trpm8 antagonist amtb n 3 aminopropyl 2 3 methylphenyl methyl  (Alomone Labs)


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    Name:
    AMTB hydrochloride
    Description:
    AMTB hydrochloride is a TRPM8 channel blocker inhibiting icilin induced human TRPM8 channel activation pIC50 6 23 selective over TRPV1 and TRPV4
    Catalog Number:
    A-305
    Price:
    55.0
    Category:
    Small Molecule
    Source:
    Synthetic
    Applications:
    0
    Purity:
    >97%
    Size:
    5 mg
    Format:
    Lyophilized/solid.
    Formula:
    C23H27ClN2O2S
    Molecular Weight:
    430.99
    Molecule Name:
    N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(thiophen-2-ylmethyl)benzamide;hydrochloride.
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    Structured Review

    Alomone Labs trpm8 antagonist amtb n 3 aminopropyl 2 3 methylphenyl methyl
    AMTB hydrochloride
    AMTB hydrochloride is a TRPM8 channel blocker inhibiting icilin induced human TRPM8 channel activation pIC50 6 23 selective over TRPV1 and TRPV4
    https://www.bioz.com/result/trpm8 antagonist amtb n 3 aminopropyl 2 3 methylphenyl methyl/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trpm8 antagonist amtb n 3 aminopropyl 2 3 methylphenyl methyl - by Bioz Stars, 2021-09
    94/100 stars

    Images

    1) Product Images from "Dysfunctional TRPM8 signalling in the vascular response to environmental cold in ageing"

    Article Title: Dysfunctional TRPM8 signalling in the vascular response to environmental cold in ageing

    Journal: bioRxiv

    doi: 10.1101/2021.05.10.443379

    Sympathetic-sensory signalling and influence of ageing (a-b) RT-PCR CT analysis shows the expression and fold change of TRPA1 and TRPM8 in young and aged sympathetic ganglia normalized to three housekeeping genes collected from the cervical and thoracic paravertebral region. (c) The western blot analysis of TRPM8 in sympathetic ganglia of young and aged mice. All results are shown as mean ± s.e.m. *p
    Figure Legend Snippet: Sympathetic-sensory signalling and influence of ageing (a-b) RT-PCR CT analysis shows the expression and fold change of TRPA1 and TRPM8 in young and aged sympathetic ganglia normalized to three housekeeping genes collected from the cervical and thoracic paravertebral region. (c) The western blot analysis of TRPM8 in sympathetic ganglia of young and aged mice. All results are shown as mean ± s.e.m. *p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Mouse Assay

    TRPA1 and TRPM8 are involved in cold-induced vascular response. Vascular responses with cold (4°C) water treatment in mice pre-treated with combined TRPA1 antagonist A967079 (100 mg kg −1 ) and TRPM8 antagonist AMTB (10 mg kg −1 ), or vehicle control (Veh - 10% DMSO, 10% Tween in saline) i.p. 30 min before cold treatment. (a-c) % change in hindpaw blood flow from baseline to 0-2min following cold treatment (maximum vasoconstriction) in mice treated with combined antagonist (a) A967079+AMTB, (b) A967079, and (c) AMTB. (d-f) Maximum vasoconstriction caused by cold water treatment in mice treated with combined antagonist (d) A967079+AMTB, (e) A967079, and (f) AMTB normalized against vehicle treated mice. (g-h) RT-PCR CT analysis shows fold change of (g) TRPA1 and (h) TRPM8 normalized to three housekeeping genes in dorsal root ganglia (DRG). (i) Representative western blot of TRPM8 in DRG of young and aged mice and densitometric analysis normalized to Tubulin (Y=young, A=aged). All results are shown as mean ± s.e.m. *p
    Figure Legend Snippet: TRPA1 and TRPM8 are involved in cold-induced vascular response. Vascular responses with cold (4°C) water treatment in mice pre-treated with combined TRPA1 antagonist A967079 (100 mg kg −1 ) and TRPM8 antagonist AMTB (10 mg kg −1 ), or vehicle control (Veh - 10% DMSO, 10% Tween in saline) i.p. 30 min before cold treatment. (a-c) % change in hindpaw blood flow from baseline to 0-2min following cold treatment (maximum vasoconstriction) in mice treated with combined antagonist (a) A967079+AMTB, (b) A967079, and (c) AMTB. (d-f) Maximum vasoconstriction caused by cold water treatment in mice treated with combined antagonist (d) A967079+AMTB, (e) A967079, and (f) AMTB normalized against vehicle treated mice. (g-h) RT-PCR CT analysis shows fold change of (g) TRPA1 and (h) TRPM8 normalized to three housekeeping genes in dorsal root ganglia (DRG). (i) Representative western blot of TRPM8 in DRG of young and aged mice and densitometric analysis normalized to Tubulin (Y=young, A=aged). All results are shown as mean ± s.e.m. *p

    Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

    TRPA1 and TRPM8 activity deteriorates with ageing (a) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of menthol (10%) and vehicle (Veh - 10% DMSO in ethanol) in ear of young and aged mice. (b) % maximum change in ear blood flow induced by menthol application in young and aged mice. (c) AUC analysis of % blood flow increase from baseline after menthol application compared to vehicle. (d) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of cinnamaldehyde (10% CA) and vehicle (10% DMSO in ethanol) in ear of young and aged mice. (e) % maximum change in ear blood flow induced by CA application in young and aged mice. (f) AUC analysis of % blood flow increase from baseline after CA application compared to vehicle. (g) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of capsaicin (10%) and vehicle (10% DMSO in ethanol) in ear of young and aged mice. (h) % maximum change in ear blood flow induced by capsaicin application in young and aged mice. (i) AUC analysis of % blood flow increase from baseline after capsaicin application compared to vehicle. All results are shown as mean ± s.e.m. *p
    Figure Legend Snippet: TRPA1 and TRPM8 activity deteriorates with ageing (a) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of menthol (10%) and vehicle (Veh - 10% DMSO in ethanol) in ear of young and aged mice. (b) % maximum change in ear blood flow induced by menthol application in young and aged mice. (c) AUC analysis of % blood flow increase from baseline after menthol application compared to vehicle. (d) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of cinnamaldehyde (10% CA) and vehicle (10% DMSO in ethanol) in ear of young and aged mice. (e) % maximum change in ear blood flow induced by CA application in young and aged mice. (f) AUC analysis of % blood flow increase from baseline after CA application compared to vehicle. (g) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of capsaicin (10%) and vehicle (10% DMSO in ethanol) in ear of young and aged mice. (h) % maximum change in ear blood flow induced by capsaicin application in young and aged mice. (i) AUC analysis of % blood flow increase from baseline after capsaicin application compared to vehicle. All results are shown as mean ± s.e.m. *p

    Techniques Used: Activity Assay, Mouse Assay

    2) Product Images from "Dysfunctional TRPM8 signalling in the vascular response to environmental cold in ageing"

    Article Title: Dysfunctional TRPM8 signalling in the vascular response to environmental cold in ageing

    Journal: bioRxiv

    doi: 10.1101/2021.05.10.443379

    Sympathetic-sensory signalling and influence of ageing (a-b) RT-PCR CT analysis shows the expression and fold change of TRPA1 and TRPM8 in young and aged sympathetic ganglia normalized to three housekeeping genes collected from the cervical and thoracic paravertebral region. (c) The western blot analysis of TRPM8 in sympathetic ganglia of young and aged mice. All results are shown as mean ± s.e.m. *p
    Figure Legend Snippet: Sympathetic-sensory signalling and influence of ageing (a-b) RT-PCR CT analysis shows the expression and fold change of TRPA1 and TRPM8 in young and aged sympathetic ganglia normalized to three housekeeping genes collected from the cervical and thoracic paravertebral region. (c) The western blot analysis of TRPM8 in sympathetic ganglia of young and aged mice. All results are shown as mean ± s.e.m. *p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Mouse Assay

    TRPA1 and TRPM8 are involved in cold-induced vascular response. Vascular responses with cold (4°C) water treatment in mice pre-treated with combined TRPA1 antagonist A967079 (100 mg kg −1 ) and TRPM8 antagonist AMTB (10 mg kg −1 ), or vehicle control (Veh - 10% DMSO, 10% Tween in saline) i.p. 30 min before cold treatment. (a-c) % change in hindpaw blood flow from baseline to 0-2min following cold treatment (maximum vasoconstriction) in mice treated with combined antagonist (a) A967079+AMTB, (b) A967079, and (c) AMTB. (d-f) Maximum vasoconstriction caused by cold water treatment in mice treated with combined antagonist (d) A967079+AMTB, (e) A967079, and (f) AMTB normalized against vehicle treated mice. (g-h) RT-PCR CT analysis shows fold change of (g) TRPA1 and (h) TRPM8 normalized to three housekeeping genes in dorsal root ganglia (DRG). (i) Representative western blot of TRPM8 in DRG of young and aged mice and densitometric analysis normalized to Tubulin (Y=young, A=aged). All results are shown as mean ± s.e.m. *p
    Figure Legend Snippet: TRPA1 and TRPM8 are involved in cold-induced vascular response. Vascular responses with cold (4°C) water treatment in mice pre-treated with combined TRPA1 antagonist A967079 (100 mg kg −1 ) and TRPM8 antagonist AMTB (10 mg kg −1 ), or vehicle control (Veh - 10% DMSO, 10% Tween in saline) i.p. 30 min before cold treatment. (a-c) % change in hindpaw blood flow from baseline to 0-2min following cold treatment (maximum vasoconstriction) in mice treated with combined antagonist (a) A967079+AMTB, (b) A967079, and (c) AMTB. (d-f) Maximum vasoconstriction caused by cold water treatment in mice treated with combined antagonist (d) A967079+AMTB, (e) A967079, and (f) AMTB normalized against vehicle treated mice. (g-h) RT-PCR CT analysis shows fold change of (g) TRPA1 and (h) TRPM8 normalized to three housekeeping genes in dorsal root ganglia (DRG). (i) Representative western blot of TRPM8 in DRG of young and aged mice and densitometric analysis normalized to Tubulin (Y=young, A=aged). All results are shown as mean ± s.e.m. *p

    Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

    TRPA1 and TRPM8 activity deteriorates with ageing (a) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of menthol (10%) and vehicle (Veh - 10% DMSO in ethanol) in ear of young and aged mice. (b) % maximum change in ear blood flow induced by menthol application in young and aged mice. (c) AUC analysis of % blood flow increase from baseline after menthol application compared to vehicle. (d) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of cinnamaldehyde (10% CA) and vehicle (10% DMSO in ethanol) in ear of young and aged mice. (e) % maximum change in ear blood flow induced by CA application in young and aged mice. (f) AUC analysis of % blood flow increase from baseline after CA application compared to vehicle. (g) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of capsaicin (10%) and vehicle (10% DMSO in ethanol) in ear of young and aged mice. (h) % maximum change in ear blood flow induced by capsaicin application in young and aged mice. (i) AUC analysis of % blood flow increase from baseline after capsaicin application compared to vehicle. All results are shown as mean ± s.e.m. *p
    Figure Legend Snippet: TRPA1 and TRPM8 activity deteriorates with ageing (a) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of menthol (10%) and vehicle (Veh - 10% DMSO in ethanol) in ear of young and aged mice. (b) % maximum change in ear blood flow induced by menthol application in young and aged mice. (c) AUC analysis of % blood flow increase from baseline after menthol application compared to vehicle. (d) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of cinnamaldehyde (10% CA) and vehicle (10% DMSO in ethanol) in ear of young and aged mice. (e) % maximum change in ear blood flow induced by CA application in young and aged mice. (f) AUC analysis of % blood flow increase from baseline after CA application compared to vehicle. (g) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of capsaicin (10%) and vehicle (10% DMSO in ethanol) in ear of young and aged mice. (h) % maximum change in ear blood flow induced by capsaicin application in young and aged mice. (i) AUC analysis of % blood flow increase from baseline after capsaicin application compared to vehicle. All results are shown as mean ± s.e.m. *p

    Techniques Used: Activity Assay, Mouse Assay

    3) Product Images from "Transient Receptor Potential Melastatin 8 (TRPM8) Channel Regulates Proliferation and Migration of Breast Cancer Cells by Activating the AMPK-ULK1 Pathway to Enhance Basal Autophagy"

    Article Title: Transient Receptor Potential Melastatin 8 (TRPM8) Channel Regulates Proliferation and Migration of Breast Cancer Cells by Activating the AMPK-ULK1 Pathway to Enhance Basal Autophagy

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2020.573127

    Upregulation of autophagy by TRPM8 in various cancer cell lines. (A, B) The construct for TRPM8 expression was transiently transfected into MCF7 for 48 h; the cells were used for WB analysis using indicated antibodies to detect autophagy-associated proteins (N = 3). (C, D) Similar WB analysis of lysates of HCT116 cells transfected with TRPM8 (N = 3). (E–H) WB analysis of lysates of MDA-MB-231 cells treated with 10 μM menthol, 2 μM icilin, 0.5 μM AMTB, or combined treatments for 48 h (N = 3). N represents the number of replicate experiments. *P
    Figure Legend Snippet: Upregulation of autophagy by TRPM8 in various cancer cell lines. (A, B) The construct for TRPM8 expression was transiently transfected into MCF7 for 48 h; the cells were used for WB analysis using indicated antibodies to detect autophagy-associated proteins (N = 3). (C, D) Similar WB analysis of lysates of HCT116 cells transfected with TRPM8 (N = 3). (E–H) WB analysis of lysates of MDA-MB-231 cells treated with 10 μM menthol, 2 μM icilin, 0.5 μM AMTB, or combined treatments for 48 h (N = 3). N represents the number of replicate experiments. *P

    Techniques Used: Construct, Expressing, Transfection, Western Blot, Multiple Displacement Amplification

    Mechanism of the stimulatory effect of TRPM8 channel function on basal autophagy. (A, B) HeLa cells were incubated with menthol (10 μM) for 48 h. The cells were lysed and assayed by western blot using indicated antibodies (N = 3). (C, D) WB analysis of HeLa cell lysates after treatment with 2 μM icilin, 0.5 μM AMTB, or their combination for 48 h (N = 3). (E, F) HEK293 cells were cotransfected with EGFP and wild type TRPM8, mutant, or a control vector. After 48 h of transfection, EGFP-positive cells were selected for recording of TRPM8 channel-mediated current. The number of cells used for current recordings: Vector (6); WT: (10); V976W (12). (G, H) HeLa cells were transfected with the constructs for the expression of wild type or mutant TRPM8. After 48 h of transfection, cells were lysed and subjected to WB analysis using indicated antibodies (N = 3). N represents the number of replicate experiments. EGFP, enhanced green fluorescent protein; Ctrl, control; *P
    Figure Legend Snippet: Mechanism of the stimulatory effect of TRPM8 channel function on basal autophagy. (A, B) HeLa cells were incubated with menthol (10 μM) for 48 h. The cells were lysed and assayed by western blot using indicated antibodies (N = 3). (C, D) WB analysis of HeLa cell lysates after treatment with 2 μM icilin, 0.5 μM AMTB, or their combination for 48 h (N = 3). (E, F) HEK293 cells were cotransfected with EGFP and wild type TRPM8, mutant, or a control vector. After 48 h of transfection, EGFP-positive cells were selected for recording of TRPM8 channel-mediated current. The number of cells used for current recordings: Vector (6); WT: (10); V976W (12). (G, H) HeLa cells were transfected with the constructs for the expression of wild type or mutant TRPM8. After 48 h of transfection, cells were lysed and subjected to WB analysis using indicated antibodies (N = 3). N represents the number of replicate experiments. EGFP, enhanced green fluorescent protein; Ctrl, control; *P

    Techniques Used: Incubation, Western Blot, Mutagenesis, Plasmid Preparation, Transfection, Construct, Expressing

    Influence of AMPK impairment on the stimulatory effect of TRPM8 on autophagy. (A, B) MCF7 cells were transiently transfected with siRNA against human AMPK and a Flag-TRPM8 construct. After 48 h of transfection, protein lysates were extracted for WB analysis to determine the effect of TRPM8 overexpression on basal autophagy in the presence of AMPK knockdown (N = 3). (C, D) WB analysis of cell lysates of MDA-MB-231 cells treatment with 2 μM icilin, 10 μM menthol, or a combination of 10 M compound C for 48 h (N = 3). N represents the number of replicate experiments. *P
    Figure Legend Snippet: Influence of AMPK impairment on the stimulatory effect of TRPM8 on autophagy. (A, B) MCF7 cells were transiently transfected with siRNA against human AMPK and a Flag-TRPM8 construct. After 48 h of transfection, protein lysates were extracted for WB analysis to determine the effect of TRPM8 overexpression on basal autophagy in the presence of AMPK knockdown (N = 3). (C, D) WB analysis of cell lysates of MDA-MB-231 cells treatment with 2 μM icilin, 10 μM menthol, or a combination of 10 M compound C for 48 h (N = 3). N represents the number of replicate experiments. *P

    Techniques Used: Transfection, Construct, Western Blot, Over Expression, Multiple Displacement Amplification

    Involvement of the AMPK-ULK1-LC3 signaling cascade in TRPM8-stimulated autophagy. (A, B) The construct for Flag-AMPK expression was transiently transfected into MCF7 cells. After 48 h of transfection, the AMPK-ULK1-LC3 signaling cascade-related proteins were detected by WB (N = 3). (C, D) MCF7 cells were transiently transfected with wild type TRPM8, mutant V976W, or control vector. After 48 h of transfection, protein lysates were used for WB analysis (N = 3). (E, F) MDA-MB-231 cells treated with 2 μM icilin, 0.5 μM AMTB, or their combination for 48 h were extracted for WB analysis (N = 3). (G, H) MCF7 cells were transfected with siRNA against human TRPM8; siRNA against TRPM8 (siTRPM8-1 and siTRPM8-2) successfully knocked down TRPM8 expression compared with that in the control scramble siRNA samples according to WB analysis using an anti-TRPM8 antibody. The AMPK-ULK1-LC3 signaling cascade-related proteins detected by WB analysis using the indicated antibodies (N = 3). N represents the number of replicate experiments. *P
    Figure Legend Snippet: Involvement of the AMPK-ULK1-LC3 signaling cascade in TRPM8-stimulated autophagy. (A, B) The construct for Flag-AMPK expression was transiently transfected into MCF7 cells. After 48 h of transfection, the AMPK-ULK1-LC3 signaling cascade-related proteins were detected by WB (N = 3). (C, D) MCF7 cells were transiently transfected with wild type TRPM8, mutant V976W, or control vector. After 48 h of transfection, protein lysates were used for WB analysis (N = 3). (E, F) MDA-MB-231 cells treated with 2 μM icilin, 0.5 μM AMTB, or their combination for 48 h were extracted for WB analysis (N = 3). (G, H) MCF7 cells were transfected with siRNA against human TRPM8; siRNA against TRPM8 (siTRPM8-1 and siTRPM8-2) successfully knocked down TRPM8 expression compared with that in the control scramble siRNA samples according to WB analysis using an anti-TRPM8 antibody. The AMPK-ULK1-LC3 signaling cascade-related proteins detected by WB analysis using the indicated antibodies (N = 3). N represents the number of replicate experiments. *P

    Techniques Used: Construct, Expressing, Transfection, Western Blot, Mutagenesis, Plasmid Preparation, Multiple Displacement Amplification

    AMPK interacts with TRPM8. (A, B) Co-IP analysis. (A) Constructs for GFP-TRPM8 and Flag-AMPK expression were transiently transfected into MCF7 cells. After 48 h of transfection, protein lysates were immunoprecipitated with an anti-GFP antibody and assayed by immunoblot with an anti-Flag antibody (lower panel). Reciprocal Co-IP with an anti-Flag antibody used for immunoprecipitation and anti-GFP used for WB analysis (upper panel) (N = 3). (B) The construct for GFP-AMPK expression was cotransfected with M8-N, M8-LI, M8-LII, or M8-C into MCF7 cells. Protein lysates were immunoprecipitated with an anti-Flag antibody and assayed by immunoblot with an anti-GFP antibody (upper). The constructs for GFP-AMPK and Flag-M8-C expression were cotransfected into MCF7 cells. Protein lysates were immunoprecipitated with an anti-GFP antibody and assayed by immunoblot with an anti-Flag antibody (upper) (N = 3). (C) GST pull-down analysis. Protein lysates of MCF7 cells transiently expressing Flag-AMPK were incubated with purified cytoplasmic C-terminus of TRPM8 GST fusion protein (GST-M8C). GST-M8C, but not control GST, successfully pulled down Flag-AMPK. PD: pull-down. The lysate was used as a positive control (N = 3). N represents the number of replicate experiments. PD, pull down; WCL, whole cell lysates.
    Figure Legend Snippet: AMPK interacts with TRPM8. (A, B) Co-IP analysis. (A) Constructs for GFP-TRPM8 and Flag-AMPK expression were transiently transfected into MCF7 cells. After 48 h of transfection, protein lysates were immunoprecipitated with an anti-GFP antibody and assayed by immunoblot with an anti-Flag antibody (lower panel). Reciprocal Co-IP with an anti-Flag antibody used for immunoprecipitation and anti-GFP used for WB analysis (upper panel) (N = 3). (B) The construct for GFP-AMPK expression was cotransfected with M8-N, M8-LI, M8-LII, or M8-C into MCF7 cells. Protein lysates were immunoprecipitated with an anti-Flag antibody and assayed by immunoblot with an anti-GFP antibody (upper). The constructs for GFP-AMPK and Flag-M8-C expression were cotransfected into MCF7 cells. Protein lysates were immunoprecipitated with an anti-GFP antibody and assayed by immunoblot with an anti-Flag antibody (upper) (N = 3). (C) GST pull-down analysis. Protein lysates of MCF7 cells transiently expressing Flag-AMPK were incubated with purified cytoplasmic C-terminus of TRPM8 GST fusion protein (GST-M8C). GST-M8C, but not control GST, successfully pulled down Flag-AMPK. PD: pull-down. The lysate was used as a positive control (N = 3). N represents the number of replicate experiments. PD, pull down; WCL, whole cell lysates.

    Techniques Used: Co-Immunoprecipitation Assay, Construct, Expressing, Transfection, Immunoprecipitation, Western Blot, Incubation, Purification, Positive Control

    RPM8 expression activates basal autophagy level. (A, B) HeLa cells were incubated with 10 M CQ for the indicated times. The cell lysates were subjected to western blot (WB) analysis using indicated antibodies (N = 3). (C, D) Construct for TRPM8 expression was transfected into HeLa cells for 48 h; the cells were used for WB analysis using indicated antibodies (N = 3). (E, F) Construct for ptfLC3 expression was cotransfected with the TRPM8 or control vector into HeLa cells for 48 h, and accumulation of yellow and red puncta were observed by fluorescence microscopy; representative confocal images are shown (scale bar, 20 μm). (G, H) WB analysis was performed in the lysates of HeLa cells transfected with TRPM8-specific siRNA to decrease TRPM8 expression (N = 3). (I, J) Yellow and red puncta in HeLa cells transfected with TRPM8 siRNA. N represents the number of replicate experiments. Ctrl, control; CQ, chloroquine; LC3, microtubule-associated protein 1 light chain 3. *P
    Figure Legend Snippet: RPM8 expression activates basal autophagy level. (A, B) HeLa cells were incubated with 10 M CQ for the indicated times. The cell lysates were subjected to western blot (WB) analysis using indicated antibodies (N = 3). (C, D) Construct for TRPM8 expression was transfected into HeLa cells for 48 h; the cells were used for WB analysis using indicated antibodies (N = 3). (E, F) Construct for ptfLC3 expression was cotransfected with the TRPM8 or control vector into HeLa cells for 48 h, and accumulation of yellow and red puncta were observed by fluorescence microscopy; representative confocal images are shown (scale bar, 20 μm). (G, H) WB analysis was performed in the lysates of HeLa cells transfected with TRPM8-specific siRNA to decrease TRPM8 expression (N = 3). (I, J) Yellow and red puncta in HeLa cells transfected with TRPM8 siRNA. N represents the number of replicate experiments. Ctrl, control; CQ, chloroquine; LC3, microtubule-associated protein 1 light chain 3. *P

    Techniques Used: Expressing, Incubation, Western Blot, Construct, Transfection, Plasmid Preparation, Fluorescence, Microscopy

    Autophagy is essential for the regulatory effect of TRPM8 on the proliferation and migration of breast cancer cells. (A, B) Cell proliferation experiments. MCF7 cells were transfected with TRPM8 constructs. After 24 h of transfection, cells were treated with 10 M CQ. (A) The in vitro colony formation assay. Cells were further cultured in growth media for 7-10 days to form the colonies. The colonies were washed with ice-cold PBS three times, stained with trypan blue, and counted (N = 3). (B) Ki67 expression was detected by immunostaining (N = 3). (C) MDA-MB-231 cells were transiently transfected with siRNA against human ATG7 (siATG7) and a Flag-TRPM8 construct. After 48 h of transfection, protein lysates were extracted for WB analysis to determine the effect of TRPM8 overexpression on basal autophagy in the presence of ATG7 knockdown (N =3). (D, E) Similar experiment was performed in MDA-MB-231 cells transfected with siATG7 and a Flag-TRPM8 construct. (N = 3). (F, G) Cell migration determined by wound healing assay. (F) MCF7 cells were transfected with the TRPM8 constructs for 24 h and scratched with a sterile 10 μl tip. After three washes with 1× PBS, the cells were cultured for 24–72 h in serum-free medium in the presence of 10 μM CQ (N = 3). (G) Similar wound healing assay as in (F) but with MDA-MB-231 cells transfected with siATG7 and a Flag-TRPM8 construct (N = 3). N represents the number of replicate experiments. *P
    Figure Legend Snippet: Autophagy is essential for the regulatory effect of TRPM8 on the proliferation and migration of breast cancer cells. (A, B) Cell proliferation experiments. MCF7 cells were transfected with TRPM8 constructs. After 24 h of transfection, cells were treated with 10 M CQ. (A) The in vitro colony formation assay. Cells were further cultured in growth media for 7-10 days to form the colonies. The colonies were washed with ice-cold PBS three times, stained with trypan blue, and counted (N = 3). (B) Ki67 expression was detected by immunostaining (N = 3). (C) MDA-MB-231 cells were transiently transfected with siRNA against human ATG7 (siATG7) and a Flag-TRPM8 construct. After 48 h of transfection, protein lysates were extracted for WB analysis to determine the effect of TRPM8 overexpression on basal autophagy in the presence of ATG7 knockdown (N =3). (D, E) Similar experiment was performed in MDA-MB-231 cells transfected with siATG7 and a Flag-TRPM8 construct. (N = 3). (F, G) Cell migration determined by wound healing assay. (F) MCF7 cells were transfected with the TRPM8 constructs for 24 h and scratched with a sterile 10 μl tip. After three washes with 1× PBS, the cells were cultured for 24–72 h in serum-free medium in the presence of 10 μM CQ (N = 3). (G) Similar wound healing assay as in (F) but with MDA-MB-231 cells transfected with siATG7 and a Flag-TRPM8 construct (N = 3). N represents the number of replicate experiments. *P

    Techniques Used: Migration, Transfection, Construct, In Vitro, Colony Assay, Cell Culture, Staining, Expressing, Immunostaining, Multiple Displacement Amplification, Western Blot, Over Expression, Wound Healing Assay

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    Alomone Labs ammtx3
    Voltage-clamp analysis of I A in WT and Kv4.3−/− SNc DA neurons. A , Voltage-clamp traces showing representative I A recordings obtained from a WT (black trace) and a Kv4.3−/− SNc DA neuron (red trace) in response to a voltage step to –40 mV (gray trace). The small residual current present in the Kv4.3−/− mice is blocked by <t>AmmTX3</t> (inset, orange trace). B , Box and whisker plot showing the distribution of values for I A amplitude in WT and Kv4.3−/− SNc DA neurons. C , Box and whisker plot showing the distribution of values for I A time constant of inactivation (I A tau) in WT and Kv4.3−/− SNc DA neurons. The green dotted rectangle highlights five Kv4.3−/− outliers displaying unusually large values for I A tau. D , Box and whisker plot showing the distribution of values for I A charge in WT and Kv4.3−/− SNc DA neurons. The green dotted rectangle highlights five Kv4.3−/− outliers displaying unusually large values for I A charge (same cells as in C ). E , left, Scatter plot showing the relationship between I A tau and charge in WT (gray dots) and Kv4.3−/− SNc DA neurons (red dots). Please note that 5 of the Kv4.3−/− measurements lie in the WT region of space (green dotted ellipse). Right, Voltage-clamp traces showing one example of the atypical I A recording (green trace, corresponding to the large green circle in the scatter plot) encountered in one of the 5 Kv4.3−/− outliers highlighted in panels C , D , compared with the typical recording obtained in Kv4.3−/− neurons (red trace, same as in panel A , corresponding to the large red circle in the scatter plot); *** p
    Ammtx3, 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
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    Alomone Labs trpm8 antagonist amtb n 3 aminopropyl 2 3 methylphenyl methyl
    Sympathetic-sensory signalling and influence of ageing (a-b) RT-PCR CT analysis shows the expression and fold change of TRPA1 and <t>TRPM8</t> in young and aged sympathetic ganglia normalized to three housekeeping genes collected from the cervical and thoracic paravertebral region. (c) The western blot analysis of TRPM8 in sympathetic ganglia of young and aged mice. All results are shown as mean ± s.e.m. *p
    Trpm8 Antagonist Amtb N 3 Aminopropyl 2 3 Methylphenyl Methyl, 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
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    Alomone Labs triton x 100
    Determination of P2Y receptors in cardiomyocytes by immunofluorescent staining and immunoblotting. Cardiomyocytes were fixed in methanol, permeabilized with 1% Triton X-100, blocked and incubated in the presence of antibodies against P2Y 2  (A) or P2Y 4  (B) receptors. For detection of the bound antibodies, cells were incubated with goat antirabbit FITC (green) following counterstaining with propidium iodide (red). Bars = 10 μm. Proteins isolated from rat cardiomyocyte homogenates were subjected to SDS-PAGE on an 11% acrylamide gel and transferred onto a nitrocellulose membrane. Filters were probed with the indicated P2Y antibodies simultaneously with the neutralizing peptide (np). Protein bands were detected with a secondary antibody coupled to peroxidase by enhanced chemiluminescence (C).
    Triton X 100, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Voltage-clamp analysis of I A in WT and Kv4.3−/− SNc DA neurons. A , Voltage-clamp traces showing representative I A recordings obtained from a WT (black trace) and a Kv4.3−/− SNc DA neuron (red trace) in response to a voltage step to –40 mV (gray trace). The small residual current present in the Kv4.3−/− mice is blocked by AmmTX3 (inset, orange trace). B , Box and whisker plot showing the distribution of values for I A amplitude in WT and Kv4.3−/− SNc DA neurons. C , Box and whisker plot showing the distribution of values for I A time constant of inactivation (I A tau) in WT and Kv4.3−/− SNc DA neurons. The green dotted rectangle highlights five Kv4.3−/− outliers displaying unusually large values for I A tau. D , Box and whisker plot showing the distribution of values for I A charge in WT and Kv4.3−/− SNc DA neurons. The green dotted rectangle highlights five Kv4.3−/− outliers displaying unusually large values for I A charge (same cells as in C ). E , left, Scatter plot showing the relationship between I A tau and charge in WT (gray dots) and Kv4.3−/− SNc DA neurons (red dots). Please note that 5 of the Kv4.3−/− measurements lie in the WT region of space (green dotted ellipse). Right, Voltage-clamp traces showing one example of the atypical I A recording (green trace, corresponding to the large green circle in the scatter plot) encountered in one of the 5 Kv4.3−/− outliers highlighted in panels C , D , compared with the typical recording obtained in Kv4.3−/− neurons (red trace, same as in panel A , corresponding to the large red circle in the scatter plot); *** p

    Journal: eNeuro

    Article Title: Refining the Identity and Role of Kv4 Channels in Mouse Substantia Nigra Dopaminergic Neurons

    doi: 10.1523/ENEURO.0207-21.2021

    Figure Lengend Snippet: Voltage-clamp analysis of I A in WT and Kv4.3−/− SNc DA neurons. A , Voltage-clamp traces showing representative I A recordings obtained from a WT (black trace) and a Kv4.3−/− SNc DA neuron (red trace) in response to a voltage step to –40 mV (gray trace). The small residual current present in the Kv4.3−/− mice is blocked by AmmTX3 (inset, orange trace). B , Box and whisker plot showing the distribution of values for I A amplitude in WT and Kv4.3−/− SNc DA neurons. C , Box and whisker plot showing the distribution of values for I A time constant of inactivation (I A tau) in WT and Kv4.3−/− SNc DA neurons. The green dotted rectangle highlights five Kv4.3−/− outliers displaying unusually large values for I A tau. D , Box and whisker plot showing the distribution of values for I A charge in WT and Kv4.3−/− SNc DA neurons. The green dotted rectangle highlights five Kv4.3−/− outliers displaying unusually large values for I A charge (same cells as in C ). E , left, Scatter plot showing the relationship between I A tau and charge in WT (gray dots) and Kv4.3−/− SNc DA neurons (red dots). Please note that 5 of the Kv4.3−/− measurements lie in the WT region of space (green dotted ellipse). Right, Voltage-clamp traces showing one example of the atypical I A recording (green trace, corresponding to the large green circle in the scatter plot) encountered in one of the 5 Kv4.3−/− outliers highlighted in panels C , D , compared with the typical recording obtained in Kv4.3−/− neurons (red trace, same as in panel A , corresponding to the large red circle in the scatter plot); *** p

    Article Snippet: AmmTX3 (1 μm , Alomone) was used to block the transient potassium current (IA) carried by Kv4 channels.

    Techniques: Mouse Assay, Whisker Assay

    Comparing the alterations in electrophysiological phenotype after acute blockade of Kv4 channels with the Kv4.3−/− mouse model. A , Current-clamp recordings showing the spontaneous pattern of activity of a WT SNc DA neuron in control condition (black trace, left) and after AmmTX3 application (red trace, right). B , left, Line and scatter plot showing the change in spontaneous firing frequency induced by AmmTX3 application in individual WT SNc DA neurons. Right, Bar plot comparing the average change in spontaneous firing frequency after AmmTX3 application (left, light colors) or Kv4.3 channel deletion (right, dark colors). C , Current-clamp recordings showing the voltage response of a WT SNc DA neuron to a hyperpolarizing current step (bottom gray traces) in control condition (left, black trace) and after AmmTX3 application (right, red trace). D , left, Line and scatter plot showing the change in rebound delay induced by AmmTX3 application in individual WT SNc DA neurons. Right, Bar plot showing the average change in rebound delay after AmmTX3 application (left, light colors) or Kv4.3 channel deletion (right, dark colors); ** p

    Journal: eNeuro

    Article Title: Refining the Identity and Role of Kv4 Channels in Mouse Substantia Nigra Dopaminergic Neurons

    doi: 10.1523/ENEURO.0207-21.2021

    Figure Lengend Snippet: Comparing the alterations in electrophysiological phenotype after acute blockade of Kv4 channels with the Kv4.3−/− mouse model. A , Current-clamp recordings showing the spontaneous pattern of activity of a WT SNc DA neuron in control condition (black trace, left) and after AmmTX3 application (red trace, right). B , left, Line and scatter plot showing the change in spontaneous firing frequency induced by AmmTX3 application in individual WT SNc DA neurons. Right, Bar plot comparing the average change in spontaneous firing frequency after AmmTX3 application (left, light colors) or Kv4.3 channel deletion (right, dark colors). C , Current-clamp recordings showing the voltage response of a WT SNc DA neuron to a hyperpolarizing current step (bottom gray traces) in control condition (left, black trace) and after AmmTX3 application (right, red trace). D , left, Line and scatter plot showing the change in rebound delay induced by AmmTX3 application in individual WT SNc DA neurons. Right, Bar plot showing the average change in rebound delay after AmmTX3 application (left, light colors) or Kv4.3 channel deletion (right, dark colors); ** p

    Article Snippet: AmmTX3 (1 μm , Alomone) was used to block the transient potassium current (IA) carried by Kv4 channels.

    Techniques: Activity Assay

    STP-SE manifests as an increase in afferent recruitment of NGFCs via enhanced E-S coupling. A. Single traces showing the effect of AmmTx3 (500 nM) on EPSC (top traces) and corresponding EPSP (bottom trace) in a given hippocampal NGFC in response to electrical stimulation of SLM afferent fibers (2 x 30Hz). B. Box plots of EPSC amplitudes (EPSC1 versus EPSC2) under baseline conditions and after AmmTx3 treatment (n=7; p values for EPSC1 and EPSC2 comparisons are 0.11 and 0.27, respectively). C. Box plots of paired pulse ratio (EPSC2 peak amplitude/EPSC1 peak amplitude) under baseline conditions and after AmmTx3 treatment (n=7; p value = 0.36). D. Box plots of EPSP summation measured as absolute Vm at peak of EPSP1 and EPSP2 under baseline conditions and after AmmTx3 treatment (n=7; =7; p values for EPSP1 and EPSP2 comparisons are 0.005 and 0.016, respectively)). E-G. Single trace examples depicting a time course of E-S coupling (SLM afferent stimulation delivered at 5 × 30 Hz) during baseline conditions and after STP-SE induction. H. Line plot depicting increased E-S coupling measured as spike probability during 10 sweeps of baseline versus 10 sweeps immediately after STP-SE induction (n=9; p value = 0.002). I. Box plot illustrating duration of enhanced E-S coupling following STP-SE induction (n=9).

    Journal: bioRxiv

    Article Title: Activity-dependent tuning of intrinsic excitability in mouse and human neurogliaform cells

    doi: 10.1101/2020.03.24.004465

    Figure Lengend Snippet: STP-SE manifests as an increase in afferent recruitment of NGFCs via enhanced E-S coupling. A. Single traces showing the effect of AmmTx3 (500 nM) on EPSC (top traces) and corresponding EPSP (bottom trace) in a given hippocampal NGFC in response to electrical stimulation of SLM afferent fibers (2 x 30Hz). B. Box plots of EPSC amplitudes (EPSC1 versus EPSC2) under baseline conditions and after AmmTx3 treatment (n=7; p values for EPSC1 and EPSC2 comparisons are 0.11 and 0.27, respectively). C. Box plots of paired pulse ratio (EPSC2 peak amplitude/EPSC1 peak amplitude) under baseline conditions and after AmmTx3 treatment (n=7; p value = 0.36). D. Box plots of EPSP summation measured as absolute Vm at peak of EPSP1 and EPSP2 under baseline conditions and after AmmTx3 treatment (n=7; =7; p values for EPSP1 and EPSP2 comparisons are 0.005 and 0.016, respectively)). E-G. Single trace examples depicting a time course of E-S coupling (SLM afferent stimulation delivered at 5 × 30 Hz) during baseline conditions and after STP-SE induction. H. Line plot depicting increased E-S coupling measured as spike probability during 10 sweeps of baseline versus 10 sweeps immediately after STP-SE induction (n=9; p value = 0.002). I. Box plot illustrating duration of enhanced E-S coupling following STP-SE induction (n=9).

    Article Snippet: In certain experiments (see results section for details) the extracellular solution was supplemented with one or a combination of the following drugs; 50μM picrotoxin (Millipore Sigma; Cat. No. 80410), 2-5μM CGP556845A (Abcam; Cat. No. ab120337), 5-10μM bicuculline methobromide (Abcam; Cat. No. ab120109), 100μM or 1-3mM 4-aminopyridine (Millipore Sigma; Cat. No. A78403), 100-200nM α -DTX (Alomone Labs; Cat No.D-350), 200 or 500nM AmmTx3 (Alomone Labs; Cat No.STA-305).

    Techniques:

    I KA in NGFCs is predominantly mediated by Kv4-subunit containing channels. A. Uniform Manifold Approximation and Projection (UMAP) plot of RNAseq data publicly available from the Allen Brain Institute (see methods for details) highlighting clusters corresponding to CGE-NGFCs (green; 3626 cells) and hippocampal pyramidal cells (blue; 2530 cells). B. Violin plots depicting comparison of mRNA levels in CGE-NGFCs that encode for subunits of I KA channels. Median values are depicted above each plot. C . Top, middle; Voltage steps and corresponding single current traces illustrating the protocol employed to isolate I KA and I KDR . Bottom; Single traces illustrating the effects of TEA (2-10 mM) and AmmTx3 (500nM) on the isolated I KA and I KDR . D. Box plots depicting inhibition of I KA and I KDR by TEA (n=8) and AmmTx3 (n=4). E. Scatterplot of median expression levels of KCND2 versus KCND3 mRNA in CGE-NGFCs and hippocampal pyramidal cells. F. Representative confocal images of KCND2 and KCND3 transcript expression in CA1 pyramidal cells revealed by RNAscope (see methods for details; scale bar = 100μm) G. Representative confocal image of the distribution of putative NGFCs (via expression of NDNF mRNA transcripts) in CA1 SLM (scale bar -= 100μM). H,I. Representative confocal images of mRNA expression of KCND2 and KNCD3 in putative NGFCs (NDNF-expressing) in CA1 SLM. (Scale bars = 50μm for top panels; 20μm for bottom panels) J. Split-violin plots depicting comparison in the expression levels of mRNA transcripts encoding known auxiliary subunits of Kv4-containing channels (KCHIPs and DPLPs) in CGE-NGFCs (green) versus hippocampal pyramidal cells (blue).

    Journal: bioRxiv

    Article Title: Activity-dependent tuning of intrinsic excitability in mouse and human neurogliaform cells

    doi: 10.1101/2020.03.24.004465

    Figure Lengend Snippet: I KA in NGFCs is predominantly mediated by Kv4-subunit containing channels. A. Uniform Manifold Approximation and Projection (UMAP) plot of RNAseq data publicly available from the Allen Brain Institute (see methods for details) highlighting clusters corresponding to CGE-NGFCs (green; 3626 cells) and hippocampal pyramidal cells (blue; 2530 cells). B. Violin plots depicting comparison of mRNA levels in CGE-NGFCs that encode for subunits of I KA channels. Median values are depicted above each plot. C . Top, middle; Voltage steps and corresponding single current traces illustrating the protocol employed to isolate I KA and I KDR . Bottom; Single traces illustrating the effects of TEA (2-10 mM) and AmmTx3 (500nM) on the isolated I KA and I KDR . D. Box plots depicting inhibition of I KA and I KDR by TEA (n=8) and AmmTx3 (n=4). E. Scatterplot of median expression levels of KCND2 versus KCND3 mRNA in CGE-NGFCs and hippocampal pyramidal cells. F. Representative confocal images of KCND2 and KCND3 transcript expression in CA1 pyramidal cells revealed by RNAscope (see methods for details; scale bar = 100μm) G. Representative confocal image of the distribution of putative NGFCs (via expression of NDNF mRNA transcripts) in CA1 SLM (scale bar -= 100μM). H,I. Representative confocal images of mRNA expression of KCND2 and KNCD3 in putative NGFCs (NDNF-expressing) in CA1 SLM. (Scale bars = 50μm for top panels; 20μm for bottom panels) J. Split-violin plots depicting comparison in the expression levels of mRNA transcripts encoding known auxiliary subunits of Kv4-containing channels (KCHIPs and DPLPs) in CGE-NGFCs (green) versus hippocampal pyramidal cells (blue).

    Article Snippet: In certain experiments (see results section for details) the extracellular solution was supplemented with one or a combination of the following drugs; 50μM picrotoxin (Millipore Sigma; Cat. No. 80410), 2-5μM CGP556845A (Abcam; Cat. No. ab120337), 5-10μM bicuculline methobromide (Abcam; Cat. No. ab120109), 100μM or 1-3mM 4-aminopyridine (Millipore Sigma; Cat. No. A78403), 100-200nM α -DTX (Alomone Labs; Cat No.D-350), 200 or 500nM AmmTx3 (Alomone Labs; Cat No.STA-305).

    Techniques: Isolation, Inhibition, Expressing

    Sympathetic-sensory signalling and influence of ageing (a-b) RT-PCR CT analysis shows the expression and fold change of TRPA1 and TRPM8 in young and aged sympathetic ganglia normalized to three housekeeping genes collected from the cervical and thoracic paravertebral region. (c) The western blot analysis of TRPM8 in sympathetic ganglia of young and aged mice. All results are shown as mean ± s.e.m. *p

    Journal: bioRxiv

    Article Title: Dysfunctional TRPM8 signalling in the vascular response to environmental cold in ageing

    doi: 10.1101/2021.05.10.443379

    Figure Lengend Snippet: Sympathetic-sensory signalling and influence of ageing (a-b) RT-PCR CT analysis shows the expression and fold change of TRPA1 and TRPM8 in young and aged sympathetic ganglia normalized to three housekeeping genes collected from the cervical and thoracic paravertebral region. (c) The western blot analysis of TRPM8 in sympathetic ganglia of young and aged mice. All results are shown as mean ± s.e.m. *p

    Article Snippet: The TRPM8 antagonist AMTB (N-(3-aminopropyl)-2-[(3-methylphenyl) methyl] oxy-N-(2-thienylmethyl) benzamide hydrochloride salt) (Alomone Labs, #A-305) was dissolved in 10% DMSO in saline.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Mouse Assay

    TRPA1 and TRPM8 are involved in cold-induced vascular response. Vascular responses with cold (4°C) water treatment in mice pre-treated with combined TRPA1 antagonist A967079 (100 mg kg −1 ) and TRPM8 antagonist AMTB (10 mg kg −1 ), or vehicle control (Veh - 10% DMSO, 10% Tween in saline) i.p. 30 min before cold treatment. (a-c) % change in hindpaw blood flow from baseline to 0-2min following cold treatment (maximum vasoconstriction) in mice treated with combined antagonist (a) A967079+AMTB, (b) A967079, and (c) AMTB. (d-f) Maximum vasoconstriction caused by cold water treatment in mice treated with combined antagonist (d) A967079+AMTB, (e) A967079, and (f) AMTB normalized against vehicle treated mice. (g-h) RT-PCR CT analysis shows fold change of (g) TRPA1 and (h) TRPM8 normalized to three housekeeping genes in dorsal root ganglia (DRG). (i) Representative western blot of TRPM8 in DRG of young and aged mice and densitometric analysis normalized to Tubulin (Y=young, A=aged). All results are shown as mean ± s.e.m. *p

    Journal: bioRxiv

    Article Title: Dysfunctional TRPM8 signalling in the vascular response to environmental cold in ageing

    doi: 10.1101/2021.05.10.443379

    Figure Lengend Snippet: TRPA1 and TRPM8 are involved in cold-induced vascular response. Vascular responses with cold (4°C) water treatment in mice pre-treated with combined TRPA1 antagonist A967079 (100 mg kg −1 ) and TRPM8 antagonist AMTB (10 mg kg −1 ), or vehicle control (Veh - 10% DMSO, 10% Tween in saline) i.p. 30 min before cold treatment. (a-c) % change in hindpaw blood flow from baseline to 0-2min following cold treatment (maximum vasoconstriction) in mice treated with combined antagonist (a) A967079+AMTB, (b) A967079, and (c) AMTB. (d-f) Maximum vasoconstriction caused by cold water treatment in mice treated with combined antagonist (d) A967079+AMTB, (e) A967079, and (f) AMTB normalized against vehicle treated mice. (g-h) RT-PCR CT analysis shows fold change of (g) TRPA1 and (h) TRPM8 normalized to three housekeeping genes in dorsal root ganglia (DRG). (i) Representative western blot of TRPM8 in DRG of young and aged mice and densitometric analysis normalized to Tubulin (Y=young, A=aged). All results are shown as mean ± s.e.m. *p

    Article Snippet: The TRPM8 antagonist AMTB (N-(3-aminopropyl)-2-[(3-methylphenyl) methyl] oxy-N-(2-thienylmethyl) benzamide hydrochloride salt) (Alomone Labs, #A-305) was dissolved in 10% DMSO in saline.

    Techniques: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

    TRPA1 and TRPM8 activity deteriorates with ageing (a) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of menthol (10%) and vehicle (Veh - 10% DMSO in ethanol) in ear of young and aged mice. (b) % maximum change in ear blood flow induced by menthol application in young and aged mice. (c) AUC analysis of % blood flow increase from baseline after menthol application compared to vehicle. (d) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of cinnamaldehyde (10% CA) and vehicle (10% DMSO in ethanol) in ear of young and aged mice. (e) % maximum change in ear blood flow induced by CA application in young and aged mice. (f) AUC analysis of % blood flow increase from baseline after CA application compared to vehicle. (g) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of capsaicin (10%) and vehicle (10% DMSO in ethanol) in ear of young and aged mice. (h) % maximum change in ear blood flow induced by capsaicin application in young and aged mice. (i) AUC analysis of % blood flow increase from baseline after capsaicin application compared to vehicle. All results are shown as mean ± s.e.m. *p

    Journal: bioRxiv

    Article Title: Dysfunctional TRPM8 signalling in the vascular response to environmental cold in ageing

    doi: 10.1101/2021.05.10.443379

    Figure Lengend Snippet: TRPA1 and TRPM8 activity deteriorates with ageing (a) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of menthol (10%) and vehicle (Veh - 10% DMSO in ethanol) in ear of young and aged mice. (b) % maximum change in ear blood flow induced by menthol application in young and aged mice. (c) AUC analysis of % blood flow increase from baseline after menthol application compared to vehicle. (d) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of cinnamaldehyde (10% CA) and vehicle (10% DMSO in ethanol) in ear of young and aged mice. (e) % maximum change in ear blood flow induced by CA application in young and aged mice. (f) AUC analysis of % blood flow increase from baseline after CA application compared to vehicle. (g) Graph shows the % mean ± s.e.m. of blood flow change from baseline in response to topical application of capsaicin (10%) and vehicle (10% DMSO in ethanol) in ear of young and aged mice. (h) % maximum change in ear blood flow induced by capsaicin application in young and aged mice. (i) AUC analysis of % blood flow increase from baseline after capsaicin application compared to vehicle. All results are shown as mean ± s.e.m. *p

    Article Snippet: The TRPM8 antagonist AMTB (N-(3-aminopropyl)-2-[(3-methylphenyl) methyl] oxy-N-(2-thienylmethyl) benzamide hydrochloride salt) (Alomone Labs, #A-305) was dissolved in 10% DMSO in saline.

    Techniques: Activity Assay, Mouse Assay

    Determination of P2Y receptors in cardiomyocytes by immunofluorescent staining and immunoblotting. Cardiomyocytes were fixed in methanol, permeabilized with 1% Triton X-100, blocked and incubated in the presence of antibodies against P2Y 2  (A) or P2Y 4  (B) receptors. For detection of the bound antibodies, cells were incubated with goat antirabbit FITC (green) following counterstaining with propidium iodide (red). Bars = 10 μm. Proteins isolated from rat cardiomyocyte homogenates were subjected to SDS-PAGE on an 11% acrylamide gel and transferred onto a nitrocellulose membrane. Filters were probed with the indicated P2Y antibodies simultaneously with the neutralizing peptide (np). Protein bands were detected with a secondary antibody coupled to peroxidase by enhanced chemiluminescence (C).

    Journal: Biochemical pharmacology

    Article Title: Involvement of uracil nucleotides in protection of cardiomyocytes from hypoxic stress

    doi: 10.1016/j.bcp.2005.01.018

    Figure Lengend Snippet: Determination of P2Y receptors in cardiomyocytes by immunofluorescent staining and immunoblotting. Cardiomyocytes were fixed in methanol, permeabilized with 1% Triton X-100, blocked and incubated in the presence of antibodies against P2Y 2 (A) or P2Y 4 (B) receptors. For detection of the bound antibodies, cells were incubated with goat antirabbit FITC (green) following counterstaining with propidium iodide (red). Bars = 10 μm. Proteins isolated from rat cardiomyocyte homogenates were subjected to SDS-PAGE on an 11% acrylamide gel and transferred onto a nitrocellulose membrane. Filters were probed with the indicated P2Y antibodies simultaneously with the neutralizing peptide (np). Protein bands were detected with a secondary antibody coupled to peroxidase by enhanced chemiluminescence (C).

    Article Snippet: Cells were fixed in absolute methanol for 10 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked in 2% normal goat serum for 10 min. Anti-P2Y2 or anti-P2Y4 antibodies (Alomone Labs) were diluted in PBS to a final concentration of 1:100 and then incubated overnight at room temperature with the cells.

    Techniques: Staining, Incubation, Isolation, SDS Page, Acrylamide Gel Assay