Journal: Journal of Biological Chemistry

Article Title: Inositol 1,4,5-Trisphosphate (IP3) Receptor Up-regulation in Hypertension Is Associated with Sensitization of Ca2+ Release and Vascular Smooth Muscle Contractility

doi: 10.1074/jbc.m113.496802

Figure Lengend Snippet: FIGURE 2. Screening of Ca2-handling proteins reveals coupled up-regu- lationofthevascularIP3RandLTCCinexperimentalmodelsofhyperten- sion. A, immunoblot analysis of IP3R1, 1C, TRPC1, TRPC4, Orai1, and STIM1 in lysates from mouse MA. MA lysate pooled from three SAL or AHT mice was added to each lane. Increased expression levels of IP3R1, 1C, and TRPC4 are detected in MA of AHT mice, whereas levels of TRPC1, Orai1, and STIM1 were similar. B, agarose gel analysis of products obtained after PCRs for IP3R1 (274 bp), 1C (185 bp), and TRPC4 (226 bp) using specific primers. -Actin (152 bp) and vWF (178 bp) were used as VSM and endothelial cell markers, respec- tively.IP3R,1C,and-actinweredetectedbothinintactMAandisolatedVSM cells as expected. In contrast, TRPC4 and vWF were detected in arteries but not isolated VSM cells, implying expression only in endothelium. C, immuno- blots comparing the expression of IP3R, 1C, PMCA, and -actin (as a loading control) between MA from normotensive Wistar Kyoto rats (WKY) and SHR and between MA from sham-operated and aortic-banded (Band) hyperten- sive rats. In each case, lysates were pooled from arteries of three or four rats. Only the IP3R and 1C proteins are up-regulated in MA from hypertensive SHR and aortic-banded rats. Blots are representative of three experiments.

Article Snippet: Forty g of protein was loaded per well, and expression of Ca2 -handling proteins was detected using specific antibodies against IP3R1 (1:1000; Millipore), 1C (1:1000; Millipore), TRPC1 (1:200; Santa Cruz Biotechnology), TRPC4 (1:200;NovusBiologicals), STIM1 (1:1000; Cell SignalingTechnology), Orai1 (1:1000; Sigma), and PMCA1 (1:500; Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Agarose Gel Electrophoresis, Isolation, Control

Journal: Oncotarget

Article Title: TRPC1/TRPC3 channels mediate lysophosphatidylcholine-induced apoptosis in cultured human coronary artery smooth muscles cells

doi: 10.18632/oncotarget.10853

Figure Lengend Snippet: A. RT-PCR (left) and Western blots (right) of TRPC isoforms in human coronary artery SMCs (passages 3, 4, and 6). B. Western blots and ratio of protein levels of TRPC1, TRPC3 or TRPC4 channels in human coronary artery SMCs transfected with 20 or 50 nmol/L siRNAs targeting TRPC1, TRPC3 or TRPC4 channels (relative to b-actin and lipofectamine 2000, n = 3 individual experiments, * P < 0.05, ** P < 0.01 vs . control siRNA or lipofectamine). C. Ca 2+ influx (data are mean ± SEM) induced by 30 μmol/L lysoPC in cells transfected with 50 nmol/L control siRNA ( n = 27), TRPC1 siRNA ( n = 27, ** P < 0.01 vs . control siRNA), TRPC3 siRNA ( n = 28, ** P < 0.01 vs . control siRNA), or TRPC4 siRNA ( n = 28). D. Co-immunoprecipitation showing the interaction between TRPC1 and TRPC3 proteins in cells treated without or with 60 μmol/L lysoPC. IP indicates the antibody used to pull down the interacting proteins. Ig G represents the negative control.

Article Snippet: Small interfering RNA (siRNA) molecules targeting human TRPC1 (sc-42664), TRPC3 (sc-42666), TRPC4 (sc-42668), and negative control (sc-37007) were purchased from Santa Cruz Biotech.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Control, Immunoprecipitation, Negative Control

Journal: Oncotarget

Article Title: TRPC1/TRPC3 channels mediate lysophosphatidylcholine-induced apoptosis in cultured human coronary artery smooth muscles cells

doi: 10.18632/oncotarget.10853

Figure Lengend Snippet: A. Flow cytometry analysis in cells transfected with 50 nM control siRNA, TRPC1 siRNA, TRPC3 siRNA or TRPC4 siRNA for 72 h and stained with PI and Annexin V-FITC after incubating with vehicle (upper panel) or 60 μmol/L lysoPC (lower panel) for 24 h (V, viability; D, death; LA, late apoptosis; EA, early apoptosis). B. Percentage of cells showing viability, early apoptosis, late apoptosis, and death analyzed by flow cytometry. Data are mean ± SEM ( n = 3, * P < 0.05, ** P < 0.01 vs . vehicle, # P < 0.05, ## P < 0.01 vs . control siRNA with lysoPC).

Article Snippet: Small interfering RNA (siRNA) molecules targeting human TRPC1 (sc-42664), TRPC3 (sc-42666), TRPC4 (sc-42668), and negative control (sc-37007) were purchased from Santa Cruz Biotech.

Techniques: Flow Cytometry, Transfection, Control, Staining

Journal: Oncotarget

Article Title: TRPC1/TRPC3 channels mediate lysophosphatidylcholine-induced apoptosis in cultured human coronary artery smooth muscles cells

doi: 10.18632/oncotarget.10853

Figure Lengend Snippet: A. Western blots of Bax, Bcl-2, caspase-3, and p-Akt in human coronary artery SMCs transfected with 50 nM control siRNA, TRPC1 siRNA, TRPC3 siRNA or TRPC4 siRNA for 72 h, and then treated with vehicle (V) or 60 μmol/L lysoPC for 3 and 6 h. B. Relative mean levels of Bax, Bcl-2, caspase-3, and p-Akt in human coronary artery SMCs with the same treatment as in A ( n = 3, * P < 0.05, ** P < 0.01 vs . vehicle, # P < 0.05, ## P < 0.01 vs . control siRNA).

Article Snippet: Small interfering RNA (siRNA) molecules targeting human TRPC1 (sc-42664), TRPC3 (sc-42666), TRPC4 (sc-42668), and negative control (sc-37007) were purchased from Santa Cruz Biotech.

Techniques: Western Blot, Transfection, Control

Journal: Andrology

Article Title: Increased expression of TRPC4 channels associated with erectile dysfunction in diabetes.

doi: 10.1111/j.2047-2927.2014.00214.x

Figure Lengend Snippet: Figure 1 RT-PCR analysis of TRPC mRNA in cor- poral smooth muscle of human (A; a: cell and b: tissue) and rat (B). TRPC1, TRPC3, TRPC4 and TRPC6 mRNA were expressed in human penile tissues, cultured human corpus smooth muscle (CSM) cells and rat CSM tissues, whereas the mRNA of the TRPC5 and TRPC7 isoforms were not. Glyceraldehyde 3-phosphate dehydrogen- ase (GAPDH) served as a loading control and RNA input internal control.

Article Snippet: Specific antibody were used with a mouse monoclonal antiTRPC1 antibody (1 : 2000 dilution; Santa Cruz, CA, USA), a mouse monoclonal anti-TRPC4 antibody (1 : 2000; Neuromab, Davis, CA, USA), a rabbit polyclonal anti-TRPC6 antibody (1 : 500; Alomone Laboratories, Jerusalem, Israel) or a monoclonal anti-b-actin antibody (1 : 5000; Abcam, Cambridge, MA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Control

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: ( A ) Calcium flux stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation): TRPC5 (closed diamonds), TRPC4beta (closed squares), TRPC4 (closed circles), TRPC6 (open squares), mock transfected cells (open circles). ( B ) Membrane depolarization stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation), markers as above. ( C ) TRPC4 current evoked by stimulation of 5 μM Englerin A, saline, or 5 μM Englerin A + 10 μM ML204 in 293T cells with Doxycyline-induced TRPC4. Currents were elicited by 200 ms voltage ramps from -100 to +100 mV, applied every 10 s from holding potential of 0 mV. ( D ) Summary of englerin A, englerin-B and ML-204 activity on membrane currents (mean +/- S.E.M.) ( E ) A-673 cell viability in the presence or absence of 50 nM englerin A and/or 50 μM ML204, a TRPC4/C5 channel blocker (mean +/- standard deviation).

Article Snippet: Cells were transfected with TRPC expressing plasmids (Origene: TRPC4beta, RC226709, TRPC6, RC210783, TRPC4 & 5 as above) using Fugene 6 according to the manufacturer’s protocol.

Techniques: Standard Deviation, Transfection, Membrane, Saline, Activity Assay

Journal: PloS one

Article Title: Short-chain 3-hydroxyacyl-coenzyme A dehydrogenase associates with a protein super-complex integrating multiple metabolic pathways.

doi: 10.1371/journal.pone.0035048

Figure Lengend Snippet: Figure 1. Western blot of SCHAD following pulldown with fructose bisphosphate aldolase. Lane 1, homogenized liver SCHAD control, Lane 2 albumin blank, Lanes 3 and 4 results from 2 separate pulldown experiments showing a normal sized mitochondrial SCHAD. doi:10.1371/journal.pone.0035048.g001

Article Snippet: According to the protocol supplied by OriGene, bait plasmid SCHAD 75–119 was tested for its autoactivation potential and ability to enter the nucleus and bind LexA operators.

Techniques: Western Blot, Control

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Normalized fold change in TRPC1/4/5 mRNA (2 –ΔΔ CT ) in TBI mice compared to sham.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques:

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Cell-type specific TRPC1, TRPC4, and TRPC5 channel upregulation in the hippocampus and cortex after CCI-TBI. (A,B) Representative Western immunoblots using (A) TRPC4 (∼120 kDa) and (B) TRPC5 (∼110 kDa) antibodies in sham and TBI cortex and hippocampus. Blots were normalized to β-actin protein (42 kDa) as loading control. (C,D) Summarized data for Western blot quantification of TRPC4 ( C , n = 7–13 animals per group) and TRPC5 ( D , n = 5–7 animals per group) from microdissected brain regions in mice 7 days after TBI. (E,F) Shown are summarized plots of percent difference in TRPC4 (E) and TRPC5 (F) protein between ipsilateral and contralateral hemispheres of microdissected regions from data in Panels (C,D) . All data bars represent the mean ± SEM. * p < 0.05 vs. sham of same subregion. † p < 0.05 vs. contralateral hemisphere of same subregion.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques: Western Blot, Control

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Normalized TRPC4/TRPC5 protein in 7-day sham and TBI mice.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques:

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: Surges in neuronal activity following CCI-TBI are TRPC4/TRPC5-mediated. (A) Representative images of parietal cortex from sham and TBI TRAP mice that were administered 4-OHT at t = 12 h before procedure. Prefix “ c ” denotes contralateral, prefix “ i ” denotes ipsilateral. Red = cFos-tdTomato; blue = DAPI. (B) Representative images of hippocampal subregions from sham and TBI TRAP mice that were administered 4-OHT at t = 12 h before procedure. (C) Summarized quantification of cFos+ neuron density (neurons/0.1 mm 3 ) in sham and TBI TRAP mice activated at the time of TBI, as in Panels (A,B) . (D) Representative images taken from sham mice, TBI mice, and TBI mice also administered M084 (10 mg/kg) (TBI + M084) that were administered 4-OHT t = 7 days after procedure. (E) Summarized quantification of cFos+ neurons in sham, TBI, and TBI + M084 mice 7 days after procedure, as in Panel (D) . All data bars represent the mean ± SEM. * p < 0.05 vs. sham. # p < 0.05 vs. TBI cDG. † p < 0.05 vs. TBI of same region. Scale bars: 100 μm.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques: Activity Assay

Journal: Frontiers in Neuroscience

Article Title: Blockade of TRPC Channels Limits Cholinergic-Driven Hyperexcitability and Seizure Susceptibility After Traumatic Brain Injury

doi: 10.3389/fnins.2021.681144

Figure Lengend Snippet: TRPC4/TRPC5 channel activation artificially prolongs Ca 2+ influx in DGGCs after CCI-TBI. (A) Cumulative probability distribution of the peak amplitude of GCaMP6f fluorescence (ΔF/F) for each DGGC from sham (control) and iTBI slices during EA (1 μM, red) or EA + M084 (10 μM, gray) application. (B) Cumulative probability distribution of the Ca 2+ influx duration (in seconds) for each DGGC from control and iTBI slices during EA or EA + M084 application. (C,D) Histogram population distribution of control DGGC Ca 2+ influx events according to peak amplitude (C) and Ca 2+ event duration (D) . (E,F) Histogram population distribution of iTBI DGGC Ca 2+ influx events according to peak amplitude (E) and Ca 2+ event duration (F) . (G) Summarized means of peak fluorescence from data as in Panel (A) . (H) Summarized means of Ca 2+ influx duration from data as in Panel (B) . Red = EA alone, gray = EA + M084. All data bars represent the mean ± SEM. * p < 0.05 vs. EA alone from same procedure condition, † p < 0.05 vs. control of same drug condition.

Article Snippet: Parietal cortex or dorsal hippocampus brain sections (−1.3 to −2.3 mm posterior to bregma) were probed with a TRPC4 mouse monoclonal antibody (1:1,000, Rockland #200-301-G54, RRID:AB_2611251 ) at 1:500 dilution and fluorescein isothiocyanate (FITC) donkey antimouse IgG (1:250, Jackson ImmunoResearch #715-095-150, RRID:AB_2340792 ).

Techniques: Activation Assay, Fluorescence, Control

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: ( A ) Volcano plot of gene expression features. X- axis indicates fold change of feature in sensitive versus refractory cell lines and Y-axis indicates statistical significance. ( B ) Waterfall plot showing TRPC4 expression levels (affymetrix microarray units) in englerin A sensitive (red) and refractory (blue) tumor cell lines.

Article Snippet: Human TRPC4 was ordered from Origene (Origene; RC218941).

Techniques: Gene Expression, Expressing, Microarray

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: (A ) Effect of TRPC4 siRNA knockdown on viability of A-498 cells in the presence of englerin A. An siRNA targeting luciferase was used as a control (mean+/- S.E.M.) Percent reduction of the TRPC4 mRNA levels are indicated in the legend, KD stands for knockdown. TRPC4 mRNA levels were normalized to peptidyl prolyl isomerase A (PPIA) mRNA levels. ( B ) Effect of TRPC4 siRNA knockdown on viability of A-673 cells in the presence of englerin A (mean +/- S.E.M.) ( C ) Effect of overexpression of TRPC4 by transient transfection on viability of HEK293T cells in the presence of englerin A. TRPC4 expression vector concentrations are indicated by different shapes ( D ) Effect of TRPC4 expression on cell viability in the presence of an englerin A in HEK293T cells engineered to express TRPC4 under control of a Doxycycline (Dox) regulated promoter (mean +/- standard deviation). 100 ng/ml Dox (black circles), 0 ng/ml Dox (open circles). ( E ) Western blot visualizing the levels of TRPC4 in the presence or absence of 100 ng/ml Dox. ( F ) Effect of PKCtheta inhibitor compound 27 on response to englerin A in A-498 cells. ( G ) Effect of PKCtheta inhibitor compound 27 on response to englerin A in A-673 cells.

Article Snippet: Human TRPC4 was ordered from Origene (Origene; RC218941).

Techniques: Knockdown, Luciferase, Control, Over Expression, Transfection, Expressing, Plasmid Preparation, Standard Deviation, Western Blot

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: ( A ) Expression of TRPC4 and TRPC5 in the cell lines from the CLiP experiment. Each circle represents a single cell line and expression levels were measured by microarray (TRPC4 probe 220818_s_at, TRPC5 probe 220552_at). The circle representing the DMS-79 cell line is indicated. ( B ) The effect of englerin A on cell viability in HEK293T cells transiently transfected with a vector expressing TRPC5 (mean +/- standard deviation).

Article Snippet: Human TRPC4 was ordered from Origene (Origene; RC218941).

Techniques: Expressing, Microarray, Transfection, Plasmid Preparation, Standard Deviation

Journal: PLoS ONE

Article Title: Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

doi: 10.1371/journal.pone.0127498

Figure Lengend Snippet: ( A ) Calcium flux stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation): TRPC5 (closed diamonds), TRPC4beta (closed squares), TRPC4 (closed circles), TRPC6 (open squares), mock transfected cells (open circles). ( B ) Membrane depolarization stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation), markers as above. ( C ) TRPC4 current evoked by stimulation of 5 μM Englerin A, saline, or 5 μM Englerin A + 10 μM ML204 in 293T cells with Doxycyline-induced TRPC4. Currents were elicited by 200 ms voltage ramps from -100 to +100 mV, applied every 10 s from holding potential of 0 mV. ( D ) Summary of englerin A, englerin-B and ML-204 activity on membrane currents (mean +/- S.E.M.) ( E ) A-673 cell viability in the presence or absence of 50 nM englerin A and/or 50 μM ML204, a TRPC4/C5 channel blocker (mean +/- standard deviation).

Article Snippet: Human TRPC4 was ordered from Origene (Origene; RC218941).

Techniques: Standard Deviation, Transfection, Membrane, Saline, Activity Assay