anti trpm2 antibodies  (Alomone Labs)


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    Name:
    Anti TRPM2 Antibody
    Description:
    Anti TRPM2 Antibody ACC 043 is a highly specific antibody directed against an intracellular epitope of mouse TRPM2 The antibody can be used in western blot immunocytochemistry and immunohistochemistry applications It has been designed to recognize TRPM2 from mouse rat and human samples
    Catalog Number:
    ACC-043
    Price:
    495.0
    Category:
    Primary Antibody
    Applications:
    Immunocytochemistry, Immunofluorescence, Immunohistochemistry, Western Blot
    Purity:
    Affinity purified on immobilized antigen.
    Immunogen:
    Synthetic peptide
    Size:
    50 mcl
    Antibody Type:
    Polyclonal Primary Antibodies
    Format:
    Lyophilized Powder
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
    Buy from Supplier


    Structured Review

    Alomone Labs anti trpm2 antibodies
    Anti TRPM2 Antibody
    Anti TRPM2 Antibody ACC 043 is a highly specific antibody directed against an intracellular epitope of mouse TRPM2 The antibody can be used in western blot immunocytochemistry and immunohistochemistry applications It has been designed to recognize TRPM2 from mouse rat and human samples
    https://www.bioz.com/result/anti trpm2 antibodies/product/Alomone Labs
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti trpm2 antibodies - by Bioz Stars, 2021-09
    88/100 stars

    Images

    1) Product Images from "The key role of transient receptor potential melastatin-2 channels in amyloid-β-induced neurovascular dysfunction"

    Article Title: The key role of transient receptor potential melastatin-2 channels in amyloid-β-induced neurovascular dysfunction

    Journal: Nature communications

    doi: 10.1038/ncomms6318

    Aβ 1–40 activates sustained TRPM2 currents in brain endothelial cells for the specificity of the immunostain) ( a ). Aβ 1–40 (Aβ) and the channel activator ADPR induce inward currents with the conductance characteristics of TRPM2 currents ( b ). The currents are blocked by the TRPM2 inhibitor 2-APB or 2-ACA, or TRPM2 knockdown ( b–d ). Aβ-induced TRPM2 currents are antagonized by the ROS scavenger MnTBAP, the NADPH oxidase peptide inhibitor gp91ds-tat, the NOS inhibitor L-NNA, the PARP inhibitor PJ34 and the PARG inhibitor ADP-HPD. ADPR-induced TRPM2 currents are unaffected by these antagonists. Data are presented as mean ± s.e.m. * P
    Figure Legend Snippet: Aβ 1–40 activates sustained TRPM2 currents in brain endothelial cells for the specificity of the immunostain) ( a ). Aβ 1–40 (Aβ) and the channel activator ADPR induce inward currents with the conductance characteristics of TRPM2 currents ( b ). The currents are blocked by the TRPM2 inhibitor 2-APB or 2-ACA, or TRPM2 knockdown ( b–d ). Aβ-induced TRPM2 currents are antagonized by the ROS scavenger MnTBAP, the NADPH oxidase peptide inhibitor gp91ds-tat, the NOS inhibitor L-NNA, the PARP inhibitor PJ34 and the PARG inhibitor ADP-HPD. ADPR-induced TRPM2 currents are unaffected by these antagonists. Data are presented as mean ± s.e.m. * P

    Techniques Used:

    Aβ 1–40 -induced neurovascular dysfunction is not observed in TRPM2-null mice Neocortical superfusion of Aβ 1–40 (Aβ) does not attenuate resting CBF ( a ), or the increase in CBF induced by whisker stimulation ( b ) or acetylcholine ( c ) in TRPM2-null mice. Responses to adenosine were not altered ( d ). Data are presented as mean ± s.e.m. * P
    Figure Legend Snippet: Aβ 1–40 -induced neurovascular dysfunction is not observed in TRPM2-null mice Neocortical superfusion of Aβ 1–40 (Aβ) does not attenuate resting CBF ( a ), or the increase in CBF induced by whisker stimulation ( b ) or acetylcholine ( c ) in TRPM2-null mice. Responses to adenosine were not altered ( d ). Data are presented as mean ± s.e.m. * P

    Techniques Used: Mouse Assay, Whisker Assay

    TRPM2 inhibition rescues the endothelial dysfunction induced by Aβ 1–40 in vivo Neocortical application of 2-APB or ACA has no effect on resting CBF ( a ), but it prevents the reduction in resting CBF induced by neocortical superfusion of Aβ 1–40 (Aβ) ( a ). 2-APB or ACA also rescues the attenuation in the CBF increase evoked by whisker stimulation ( b ) or acetylcholine ( c ) induced by Aβ 1–40 or observed in tg2576 mice. The CBF increase induced by adenosine is unaffected ( d ). Data are presented as mean ± s.e.m. * P
    Figure Legend Snippet: TRPM2 inhibition rescues the endothelial dysfunction induced by Aβ 1–40 in vivo Neocortical application of 2-APB or ACA has no effect on resting CBF ( a ), but it prevents the reduction in resting CBF induced by neocortical superfusion of Aβ 1–40 (Aβ) ( a ). 2-APB or ACA also rescues the attenuation in the CBF increase evoked by whisker stimulation ( b ) or acetylcholine ( c ) induced by Aβ 1–40 or observed in tg2576 mice. The CBF increase induced by adenosine is unaffected ( d ). Data are presented as mean ± s.e.m. * P

    Techniques Used: Inhibition, In Vivo, Whisker Assay, Mouse Assay

    Presumed signalling pathways through which Aβ 1–40 activates endothelial TRPM2 channels Aβ 1–40 (Aβ) activates the innate immunity receptor CD36 leading to production of superoxide via NADPH oxidase. Superoxide reacts with NO, made continuously in endothelial cells, to form peroxynitrite (PN). PN induces DNA damage, which, in turn, activates PARP. ADPR formation by PARG cleavage of PAR activates the Nudix (Nu) domain of TRPM2 leading to massive increases in intracellular Ca 2+ , which induce endothelial dysfunction. However, the involvement of other TRPM2-permeable anions, such as Na + , cannot be ruled out. In a multicellular context, for example, in vivo , PN, a diffusible agent, could also be produced by other vascular cells, and diffuse into endothelial cells to activate this pathway.
    Figure Legend Snippet: Presumed signalling pathways through which Aβ 1–40 activates endothelial TRPM2 channels Aβ 1–40 (Aβ) activates the innate immunity receptor CD36 leading to production of superoxide via NADPH oxidase. Superoxide reacts with NO, made continuously in endothelial cells, to form peroxynitrite (PN). PN induces DNA damage, which, in turn, activates PARP. ADPR formation by PARG cleavage of PAR activates the Nudix (Nu) domain of TRPM2 leading to massive increases in intracellular Ca 2+ , which induce endothelial dysfunction. However, the involvement of other TRPM2-permeable anions, such as Na + , cannot be ruled out. In a multicellular context, for example, in vivo , PN, a diffusible agent, could also be produced by other vascular cells, and diffuse into endothelial cells to activate this pathway.

    Techniques Used: In Vivo, Produced

    Aβ 1–40 induces large and sustained increases in intracellular Ca2þ via TRPM2 channels in brain endothelial cells The Aβ 1–40 are attenuated by the mechanistically distinct TRPM2 inhibitors 2-APB and ACA ( a,c ) or by TRPM2 knockdown using siRNA, but not control siRNA (control si) ( b,d ). Data are presented as mean ± s.e.m. * P
    Figure Legend Snippet: Aβ 1–40 induces large and sustained increases in intracellular Ca2þ via TRPM2 channels in brain endothelial cells The Aβ 1–40 are attenuated by the mechanistically distinct TRPM2 inhibitors 2-APB and ACA ( a,c ) or by TRPM2 knockdown using siRNA, but not control siRNA (control si) ( b,d ). Data are presented as mean ± s.e.m. * P

    Techniques Used:

    2) Product Images from "Prolactin protects retinal pigment epithelium by inhibiting sirtuin 2-dependent cell death"

    Article Title: Prolactin protects retinal pigment epithelium by inhibiting sirtuin 2-dependent cell death

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2016.03.048

    Schematic depiction of the antiapoptotic conditions including the PRL signaling pathway in young RPE versus the proapoptotic conditions in aging RPE. Young RPE cells express PRL and its receptor (PRLR) that (1) signal for the synthesis of reduced glutathione (GSH) that transforms two molecules of hydrogen peroxide (H 2 O 2 ) into water and (2) induces the transcription of catalase, which also processes H 2 O 2 into water; both of these processes limit ROS levels. This antioxidant pathway is more prominent than the pro-oxidant pathway comprised by the TRPM2-mediated intracellular Ca 2+ rise induced by the metabolite 2′O-acetyl-ADP-ribose (O-Ac-ADPR) that results from the deacetylation (removal of Ac group) of various substrates by SIRT2. The viability of RPE is therefore not jeopardized. In contrast, in aged RPE cells, the PRL signaling pathway is blunted. Therefore, the pro-oxidant pathway predominates over the antioxidant one. High levels of ROS, illustrated by increased H 2 O 2 , promote the up-regulation of SIRT2 which, in turn, activates TRPM2 by producing O-Ac-ADPR. The subsequent sustained rise in intracellular Ca 2+ levels can depolarize mitochondria ( Berridge et al., 2000 ), which causes apoptosis. RPE from PRLR null ( −/− ) mice emphasizes these features.
    Figure Legend Snippet: Schematic depiction of the antiapoptotic conditions including the PRL signaling pathway in young RPE versus the proapoptotic conditions in aging RPE. Young RPE cells express PRL and its receptor (PRLR) that (1) signal for the synthesis of reduced glutathione (GSH) that transforms two molecules of hydrogen peroxide (H 2 O 2 ) into water and (2) induces the transcription of catalase, which also processes H 2 O 2 into water; both of these processes limit ROS levels. This antioxidant pathway is more prominent than the pro-oxidant pathway comprised by the TRPM2-mediated intracellular Ca 2+ rise induced by the metabolite 2′O-acetyl-ADP-ribose (O-Ac-ADPR) that results from the deacetylation (removal of Ac group) of various substrates by SIRT2. The viability of RPE is therefore not jeopardized. In contrast, in aged RPE cells, the PRL signaling pathway is blunted. Therefore, the pro-oxidant pathway predominates over the antioxidant one. High levels of ROS, illustrated by increased H 2 O 2 , promote the up-regulation of SIRT2 which, in turn, activates TRPM2 by producing O-Ac-ADPR. The subsequent sustained rise in intracellular Ca 2+ levels can depolarize mitochondria ( Berridge et al., 2000 ), which causes apoptosis. RPE from PRLR null ( −/− ) mice emphasizes these features.

    Techniques Used: Mouse Assay

    PRL maintains human RPE cell survival by inhibiting the oxidant-induced SIRT2-dependent induction of TRPM2-mediated intracellular Ca 2+ increase. (a) RT-PCR and (b) Western-blotting of TRPM2 in ARPE-19 cell lysates. (a) TBP was used as a positive control. bp, DNA ladder. RT-PCR was performed in RNA extracted from three independent cell cultures (N = 3). (b) ARPE-19 cells were untreated (Ctl), subjected to lipofectamin alone (Lipo.) or transfected with siRNA against TRPM2 (siRNA) or scramble sequence (Scr.). TRPM2 siRNA efficiently reduced TRPM2 expression as observed by immunoblotting using an anti-TRPM2 antibody that labeled a protein at the expected molecular weight for TRPM2 (171 kDa). β-Actin served as loading control. Extracts from three independent ARPE-19 cell cultures in each condition were analyzed (N = 3). (c, d) Measurement of the change in intracellular Ca 2+ measured by the change in fluo-8 fluorescence (Δ fluorescence) in ARPE19 cells exposed to (c) H 2 O 2 (100 μM) or (d) piceatannol (10 μM) while TRPM2 was blocked by siRNA against TRPM2 or PRL was applied (hPRL, 100 pmol/l, 15-min pretreatment). Scramble sequence (siRNA ctl) was used as a negative control for siRNA against TRPM2. Treatments with H 2 O 2 or piceatannol began at time = 0 s ( n = 170–190 cells; N = 3 independent replicates). (e) Ca 2+ -dependent fluorescence change in ARPE19 cells 180 s after application of SIRT2 inhibitor AGK2 (10 μM) combined or not with H 2 O 2 (100 μM) ( n = 160–190 cells; N = 3 independent replicates). (f) Effect of TRPM2 inhibition on survival of ARPE-19 subjected to a 24-h H 2 O 2 insult (100 μM) by MTT assay. ARPE-19 cells were untreated (Ctl), treated with lipofectamin alone (Lipo.) or transfected with siRNA against TRPM2 or the scramble sequence (Scr.) 24 h prior initiating the MTT assay. In (c–e), signals were normalized by subtracting the Fluo-8 Δ fluorescence to the one in untreated conditions. All bar plots, mean plus S.E.M.; P values: ANOVA and Bonferroni post-hoc test.
    Figure Legend Snippet: PRL maintains human RPE cell survival by inhibiting the oxidant-induced SIRT2-dependent induction of TRPM2-mediated intracellular Ca 2+ increase. (a) RT-PCR and (b) Western-blotting of TRPM2 in ARPE-19 cell lysates. (a) TBP was used as a positive control. bp, DNA ladder. RT-PCR was performed in RNA extracted from three independent cell cultures (N = 3). (b) ARPE-19 cells were untreated (Ctl), subjected to lipofectamin alone (Lipo.) or transfected with siRNA against TRPM2 (siRNA) or scramble sequence (Scr.). TRPM2 siRNA efficiently reduced TRPM2 expression as observed by immunoblotting using an anti-TRPM2 antibody that labeled a protein at the expected molecular weight for TRPM2 (171 kDa). β-Actin served as loading control. Extracts from three independent ARPE-19 cell cultures in each condition were analyzed (N = 3). (c, d) Measurement of the change in intracellular Ca 2+ measured by the change in fluo-8 fluorescence (Δ fluorescence) in ARPE19 cells exposed to (c) H 2 O 2 (100 μM) or (d) piceatannol (10 μM) while TRPM2 was blocked by siRNA against TRPM2 or PRL was applied (hPRL, 100 pmol/l, 15-min pretreatment). Scramble sequence (siRNA ctl) was used as a negative control for siRNA against TRPM2. Treatments with H 2 O 2 or piceatannol began at time = 0 s ( n = 170–190 cells; N = 3 independent replicates). (e) Ca 2+ -dependent fluorescence change in ARPE19 cells 180 s after application of SIRT2 inhibitor AGK2 (10 μM) combined or not with H 2 O 2 (100 μM) ( n = 160–190 cells; N = 3 independent replicates). (f) Effect of TRPM2 inhibition on survival of ARPE-19 subjected to a 24-h H 2 O 2 insult (100 μM) by MTT assay. ARPE-19 cells were untreated (Ctl), treated with lipofectamin alone (Lipo.) or transfected with siRNA against TRPM2 or the scramble sequence (Scr.) 24 h prior initiating the MTT assay. In (c–e), signals were normalized by subtracting the Fluo-8 Δ fluorescence to the one in untreated conditions. All bar plots, mean plus S.E.M.; P values: ANOVA and Bonferroni post-hoc test.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control, CTL Assay, Transfection, Sequencing, Expressing, Labeling, Molecular Weight, Fluorescence, Negative Control, Inhibition, MTT Assay

    Related Articles

    Western Blot:

    Article Title: The key role of transient receptor potential melastatin-2 channels in amyloid-β-induced neurovascular dysfunction
    Article Snippet: .. The reduction of TRPM2 protein in whole-cell lysates was confirmed with western blot analysis using anti-TRPM2 antibodies (1:200; ant-rabbit; alomone labs). ..

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    Alomone Labs anti trpm2 antibodies
    Aβ 1–40 activates sustained <t>TRPM2</t> currents in brain endothelial cells for the specificity of the immunostain) ( a ). Aβ 1–40 (Aβ) and the channel activator ADPR induce inward currents with the conductance characteristics of TRPM2 currents ( b ). The currents are blocked by the TRPM2 inhibitor 2-APB or 2-ACA, or TRPM2 knockdown ( b–d ). Aβ-induced TRPM2 currents are antagonized by the ROS scavenger MnTBAP, the NADPH oxidase peptide inhibitor gp91ds-tat, the NOS inhibitor L-NNA, the PARP inhibitor PJ34 and the PARG inhibitor ADP-HPD. ADPR-induced TRPM2 currents are unaffected by these antagonists. Data are presented as mean ± s.e.m. * P
    Anti Trpm2 Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti trpm2 antibodies/product/Alomone Labs
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti trpm2 antibodies - by Bioz Stars, 2021-09
    88/100 stars
      Buy from Supplier

    93
    Alomone Labs anti trpm2 atto 594
    Aβ 1–40 activates sustained <t>TRPM2</t> currents in brain endothelial cells for the specificity of the immunostain) ( a ). Aβ 1–40 (Aβ) and the channel activator ADPR induce inward currents with the conductance characteristics of TRPM2 currents ( b ). The currents are blocked by the TRPM2 inhibitor 2-APB or 2-ACA, or TRPM2 knockdown ( b–d ). Aβ-induced TRPM2 currents are antagonized by the ROS scavenger MnTBAP, the NADPH oxidase peptide inhibitor gp91ds-tat, the NOS inhibitor L-NNA, the PARP inhibitor PJ34 and the PARG inhibitor ADP-HPD. ADPR-induced TRPM2 currents are unaffected by these antagonists. Data are presented as mean ± s.e.m. * P
    Anti Trpm2 Atto 594, 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
    https://www.bioz.com/result/anti trpm2 atto 594/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti trpm2 atto 594 - by Bioz Stars, 2021-09
    93/100 stars
      Buy from Supplier

    Image Search Results


    Aβ 1–40 activates sustained TRPM2 currents in brain endothelial cells for the specificity of the immunostain) ( a ). Aβ 1–40 (Aβ) and the channel activator ADPR induce inward currents with the conductance characteristics of TRPM2 currents ( b ). The currents are blocked by the TRPM2 inhibitor 2-APB or 2-ACA, or TRPM2 knockdown ( b–d ). Aβ-induced TRPM2 currents are antagonized by the ROS scavenger MnTBAP, the NADPH oxidase peptide inhibitor gp91ds-tat, the NOS inhibitor L-NNA, the PARP inhibitor PJ34 and the PARG inhibitor ADP-HPD. ADPR-induced TRPM2 currents are unaffected by these antagonists. Data are presented as mean ± s.e.m. * P

    Journal: Nature communications

    Article Title: The key role of transient receptor potential melastatin-2 channels in amyloid-β-induced neurovascular dysfunction

    doi: 10.1038/ncomms6318

    Figure Lengend Snippet: Aβ 1–40 activates sustained TRPM2 currents in brain endothelial cells for the specificity of the immunostain) ( a ). Aβ 1–40 (Aβ) and the channel activator ADPR induce inward currents with the conductance characteristics of TRPM2 currents ( b ). The currents are blocked by the TRPM2 inhibitor 2-APB or 2-ACA, or TRPM2 knockdown ( b–d ). Aβ-induced TRPM2 currents are antagonized by the ROS scavenger MnTBAP, the NADPH oxidase peptide inhibitor gp91ds-tat, the NOS inhibitor L-NNA, the PARP inhibitor PJ34 and the PARG inhibitor ADP-HPD. ADPR-induced TRPM2 currents are unaffected by these antagonists. Data are presented as mean ± s.e.m. * P

    Article Snippet: The reduction of TRPM2 protein in whole-cell lysates was confirmed with western blot analysis using anti-TRPM2 antibodies (1:200; ant-rabbit; alomone labs).

    Techniques:

    Aβ 1–40 -induced neurovascular dysfunction is not observed in TRPM2-null mice Neocortical superfusion of Aβ 1–40 (Aβ) does not attenuate resting CBF ( a ), or the increase in CBF induced by whisker stimulation ( b ) or acetylcholine ( c ) in TRPM2-null mice. Responses to adenosine were not altered ( d ). Data are presented as mean ± s.e.m. * P

    Journal: Nature communications

    Article Title: The key role of transient receptor potential melastatin-2 channels in amyloid-β-induced neurovascular dysfunction

    doi: 10.1038/ncomms6318

    Figure Lengend Snippet: Aβ 1–40 -induced neurovascular dysfunction is not observed in TRPM2-null mice Neocortical superfusion of Aβ 1–40 (Aβ) does not attenuate resting CBF ( a ), or the increase in CBF induced by whisker stimulation ( b ) or acetylcholine ( c ) in TRPM2-null mice. Responses to adenosine were not altered ( d ). Data are presented as mean ± s.e.m. * P

    Article Snippet: The reduction of TRPM2 protein in whole-cell lysates was confirmed with western blot analysis using anti-TRPM2 antibodies (1:200; ant-rabbit; alomone labs).

    Techniques: Mouse Assay, Whisker Assay

    TRPM2 inhibition rescues the endothelial dysfunction induced by Aβ 1–40 in vivo Neocortical application of 2-APB or ACA has no effect on resting CBF ( a ), but it prevents the reduction in resting CBF induced by neocortical superfusion of Aβ 1–40 (Aβ) ( a ). 2-APB or ACA also rescues the attenuation in the CBF increase evoked by whisker stimulation ( b ) or acetylcholine ( c ) induced by Aβ 1–40 or observed in tg2576 mice. The CBF increase induced by adenosine is unaffected ( d ). Data are presented as mean ± s.e.m. * P

    Journal: Nature communications

    Article Title: The key role of transient receptor potential melastatin-2 channels in amyloid-β-induced neurovascular dysfunction

    doi: 10.1038/ncomms6318

    Figure Lengend Snippet: TRPM2 inhibition rescues the endothelial dysfunction induced by Aβ 1–40 in vivo Neocortical application of 2-APB or ACA has no effect on resting CBF ( a ), but it prevents the reduction in resting CBF induced by neocortical superfusion of Aβ 1–40 (Aβ) ( a ). 2-APB or ACA also rescues the attenuation in the CBF increase evoked by whisker stimulation ( b ) or acetylcholine ( c ) induced by Aβ 1–40 or observed in tg2576 mice. The CBF increase induced by adenosine is unaffected ( d ). Data are presented as mean ± s.e.m. * P

    Article Snippet: The reduction of TRPM2 protein in whole-cell lysates was confirmed with western blot analysis using anti-TRPM2 antibodies (1:200; ant-rabbit; alomone labs).

    Techniques: Inhibition, In Vivo, Whisker Assay, Mouse Assay

    Presumed signalling pathways through which Aβ 1–40 activates endothelial TRPM2 channels Aβ 1–40 (Aβ) activates the innate immunity receptor CD36 leading to production of superoxide via NADPH oxidase. Superoxide reacts with NO, made continuously in endothelial cells, to form peroxynitrite (PN). PN induces DNA damage, which, in turn, activates PARP. ADPR formation by PARG cleavage of PAR activates the Nudix (Nu) domain of TRPM2 leading to massive increases in intracellular Ca 2+ , which induce endothelial dysfunction. However, the involvement of other TRPM2-permeable anions, such as Na + , cannot be ruled out. In a multicellular context, for example, in vivo , PN, a diffusible agent, could also be produced by other vascular cells, and diffuse into endothelial cells to activate this pathway.

    Journal: Nature communications

    Article Title: The key role of transient receptor potential melastatin-2 channels in amyloid-β-induced neurovascular dysfunction

    doi: 10.1038/ncomms6318

    Figure Lengend Snippet: Presumed signalling pathways through which Aβ 1–40 activates endothelial TRPM2 channels Aβ 1–40 (Aβ) activates the innate immunity receptor CD36 leading to production of superoxide via NADPH oxidase. Superoxide reacts with NO, made continuously in endothelial cells, to form peroxynitrite (PN). PN induces DNA damage, which, in turn, activates PARP. ADPR formation by PARG cleavage of PAR activates the Nudix (Nu) domain of TRPM2 leading to massive increases in intracellular Ca 2+ , which induce endothelial dysfunction. However, the involvement of other TRPM2-permeable anions, such as Na + , cannot be ruled out. In a multicellular context, for example, in vivo , PN, a diffusible agent, could also be produced by other vascular cells, and diffuse into endothelial cells to activate this pathway.

    Article Snippet: The reduction of TRPM2 protein in whole-cell lysates was confirmed with western blot analysis using anti-TRPM2 antibodies (1:200; ant-rabbit; alomone labs).

    Techniques: In Vivo, Produced

    Aβ 1–40 induces large and sustained increases in intracellular Ca2þ via TRPM2 channels in brain endothelial cells The Aβ 1–40 are attenuated by the mechanistically distinct TRPM2 inhibitors 2-APB and ACA ( a,c ) or by TRPM2 knockdown using siRNA, but not control siRNA (control si) ( b,d ). Data are presented as mean ± s.e.m. * P

    Journal: Nature communications

    Article Title: The key role of transient receptor potential melastatin-2 channels in amyloid-β-induced neurovascular dysfunction

    doi: 10.1038/ncomms6318

    Figure Lengend Snippet: Aβ 1–40 induces large and sustained increases in intracellular Ca2þ via TRPM2 channels in brain endothelial cells The Aβ 1–40 are attenuated by the mechanistically distinct TRPM2 inhibitors 2-APB and ACA ( a,c ) or by TRPM2 knockdown using siRNA, but not control siRNA (control si) ( b,d ). Data are presented as mean ± s.e.m. * P

    Article Snippet: The reduction of TRPM2 protein in whole-cell lysates was confirmed with western blot analysis using anti-TRPM2 antibodies (1:200; ant-rabbit; alomone labs).

    Techniques:

    Schematic depiction of the antiapoptotic conditions including the PRL signaling pathway in young RPE versus the proapoptotic conditions in aging RPE. Young RPE cells express PRL and its receptor (PRLR) that (1) signal for the synthesis of reduced glutathione (GSH) that transforms two molecules of hydrogen peroxide (H 2 O 2 ) into water and (2) induces the transcription of catalase, which also processes H 2 O 2 into water; both of these processes limit ROS levels. This antioxidant pathway is more prominent than the pro-oxidant pathway comprised by the TRPM2-mediated intracellular Ca 2+ rise induced by the metabolite 2′O-acetyl-ADP-ribose (O-Ac-ADPR) that results from the deacetylation (removal of Ac group) of various substrates by SIRT2. The viability of RPE is therefore not jeopardized. In contrast, in aged RPE cells, the PRL signaling pathway is blunted. Therefore, the pro-oxidant pathway predominates over the antioxidant one. High levels of ROS, illustrated by increased H 2 O 2 , promote the up-regulation of SIRT2 which, in turn, activates TRPM2 by producing O-Ac-ADPR. The subsequent sustained rise in intracellular Ca 2+ levels can depolarize mitochondria ( Berridge et al., 2000 ), which causes apoptosis. RPE from PRLR null ( −/− ) mice emphasizes these features.

    Journal: EBioMedicine

    Article Title: Prolactin protects retinal pigment epithelium by inhibiting sirtuin 2-dependent cell death

    doi: 10.1016/j.ebiom.2016.03.048

    Figure Lengend Snippet: Schematic depiction of the antiapoptotic conditions including the PRL signaling pathway in young RPE versus the proapoptotic conditions in aging RPE. Young RPE cells express PRL and its receptor (PRLR) that (1) signal for the synthesis of reduced glutathione (GSH) that transforms two molecules of hydrogen peroxide (H 2 O 2 ) into water and (2) induces the transcription of catalase, which also processes H 2 O 2 into water; both of these processes limit ROS levels. This antioxidant pathway is more prominent than the pro-oxidant pathway comprised by the TRPM2-mediated intracellular Ca 2+ rise induced by the metabolite 2′O-acetyl-ADP-ribose (O-Ac-ADPR) that results from the deacetylation (removal of Ac group) of various substrates by SIRT2. The viability of RPE is therefore not jeopardized. In contrast, in aged RPE cells, the PRL signaling pathway is blunted. Therefore, the pro-oxidant pathway predominates over the antioxidant one. High levels of ROS, illustrated by increased H 2 O 2 , promote the up-regulation of SIRT2 which, in turn, activates TRPM2 by producing O-Ac-ADPR. The subsequent sustained rise in intracellular Ca 2+ levels can depolarize mitochondria ( Berridge et al., 2000 ), which causes apoptosis. RPE from PRLR null ( −/− ) mice emphasizes these features.

    Article Snippet: Anti-human PRL rabbit polyclonal antibody IC5 and anti-TRPM2 (#ACC-043) were kind gifts from the National Hormone and Pituitary Program (UCLA Medical School, Torrance, CA) and Alomone Labs, respectively.

    Techniques: Mouse Assay

    PRL maintains human RPE cell survival by inhibiting the oxidant-induced SIRT2-dependent induction of TRPM2-mediated intracellular Ca 2+ increase. (a) RT-PCR and (b) Western-blotting of TRPM2 in ARPE-19 cell lysates. (a) TBP was used as a positive control. bp, DNA ladder. RT-PCR was performed in RNA extracted from three independent cell cultures (N = 3). (b) ARPE-19 cells were untreated (Ctl), subjected to lipofectamin alone (Lipo.) or transfected with siRNA against TRPM2 (siRNA) or scramble sequence (Scr.). TRPM2 siRNA efficiently reduced TRPM2 expression as observed by immunoblotting using an anti-TRPM2 antibody that labeled a protein at the expected molecular weight for TRPM2 (171 kDa). β-Actin served as loading control. Extracts from three independent ARPE-19 cell cultures in each condition were analyzed (N = 3). (c, d) Measurement of the change in intracellular Ca 2+ measured by the change in fluo-8 fluorescence (Δ fluorescence) in ARPE19 cells exposed to (c) H 2 O 2 (100 μM) or (d) piceatannol (10 μM) while TRPM2 was blocked by siRNA against TRPM2 or PRL was applied (hPRL, 100 pmol/l, 15-min pretreatment). Scramble sequence (siRNA ctl) was used as a negative control for siRNA against TRPM2. Treatments with H 2 O 2 or piceatannol began at time = 0 s ( n = 170–190 cells; N = 3 independent replicates). (e) Ca 2+ -dependent fluorescence change in ARPE19 cells 180 s after application of SIRT2 inhibitor AGK2 (10 μM) combined or not with H 2 O 2 (100 μM) ( n = 160–190 cells; N = 3 independent replicates). (f) Effect of TRPM2 inhibition on survival of ARPE-19 subjected to a 24-h H 2 O 2 insult (100 μM) by MTT assay. ARPE-19 cells were untreated (Ctl), treated with lipofectamin alone (Lipo.) or transfected with siRNA against TRPM2 or the scramble sequence (Scr.) 24 h prior initiating the MTT assay. In (c–e), signals were normalized by subtracting the Fluo-8 Δ fluorescence to the one in untreated conditions. All bar plots, mean plus S.E.M.; P values: ANOVA and Bonferroni post-hoc test.

    Journal: EBioMedicine

    Article Title: Prolactin protects retinal pigment epithelium by inhibiting sirtuin 2-dependent cell death

    doi: 10.1016/j.ebiom.2016.03.048

    Figure Lengend Snippet: PRL maintains human RPE cell survival by inhibiting the oxidant-induced SIRT2-dependent induction of TRPM2-mediated intracellular Ca 2+ increase. (a) RT-PCR and (b) Western-blotting of TRPM2 in ARPE-19 cell lysates. (a) TBP was used as a positive control. bp, DNA ladder. RT-PCR was performed in RNA extracted from three independent cell cultures (N = 3). (b) ARPE-19 cells were untreated (Ctl), subjected to lipofectamin alone (Lipo.) or transfected with siRNA against TRPM2 (siRNA) or scramble sequence (Scr.). TRPM2 siRNA efficiently reduced TRPM2 expression as observed by immunoblotting using an anti-TRPM2 antibody that labeled a protein at the expected molecular weight for TRPM2 (171 kDa). β-Actin served as loading control. Extracts from three independent ARPE-19 cell cultures in each condition were analyzed (N = 3). (c, d) Measurement of the change in intracellular Ca 2+ measured by the change in fluo-8 fluorescence (Δ fluorescence) in ARPE19 cells exposed to (c) H 2 O 2 (100 μM) or (d) piceatannol (10 μM) while TRPM2 was blocked by siRNA against TRPM2 or PRL was applied (hPRL, 100 pmol/l, 15-min pretreatment). Scramble sequence (siRNA ctl) was used as a negative control for siRNA against TRPM2. Treatments with H 2 O 2 or piceatannol began at time = 0 s ( n = 170–190 cells; N = 3 independent replicates). (e) Ca 2+ -dependent fluorescence change in ARPE19 cells 180 s after application of SIRT2 inhibitor AGK2 (10 μM) combined or not with H 2 O 2 (100 μM) ( n = 160–190 cells; N = 3 independent replicates). (f) Effect of TRPM2 inhibition on survival of ARPE-19 subjected to a 24-h H 2 O 2 insult (100 μM) by MTT assay. ARPE-19 cells were untreated (Ctl), treated with lipofectamin alone (Lipo.) or transfected with siRNA against TRPM2 or the scramble sequence (Scr.) 24 h prior initiating the MTT assay. In (c–e), signals were normalized by subtracting the Fluo-8 Δ fluorescence to the one in untreated conditions. All bar plots, mean plus S.E.M.; P values: ANOVA and Bonferroni post-hoc test.

    Article Snippet: Anti-human PRL rabbit polyclonal antibody IC5 and anti-TRPM2 (#ACC-043) were kind gifts from the National Hormone and Pituitary Program (UCLA Medical School, Torrance, CA) and Alomone Labs, respectively.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control, CTL Assay, Transfection, Sequencing, Expressing, Labeling, Molecular Weight, Fluorescence, Negative Control, Inhibition, MTT Assay