trpm7  (Alomone Labs)


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    Structured Review

    Alomone Labs trpm7
    Possible association of zinc with neuronal death after pilocarpine-induced SE. This schematic drawing represents several chain reactions that may occur after carvacrol and 2-APB treatment in pilocarpine-induced SE. ( A ) These are the possible cellular pathways through which neuronal death occurs after pilocarpine-induced SE. ( B ) Blocking <t>TRPM7</t> by carvacrol and 2-APB can inhibit several chain reactions that are thought to occur following pilocarpine-induced SE.
    Trpm7, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trpm7/product/Alomone Labs
    Average 93 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    trpm7 - by Bioz Stars, 2022-12
    93/100 stars

    Images

    1) Product Images from "The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity"

    Article Title: The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21217897

    Possible association of zinc with neuronal death after pilocarpine-induced SE. This schematic drawing represents several chain reactions that may occur after carvacrol and 2-APB treatment in pilocarpine-induced SE. ( A ) These are the possible cellular pathways through which neuronal death occurs after pilocarpine-induced SE. ( B ) Blocking TRPM7 by carvacrol and 2-APB can inhibit several chain reactions that are thought to occur following pilocarpine-induced SE.
    Figure Legend Snippet: Possible association of zinc with neuronal death after pilocarpine-induced SE. This schematic drawing represents several chain reactions that may occur after carvacrol and 2-APB treatment in pilocarpine-induced SE. ( A ) These are the possible cellular pathways through which neuronal death occurs after pilocarpine-induced SE. ( B ) Blocking TRPM7 by carvacrol and 2-APB can inhibit several chain reactions that are thought to occur following pilocarpine-induced SE.

    Techniques Used: Blocking Assay

    2-APB treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 5–6 from each seizure group). * p
    Figure Legend Snippet: 2-APB treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 5–6 from each seizure group). * p

    Techniques Used: Over Expression, Immunofluorescence

    Carvacrol treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 7 from each seizure group). * p
    Figure Legend Snippet: Carvacrol treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 7 from each seizure group). * p

    Techniques Used: Over Expression, Immunofluorescence

    2) Product Images from "The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity"

    Article Title: The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21217897

    Possible association of zinc with neuronal death after pilocarpine-induced SE. This schematic drawing represents several chain reactions that may occur after carvacrol and 2-APB treatment in pilocarpine-induced SE. ( A ) These are the possible cellular pathways through which neuronal death occurs after pilocarpine-induced SE. ( B ) Blocking TRPM7 by carvacrol and 2-APB can inhibit several chain reactions that are thought to occur following pilocarpine-induced SE.
    Figure Legend Snippet: Possible association of zinc with neuronal death after pilocarpine-induced SE. This schematic drawing represents several chain reactions that may occur after carvacrol and 2-APB treatment in pilocarpine-induced SE. ( A ) These are the possible cellular pathways through which neuronal death occurs after pilocarpine-induced SE. ( B ) Blocking TRPM7 by carvacrol and 2-APB can inhibit several chain reactions that are thought to occur following pilocarpine-induced SE.

    Techniques Used: Blocking Assay

    2-APB treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 5–6 from each seizure group). * p
    Figure Legend Snippet: 2-APB treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 5–6 from each seizure group). * p

    Techniques Used: Over Expression, Immunofluorescence

    Carvacrol treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 7 from each seizure group). * p
    Figure Legend Snippet: Carvacrol treatment reduces TRPM7 overexpression, zinc accumulation, and neuronal degeneration after seizure. ( A ) Representative images showing TRPM7 immunoreactivity (green) in the CA1 of the hippocampus. Nuclei are counterstained with DAPI (blue). Scale bar = 20 µm. ( B ) The bar graph representing the immunofluorescence intensity of TRPM7 as determined in the same hippocampal region (mean ± SEM; n = 5 from each sham group, n = 7 from each seizure group). * p

    Techniques Used: Over Expression, Immunofluorescence

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    Alomone Labs anti trpm7 antibody
    CNNMs interact with <t>TRPM7</t> and regulate its activity. (A) HA-TRPM7 was coexpressed with FLAG-tagged CNNM1-4 in HEK-293T cells, and the channel was immunoprecipitated with HA-agarose. CNNM1 and CNNM3 strongly interacted with TRPM7. CNNM2 and CNNM4 also interacted with TRPM7, but lowered TRPM7 expression nonspecifically. CNNMs were not coimmunoprecipitated from HEK-293T cells N.T. with HA-TRPM7. Supplementation of the growth medium with 20 mM MgCl 2 boosted TRPM7 expression, revealing the channel’s capacity to interact with CNNM2 and CNNM4. (B) FLAG-tagged TRPM7 robustly coimmunoprecipitated native CNNM3 and CNNM4, whereas FLAG-TRPM2 did not. (C) A Zinc influx assay using the FluoZin-3 Zn 2 + indicator was used to monitor TRPM7 function in intact 293-TRPM7 cells (293-M7 cells), which express FLAG-tagged TRPM7 upon tetracycline treatment. Coexpression of CNNMs with TRPM7 increased intracellular free Zn 2 + more than expression of TRPM7 alone. The T-REx-293 cell line (293), which expresses the Tet Repressor protein, was used as the negative control. Shown are images taken at a time point between 5 and 10 minutes after application of 30 μM ZnCl 2 . White scale bar = 100 μM. (D) Quantification of the results from (C). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (E) Western blot showing expression of TRPM7 in the sample from (C). (F) Separate time course measurements were also acquired to further demonstrate that cells coexpressing CNNMs with TRPM7 have a higher rate of Zn 2+ influx compared to 293-TRPM7 cells expressing TRPM7 or CNNMs alone. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data . HBSS, Hanks’ balanced salt solution; N.T., not transfected.
    Anti Trpm7 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
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    anti trpm7 antibody - by Bioz Stars, 2022-12
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    CNNMs interact with TRPM7 and regulate its activity. (A) HA-TRPM7 was coexpressed with FLAG-tagged CNNM1-4 in HEK-293T cells, and the channel was immunoprecipitated with HA-agarose. CNNM1 and CNNM3 strongly interacted with TRPM7. CNNM2 and CNNM4 also interacted with TRPM7, but lowered TRPM7 expression nonspecifically. CNNMs were not coimmunoprecipitated from HEK-293T cells N.T. with HA-TRPM7. Supplementation of the growth medium with 20 mM MgCl 2 boosted TRPM7 expression, revealing the channel’s capacity to interact with CNNM2 and CNNM4. (B) FLAG-tagged TRPM7 robustly coimmunoprecipitated native CNNM3 and CNNM4, whereas FLAG-TRPM2 did not. (C) A Zinc influx assay using the FluoZin-3 Zn 2 + indicator was used to monitor TRPM7 function in intact 293-TRPM7 cells (293-M7 cells), which express FLAG-tagged TRPM7 upon tetracycline treatment. Coexpression of CNNMs with TRPM7 increased intracellular free Zn 2 + more than expression of TRPM7 alone. The T-REx-293 cell line (293), which expresses the Tet Repressor protein, was used as the negative control. Shown are images taken at a time point between 5 and 10 minutes after application of 30 μM ZnCl 2 . White scale bar = 100 μM. (D) Quantification of the results from (C). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (E) Western blot showing expression of TRPM7 in the sample from (C). (F) Separate time course measurements were also acquired to further demonstrate that cells coexpressing CNNMs with TRPM7 have a higher rate of Zn 2+ influx compared to 293-TRPM7 cells expressing TRPM7 or CNNMs alone. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data . HBSS, Hanks’ balanced salt solution; N.T., not transfected.

    Journal: PLoS Biology

    Article Title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

    doi: 10.1371/journal.pbio.3001496

    Figure Lengend Snippet: CNNMs interact with TRPM7 and regulate its activity. (A) HA-TRPM7 was coexpressed with FLAG-tagged CNNM1-4 in HEK-293T cells, and the channel was immunoprecipitated with HA-agarose. CNNM1 and CNNM3 strongly interacted with TRPM7. CNNM2 and CNNM4 also interacted with TRPM7, but lowered TRPM7 expression nonspecifically. CNNMs were not coimmunoprecipitated from HEK-293T cells N.T. with HA-TRPM7. Supplementation of the growth medium with 20 mM MgCl 2 boosted TRPM7 expression, revealing the channel’s capacity to interact with CNNM2 and CNNM4. (B) FLAG-tagged TRPM7 robustly coimmunoprecipitated native CNNM3 and CNNM4, whereas FLAG-TRPM2 did not. (C) A Zinc influx assay using the FluoZin-3 Zn 2 + indicator was used to monitor TRPM7 function in intact 293-TRPM7 cells (293-M7 cells), which express FLAG-tagged TRPM7 upon tetracycline treatment. Coexpression of CNNMs with TRPM7 increased intracellular free Zn 2 + more than expression of TRPM7 alone. The T-REx-293 cell line (293), which expresses the Tet Repressor protein, was used as the negative control. Shown are images taken at a time point between 5 and 10 minutes after application of 30 μM ZnCl 2 . White scale bar = 100 μM. (D) Quantification of the results from (C). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (E) Western blot showing expression of TRPM7 in the sample from (C). (F) Separate time course measurements were also acquired to further demonstrate that cells coexpressing CNNMs with TRPM7 have a higher rate of Zn 2+ influx compared to 293-TRPM7 cells expressing TRPM7 or CNNMs alone. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data . HBSS, Hanks’ balanced salt solution; N.T., not transfected.

    Article Snippet: Native TRPM7 was immunoprecipitated overnight using 2 μg Anti-TRPM7 antibody from Alomone Labs (#ACC-047; Jerusalem, Israel) bound to 40 μL Protein A agarose (Santa Cruz Biotechnology, Dallas, Texas, USA).

    Techniques: Activity Assay, Immunoprecipitation, Expressing, Negative Control, Western Blot, Fluorescence, Transfection

    Functional assessment of CNNMs on TRPM7 channel activity. TRPM7 currents were recorded in 293-TRPM7 cells ( n = 30), 293-M7-ΔCNNM3/4 cells ( n = 30), and 293-M7-ΔCNNM3/4 cells ( n = 40) with stable episomal expression of CNNM4 to keep CNNM4 protein levels low so that it would not interfere with TRPM7 protein expression. (A) Western blot demonstrating CNNM4 expression in the cell groups used in (B–D). (B) Representative TRPM7 whole-cell currents from the 3 cell lines recorded with an internal pipette solution containing 0 Mg 2+ /Mg-ATP to achieve full TRPM7 channel activity. (C) Average current density of the different groups from (A). (D) Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in cells from (B,C). White scale bar = 100 μM. Shown are images taken at a time point 5 to 10 minutes after application of 30 μM ZnCl 2 . (E) Quantification of results from (D). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (F) Native TRPM7 currents were recorded in the T-REx-293 Cell Line (293), which was the parental cell line of 293-ΔCNNM3/4 cells. Shown are representative TRPM7 whole-cell currents from the 2 cell lines recorded with an internal pipette solution containing 0 Mg 2+ /Mg-ATP to achieve full TRPM7 channel activity. (G) Average current density of the different groups from (F) ( n = 16 per group). Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data .

    Journal: PLoS Biology

    Article Title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

    doi: 10.1371/journal.pbio.3001496

    Figure Lengend Snippet: Functional assessment of CNNMs on TRPM7 channel activity. TRPM7 currents were recorded in 293-TRPM7 cells ( n = 30), 293-M7-ΔCNNM3/4 cells ( n = 30), and 293-M7-ΔCNNM3/4 cells ( n = 40) with stable episomal expression of CNNM4 to keep CNNM4 protein levels low so that it would not interfere with TRPM7 protein expression. (A) Western blot demonstrating CNNM4 expression in the cell groups used in (B–D). (B) Representative TRPM7 whole-cell currents from the 3 cell lines recorded with an internal pipette solution containing 0 Mg 2+ /Mg-ATP to achieve full TRPM7 channel activity. (C) Average current density of the different groups from (A). (D) Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in cells from (B,C). White scale bar = 100 μM. Shown are images taken at a time point 5 to 10 minutes after application of 30 μM ZnCl 2 . (E) Quantification of results from (D). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (F) Native TRPM7 currents were recorded in the T-REx-293 Cell Line (293), which was the parental cell line of 293-ΔCNNM3/4 cells. Shown are representative TRPM7 whole-cell currents from the 2 cell lines recorded with an internal pipette solution containing 0 Mg 2+ /Mg-ATP to achieve full TRPM7 channel activity. (G) Average current density of the different groups from (F) ( n = 16 per group). Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data .

    Article Snippet: Native TRPM7 was immunoprecipitated overnight using 2 μg Anti-TRPM7 antibody from Alomone Labs (#ACC-047; Jerusalem, Israel) bound to 40 μL Protein A agarose (Santa Cruz Biotechnology, Dallas, Texas, USA).

    Techniques: Functional Assay, Activity Assay, Expressing, Western Blot, Transferring

    CNNMs have TRPM7-dependent and independent activities. (A) A 25 Mg uptake assay was employed to assay TRPM7 channel function and its dependence on CNNMs. Moreover, 293-TRPM7 cells have significantly higher 25 Mg uptake than the negative control T-REx-293 cell line (293), which expresses the Tet Repressor protein. Compared to 293-TRPM7 cells, 293-M7-ΔCNNM3/4 exhibited significantly lower 25 Mg uptake, which was increased by reexpression of CNNM4. * = p

    Journal: PLoS Biology

    Article Title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

    doi: 10.1371/journal.pbio.3001496

    Figure Lengend Snippet: CNNMs have TRPM7-dependent and independent activities. (A) A 25 Mg uptake assay was employed to assay TRPM7 channel function and its dependence on CNNMs. Moreover, 293-TRPM7 cells have significantly higher 25 Mg uptake than the negative control T-REx-293 cell line (293), which expresses the Tet Repressor protein. Compared to 293-TRPM7 cells, 293-M7-ΔCNNM3/4 exhibited significantly lower 25 Mg uptake, which was increased by reexpression of CNNM4. * = p

    Article Snippet: Native TRPM7 was immunoprecipitated overnight using 2 μg Anti-TRPM7 antibody from Alomone Labs (#ACC-047; Jerusalem, Israel) bound to 40 μL Protein A agarose (Santa Cruz Biotechnology, Dallas, Texas, USA).

    Techniques: Negative Control

    CNNMs are required for TRPM7-mediated Zn 2+ influx. (A) A Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in intact cells under the same conditions described in Fig 1 . KO of CNNM3 , CNNM4 , and both CNNM3 and CNNM4 from 293-TRPM7 cells (293-M7-ΔCNNM3, 293-M7-ΔCNNM4, and 293-M7-ΔCNNM3/4) reduced TRPM7 channel function, which could be rescued to varying degrees by reexpression of CNNM3 and/or CNNM4. The T-REx-293 cell line (293), which expresses the Tet Repressor protein, was used as the negative control. All the cells in the assay were treated with tetracycline to induce TRPM7 expression. White scale bar = 100 μM. (B) Quantification of the results from (A). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. Images taken 5 to 10 minutes after application of 30 μM ZnCl 2 . (C) Separate time course measurements were also acquired to demonstrate that 293-TRPM7 cells lacking CNNMs have a reduced rate of Zn 2+ influx compared to 293-TRPM7 expressing TRPM7. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. (D) CRISPR/Cas-9 was used to KO CNNM3 , CNNM4 , and both CNNM3 4 from 293-TRPM7 cells, which express TRPM7. Western blotting of 2 independent clones showed specific KO of CNNM isoforms and similar expression of TRPM7 among the lines. Note that the antibody used to detected CNNM4 detects an unrelated protein as indicated. (E) Cell surface biotinylation of 293-TRPM7 WT and 293-M7-ΔCNNM3/4 cells demonstrated that surface level of TRPM7 in the 2 cell lines is similar. Shown are SA purified (SA purified) proteins, the input lysate for each cell type, and the proteins not bound to SA (SA unbound). (F) Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in intact cells. Shown are images taken at a time point between 5 and 10 minutes after application of 30 μM ZnCl 2 . Overexpression of PRL-2 stimulates TRPM7 channels function, which requires to varying degrees CNNM3 and CNNM4. White scale bar = 100 μM. (G) Western blot demonstrating expression of TRPM7 (Anti-FLAG) and PRL-2 under the conditions described in (F). (H) Quantification of the results from (F). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (I) Separate time course measurements were also acquired to demonstrate that PRL overexpression stimulates an increased rate of Zn 2+ influx compared to 293-TRPM7 expressing TRPM7 and that CNNM3 and CNNM4 are required for this activity. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data . HBSS, Hanks’ balanced salt solution; KO, knockout; PRL, phosphatase of regenerating liver; PRL-2, phosphatase of regenerating liver 2; SA, streptavidin agarose.

    Journal: PLoS Biology

    Article Title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

    doi: 10.1371/journal.pbio.3001496

    Figure Lengend Snippet: CNNMs are required for TRPM7-mediated Zn 2+ influx. (A) A Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in intact cells under the same conditions described in Fig 1 . KO of CNNM3 , CNNM4 , and both CNNM3 and CNNM4 from 293-TRPM7 cells (293-M7-ΔCNNM3, 293-M7-ΔCNNM4, and 293-M7-ΔCNNM3/4) reduced TRPM7 channel function, which could be rescued to varying degrees by reexpression of CNNM3 and/or CNNM4. The T-REx-293 cell line (293), which expresses the Tet Repressor protein, was used as the negative control. All the cells in the assay were treated with tetracycline to induce TRPM7 expression. White scale bar = 100 μM. (B) Quantification of the results from (A). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. Images taken 5 to 10 minutes after application of 30 μM ZnCl 2 . (C) Separate time course measurements were also acquired to demonstrate that 293-TRPM7 cells lacking CNNMs have a reduced rate of Zn 2+ influx compared to 293-TRPM7 expressing TRPM7. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. (D) CRISPR/Cas-9 was used to KO CNNM3 , CNNM4 , and both CNNM3 4 from 293-TRPM7 cells, which express TRPM7. Western blotting of 2 independent clones showed specific KO of CNNM isoforms and similar expression of TRPM7 among the lines. Note that the antibody used to detected CNNM4 detects an unrelated protein as indicated. (E) Cell surface biotinylation of 293-TRPM7 WT and 293-M7-ΔCNNM3/4 cells demonstrated that surface level of TRPM7 in the 2 cell lines is similar. Shown are SA purified (SA purified) proteins, the input lysate for each cell type, and the proteins not bound to SA (SA unbound). (F) Zinc influx assay using the FluoZin-3 Zn 2+ indicator was used to monitor TRPM7 function in intact cells. Shown are images taken at a time point between 5 and 10 minutes after application of 30 μM ZnCl 2 . Overexpression of PRL-2 stimulates TRPM7 channels function, which requires to varying degrees CNNM3 and CNNM4. White scale bar = 100 μM. (G) Western blot demonstrating expression of TRPM7 (Anti-FLAG) and PRL-2 under the conditions described in (F). (H) Quantification of the results from (F). A total of 100 cells were randomly selected for quantification. n = 100. * indicates a p -value of less than 0.05. (I) Separate time course measurements were also acquired to demonstrate that PRL overexpression stimulates an increased rate of Zn 2+ influx compared to 293-TRPM7 expressing TRPM7 and that CNNM3 and CNNM4 are required for this activity. HBSS media was replaced with HBSS containing 30 μM ZnCl 2 for the period indicated. The fluorescence intensity of the cells (mean of 50 cells) were quantified for each time point. Unprocessed images of blots are shown in S1 Raw Images . The underlying data for this figure can be found in S1 Data . HBSS, Hanks’ balanced salt solution; KO, knockout; PRL, phosphatase of regenerating liver; PRL-2, phosphatase of regenerating liver 2; SA, streptavidin agarose.

    Article Snippet: Native TRPM7 was immunoprecipitated overnight using 2 μg Anti-TRPM7 antibody from Alomone Labs (#ACC-047; Jerusalem, Israel) bound to 40 μL Protein A agarose (Santa Cruz Biotechnology, Dallas, Texas, USA).

    Techniques: Negative Control, Expressing, Fluorescence, CRISPR, Western Blot, Clone Assay, Purification, Over Expression, Activity Assay, Knock-Out

    Immunofluorescence images suggesting the presence of TRPM6 and TRPM7 proteins in pig cardiomyocytes from different cardiac chamber walls. ( A – D ) Immunofluorescence of TRPM7 (left) and TRPM6 (right) in the left atrium (LA), right atrium (RA), left ventricle (LV), and right ventricle (RV) cardiomyocytes when using Alexa Fluor 488 for the TRPM7 and TRPM6 proteins (stained in green), Alexa Fluor 546 for the F-actin cytoskeleton (stained in red), and Hoechst 33342 for the nuclei (stained in blue). Scale bars indicate 20 µm. ( E ) Example of a negative control, where the primary antibody for TRPM6 and/or TRPM7 is not added, but the cardiomyocyte was subjected to Hoechst 33342 and Alexa Fluor 546. Under such conditions, only immunofluorescence of the nuclei (stained in blue) and F actin cytoskeleton (stained in red) is detected. Note: same cardiomyocyte in the left and right (merged image) panels ( F ) Quantification of the staining intensity of the immunodetected fluorescence of TRPM7 and TRPM6 in the four cardiac chamber walls: LA, RA, LV, and RV. The mean data is provided in arbitrary units (a.u.) (see Table 1 ). A blinded study design (with the origin or treatment of cells unknown to the investigator) was used for the detection of immunofluorescence during the various experimental conditions.

    Journal: International Journal of Molecular Sciences

    Article Title: Modulation of the Cardiac Myocyte Action Potential by the Magnesium-Sensitive TRPM6 and TRPM7-like Current

    doi: 10.3390/ijms22168744

    Figure Lengend Snippet: Immunofluorescence images suggesting the presence of TRPM6 and TRPM7 proteins in pig cardiomyocytes from different cardiac chamber walls. ( A – D ) Immunofluorescence of TRPM7 (left) and TRPM6 (right) in the left atrium (LA), right atrium (RA), left ventricle (LV), and right ventricle (RV) cardiomyocytes when using Alexa Fluor 488 for the TRPM7 and TRPM6 proteins (stained in green), Alexa Fluor 546 for the F-actin cytoskeleton (stained in red), and Hoechst 33342 for the nuclei (stained in blue). Scale bars indicate 20 µm. ( E ) Example of a negative control, where the primary antibody for TRPM6 and/or TRPM7 is not added, but the cardiomyocyte was subjected to Hoechst 33342 and Alexa Fluor 546. Under such conditions, only immunofluorescence of the nuclei (stained in blue) and F actin cytoskeleton (stained in red) is detected. Note: same cardiomyocyte in the left and right (merged image) panels ( F ) Quantification of the staining intensity of the immunodetected fluorescence of TRPM7 and TRPM6 in the four cardiac chamber walls: LA, RA, LV, and RV. The mean data is provided in arbitrary units (a.u.) (see Table 1 ). A blinded study design (with the origin or treatment of cells unknown to the investigator) was used for the detection of immunofluorescence during the various experimental conditions.

    Article Snippet: The TRPM6 and TRPM7 antibodies were obtained from the same company in order to minimize the possibility of cross-reactivity during immunolabeling.

    Techniques: Immunofluorescence, Staining, Negative Control, Fluorescence

    Comparison of the expression of TRPM6 and TRPM7 in left ventricular cardiomyocytes incubated for 2 h vs. 12 h in extracellular solutions with ( A , B ) and without ( C , D ) divalent cations (DV and DVF, respectively). ( A , C ) The cardiomyocytes were fixed after 2 h (filled columns) or 12 h (unfilled columns) of cell isolation: Alexa Fluor 488 for the TRPM7 and TRPM6 proteins (stained in green), Alexa Fluor 546 for the F-actin cytoskeleton (stained in red), and Hoechst 33342 for the nuclei (stained in blue). Scale bars indicate 20 µm. ( B , D ) Quantification of the intensity of the fluorescence expressed in arbitrary units (a.u.). # p

    Journal: International Journal of Molecular Sciences

    Article Title: Modulation of the Cardiac Myocyte Action Potential by the Magnesium-Sensitive TRPM6 and TRPM7-like Current

    doi: 10.3390/ijms22168744

    Figure Lengend Snippet: Comparison of the expression of TRPM6 and TRPM7 in left ventricular cardiomyocytes incubated for 2 h vs. 12 h in extracellular solutions with ( A , B ) and without ( C , D ) divalent cations (DV and DVF, respectively). ( A , C ) The cardiomyocytes were fixed after 2 h (filled columns) or 12 h (unfilled columns) of cell isolation: Alexa Fluor 488 for the TRPM7 and TRPM6 proteins (stained in green), Alexa Fluor 546 for the F-actin cytoskeleton (stained in red), and Hoechst 33342 for the nuclei (stained in blue). Scale bars indicate 20 µm. ( B , D ) Quantification of the intensity of the fluorescence expressed in arbitrary units (a.u.). # p

    Article Snippet: The TRPM6 and TRPM7 antibodies were obtained from the same company in order to minimize the possibility of cross-reactivity during immunolabeling.

    Techniques: Expressing, Incubation, Cell Isolation, Staining, Fluorescence