trpm4 (Alomone Labs)

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Alomone Labs trpm4

Inhibition of <t>TRPM4</t> channels with 9-phenanthrol attenuates spontaneous phasic and tonic contractions of human DSM isolated strips. A : representative myograph recording demonstrating inhibition of spontaneous phasic contractions and tone of a human DSM isolated strip by 30 μM 9-phenanthrol. B : summary data illustrating decrease in spontaneous phasic contraction amplitude, muscle force integral, frequency, duration, and tone of human DSM strips by 30 μM 9-phenanthrol ( n = 11, N = 5). *** P

Trpm4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/trpm4/product/Alomone Labs
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99

trpm4 - by Bioz Stars, 2022-05

94/100 stars

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1) Product Images from "Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function"

Article Title: Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00270.2015

Inhibition of TRPM4 channels with 9-phenanthrol attenuates spontaneous phasic and tonic contractions of human DSM isolated strips. A : representative myograph recording demonstrating inhibition of spontaneous phasic contractions and tone of a human DSM isolated strip by 30 μM 9-phenanthrol. B : summary data illustrating decrease in spontaneous phasic contraction amplitude, muscle force integral, frequency, duration, and tone of human DSM strips by 30 μM 9-phenanthrol ( n = 11, N = 5). *** P

Figure Legend Snippet: Inhibition of TRPM4 channels with 9-phenanthrol attenuates spontaneous phasic and tonic contractions of human DSM isolated strips. A : representative myograph recording demonstrating inhibition of spontaneous phasic contractions and tone of a human DSM isolated strip by 30 μM 9-phenanthrol. B : summary data illustrating decrease in spontaneous phasic contraction amplitude, muscle force integral, frequency, duration, and tone of human DSM strips by 30 μM 9-phenanthrol ( n = 11, N = 5). *** P

Techniques Used: Inhibition, Isolation, Stripping Membranes

Inhibition of TRPM4 channels with 9-phenanthrol hyperpolarizes the resting membrane potential (RMP) in human DSM cells. A : representative current-clamp recording illustrating hyperpolarizing effects of 30 μM 9-phenanthrol on RMP in a human DSM cell. B : summary data illustrating hyperpolarizing effects of 30 μM 9-phenanthrol on the RMP in human DSM cells ( n = 4, N = 3). * P

Figure Legend Snippet: Inhibition of TRPM4 channels with 9-phenanthrol hyperpolarizes the resting membrane potential (RMP) in human DSM cells. A : representative current-clamp recording illustrating hyperpolarizing effects of 30 μM 9-phenanthrol on RMP in a human DSM cell. B : summary data illustrating hyperpolarizing effects of 30 μM 9-phenanthrol on the RMP in human DSM cells ( n = 4, N = 3). * P

Techniques Used: Inhibition

Western blot, immunohistochemical, and immunocytochemical detections of TRPM4 channel protein in human DSM tissue and DSM single cells. A : Western blot showing TRPM4 channel protein expression in human DSM tissue. Arrow indicates ∼134-kDa band, consistent with expected molecular mass of TRPM4 channel protein. Lack of an immunoreactive band in the presence of competing peptide (CP) confirmed specificity of the primary antibody. HEK-293 cell lysate was used as a positive control. B : confocal images showing immunohistochemical detection of TRPM4 channel protein expression in human DSM tissue. Red staining ( bottom left ) indicates TRPM4 channels; blue staining indicates cell nuclei ( top left ); green staining indicates α-smooth muscle actin (α-SMA, top right ); merged image ( bottom right ) illustrates overlap of all 3 images. C : confocal images illustrating immunocytochemical detection of TRPM4 channel protein expression in isolated human DSM cells. Red staining ( bottom left ) indicates TRPM4 channels; blue staining indicates cell nucleus ( top left ); green staining indicates α-SMA ( top right ); merged image ( bottom right ) illustrates overlap of all 3 images. Results were verified in 4 separate experiments using DSM whole tissue or multiple DSM cells isolated from 4 patients. D and E : immunohistochemistry and immunocytochemistry experiments, respectively. In control experiments, no staining was visible after absorption of the primary antibody with a competing peptide (+CP control). Merged image ( bottom right ) illustrates overlap of all 3 images.

Figure Legend Snippet: Western blot, immunohistochemical, and immunocytochemical detections of TRPM4 channel protein in human DSM tissue and DSM single cells. A : Western blot showing TRPM4 channel protein expression in human DSM tissue. Arrow indicates ∼134-kDa band, consistent with expected molecular mass of TRPM4 channel protein. Lack of an immunoreactive band in the presence of competing peptide (CP) confirmed specificity of the primary antibody. HEK-293 cell lysate was used as a positive control. B : confocal images showing immunohistochemical detection of TRPM4 channel protein expression in human DSM tissue. Red staining ( bottom left ) indicates TRPM4 channels; blue staining indicates cell nuclei ( top left ); green staining indicates α-smooth muscle actin (α-SMA, top right ); merged image ( bottom right ) illustrates overlap of all 3 images. C : confocal images illustrating immunocytochemical detection of TRPM4 channel protein expression in isolated human DSM cells. Red staining ( bottom left ) indicates TRPM4 channels; blue staining indicates cell nucleus ( top left ); green staining indicates α-SMA ( top right ); merged image ( bottom right ) illustrates overlap of all 3 images. Results were verified in 4 separate experiments using DSM whole tissue or multiple DSM cells isolated from 4 patients. D and E : immunohistochemistry and immunocytochemistry experiments, respectively. In control experiments, no staining was visible after absorption of the primary antibody with a competing peptide (+CP control). Merged image ( bottom right ) illustrates overlap of all 3 images.

Techniques Used: Western Blot, Immunohistochemistry, Expressing, Positive Control, Staining, Isolation, Immunocytochemistry

Inhibition of TRPM4 channels with 9-phenanthrol decreases the amplitude of 3.5- to 50-Hz EFS-induced contractions of human DSM isolated strips. A : representative recording of EFS-induced contractions (stimulation frequency = 3.5–50 Hz) in the absence of 9-phenanthrol (control) and 10 min after application of 30 μM 9-phenanthrol. B : frequency-response curves in the presence or absence of 30 μM 9-phenanthrol illustrating a decrease in amplitude of EFS-induced contractions of human DSM isolated strips ( n = 22, N = 16). ** P

Figure Legend Snippet: Inhibition of TRPM4 channels with 9-phenanthrol decreases the amplitude of 3.5- to 50-Hz EFS-induced contractions of human DSM isolated strips. A : representative recording of EFS-induced contractions (stimulation frequency = 3.5–50 Hz) in the absence of 9-phenanthrol (control) and 10 min after application of 30 μM 9-phenanthrol. B : frequency-response curves in the presence or absence of 30 μM 9-phenanthrol illustrating a decrease in amplitude of EFS-induced contractions of human DSM isolated strips ( n = 22, N = 16). ** P

Techniques Used: Inhibition, Isolation

Transient receptor potential melastatin 4 (TRPM4) channel mRNA expression in human detrusor smooth muscle (DSM) whole tissue and native freshly isolated human DSM single cells. Gel electrophoresis imaging illustrates RT-PCR detection of TRPM4 channel mRNA transcripts (196 bp) in DSM whole tissue and DSM single cells ( n = 4, N = 4). Human brain and prostate samples were used as positive controls (+). No products were observed in the negative control (−RT), in which reverse transcriptase (RT) was not added to the reaction.

Figure Legend Snippet: Transient receptor potential melastatin 4 (TRPM4) channel mRNA expression in human detrusor smooth muscle (DSM) whole tissue and native freshly isolated human DSM single cells. Gel electrophoresis imaging illustrates RT-PCR detection of TRPM4 channel mRNA transcripts (196 bp) in DSM whole tissue and DSM single cells ( n = 4, N = 4). Human brain and prostate samples were used as positive controls (+). No products were observed in the negative control (−RT), in which reverse transcriptase (RT) was not added to the reaction.

Techniques Used: Expressing, Isolation, Nucleic Acid Electrophoresis, Imaging, Reverse Transcription Polymerase Chain Reaction, Negative Control

Inhibition of transient inward cationic currents (TICCs) by the TRPM4 channel-selective inhibitor 9-phenanthrol in freshly isolated human DSM cells. A : original recording illustrating the inhibitory effect of 30 μM 9-phenanthrol on TICC activity in a human DSM single cell recorded at −70 mV. B : summary data illustrating inhibitory effects of 30 μM 9-phenanthrol on TICCs, analyzed as total open channel probability ( NP o ) before and after application of 30 μM 9-phenanthrol ( n = 15, N = 9). * P

Figure Legend Snippet: Inhibition of transient inward cationic currents (TICCs) by the TRPM4 channel-selective inhibitor 9-phenanthrol in freshly isolated human DSM cells. A : original recording illustrating the inhibitory effect of 30 μM 9-phenanthrol on TICC activity in a human DSM single cell recorded at −70 mV. B : summary data illustrating inhibitory effects of 30 μM 9-phenanthrol on TICCs, analyzed as total open channel probability ( NP o ) before and after application of 30 μM 9-phenanthrol ( n = 15, N = 9). * P

Techniques Used: Inhibition, Isolation, Activity Assay

Inhibition of TRPM4 channels with 9-phenanthrol attenuates voltage step-induced whole cell currents in human DSM cells. A : representative recordings illustrate the inhibitory effect of 30 μM 9-phenanthrol on the amplitude of the voltage step-induced whole cell current in human DSM cell. Inhibitory effect of 9-phenanthrol was reversed after washout of the human DSM cell with fresh 9-phenanthrol-free bath solution. B : summary data of current-voltage relationships in the absence (control), in the presence, and after washout of 30 μM 9-phenanthrol ( n = 7, N = 7). * P

Figure Legend Snippet: Inhibition of TRPM4 channels with 9-phenanthrol attenuates voltage step-induced whole cell currents in human DSM cells. A : representative recordings illustrate the inhibitory effect of 30 μM 9-phenanthrol on the amplitude of the voltage step-induced whole cell current in human DSM cell. Inhibitory effect of 9-phenanthrol was reversed after washout of the human DSM cell with fresh 9-phenanthrol-free bath solution. B : summary data of current-voltage relationships in the absence (control), in the presence, and after washout of 30 μM 9-phenanthrol ( n = 7, N = 7). * P

Techniques Used: Inhibition

Inhibition of TRPM4 channels with 9-phenanthrol significantly reduces carbachol-induced phasic and tonic contractions of human DSM isolated strips. A : representative myograph recording obtained from a human DSM strip depicting the inhibitory effect of 30 μM 9-phenanthrol on 0.1 μM carbachol-induced phasic contractions. B : summary data illustrating inhibitory effects of 30 μM 9-phenanthrol on amplitude, muscle force integral, frequency, duration, and tone of carbachol-induced contractions of human DSM isolated strips ( n = 17, N = 5). *** P

Figure Legend Snippet: Inhibition of TRPM4 channels with 9-phenanthrol significantly reduces carbachol-induced phasic and tonic contractions of human DSM isolated strips. A : representative myograph recording obtained from a human DSM strip depicting the inhibitory effect of 30 μM 9-phenanthrol on 0.1 μM carbachol-induced phasic contractions. B : summary data illustrating inhibitory effects of 30 μM 9-phenanthrol on amplitude, muscle force integral, frequency, duration, and tone of carbachol-induced contractions of human DSM isolated strips ( n = 17, N = 5). *** P

Techniques Used: Inhibition, Isolation, Stripping Membranes

2) Product Images from "Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCDc14 Cells"

Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCDc14 Cells

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2021.627875

Application of MβCD to the cytoplasmic bath decreases TRPM4 channel activity which is reversed by exogenous cholesterol. (A) A representative single channel recording from an inside-out patch shows TRPM4 activity before and after replacement of control cytoplasmic bath solution first with a solution containing 0.5 mM MβCD and then with a solution containing 30 μg/ml cholesterol. “C” indicates channel at the closed state. “O” indicates single-level openings. “I, II, and III” are zoom-ins of the single-channel recording. (B) Summary plots of TRPM4 channel P O under each indicted condition. n = 5 paired experiments, ** P

Figure Legend Snippet: Application of MβCD to the cytoplasmic bath decreases TRPM4 channel activity which is reversed by exogenous cholesterol. (A) A representative single channel recording from an inside-out patch shows TRPM4 activity before and after replacement of control cytoplasmic bath solution first with a solution containing 0.5 mM MβCD and then with a solution containing 30 μg/ml cholesterol. “C” indicates channel at the closed state. “O” indicates single-level openings. “I, II, and III” are zoom-ins of the single-channel recording. (B) Summary plots of TRPM4 channel P O under each indicted condition. n = 5 paired experiments, ** P

Techniques Used: Activity Assay

Treatment of cells with cholesterol increases, but with lovastatin decreases, TRPM4 channel activity by regulating its sensitivity to Ca 2+ in mpkCCD c14 Cells. (A) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing different concentrations of free Ca 2+ . The cells were treated with 30 μg/ml cholesterol for 48 hrs. From top to bottom: 1 μM, 10 μM, 200 μM, 1 mM, and 5 mM free bath Ca 2+ . “C” indicates channel at the closed state; “O” indicates single-level openings. (B) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing different concentrations of free Ca 2+ . The cells were treated with 5 μM lovastatin for 48 hrs. From top to bottom: 10 μM, 200 μM, 1 mM, 5 mM and 10 mM free bath Ca 2+ . “C” indicates channel at the closed state; “O” indicates single-level openings. (C) The effect of membrane cholesterol on Ca 2+ -dependence of channel opening. Channel P O was plotted as a function of free Ca 2+ concentration in the bath. P o values are shown for patches either with exogenous cholesterol treatment (black line) or lovastatin treatment (red line). n = 4–7 cells for different data points.

Figure Legend Snippet: Treatment of cells with cholesterol increases, but with lovastatin decreases, TRPM4 channel activity by regulating its sensitivity to Ca 2+ in mpkCCD c14 Cells. (A) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing different concentrations of free Ca 2+ . The cells were treated with 30 μg/ml cholesterol for 48 hrs. From top to bottom: 1 μM, 10 μM, 200 μM, 1 mM, and 5 mM free bath Ca 2+ . “C” indicates channel at the closed state; “O” indicates single-level openings. (B) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing different concentrations of free Ca 2+ . The cells were treated with 5 μM lovastatin for 48 hrs. From top to bottom: 10 μM, 200 μM, 1 mM, 5 mM and 10 mM free bath Ca 2+ . “C” indicates channel at the closed state; “O” indicates single-level openings. (C) The effect of membrane cholesterol on Ca 2+ -dependence of channel opening. Channel P O was plotted as a function of free Ca 2+ concentration in the bath. P o values are shown for patches either with exogenous cholesterol treatment (black line) or lovastatin treatment (red line). n = 4–7 cells for different data points.

Techniques Used: Activity Assay, Concentration Assay

PI(4, 5)P 2 stimulates TRPM4 via a physical interaction. (A) A representative single channel recording shows that diC8-PI(4,5)P 2 significantly increased TRPM4 activity. (B) Summary plots of TRPM4 channel P O under each indicated conditions. n = 5 paired experiments. ** P

Figure Legend Snippet: PI(4, 5)P 2 stimulates TRPM4 via a physical interaction. (A) A representative single channel recording shows that diC8-PI(4,5)P 2 significantly increased TRPM4 activity. (B) Summary plots of TRPM4 channel P O under each indicated conditions. n = 5 paired experiments. ** P

Techniques Used: Activity Assay

Cholesterol stimulates TRPM4 via a PI(4, 5)P 2 -dependent mechanism. (A) A representative single channel recording shows that treatment of mpkCCD c14 cells with 20 nM wortmannin had no effect on cholesterol-induced TRPM4 channel activity. (B) Summary plots of TRPM4 channel P O under each indicated conditions. n = 6 paired experiments, ** P

Figure Legend Snippet: Cholesterol stimulates TRPM4 via a PI(4, 5)P 2 -dependent mechanism. (A) A representative single channel recording shows that treatment of mpkCCD c14 cells with 20 nM wortmannin had no effect on cholesterol-induced TRPM4 channel activity. (B) Summary plots of TRPM4 channel P O under each indicated conditions. n = 6 paired experiments, ** P

Techniques Used: Activity Assay

TRPM4 channels are mainly located in lipid rafts. (A) Representative confocal microscopy images indicate that majority of TRPM4 (green) was co-localized with cholera toxin B (red) in the apical membrane; white rectangular box indicate zoomed-in areas shown in the Zoom-in panels. Data represent five individual experiments showing consistent results (B) Majority of TRPM4 was detected in low-density regions in sucrose gradient experiments; Caveolin-1 was used as a control protein that is known to be located in lipid rafts. Data represent three individual experiments showing consistent results.

Figure Legend Snippet: TRPM4 channels are mainly located in lipid rafts. (A) Representative confocal microscopy images indicate that majority of TRPM4 (green) was co-localized with cholera toxin B (red) in the apical membrane; white rectangular box indicate zoomed-in areas shown in the Zoom-in panels. Data represent five individual experiments showing consistent results (B) Majority of TRPM4 was detected in low-density regions in sucrose gradient experiments; Caveolin-1 was used as a control protein that is known to be located in lipid rafts. Data represent three individual experiments showing consistent results.

Techniques Used: Confocal Microscopy

Treatment with cholesterol or lovastatin does not alter expression levels of TRPM4 in mpkCCDc14 Cells. (A) Representative confocal microscopy images of mpkCCDc14 cells stained with TRPM4 antibody under each indicated conditions. (B) Summary plots of fluorescence intensity of TRPM4. Data are from 24 cells in four sets of separate experiments. (C) Representative Western blots from cell-surface biotinylated and the total proteins of TRPM4 protein. (D) Summary plots of relative expression of TRPM4. Cells were either under control conditions or treated with 30 μg/ml cholesterol alone, 30 μg/ml cholesterol plus 5 μM lovastatin, or 5 μM lovastatin alone for 48 hrs, respectively. n = 5. (E) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing 5 mM CaCl 2 , followed by a bath solution with 10 mM EGTA and no calcium. Cells were either under control conditions or treated with 30 μg/ml cholesterol, or 5 μM lovastatin for 48 hrs, respectively. (F) Summary plots of the number of active channels in the patches under each indicated condition. n = 5 for control cells, n = 5 for cells treated with cholesterol, n = 4 for cells treated with lovastatin.

Figure Legend Snippet: Treatment with cholesterol or lovastatin does not alter expression levels of TRPM4 in mpkCCDc14 Cells. (A) Representative confocal microscopy images of mpkCCDc14 cells stained with TRPM4 antibody under each indicated conditions. (B) Summary plots of fluorescence intensity of TRPM4. Data are from 24 cells in four sets of separate experiments. (C) Representative Western blots from cell-surface biotinylated and the total proteins of TRPM4 protein. (D) Summary plots of relative expression of TRPM4. Cells were either under control conditions or treated with 30 μg/ml cholesterol alone, 30 μg/ml cholesterol plus 5 μM lovastatin, or 5 μM lovastatin alone for 48 hrs, respectively. n = 5. (E) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing 5 mM CaCl 2 , followed by a bath solution with 10 mM EGTA and no calcium. Cells were either under control conditions or treated with 30 μg/ml cholesterol, or 5 μM lovastatin for 48 hrs, respectively. (F) Summary plots of the number of active channels in the patches under each indicated condition. n = 5 for control cells, n = 5 for cells treated with cholesterol, n = 4 for cells treated with lovastatin.

Techniques Used: Expressing, Confocal Microscopy, Staining, Fluorescence, Western Blot

3) Product Images from "Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function"

Article Title: Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00270.2015

Techniques Used: Inhibition, Isolation, Stripping Membranes

Techniques Used: Inhibition

Techniques Used: Western Blot, Immunohistochemistry, Expressing, Positive Control, Staining, Isolation, Immunocytochemistry

Techniques Used: Inhibition, Isolation

Techniques Used: Expressing, Isolation, Nucleic Acid Electrophoresis, Imaging, Reverse Transcription Polymerase Chain Reaction, Negative Control

Techniques Used: Inhibition, Isolation, Activity Assay

Techniques Used: Inhibition

Techniques Used: Inhibition, Isolation, Stripping Membranes

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anti trpm4 antibody (Alomone Labs)

Alomone Labs anti trpm4 antibody

Anti Trpm4 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti trpm4 antibody/product/Alomone Labs
Average 94 stars, based on 1 article reviews
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anti trpm4 antibody - by Bioz Stars, 2022-05

94/100 stars

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Image Search Results

Journal: PLoS ONE

Article Title: Transient Receptor Potential Melastatin-4 Is Involved in Hypoxia-Reoxygenation Injury in the Cardiomyocytes

doi: 10.1371/journal.pone.0121703

Figure Lengend Snippet: Immunofluorescence imaging of TRPM4 in the healthy rat heart. (A) Adult rat heart sections were labeled with anti-TRPM4 antibodies (green) and stained with Hoechst 33342 for the nuclei (blue). The green and blue fluorescence images were overlaid with the DIC image. Staining specificity was confirmed by the lack of signal when anti-TRPM4 antibodies were preincubated with the antigenic blocking peptide (ab65597). Scale bar: 100 μm. (B) Double immunofluorescence showing TRPM4 (green) and SERCA2 (red) expression in ventricular cardiomyocytes. Scale bar: 20 μm. The overlaid image (left bottom) reveals alternating positions for the TRPM4 and SERCA2 proteins on the longitudinal axis of cardiomyocytes. Arrowheads indicate the intercalated disc. Fluorescence intensity profile (right bottom) confirmed the alternating expression of TRPM4 and SERCA2.

Article Snippet: Similar results were obtained using a different anti-TRPM4 antibody.

Techniques: Immunofluorescence, Imaging, Labeling, Staining, Fluorescence, Blocking Assay, Expressing

Knockdown of TRPM4 prevents cell death caused by H 2 O 2 challenge in H9c2 cardiomyocytes. (A) Quantitative RT-PCR confirming the gene silencing of TRPM4. siNEG, cells transfected with control siRNA; siTRPM4, cells transfected with TRPM4-targeting siRNA. n = 5 for each group. (B) Confirmation of suppressed TRPM4 protein expression by immunocytochemistry 48 h after siRNA transfection. Green, anti-TRPM4, Blue, Hoechst 33342 dye (nuclei). The fluorescent images were overlaid with DIC images of the cultures. (C) Confirmation of suppressed TRPM4 protein expression by Western blot 24 h after siRNA transfection. (D) Impact of gene silencing on the loss of viability induced by 200 μM H 2 O 2 . Cell viability was measured by the MTT assay. n = 5 for each group. (E) Impact of TRPM4 knockdown on the hypoxia/reoxygenation (H/R) challenge. Cell viability was measured by the MTT assay. n = 6 for each group. Statistical analysis was performed using Dunnett’s test as post hoc. *: p

Journal: PLoS ONE

Article Title: Transient Receptor Potential Melastatin-4 Is Involved in Hypoxia-Reoxygenation Injury in the Cardiomyocytes

doi: 10.1371/journal.pone.0121703

Figure Lengend Snippet: Knockdown of TRPM4 prevents cell death caused by H 2 O 2 challenge in H9c2 cardiomyocytes. (A) Quantitative RT-PCR confirming the gene silencing of TRPM4. siNEG, cells transfected with control siRNA; siTRPM4, cells transfected with TRPM4-targeting siRNA. n = 5 for each group. (B) Confirmation of suppressed TRPM4 protein expression by immunocytochemistry 48 h after siRNA transfection. Green, anti-TRPM4, Blue, Hoechst 33342 dye (nuclei). The fluorescent images were overlaid with DIC images of the cultures. (C) Confirmation of suppressed TRPM4 protein expression by Western blot 24 h after siRNA transfection. (D) Impact of gene silencing on the loss of viability induced by 200 μM H 2 O 2 . Cell viability was measured by the MTT assay. n = 5 for each group. (E) Impact of TRPM4 knockdown on the hypoxia/reoxygenation (H/R) challenge. Cell viability was measured by the MTT assay. n = 6 for each group. Statistical analysis was performed using Dunnett’s test as post hoc. *: p

Article Snippet: Similar results were obtained using a different anti-TRPM4 antibody.

Techniques: Quantitative RT-PCR, Transfection, Expressing, Immunocytochemistry, Western Blot, MTT Assay

Detection of TRPM4 protein using western blotting. TRPM4 protein expression was significantly lower in TRPM4 KO than in WT cells. β -actin was used as loading control. The TRPM4 expression level was normalized by dividing the band intensity of TRPM4 by that of β -actin. Chart represents average of three independent experiments. Error bars represent SEM. **** p

Journal: MethodsX

Article Title: Production of TRPM4 knockout cell line using rat cardiomyocyte H9c2

doi: 10.1016/j.mex.2021.101404

Figure Lengend Snippet: Detection of TRPM4 protein using western blotting. TRPM4 protein expression was significantly lower in TRPM4 KO than in WT cells. β -actin was used as loading control. The TRPM4 expression level was normalized by dividing the band intensity of TRPM4 by that of β -actin. Chart represents average of three independent experiments. Error bars represent SEM. **** p

Article Snippet: Then the membranes were incubated overnight at 4 °C with primary antibodies: mouse anti-β-Actin antibody (1:1,000; Cell signaling technology, Danvers, MA, USA; Cat. no: 3700S) for the lower molecular weight part and rabbit anti-TRPM4 antibody (1:400; Alomone Labs, Jerusalem, Israel; Cat. no: ACC-044) for the higher molecular weight part.

Techniques: Western Blot, Expressing

Comparison of DNA sequence between the WT and TRPM4 KO cells . Left: Sequencing chromatographs of genomic DNA samples obtained from WT and TRPM4 KO cells. The numbers indicate locations in the rat chromosomal DNA (accession: NC_005100.4). The guanine at site 101,316,729 (arrowhead) is missing in the DNA of TRPM4 KO cells. Right: Sequencing results of 8 samples (reverse complemented). The cytosine residue between thymine and cytosine is missing in TRPM4 KO 4 and TRPM4 KO 5 samples.

Journal: MethodsX

Article Title: Production of TRPM4 knockout cell line using rat cardiomyocyte H9c2

doi: 10.1016/j.mex.2021.101404

Figure Lengend Snippet: Comparison of DNA sequence between the WT and TRPM4 KO cells . Left: Sequencing chromatographs of genomic DNA samples obtained from WT and TRPM4 KO cells. The numbers indicate locations in the rat chromosomal DNA (accession: NC_005100.4). The guanine at site 101,316,729 (arrowhead) is missing in the DNA of TRPM4 KO cells. Right: Sequencing results of 8 samples (reverse complemented). The cytosine residue between thymine and cytosine is missing in TRPM4 KO 4 and TRPM4 KO 5 samples.

Techniques: Sequencing

Inhibition of I shear by the blockers of TRPM4 channel

Journal: The Journal of Physiology

Article Title: Shear stress activates monovalent cation channel transient receptor potential melastatin subfamily 4 in rat atrial myocytes via type 2 inositol 1,4,5‐trisphosphate receptors and Ca2+ release

doi: 10.1113/JP270887

Figure Lengend Snippet: Inhibition of I shear by the blockers of TRPM4 channel

Article Snippet: The cells were incubated in PBS containing 1% BSA (Sigma) for 1 h to block non‐specific labelling, and then incubated with anti‐TRPM4 [rabbit polyclonal IgG, dilution 1:200; epitope, EKEQSWIPKIFKK(C), corresponding to amino acid residues 5–17 of N‐terminus in human TRPM4; Intracellular; ACC‐044, Alomone Labs, Jerusalem, Israel] and anti‐IP3 R2 (goat polyclonal IgG, 2 μg ml−1 ; Santa Cruz Biotechnology Inc.) overnight at 4°C.

Techniques: Inhibition

Journal: The Journal of Physiology

doi: 10.1113/JP270887

Figure Lengend Snippet: Inhibition of I shear by KD of TRPM4

Techniques: Inhibition

Expression of TRPM4 in rat atrial myocytes and its specific co‐localization with IP 3 R2

Journal: The Journal of Physiology

doi: 10.1113/JP270887

Figure Lengend Snippet: Expression of TRPM4 in rat atrial myocytes and its specific co‐localization with IP 3 R2

Techniques: Expressing

Inhibition of I shear by intracellular dialysis with anti‐TRPM4 antibody

Journal: The Journal of Physiology

doi: 10.1113/JP270887

Figure Lengend Snippet: Inhibition of I shear by intracellular dialysis with anti‐TRPM4 antibody

Techniques: Inhibition

No role of IP 3 R2 in TRPM4 subcellular localization

Journal: The Journal of Physiology

doi: 10.1113/JP270887

Figure Lengend Snippet: No role of IP 3 R2 in TRPM4 subcellular localization

Techniques:

Co‐localization of IP3 R2 and TRPM4 at the cell periphery

Journal: The Journal of Physiology

doi: 10.1113/JP270887

Figure Lengend Snippet: Co‐localization of IP3 R2 and TRPM4 at the cell periphery

Techniques:

TRPM4 is expressed in pyramidal neurons and interneurons in layers 2/3 of the mPFC. Confocal images from double immunofluorescence labeling for (A) TRPM4 (green), (B) Neurogranin (red), and (C) the merged signals in a coronal mouse brain section at P35. Confocal images from double immunofluorescence labeling for (D) TRPM4 (green), (E) GAD67 (red), and (F) the merge of both signals in a coronal mouse brain section at P35. Arrows point to expression on interneurons. Scale bar = 40 μm.

Journal: Frontiers in Cellular Neuroscience

Article Title: Subcellular Localization and Activity of TRPM4 in Medial Prefrontal Cortex Layer 2/3

doi: 10.3389/fncel.2018.00012

Figure Lengend Snippet: TRPM4 is expressed in pyramidal neurons and interneurons in layers 2/3 of the mPFC. Confocal images from double immunofluorescence labeling for (A) TRPM4 (green), (B) Neurogranin (red), and (C) the merged signals in a coronal mouse brain section at P35. Confocal images from double immunofluorescence labeling for (D) TRPM4 (green), (E) GAD67 (red), and (F) the merge of both signals in a coronal mouse brain section at P35. Arrows point to expression on interneurons. Scale bar = 40 μm.

Article Snippet: Primary Antibodies Two different rabbit polyclonal antibodies against TRPM4, ACC-044 ( RRID:AB_2040250 ) and ab104572 ( RRID:AB_10712148 ), were obtained from Alomone (Israel) and Abcam (United States) respectively.

Techniques: Immunofluorescence, Labeling, Expressing

Neuronal expression of TRPM4 in mouse prefrontal cortex (PFC). (A) Panoramic view of the frontal cortex showing double immunofluorescence labeling for TRPM4 (green) and NeuN (red) in a coronal mouse brain section at P35. The inset shows a coronal map of this area. (B) Confocal images of layers 1 and 2/3 of mPFC showing the expression of TRPM4 (top), NeuN (middle), and the merge of both signals (bottom) showing the expression in the soma and neurites (arrows). Scale bar: A = 300 μm; B = 40 μm. (C) Representative I/V ramps (–80 to 80 mV, 0.16 mV/ms) recorded in the soma of pyramidal neurons from layer 2/3 at mPFC. Black traces represent the basal current, red traces represent the effect of 10 μM 9-Ph (whole bath perfusion), and blue traces represent intracellular EGTA (10 mM). The right panel shows the quantification of the current at –80 mV (arrow) ( n = 8). All experiments were performed in the presence of glutamatergic (CNQX, AP5) and GABAergic blockers (Picrotoxin).

Journal: Frontiers in Cellular Neuroscience

Article Title: Subcellular Localization and Activity of TRPM4 in Medial Prefrontal Cortex Layer 2/3

doi: 10.3389/fncel.2018.00012

Figure Lengend Snippet: Neuronal expression of TRPM4 in mouse prefrontal cortex (PFC). (A) Panoramic view of the frontal cortex showing double immunofluorescence labeling for TRPM4 (green) and NeuN (red) in a coronal mouse brain section at P35. The inset shows a coronal map of this area. (B) Confocal images of layers 1 and 2/3 of mPFC showing the expression of TRPM4 (top), NeuN (middle), and the merge of both signals (bottom) showing the expression in the soma and neurites (arrows). Scale bar: A = 300 μm; B = 40 μm. (C) Representative I/V ramps (–80 to 80 mV, 0.16 mV/ms) recorded in the soma of pyramidal neurons from layer 2/3 at mPFC. Black traces represent the basal current, red traces represent the effect of 10 μM 9-Ph (whole bath perfusion), and blue traces represent intracellular EGTA (10 mM). The right panel shows the quantification of the current at –80 mV (arrow) ( n = 8). All experiments were performed in the presence of glutamatergic (CNQX, AP5) and GABAergic blockers (Picrotoxin).

Techniques: Expressing, Immunofluorescence, Labeling

TRPM4 expression is restricted to the somatodendritic region of the pyramidal neurons at mPFC layers 2/3. (A) Confocal images from triple immunofluorescence labeling for TRPM4 (green), MAP2 (blue), and AnkG (orange) in the mPFC in a coronal mouse brain section at P35. Magnification of layers 2/3 showing (B) TRPM4, (C) MAP2, (D) AnkG and (E) merge of the three signals, white arrows indicate TRPM4 and MAP2 co-expression in proximal dendrites, yellow arrows indicate AnkG expression in the AIS, which does not contain detectable TRPM4 immunolabeling. Scale bar: A = 20 μm; B–E = 10 μm. (F) Schematic figure showing the areas of drug perfusion and a DIC picture of the recorded area. Representative I/V curves (–80 to 80 mV, 0.16 mV/ms) recorded in the soma using local somatic (G) , medial, (H) , and distal dendrite (I) drug perfusion; black traces represent the basal current, red traces represent the effect of 10 μM 9-Ph. Right panels showed the quantification of the currents at –80 mV (arrow) ( n = 7). All experiments were performed in the presence of glutamatergic (CNQX, AP5) and GABAergic blockers (Picrotoxin).

Journal: Frontiers in Cellular Neuroscience

Article Title: Subcellular Localization and Activity of TRPM4 in Medial Prefrontal Cortex Layer 2/3

doi: 10.3389/fncel.2018.00012

Figure Lengend Snippet: TRPM4 expression is restricted to the somatodendritic region of the pyramidal neurons at mPFC layers 2/3. (A) Confocal images from triple immunofluorescence labeling for TRPM4 (green), MAP2 (blue), and AnkG (orange) in the mPFC in a coronal mouse brain section at P35. Magnification of layers 2/3 showing (B) TRPM4, (C) MAP2, (D) AnkG and (E) merge of the three signals, white arrows indicate TRPM4 and MAP2 co-expression in proximal dendrites, yellow arrows indicate AnkG expression in the AIS, which does not contain detectable TRPM4 immunolabeling. Scale bar: A = 20 μm; B–E = 10 μm. (F) Schematic figure showing the areas of drug perfusion and a DIC picture of the recorded area. Representative I/V curves (–80 to 80 mV, 0.16 mV/ms) recorded in the soma using local somatic (G) , medial, (H) , and distal dendrite (I) drug perfusion; black traces represent the basal current, red traces represent the effect of 10 μM 9-Ph. Right panels showed the quantification of the currents at –80 mV (arrow) ( n = 7). All experiments were performed in the presence of glutamatergic (CNQX, AP5) and GABAergic blockers (Picrotoxin).

Techniques: Expressing, Immunofluorescence, Labeling, Immunolabeling

TRPM4 expression at P0 and P7 in the mPFC. (A) Panoramic view of the frontal cortex in a coronal mouse brain section at P0, showing double immunofluorescence labeling or TRPM4 (green) and NeuN (orange). The inset shows a coronal map of the area. (B) Confocal images from the mPFC at P0 showing TRPM4 (top panel), NeuN (middle), and the merged signals (bottom). (C) Panoramic view of the frontal cortex in a coronal mouse brain section at P7, showing double immunofluorescence labeling for TRPM4 (green) and NeuN (orange), inset shows a coronal map of the area. (D) Confocal images from cortical layers of mPFC at P7 showing TRPM4 (top), NeuN (middle) and merged signals (bottom). Arrow shows expression in soma and neurites. Scale bar: A,C = 300 μm; B,D = 40 μm. (E) Representative I/V curve (–80 to 80 mV, 0.16 mV/ms) in perforated-patch configuration indicating the effect of 10 μM 9-Ph (whole bath perfusion). Black traces represent the basal current, red traces represent the effect of 10 μM 9-Ph and blue traces represent the effect of 10 mM EGTA. Right panel showed the quantification of the current at –80 mV (arrow) in P7 mice ( n = 8). All experiments were performed in the presence of glutamatergic (CNQX, AP5) and GABAergic blockers (Picrotoxin).

Journal: Frontiers in Cellular Neuroscience

Article Title: Subcellular Localization and Activity of TRPM4 in Medial Prefrontal Cortex Layer 2/3

doi: 10.3389/fncel.2018.00012

Figure Lengend Snippet: TRPM4 expression at P0 and P7 in the mPFC. (A) Panoramic view of the frontal cortex in a coronal mouse brain section at P0, showing double immunofluorescence labeling or TRPM4 (green) and NeuN (orange). The inset shows a coronal map of the area. (B) Confocal images from the mPFC at P0 showing TRPM4 (top panel), NeuN (middle), and the merged signals (bottom). (C) Panoramic view of the frontal cortex in a coronal mouse brain section at P7, showing double immunofluorescence labeling for TRPM4 (green) and NeuN (orange), inset shows a coronal map of the area. (D) Confocal images from cortical layers of mPFC at P7 showing TRPM4 (top), NeuN (middle) and merged signals (bottom). Arrow shows expression in soma and neurites. Scale bar: A,C = 300 μm; B,D = 40 μm. (E) Representative I/V curve (–80 to 80 mV, 0.16 mV/ms) in perforated-patch configuration indicating the effect of 10 μM 9-Ph (whole bath perfusion). Black traces represent the basal current, red traces represent the effect of 10 μM 9-Ph and blue traces represent the effect of 10 mM EGTA. Right panel showed the quantification of the current at –80 mV (arrow) in P7 mice ( n = 8). All experiments were performed in the presence of glutamatergic (CNQX, AP5) and GABAergic blockers (Picrotoxin).

Techniques: Expressing, Immunofluorescence, Labeling, Mouse Assay

TRPM4 expression at P14 and P90 in the mPFC. (A) Panoramic view of the frontal cortex in a coronal mouse brain section at P14, showing double-immunofluorescence labeling for TRPM4 (green) and NeuN (orange). The inset shows a coronal map of the area. (B) Confocal images from the mPFC at P14 showing TRPM4 (top panel), NeuN (middle), and the merged signals (bottom). (C) Panoramic view of the frontal cortex in a coronal mouse brain section at P90, showing double immunofluorescence labeling for TRPM4 (green) and NeuN (orange), inset shows a coronal map of the area. (D) Confocal images from cortical layers of mPFC at P90 showing TRPM4 (top), NeuN (middle), and the merged signals (bottom). (E) Representative I/V curves (–80 to 80 mV, 0.16 mV/ms) in perforated-patch configuration showing the effect of 10 μM 9-Ph (whole bath perfusion), right panel showed the quantification of the current at –80 mV (arrow) in P14 ( n = 7). (F) Representative I/V curve (–80 to 80 mV, 0.16 mV/ms) in perforated-patch configuration indicating the effect of 10 μM 9-Ph (whole bath perfusion), right panel showed the quantification of the current at P90 ( n = 8). The arrows in all images shows expression in soma and neurites, black arrows in the plots show the voltage at which currents were measured (–80 mV). Black traces represent the basal current, red traces represent the effect of 10 μM 9-Ph and blue traces represent the effect of 10 mM EGTA. All experiments were performed in the presence of glutamatergic (CNQX, AP5) and GABAergic blockers (Picrotoxin). Scale bar: A,C = 300 μm; B,D = 40 μm.

Journal: Frontiers in Cellular Neuroscience

Article Title: Subcellular Localization and Activity of TRPM4 in Medial Prefrontal Cortex Layer 2/3

doi: 10.3389/fncel.2018.00012

Figure Lengend Snippet: TRPM4 expression at P14 and P90 in the mPFC. (A) Panoramic view of the frontal cortex in a coronal mouse brain section at P14, showing double-immunofluorescence labeling for TRPM4 (green) and NeuN (orange). The inset shows a coronal map of the area. (B) Confocal images from the mPFC at P14 showing TRPM4 (top panel), NeuN (middle), and the merged signals (bottom). (C) Panoramic view of the frontal cortex in a coronal mouse brain section at P90, showing double immunofluorescence labeling for TRPM4 (green) and NeuN (orange), inset shows a coronal map of the area. (D) Confocal images from cortical layers of mPFC at P90 showing TRPM4 (top), NeuN (middle), and the merged signals (bottom). (E) Representative I/V curves (–80 to 80 mV, 0.16 mV/ms) in perforated-patch configuration showing the effect of 10 μM 9-Ph (whole bath perfusion), right panel showed the quantification of the current at –80 mV (arrow) in P14 ( n = 7). (F) Representative I/V curve (–80 to 80 mV, 0.16 mV/ms) in perforated-patch configuration indicating the effect of 10 μM 9-Ph (whole bath perfusion), right panel showed the quantification of the current at P90 ( n = 8). The arrows in all images shows expression in soma and neurites, black arrows in the plots show the voltage at which currents were measured (–80 mV). Black traces represent the basal current, red traces represent the effect of 10 μM 9-Ph and blue traces represent the effect of 10 mM EGTA. All experiments were performed in the presence of glutamatergic (CNQX, AP5) and GABAergic blockers (Picrotoxin). Scale bar: A,C = 300 μm; B,D = 40 μm.

Techniques: Expressing, Immunofluorescence, Labeling