trpm8  (Alomone Labs)


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    Structured Review

    Alomone Labs trpm8
    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, 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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    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trpm8 - by Bioz Stars, 2022-07
    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 "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

    5) 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

    6) 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: eLife

    doi: 10.7554/eLife.70153

    Proposed cold-induced vasoconstriction signalling pathway in young and aged mice. The local cold exposure produces rapid vasoconstriction which is significantly blunted in the aged mice (see blood flow graph at top centre). Cold water (4 °C) exposure to hindpaw activates the cold receptors TRPA1 and TRPM8 in sympathetic nerves, which leads to increased intracellular calcium and release of NA. This signalling, however, is significantly downregulated in aged mice due to diminished expression of TRPA1/TRPM8 in sympathetic nerves. NA acts on the post-synaptic α 2c adrenergic receptors on smooth muscle cells to mediate vasoconstriction. However, α 2c adrenergic receptor are also significantly diminished in aged mice, which leads to reduced signalling. All these factors contribute to an attenuated vascular cold response in aged mice compared to young mice.
    Figure Legend Snippet: Proposed cold-induced vasoconstriction signalling pathway in young and aged mice. The local cold exposure produces rapid vasoconstriction which is significantly blunted in the aged mice (see blood flow graph at top centre). Cold water (4 °C) exposure to hindpaw activates the cold receptors TRPA1 and TRPM8 in sympathetic nerves, which leads to increased intracellular calcium and release of NA. This signalling, however, is significantly downregulated in aged mice due to diminished expression of TRPA1/TRPM8 in sympathetic nerves. NA acts on the post-synaptic α 2c adrenergic receptors on smooth muscle cells to mediate vasoconstriction. However, α 2c adrenergic receptor are also significantly diminished in aged mice, which leads to reduced signalling. All these factors contribute to an attenuated vascular cold response in aged mice compared to young mice.

    Techniques Used: Mouse Assay, Expressing

    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 0–2 min 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 normalised against vehicle treated mice. (n = 5–10) ( g–h ) RT-PCR CT analysis shows fold change of ( g ) Trpa1 and ( h ) Trpm8 normalised to three housekeeping genes in DRG. (n = 14–15) ( i ) Representative western blot of TRPM8 in DRG of young and aged mice and densitometric analysis normalised to Tubulin (Y = young, A = aged) (n = 9). 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 0–2 min 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 normalised against vehicle treated mice. (n = 5–10) ( g–h ) RT-PCR CT analysis shows fold change of ( g ) Trpa1 and ( h ) Trpm8 normalised to three housekeeping genes in DRG. (n = 14–15) ( i ) Representative western blot of TRPM8 in DRG of young and aged mice and densitometric analysis normalised to Tubulin (Y = young, A = aged) (n = 9). All results are shown as mean ± s.e.m. *p

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

    7) 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

    8) 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

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  • 95
    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: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti trpm8/product/Alomone Labs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti trpm8 - by Bioz Stars, 2022-07
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    94
    Boster Bio anti trpm8 extracellular antibody
    Identification of novel <t>TRPM8-interacting</t> partners by Co-IP assay. ( A ) Co-IP assay coupled with Coomassie staining. The lysates from MCF7 cells transfected with control vector or Flag-TRPM8 construct were precipitated with an anti-Flag antibody and subjected to Coomassie staining. ( B-D ) The relevant ~ 60 kD band (shown with an arrow) as in A was selected for mass spectrometric (MS) assay in combination with the NCBI Blast. Peptide sequences of MS assay are indicated in B (BLK), C (LCK), and D (LYN). ( E ) GST pull-down coupled with Coomassie staining. Purified GST alone or GST-M8C proteins expressing in E.coli BL21 bacteria were incubated with the lysates of MCF7 cells and subjected to Coomassie staining. The relevant band of ~ 30 kD protein (shown with an arrow) was selected for MS analysis. ( F ) MS imaging of 14−3−3ζ as a novel partner of TRPM8 in combination with the NCBI blast (peptide sequences are indicated).
    Anti Trpm8 Extracellular Antibody, supplied by Boster Bio, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti trpm8 extracellular antibody/product/Boster Bio
    Average 94 stars, based on 1 article reviews
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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

    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

    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

    Identification of novel TRPM8-interacting partners by Co-IP assay. ( A ) Co-IP assay coupled with Coomassie staining. The lysates from MCF7 cells transfected with control vector or Flag-TRPM8 construct were precipitated with an anti-Flag antibody and subjected to Coomassie staining. ( B-D ) The relevant ~ 60 kD band (shown with an arrow) as in A was selected for mass spectrometric (MS) assay in combination with the NCBI Blast. Peptide sequences of MS assay are indicated in B (BLK), C (LCK), and D (LYN). ( E ) GST pull-down coupled with Coomassie staining. Purified GST alone or GST-M8C proteins expressing in E.coli BL21 bacteria were incubated with the lysates of MCF7 cells and subjected to Coomassie staining. The relevant band of ~ 30 kD protein (shown with an arrow) was selected for MS analysis. ( F ) MS imaging of 14−3−3ζ as a novel partner of TRPM8 in combination with the NCBI blast (peptide sequences are indicated).

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: Identification of novel TRPM8-interacting partners by Co-IP assay. ( A ) Co-IP assay coupled with Coomassie staining. The lysates from MCF7 cells transfected with control vector or Flag-TRPM8 construct were precipitated with an anti-Flag antibody and subjected to Coomassie staining. ( B-D ) The relevant ~ 60 kD band (shown with an arrow) as in A was selected for mass spectrometric (MS) assay in combination with the NCBI Blast. Peptide sequences of MS assay are indicated in B (BLK), C (LCK), and D (LYN). ( E ) GST pull-down coupled with Coomassie staining. Purified GST alone or GST-M8C proteins expressing in E.coli BL21 bacteria were incubated with the lysates of MCF7 cells and subjected to Coomassie staining. The relevant band of ~ 30 kD protein (shown with an arrow) was selected for MS analysis. ( F ) MS imaging of 14−3−3ζ as a novel partner of TRPM8 in combination with the NCBI blast (peptide sequences are indicated).

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Co-Immunoprecipitation Assay, Staining, Transfection, Plasmid Preparation, Construct, Purification, Expressing, Incubation, Imaging

    The effect of LCK mutants on TRPM8 phosphotyrosine. ( A-B ) Expression constructs for Flag-WT-M8 or Flag-M8-Y1022F were co-transfected with control vector, HA-tagged wild type, or mutant LCK into HEK293T cells. The cells were harvested for IP with an anti-Flag antibody and WB with the indicated antibodies to detect the level of TRPM8 phosphotyrosine. **, P

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: The effect of LCK mutants on TRPM8 phosphotyrosine. ( A-B ) Expression constructs for Flag-WT-M8 or Flag-M8-Y1022F were co-transfected with control vector, HA-tagged wild type, or mutant LCK into HEK293T cells. The cells were harvested for IP with an anti-Flag antibody and WB with the indicated antibodies to detect the level of TRPM8 phosphotyrosine. **, P

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

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

    The role of Y1022 on TRPM8 on tumor cell proliferation, migration and tumorigenesis. ( A-D ) Cell proliferation assays in vitro. ( A-B ) The RFP labeled cell lines of PANC-1 or AsPC-1 cells stably expressing control vector, WT-TRPM8, or mutant TRPM8-Y1022F were constructed and used for EdU incorporation assays (Upper panel) and Ki67 immunofluorescence (Lower panel). Scale bars, 100 μm. ( C-D ) Colony formation assays were performed in RFP labeled PANC-1 stably maintained cells. Scale bars, 100 μm. ( E-I ) Cell migration assays. ( E-F ) Wound-healing assay was performed in RFP labeled AsPC-1 stably maintained cells. ( G-H ) Transwell assay was performed in RFP labeled PANC-1 stably maintained cells. Scale bars, 100 μm. ( I-L ) Animal xenotransplantation engraftment experiments. ( I ) Representative confocal microscopy images of 6 days xenotransplantation of zebrafish injecting with RFP labeled PANC-1 stably maintained cells. Scale bars, 200 μm. ( J ) Imaging of tumors excised from the mice subcutaneously injecting RFP labeled PANC-1 stably maintained cells by growth for 5 weeks. ( K ) Quantification of the expression of TRPM8 mRNA in ( J ). ( L ) Weights of the excised tumors in each group in ( J ). ( M ) Growth curves showing the changes in the tumor volume in mice in different groups every 5 days from the injection. ( N ) Representative H E staining images and immunohistochemical images of Ki67 in excised tumors tissues. Scale bars, 20 μm. ( O ) Quantification of Ki67 expression in ( N ). **, P

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: The role of Y1022 on TRPM8 on tumor cell proliferation, migration and tumorigenesis. ( A-D ) Cell proliferation assays in vitro. ( A-B ) The RFP labeled cell lines of PANC-1 or AsPC-1 cells stably expressing control vector, WT-TRPM8, or mutant TRPM8-Y1022F were constructed and used for EdU incorporation assays (Upper panel) and Ki67 immunofluorescence (Lower panel). Scale bars, 100 μm. ( C-D ) Colony formation assays were performed in RFP labeled PANC-1 stably maintained cells. Scale bars, 100 μm. ( E-I ) Cell migration assays. ( E-F ) Wound-healing assay was performed in RFP labeled AsPC-1 stably maintained cells. ( G-H ) Transwell assay was performed in RFP labeled PANC-1 stably maintained cells. Scale bars, 100 μm. ( I-L ) Animal xenotransplantation engraftment experiments. ( I ) Representative confocal microscopy images of 6 days xenotransplantation of zebrafish injecting with RFP labeled PANC-1 stably maintained cells. Scale bars, 200 μm. ( J ) Imaging of tumors excised from the mice subcutaneously injecting RFP labeled PANC-1 stably maintained cells by growth for 5 weeks. ( K ) Quantification of the expression of TRPM8 mRNA in ( J ). ( L ) Weights of the excised tumors in each group in ( J ). ( M ) Growth curves showing the changes in the tumor volume in mice in different groups every 5 days from the injection. ( N ) Representative H E staining images and immunohistochemical images of Ki67 in excised tumors tissues. Scale bars, 20 μm. ( O ) Quantification of Ki67 expression in ( N ). **, P

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Migration, In Vitro, Labeling, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Construct, Immunofluorescence, Wound Healing Assay, Transwell Assay, Confocal Microscopy, Imaging, Mouse Assay, Injection, Staining, Immunohistochemistry

    Schematic diagram of the biological role of the LCK-14−3−3ζ-TRPM8 axis in egulation of TRPM8 function and LCK activity.

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: Schematic diagram of the biological role of the LCK-14−3−3ζ-TRPM8 axis in egulation of TRPM8 function and LCK activity.

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Activity Assay

    Functional effects of LCK on TRPM8 in HEK293T cells. ( A-E ) Electrophysiological analysis of LCK on whole-cell TRPM8 currents (I TRPM s) by patch-clamping experiments. ( A-C ) Expression constructs for Flag-TRPM8 and pEGFP-N1 were co-transfected with HA-BLK, HA-LCK, HA-LYN, or control vector into HEK293T cells. EGFP-positive cells were selected for recording I TRPM8 (n=5~20 cells per group). The voltage clamp protocol is shown in the inset of figure. ( A ) Representative imaging of I TRPM8 . ( B ) The relationship of average I TRPM8 density (I TRPM8 normalized to cell capacitance) and voltage. ( C ) Quantification of peak I TRPM8 density on +80 mV as in B . ( D-E ) Similar I TRPM8 recordings as in A but HEK293T cells co-expressing Flag-TRPM8 and pEGFP-N1 with siLCK or negative scramble siRNAs. (n=5~20 cells per group). ( D ) The relationship of average I TRPM8 density and voltage. ( E ) Quantification of peak I TRPM8 density on +80 mV as in D . ( F-I ) Cell-surface biotinylation assays for detecting TRPM8 PM expression. ( F ) Representative WB images of TRPM8 on the PM and total lysates from HEK293T cells co-transfected with or without HA-LCK. ( G ) Quantification of PM and total protein expression levels of TRPM8 in ( F ). ( H-I ) Similar experiments in F and G but cells co-expressing with siLCK or negative scramble siRNAs. ITGA5 (Intergrin α5) was used as a loading control for PM proteins. ***, P

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: Functional effects of LCK on TRPM8 in HEK293T cells. ( A-E ) Electrophysiological analysis of LCK on whole-cell TRPM8 currents (I TRPM s) by patch-clamping experiments. ( A-C ) Expression constructs for Flag-TRPM8 and pEGFP-N1 were co-transfected with HA-BLK, HA-LCK, HA-LYN, or control vector into HEK293T cells. EGFP-positive cells were selected for recording I TRPM8 (n=5~20 cells per group). The voltage clamp protocol is shown in the inset of figure. ( A ) Representative imaging of I TRPM8 . ( B ) The relationship of average I TRPM8 density (I TRPM8 normalized to cell capacitance) and voltage. ( C ) Quantification of peak I TRPM8 density on +80 mV as in B . ( D-E ) Similar I TRPM8 recordings as in A but HEK293T cells co-expressing Flag-TRPM8 and pEGFP-N1 with siLCK or negative scramble siRNAs. (n=5~20 cells per group). ( D ) The relationship of average I TRPM8 density and voltage. ( E ) Quantification of peak I TRPM8 density on +80 mV as in D . ( F-I ) Cell-surface biotinylation assays for detecting TRPM8 PM expression. ( F ) Representative WB images of TRPM8 on the PM and total lysates from HEK293T cells co-transfected with or without HA-LCK. ( G ) Quantification of PM and total protein expression levels of TRPM8 in ( F ). ( H-I ) Similar experiments in F and G but cells co-expressing with siLCK or negative scramble siRNAs. ITGA5 (Intergrin α5) was used as a loading control for PM proteins. ***, P

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Functional Assay, Expressing, Construct, Transfection, Plasmid Preparation, Imaging, Western Blot

    LCK activity regulated by TRPM8 phosphotyrosine. ( A-B ) LCK phosphorylation assay. PANC-1 cells were co-transfected HA-LCK with control vector, WT-TRPM8 or mutant TRPM8-Y1022 with the indicated antibodies as shown in ( A) , and quantification of the level of LCK phosphorylation shown in ( B ). ( C-F ) LCK ubiquitination assay. ( C-D ) Expression constructs for HA-LCK and Myc-Ub were co-transfected with control vector, WT-TRPM8 or mutant TRPM8-Y1022 into AsPC-1 cells. The cells were treated with 10 μM MG132 for 6 h before harvest and used for IP with an anti-HA antibody and WB with the indicated antibodies. ( E-F ) Expression constructs for Myc-Ub and WT-TRPM8 or mutant TRPM8-Y1022F were co-transfected with HA-tagged wild type or mutant LCK into PANC-1 cells. The cells were treated with 10 μM MG132 before harvest and used for IP with an anti-HA antibody and WB with the indicated antibodies. ***, P

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: LCK activity regulated by TRPM8 phosphotyrosine. ( A-B ) LCK phosphorylation assay. PANC-1 cells were co-transfected HA-LCK with control vector, WT-TRPM8 or mutant TRPM8-Y1022 with the indicated antibodies as shown in ( A) , and quantification of the level of LCK phosphorylation shown in ( B ). ( C-F ) LCK ubiquitination assay. ( C-D ) Expression constructs for HA-LCK and Myc-Ub were co-transfected with control vector, WT-TRPM8 or mutant TRPM8-Y1022 into AsPC-1 cells. The cells were treated with 10 μM MG132 for 6 h before harvest and used for IP with an anti-HA antibody and WB with the indicated antibodies. ( E-F ) Expression constructs for Myc-Ub and WT-TRPM8 or mutant TRPM8-Y1022F were co-transfected with HA-tagged wild type or mutant LCK into PANC-1 cells. The cells were treated with 10 μM MG132 before harvest and used for IP with an anti-HA antibody and WB with the indicated antibodies. ***, P

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Activity Assay, Phosphorylation Assay, Transfection, Plasmid Preparation, Mutagenesis, Ubiquitin Assay, Expressing, Construct, Western Blot

    The effect of LCK or TRPM8-Y1022F on Ser/Thr phosphorylation of TRPM8 and LCK on mutant TRPM8-Y1022F expression on the PM. ( A-B ) HEK293T cells were transfected with Flag-WT-TRPM8 or Flag-M8-Y1022F, and harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies. ( C-D ) Flag-TRPM8 were co-transfected with or without HA-LCK into HEK293 cells. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies. ( E ) WB imaging of TRPM8 in the PM and total lysates from PANC-1 cells co-transfected Flag-WT-TRPM8 or Flag-TRPM8-Y1022F, in the presence or absence of HA-LCK. ( F ) Quantification of PM and total protein expression levels of TRPM8 in ( E ). ( G ) Representative confocal imaging of PANC-1 cells co-expressing mcherry-LCK with EGFP-WT-TRPM8 or EGFP-TRPM8-Y1022F. DAPI (1 μg/ml) was used for nuclei staining. ***, P

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: The effect of LCK or TRPM8-Y1022F on Ser/Thr phosphorylation of TRPM8 and LCK on mutant TRPM8-Y1022F expression on the PM. ( A-B ) HEK293T cells were transfected with Flag-WT-TRPM8 or Flag-M8-Y1022F, and harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies. ( C-D ) Flag-TRPM8 were co-transfected with or without HA-LCK into HEK293 cells. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies. ( E ) WB imaging of TRPM8 in the PM and total lysates from PANC-1 cells co-transfected Flag-WT-TRPM8 or Flag-TRPM8-Y1022F, in the presence or absence of HA-LCK. ( F ) Quantification of PM and total protein expression levels of TRPM8 in ( E ). ( G ) Representative confocal imaging of PANC-1 cells co-expressing mcherry-LCK with EGFP-WT-TRPM8 or EGFP-TRPM8-Y1022F. DAPI (1 μg/ml) was used for nuclei staining. ***, P

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Mutagenesis, Expressing, Transfection, Western Blot, Imaging, Staining

    The multimerization, but not intracellular N-C binding of TRPM8, regulated by LCK. ( A-D ) The effect of LCK on the intracellular N-C binding of TRPM8. ( A-B ) Expression constructs for Flag-tagged N-terminus of TRPM8 (Flag-M8N) and GFP-M8C were co-transfected with HA-LCK or control vector into HEK293T cells, before harvest for treatment with 10 μM PP2 for 24 h. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the intracellular N-C binding of TRPM8. ( C-D ) Similar experiments in A and B but cells co-expressing Flag-M8N and GFP-M8C along with siLCK. ( E-L ) The effect of LCK on the multimerization of TRPM8. ( E-F ) Expression constructs for Flag-TRPM8 were co-transfected with HA-LCK or control vector into PANC-1 cells, before harvest for treatment with 1 μM DSS for 30 min, a crosslinking agent. The cell lysates were subjected to WB assay with the indicated antibodies to detect the level of TRPM8 multimerization. ( G-H ) Similar experiments in E and F but cells co-expressing Flag-TRPM8 along with siLCK. ( I-J ) Flag-TRPM8 and GFP-TRPM8 were co-transfected with or without HA-LCK into AsPC-1 cells. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the binding of intermolecular TRPM8. ( K-L ) Similar experiments in I and J but cells co-expressing Flag-TRPM8 and GFP-TRPM8 along with siLCK. **, P

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: The multimerization, but not intracellular N-C binding of TRPM8, regulated by LCK. ( A-D ) The effect of LCK on the intracellular N-C binding of TRPM8. ( A-B ) Expression constructs for Flag-tagged N-terminus of TRPM8 (Flag-M8N) and GFP-M8C were co-transfected with HA-LCK or control vector into HEK293T cells, before harvest for treatment with 10 μM PP2 for 24 h. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the intracellular N-C binding of TRPM8. ( C-D ) Similar experiments in A and B but cells co-expressing Flag-M8N and GFP-M8C along with siLCK. ( E-L ) The effect of LCK on the multimerization of TRPM8. ( E-F ) Expression constructs for Flag-TRPM8 were co-transfected with HA-LCK or control vector into PANC-1 cells, before harvest for treatment with 1 μM DSS for 30 min, a crosslinking agent. The cell lysates were subjected to WB assay with the indicated antibodies to detect the level of TRPM8 multimerization. ( G-H ) Similar experiments in E and F but cells co-expressing Flag-TRPM8 along with siLCK. ( I-J ) Flag-TRPM8 and GFP-TRPM8 were co-transfected with or without HA-LCK into AsPC-1 cells. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the binding of intermolecular TRPM8. ( K-L ) Similar experiments in I and J but cells co-expressing Flag-TRPM8 and GFP-TRPM8 along with siLCK. **, P

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Binding Assay, Expressing, Construct, Transfection, Plasmid Preparation, Western Blot

    14−3−3ζ interaction with TRPM8 for regulating TRPM8 multimerization. ( A ) GST pull-down assay. Purified GST alone or GST-M8C fusion proteins expressing in E.coli BL21 bacteria were incubated with the lysates from HEK293T cells expressing GFP-14−3−3ζ constructs and subjected to WB assay. ( B ) Co-IP assay. The lysates of native PANC-1 cells were added to an anti-14−3−3 antibody for IP and then subjected to WB assay with an anti-TRPM8 antibody. ( C-D ) Expression constructs for Flag-TRPM8 with or without GFP-14−3−3ζ were transfected into PANC-1 cells, before harvest for treatment with 1 μM DSS for 30 min. The lysates were subjected to WB assay with the indicated antibodies to detect the level of TRPM8 multimerization. ( E-F ) Similar experiments in C and D but cells co-expressing Flag-TRPM8 along with human 14−3−3ζ-specific siRNAs (si14−3−3ζ#1, #2, or #3). ( G-H ) AsPC-1 cells were co-transfected Flag-TRPM8 and GFP-TRPM8 with or without si14−3−3ζ, and then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the binding of intermolecular TRPM8. **, P

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: 14−3−3ζ interaction with TRPM8 for regulating TRPM8 multimerization. ( A ) GST pull-down assay. Purified GST alone or GST-M8C fusion proteins expressing in E.coli BL21 bacteria were incubated with the lysates from HEK293T cells expressing GFP-14−3−3ζ constructs and subjected to WB assay. ( B ) Co-IP assay. The lysates of native PANC-1 cells were added to an anti-14−3−3 antibody for IP and then subjected to WB assay with an anti-TRPM8 antibody. ( C-D ) Expression constructs for Flag-TRPM8 with or without GFP-14−3−3ζ were transfected into PANC-1 cells, before harvest for treatment with 1 μM DSS for 30 min. The lysates were subjected to WB assay with the indicated antibodies to detect the level of TRPM8 multimerization. ( E-F ) Similar experiments in C and D but cells co-expressing Flag-TRPM8 along with human 14−3−3ζ-specific siRNAs (si14−3−3ζ#1, #2, or #3). ( G-H ) AsPC-1 cells were co-transfected Flag-TRPM8 and GFP-TRPM8 with or without si14−3−3ζ, and then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the binding of intermolecular TRPM8. **, P

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Pull Down Assay, Purification, Expressing, Incubation, Construct, Western Blot, Co-Immunoprecipitation Assay, Transfection, Binding Assay

    Effect of the compounds for PP2 and sodium orthovanadate, and LCK on TRPM8 phosphotyrosine. ( A-B ) HeLa cells expressing Flag-TRPM8 were incubated with DMSO dissolving different concentrations of PP2 (in μM, 0, 2.5, 10, 20) for 24 h before harvest, lysed and immunoprecipitated with an anti-Flag antibody. The samples were then analyzed by immunoblotting with the anti-Flag and p-Tyr antibodies to detect the level of TRPM8 phosphotyrosine. ( C-D ) Similar experiments in A and B but treatment with 10 μM PP2, 1 mM Na3VO4, or their combination. Na3VO4, sodium orthovanadate. PP2, 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl) pyrazolo[3,4-d] pyrimidine, a Src family kinases inhibitor. ( E-F ) Expression constructs for Flag-TRPM8 were co-transfected into PANC-1 cells with HA-BLK, HA-LCK, or HA-LYN, respectively. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the anti-Flag and p-Tyr antibodies to detect the level of TRPM8 phosphotyrosine. ( G-H ) Similar experiments in E and F but cells expressing with various amounts of HA-LCK. ( I-J ) Similar experiments in E and F but cells expressing with human LCK-specific siRNAs (siLCK#1 or #2) or negative scramble siRNAs. ( K-L ) HEK293T cells were co-transfected with GFP-M8C with HA-LCK or control vector, then harvested for IP with an anti-GFP antibodies, and WB assay with the anti-GFP and p-Tyr antibodies to detect the level of M8C phosphotyrosine. ( M ) Kinase assay in vitro. Purified GST alone or GST-M8C fusion proteins expressing in E.coli bacteria and HA-LCK immunoprecipitated with anti-HA antibody from HEK293T cells expressing HA-LCK constructs were mixed with or without 1 mM ATP in kinase assay buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 10 mM MnCl 2 ), and the reaction mixtures were then terminated, followed by WB assay with the indicated antibodies. ***, P

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: Effect of the compounds for PP2 and sodium orthovanadate, and LCK on TRPM8 phosphotyrosine. ( A-B ) HeLa cells expressing Flag-TRPM8 were incubated with DMSO dissolving different concentrations of PP2 (in μM, 0, 2.5, 10, 20) for 24 h before harvest, lysed and immunoprecipitated with an anti-Flag antibody. The samples were then analyzed by immunoblotting with the anti-Flag and p-Tyr antibodies to detect the level of TRPM8 phosphotyrosine. ( C-D ) Similar experiments in A and B but treatment with 10 μM PP2, 1 mM Na3VO4, or their combination. Na3VO4, sodium orthovanadate. PP2, 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl) pyrazolo[3,4-d] pyrimidine, a Src family kinases inhibitor. ( E-F ) Expression constructs for Flag-TRPM8 were co-transfected into PANC-1 cells with HA-BLK, HA-LCK, or HA-LYN, respectively. The cells were then harvested for IP with an anti-Flag antibody and WB assay with the anti-Flag and p-Tyr antibodies to detect the level of TRPM8 phosphotyrosine. ( G-H ) Similar experiments in E and F but cells expressing with various amounts of HA-LCK. ( I-J ) Similar experiments in E and F but cells expressing with human LCK-specific siRNAs (siLCK#1 or #2) or negative scramble siRNAs. ( K-L ) HEK293T cells were co-transfected with GFP-M8C with HA-LCK or control vector, then harvested for IP with an anti-GFP antibodies, and WB assay with the anti-GFP and p-Tyr antibodies to detect the level of M8C phosphotyrosine. ( M ) Kinase assay in vitro. Purified GST alone or GST-M8C fusion proteins expressing in E.coli bacteria and HA-LCK immunoprecipitated with anti-HA antibody from HEK293T cells expressing HA-LCK constructs were mixed with or without 1 mM ATP in kinase assay buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 10 mM MnCl 2 ), and the reaction mixtures were then terminated, followed by WB assay with the indicated antibodies. ***, P

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Expressing, Incubation, Immunoprecipitation, Construct, Transfection, Western Blot, Plasmid Preparation, Kinase Assay, In Vitro, Purification

    Identification of phosphotyrosine on TRPM8 at position 1022 regulated by LCK. ( A ) MS imaging of phosphotyrosine site of TRPM8 in combination with the NCBI blast (peptide sequences are indicated). ( B ) Amino acid sequence alignment showing that tyrosine at position 1022 is highly conserved among multiple species. ( C-D ) Expression constructs for Flag-tagged wild type TRPM8 (Flag-WT-M8) or mutant Y1022F (Flag-M8-Y1022F) were transfected with or without HA-LCK into HEK293T cells, before harvest for treatment with 10 μM saracatinib for 24 h. The lysates were then used for IP with an anti-Flag antibody and then subjected to WB assay with the indicated antibodies to detect the level of TRPM8 phosphotyrosine. ( E-F ) Kinase assay in vitro. Purified GST alone, GST tagged wild type or mutant of C-terminus of TRPM8 fusion proteins expressing in E.coli bacteria were mixed with HA-LCK immunoprecipitated with anti-HA antibody from HEK293T cells expressing HA-LCK construct, 1 mM ATP or their combination in kinase assay buffer to determine the level of M8C phosphotyrosine. ( G ) Relationship of test potential and averaged densities of I TRP M8 recorded from HEK293T cells co-transfected Flag-WT-M8 or Flag-M8-Y1022F with pEGFP-N1. ( H ) Peak current density on +80 mV as in G (n=15~20 cells per group). ( I-J ) Expression constructs for Flag-WT-M8 or Flag-M8-Y1022F were co-transfected with or without HA-LCK into PANC-1 cells, before harvest for treatment with 1 μM DSS for 30 min for WB to detect the level of TRPM8 multimerization. ( K-L ) Flag-WT-M8 or Flag-M8-Y1022F along with GFP-tagged wild type TRPM8 (GFP-WT-M8) were co-transfected with or without HA-LCK into AsPC-1 cells to determine the binding of intermolecular TRPM8. ***, P

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: Identification of phosphotyrosine on TRPM8 at position 1022 regulated by LCK. ( A ) MS imaging of phosphotyrosine site of TRPM8 in combination with the NCBI blast (peptide sequences are indicated). ( B ) Amino acid sequence alignment showing that tyrosine at position 1022 is highly conserved among multiple species. ( C-D ) Expression constructs for Flag-tagged wild type TRPM8 (Flag-WT-M8) or mutant Y1022F (Flag-M8-Y1022F) were transfected with or without HA-LCK into HEK293T cells, before harvest for treatment with 10 μM saracatinib for 24 h. The lysates were then used for IP with an anti-Flag antibody and then subjected to WB assay with the indicated antibodies to detect the level of TRPM8 phosphotyrosine. ( E-F ) Kinase assay in vitro. Purified GST alone, GST tagged wild type or mutant of C-terminus of TRPM8 fusion proteins expressing in E.coli bacteria were mixed with HA-LCK immunoprecipitated with anti-HA antibody from HEK293T cells expressing HA-LCK construct, 1 mM ATP or their combination in kinase assay buffer to determine the level of M8C phosphotyrosine. ( G ) Relationship of test potential and averaged densities of I TRP M8 recorded from HEK293T cells co-transfected Flag-WT-M8 or Flag-M8-Y1022F with pEGFP-N1. ( H ) Peak current density on +80 mV as in G (n=15~20 cells per group). ( I-J ) Expression constructs for Flag-WT-M8 or Flag-M8-Y1022F were co-transfected with or without HA-LCK into PANC-1 cells, before harvest for treatment with 1 μM DSS for 30 min for WB to detect the level of TRPM8 multimerization. ( K-L ) Flag-WT-M8 or Flag-M8-Y1022F along with GFP-tagged wild type TRPM8 (GFP-WT-M8) were co-transfected with or without HA-LCK into AsPC-1 cells to determine the binding of intermolecular TRPM8. ***, P

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Imaging, Sequencing, Expressing, Construct, Mutagenesis, Transfection, Western Blot, Kinase Assay, In Vitro, Purification, Immunoprecipitation, Binding Assay

    Src family kinases for BLK, LCK, and LYN interaction with TRPM8. ( A ) GST pull-down assays to assess the interaction between purified GST-tagged C-terminus of TRPM8 (GST-M8C) expressing in E.coli BL21 bacteria and different HA-tagged Src family kinases expressing in HEK293T cells. Western blotting (WB) was performed using the indicated antibodies. ( B-D ) Co-immunoprecipitation (Co-IP) assays. ( B ) HeLa cells were transfected with the indicated constructs along with Flag-tagged full-length TRPM8 (Flag-TRPM8). Immunoprecipitation (IP) was performed with an anti-HA or anti-Flag antibody, and the samples were analyzed by immunoblotting with the indicated antibodies. ( C ) Similar Co-IP in B but with protein extracts from HeLa cells co-transfected with the indicated constructs along with GFP-tagged C-terminus of TRPM8 (GFP-M8C). ( D ) Co-IP as in B and C but with protein extracts from native PANC-1 cells. ( E ) Representative confocal imaging of co-localization of mcherry-LCK and GFP-TRPM8 in HeLa cells. Overlay images show co-localization of green signals (TRPM8) and red signals (LCK), which generated yellow signals in HeLa. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. ( F ) Assay of the interaction in vitro between purified His-LCK fusion and GST-tagged C-terminus of TRPM8 (GST-M8C) from E.coli bacteria. ( G-H ) HEK293T cells co-expressing HA-LCK constructs with a series of mutant Flag-tagged cytoplasmic domain of TRPM8 were harvested for Co-IP assays. All studies were repeated at least three times. GFP, green fluorescent protein. All studies were repeated at least three times.

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: Src family kinases for BLK, LCK, and LYN interaction with TRPM8. ( A ) GST pull-down assays to assess the interaction between purified GST-tagged C-terminus of TRPM8 (GST-M8C) expressing in E.coli BL21 bacteria and different HA-tagged Src family kinases expressing in HEK293T cells. Western blotting (WB) was performed using the indicated antibodies. ( B-D ) Co-immunoprecipitation (Co-IP) assays. ( B ) HeLa cells were transfected with the indicated constructs along with Flag-tagged full-length TRPM8 (Flag-TRPM8). Immunoprecipitation (IP) was performed with an anti-HA or anti-Flag antibody, and the samples were analyzed by immunoblotting with the indicated antibodies. ( C ) Similar Co-IP in B but with protein extracts from HeLa cells co-transfected with the indicated constructs along with GFP-tagged C-terminus of TRPM8 (GFP-M8C). ( D ) Co-IP as in B and C but with protein extracts from native PANC-1 cells. ( E ) Representative confocal imaging of co-localization of mcherry-LCK and GFP-TRPM8 in HeLa cells. Overlay images show co-localization of green signals (TRPM8) and red signals (LCK), which generated yellow signals in HeLa. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. ( F ) Assay of the interaction in vitro between purified His-LCK fusion and GST-tagged C-terminus of TRPM8 (GST-M8C) from E.coli bacteria. ( G-H ) HEK293T cells co-expressing HA-LCK constructs with a series of mutant Flag-tagged cytoplasmic domain of TRPM8 were harvested for Co-IP assays. All studies were repeated at least three times. GFP, green fluorescent protein. All studies were repeated at least three times.

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Purification, Expressing, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Construct, Imaging, Generated, Staining, In Vitro, Mutagenesis

    The effect of TRPM8 on LCK phosphorylation. ( A ) WB imaging of HEK293T cells individually transfected with control vector, HA-tagged wild type, or mutant LCK with the indicated antibodies. ( B-C ) PANC-1 cells were co-transfected HA-LCK with or without Flag-TRPM8, and then harvested for IP with an anti-HA antibody and WB assay with the indicated antibodies. ( D-E ) PANC-1 cells were co-transfected HA-LCK with or without Flag-TRPM8, before harvest for treatment with 10 μM saracatinib for 24 h. The lysates were subjected to WB assay with the indicated antibodies. ***, P

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: The effect of TRPM8 on LCK phosphorylation. ( A ) WB imaging of HEK293T cells individually transfected with control vector, HA-tagged wild type, or mutant LCK with the indicated antibodies. ( B-C ) PANC-1 cells were co-transfected HA-LCK with or without Flag-TRPM8, and then harvested for IP with an anti-HA antibody and WB assay with the indicated antibodies. ( D-E ) PANC-1 cells were co-transfected HA-LCK with or without Flag-TRPM8, before harvest for treatment with 10 μM saracatinib for 24 h. The lysates were subjected to WB assay with the indicated antibodies. ***, P

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Western Blot, Imaging, Transfection, Plasmid Preparation, Mutagenesis

    14−3−3ζ involved in the regulation of LCK on the multimerization of TRPM8. ( A-B ) PANC-1 cells were co-transfected Flag-TRPM8 with HA-LCK or control vector and then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the binding of TRPM8 and 14−3−3ζ. ( C-D ) Similar experiments in A and B but cells co-expressing Flag-TRPM8 along with siLCK. ( E-F ) Expression constructs for Flag-WT-TRPM8 or Flag-TRPM8-Y1022F along with GFP-TRPM8 were co-transfected with or without HA-LCK into HEK293T cells to determine the binding of intermolecular TRPM8. ( G-H ) PANC-1 cells were co-transfected with Flag-TRPM8, HA-LCK, si14−3−3ζ (#1, #2 or #3) or their combination, before harvest for treatment with 1 μM DSS for 30 min, and subjected to WB assay to detect the level of TRPM8 multimerization. ( I-J ) AsPC-1 cells were co-transfected with Flag-TRPM8 and GFP-TRPM8, HA-LCK, si14−3−3ζ or their combination, and then harvested for IP with an anti-Flag antibody and subjected to determine the binding of intermolecular TRPM8. ***, P

    Journal: bioRxiv

    Article Title: LCK-14-3-3ζ-TRPM8 axis for regulating TRPM8 function/assembly promotes pancreatic cancer malignancy

    doi: 10.1101/2022.01.26.477835

    Figure Lengend Snippet: 14−3−3ζ involved in the regulation of LCK on the multimerization of TRPM8. ( A-B ) PANC-1 cells were co-transfected Flag-TRPM8 with HA-LCK or control vector and then harvested for IP with an anti-Flag antibody and WB assay with the indicated antibodies to determine the binding of TRPM8 and 14−3−3ζ. ( C-D ) Similar experiments in A and B but cells co-expressing Flag-TRPM8 along with siLCK. ( E-F ) Expression constructs for Flag-WT-TRPM8 or Flag-TRPM8-Y1022F along with GFP-TRPM8 were co-transfected with or without HA-LCK into HEK293T cells to determine the binding of intermolecular TRPM8. ( G-H ) PANC-1 cells were co-transfected with Flag-TRPM8, HA-LCK, si14−3−3ζ (#1, #2 or #3) or their combination, before harvest for treatment with 1 μM DSS for 30 min, and subjected to WB assay to detect the level of TRPM8 multimerization. ( I-J ) AsPC-1 cells were co-transfected with Flag-TRPM8 and GFP-TRPM8, HA-LCK, si14−3−3ζ or their combination, and then harvested for IP with an anti-Flag antibody and subjected to determine the binding of intermolecular TRPM8. ***, P

    Article Snippet: Antibodies and reagentsA rabbit anti-LCK (#12477, PTGCN, China), anti-GFP (#50430, PTGCN), anti-14-3-3 (#14503,PTGCN), anti-Phosphotyrosine (p-Tyr) (Sigma-Aldrich), anti-Phosphothreonine (Cell Signaling Technology) Anti-Phosphoserine (Abcam, ab9332), anti-phospho-Lck-Y505 (pY505) (#MAB7500, R & D), anti-TRPM8 (#ACC-049, Alomone, Israel), anti-LC3B (#18725, PTGCN, China), anti-SQSTM1/p62 (#BM4385, Boster, China), anti-ULK1 (#20986, PTGCN), anti-phospho-ULK1 (Ser317) (#12753, Cell Signaling Technology), anti-AMPKa1 (#BM4202, Boster), and anti-phospho-AMPKa (Thr172) antibodies (#2535, Cell Signaling Technology), and mouse anti-phospho-Lck-Y394 (pY394) (#2751, Cell Signaling Technology) antibodies were used at a dilution factor of 1:1000.

    Techniques: Transfection, Plasmid Preparation, Western Blot, Binding Assay, Expressing, Construct