFP) or DN of TRPC3 (Ad-DN-TRPC3) were used to infect MDA-MB-231 for 48 h. Infection efficiency was determined by the percentage of cells with GFP fluorescence and was typically assessed to be 90–95%; ( B ) DN of TRPC3 attenuated cell proliferation as measured by MTT assay performed at 24 and 48 h after adenoviruses withdrawal. OD570 values of viable cells were compared between Ad-GFP and Ad-DN-TRPC3-infected group at different time points. Values are mean ± SEM ( n = 3). * p
    Figure Legend Snippet: Dominant negative (DN) of TRPC3 attenuated proliferation, induced apoptosis and sensitized cell death to chemotherapeutic agents in MDA-MB-231. ( A ) recombinant adenoviruses (Ad) harboring GFP (Ad-GFP) or DN of TRPC3 (Ad-DN-TRPC3) were used to infect MDA-MB-231 for 48 h. Infection efficiency was determined by the percentage of cells with GFP fluorescence and was typically assessed to be 90–95%; ( B ) DN of TRPC3 attenuated cell proliferation as measured by MTT assay performed at 24 and 48 h after adenoviruses withdrawal. OD570 values of viable cells were compared between Ad-GFP and Ad-DN-TRPC3-infected group at different time points. Values are mean ± SEM ( n = 3). * p

    Techniques Used: Dominant Negative Mutation, Multiple Displacement Amplification, Recombinant, Infection, Fluorescence, MTT Assay

    Blocking TRPC3 in MDA-MB-231 downregulated RASA4 expression on the plasma membrane. ( A ) representative confocal images showing the subcellular distribution of RASA4 (green) in MDA-MB-231. Cells were fixed after DMSO/ Pyr3 treatment for 1 h. Merging fluorescence image with bright field image suggested that RASA4 expression on the plasma membrane (indicated by white arrows) was decreased in most of the Pyr3-treated cells. Scale bar: 20 μm; ( B ) subcellular fractionation followed by Western blot analysis confirmed that blocking TRPC3 by Pyr3 (1.0 μM for 1 h) significantly decreased the expression of RASA4 proteins in the membrane fraction of MDA-MB-231 when compared to DMSO control group. Na/K-ATPase α1 was used as a membrane protein marker and β-tubulin was used as a cytosolic protein marker. Band density (total RASA4 normalized to Na/K-ATPase α1) was calculated by Image J software. Values are mean ± SEM ( n = 3). ** p
    Figure Legend Snippet: Blocking TRPC3 in MDA-MB-231 downregulated RASA4 expression on the plasma membrane. ( A ) representative confocal images showing the subcellular distribution of RASA4 (green) in MDA-MB-231. Cells were fixed after DMSO/ Pyr3 treatment for 1 h. Merging fluorescence image with bright field image suggested that RASA4 expression on the plasma membrane (indicated by white arrows) was decreased in most of the Pyr3-treated cells. Scale bar: 20 μm; ( B ) subcellular fractionation followed by Western blot analysis confirmed that blocking TRPC3 by Pyr3 (1.0 μM for 1 h) significantly decreased the expression of RASA4 proteins in the membrane fraction of MDA-MB-231 when compared to DMSO control group. Na/K-ATPase α1 was used as a membrane protein marker and β-tubulin was used as a cytosolic protein marker. Band density (total RASA4 normalized to Na/K-ATPase α1) was calculated by Image J software. Values are mean ± SEM ( n = 3). ** p

    Techniques Used: Blocking Assay, Multiple Displacement Amplification, Expressing, Fluorescence, Fractionation, Western Blot, Marker, Software

    TRPC3 was over-expressed on the plasma membrane of MDA-MB-231. ( A ) representative Western blots showing the expression of TRPC3 in MCF-7 and MDA-MB-231. TRPC3 protein (~100 kDa) was expressed in both MCF-7 and MDA-MB-231, whereas TRPC3 protein represented by the band between 140 and 180 kDa was over-expressed in MDA-MB-231. Membranes were incubated with two different TRPC3 antibodies (Alomone Labs, Jerusalem, Israel and Santa Cruz, Dallas, TX, USA) and consistent expression patterns were detected. β-tubulin was used as an internal control. Corresponding bands became faded or disappeared when the membrane was incubated with TRPC3 antibody pre-incubated with its corresponding peptide antigen (Alomone Labs), suggesting the specificity of the bands. ( B ) representative confocal images showing the subcellular localization of TRPC3 (green) in MCF-7 and MDA-MB-231. Cells were incubated with two different TRPC3 antibodies (Abcam, Cambridge, UK and Abnova, Taipei, Taiwan). Nuclei were stained with DAPI (blue). Merging fluorescence images with bright field images revealed that TRPC3 was over-expressed on the plasma membrane of MDA-MB-231 when compared to MCF-7. Plasma membrane positions were indicated by white arrows. Scale bar: 20 μm. ( C ) subcellular fractionation followed by Western blot analysis confirmed that the over-expressed TRPC3 protein represented by the band between 140 and 180 kDa was enriched in the membrane fraction of MDA-MB-231. Na/K-ATPase α1 was used as a membrane protein marker and β-tubulin was used as a cytosolic protein marker.
    Figure Legend Snippet: TRPC3 was over-expressed on the plasma membrane of MDA-MB-231. ( A ) representative Western blots showing the expression of TRPC3 in MCF-7 and MDA-MB-231. TRPC3 protein (~100 kDa) was expressed in both MCF-7 and MDA-MB-231, whereas TRPC3 protein represented by the band between 140 and 180 kDa was over-expressed in MDA-MB-231. Membranes were incubated with two different TRPC3 antibodies (Alomone Labs, Jerusalem, Israel and Santa Cruz, Dallas, TX, USA) and consistent expression patterns were detected. β-tubulin was used as an internal control. Corresponding bands became faded or disappeared when the membrane was incubated with TRPC3 antibody pre-incubated with its corresponding peptide antigen (Alomone Labs), suggesting the specificity of the bands. ( B ) representative confocal images showing the subcellular localization of TRPC3 (green) in MCF-7 and MDA-MB-231. Cells were incubated with two different TRPC3 antibodies (Abcam, Cambridge, UK and Abnova, Taipei, Taiwan). Nuclei were stained with DAPI (blue). Merging fluorescence images with bright field images revealed that TRPC3 was over-expressed on the plasma membrane of MDA-MB-231 when compared to MCF-7. Plasma membrane positions were indicated by white arrows. Scale bar: 20 μm. ( C ) subcellular fractionation followed by Western blot analysis confirmed that the over-expressed TRPC3 protein represented by the band between 140 and 180 kDa was enriched in the membrane fraction of MDA-MB-231. Na/K-ATPase α1 was used as a membrane protein marker and β-tubulin was used as a cytosolic protein marker.

    Techniques Used: Multiple Displacement Amplification, Western Blot, Expressing, Incubation, Staining, Fluorescence, Fractionation, Marker

    A schematic diagram explaining how TRPC3 acts as an anti-apoptotic regulator via RASA4-MAPK pathway in MDA-MB-231. TNBC cell line MDA-MB-231 overexpresses TRPC3 on the plasma membrane. Functional presence of TRPC3 regulates calcium entry across the plasma membrane into the cytosol. TRPC3 is oncogenic in MDA-MB-231 with suppression of ERK1/2 phosphorylation. TRPC3 blocker Pyr3 and DN of TRPC3 inhibit cell proliferation and induce apoptosis in a caspase-dependent manner. Blocking TRPC3 activates MAPK pathways in MDA-MB-231. RASA4, a Ca 2+ -promoted Ras-MAPK pathway suppressor, is located on the plasma membrane of MDA-MB-231 where it inhibits Ras-MAPK pathway. Ca 2+ influx through TRPC3 channel sustains the expression of RASA4 on the cell plasma membrane. Blocking TRPC3 decreases the cytosolic Ca 2+ level; this, in turn, decreases the amount of RASA4 on the plasma membrane, with concomitant activation of MAPK pathway. Taken together, functional TRPC3 channels over-expressed on the plasma membrane contribute to the apoptosis resistance of MDA-MB-231 cells through regulating Ca 2+ -dependent signaling cascade. Our study suggests that TRPC3 can be exploited as a potential molecular-based therapeutic target for TNBC.
    Figure Legend Snippet: A schematic diagram explaining how TRPC3 acts as an anti-apoptotic regulator via RASA4-MAPK pathway in MDA-MB-231. TNBC cell line MDA-MB-231 overexpresses TRPC3 on the plasma membrane. Functional presence of TRPC3 regulates calcium entry across the plasma membrane into the cytosol. TRPC3 is oncogenic in MDA-MB-231 with suppression of ERK1/2 phosphorylation. TRPC3 blocker Pyr3 and DN of TRPC3 inhibit cell proliferation and induce apoptosis in a caspase-dependent manner. Blocking TRPC3 activates MAPK pathways in MDA-MB-231. RASA4, a Ca 2+ -promoted Ras-MAPK pathway suppressor, is located on the plasma membrane of MDA-MB-231 where it inhibits Ras-MAPK pathway. Ca 2+ influx through TRPC3 channel sustains the expression of RASA4 on the cell plasma membrane. Blocking TRPC3 decreases the cytosolic Ca 2+ level; this, in turn, decreases the amount of RASA4 on the plasma membrane, with concomitant activation of MAPK pathway. Taken together, functional TRPC3 channels over-expressed on the plasma membrane contribute to the apoptosis resistance of MDA-MB-231 cells through regulating Ca 2+ -dependent signaling cascade. Our study suggests that TRPC3 can be exploited as a potential molecular-based therapeutic target for TNBC.

    Techniques Used: Multiple Displacement Amplification, Functional Assay, Blocking Assay, Expressing, Activation Assay

    22) Product Images from "TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *"

    Article Title: TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M710231200

    Schematic model of TRPC3 transmembrane domains and protein binding sites. Predicted transmembrane domains, calcium entry pore, IP 3 R, and PLCγ SH2 binding sites and deleted and substituted sites in TRPC3-DEL and TRPC3-SUB are shown. aa , amino
    Figure Legend Snippet: Schematic model of TRPC3 transmembrane domains and protein binding sites. Predicted transmembrane domains, calcium entry pore, IP 3 R, and PLCγ SH2 binding sites and deleted and substituted sites in TRPC3-DEL and TRPC3-SUB are shown. aa , amino

    Techniques Used: Protein Binding, Binding Assay

    Association of TRPC3 IP 3 R binding mutants with IP 3 R. IP 3 R type II and V5-TRPC3, V5-TRPC3-DEL, or V5-TRPC3-SUB were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-V5 or anti-IP 3 R antibodies or normal rabbit
    Figure Legend Snippet: Association of TRPC3 IP 3 R binding mutants with IP 3 R. IP 3 R type II and V5-TRPC3, V5-TRPC3-DEL, or V5-TRPC3-SUB were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-V5 or anti-IP 3 R antibodies or normal rabbit

    Techniques Used: Binding Assay, Immunoprecipitation

    Dose response and time course of [Ca 2+ ] i after Epo stimulation of HEK 293T cells transfected with TRPC3 and Epo-R. A , Epo dose response. HEK 293T cells transfected with TRPC3 and Epo-R were stimulated with 0–40 units/ml Epo. [Ca 2+ ] i was measured
    Figure Legend Snippet: Dose response and time course of [Ca 2+ ] i after Epo stimulation of HEK 293T cells transfected with TRPC3 and Epo-R. A , Epo dose response. HEK 293T cells transfected with TRPC3 and Epo-R were stimulated with 0–40 units/ml Epo. [Ca 2+ ] i was measured

    Techniques Used: Transfection

    Requirement for external calcium in the Epo-stimulated calcium increase in HEK 293T cells. Fura Red-loaded HEK 293T cells were transfected with BFP-TRPC3 and Epo-R. A , cells were treated with 40 units/ml Epo in the presence (0.68 m m ) or absence (2
    Figure Legend Snippet: Requirement for external calcium in the Epo-stimulated calcium increase in HEK 293T cells. Fura Red-loaded HEK 293T cells were transfected with BFP-TRPC3 and Epo-R. A , cells were treated with 40 units/ml Epo in the presence (0.68 m m ) or absence (2

    Techniques Used: Transfection

    Interaction of TRPC3 with PLCγ SH2 binding site substitutions with PLCγ. PLCγ and V5-TRPC3, V5-TRPC3-F4, or V5-TRPC3-Y226F were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-PLCγ
    Figure Legend Snippet: Interaction of TRPC3 with PLCγ SH2 binding site substitutions with PLCγ. PLCγ and V5-TRPC3, V5-TRPC3-F4, or V5-TRPC3-Y226F were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-PLCγ

    Techniques Used: Binding Assay, Immunoprecipitation

    Western blot of HEK 293T cells transfected with siRNA targeted to PLC γ. Lysates were prepared from HEK 293T cells transfected ( Tx ' d ) with or without BFP-TRPC3 and Epo-R, and siRNA was targeted to PLCγ or control siRNA. Blots were probed
    Figure Legend Snippet: Western blot of HEK 293T cells transfected with siRNA targeted to PLC γ. Lysates were prepared from HEK 293T cells transfected ( Tx ' d ) with or without BFP-TRPC3 and Epo-R, and siRNA was targeted to PLCγ or control siRNA. Blots were probed

    Techniques Used: Western Blot, Transfection, Planar Chromatography

    Endogenous expression of TRPC3 in human hematopoietic cells. Western blotting was performed on lysates from UT-7 and TF-1 Epo-responsive cell lines, from CD34 + cells and from day 10 and 14 BFU-E-derived erythroblasts. Equivalent amounts of protein
    Figure Legend Snippet: Endogenous expression of TRPC3 in human hematopoietic cells. Western blotting was performed on lysates from UT-7 and TF-1 Epo-responsive cell lines, from CD34 + cells and from day 10 and 14 BFU-E-derived erythroblasts. Equivalent amounts of protein

    Techniques Used: Expressing, Western Blot, Derivative Assay

    Plasma membrane externalization of TRPC3 detected by cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-DEL, V5-TRPC3-SUB, or V5-TRPC3-F4 and Epo-R. Lysates were prepared, and immunoprecipitation
    Figure Legend Snippet: Plasma membrane externalization of TRPC3 detected by cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-DEL, V5-TRPC3-SUB, or V5-TRPC3-F4 and Epo-R. Lysates were prepared, and immunoprecipitation

    Techniques Used: Expressing, Immunoprecipitation

    Association of TRPC3 and TRPC3-F4 with PLCγ or Epo-R. A , PLCγ and V5-TRPC3 or V5-TRPC3-F4 were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-PLCγ or anti-V5 antibodies or normal rabbit
    Figure Legend Snippet: Association of TRPC3 and TRPC3-F4 with PLCγ or Epo-R. A , PLCγ and V5-TRPC3 or V5-TRPC3-F4 were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-PLCγ or anti-V5 antibodies or normal rabbit

    Techniques Used: Immunoprecipitation

    23) Product Images from "Dysregulation of Ca2+ signaling in astrocytes from mice lacking amyloid precursor protein"

    Article Title: Dysregulation of Ca2+ signaling in astrocytes from mice lacking amyloid precursor protein

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00379.2010

    Expression of C-type transient receptor potential channels (TRPCs), STIM1, and Orai1 in primary cultured WT and APP KO astrocytes. A – L : Western blot analysis of TRPC1 ( A and B ), TRPC4 ( C and D ), TRPC5 ( E and F ), TRPC3 ( G and H ), STIM1 ( I and
    Figure Legend Snippet: Expression of C-type transient receptor potential channels (TRPCs), STIM1, and Orai1 in primary cultured WT and APP KO astrocytes. A – L : Western blot analysis of TRPC1 ( A and B ), TRPC4 ( C and D ), TRPC5 ( E and F ), TRPC3 ( G and H ), STIM1 ( I and

    Techniques Used: Expressing, Cell Culture, Western Blot

    24) Product Images from "Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein kinase G"

    Article Title: Regulation of canonical transient receptor potential isoform 3 (TRPC3) channel by protein kinase G

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

    doi: 10.1073/pnas.0304471101

    Effect of TRPC3 overexpression on store-operated Ca 2+ influx in PKG-HEK cells. ( A ) Immunoblots with an anti-PGK antibody showed that PKG-HEK cells, a HEK293 cell line stably transfected with PKG construct, expressed a much higher level of PKG compared
    Figure Legend Snippet: Effect of TRPC3 overexpression on store-operated Ca 2+ influx in PKG-HEK cells. ( A ) Immunoblots with an anti-PGK antibody showed that PKG-HEK cells, a HEK293 cell line stably transfected with PKG construct, expressed a much higher level of PKG compared

    Techniques Used: Over Expression, Western Blot, Stable Transfection, Transfection, Construct

    Effect of cGMP, KT5823 on TRPC3-mediated store-operated Ca 2+ influx in HEK293 cells without PKG transfection. 8-BrcGMP (2 mM) and KT5823 (1 μM) had no effect on the store-operated Ca 2+ influx in the HEK293 cells that were transiently transfected
    Figure Legend Snippet: Effect of cGMP, KT5823 on TRPC3-mediated store-operated Ca 2+ influx in HEK293 cells without PKG transfection. 8-BrcGMP (2 mM) and KT5823 (1 μM) had no effect on the store-operated Ca 2+ influx in the HEK293 cells that were transiently transfected

    Techniques Used: Transfection

    Effect of cGMP, KT5823, and H8 on TRPC3-mediated store-operated Ca 2+ influx in PKG-HEK cells. ( A ) Store-operated Ca 2+ influx in TRPC3-transfected PKG-HEK cells. Shown is the mean ± SE ( n = 14 experiments). ( B ) Store-operated Ca 2+ influx in PKG-HEK
    Figure Legend Snippet: Effect of cGMP, KT5823, and H8 on TRPC3-mediated store-operated Ca 2+ influx in PKG-HEK cells. ( A ) Store-operated Ca 2+ influx in TRPC3-transfected PKG-HEK cells. Shown is the mean ± SE ( n = 14 experiments). ( B ) Store-operated Ca 2+ influx in PKG-HEK

    Techniques Used: Transfection

    Direct PKG phosphorylation on TRPC3 proteins. ( A ) Immunoblot of TRPC3 proteins immunoprecipitated by an anti-TRPC3 (Alomone Laboratories). The antibody used for immunoblot was a second anti-TRPC3 antibody (A-15, Santa Cruz Biotechnology). ( B ) In vitro
    Figure Legend Snippet: Direct PKG phosphorylation on TRPC3 proteins. ( A ) Immunoblot of TRPC3 proteins immunoprecipitated by an anti-TRPC3 (Alomone Laboratories). The antibody used for immunoblot was a second anti-TRPC3 antibody (A-15, Santa Cruz Biotechnology). ( B ) In vitro

    Techniques Used: Immunoprecipitation, In Vitro

    25) 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

    Interaction of V5-TRPC3 and V5-TRPC3-C6TRP with Epo-R and PLCγ. A , HEK 293T cells were transfected ( Tx'd ) with Epo-R and V5-TRPC3 or V5-TRPC3-C6TRP. Lysates were immunoprecipitated ( IP ) with anti-V5, anti-Epo-R antibodies, or normal rabbit serum ( NS ). Western blots ( WB ) of lysates and immunoprecipitates were probed with anti-V5-HRP or anti-Epo-R and appropriate secondary antibodies. Western blotting of lysates demonstrated equivalent input from each construct. A representative result of six experiments is shown. Band intensities in A were quantitated with densitometry, and the mean ± S.E. ( error bars ) ratio of Epo-R/channel, normalized to wild type TRPC3, from six experiments is shown. There was no significant difference in interaction of Epo-R with TRPC3 compared with TRPC3-C6TRP. B , HEK 293T cells were transfected with PLCγ and V5-TRPC3 or V5-TRPC3-C6TRP. Lysates were immunoprecipitated with anti-V5, anti-PLCγ antibodies, or normal rabbit serum ( NS ). Western blots of lysates and immunoprecipitates were probed with anti-V5-HRP or anti-PLCγ and appropriate secondary antibodies. A representative result of four experiments is shown. Band intensities in B were quantitated with densitometry, and mean ± S.E. ratio of PLCγ/channel, normalized to TRPC3, from four experiments is shown. *, significant difference in the ratio compared with V5-TRPC3 ( p
    Figure Legend Snippet: Interaction of V5-TRPC3 and V5-TRPC3-C6TRP with Epo-R and PLCγ. A , HEK 293T cells were transfected ( Tx'd ) with Epo-R and V5-TRPC3 or V5-TRPC3-C6TRP. Lysates were immunoprecipitated ( IP ) with anti-V5, anti-Epo-R antibodies, or normal rabbit serum ( NS ). Western blots ( WB ) of lysates and immunoprecipitates were probed with anti-V5-HRP or anti-Epo-R and appropriate secondary antibodies. Western blotting of lysates demonstrated equivalent input from each construct. A representative result of six experiments is shown. Band intensities in A were quantitated with densitometry, and the mean ± S.E. ( error bars ) ratio of Epo-R/channel, normalized to wild type TRPC3, from six experiments is shown. There was no significant difference in interaction of Epo-R with TRPC3 compared with TRPC3-C6TRP. B , HEK 293T cells were transfected with PLCγ and V5-TRPC3 or V5-TRPC3-C6TRP. Lysates were immunoprecipitated with anti-V5, anti-PLCγ antibodies, or normal rabbit serum ( NS ). Western blots of lysates and immunoprecipitates were probed with anti-V5-HRP or anti-PLCγ and appropriate secondary antibodies. A representative result of four experiments is shown. Band intensities in B were quantitated with densitometry, and mean ± S.E. ratio of PLCγ/channel, normalized to TRPC3, from four experiments is shown. *, significant difference in the ratio compared with V5-TRPC3 ( p

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Construct

    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

    Interaction of FLAG-TRPC3 and FLAG-TRPC3-C6TRP with Epo-R and PLCγ. A , HEK 293T cells were transfected ( Tx'd ) with Epo-R and FLAG-TRPC3 or FLAG-TRPC3-C6TRP. Lysates were immunoprecipitated ( IP ) with anti-FLAG-agarose. Western blots ( WB ) of lysates and immunoprecipitates were probed with anti-FLAG or anti-Epo-R and appropriate secondary antibodies. A representative result of seven experiments is shown. B , HEK 293T cells were transfected with PLCγ and FLAG-TRPC3 or FLAG-TRPC3-C6TRP. Lysates were immunoprecipitated with anti-FLAG-agarose. Western blots of lysates and immunoprecipitates were probed with anti-FLAG or anti-PLCγ and appropriate secondary antibodies. A representative result of three experiments is shown. C and D , intensity of bands was quantitated with densitometry, and the mean ± S.E. ( error bars ) ratio of Epo-R/channel ( C ) or PLCγ/channel ( D ), normalized to FLAG-TRPC3, for seven or three experiments, respectively, is shown. *, significant difference in the ratio compared with TRPC3 ( p
    Figure Legend Snippet: Interaction of FLAG-TRPC3 and FLAG-TRPC3-C6TRP with Epo-R and PLCγ. A , HEK 293T cells were transfected ( Tx'd ) with Epo-R and FLAG-TRPC3 or FLAG-TRPC3-C6TRP. Lysates were immunoprecipitated ( IP ) with anti-FLAG-agarose. Western blots ( WB ) of lysates and immunoprecipitates were probed with anti-FLAG or anti-Epo-R and appropriate secondary antibodies. A representative result of seven experiments is shown. B , HEK 293T cells were transfected with PLCγ and FLAG-TRPC3 or FLAG-TRPC3-C6TRP. Lysates were immunoprecipitated with anti-FLAG-agarose. Western blots of lysates and immunoprecipitates were probed with anti-FLAG or anti-PLCγ and appropriate secondary antibodies. A representative result of three experiments is shown. C and D , intensity of bands was quantitated with densitometry, and the mean ± S.E. ( error bars ) ratio of Epo-R/channel ( C ) or PLCγ/channel ( D ), normalized to FLAG-TRPC3, for seven or three experiments, respectively, is shown. *, significant difference in the ratio compared with TRPC3 ( p

    Techniques Used: Transfection, Immunoprecipitation, Western Blot

    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

    26) Product Images from "Canonical Transient Receptor Potential Channel (TRPC) 3 and TRPC6 Associate with Large-Conductance Ca2+-Activated K+ (BKCa) Channels: Role in BKCa Trafficking to the Surface of Cultured Podocytes"

    Article Title: Canonical Transient Receptor Potential Channel (TRPC) 3 and TRPC6 Associate with Large-Conductance Ca2+-Activated K+ (BKCa) Channels: Role in BKCa Trafficking to the Surface of Cultured Podocytes

    Journal:

    doi: 10.1124/mol.108.051912

    Biochemical interaction between TRPC3 and BK Ca channels. A, coimmunoprecipitation of TRPC3 and Slo1 channels in podocyte lysates. Slo1 channels can be detected in immunoprecipitates prepared using an antibody against TRPC6 (left), and TRPC6 channels
    Figure Legend Snippet: Biochemical interaction between TRPC3 and BK Ca channels. A, coimmunoprecipitation of TRPC3 and Slo1 channels in podocyte lysates. Slo1 channels can be detected in immunoprecipitates prepared using an antibody against TRPC6 (left), and TRPC6 channels

    Techniques Used:

    Coexpression of TRPC3 does not affect the surface expression of myc-tagged Slo1 VEDEC channels in HEK293T cells. A, cell-surface biotinylation assays show no effect on surface expression of myc-tagged Slo1 VEDEC in HEK293T cells coexpressing TRPC3. B,
    Figure Legend Snippet: Coexpression of TRPC3 does not affect the surface expression of myc-tagged Slo1 VEDEC channels in HEK293T cells. A, cell-surface biotinylation assays show no effect on surface expression of myc-tagged Slo1 VEDEC in HEK293T cells coexpressing TRPC3. B,

    Techniques Used: Expressing

    Coexpression of TRPC3 does not affect the number of functional cell surface BK Ca channels in HEK293T cells expressing Slo1 VEDEC . A, typical families of voltage-evoked outward currents observed in whole-cell recordings using recording electrodes containing
    Figure Legend Snippet: Coexpression of TRPC3 does not affect the number of functional cell surface BK Ca channels in HEK293T cells expressing Slo1 VEDEC . A, typical families of voltage-evoked outward currents observed in whole-cell recordings using recording electrodes containing

    Techniques Used: Functional Assay, Expressing

    Colocalization of TRPC3 and Slo1 channels. Confocal analyses of native channels in podocytes (A) and of HEK293T cells podocytes. There is less colocalization in HEK293T cells, as signal from TRPC3 channels is intense on the cell periphery, where signal
    Figure Legend Snippet: Colocalization of TRPC3 and Slo1 channels. Confocal analyses of native channels in podocytes (A) and of HEK293T cells podocytes. There is less colocalization in HEK293T cells, as signal from TRPC3 channels is intense on the cell periphery, where signal

    Techniques Used:

    Application of specific siRNAs cause knockdown of TRPC6 and TRPC3 channels in podocytes. Control cells were treated with a nonspecific siRNA. A, effectiveness of knockdown was demonstrated by immunoblot analysis and densitometric analysis using antibodies
    Figure Legend Snippet: Application of specific siRNAs cause knockdown of TRPC6 and TRPC3 channels in podocytes. Control cells were treated with a nonspecific siRNA. A, effectiveness of knockdown was demonstrated by immunoblot analysis and densitometric analysis using antibodies

    Techniques Used:

    Knockdown of TRPC6 reduces surface expression of endogenous Slo1 channels in podocytes. Cell-surface biotinylation assays show a reduction in surface expression of Slo1 48 h after siRNA knockdown of TRPC6 (A) but not after knockdown of TRPC3 (B). Bar
    Figure Legend Snippet: Knockdown of TRPC6 reduces surface expression of endogenous Slo1 channels in podocytes. Cell-surface biotinylation assays show a reduction in surface expression of Slo1 48 h after siRNA knockdown of TRPC6 (A) but not after knockdown of TRPC3 (B). Bar

    Techniques Used: Expressing

    27) Product Images from "Transient Receptor Potential Channel Activation Causes a Novel Form of [Ca 2+]i Oscillations and Is Not Involved in Capacitative Ca 2+ Entry in Glial Cells"

    Article Title: Transient Receptor Potential Channel Activation Causes a Novel Form of [Ca 2+]i Oscillations and Is Not Involved in Capacitative Ca 2+ Entry in Glial Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.23-11-04737.2003

    Effect of anti-TRPC3 antisense oligonucleotides on OAG-induced [Ca 2 + ] i oscillations. Astrocytes were treated for 36 hr with vehicle, and 100 μg/ml TRPC3 sense or antisense oligonucleotides were loaded with fura-2 and then exposed to 100 μ
    Figure Legend Snippet: Effect of anti-TRPC3 antisense oligonucleotides on OAG-induced [Ca 2 + ] i oscillations. Astrocytes were treated for 36 hr with vehicle, and 100 μg/ml TRPC3 sense or antisense oligonucleotides were loaded with fura-2 and then exposed to 100 μ

    Techniques Used:

    28) 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:

    FRAP. A, localization of GFP-TRPC3 in HEK 293T cells transfected with GFP-TRPC3 and Epo-R examined with confocal microscopy and differential interference contrast ( DIC ). B, representative FRAP experiment in HEK 293T expressing Epo-R and GFP-TRPC3. Upper panels show untreated cells before photobleaching and then at 10, 40, and 80 s after photobleaching. Lower panels show recovery of cells photobleached in the presence of Epo (40 units/ml). Area of membrane bleached is shown in prominent white lines. C, schema showing calculation of half-time ( t ½ ) and the mobile fraction. When the region is bleached, fluorescence decreases from F i , the initial fluorescence value, to F o at time t o immediately after photobleaching. Fluorescence recovers over time until it reaches plateau value F p . t ½ is the time to recovery of half of plateau fluorescence ( F p ). a = the immobile fraction, b = the mobile fraction, calculated from F i i , F p , and F o . D, graph from a representative FRAP experiment showing relative fluorescence recovery after photobleaching (IgorPro analysis). E , t ½ (in seconds) of fluorescence recovery with or without Epo stimulation ( n = number of cells). F, mobile fraction with and without Epo stimulation ( n = number of cells). Mean ± S.E. calculated using one-way analysis of variance ( * , p ≤ 0.001).
    Figure Legend Snippet: FRAP. A, localization of GFP-TRPC3 in HEK 293T cells transfected with GFP-TRPC3 and Epo-R examined with confocal microscopy and differential interference contrast ( DIC ). B, representative FRAP experiment in HEK 293T expressing Epo-R and GFP-TRPC3. Upper panels show untreated cells before photobleaching and then at 10, 40, and 80 s after photobleaching. Lower panels show recovery of cells photobleached in the presence of Epo (40 units/ml). Area of membrane bleached is shown in prominent white lines. C, schema showing calculation of half-time ( t ½ ) and the mobile fraction. When the region is bleached, fluorescence decreases from F i , the initial fluorescence value, to F o at time t o immediately after photobleaching. Fluorescence recovers over time until it reaches plateau value F p . t ½ is the time to recovery of half of plateau fluorescence ( F p ). a = the immobile fraction, b = the mobile fraction, calculated from F i i , F p , and F o . D, graph from a representative FRAP experiment showing relative fluorescence recovery after photobleaching (IgorPro analysis). E , t ½ (in seconds) of fluorescence recovery with or without Epo stimulation ( n = number of cells). F, mobile fraction with and without Epo stimulation ( n = number of cells). Mean ± S.E. calculated using one-way analysis of variance ( * , p ≤ 0.001).

    Techniques Used: Transfection, Confocal Microscopy, Expressing, Fluorescence

    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

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    Alomone Labs anti trpc3
    Schematic model of <t>TRPC3</t> transmembrane domains and protein binding sites. Predicted transmembrane domains, calcium entry pore, IP 3 R, and PLCγ SH2 binding sites and deleted and substituted sites in TRPC3-DEL and TRPC3-SUB are shown. aa , amino
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    Schematic model of TRPC3 transmembrane domains and protein binding sites. Predicted transmembrane domains, calcium entry pore, IP 3 R, and PLCγ SH2 binding sites and deleted and substituted sites in TRPC3-DEL and TRPC3-SUB are shown. aa , amino

    Journal: The Journal of Biological Chemistry

    Article Title: TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *

    doi: 10.1074/jbc.M710231200

    Figure Lengend Snippet: Schematic model of TRPC3 transmembrane domains and protein binding sites. Predicted transmembrane domains, calcium entry pore, IP 3 R, and PLCγ SH2 binding sites and deleted and substituted sites in TRPC3-DEL and TRPC3-SUB are shown. aa , amino

    Article Snippet: Sample buffer (3×) was added to the pellets, and the samples were heated at 60 °C for 30 min. Western blotting was performed as described above, and blots were probed with anti-V5-HRP or anti-Epo-R, anti-PLCγ1, anti-IP3 R type II, anti-TRPC3, or anti-actin antibodies, followed by the appropriate HRP-conjugated secondary antibodies and ECL.

    Techniques: Protein Binding, Binding Assay

    Association of TRPC3 IP 3 R binding mutants with IP 3 R. IP 3 R type II and V5-TRPC3, V5-TRPC3-DEL, or V5-TRPC3-SUB were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-V5 or anti-IP 3 R antibodies or normal rabbit

    Journal: The Journal of Biological Chemistry

    Article Title: TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *

    doi: 10.1074/jbc.M710231200

    Figure Lengend Snippet: Association of TRPC3 IP 3 R binding mutants with IP 3 R. IP 3 R type II and V5-TRPC3, V5-TRPC3-DEL, or V5-TRPC3-SUB were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-V5 or anti-IP 3 R antibodies or normal rabbit

    Article Snippet: Sample buffer (3×) was added to the pellets, and the samples were heated at 60 °C for 30 min. Western blotting was performed as described above, and blots were probed with anti-V5-HRP or anti-Epo-R, anti-PLCγ1, anti-IP3 R type II, anti-TRPC3, or anti-actin antibodies, followed by the appropriate HRP-conjugated secondary antibodies and ECL.

    Techniques: Binding Assay, Immunoprecipitation

    Dose response and time course of [Ca 2+ ] i after Epo stimulation of HEK 293T cells transfected with TRPC3 and Epo-R. A , Epo dose response. HEK 293T cells transfected with TRPC3 and Epo-R were stimulated with 0–40 units/ml Epo. [Ca 2+ ] i was measured

    Journal: The Journal of Biological Chemistry

    Article Title: TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *

    doi: 10.1074/jbc.M710231200

    Figure Lengend Snippet: Dose response and time course of [Ca 2+ ] i after Epo stimulation of HEK 293T cells transfected with TRPC3 and Epo-R. A , Epo dose response. HEK 293T cells transfected with TRPC3 and Epo-R were stimulated with 0–40 units/ml Epo. [Ca 2+ ] i was measured

    Article Snippet: Sample buffer (3×) was added to the pellets, and the samples were heated at 60 °C for 30 min. Western blotting was performed as described above, and blots were probed with anti-V5-HRP or anti-Epo-R, anti-PLCγ1, anti-IP3 R type II, anti-TRPC3, or anti-actin antibodies, followed by the appropriate HRP-conjugated secondary antibodies and ECL.

    Techniques: Transfection

    Requirement for external calcium in the Epo-stimulated calcium increase in HEK 293T cells. Fura Red-loaded HEK 293T cells were transfected with BFP-TRPC3 and Epo-R. A , cells were treated with 40 units/ml Epo in the presence (0.68 m m ) or absence (2

    Journal: The Journal of Biological Chemistry

    Article Title: TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *

    doi: 10.1074/jbc.M710231200

    Figure Lengend Snippet: Requirement for external calcium in the Epo-stimulated calcium increase in HEK 293T cells. Fura Red-loaded HEK 293T cells were transfected with BFP-TRPC3 and Epo-R. A , cells were treated with 40 units/ml Epo in the presence (0.68 m m ) or absence (2

    Article Snippet: Sample buffer (3×) was added to the pellets, and the samples were heated at 60 °C for 30 min. Western blotting was performed as described above, and blots were probed with anti-V5-HRP or anti-Epo-R, anti-PLCγ1, anti-IP3 R type II, anti-TRPC3, or anti-actin antibodies, followed by the appropriate HRP-conjugated secondary antibodies and ECL.

    Techniques: Transfection

    Interaction of TRPC3 with PLCγ SH2 binding site substitutions with PLCγ. PLCγ and V5-TRPC3, V5-TRPC3-F4, or V5-TRPC3-Y226F were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-PLCγ

    Journal: The Journal of Biological Chemistry

    Article Title: TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *

    doi: 10.1074/jbc.M710231200

    Figure Lengend Snippet: Interaction of TRPC3 with PLCγ SH2 binding site substitutions with PLCγ. PLCγ and V5-TRPC3, V5-TRPC3-F4, or V5-TRPC3-Y226F were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-PLCγ

    Article Snippet: Sample buffer (3×) was added to the pellets, and the samples were heated at 60 °C for 30 min. Western blotting was performed as described above, and blots were probed with anti-V5-HRP or anti-Epo-R, anti-PLCγ1, anti-IP3 R type II, anti-TRPC3, or anti-actin antibodies, followed by the appropriate HRP-conjugated secondary antibodies and ECL.

    Techniques: Binding Assay, Immunoprecipitation

    Western blot of HEK 293T cells transfected with siRNA targeted to PLC γ. Lysates were prepared from HEK 293T cells transfected ( Tx ' d ) with or without BFP-TRPC3 and Epo-R, and siRNA was targeted to PLCγ or control siRNA. Blots were probed

    Journal: The Journal of Biological Chemistry

    Article Title: TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *

    doi: 10.1074/jbc.M710231200

    Figure Lengend Snippet: Western blot of HEK 293T cells transfected with siRNA targeted to PLC γ. Lysates were prepared from HEK 293T cells transfected ( Tx ' d ) with or without BFP-TRPC3 and Epo-R, and siRNA was targeted to PLCγ or control siRNA. Blots were probed

    Article Snippet: Sample buffer (3×) was added to the pellets, and the samples were heated at 60 °C for 30 min. Western blotting was performed as described above, and blots were probed with anti-V5-HRP or anti-Epo-R, anti-PLCγ1, anti-IP3 R type II, anti-TRPC3, or anti-actin antibodies, followed by the appropriate HRP-conjugated secondary antibodies and ECL.

    Techniques: Western Blot, Transfection, Planar Chromatography

    Endogenous expression of TRPC3 in human hematopoietic cells. Western blotting was performed on lysates from UT-7 and TF-1 Epo-responsive cell lines, from CD34 + cells and from day 10 and 14 BFU-E-derived erythroblasts. Equivalent amounts of protein

    Journal: The Journal of Biological Chemistry

    Article Title: TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *

    doi: 10.1074/jbc.M710231200

    Figure Lengend Snippet: Endogenous expression of TRPC3 in human hematopoietic cells. Western blotting was performed on lysates from UT-7 and TF-1 Epo-responsive cell lines, from CD34 + cells and from day 10 and 14 BFU-E-derived erythroblasts. Equivalent amounts of protein

    Article Snippet: Sample buffer (3×) was added to the pellets, and the samples were heated at 60 °C for 30 min. Western blotting was performed as described above, and blots were probed with anti-V5-HRP or anti-Epo-R, anti-PLCγ1, anti-IP3 R type II, anti-TRPC3, or anti-actin antibodies, followed by the appropriate HRP-conjugated secondary antibodies and ECL.

    Techniques: Expressing, Western Blot, Derivative Assay

    Plasma membrane externalization of TRPC3 detected by cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-DEL, V5-TRPC3-SUB, or V5-TRPC3-F4 and Epo-R. Lysates were prepared, and immunoprecipitation

    Journal: The Journal of Biological Chemistry

    Article Title: TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *

    doi: 10.1074/jbc.M710231200

    Figure Lengend Snippet: Plasma membrane externalization of TRPC3 detected by cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-DEL, V5-TRPC3-SUB, or V5-TRPC3-F4 and Epo-R. Lysates were prepared, and immunoprecipitation

    Article Snippet: Sample buffer (3×) was added to the pellets, and the samples were heated at 60 °C for 30 min. Western blotting was performed as described above, and blots were probed with anti-V5-HRP or anti-Epo-R, anti-PLCγ1, anti-IP3 R type II, anti-TRPC3, or anti-actin antibodies, followed by the appropriate HRP-conjugated secondary antibodies and ECL.

    Techniques: Expressing, Immunoprecipitation

    Association of TRPC3 and TRPC3-F4 with PLCγ or Epo-R. A , PLCγ and V5-TRPC3 or V5-TRPC3-F4 were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-PLCγ or anti-V5 antibodies or normal rabbit

    Journal: The Journal of Biological Chemistry

    Article Title: TRPC3 Is the Erythropoietin-regulated Calcium Channel in Human Erythroid Cells *

    doi: 10.1074/jbc.M710231200

    Figure Lengend Snippet: Association of TRPC3 and TRPC3-F4 with PLCγ or Epo-R. A , PLCγ and V5-TRPC3 or V5-TRPC3-F4 were expressed in HEK 293T cells. Immunoprecipitation ( IP ) was performed on lysates with anti-PLCγ or anti-V5 antibodies or normal rabbit

    Article Snippet: Sample buffer (3×) was added to the pellets, and the samples were heated at 60 °C for 30 min. Western blotting was performed as described above, and blots were probed with anti-V5-HRP or anti-Epo-R, anti-PLCγ1, anti-IP3 R type II, anti-TRPC3, or anti-actin antibodies, followed by the appropriate HRP-conjugated secondary antibodies and ECL.

    Techniques: Immunoprecipitation

    A reduction in Ca 2+ transients in response to membrane depolarization, and the disruption of the binding between endogenous MG29 and TRPC3 in mouse primary skeletal myotubes expressing Δ116-MG29

    Journal: Biochemical and biophysical research communications

    Article Title: Interaction between mitsugumin 29 and TRPC3 participates in regulating Ca2+ transients in skeletal muscle

    doi: 10.1016/j.bbrc.2015.06.096

    Figure Lengend Snippet: A reduction in Ca 2+ transients in response to membrane depolarization, and the disruption of the binding between endogenous MG29 and TRPC3 in mouse primary skeletal myotubes expressing Δ116-MG29

    Article Snippet: For immunoblot assays, various antibodies were used: anti-RyR1, anti-DHPR, anti-SERCA1a, anti-MG29, anti-JP1, anti-JP2, and anti-GST antibodies (1:1,000) from Thermo Scientific Inc. (Rockford, IL, USA), anti-TRPC3 and anti-TRPC4 antibodies (1:800) from Alomone Laboratories (Jerusalem 9104201, Israel), and anti-Orai1, anti-STIM1, and anti-α-actin antibodies (1:1,000) from Abcam (Cambridge, MA, USA).

    Techniques: Binding Assay, Expressing

    Co-immunoprecipitation of TRPC3 with each MG29 portion

    Journal: Biochemical and biophysical research communications

    Article Title: Interaction between mitsugumin 29 and TRPC3 participates in regulating Ca2+ transients in skeletal muscle

    doi: 10.1016/j.bbrc.2015.06.096

    Figure Lengend Snippet: Co-immunoprecipitation of TRPC3 with each MG29 portion

    Article Snippet: For immunoblot assays, various antibodies were used: anti-RyR1, anti-DHPR, anti-SERCA1a, anti-MG29, anti-JP1, anti-JP2, and anti-GST antibodies (1:1,000) from Thermo Scientific Inc. (Rockford, IL, USA), anti-TRPC3 and anti-TRPC4 antibodies (1:800) from Alomone Laboratories (Jerusalem 9104201, Israel), and anti-Orai1, anti-STIM1, and anti-α-actin antibodies (1:1,000) from Abcam (Cambridge, MA, USA).

    Techniques: Immunoprecipitation

    TRPC3 forms a stable ternary complex with Nox2 and p22 phox . ( a , b ) Expression of Nox2 and p22 phox proteins in HEK293 cells that express a different combination of TRPC3-GFP and GFP. Results of a quantitative analysis are shown in ( b ) (n = 3). ( c ) Nox2 mRNA amounts in HEK293 cells co-expressing Nox2 with GFP or TRPC3-GFP (n = 3). ( d ) Increased Nox2 and p22 phox protein in HEK293 cells co-expressing pore-dead mutant of TRPC3 (n = 3). ( e ) Interaction of TRPC3 with Nox2 in HEK293 cells. Immunoprecipitation was performed using an anti-flag antibody. ( f ) Nox2 protein expression in HEK293 cells expressing TRPC3 alone or co-expressing TRPC3 and TRPC6 (n = 3). Error bars, s.e.m. *P

    Journal: Scientific Reports

    Article Title: TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling

    doi: 10.1038/srep37001

    Figure Lengend Snippet: TRPC3 forms a stable ternary complex with Nox2 and p22 phox . ( a , b ) Expression of Nox2 and p22 phox proteins in HEK293 cells that express a different combination of TRPC3-GFP and GFP. Results of a quantitative analysis are shown in ( b ) (n = 3). ( c ) Nox2 mRNA amounts in HEK293 cells co-expressing Nox2 with GFP or TRPC3-GFP (n = 3). ( d ) Increased Nox2 and p22 phox protein in HEK293 cells co-expressing pore-dead mutant of TRPC3 (n = 3). ( e ) Interaction of TRPC3 with Nox2 in HEK293 cells. Immunoprecipitation was performed using an anti-flag antibody. ( f ) Nox2 protein expression in HEK293 cells expressing TRPC3 alone or co-expressing TRPC3 and TRPC6 (n = 3). Error bars, s.e.m. *P

    Article Snippet: The following primary antibodies were used: GAPDH (sc-25778), gp91phox (sc-130543), p47phox (sc-17845) and p22phox (sc-20781) from Santa Cruz Biotechnology, flag M2-HRP (A8592) from Sigma Aldrich, GFP (CHIP grade, ab290) from Abcam, myc-tag (05–742) from Merck, phospho p47phox (p-Ser370) (A1171) from Assay Bio Tech, and TRPC3 (ACC-016) from Alomone Labs.

    Techniques: Expressing, Mutagenesis, Immunoprecipitation

    TRPC3 plays a critical role in Mechanical stretch-induced ROS production. ( a , b ) Effects of siRNA targeting TRPC1, C3 or C6 on mechanical stretch (MS)-induced ROS production (n = 3). ( c ) mRNA expression of either TRPC1 or TRPC6 in NRCM transfected with siRNAs against either TRPC1 or TRPC6, respectively (n = 3). ( d,e ) Time courses of MS-induced ROS production in NRCMs treated with GsMTx-4 (1 μM; ( d ) or TRPV4 inhibitor (RN1734, 50 μM; ( e ) Reagents were added to cells 5 min before MS (n = 3). ( f ) MS-induced ROS production in TRPC(1–7)-deficient MEF cells expressing TRPC3, TRPC6, TRPC7, or LacZ (n = 30). Data are representative of three independent experiments. ( g–i ) Effect of TRPC3 siRNA on the protein abundances of TRPC3 ( h ) and Nox2 ( i ) protein expressions in NRCMs (n = 3). Error bars, s.e.m. *P

    Journal: Scientific Reports

    Article Title: TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling

    doi: 10.1038/srep37001

    Figure Lengend Snippet: TRPC3 plays a critical role in Mechanical stretch-induced ROS production. ( a , b ) Effects of siRNA targeting TRPC1, C3 or C6 on mechanical stretch (MS)-induced ROS production (n = 3). ( c ) mRNA expression of either TRPC1 or TRPC6 in NRCM transfected with siRNAs against either TRPC1 or TRPC6, respectively (n = 3). ( d,e ) Time courses of MS-induced ROS production in NRCMs treated with GsMTx-4 (1 μM; ( d ) or TRPV4 inhibitor (RN1734, 50 μM; ( e ) Reagents were added to cells 5 min before MS (n = 3). ( f ) MS-induced ROS production in TRPC(1–7)-deficient MEF cells expressing TRPC3, TRPC6, TRPC7, or LacZ (n = 30). Data are representative of three independent experiments. ( g–i ) Effect of TRPC3 siRNA on the protein abundances of TRPC3 ( h ) and Nox2 ( i ) protein expressions in NRCMs (n = 3). Error bars, s.e.m. *P

    Article Snippet: The following primary antibodies were used: GAPDH (sc-25778), gp91phox (sc-130543), p47phox (sc-17845) and p22phox (sc-20781) from Santa Cruz Biotechnology, flag M2-HRP (A8592) from Sigma Aldrich, GFP (CHIP grade, ab290) from Abcam, myc-tag (05–742) from Merck, phospho p47phox (p-Ser370) (A1171) from Assay Bio Tech, and TRPC3 (ACC-016) from Alomone Labs.

    Techniques: Mass Spectrometry, Expressing, Transfection

    TRPC3 prevents Nox2 protein from proteasomal degradation. ( a–e ) Abundances of Nox2 protein ( a , b ) and mRNAs of TRPC3 ( c ), Nox2 ( d ), and p22 phox ( e ) in NRCM transfected with siRNAs targeting TRPC3 with or without MG132. Cells were treated with siRNAs and MG132 (1 μM) simultaneously (n = 3). ( f , g ) Effect of siRNA targeting TRPC3 on Nox2 protein abundance in cell surface (Surface) and total lysates (Total) from NRCMs (n = 3). GAPDH was used as an internal control. Error bars, s.e.m. *P

    Journal: Scientific Reports

    Article Title: TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling

    doi: 10.1038/srep37001

    Figure Lengend Snippet: TRPC3 prevents Nox2 protein from proteasomal degradation. ( a–e ) Abundances of Nox2 protein ( a , b ) and mRNAs of TRPC3 ( c ), Nox2 ( d ), and p22 phox ( e ) in NRCM transfected with siRNAs targeting TRPC3 with or without MG132. Cells were treated with siRNAs and MG132 (1 μM) simultaneously (n = 3). ( f , g ) Effect of siRNA targeting TRPC3 on Nox2 protein abundance in cell surface (Surface) and total lysates (Total) from NRCMs (n = 3). GAPDH was used as an internal control. Error bars, s.e.m. *P

    Article Snippet: The following primary antibodies were used: GAPDH (sc-25778), gp91phox (sc-130543), p47phox (sc-17845) and p22phox (sc-20781) from Santa Cruz Biotechnology, flag M2-HRP (A8592) from Sigma Aldrich, GFP (CHIP grade, ab290) from Abcam, myc-tag (05–742) from Merck, phospho p47phox (p-Ser370) (A1171) from Assay Bio Tech, and TRPC3 (ACC-016) from Alomone Labs.

    Techniques: Transfection

    TRPC3 forms a stable ternary complex with Nox2 and p22 phox proteins in endogenously p22 phox -absent CHO cells. ( a ) Expression of Nox2 and p22 phox proteins in CHO cells that express a different combination of TRPC3-GFP and GFP. ( b ) Results of quantitative analysis (n = 3). ( c ) Expression of Nox2 and p22 phox co-expressed with either GFP or TRPC3-GFP in MG132 (10 μM)-treated CHO cells. ( d ) Graphs depict the relative expression of either Nox2 or p22 phox protein to that in non-treated cells. Band intensities were normalized by GAPDH. ( e–g ) Interaction of TRPC3 with p22 phox and Nox2 in CHO cells. ( h ) Localization of Nox2 in CHO cells co-expressing Nox2 with TRPC3-GFP (or GFP-F). Error bars, s.e.m. *P

    Journal: Scientific Reports

    Article Title: TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling

    doi: 10.1038/srep37001

    Figure Lengend Snippet: TRPC3 forms a stable ternary complex with Nox2 and p22 phox proteins in endogenously p22 phox -absent CHO cells. ( a ) Expression of Nox2 and p22 phox proteins in CHO cells that express a different combination of TRPC3-GFP and GFP. ( b ) Results of quantitative analysis (n = 3). ( c ) Expression of Nox2 and p22 phox co-expressed with either GFP or TRPC3-GFP in MG132 (10 μM)-treated CHO cells. ( d ) Graphs depict the relative expression of either Nox2 or p22 phox protein to that in non-treated cells. Band intensities were normalized by GAPDH. ( e–g ) Interaction of TRPC3 with p22 phox and Nox2 in CHO cells. ( h ) Localization of Nox2 in CHO cells co-expressing Nox2 with TRPC3-GFP (or GFP-F). Error bars, s.e.m. *P

    Article Snippet: The following primary antibodies were used: GAPDH (sc-25778), gp91phox (sc-130543), p47phox (sc-17845) and p22phox (sc-20781) from Santa Cruz Biotechnology, flag M2-HRP (A8592) from Sigma Aldrich, GFP (CHIP grade, ab290) from Abcam, myc-tag (05–742) from Merck, phospho p47phox (p-Ser370) (A1171) from Assay Bio Tech, and TRPC3 (ACC-016) from Alomone Labs.

    Techniques: Expressing

    Formation of a TRPC3/Nox2 complex promotes TRPC3 channel activity through stabilization at the plasma membrane. ( a ) Effect of Nox2 siRNA on expression of TRPC3 in NRCMs (n = 3). ( b ) Representative images showing the levels of TRPC3-GFP and GFP expression in HEK293 cells co-expressing p22 phox or Nox2 (n = 3). ( c ) Expression of TRPC3-GFP mRNA in HEK293 cells co-expressing p22 phox or Nox2 (n = 3). ( d–f ) Representative time courses of TRPC3 currents ( d ) and the current-voltage (I-V) relationships ( e ) and peak TRPC3 current densities at −60 mV ( f ) induced by 100 μM carbachol (CCh) in HEK293 cells expressing TRPC3-mCherry alone or with p22 phox , Nox2, both p22 phox and Nox2, or Nox2 treated with DPI. DPI (0.3 μM) was treated 1 min before CCh stimulation. ( g , h ) Representative Ca 2+ responses in the presence ( g ) or absence ( h ) of pyrazole-3 (Pyr3, 1 μM) upon mechanical stretch (MS) application. ( i ) Peak Ca 2+ increases after MS in NRCMs treated with (n = 61) or without Pyr3 (n = 78). ( j ) Changes of minimal [Ca 2+ ] i before and after MS application. Minimal [Ca 2+ ] i from Ca 2+ responses in every 1 min were analyzed and represented as diastolic [Ca 2+ ] i . ( k ) Schematic images showing phosphorylation of p47 phox via TRPC3-PKCβ activation induced by MS in the heart. ( l–n ) Effects of TRPC3 ( l , m ) or PKCβ ( n) ; 10 μM Gö6976) inhibitors on p47 phox phosphorylation induced by MS in NRCMs (n = 3). ( o ) MS-induced ROS generation in NRCMs treated with a PKCβ inhibitor (n = 3). ( p ) Co-immunoprecipitation of TRPC3 with PKCβ, Nox2 and p22 phox in mouse hearts 1week after TAC operation (n = 3). Error bars, s.e.m.*P

    Journal: Scientific Reports

    Article Title: TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling

    doi: 10.1038/srep37001

    Figure Lengend Snippet: Formation of a TRPC3/Nox2 complex promotes TRPC3 channel activity through stabilization at the plasma membrane. ( a ) Effect of Nox2 siRNA on expression of TRPC3 in NRCMs (n = 3). ( b ) Representative images showing the levels of TRPC3-GFP and GFP expression in HEK293 cells co-expressing p22 phox or Nox2 (n = 3). ( c ) Expression of TRPC3-GFP mRNA in HEK293 cells co-expressing p22 phox or Nox2 (n = 3). ( d–f ) Representative time courses of TRPC3 currents ( d ) and the current-voltage (I-V) relationships ( e ) and peak TRPC3 current densities at −60 mV ( f ) induced by 100 μM carbachol (CCh) in HEK293 cells expressing TRPC3-mCherry alone or with p22 phox , Nox2, both p22 phox and Nox2, or Nox2 treated with DPI. DPI (0.3 μM) was treated 1 min before CCh stimulation. ( g , h ) Representative Ca 2+ responses in the presence ( g ) or absence ( h ) of pyrazole-3 (Pyr3, 1 μM) upon mechanical stretch (MS) application. ( i ) Peak Ca 2+ increases after MS in NRCMs treated with (n = 61) or without Pyr3 (n = 78). ( j ) Changes of minimal [Ca 2+ ] i before and after MS application. Minimal [Ca 2+ ] i from Ca 2+ responses in every 1 min were analyzed and represented as diastolic [Ca 2+ ] i . ( k ) Schematic images showing phosphorylation of p47 phox via TRPC3-PKCβ activation induced by MS in the heart. ( l–n ) Effects of TRPC3 ( l , m ) or PKCβ ( n) ; 10 μM Gö6976) inhibitors on p47 phox phosphorylation induced by MS in NRCMs (n = 3). ( o ) MS-induced ROS generation in NRCMs treated with a PKCβ inhibitor (n = 3). ( p ) Co-immunoprecipitation of TRPC3 with PKCβ, Nox2 and p22 phox in mouse hearts 1week after TAC operation (n = 3). Error bars, s.e.m.*P

    Article Snippet: The following primary antibodies were used: GAPDH (sc-25778), gp91phox (sc-130543), p47phox (sc-17845) and p22phox (sc-20781) from Santa Cruz Biotechnology, flag M2-HRP (A8592) from Sigma Aldrich, GFP (CHIP grade, ab290) from Abcam, myc-tag (05–742) from Merck, phospho p47phox (p-Ser370) (A1171) from Assay Bio Tech, and TRPC3 (ACC-016) from Alomone Labs.

    Techniques: Activity Assay, Expressing, Mass Spectrometry, Activation Assay, Immunoprecipitation

    TRPC3 deletion suppresses TAC-induced LV dysfunction and dilation through Nox2 inhibition. ( a ) Left ventricular end-diastolic pressure (LVEDP; left) and dP/dT max (right) in TAC-operated TRPC3 (+/+) (n = 13) and TRPC3 (−/−) (n = 12) mice 6 week post-operation. ( b ) Myocardial malondialdehyde concentrations 1 week after TAC (n = 4). ( c ) Abundance of Nox2 protein in TRPC3 (+/+) and TRPC3 (−/−) hearts 1 week after TAC (n = 3). ( d ) Representative immunofluorescence images of TRPC3, p22 phox , and caveolin-3 (Cav-3) in adult mouse cardiomyocytes isolated from muscle LIM protein-deficient hearts. ( e ) Representative immunofluorescence images of p22 phox in adult mouse cardiomyocytes: green, anti-p22 phox ; blue, DAPI. ( f ) Relative abundances of p22 phox and Nox2 mRNA in mouse hearts 1 week after TAC (n = 4). ( g ) Abundance of Nox2 protein in TRPC6 (+/+) and TRPC6 (−/−) hearts 1 week after TAC (n = 3). Error bars, s.e.m. *P

    Journal: Scientific Reports

    Article Title: TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling

    doi: 10.1038/srep37001

    Figure Lengend Snippet: TRPC3 deletion suppresses TAC-induced LV dysfunction and dilation through Nox2 inhibition. ( a ) Left ventricular end-diastolic pressure (LVEDP; left) and dP/dT max (right) in TAC-operated TRPC3 (+/+) (n = 13) and TRPC3 (−/−) (n = 12) mice 6 week post-operation. ( b ) Myocardial malondialdehyde concentrations 1 week after TAC (n = 4). ( c ) Abundance of Nox2 protein in TRPC3 (+/+) and TRPC3 (−/−) hearts 1 week after TAC (n = 3). ( d ) Representative immunofluorescence images of TRPC3, p22 phox , and caveolin-3 (Cav-3) in adult mouse cardiomyocytes isolated from muscle LIM protein-deficient hearts. ( e ) Representative immunofluorescence images of p22 phox in adult mouse cardiomyocytes: green, anti-p22 phox ; blue, DAPI. ( f ) Relative abundances of p22 phox and Nox2 mRNA in mouse hearts 1 week after TAC (n = 4). ( g ) Abundance of Nox2 protein in TRPC6 (+/+) and TRPC6 (−/−) hearts 1 week after TAC (n = 3). Error bars, s.e.m. *P

    Article Snippet: The following primary antibodies were used: GAPDH (sc-25778), gp91phox (sc-130543), p47phox (sc-17845) and p22phox (sc-20781) from Santa Cruz Biotechnology, flag M2-HRP (A8592) from Sigma Aldrich, GFP (CHIP grade, ab290) from Abcam, myc-tag (05–742) from Merck, phospho p47phox (p-Ser370) (A1171) from Assay Bio Tech, and TRPC3 (ACC-016) from Alomone Labs.

    Techniques: Inhibition, Mouse Assay, Immunofluorescence, Isolation

    Physical interaction between TRPC3 and Nox2 is critical for stabilization of Nox2. ( a ) Schematic illustration of TRPC3 terminal deletion mutants. ( b , c ) Expression of Nox2 and p22 phox co-expressed with TRPC3 deletion mutants in HEK293 cells (n = 3). ( d ) OAG-induced ROS production in NRCMs expressing Nox2-interacting TRPC3 C-terminal fragment (C3-C fragment) (n = 20–28). ( e ) Co-immunoprecipitation of TRPC3 with Nox2 in the presence or absence of C3-C fragment. Representative blot from three independent experiments was shown. ( f ) ATP (100 μM)-induced Ca 2+ responses in HEK293 cells expressing TRPC3 with or without C3-C fragment (n = 35–51). Timing of solution exchanges were indicated by horizontal bars above the graph. ( g ) Model of the regulation of TRPC3-Nox2 stability and induction of LV dysfunction induced by diastolic stretch of cardiomyocytes. Error bars, s.e.m.*P

    Journal: Scientific Reports

    Article Title: TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling

    doi: 10.1038/srep37001

    Figure Lengend Snippet: Physical interaction between TRPC3 and Nox2 is critical for stabilization of Nox2. ( a ) Schematic illustration of TRPC3 terminal deletion mutants. ( b , c ) Expression of Nox2 and p22 phox co-expressed with TRPC3 deletion mutants in HEK293 cells (n = 3). ( d ) OAG-induced ROS production in NRCMs expressing Nox2-interacting TRPC3 C-terminal fragment (C3-C fragment) (n = 20–28). ( e ) Co-immunoprecipitation of TRPC3 with Nox2 in the presence or absence of C3-C fragment. Representative blot from three independent experiments was shown. ( f ) ATP (100 μM)-induced Ca 2+ responses in HEK293 cells expressing TRPC3 with or without C3-C fragment (n = 35–51). Timing of solution exchanges were indicated by horizontal bars above the graph. ( g ) Model of the regulation of TRPC3-Nox2 stability and induction of LV dysfunction induced by diastolic stretch of cardiomyocytes. Error bars, s.e.m.*P

    Article Snippet: The following primary antibodies were used: GAPDH (sc-25778), gp91phox (sc-130543), p47phox (sc-17845) and p22phox (sc-20781) from Santa Cruz Biotechnology, flag M2-HRP (A8592) from Sigma Aldrich, GFP (CHIP grade, ab290) from Abcam, myc-tag (05–742) from Merck, phospho p47phox (p-Ser370) (A1171) from Assay Bio Tech, and TRPC3 (ACC-016) from Alomone Labs.

    Techniques: Expressing, Immunoprecipitation

    Immunostaining for transient receptor potential proteins TRPC1, TRPC3, TRPC4, and TRPC6/7 in rat lungs. Expression of each TRPC isoform in endothelium was confirmed in small muscular or partially muscularized extraalveolar vessels ( A ) and conduit artery

    Journal:

    Article Title: Resistance to Store Depletion-induced Endothelial Injury in Rat Lung after Chronic Heart Failure

    doi: 10.1164/rccm.200506-847OC

    Figure Lengend Snippet: Immunostaining for transient receptor potential proteins TRPC1, TRPC3, TRPC4, and TRPC6/7 in rat lungs. Expression of each TRPC isoform in endothelium was confirmed in small muscular or partially muscularized extraalveolar vessels ( A ) and conduit artery

    Article Snippet: Expression of putative SOCs, including TRPC1, TRPC3, and TRPC4, was downregulated in fistulas compared with shams, whereas expression of SERCA isoforms was not altered.

    Techniques: Immunostaining, Expressing