trpm8  (Alomone Labs)


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    Name:
    Anti TRPM8 extracellular Antibody
    Description:
    Anti TRPM8 extracellular Antibody ACC 049 is a highly specific antibody directed against an epitope of the human protein The antibody can be used in western blot immunoprecipitation immunohistochemistry immunocytochemistry and neutralization applications It has been designed to recognize TRPM8 from human rat and mouse samples
    Catalog Number:
    ACC-049
    Price:
    397.0
    Category:
    Primary Antibody
    Applications:
    Immunocytochemistry, Immunofluorescence, Immunohistochemistry, Immunoprecipitation, Live Cell Imaging, Neutralization, Western Blot
    Purity:
    Affinity purified on immobilized antigen.
    Immunogen:
    Synthetic peptide
    Size:
    25 mcl
    Antibody Type:
    Polyclonal Primary Antibodies
    Format:
    Lyophilized Powder
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Alomone Labs trpm8
    Anti TRPM8 extracellular Antibody
    Anti TRPM8 extracellular Antibody ACC 049 is a highly specific antibody directed against an epitope of the human protein The antibody can be used in western blot immunoprecipitation immunohistochemistry immunocytochemistry and neutralization applications It has been designed to recognize TRPM8 from human rat and mouse samples
    https://www.bioz.com/result/trpm8/product/Alomone Labs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trpm8 - by Bioz Stars, 2021-09
    95/100 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 "Wu-Tou Decoction Inhibits Chronic Inflammatory Pain in Mice: Participation of TRPV1 and TRPA1 Ion Channels"

    Article Title: Wu-Tou Decoction Inhibits Chronic Inflammatory Pain in Mice: Participation of TRPV1 and TRPA1 Ion Channels

    Journal: BioMed Research International

    doi: 10.1155/2015/328707

    Effect of WTD on the activities of TRPV1 (a), TRPA1 (b), and TRPM8 (c) ion channels in mice. (a) Effect of WTD (6.30 g/kg, p.o.) and the TRPV1 antagonist AMG9810 (30 mg/kg, i.p.) on capsaicin-induced (2 μ g/paw) nociception. (b) Effect of WTD (6.30 g/kg, p.o.) and the TRPA1 antagonist camphor (7.6 mg/kg, s.c.) on cinnamaldehyde-induced (1.3 μ g/paw) nociception. (c) Effect of WTD (6.30 g/kg, p.o.) on icilin-induced (50 mg/kg, i.p.) jumping and WDS behaviors. Data are represented as the mean ± SEM ( n = 6). *** P
    Figure Legend Snippet: Effect of WTD on the activities of TRPV1 (a), TRPA1 (b), and TRPM8 (c) ion channels in mice. (a) Effect of WTD (6.30 g/kg, p.o.) and the TRPV1 antagonist AMG9810 (30 mg/kg, i.p.) on capsaicin-induced (2 μ g/paw) nociception. (b) Effect of WTD (6.30 g/kg, p.o.) and the TRPA1 antagonist camphor (7.6 mg/kg, s.c.) on cinnamaldehyde-induced (1.3 μ g/paw) nociception. (c) Effect of WTD (6.30 g/kg, p.o.) on icilin-induced (50 mg/kg, i.p.) jumping and WDS behaviors. Data are represented as the mean ± SEM ( n = 6). *** P

    Techniques Used: Mouse Assay

    Effect of WTD on the expression of TRPV1, TRPA1, and TRPM8 in DRGs of inflammatory pain mice by immunohistochemical staining. Mice were orally administrated with WTD (1.58, 3.15, and 6.30 g/kg, resp.) or water daily for 15 days. (a) Localization of positive TRPV1, TRPA1, and TRPM8 neurons in DRGs of mice from control, CFA, and WTD groups, respectively. ((b) and (c)) The numbers of TRPV1- and TRPA1-positive neurons significantly increased in DRGs in CFA group, while WTD significantly reduced their expression. (c) No significant difference of the number of TRPM8-positive neurons was observed among the five groups. Data are represented as the mean ± SEM ( n = 8). ## P
    Figure Legend Snippet: Effect of WTD on the expression of TRPV1, TRPA1, and TRPM8 in DRGs of inflammatory pain mice by immunohistochemical staining. Mice were orally administrated with WTD (1.58, 3.15, and 6.30 g/kg, resp.) or water daily for 15 days. (a) Localization of positive TRPV1, TRPA1, and TRPM8 neurons in DRGs of mice from control, CFA, and WTD groups, respectively. ((b) and (c)) The numbers of TRPV1- and TRPA1-positive neurons significantly increased in DRGs in CFA group, while WTD significantly reduced their expression. (c) No significant difference of the number of TRPM8-positive neurons was observed among the five groups. Data are represented as the mean ± SEM ( n = 8). ## P

    Techniques Used: Expressing, Mouse Assay, Immunohistochemistry, Staining

    Effect of WTD on the expression of TRPV1 (a), TRPA1 (b), and TRPM8 (c) in skins of injured paw of inflammatory pain mice by western blot. Mice were orally administrated with WTD (1.58, 3.15, and 6.30 g/kg, resp.), ibuprofen (0.14 g/kg), or water daily for 15 days. The protein expression of TRPV1 and TRPA1 significantly increased in CFA group, while WTD dose-dependently decreased their expression. No significant difference of TRPM8 protein expression was observed among the five groups. Data are represented as the mean ± SEM ( n = 4). ## P
    Figure Legend Snippet: Effect of WTD on the expression of TRPV1 (a), TRPA1 (b), and TRPM8 (c) in skins of injured paw of inflammatory pain mice by western blot. Mice were orally administrated with WTD (1.58, 3.15, and 6.30 g/kg, resp.), ibuprofen (0.14 g/kg), or water daily for 15 days. The protein expression of TRPV1 and TRPA1 significantly increased in CFA group, while WTD dose-dependently decreased their expression. No significant difference of TRPM8 protein expression was observed among the five groups. Data are represented as the mean ± SEM ( n = 4). ## P

    Techniques Used: Expressing, Mouse Assay, Western Blot

    3) 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

    4) Product Images from "Calcium cytotoxicity sensitizes prostate cancer cells to standard-of-care treatments for locally advanced tumors"

    Article Title: Calcium cytotoxicity sensitizes prostate cancer cells to standard-of-care treatments for locally advanced tumors

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-03256-5

    TRPM8 immunoscoring predicts X-rays + WS-12 efficacy. a TRPM8 immunostaining of BM-18 PDX. Scale bars, 100 μm. b Western blotting analysis shows comparable expression levels of TRPM8 in BM-18 and RWPE-1 M8 cells. c Immunofluorescence images showing co-staining of Ki-67 (green, upper panel) or Cleaved Caspase-3 (green, lower panel) with CK8 (red) and DAPI (blue). d Percentage of Ki-67 positive cells on a total of 30,000 cells in at least five different areas of the sample. Scale bars, 50 μm. e Western blotting analysis in BM-18 PDX tissues slices upon WS-12 (1 μM, 48 h), X-rays (10 Gy), or X-ray + WS-12 treatments showing molecular hallmarks of apoptotic cell death (Caspase-3 and PARP cleavage). Error bars, mean ± SD. Data were analyzed using a two-tailed Student’s t -test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. f Representative flow cytometry analysis of apoptotic cell death by Annexin-V/Sytox-Green labeling in LNCaP FGC M8 cells treated with WS-12 (1 μM), docetaxel (5 nM), enzalutamide (1 μM), WS-12 + docetaxel, or WS-12 + enzalutamide for 48 h. Untreated cells were used as control. g Quantification of dying cells in LNCaP FGC expressing endogenous (WT), increased (M8) or knocked-out (CAS) levels of TRPM8 treated as indicated in f . h Western blotting analysis of the indicated samples showing CaMKIIα activation (phosphorylation of Thr286) following WS-12 treatment of LNCaP FGC WT and M8 cells and the molecular signature of apoptotic cell death (Caspase-3 and PARP cleavage) upon treatment with combination of WS-12 with docetaxel or enzalutamide. Error bars, mean ± SD. Experiments were performed in quadruplicate; data were analyzed using a two-way ANOVA test. ** P ≤ 0.01.
    Figure Legend Snippet: TRPM8 immunoscoring predicts X-rays + WS-12 efficacy. a TRPM8 immunostaining of BM-18 PDX. Scale bars, 100 μm. b Western blotting analysis shows comparable expression levels of TRPM8 in BM-18 and RWPE-1 M8 cells. c Immunofluorescence images showing co-staining of Ki-67 (green, upper panel) or Cleaved Caspase-3 (green, lower panel) with CK8 (red) and DAPI (blue). d Percentage of Ki-67 positive cells on a total of 30,000 cells in at least five different areas of the sample. Scale bars, 50 μm. e Western blotting analysis in BM-18 PDX tissues slices upon WS-12 (1 μM, 48 h), X-rays (10 Gy), or X-ray + WS-12 treatments showing molecular hallmarks of apoptotic cell death (Caspase-3 and PARP cleavage). Error bars, mean ± SD. Data were analyzed using a two-tailed Student’s t -test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. f Representative flow cytometry analysis of apoptotic cell death by Annexin-V/Sytox-Green labeling in LNCaP FGC M8 cells treated with WS-12 (1 μM), docetaxel (5 nM), enzalutamide (1 μM), WS-12 + docetaxel, or WS-12 + enzalutamide for 48 h. Untreated cells were used as control. g Quantification of dying cells in LNCaP FGC expressing endogenous (WT), increased (M8) or knocked-out (CAS) levels of TRPM8 treated as indicated in f . h Western blotting analysis of the indicated samples showing CaMKIIα activation (phosphorylation of Thr286) following WS-12 treatment of LNCaP FGC WT and M8 cells and the molecular signature of apoptotic cell death (Caspase-3 and PARP cleavage) upon treatment with combination of WS-12 with docetaxel or enzalutamide. Error bars, mean ± SD. Experiments were performed in quadruplicate; data were analyzed using a two-way ANOVA test. ** P ≤ 0.01.

    Techniques Used: Immunostaining, Western Blot, Expressing, Immunofluorescence, Staining, Two Tailed Test, Flow Cytometry, Labeling, Activation Assay

    Proposed model for therapy resistance bypass in PCa cells. The scheme shows the lethal synergy between standard-of-care therapies and Ca 2+ cytotoxicity induced by potent TRPM8 agonists in PCa cells expressing increased amounts of the channel (BioRender.com).
    Figure Legend Snippet: Proposed model for therapy resistance bypass in PCa cells. The scheme shows the lethal synergy between standard-of-care therapies and Ca 2+ cytotoxicity induced by potent TRPM8 agonists in PCa cells expressing increased amounts of the channel (BioRender.com).

    Techniques Used: Expressing

    TRPM8 expression in human PCa. a TCGA RNA-seq dataset showing TRPM8 expression levels in normal tissues and related primary tumors. b TCGA, SU2C, and Beltran RNA-seq datasets analysis stating TRPM8 expression levels in benign prostate tissue, primary PCa and castration resistant metastatic adeno-PCa. Data were analyzed using a two-tailed Wilcoxon–Mann–Whitney test with a significance level set at 5%. c TRPM8 mRNA levels in 52 matched normal and adjacent PCa samples showing increased expression of TRPM8 in PCa compared to adjacent normal tissue in 36 cases, reduction in 5 and comparable levels in 11. d Relative amount of PM-associated 6TM (full length) and ER-associated 4TM TRPM8 transcript isoforms in 52 matched normal (N) and primary PCa (PCa) samples, as retrieved in TCGA RNA-seq dataset. e TRPM8 immunostaining score of a commercially available PCa tissue microarray (TMA). TRPM8 immunostaining was scored as weak (0), moderate (1), high (2), or very high (3) on 5 normal prostate cores and 171 PCa cores representing 57 different cases (3 cores × tumor). Representative images of scored normal prostate tissue and prostate adenocarcinoma cores are shown. Results are presented as percentage of tumors scored 0-to-3 respect to tumor stage. Stage I: score 1 = 36%; score 2 = 36%; score 3 = 28%; stage II: score 1 = 9%, score 2 = 64%, score 3 = 27%; stage III: score 1 = 8%; score 2 = 38%; score 3 54%; stage IV: score 1 = 12%, score 2 = 25%, score 3 = 63%. Scale bars, 100 μm. f TRPM8 immunostaining of matched primary PCa (A, B) and hormone naïve lymph node metastases (a, b). Scale bars, 100 μm.
    Figure Legend Snippet: TRPM8 expression in human PCa. a TCGA RNA-seq dataset showing TRPM8 expression levels in normal tissues and related primary tumors. b TCGA, SU2C, and Beltran RNA-seq datasets analysis stating TRPM8 expression levels in benign prostate tissue, primary PCa and castration resistant metastatic adeno-PCa. Data were analyzed using a two-tailed Wilcoxon–Mann–Whitney test with a significance level set at 5%. c TRPM8 mRNA levels in 52 matched normal and adjacent PCa samples showing increased expression of TRPM8 in PCa compared to adjacent normal tissue in 36 cases, reduction in 5 and comparable levels in 11. d Relative amount of PM-associated 6TM (full length) and ER-associated 4TM TRPM8 transcript isoforms in 52 matched normal (N) and primary PCa (PCa) samples, as retrieved in TCGA RNA-seq dataset. e TRPM8 immunostaining score of a commercially available PCa tissue microarray (TMA). TRPM8 immunostaining was scored as weak (0), moderate (1), high (2), or very high (3) on 5 normal prostate cores and 171 PCa cores representing 57 different cases (3 cores × tumor). Representative images of scored normal prostate tissue and prostate adenocarcinoma cores are shown. Results are presented as percentage of tumors scored 0-to-3 respect to tumor stage. Stage I: score 1 = 36%; score 2 = 36%; score 3 = 28%; stage II: score 1 = 9%, score 2 = 64%, score 3 = 27%; stage III: score 1 = 8%; score 2 = 38%; score 3 54%; stage IV: score 1 = 12%, score 2 = 25%, score 3 = 63%. Scale bars, 100 μm. f TRPM8 immunostaining of matched primary PCa (A, B) and hormone naïve lymph node metastases (a, b). Scale bars, 100 μm.

    Techniques Used: Expressing, RNA Sequencing Assay, Two Tailed Test, MANN-WHITNEY, Immunostaining, Microarray

    Modeling different levels of TRPM8 in RWPE-1 and LNCaP FGC prostate cell lines. a Western blot analysis and quantification of full-length 6TM TRPM8 amount in RWPE-1 and LNCaP FGC cell lines expressing endogenous (WT), overexpressed (M8) or knocked-out (CAS) levels of the protein. b Immunofluorescence analysis and quantification of PM-associated full-length 6TM TRPM8 in RWPE-1 and LNCaP FGC cell lines with endogenous (WT), overexpressed (M8), or knocked-out (CAS) levels of the channel. For quantification of PM TRPM8 positive cells a total of 6000 cells were counted from different fields. Scale bar, 5 μm. c–e Morphology ( c ), growth ( d ), and cell death ( e ) analyses of RWPE-1 and LNCaP FGC cell lines with endogenous (WT), overexpressed (M8), or knocked-out (CAS) levels of TRPM8. Error bars, mean ± SD. Experiments were performed in triplicate; data were analyzed using a two-tailed Student’s t -test. ** P ≤ 0.01; *** P ≤ 0.001.
    Figure Legend Snippet: Modeling different levels of TRPM8 in RWPE-1 and LNCaP FGC prostate cell lines. a Western blot analysis and quantification of full-length 6TM TRPM8 amount in RWPE-1 and LNCaP FGC cell lines expressing endogenous (WT), overexpressed (M8) or knocked-out (CAS) levels of the protein. b Immunofluorescence analysis and quantification of PM-associated full-length 6TM TRPM8 in RWPE-1 and LNCaP FGC cell lines with endogenous (WT), overexpressed (M8), or knocked-out (CAS) levels of the channel. For quantification of PM TRPM8 positive cells a total of 6000 cells were counted from different fields. Scale bar, 5 μm. c–e Morphology ( c ), growth ( d ), and cell death ( e ) analyses of RWPE-1 and LNCaP FGC cell lines with endogenous (WT), overexpressed (M8), or knocked-out (CAS) levels of TRPM8. Error bars, mean ± SD. Experiments were performed in triplicate; data were analyzed using a two-tailed Student’s t -test. ** P ≤ 0.01; *** P ≤ 0.001.

    Techniques Used: Western Blot, Expressing, Immunofluorescence, Two Tailed Test

    RWPE-1 response to TRPM8 agonist WS-12. a Cell death response by FACS (Annexin-V; Sytox-Green) in RWPE-1 cells expressing endogenous, increased (M8) or knocked-out (CAS) TRPM8 levels following 12 h WS-12 (1 μM) administration. Quantification is reported as percentage of total cells (lower panel). b Western blotting analysis showing molecular signature of apoptotic cell death (Caspase-3 and PARP cleavage). Staurosporine was used as positive control. Error bars, mean ± SD. Experiments were performed in triplicate; data were analyzed using a two-tailed Student’s t -test. *** P ≤ 0.001.
    Figure Legend Snippet: RWPE-1 response to TRPM8 agonist WS-12. a Cell death response by FACS (Annexin-V; Sytox-Green) in RWPE-1 cells expressing endogenous, increased (M8) or knocked-out (CAS) TRPM8 levels following 12 h WS-12 (1 μM) administration. Quantification is reported as percentage of total cells (lower panel). b Western blotting analysis showing molecular signature of apoptotic cell death (Caspase-3 and PARP cleavage). Staurosporine was used as positive control. Error bars, mean ± SD. Experiments were performed in triplicate; data were analyzed using a two-tailed Student’s t -test. *** P ≤ 0.001.

    Techniques Used: FACS, Expressing, Western Blot, Positive Control, Two Tailed Test

    TRPM8 channel activity in RWPE-1 prostate cells. a Representative images (upper panels) and traces (lower panels) showing [Ca 2+ ] i changes under control solution (CTR) or upon perfusion with menthol (1 mM), WS-12 (1 μM), or icilin (10 μM) on RWPE-1 cells. Time of drugs exposure is indicated by the bar on top of the traces. Quantification of [Ca 2+ ] i peaks measured upon perfusion with TRPM8 activators is reported on the right panel ( n = number of analyzed cells). The inset graph indicates the quantification of the total % of cells responsive to tested drugs. b Representative images (left panels) and traces (middle panels) showing [Ca 2+ ] i under control solution (CTR), upon perfusion with menthol (1 mM), WS-12 (1 μM), or icilin (10 μM), or after drugs washout on RWPE-1 M8 cells. Time of drugs exposure is indicated by the bar on top of the traces. Quantification of the [Ca 2+ ] i peaks measured upon perfusion with TRPM8 activators is reported on the right panel ( n = number of analyzed cells). The inset graph indicates the percentage of cells responsive to the drugs tested. c Left panel, representative traces of currents evoked by a 100 ms voltage ramp ranging from −100 mV to +100 mV applied every 4 s in control solution (CTL), during application of menthol (500 μM) or after drug washout (W). Right panel, representative time-courses of currents recorded at +80 mV (blue symbols) or −80 mV (red symbols) in single RWPE-1 M8 cells upon exposure to menthol (500 μM). Time of menthol exposure is indicated by the line on top of the traces. d Western blotting analysis with two independent antibodies (D21E4 and 22B1) showing CaMKIIα activation (phosphorylation of Thr286) following WS-12 treatment of RWPE-1 M8 cells. Cells treated with ionomycin were used as positive control for calcium dependent CaMKIIα phosphorylation. Error bars, mean ± SEM. Experiments were performed in at least three experimental sessions; data were analyzed using a two-tailed Student’s t -test. * P ≤ 0.05.
    Figure Legend Snippet: TRPM8 channel activity in RWPE-1 prostate cells. a Representative images (upper panels) and traces (lower panels) showing [Ca 2+ ] i changes under control solution (CTR) or upon perfusion with menthol (1 mM), WS-12 (1 μM), or icilin (10 μM) on RWPE-1 cells. Time of drugs exposure is indicated by the bar on top of the traces. Quantification of [Ca 2+ ] i peaks measured upon perfusion with TRPM8 activators is reported on the right panel ( n = number of analyzed cells). The inset graph indicates the quantification of the total % of cells responsive to tested drugs. b Representative images (left panels) and traces (middle panels) showing [Ca 2+ ] i under control solution (CTR), upon perfusion with menthol (1 mM), WS-12 (1 μM), or icilin (10 μM), or after drugs washout on RWPE-1 M8 cells. Time of drugs exposure is indicated by the bar on top of the traces. Quantification of the [Ca 2+ ] i peaks measured upon perfusion with TRPM8 activators is reported on the right panel ( n = number of analyzed cells). The inset graph indicates the percentage of cells responsive to the drugs tested. c Left panel, representative traces of currents evoked by a 100 ms voltage ramp ranging from −100 mV to +100 mV applied every 4 s in control solution (CTL), during application of menthol (500 μM) or after drug washout (W). Right panel, representative time-courses of currents recorded at +80 mV (blue symbols) or −80 mV (red symbols) in single RWPE-1 M8 cells upon exposure to menthol (500 μM). Time of menthol exposure is indicated by the line on top of the traces. d Western blotting analysis with two independent antibodies (D21E4 and 22B1) showing CaMKIIα activation (phosphorylation of Thr286) following WS-12 treatment of RWPE-1 M8 cells. Cells treated with ionomycin were used as positive control for calcium dependent CaMKIIα phosphorylation. Error bars, mean ± SEM. Experiments were performed in at least three experimental sessions; data were analyzed using a two-tailed Student’s t -test. * P ≤ 0.05.

    Techniques Used: Activity Assay, Western Blot, Activation Assay, Positive Control, Two Tailed Test

    5) Product Images from "Forsythoside A exerts antipyretic effect on yeast-induced pyrexia mice via inhibiting transient receptor potential vanilloid 1 function"

    Article Title: Forsythoside A exerts antipyretic effect on yeast-induced pyrexia mice via inhibiting transient receptor potential vanilloid 1 function

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.18045

    FT-Ainhibits TRPV1 expression while increases TRPA1 and TRPM8 expression in the hypothalamus and DRG of the mice with yeast-induced pyrexia. Expressions ofTRPV1, TRPA1 and TRPM8 were evaluated in the hypothalamus (A) and DRG (B) by Western blotting. Values are Mean ± SD of 5 mice per group. # P
    Figure Legend Snippet: FT-Ainhibits TRPV1 expression while increases TRPA1 and TRPM8 expression in the hypothalamus and DRG of the mice with yeast-induced pyrexia. Expressions ofTRPV1, TRPA1 and TRPM8 were evaluated in the hypothalamus (A) and DRG (B) by Western blotting. Values are Mean ± SD of 5 mice per group. # P

    Techniques Used: Expressing, Mouse Assay, Western Blot

    6) Product Images from "Wu-Tou Decoction Inhibits Chronic Inflammatory Pain in Mice: Participation of TRPV1 and TRPA1 Ion Channels"

    Article Title: Wu-Tou Decoction Inhibits Chronic Inflammatory Pain in Mice: Participation of TRPV1 and TRPA1 Ion Channels

    Journal: BioMed Research International

    doi: 10.1155/2015/328707

    Effect of WTD on the activities of TRPV1 (a), TRPA1 (b), and TRPM8 (c) ion channels in mice. (a) Effect of WTD (6.30 g/kg, p.o.) and the TRPV1 antagonist AMG9810 (30 mg/kg, i.p.) on capsaicin-induced (2 μ g/paw) nociception. (b) Effect of WTD (6.30 g/kg, p.o.) and the TRPA1 antagonist camphor (7.6 mg/kg, s.c.) on cinnamaldehyde-induced (1.3 μ g/paw) nociception. (c) Effect of WTD (6.30 g/kg, p.o.) on icilin-induced (50 mg/kg, i.p.) jumping and WDS behaviors. Data are represented as the mean ± SEM ( n = 6). *** P
    Figure Legend Snippet: Effect of WTD on the activities of TRPV1 (a), TRPA1 (b), and TRPM8 (c) ion channels in mice. (a) Effect of WTD (6.30 g/kg, p.o.) and the TRPV1 antagonist AMG9810 (30 mg/kg, i.p.) on capsaicin-induced (2 μ g/paw) nociception. (b) Effect of WTD (6.30 g/kg, p.o.) and the TRPA1 antagonist camphor (7.6 mg/kg, s.c.) on cinnamaldehyde-induced (1.3 μ g/paw) nociception. (c) Effect of WTD (6.30 g/kg, p.o.) on icilin-induced (50 mg/kg, i.p.) jumping and WDS behaviors. Data are represented as the mean ± SEM ( n = 6). *** P

    Techniques Used: Mouse Assay

    Effect of WTD on the expression of TRPV1, TRPA1, and TRPM8 in DRGs of inflammatory pain mice by immunohistochemical staining. Mice were orally administrated with WTD (1.58, 3.15, and 6.30 g/kg, resp.) or water daily for 15 days. (a) Localization of positive TRPV1, TRPA1, and TRPM8 neurons in DRGs of mice from control, CFA, and WTD groups, respectively. ((b) and (c)) The numbers of TRPV1- and TRPA1-positive neurons significantly increased in DRGs in CFA group, while WTD significantly reduced their expression. (c) No significant difference of the number of TRPM8-positive neurons was observed among the five groups. Data are represented as the mean ± SEM ( n = 8). ## P
    Figure Legend Snippet: Effect of WTD on the expression of TRPV1, TRPA1, and TRPM8 in DRGs of inflammatory pain mice by immunohistochemical staining. Mice were orally administrated with WTD (1.58, 3.15, and 6.30 g/kg, resp.) or water daily for 15 days. (a) Localization of positive TRPV1, TRPA1, and TRPM8 neurons in DRGs of mice from control, CFA, and WTD groups, respectively. ((b) and (c)) The numbers of TRPV1- and TRPA1-positive neurons significantly increased in DRGs in CFA group, while WTD significantly reduced their expression. (c) No significant difference of the number of TRPM8-positive neurons was observed among the five groups. Data are represented as the mean ± SEM ( n = 8). ## P

    Techniques Used: Expressing, Mouse Assay, Immunohistochemistry, Staining

    Effect of WTD on the expression of TRPV1 (a), TRPA1 (b), and TRPM8 (c) in skins of injured paw of inflammatory pain mice by western blot. Mice were orally administrated with WTD (1.58, 3.15, and 6.30 g/kg, resp.), ibuprofen (0.14 g/kg), or water daily for 15 days. The protein expression of TRPV1 and TRPA1 significantly increased in CFA group, while WTD dose-dependently decreased their expression. No significant difference of TRPM8 protein expression was observed among the five groups. Data are represented as the mean ± SEM ( n = 4). ## P
    Figure Legend Snippet: Effect of WTD on the expression of TRPV1 (a), TRPA1 (b), and TRPM8 (c) in skins of injured paw of inflammatory pain mice by western blot. Mice were orally administrated with WTD (1.58, 3.15, and 6.30 g/kg, resp.), ibuprofen (0.14 g/kg), or water daily for 15 days. The protein expression of TRPV1 and TRPA1 significantly increased in CFA group, while WTD dose-dependently decreased their expression. No significant difference of TRPM8 protein expression was observed among the five groups. Data are represented as the mean ± SEM ( n = 4). ## P

    Techniques Used: Expressing, Mouse Assay, Western Blot

    Related Articles

    Expressing:

    Article Title: TRPM8 protein expression in hormone naïve local and lymph node metastatic prostate cancer
    Article Snippet: .. Furthermore, the ACC-049 antibody confirmed the lack of TRPM8 expression in the metastatic PCa cell line PC-3 , , while it detected an increased amount of TRPM8 in the RWPE-1 M8 cells, which was obtained by stable insertion in their genome of exogenous cDNA encoding the channel ( ). ..

    Immunocytochemistry:

    Article Title: TRPM8 protein expression in hormone naïve local and lymph node metastatic prostate cancer
    Article Snippet: .. Next, the Alomone ACC-049 antibody was tested for the ability to detect TRPM8 by immunocytochemistry in RWPE-1, RWPE-1 M8, LNCaP, and PC3 aldehyde fixed-paraffin embedded cellular pellets. ..

    Article Title: TRPM8 protein expression in hormone naïve local and lymph node metastatic prostate cancer
    Article Snippet: .. A combination of western blot studies and immunocytochemistry analyses demonstrated the specificity of the Alomone Labs ACC-049 antibody. ..

    Immunohistochemistry:

    Article Title: TRPM8 protein expression in hormone naïve local and lymph node metastatic prostate cancer
    Article Snippet: .. Finally, by immunohistochemistry, ACC-049 antibody marked the epithelial compartment of the gland in human prostate specimens, with cancer cells (lumens without outer layer of HMWCKs positive cells) more strongly stained than benign (lumens with an outer layer of HMWCKs positive cells) ( ). ..

    Article Title: TRPM8 protein expression in hormone naïve local and lymph node metastatic prostate cancer
    Article Snippet: .. SPECIFICITY OF TRPM8 ANTIBODY ACC049 The reliability of immunohistochemical studies critically depends on the specificity of the antibodies. ..

    Article Title: TRPM8 protein expression in hormone naïve local and lymph node metastatic prostate cancer
    Article Snippet: .. To establish a consistent immunohistochemical procedure to score TRPM8 levels in prostate specimens, we meticulously tested a panel of commercially available TRPM8 antibodies. ..

    Staining:

    Article Title: TRPM8 protein expression in hormone naïve local and lymph node metastatic prostate cancer
    Article Snippet: .. Finally, by immunohistochemistry, ACC-049 antibody marked the epithelial compartment of the gland in human prostate specimens, with cancer cells (lumens without outer layer of HMWCKs positive cells) more strongly stained than benign (lumens with an outer layer of HMWCKs positive cells) ( ). ..

    Western Blot:

    Article Title: TRPM8 protein expression in hormone naïve local and lymph node metastatic prostate cancer
    Article Snippet: .. A combination of western blot studies and immunocytochemistry analyses demonstrated the specificity of the Alomone Labs ACC-049 antibody. ..

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    Alomone Labs anti trpm8
    Effects of cold on <t>TRPM8</t> channel currents in Du 145 cells with and without TRPM8 (mean ± SD; n = 3 independent experiments). After whole-cell configuration (W.C.), the temperature of the patch chamber was gradually lowered from 27 °C to 15 °C by using a heat-controlled path-chamber and the currents were recorded in the patched cells. A) Recording from a Du 145 M8KO cell with cold stimulation. B) Recording from a Du 145 WT cell with cold stimulation and ACA inhibition. Corresponding I/V- relation (red traces) of currents recorded at the indicated time points 1 and 2. C ) Current densities after cold exposure. Cold-evoked TRPM8 currents were blocked by extracellular ACA (0.025 mM). ( a p
    Anti Trpm8, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs affinity purified rabbit anti trpm8 polyclonal antibody
    Rap1 is a TRPM8-interacting protein. (A) Representative immunoprecipitation experiments. Expression vectors encoding Rap1-WT-HA were transfected into HEK-overexpressing TRPM8- or TRPM8 Y905A -tagged cells. Cell lysates were immunoprecipitated (IP) with an anti–HA antibody and immunoblotted with antibodies against TRPM8 and HA. Images in C and D are representative of three independent experiments. (B) In situ detection of endogenous TRPM8/Rap1 interaction in HMECs. TRPM8/Rap1 complexes were monitored using PLA using <t>anti–TRPM8</t> and Rap1 antibodies followed by staining with proximity probes, ligation, and localized rolling-circle amplification. HMECs silenced for TRPM8 (siTRPM8) or not (siCNTRL), in the presence or absence of 10 µM icilin (10 min treatment). Close locations between the two proteins were observed as red fluorescent dots and DAPI-stained nuclei as blue. (Bii) Puncta density quantified as mean ± SEM puncta per cell. *, P ≤ 0.05; **, P ≤ 0.001 (Student’s t test). (Ci) TRPM8 N-terminal tail (GST-Nt), C-terminal tail (GST-Ct), or GST were incubated with lysates of HEK cells overexpressing Rap1-WT-GFP, Rap1-N17-GFP, or Rap1-V12 and precipitated using GST. Western blotting was performed with anti–GFP antibody. One representative experiment of three is shown. (Cii) Quantification of Rap1/TRPM8 tails normalized over the input. Data are expressed as mean ± SEM. *, P
    Affinity Purified Rabbit Anti Trpm8 Polyclonal Antibody, 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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    Effects of cold on TRPM8 channel currents in Du 145 cells with and without TRPM8 (mean ± SD; n = 3 independent experiments). After whole-cell configuration (W.C.), the temperature of the patch chamber was gradually lowered from 27 °C to 15 °C by using a heat-controlled path-chamber and the currents were recorded in the patched cells. A) Recording from a Du 145 M8KO cell with cold stimulation. B) Recording from a Du 145 WT cell with cold stimulation and ACA inhibition. Corresponding I/V- relation (red traces) of currents recorded at the indicated time points 1 and 2. C ) Current densities after cold exposure. Cold-evoked TRPM8 currents were blocked by extracellular ACA (0.025 mM). ( a p

    Journal: Redox Biology

    Article Title: Menthol evokes Ca2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

    doi: 10.1016/j.redox.2017.10.009

    Figure Lengend Snippet: Effects of cold on TRPM8 channel currents in Du 145 cells with and without TRPM8 (mean ± SD; n = 3 independent experiments). After whole-cell configuration (W.C.), the temperature of the patch chamber was gradually lowered from 27 °C to 15 °C by using a heat-controlled path-chamber and the currents were recorded in the patched cells. A) Recording from a Du 145 M8KO cell with cold stimulation. B) Recording from a Du 145 WT cell with cold stimulation and ACA inhibition. Corresponding I/V- relation (red traces) of currents recorded at the indicated time points 1 and 2. C ) Current densities after cold exposure. Cold-evoked TRPM8 currents were blocked by extracellular ACA (0.025 mM). ( a p

    Article Snippet: Antibodies used and their dilutions were: anti-TRPM8 (1:200; rabbit polyclonal, Alomone Labs #ACC-049) and anti-GAPDH (1∶10,000, rabbit polyclonal, Sigma Aldrich, # SAB2100894) and anti-rabbit-HRP (1:10,000; goat secondary, Sigma-Aldrich, #A5420).

    Techniques: Inhibition

    Detection of TRPM8 transcripts and protein in prostate and breast cancer cell lines. A ) Signals for TRPM8 mRNA were found in cell lines derived from prostate cancer-derived (lanes 1–3) and breast cancer-derived (lanes 4–6) cell lines. The normalization with the GAPDH housekeeping control allowed to semi-quantitatively assessing the abundance of the different transcripts present in each sample. B) Signals for TRPM8 protein were detected in all cell lines; the ones for MCF7 and BT-474 were weaker than for the other cell lines (expected mass: 129 kDa). The GAPDH protein signal was used as a loading control. C) Densitometry analysis. The values of TRPM8 expression for mRNA (filled columns) and protein (empty columns) levels were normalized to the values of GAPDH controls.

    Journal: Redox Biology

    Article Title: Menthol evokes Ca2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

    doi: 10.1016/j.redox.2017.10.009

    Figure Lengend Snippet: Detection of TRPM8 transcripts and protein in prostate and breast cancer cell lines. A ) Signals for TRPM8 mRNA were found in cell lines derived from prostate cancer-derived (lanes 1–3) and breast cancer-derived (lanes 4–6) cell lines. The normalization with the GAPDH housekeeping control allowed to semi-quantitatively assessing the abundance of the different transcripts present in each sample. B) Signals for TRPM8 protein were detected in all cell lines; the ones for MCF7 and BT-474 were weaker than for the other cell lines (expected mass: 129 kDa). The GAPDH protein signal was used as a loading control. C) Densitometry analysis. The values of TRPM8 expression for mRNA (filled columns) and protein (empty columns) levels were normalized to the values of GAPDH controls.

    Article Snippet: Antibodies used and their dilutions were: anti-TRPM8 (1:200; rabbit polyclonal, Alomone Labs #ACC-049) and anti-GAPDH (1∶10,000, rabbit polyclonal, Sigma Aldrich, # SAB2100894) and anti-rabbit-HRP (1:10,000; goat secondary, Sigma-Aldrich, #A5420).

    Techniques: Derivative Assay, Expressing

    The effect of different Ca 2+ signal modulators on menthol-evoked responses. A)- L) Single-cell (colored traces) and average fluorescence (grey traces) recordings from time-lapse videos show changes in [Ca 2+ ] cyt . Bars represent standard deviations (SD). Each figure represents the results of one representative experiment out of three with similar results. A) ATMB hydrochloride , a TRPM8 blocker administered before menthol had no effect on menthol-evoked Ca 2+ response B ) BCTC, a putative TRPM8 blocker, administered before menthol had no effect on menthol-evoked Ca 2+ responses. C) CapZ, an another putative TRPM8 blocker had no effect on menthol-evoked Ca 2+ responses. D ) Removing the extracellular Ca 2+ ions strongly reduced the menthol-evoked responses E) Cells pre-treated with 50 µM U-73122 did not show oscillations. F) Overexpression of InsP 3 −5-phosphatase hydrolyzing InsP 3 with concomitant BFP expression in transfected cells inhibited menthol-induced oscillations compared to the non-transfected cells. Blue and yellow traces represent average fluorescence recordings from cells with or without InsP 3 −5-phosphatase from the same culture dish, respectively. Asterisks represent significant differences, p

    Journal: Redox Biology

    Article Title: Menthol evokes Ca2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

    doi: 10.1016/j.redox.2017.10.009

    Figure Lengend Snippet: The effect of different Ca 2+ signal modulators on menthol-evoked responses. A)- L) Single-cell (colored traces) and average fluorescence (grey traces) recordings from time-lapse videos show changes in [Ca 2+ ] cyt . Bars represent standard deviations (SD). Each figure represents the results of one representative experiment out of three with similar results. A) ATMB hydrochloride , a TRPM8 blocker administered before menthol had no effect on menthol-evoked Ca 2+ response B ) BCTC, a putative TRPM8 blocker, administered before menthol had no effect on menthol-evoked Ca 2+ responses. C) CapZ, an another putative TRPM8 blocker had no effect on menthol-evoked Ca 2+ responses. D ) Removing the extracellular Ca 2+ ions strongly reduced the menthol-evoked responses E) Cells pre-treated with 50 µM U-73122 did not show oscillations. F) Overexpression of InsP 3 −5-phosphatase hydrolyzing InsP 3 with concomitant BFP expression in transfected cells inhibited menthol-induced oscillations compared to the non-transfected cells. Blue and yellow traces represent average fluorescence recordings from cells with or without InsP 3 −5-phosphatase from the same culture dish, respectively. Asterisks represent significant differences, p

    Article Snippet: Antibodies used and their dilutions were: anti-TRPM8 (1:200; rabbit polyclonal, Alomone Labs #ACC-049) and anti-GAPDH (1∶10,000, rabbit polyclonal, Sigma Aldrich, # SAB2100894) and anti-rabbit-HRP (1:10,000; goat secondary, Sigma-Aldrich, #A5420).

    Techniques: Fluorescence, Over Expression, Expressing, Transfection

    Menthol-evoked responses in HEK-293 cells. A) Time-lapse image series of Ca 2+ waves. The acquisition rate was set to 3 s. Blue and red colors depict lower and higher fluorescence intensities, respectively. A Ca 2+ wave in HEK-293 cells was evoked by administration of 500 µM menthol. Bar represent 100 µm. B) Single-cell (colored traces) and average fluorescence (grey traces) recordings from time-lapse videos show changes in [Ca 2+ ] cyt . Bars represent standard deviations (SD). A Ca 2+ response in HEK-293 cells was evoked by administration of 500 µM menthol. C ) Non-transfected HEK-293 cells didn’t respond to 50 µM menthol, but responded, if cells were transfected with a plasmid encoding the human TRPM8 receptor (yellow trace and green traces, respectively). D) Transfected HEK-293 cells showed only very small responses to 50 µM menthol in the absence of extracellular Ca 2+ ions; see magnification of the traces in absence of extracellular Ca 2+ in the boxed red area. This indicates that essentially TRPM8 PM channels are involved in the Ca 2+ responses. C-D ) Traces show average fluorescence values with SD. Asterisks represent significant differences, p

    Journal: Redox Biology

    Article Title: Menthol evokes Ca2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

    doi: 10.1016/j.redox.2017.10.009

    Figure Lengend Snippet: Menthol-evoked responses in HEK-293 cells. A) Time-lapse image series of Ca 2+ waves. The acquisition rate was set to 3 s. Blue and red colors depict lower and higher fluorescence intensities, respectively. A Ca 2+ wave in HEK-293 cells was evoked by administration of 500 µM menthol. Bar represent 100 µm. B) Single-cell (colored traces) and average fluorescence (grey traces) recordings from time-lapse videos show changes in [Ca 2+ ] cyt . Bars represent standard deviations (SD). A Ca 2+ response in HEK-293 cells was evoked by administration of 500 µM menthol. C ) Non-transfected HEK-293 cells didn’t respond to 50 µM menthol, but responded, if cells were transfected with a plasmid encoding the human TRPM8 receptor (yellow trace and green traces, respectively). D) Transfected HEK-293 cells showed only very small responses to 50 µM menthol in the absence of extracellular Ca 2+ ions; see magnification of the traces in absence of extracellular Ca 2+ in the boxed red area. This indicates that essentially TRPM8 PM channels are involved in the Ca 2+ responses. C-D ) Traces show average fluorescence values with SD. Asterisks represent significant differences, p

    Article Snippet: Antibodies used and their dilutions were: anti-TRPM8 (1:200; rabbit polyclonal, Alomone Labs #ACC-049) and anti-GAPDH (1∶10,000, rabbit polyclonal, Sigma Aldrich, # SAB2100894) and anti-rabbit-HRP (1:10,000; goat secondary, Sigma-Aldrich, #A5420).

    Techniques: Fluorescence, Transfection, IF-cells, Plasmid Preparation

    Rap1 is a TRPM8-interacting protein. (A) Representative immunoprecipitation experiments. Expression vectors encoding Rap1-WT-HA were transfected into HEK-overexpressing TRPM8- or TRPM8 Y905A -tagged cells. Cell lysates were immunoprecipitated (IP) with an anti–HA antibody and immunoblotted with antibodies against TRPM8 and HA. Images in C and D are representative of three independent experiments. (B) In situ detection of endogenous TRPM8/Rap1 interaction in HMECs. TRPM8/Rap1 complexes were monitored using PLA using anti–TRPM8 and Rap1 antibodies followed by staining with proximity probes, ligation, and localized rolling-circle amplification. HMECs silenced for TRPM8 (siTRPM8) or not (siCNTRL), in the presence or absence of 10 µM icilin (10 min treatment). Close locations between the two proteins were observed as red fluorescent dots and DAPI-stained nuclei as blue. (Bii) Puncta density quantified as mean ± SEM puncta per cell. *, P ≤ 0.05; **, P ≤ 0.001 (Student’s t test). (Ci) TRPM8 N-terminal tail (GST-Nt), C-terminal tail (GST-Ct), or GST were incubated with lysates of HEK cells overexpressing Rap1-WT-GFP, Rap1-N17-GFP, or Rap1-V12 and precipitated using GST. Western blotting was performed with anti–GFP antibody. One representative experiment of three is shown. (Cii) Quantification of Rap1/TRPM8 tails normalized over the input. Data are expressed as mean ± SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: TRPM8 inhibits endothelial cell migration via a non-channel function by trapping the small GTPase Rap1

    doi: 10.1083/jcb.201506024

    Figure Lengend Snippet: Rap1 is a TRPM8-interacting protein. (A) Representative immunoprecipitation experiments. Expression vectors encoding Rap1-WT-HA were transfected into HEK-overexpressing TRPM8- or TRPM8 Y905A -tagged cells. Cell lysates were immunoprecipitated (IP) with an anti–HA antibody and immunoblotted with antibodies against TRPM8 and HA. Images in C and D are representative of three independent experiments. (B) In situ detection of endogenous TRPM8/Rap1 interaction in HMECs. TRPM8/Rap1 complexes were monitored using PLA using anti–TRPM8 and Rap1 antibodies followed by staining with proximity probes, ligation, and localized rolling-circle amplification. HMECs silenced for TRPM8 (siTRPM8) or not (siCNTRL), in the presence or absence of 10 µM icilin (10 min treatment). Close locations between the two proteins were observed as red fluorescent dots and DAPI-stained nuclei as blue. (Bii) Puncta density quantified as mean ± SEM puncta per cell. *, P ≤ 0.05; **, P ≤ 0.001 (Student’s t test). (Ci) TRPM8 N-terminal tail (GST-Nt), C-terminal tail (GST-Ct), or GST were incubated with lysates of HEK cells overexpressing Rap1-WT-GFP, Rap1-N17-GFP, or Rap1-V12 and precipitated using GST. Western blotting was performed with anti–GFP antibody. One representative experiment of three is shown. (Cii) Quantification of Rap1/TRPM8 tails normalized over the input. Data are expressed as mean ± SEM. *, P

    Article Snippet: They were then incubated overnight at 4°C with PBS/5% nonimmunized serum containing a 1:200 dilution of the primary affinity-purified rabbit anti–TRPM8 polyclonal antibody (Alomone) and primary mouse anti–calnexin antibody.

    Techniques: Immunoprecipitation, Expressing, Transfection, In Situ, Proximity Ligation Assay, Staining, Ligation, Amplification, Incubation, Western Blot

    TCAF expression profile in prostate cancer. (A and B) Analysis of TRPM8, TCAF1, and TCAF2 mRNA expression levels by qPCR in healthy and cancerous human prostate samples (A), as well as in localized and metastatic human prostate cancer specimens (B). Values are calculated relative to 18S rRNA expression and presented as means ± SEM (error bars; n = 7–10; *, P

    Journal: The Journal of Cell Biology

    Article Title: TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

    doi: 10.1083/jcb.201402076

    Figure Lengend Snippet: TCAF expression profile in prostate cancer. (A and B) Analysis of TRPM8, TCAF1, and TCAF2 mRNA expression levels by qPCR in healthy and cancerous human prostate samples (A), as well as in localized and metastatic human prostate cancer specimens (B). Values are calculated relative to 18S rRNA expression and presented as means ± SEM (error bars; n = 7–10; *, P

    Article Snippet: Finally, biotinylated proteins were eluted with SDS-PAGE loading buffer, separated on 10% wt/vol SDS-PAGE gel, and analyzed by immunoblotting as described for the immunoprecipitation assays with rabbit anti-TRPM8 antibody (1:1,500; Alomone Labs Ltd).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    TCAF functional role in prostate cancer cell migration. (A) Representative migration plots of control (top left) and TRPM8-overexpressing cells (top right) or TRPM8 cotransfected with wild-type TCAF1 (bottom left) or truncated TCAF1 lacking the PI3K domain (TCAF1 ΔPI3K, bottom right). Each line represents the migration of one cell within a 10-h period. The data shown are from a single representative experiment out of three repeats. For the experiment shown: CNTRL n = 28, TRPM8 n = 58, TRPM8/TCAF1 n = 69, TRPM8/TCAF1ΔPI3K n = 98. (B and C) Bar graphs showing the quantification of cell speed in control nontransfected prostate cancer cells (CNTRL), TRPM8-overexpressing cells (TRPM8), TCAF1-overexpressing cells (TCAF1), TRPM8- and TCAF1-overexpressing cells (TRPM8/TCAF1), and TRPM8 and TCAF1 lacking the PI3K domain (TCAF1 ΔPI3K ) in the presence or absence of 10 µM icilin. (D–F) Bar graphs showing the quantification of cell speed (D) and the migratory persistence index (E, calculated as indicated in the online materials) in control cells and TRPM8-overexpressing cells in which TCAF1 or TCAF2 are down-regulated (TRPM8/siTCAF1), in the presence or absence of 10 µM icilin (F). Each graph represents the quantification of pooled independent experiments (at least three experiments for each experimental condition). For each experiment, at least 60 cells were followed per condition (*, P

    Journal: The Journal of Cell Biology

    Article Title: TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

    doi: 10.1083/jcb.201402076

    Figure Lengend Snippet: TCAF functional role in prostate cancer cell migration. (A) Representative migration plots of control (top left) and TRPM8-overexpressing cells (top right) or TRPM8 cotransfected with wild-type TCAF1 (bottom left) or truncated TCAF1 lacking the PI3K domain (TCAF1 ΔPI3K, bottom right). Each line represents the migration of one cell within a 10-h period. The data shown are from a single representative experiment out of three repeats. For the experiment shown: CNTRL n = 28, TRPM8 n = 58, TRPM8/TCAF1 n = 69, TRPM8/TCAF1ΔPI3K n = 98. (B and C) Bar graphs showing the quantification of cell speed in control nontransfected prostate cancer cells (CNTRL), TRPM8-overexpressing cells (TRPM8), TCAF1-overexpressing cells (TCAF1), TRPM8- and TCAF1-overexpressing cells (TRPM8/TCAF1), and TRPM8 and TCAF1 lacking the PI3K domain (TCAF1 ΔPI3K ) in the presence or absence of 10 µM icilin. (D–F) Bar graphs showing the quantification of cell speed (D) and the migratory persistence index (E, calculated as indicated in the online materials) in control cells and TRPM8-overexpressing cells in which TCAF1 or TCAF2 are down-regulated (TRPM8/siTCAF1), in the presence or absence of 10 µM icilin (F). Each graph represents the quantification of pooled independent experiments (at least three experiments for each experimental condition). For each experiment, at least 60 cells were followed per condition (*, P

    Article Snippet: Finally, biotinylated proteins were eluted with SDS-PAGE loading buffer, separated on 10% wt/vol SDS-PAGE gel, and analyzed by immunoblotting as described for the immunoprecipitation assays with rabbit anti-TRPM8 antibody (1:1,500; Alomone Labs Ltd).

    Techniques: Functional Assay, Migration

    TCAF1 interaction with TRPM8 introduces new kinetic states. (A) Representative trace fragments of control and TCAF1-bound TRPM8 activity stimulated by cold (T = 20°C, controlled by room thermostat) or cold + 100 µM menthol, as indicated. (B) Representative distributions of closed (left) and open ( Chetrite et al., 2000 ) dwell times in the traces of control and TCAF1-bound TRPM8 activity stimulated as indicated. The histogram shows the distribution of dwell times. The thick continuous line represents the cumulative best fit corresponding to the kinetic model used (C) and broken lines represent individual components of the fit. (C) Kinetic models providing the best fit to the control and TCAF1-bound TRPM8 activity. The area of each circle is proportional to the log of total time spent the corresponding state. Kinetic rates are indicated as numbers associated with the corresponding arrows. (D) Energy landscapes calculated from the corresponding kinetic models. Arrows point to significantly shifted energies in corresponding states caused by menthol stimulation. Note that contrary to the effects of different modes of TRPM8 stimulation (shifts in state energies), the interaction with TCAF1 leads to the appearance of new kinetic states.

    Journal: The Journal of Cell Biology

    Article Title: TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

    doi: 10.1083/jcb.201402076

    Figure Lengend Snippet: TCAF1 interaction with TRPM8 introduces new kinetic states. (A) Representative trace fragments of control and TCAF1-bound TRPM8 activity stimulated by cold (T = 20°C, controlled by room thermostat) or cold + 100 µM menthol, as indicated. (B) Representative distributions of closed (left) and open ( Chetrite et al., 2000 ) dwell times in the traces of control and TCAF1-bound TRPM8 activity stimulated as indicated. The histogram shows the distribution of dwell times. The thick continuous line represents the cumulative best fit corresponding to the kinetic model used (C) and broken lines represent individual components of the fit. (C) Kinetic models providing the best fit to the control and TCAF1-bound TRPM8 activity. The area of each circle is proportional to the log of total time spent the corresponding state. Kinetic rates are indicated as numbers associated with the corresponding arrows. (D) Energy landscapes calculated from the corresponding kinetic models. Arrows point to significantly shifted energies in corresponding states caused by menthol stimulation. Note that contrary to the effects of different modes of TRPM8 stimulation (shifts in state energies), the interaction with TCAF1 leads to the appearance of new kinetic states.

    Article Snippet: Finally, biotinylated proteins were eluted with SDS-PAGE loading buffer, separated on 10% wt/vol SDS-PAGE gel, and analyzed by immunoblotting as described for the immunoprecipitation assays with rabbit anti-TRPM8 antibody (1:1,500; Alomone Labs Ltd).

    Techniques: Activity Assay

    TCAF1 exhibits a PI3K homology domain that is critical for the TCAF1 modulation of TRPM8 activity. (A) Mean time course of cold- (22°C), icilin- (10 µM), and menthol-activated (500 µM) I TRPM8 in LNCaP cells pretreated (closed circles) or not (ctrl, open circles) for 15 min with 1 µM wortmannin. For pretreated cells, experiments were performed in the continuous presence of wortmannin at the same concentration. Currents were monitored at +100 mV, and results are presented as means ± SEM (error bars). (B) GST pull-down assay between [ 35 S]methionine-labeled TCAF1 or TCAF1 ΔPI3K protein and GST or GST fused to the TRPM8 N-terminal tail (GST-M8N) or C-terminal tail (GST-M8C). A schematic representation of TCAF1 and TCAF2 protein sequences and the putative PI3K site is shown. (C) Time course of mean intracellular Ca 2+ concentration in LNCaP cells transfected with TRPM8 and an empty vector (ctrl, open circles), wild-type TCAF1 (TCAF1, black closed circles), or a truncated TCAF1 lacking the PI3K domain (TCAF1 ΔPI3K , gray closed circles). Cells were loaded with Fura 2-AM, exposed to 10 µM icilin, and variations in [Ca 2+ ]i were monitored using an imaging system. Results are presented as means ± SEM (error bars). (inset) Histogram summarizing calcium imaging results (**, P

    Journal: The Journal of Cell Biology

    Article Title: TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

    doi: 10.1083/jcb.201402076

    Figure Lengend Snippet: TCAF1 exhibits a PI3K homology domain that is critical for the TCAF1 modulation of TRPM8 activity. (A) Mean time course of cold- (22°C), icilin- (10 µM), and menthol-activated (500 µM) I TRPM8 in LNCaP cells pretreated (closed circles) or not (ctrl, open circles) for 15 min with 1 µM wortmannin. For pretreated cells, experiments were performed in the continuous presence of wortmannin at the same concentration. Currents were monitored at +100 mV, and results are presented as means ± SEM (error bars). (B) GST pull-down assay between [ 35 S]methionine-labeled TCAF1 or TCAF1 ΔPI3K protein and GST or GST fused to the TRPM8 N-terminal tail (GST-M8N) or C-terminal tail (GST-M8C). A schematic representation of TCAF1 and TCAF2 protein sequences and the putative PI3K site is shown. (C) Time course of mean intracellular Ca 2+ concentration in LNCaP cells transfected with TRPM8 and an empty vector (ctrl, open circles), wild-type TCAF1 (TCAF1, black closed circles), or a truncated TCAF1 lacking the PI3K domain (TCAF1 ΔPI3K , gray closed circles). Cells were loaded with Fura 2-AM, exposed to 10 µM icilin, and variations in [Ca 2+ ]i were monitored using an imaging system. Results are presented as means ± SEM (error bars). (inset) Histogram summarizing calcium imaging results (**, P

    Article Snippet: Finally, biotinylated proteins were eluted with SDS-PAGE loading buffer, separated on 10% wt/vol SDS-PAGE gel, and analyzed by immunoblotting as described for the immunoprecipitation assays with rabbit anti-TRPM8 antibody (1:1,500; Alomone Labs Ltd).

    Techniques: Activity Assay, Concentration Assay, Pull Down Assay, Labeling, Transfection, Plasmid Preparation, Imaging

    TCAF1 and TCAF2 interact and colocalize with TRPM8. (A) GST pull-down assay using [ 35 S]methionine-labeled TCAF1 or TCAF2 protein and GST or GST fused to the TRPM8 N-terminal tail (GST-M8N) or C-terminal tail (GST-M8C). (B) Cells transfected with HA-tagged TCAF1 or myc-tagged TCAF2 with (HEK-M8) or without (HEK) his-tagged full-length TRPM8, and total lysates (TL) were used for immunoprecipitation and subsequent immunoblot analysis. Immunoprecipitation of the channel was confirmed by immunoblotting for TRPM8, and the coimmunoprecipitation of TCAF1 or TCAF2 proteins was detected using an anti-HA or anti-myc antibody, respectively, on the immunoprecipitated complex (IP). No coimmunoprecipitation was detected in control immunoblots for actin. The black lines on the HA-TCAF1 blots indicate the removal of intervening lanes for presentation purposes. (C–F) TCAF1/2-to-TRPM8 binding was assessed by measuring FRET with TM FLIM in HEK293 cells after a 24-h transfection. The TCSPC approach was chosen to achieve a good spatial resolution, which would allow discriminating TRPM8-ER localization. (C) An example of concomitant expression of TRPM8-mTurquoise2 (cyan) and TCAF1-SYFP2 (yellow). (D and E) Representative FRET-FLIM images for TRPM8-mTurquoise alone and for TRPM8-mTurquoise + TCAF1-SYFP2, respectively. Grayscale-coded fluorescence images of the donor of FRET (left images in D and E; fluorescence intensity [FI] is given in arbitrary units [a.u.]) were analyzed by the phasor plot method (Phasor) to compute FLIM images (τ mean ) showing the mean lifetime (ns; right images of D and E). The outlines define the regions of interest for which mean lifetime of the donor of fluorescence was calculated. Bars, 10 µm. (F) Chart representing the mean lifetime of TRPM8-mTurquoise2 alone, coexpressed with the negative control SYFP2, or with TCAF1-SYFP2 or TCAF2-SYFP2. FRET efficiency (E FRET ) is shown on the top of the chart. Values are presented as means ± SD (error bars). ***, P

    Journal: The Journal of Cell Biology

    Article Title: TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

    doi: 10.1083/jcb.201402076

    Figure Lengend Snippet: TCAF1 and TCAF2 interact and colocalize with TRPM8. (A) GST pull-down assay using [ 35 S]methionine-labeled TCAF1 or TCAF2 protein and GST or GST fused to the TRPM8 N-terminal tail (GST-M8N) or C-terminal tail (GST-M8C). (B) Cells transfected with HA-tagged TCAF1 or myc-tagged TCAF2 with (HEK-M8) or without (HEK) his-tagged full-length TRPM8, and total lysates (TL) were used for immunoprecipitation and subsequent immunoblot analysis. Immunoprecipitation of the channel was confirmed by immunoblotting for TRPM8, and the coimmunoprecipitation of TCAF1 or TCAF2 proteins was detected using an anti-HA or anti-myc antibody, respectively, on the immunoprecipitated complex (IP). No coimmunoprecipitation was detected in control immunoblots for actin. The black lines on the HA-TCAF1 blots indicate the removal of intervening lanes for presentation purposes. (C–F) TCAF1/2-to-TRPM8 binding was assessed by measuring FRET with TM FLIM in HEK293 cells after a 24-h transfection. The TCSPC approach was chosen to achieve a good spatial resolution, which would allow discriminating TRPM8-ER localization. (C) An example of concomitant expression of TRPM8-mTurquoise2 (cyan) and TCAF1-SYFP2 (yellow). (D and E) Representative FRET-FLIM images for TRPM8-mTurquoise alone and for TRPM8-mTurquoise + TCAF1-SYFP2, respectively. Grayscale-coded fluorescence images of the donor of FRET (left images in D and E; fluorescence intensity [FI] is given in arbitrary units [a.u.]) were analyzed by the phasor plot method (Phasor) to compute FLIM images (τ mean ) showing the mean lifetime (ns; right images of D and E). The outlines define the regions of interest for which mean lifetime of the donor of fluorescence was calculated. Bars, 10 µm. (F) Chart representing the mean lifetime of TRPM8-mTurquoise2 alone, coexpressed with the negative control SYFP2, or with TCAF1-SYFP2 or TCAF2-SYFP2. FRET efficiency (E FRET ) is shown on the top of the chart. Values are presented as means ± SD (error bars). ***, P

    Article Snippet: Finally, biotinylated proteins were eluted with SDS-PAGE loading buffer, separated on 10% wt/vol SDS-PAGE gel, and analyzed by immunoblotting as described for the immunoprecipitation assays with rabbit anti-TRPM8 antibody (1:1,500; Alomone Labs Ltd).

    Techniques: Pull Down Assay, Labeling, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Expressing, Fluorescence, Negative Control

    TCAF1 and TCAF2 proteins have opposing effects on TRPM8 activity. (A) Reverse-transcription PCR showing the specific decrease of TCAF1 (top) and TCAF2 (bottom) bands after cell transfection with 100 nM of the respective siRNAs. Actin was used to normalize relative expression, and siRNA to luciferase was used as a negative silencing control (siLuc). The white line on the TCAF1 gel indicates the removal of intervening lanes for presentation purposes. (B) Western blot analysis confirming the siRNA effect on HA-TCAF1– (top) and myc-TCAF2–transfected cells (bottom). Calnexin (Clnx) was used as a loading control. (C and D) Cell surface biotinylation analysis of TRPM8-transfected cells cotransfected with the empty vector (CTRL), TCAF1, or TCAF2 (C), or co-treated with siLuc, siTCAF1, or siTCAF2 (D). TRPM8 expression was analyzed by immunoblotting the plasma membrane fraction (TRPM8 PM ) or total cell lysates (TRPM8 TL ). Shown is the mean time course of cold- (22°C), icilin- (10 µM), and menthol-activated (500 µM) I TRPM8 in LNCaP cells transiently transfected with TRPM8 and treated with siLuc (open circles), siTCAF1 (E), or siTCAF2 (F; closed circles). Values are expressed as means ± SEM (error bars). (G) Single traces of menthol-evoked currents recorded in a representative LNCaP cell (out of five different cells per condition) transfected with TRPM8 and either an empty vector (ctrl), TCAF1, or TCAF2. Stimulation protocol is presented in the top panel. The corresponding IV relationships are presented in H.

    Journal: The Journal of Cell Biology

    Article Title: TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

    doi: 10.1083/jcb.201402076

    Figure Lengend Snippet: TCAF1 and TCAF2 proteins have opposing effects on TRPM8 activity. (A) Reverse-transcription PCR showing the specific decrease of TCAF1 (top) and TCAF2 (bottom) bands after cell transfection with 100 nM of the respective siRNAs. Actin was used to normalize relative expression, and siRNA to luciferase was used as a negative silencing control (siLuc). The white line on the TCAF1 gel indicates the removal of intervening lanes for presentation purposes. (B) Western blot analysis confirming the siRNA effect on HA-TCAF1– (top) and myc-TCAF2–transfected cells (bottom). Calnexin (Clnx) was used as a loading control. (C and D) Cell surface biotinylation analysis of TRPM8-transfected cells cotransfected with the empty vector (CTRL), TCAF1, or TCAF2 (C), or co-treated with siLuc, siTCAF1, or siTCAF2 (D). TRPM8 expression was analyzed by immunoblotting the plasma membrane fraction (TRPM8 PM ) or total cell lysates (TRPM8 TL ). Shown is the mean time course of cold- (22°C), icilin- (10 µM), and menthol-activated (500 µM) I TRPM8 in LNCaP cells transiently transfected with TRPM8 and treated with siLuc (open circles), siTCAF1 (E), or siTCAF2 (F; closed circles). Values are expressed as means ± SEM (error bars). (G) Single traces of menthol-evoked currents recorded in a representative LNCaP cell (out of five different cells per condition) transfected with TRPM8 and either an empty vector (ctrl), TCAF1, or TCAF2. Stimulation protocol is presented in the top panel. The corresponding IV relationships are presented in H.

    Article Snippet: Finally, biotinylated proteins were eluted with SDS-PAGE loading buffer, separated on 10% wt/vol SDS-PAGE gel, and analyzed by immunoblotting as described for the immunoprecipitation assays with rabbit anti-TRPM8 antibody (1:1,500; Alomone Labs Ltd).

    Techniques: Activity Assay, Polymerase Chain Reaction, Transfection, Expressing, Luciferase, Western Blot, Plasmid Preparation

    Bayesian phylogenetic tree of the vertebrate TCAF1 / TCAF2 / TCAF3 genes and tissue expression pattern of the human homologues. (A) The maximum likelihood tree was similar but for one node: the cow and horse FAM115C sequences were sister groups. The phylogenetic reconstruction displays two duplication events, one during the early period of mammalian evolution and the other during rodent evolution. The scale bar represents the number of expected changes per site. (B) Analysis of TRPM8, TCAF1, and TCAF2 mRNA expression levels by qPCR in several human tissues. Values are expressed relative to 18S rRNA expression, and further normalized to TRPM8 levels in the prostate. Values are presented as means of three experimental repeats ± SEM (error bars; n = 3 for a single experiment).

    Journal: The Journal of Cell Biology

    Article Title: TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

    doi: 10.1083/jcb.201402076

    Figure Lengend Snippet: Bayesian phylogenetic tree of the vertebrate TCAF1 / TCAF2 / TCAF3 genes and tissue expression pattern of the human homologues. (A) The maximum likelihood tree was similar but for one node: the cow and horse FAM115C sequences were sister groups. The phylogenetic reconstruction displays two duplication events, one during the early period of mammalian evolution and the other during rodent evolution. The scale bar represents the number of expected changes per site. (B) Analysis of TRPM8, TCAF1, and TCAF2 mRNA expression levels by qPCR in several human tissues. Values are expressed relative to 18S rRNA expression, and further normalized to TRPM8 levels in the prostate. Values are presented as means of three experimental repeats ± SEM (error bars; n = 3 for a single experiment).

    Article Snippet: Finally, biotinylated proteins were eluted with SDS-PAGE loading buffer, separated on 10% wt/vol SDS-PAGE gel, and analyzed by immunoblotting as described for the immunoprecipitation assays with rabbit anti-TRPM8 antibody (1:1,500; Alomone Labs Ltd).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    The menthol-induced response of endogenous TRPM8 is modulated by TCAF1 and TCAF2. (A–G) Changes of [Ca 2+ ] i in response to the activation of TRPM8 with 200 µM menthol were monitored using an x-y time-series imaging of fluo-4 fluorescence in control LNCaP cells (A), LNCaP cells overexpressing TCAF1 (B) or TCAF2 (C), and LNCaP cells pretreated with siLuc (E), siTCAF1 (F), or siTCAF2 (G). Images were acquired at 0.6 Hz from confocal optical slices

    Journal: The Journal of Cell Biology

    Article Title: TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

    doi: 10.1083/jcb.201402076

    Figure Lengend Snippet: The menthol-induced response of endogenous TRPM8 is modulated by TCAF1 and TCAF2. (A–G) Changes of [Ca 2+ ] i in response to the activation of TRPM8 with 200 µM menthol were monitored using an x-y time-series imaging of fluo-4 fluorescence in control LNCaP cells (A), LNCaP cells overexpressing TCAF1 (B) or TCAF2 (C), and LNCaP cells pretreated with siLuc (E), siTCAF1 (F), or siTCAF2 (G). Images were acquired at 0.6 Hz from confocal optical slices

    Article Snippet: Finally, biotinylated proteins were eluted with SDS-PAGE loading buffer, separated on 10% wt/vol SDS-PAGE gel, and analyzed by immunoblotting as described for the immunoprecipitation assays with rabbit anti-TRPM8 antibody (1:1,500; Alomone Labs Ltd).

    Techniques: Activation Assay, Imaging, Fluorescence

    (A) Western blotting analysis with Alomone ACC-049 antibody detecting endogenous full length TRPM8 protein in both RWPE1 and LNCaP prostate cell lines. Antibody specificity was confirmed through TRPM8 knockdown. RWPE1 and LNCaP prostate cells were transfected with non-targeting (siCTR) or TRPM8 targeting (siRNA1 and siRNA2) small interfering RNA molecules as indicated. (B) Western blotting analysis with Alomone ACC049 antibody detecting endogenous amount of full length TRPM8 protein in RWPE1 and LNCaP but not in PC3 prostate cell lines. Increased levels of the channel are detectable in RWPE1 stably overexpressing exogenous TRPM8. (C) Immunohistochemistry of TRPM8 on paraffin embedded RWPE1, RWPE1 M8, LNCaP and PC3 cell pellets matching the biochemical data. Scale bars, 100 μm.

    Journal: Pathologica

    Article Title: TRPM8 protein expression in hormone naïve local and lymph node metastatic prostate cancer

    doi: 10.32074/1591-951X-262

    Figure Lengend Snippet: (A) Western blotting analysis with Alomone ACC-049 antibody detecting endogenous full length TRPM8 protein in both RWPE1 and LNCaP prostate cell lines. Antibody specificity was confirmed through TRPM8 knockdown. RWPE1 and LNCaP prostate cells were transfected with non-targeting (siCTR) or TRPM8 targeting (siRNA1 and siRNA2) small interfering RNA molecules as indicated. (B) Western blotting analysis with Alomone ACC049 antibody detecting endogenous amount of full length TRPM8 protein in RWPE1 and LNCaP but not in PC3 prostate cell lines. Increased levels of the channel are detectable in RWPE1 stably overexpressing exogenous TRPM8. (C) Immunohistochemistry of TRPM8 on paraffin embedded RWPE1, RWPE1 M8, LNCaP and PC3 cell pellets matching the biochemical data. Scale bars, 100 μm.

    Article Snippet: Next, the Alomone ACC-049 antibody was tested for the ability to detect TRPM8 by immunocytochemistry in RWPE-1, RWPE-1 M8, LNCaP, and PC3 aldehyde fixed-paraffin embedded cellular pellets.

    Techniques: Western Blot, Transfection, Small Interfering RNA, Stable Transfection, Immunohistochemistry