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Novus Biologicals rabbit polyclonal anti trpm8
Rabbit Polyclonal Anti Trpm8, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 10 article reviews

rabbit polyclonal anti trpm8 - by Bioz Stars, 2026-04

92/100 stars

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Image Search Results

TrkA- and TRPM8 expression and morphology of DRG neurons in culture . (A&B) Small diameter DRG neurons in culture express the TrkA receptor and TRPM8 channels. Immunofluoresence images of 12 hr DRG cultures stained with (A) an anti-TrkA (green), (B) an anti-TRPM8 (green) antibodies, and DAPI (blue). Analysis of somatic diameters displaying immunoreactivity (closed circles) for (C) TrkA and (D) TRPM8 show a significant bias of TrkA and TRPM8 immunoreactivity (closed circles) in smaller neurons (<15 μm) compared with TrkA and TRPM8 negative neurons (open circles). (n = 326 and 223 cells counted respectively, scale bar is 20 μm). (E) TRPM8 immunoreactivity in culture. DRG neurons displaying effusive growth cone morphologies (suitable of calcium imaging experiments) were TRPM8 positive (scale bar is 50 μm).

Journal: Molecular Neurodegeneration

Article Title: TRPM8 and Na v 1.8 sodium channels are required for transthyretin-induced calcium influx in growth cones of small-diameter TrkA-positive sensory neurons

doi: 10.1186/1750-1326-6-19

Figure Lengend Snippet: TrkA- and TRPM8 expression and morphology of DRG neurons in culture . (A&B) Small diameter DRG neurons in culture express the TrkA receptor and TRPM8 channels. Immunofluoresence images of 12 hr DRG cultures stained with (A) an anti-TrkA (green), (B) an anti-TRPM8 (green) antibodies, and DAPI (blue). Analysis of somatic diameters displaying immunoreactivity (closed circles) for (C) TrkA and (D) TRPM8 show a significant bias of TrkA and TRPM8 immunoreactivity (closed circles) in smaller neurons (<15 μm) compared with TrkA and TRPM8 negative neurons (open circles). (n = 326 and 223 cells counted respectively, scale bar is 20 μm). (E) TRPM8 immunoreactivity in culture. DRG neurons displaying effusive growth cone morphologies (suitable of calcium imaging experiments) were TRPM8 positive (scale bar is 50 μm).

Article Snippet: The primary antibodies, a polyclonal rabbit anti-TRPM8 (extracellular) (1:1000, Alomone Labs, Israel) or polyclonal rabbit anti-TrkA (1:1000, Abcam, Cambridge, UK) were added to coverslips and incubated for 4 hr at 22°C.

Techniques: Expressing, Staining, Imaging

siRNA silencing of TRPM8 expression . Figure shows the level of TRPM8 immunoreactivity as determined by immunocytochemistry and by western blotting after treatment with siRNA oligonucleotides. (A) TRPM8 staining of a DRG growth cone treated with a non-specific control siRNA or with a specific TRPM8 siRNA oligonucleotide (#57381). The dotted line describes outline of the growth cone in the TRPM8 siRNA culture. (B) Quantitative analysis of TRPM8 immunocytochemistry shows that there was a significant decrease in TRPM8 expression in the presence of the TRPM8 siRNA (#57381, n = 73 growth cones) compared with the non-specific control siRNA (n = 56 growth cones). (C,D) Western blot and quantitation of TRPM8 immunoreactivity after treatment with 3 different TRPM8 siRNA oligonucleotides and a non-specific control siRNA. (C) Extracts from siRNA-treated DRG cultures were applied to SDS gels and probed with an anti-TRPM8 antibody. (D) Quantitative analysis of blots revealed significant knockdown of TRPM8 protein expression compared to cultures treated with a non-specific control siRNA. TRPM8 siRNA levels were normalised to GAPDH expression. Figure shows 60-70% knockdown achieved with 3 separate TRPM8 oligonucleotides. Values are means of 9 replicate immunoblot lanes over 3 separate experiments. Significant differences from control are depicted as: ** p < 0.005 as determined by a Mann-Whitney U-test. Error bars show means ± SEM

Journal: Molecular Neurodegeneration

Article Title: TRPM8 and Na v 1.8 sodium channels are required for transthyretin-induced calcium influx in growth cones of small-diameter TrkA-positive sensory neurons

doi: 10.1186/1750-1326-6-19

Figure Lengend Snippet: siRNA silencing of TRPM8 expression . Figure shows the level of TRPM8 immunoreactivity as determined by immunocytochemistry and by western blotting after treatment with siRNA oligonucleotides. (A) TRPM8 staining of a DRG growth cone treated with a non-specific control siRNA or with a specific TRPM8 siRNA oligonucleotide (#57381). The dotted line describes outline of the growth cone in the TRPM8 siRNA culture. (B) Quantitative analysis of TRPM8 immunocytochemistry shows that there was a significant decrease in TRPM8 expression in the presence of the TRPM8 siRNA (#57381, n = 73 growth cones) compared with the non-specific control siRNA (n = 56 growth cones). (C,D) Western blot and quantitation of TRPM8 immunoreactivity after treatment with 3 different TRPM8 siRNA oligonucleotides and a non-specific control siRNA. (C) Extracts from siRNA-treated DRG cultures were applied to SDS gels and probed with an anti-TRPM8 antibody. (D) Quantitative analysis of blots revealed significant knockdown of TRPM8 protein expression compared to cultures treated with a non-specific control siRNA. TRPM8 siRNA levels were normalised to GAPDH expression. Figure shows 60-70% knockdown achieved with 3 separate TRPM8 oligonucleotides. Values are means of 9 replicate immunoblot lanes over 3 separate experiments. Significant differences from control are depicted as: ** p < 0.005 as determined by a Mann-Whitney U-test. Error bars show means ± SEM

Techniques: Expressing, Immunocytochemistry, Western Blot, Staining, Quantitation Assay, MANN-WHITNEY

Effect of TRPM8 knockdown on TTR-induced calcium influx . TRPM8 channels are necessary for L55P-induced calcium influx. Representative images of DRG neurons loaded with (A) non-specific control siRNA elicited a greater L55P-induced calcium influx (ΔF/F 0 ) than growth cones loaded with (B) specific TRPM8 siRNA (#57381). (C) Pooled results show calcium influx (ΔF/F 0 ) in response to L55P for control siRNA (closed circles, n = 12) and a specific TRPM8 siRNA (open circles, n = 16) from 3 separate experiments. (D) Using 3 different TRPM8 siRNA oligonucleotides results in a similar reduction in L55P-mediated calcium entry. Significant differences from control are depicted as: *p < 0.05; **p < 0.005; Mann-Whitney U-test. Error bars indicate mean ± SEM. Scale bar is 5 μm.

Journal: Molecular Neurodegeneration

Article Title: TRPM8 and Na v 1.8 sodium channels are required for transthyretin-induced calcium influx in growth cones of small-diameter TrkA-positive sensory neurons

doi: 10.1186/1750-1326-6-19

Figure Lengend Snippet: Effect of TRPM8 knockdown on TTR-induced calcium influx . TRPM8 channels are necessary for L55P-induced calcium influx. Representative images of DRG neurons loaded with (A) non-specific control siRNA elicited a greater L55P-induced calcium influx (ΔF/F 0 ) than growth cones loaded with (B) specific TRPM8 siRNA (#57381). (C) Pooled results show calcium influx (ΔF/F 0 ) in response to L55P for control siRNA (closed circles, n = 12) and a specific TRPM8 siRNA (open circles, n = 16) from 3 separate experiments. (D) Using 3 different TRPM8 siRNA oligonucleotides results in a similar reduction in L55P-mediated calcium entry. Significant differences from control are depicted as: *p < 0.05; **p < 0.005; Mann-Whitney U-test. Error bars indicate mean ± SEM. Scale bar is 5 μm.

Techniques: MANN-WHITNEY

a Representative RT-PCR analysis of mRNA expression for trpm1-trpm8b in the whole embryo and tail at stage 42/43 ( N = 3 independent samples). trpm6 mRNA was detected slightly in 1 of 3 independent replicates. b Immunohistochemistry against TRPM8 and Tyrosinase related protein 1 (Tyrp-1) in consecutive sections (schematic). DAPI staining (blue) was used to visualize cell nuclei and facilitate overlapping of adjacent sections. Boxed areas are shown enlarged (c’ and c”). Immunolabel displays co-localization of Tyrp-1 (arrows) in melanophores (M) of the skin (S) with Trpm8. Scale bar = 100 µm.

Journal: Communications Biology

Article Title: TRPM8 thermosensation in poikilotherms mediates both skin colour and locomotor performance responses to cold temperature

doi: 10.1038/s42003-023-04489-8

Figure Lengend Snippet: a Representative RT-PCR analysis of mRNA expression for trpm1-trpm8b in the whole embryo and tail at stage 42/43 ( N = 3 independent samples). trpm6 mRNA was detected slightly in 1 of 3 independent replicates. b Immunohistochemistry against TRPM8 and Tyrosinase related protein 1 (Tyrp-1) in consecutive sections (schematic). DAPI staining (blue) was used to visualize cell nuclei and facilitate overlapping of adjacent sections. Boxed areas are shown enlarged (c’ and c”). Immunolabel displays co-localization of Tyrp-1 (arrows) in melanophores (M) of the skin (S) with Trpm8. Scale bar = 100 µm.

Article Snippet: To verify that Trpm8 protein was expressed by skin melanophores we used an anti-human TRPM8 (rabbit polyclonal; 1/1000 dilution; antibodies-online.com).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Staining, Immunolabeling

a Pigmentation index for stage 42/43 Xenopus tadpoles treated at 24 °C for 30 min at the indicated concentrations of TRPA1 (AITC) and TRPM8 (menthol; WS12) agonists. Reversibility (Rev) 6 h after removing the WS12. b , c A TRPM8 antagonist (PF05105679) blocks melanosome aggregation induced by cool temperature (6 °C) ( b ), while the TRPM4 blocker CBA has no effect ( c ); Each dot represented the measurement in one tadpole, and the bar is the mean with 95% confidence interval. n = 9 embryos; N = 3 independent experiments; ** p < 0.01, *** p < 0.001; **** p < 0.0001; ANOVA followed by Bonferroni’s test.

Journal: Communications Biology

Article Title: TRPM8 thermosensation in poikilotherms mediates both skin colour and locomotor performance responses to cold temperature

doi: 10.1038/s42003-023-04489-8

Figure Lengend Snippet: a Pigmentation index for stage 42/43 Xenopus tadpoles treated at 24 °C for 30 min at the indicated concentrations of TRPA1 (AITC) and TRPM8 (menthol; WS12) agonists. Reversibility (Rev) 6 h after removing the WS12. b , c A TRPM8 antagonist (PF05105679) blocks melanosome aggregation induced by cool temperature (6 °C) ( b ), while the TRPM4 blocker CBA has no effect ( c ); Each dot represented the measurement in one tadpole, and the bar is the mean with 95% confidence interval. n = 9 embryos; N = 3 independent experiments; ** p < 0.01, *** p < 0.001; **** p < 0.0001; ANOVA followed by Bonferroni’s test.

Article Snippet: To verify that Trpm8 protein was expressed by skin melanophores we used an anti-human TRPM8 (rabbit polyclonal; 1/1000 dilution; antibodies-online.com).

Techniques:

Sequence comparison within the S3 region of selected voltage-gated as well as voltage-independent cation channels, including TRPM8 and TRPM2. The amino acid sequences are shown in single letter code. The highly conserved N-x-x-D motif is highlighted with the outer pair of amino acid residues labeled in red and the inner pair in orange. Further highly conserved amino acid residues upstream of the N-x-x-D-motif are given in bold letters. The glycine residue at position 805 in the sequence of human TRPM8 which is crucial for the icilin sensitivity of the channel is marked in blue. Accession numbers are as follows human TRPM8: Q7Z2W7; human TRPM2: O94759; human TRPA1: O75762; human TRPC3: Q13507, Shaker H4 (KCNAS_DROME): P08510; human sodium channel type 2 alpha subunit (SCN2A), domain 4: Q99250; voltage-gated sodium channel from Bacillus halodurans (NaChBac): Q9KCR8; human L-type calcium channel subunit alpha 1C (CACNA1C) domain 4: Q13936.

Journal: PLoS ONE

Article Title: Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

doi: 10.1371/journal.pone.0049877

Figure Lengend Snippet: Sequence comparison within the S3 region of selected voltage-gated as well as voltage-independent cation channels, including TRPM8 and TRPM2. The amino acid sequences are shown in single letter code. The highly conserved N-x-x-D motif is highlighted with the outer pair of amino acid residues labeled in red and the inner pair in orange. Further highly conserved amino acid residues upstream of the N-x-x-D-motif are given in bold letters. The glycine residue at position 805 in the sequence of human TRPM8 which is crucial for the icilin sensitivity of the channel is marked in blue. Accession numbers are as follows human TRPM8: Q7Z2W7; human TRPM2: O94759; human TRPA1: O75762; human TRPC3: Q13507, Shaker H4 (KCNAS_DROME): P08510; human sodium channel type 2 alpha subunit (SCN2A), domain 4: Q99250; voltage-gated sodium channel from Bacillus halodurans (NaChBac): Q9KCR8; human L-type calcium channel subunit alpha 1C (CACNA1C) domain 4: Q13936.

Article Snippet: After protein transfer to an activated PVDF-membrane and blocking (5% dry-milk in PBS), expression of wild-type or mutant hTRPM8 protein was determined by detection of immunoreactive products with a monoclonal rabbit-anti-human TRPM8 antibody (Epitomics; 1∶1000 in 5% dry-milk) directed against an extracellular domain or alternatively with a polyclonal rabbit-anti-human TRPM8 antibody (Abgent; 1∶500 in 5% dry-milk) directed against amino acids 1075–1104 of the C-terminus.

Techniques: Sequencing, Labeling

Variations of the N-x-x-D motif and corresponding nomenclature of channel variants examined in the study.

Journal: PLoS ONE

Article Title: Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

doi: 10.1371/journal.pone.0049877

Figure Lengend Snippet: Variations of the N-x-x-D motif and corresponding nomenclature of channel variants examined in the study.

Techniques:

The variants were wild-type, D802N, N799D, and N799D+D802N. ( A ), Current densities (representing mean ± S.E.M of 26–31 independent experiments) obtained at room temperature during voltage ramps between −150 mV and +150mV applied over 200 ms. Note that only the voltage range from 0 to +150 mV is shown because currents are sizeable exclusively in the outward direction ( B ), Whole-cell patch clamp measurement on HEK-293 cells expressing wild-type TRPM8 during stimulation with menthol (100 µM) or ice-cold bath solution as indicated by the horizontal bars. Between the two stimulations an intermediate wash-step with standard bath solution at room temperature was performed. The holding potential was −60 mV. ( C ), Similar experiment as shown in panel B on cells expressing the TRPM8 variant N799D. Note the different scaling of the ordinates in panels B and C. The inset shows the corresponding current-voltage relation of N799D in comparison to wild-type in the presence of menthol. ( D ), Mean inward current densities of the TRPM8 variants in response to menthol or cold obtained at a holding potential of −60 mV. Note that in double stimulation experiments only the data from the first stimulation were used for the statistical analysis. Each column represents mean ± S.E.M. of 10–16 independent experiments. Values of ***p<0.001 were considered extremely significant.

Journal: PLoS ONE

Article Title: Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

doi: 10.1371/journal.pone.0049877

Figure Lengend Snippet: The variants were wild-type, D802N, N799D, and N799D+D802N. ( A ), Current densities (representing mean ± S.E.M of 26–31 independent experiments) obtained at room temperature during voltage ramps between −150 mV and +150mV applied over 200 ms. Note that only the voltage range from 0 to +150 mV is shown because currents are sizeable exclusively in the outward direction ( B ), Whole-cell patch clamp measurement on HEK-293 cells expressing wild-type TRPM8 during stimulation with menthol (100 µM) or ice-cold bath solution as indicated by the horizontal bars. Between the two stimulations an intermediate wash-step with standard bath solution at room temperature was performed. The holding potential was −60 mV. ( C ), Similar experiment as shown in panel B on cells expressing the TRPM8 variant N799D. Note the different scaling of the ordinates in panels B and C. The inset shows the corresponding current-voltage relation of N799D in comparison to wild-type in the presence of menthol. ( D ), Mean inward current densities of the TRPM8 variants in response to menthol or cold obtained at a holding potential of −60 mV. Note that in double stimulation experiments only the data from the first stimulation were used for the statistical analysis. Each column represents mean ± S.E.M. of 10–16 independent experiments. Values of ***p<0.001 were considered extremely significant.

Techniques: Patch Clamp, Expressing, Variant Assay

$( A ), Maximum increase in the F 340 /F 380 ratio of each variant in response to 300 µM menthol or ice-cold bath solution. In double stimulation experiments only the data from the first stimulation were used for statistical analysis. The miniscule increases in fluorescence observed in mock-transfected controls are subtracted. Each column represents mean ± S.E.M. of 8–17 independent experiments. Significant differences to control are indicated with asterisks. Values of ***p<0.001 were considered extremely significant. ( B ), Western blot of TRPM8 protein on plasma membrane fractions prepared by the differential centrifugation method (“low speed fraction” ref. to ) of HEK-293 cells expressing various TRPM8 variants. Note the dual bands, shown in previous studies to indicate the glycosylated and non-glycosylated channel protein. The glycosylated form is absent in the weakly functional channel variant N799D. As negative control a plasma membrane fraction of mock-transfected HEK-293cells is included.$

Journal: PLoS ONE

Article Title: Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

doi: 10.1371/journal.pone.0049877

Figure Lengend Snippet: ( A ), Maximum increase in the F 340 /F 380 ratio of each variant in response to 300 µM menthol or ice-cold bath solution. In double stimulation experiments only the data from the first stimulation were used for statistical analysis. The miniscule increases in fluorescence observed in mock-transfected controls are subtracted. Each column represents mean ± S.E.M. of 8–17 independent experiments. Significant differences to control are indicated with asterisks. Values of ***p<0.001 were considered extremely significant. ( B ), Western blot of TRPM8 protein on plasma membrane fractions prepared by the differential centrifugation method (“low speed fraction” ref. to ) of HEK-293 cells expressing various TRPM8 variants. Note the dual bands, shown in previous studies to indicate the glycosylated and non-glycosylated channel protein. The glycosylated form is absent in the weakly functional channel variant N799D. As negative control a plasma membrane fraction of mock-transfected HEK-293cells is included.

Techniques: Variant Assay, Fluorescence, Transfection, Western Blot, Centrifugation, Expressing, Functional Assay, Negative Control

Whole-cell patch clamp measurements on HEK-293 cells expressing wild-type TRPM2 ( A ) or TRPM2-variant N869D ( B ). Stimulation was performed with ADPR (600 µM in the absence of intracellular Ca 2+ ) infused into the cell through the patch pipette. The holding potential was −60 mV. Insets show the current-voltage relations obtained during voltage ramps within the indicated voltage ranges. Inward currents were repeatedly blocked with NMDG. The TRPM2 variant N869D is analogous to the virtually non-functional TRPM8 variant N799D.

Journal: PLoS ONE

Article Title: Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

doi: 10.1371/journal.pone.0049877

Figure Lengend Snippet: Whole-cell patch clamp measurements on HEK-293 cells expressing wild-type TRPM2 ( A ) or TRPM2-variant N869D ( B ). Stimulation was performed with ADPR (600 µM in the absence of intracellular Ca 2+ ) infused into the cell through the patch pipette. The holding potential was −60 mV. Insets show the current-voltage relations obtained during voltage ramps within the indicated voltage ranges. Inward currents were repeatedly blocked with NMDG. The TRPM2 variant N869D is analogous to the virtually non-functional TRPM8 variant N799D.

Techniques: Patch Clamp, Expressing, Variant Assay, Transferring, Functional Assay

Whole-cell patch clamp measurements on HEK-293 cells expressing variants of TRPM2 and TRPM8 where the inner pair of residues of the N-x-x-D motif is reciprocally exchanged. ( A ), TRPM8 variant V800K+M801L first stimulated with ice-cold bath solution and after an intermediate wash-step with standard bath solution stimulated with 100 µM menthol (as indicated by horizontal bars). ( B ), TRPM2 variant K870V+L871M stimulated by infusion of 600 µM ADPR into the cell through the patch pipette and in the absence of intracellular Ca 2+ . Insets show the corresponding current-voltage relations during stimulation.

Journal: PLoS ONE

Article Title: Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2

doi: 10.1371/journal.pone.0049877

Figure Lengend Snippet: Whole-cell patch clamp measurements on HEK-293 cells expressing variants of TRPM2 and TRPM8 where the inner pair of residues of the N-x-x-D motif is reciprocally exchanged. ( A ), TRPM8 variant V800K+M801L first stimulated with ice-cold bath solution and after an intermediate wash-step with standard bath solution stimulated with 100 µM menthol (as indicated by horizontal bars). ( B ), TRPM2 variant K870V+L871M stimulated by infusion of 600 µM ADPR into the cell through the patch pipette and in the absence of intracellular Ca 2+ . Insets show the corresponding current-voltage relations during stimulation.

Techniques: Patch Clamp, Expressing, Variant Assay, Transferring

The expression of TRPM8 in osteosarcoma tissues and cell lines. A : TRPM8 is over-expressed in osteosarcoma tissues when compared with osteochondroma tissues used as normal control (magnification x400), arrows indicate the positive staining cells. Negative controls for immunostaining were performed in the absence of anti-TRPM8 antibody. The correlation of TRPM8 expression in osteosarcoma and osteochondroma specimens was assessed by chi-square test. B : The expression of TRPM8 in osteosarcoma cell lines was detected by western blot (a) and its abundance was expressed as normalized values over BMSCs (b). C : The knockdown efficiency of siRNA targeted on TRPM8 in MG-63 and U2OS cells. (a) Ca 2+ imaging indicated that the response to menthol was clearly diminished in siTRPM8 cells and the knockdown of TRPM8 leads to impaired regulation of intracellular Ca 2+ concentration. The fluorescence intensity in y -axis represents intracellular Ca 2+ concentration. (b) The time course and dose dependent manner of TRPM8 siRNA in MG-63 and U2OS cells. (c) The western blot data in (b) was quantified and the results were expressed in histograms.

Journal: International Journal of Biological Sciences

Article Title: Knockdown of TRPM8 Suppresses Cancer Malignancy and Enhances Epirubicin-induced Apoptosis in Human Osteosarcoma Cells

doi: 10.7150/ijbs.7738

Figure Lengend Snippet: The expression of TRPM8 in osteosarcoma tissues and cell lines. A : TRPM8 is over-expressed in osteosarcoma tissues when compared with osteochondroma tissues used as normal control (magnification x400), arrows indicate the positive staining cells. Negative controls for immunostaining were performed in the absence of anti-TRPM8 antibody. The correlation of TRPM8 expression in osteosarcoma and osteochondroma specimens was assessed by chi-square test. B : The expression of TRPM8 in osteosarcoma cell lines was detected by western blot (a) and its abundance was expressed as normalized values over BMSCs (b). C : The knockdown efficiency of siRNA targeted on TRPM8 in MG-63 and U2OS cells. (a) Ca 2+ imaging indicated that the response to menthol was clearly diminished in siTRPM8 cells and the knockdown of TRPM8 leads to impaired regulation of intracellular Ca 2+ concentration. The fluorescence intensity in y -axis represents intracellular Ca 2+ concentration. (b) The time course and dose dependent manner of TRPM8 siRNA in MG-63 and U2OS cells. (c) The western blot data in (b) was quantified and the results were expressed in histograms.

Article Snippet: After antigen retrieval by microwave, new born calf serum was added as blocking agent, and10min later, rabbit polyclonal anti-TRPM8 antibody (1:100, Cat #: ACC-049, Alomone Labs, Jerusalem, Israel) was added to incubate overnight (4°C) and then anti-rabbit IgG (BOSTER, China) was added to incubate for 20min at room temperature.

Techniques: Expressing, Staining, Immunostaining, Western Blot, Imaging, Concentration Assay, Fluorescence

Knockdown of TRPM8 suppressed growth of MG-63 and U2OS cells. A : The effect of TRPM8 knockdown on MG-63 (a) and U2OS (b) cell proliferation was measured by CCK8 assay. Growth of siTRPM8 cells was significantly suppressed at day 2 compared with Parental and siCON cells. One-Way ANOVA was used for the data analysis. ** P <0.01. OD in y -axis means A450nm and represents cell growth; bars, SD. B : Parental, siCON and siTRPM8 cells of MG-63 (a) and U2OS (b) were immunostained with anti-Ki67 antibody (green) and Hoechst33258 (blue), scale bars = 50 μm.

Journal: International Journal of Biological Sciences

Article Title: Knockdown of TRPM8 Suppresses Cancer Malignancy and Enhances Epirubicin-induced Apoptosis in Human Osteosarcoma Cells

doi: 10.7150/ijbs.7738

Figure Lengend Snippet: Knockdown of TRPM8 suppressed growth of MG-63 and U2OS cells. A : The effect of TRPM8 knockdown on MG-63 (a) and U2OS (b) cell proliferation was measured by CCK8 assay. Growth of siTRPM8 cells was significantly suppressed at day 2 compared with Parental and siCON cells. One-Way ANOVA was used for the data analysis. ** P <0.01. OD in y -axis means A450nm and represents cell growth; bars, SD. B : Parental, siCON and siTRPM8 cells of MG-63 (a) and U2OS (b) were immunostained with anti-Ki67 antibody (green) and Hoechst33258 (blue), scale bars = 50 μm.

Techniques: CCK-8 Assay

Knockdown of TRPM8 induced G 0 /G 1 arrest and decreased the level of phospho-p44/p42. A : (a) Knockdown of TRPM8 induced G 0 /G 1 arrest. 48h after the indicated transfection, cells were harvested and cell cycle was examined. (b) Compared with Parental and siCON cells, the accumulation of cells in G 0 /G 1 phase was significantly increased in siTRPM8 cells, ** P <0.01. B : (a) Knockdown of TRPM8 suppressed the Akt-GSK-3β pathway and decreased the expression of cyclinD1 and Cdk4. 48h after the indicated transfection, cells were harvested and western blot was performed. (b) The western blot results were quantified and statistic analysis was performed, * P <0.05. C : (a) Knockdown of TRPM8 decreased the level of phospho-p44/p42. (b) The western blot results were quantified and statistic analysis was performed. * P <0.05. All the data in this figure was analyzed by One-Way ANOVA.

Journal: International Journal of Biological Sciences

Article Title: Knockdown of TRPM8 Suppresses Cancer Malignancy and Enhances Epirubicin-induced Apoptosis in Human Osteosarcoma Cells

doi: 10.7150/ijbs.7738

Figure Lengend Snippet: Knockdown of TRPM8 induced G 0 /G 1 arrest and decreased the level of phospho-p44/p42. A : (a) Knockdown of TRPM8 induced G 0 /G 1 arrest. 48h after the indicated transfection, cells were harvested and cell cycle was examined. (b) Compared with Parental and siCON cells, the accumulation of cells in G 0 /G 1 phase was significantly increased in siTRPM8 cells, ** P <0.01. B : (a) Knockdown of TRPM8 suppressed the Akt-GSK-3β pathway and decreased the expression of cyclinD1 and Cdk4. 48h after the indicated transfection, cells were harvested and western blot was performed. (b) The western blot results were quantified and statistic analysis was performed, * P <0.05. C : (a) Knockdown of TRPM8 decreased the level of phospho-p44/p42. (b) The western blot results were quantified and statistic analysis was performed. * P <0.05. All the data in this figure was analyzed by One-Way ANOVA.

Techniques: Transfection, Expressing, Western Blot

Knockdown of TRPM8 inhibited cell migration and invasion. A : Knockdown of TRPM8 inhibited cell migration in MG-63 (a) and U2OS (b) cells (magnification x200). (c) The migration rate was expressed as a percentage with the value of Parental cells being 100% and the migration was significantly suppressed in siTRPM8 cells, ** P <0.01. B : Knockdown of TRPM8 inhibited cell invasion. (a) The invaded siTRPM8 cells were obviously less than the invaded Parental and siCON cells (magnification x200). (b) Values are presented as invaded cells/field, ** P <0.01. C : (a) Knockdown of TRPM8 suppressed the expression of pFAK and MMP-2. (b) The western blot results were quantified and statistic analysis was performed, ** P <0.01, * P <0.05. All the data in this figure was analyzed by One-Way ANOVA.

Journal: International Journal of Biological Sciences

Article Title: Knockdown of TRPM8 Suppresses Cancer Malignancy and Enhances Epirubicin-induced Apoptosis in Human Osteosarcoma Cells

doi: 10.7150/ijbs.7738

Figure Lengend Snippet: Knockdown of TRPM8 inhibited cell migration and invasion. A : Knockdown of TRPM8 inhibited cell migration in MG-63 (a) and U2OS (b) cells (magnification x200). (c) The migration rate was expressed as a percentage with the value of Parental cells being 100% and the migration was significantly suppressed in siTRPM8 cells, ** P <0.01. B : Knockdown of TRPM8 inhibited cell invasion. (a) The invaded siTRPM8 cells were obviously less than the invaded Parental and siCON cells (magnification x200). (b) Values are presented as invaded cells/field, ** P <0.01. C : (a) Knockdown of TRPM8 suppressed the expression of pFAK and MMP-2. (b) The western blot results were quantified and statistic analysis was performed, ** P <0.01, * P <0.05. All the data in this figure was analyzed by One-Way ANOVA.

Techniques: Migration, Expressing, Western Blot

Knockdown of TRPM8 enhanced epirubicin-induced apoptosis. A : Knockdown of TRPM8 significantly reduced the viability of MG-63 (a) and U2OS (b) cells, ** P <0.01. B : (a) Cell apoptosis was analyzed by Annexin V assay. (b) Knockdown of TRPM8 enhanced epirubicin-induced apoptosis, ** P <0.01. C : (a) The Hoechst 33258 staining assay revealed that down regulation of TRPM8 facilitated cell apoptosis in MG-63 and U2OS cells. Arrows indicate apoptotic cells. (b) The quantitative data showed the percentage of apoptotic cells in EPI-treated MG-63 and U2OS cells, ** P <0.01. All the data in this figure was analyzed by One-Way ANOVA.

Journal: International Journal of Biological Sciences

Article Title: Knockdown of TRPM8 Suppresses Cancer Malignancy and Enhances Epirubicin-induced Apoptosis in Human Osteosarcoma Cells

doi: 10.7150/ijbs.7738

Figure Lengend Snippet: Knockdown of TRPM8 enhanced epirubicin-induced apoptosis. A : Knockdown of TRPM8 significantly reduced the viability of MG-63 (a) and U2OS (b) cells, ** P <0.01. B : (a) Cell apoptosis was analyzed by Annexin V assay. (b) Knockdown of TRPM8 enhanced epirubicin-induced apoptosis, ** P <0.01. C : (a) The Hoechst 33258 staining assay revealed that down regulation of TRPM8 facilitated cell apoptosis in MG-63 and U2OS cells. Arrows indicate apoptotic cells. (b) The quantitative data showed the percentage of apoptotic cells in EPI-treated MG-63 and U2OS cells, ** P <0.01. All the data in this figure was analyzed by One-Way ANOVA.

Techniques: Annexin V Assay, Staining

The changes of p-p44/p42 and pJNK. A : After transfected with siCON and siTRPM8, MG-63 (a) and U2OS (b) cells were treated with 500ng/ml EPI for the indicated time and then western blot was performed to investigate the p-p44/p42 and p44/p42. (c) The results of the western blot were quantified and expressed in histograms. B : (a) After 48h EPI treatment, p-p44/p42 was analyzed in each sample by western blot. (b) The quantitative data of the western blot and statistic analysis was performed by One-Way ANOVA, ** P <0.01, * P <0.05. C : Cells were prepared as indicated in material and methods, and p-JNK and JNK was analyzed by western blot. Knockdown of TRPM8 had no influence on the phosphorylation of JNK (a); however, after EPI treatment it enhanced the phosphorylation of JNK (b). (c) The results of western blot in (b) were quantified and One-Way ANOVA was applied for the statistic analysis, * P <0.05. D : (a) p-MEK, MEK and MKP-1 was investigated by western blot in Parental, siCON and siTRPM8 cells. (b) The results of western blot were quantified and One-Way ANOVA was applied for the statistic analysis, * P <0.05.

Journal: International Journal of Biological Sciences

Article Title: Knockdown of TRPM8 Suppresses Cancer Malignancy and Enhances Epirubicin-induced Apoptosis in Human Osteosarcoma Cells

doi: 10.7150/ijbs.7738

Figure Lengend Snippet: The changes of p-p44/p42 and pJNK. A : After transfected with siCON and siTRPM8, MG-63 (a) and U2OS (b) cells were treated with 500ng/ml EPI for the indicated time and then western blot was performed to investigate the p-p44/p42 and p44/p42. (c) The results of the western blot were quantified and expressed in histograms. B : (a) After 48h EPI treatment, p-p44/p42 was analyzed in each sample by western blot. (b) The quantitative data of the western blot and statistic analysis was performed by One-Way ANOVA, ** P <0.01, * P <0.05. C : Cells were prepared as indicated in material and methods, and p-JNK and JNK was analyzed by western blot. Knockdown of TRPM8 had no influence on the phosphorylation of JNK (a); however, after EPI treatment it enhanced the phosphorylation of JNK (b). (c) The results of western blot in (b) were quantified and One-Way ANOVA was applied for the statistic analysis, * P <0.05. D : (a) p-MEK, MEK and MKP-1 was investigated by western blot in Parental, siCON and siTRPM8 cells. (b) The results of western blot were quantified and One-Way ANOVA was applied for the statistic analysis, * P <0.05.

Techniques: Transfection, Western Blot

The expression of TRPM8 in osteosarcoma and osteochondroma specimens

Journal: International Journal of Biological Sciences

Article Title: Knockdown of TRPM8 Suppresses Cancer Malignancy and Enhances Epirubicin-induced Apoptosis in Human Osteosarcoma Cells

doi: 10.7150/ijbs.7738

Figure Lengend Snippet: The expression of TRPM8 in osteosarcoma and osteochondroma specimens

Techniques: Expressing

Alignment of the third extracellular loop sequences of the human, rat and mouse TRPM8 channel and the human TRPA1 and TRPV1 channels. The red line indicates the epitope sequence that ACC-049 was generated against.

Journal: PLoS ONE

Article Title: Antibodies to the Extracellular Pore Loop of TRPM8 Act as Antagonists of Channel Activation

doi: 10.1371/journal.pone.0107151

Figure Lengend Snippet: Alignment of the third extracellular loop sequences of the human, rat and mouse TRPM8 channel and the human TRPA1 and TRPV1 channels. The red line indicates the epitope sequence that ACC-049 was generated against.

Article Snippet: ACC-049, a rabbit polyclonal TRPM8 antibody generated against an epitope in the third extracellular loop near the pore region of human TRPM8 was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Sequencing, Generated

Specificity of ACC-049 (2.5 µM) for blocking human TRPM8 activation induced by the specific natural agonist cold (A) or synthetic agonist icilin (D). No effect of ACC-049 on noxious cold induced human TRPA1 (B) or heat induced TRPV1 activation (C). Small molecule antagonists AMG9090 and AMG6541 are the positive control for TRPA1 (B) or TRPV1 (C) blockage, respectively. Note the near complete blockade of TRPM8 activation by ACC-049 at 2.5 µM, similar to that by the positive small molecule antagonist control M8-B (A). Neither control IgG, nor peptide-absorbed ACC-049, or peptide alone blocked activation of any of the channels tested (A–D). Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). Agonist induced 45 Ca 2+ uptake in the absence of antibodies (no Ab) was considered as 100 percent and wells with small molecule antagonists plus 45 Ca 2+ were set as zero percent.

Journal: PLoS ONE

Article Title: Antibodies to the Extracellular Pore Loop of TRPM8 Act as Antagonists of Channel Activation

doi: 10.1371/journal.pone.0107151

Figure Lengend Snippet: Specificity of ACC-049 (2.5 µM) for blocking human TRPM8 activation induced by the specific natural agonist cold (A) or synthetic agonist icilin (D). No effect of ACC-049 on noxious cold induced human TRPA1 (B) or heat induced TRPV1 activation (C). Small molecule antagonists AMG9090 and AMG6541 are the positive control for TRPA1 (B) or TRPV1 (C) blockage, respectively. Note the near complete blockade of TRPM8 activation by ACC-049 at 2.5 µM, similar to that by the positive small molecule antagonist control M8-B (A). Neither control IgG, nor peptide-absorbed ACC-049, or peptide alone blocked activation of any of the channels tested (A–D). Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). Agonist induced 45 Ca 2+ uptake in the absence of antibodies (no Ab) was considered as 100 percent and wells with small molecule antagonists plus 45 Ca 2+ were set as zero percent.

Techniques: Blocking Assay, Activation Assay, Positive Control

Concentration dependent antagonism of cold activation (10°C) of the human (A), rat (B), or mouse (C) TRPM8 channels by ACC-049, control IgG and M8-B measured by 45 calcium uptake. Note the right shifted concentration response of ACC-049 on human TRPM8 (A) compared to rat (B) or mouse (C) TRPM8, while the small molecule antagonist positive control M8-B exhibited comparable responses on TRPM8 channels of all species tested (A–C). Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). Cold induced 45 Ca 2+ uptake was considered as 100 percent and wells with M8-B at 1 µM plus 45 Ca 2+ were set as zero percent.

Journal: PLoS ONE

Article Title: Antibodies to the Extracellular Pore Loop of TRPM8 Act as Antagonists of Channel Activation

doi: 10.1371/journal.pone.0107151

Figure Lengend Snippet: Concentration dependent antagonism of cold activation (10°C) of the human (A), rat (B), or mouse (C) TRPM8 channels by ACC-049, control IgG and M8-B measured by 45 calcium uptake. Note the right shifted concentration response of ACC-049 on human TRPM8 (A) compared to rat (B) or mouse (C) TRPM8, while the small molecule antagonist positive control M8-B exhibited comparable responses on TRPM8 channels of all species tested (A–C). Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). Cold induced 45 Ca 2+ uptake was considered as 100 percent and wells with M8-B at 1 µM plus 45 Ca 2+ were set as zero percent.

Techniques: Concentration Assay, Activation Assay, Positive Control

IC 50 values (nM) of cold, icilin, and menthol induced human, rat, or mouse TRPM8 channel activation by ACC-049.

Journal: PLoS ONE

Article Title: Antibodies to the Extracellular Pore Loop of TRPM8 Act as Antagonists of Channel Activation

doi: 10.1371/journal.pone.0107151

Figure Lengend Snippet: IC 50 values (nM) of cold, icilin, and menthol induced human, rat, or mouse TRPM8 channel activation by ACC-049.

Techniques: Activation Assay

Concentration dependent antagonism of icilin induced activation of the human (A), rat (B) or mouse (C) TRPM8 channels by ACC-049, control IgG and M8-B measured by 45 calcium uptake. Note the right shifted concentration response of ACC-049 on human TRPM8 (A) compared to rat (B) or mouse (C) TRPM8, while the small molecule antagonist positive control M8-B exhibited comparable responses on TRPM8 channels of all species tested (A–C). Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). Icilin induced 45 Ca 2+ uptake was considered as 100 percent and wells with only assay buffer plus 45 Ca 2+ were set as zero percent.

Journal: PLoS ONE

Article Title: Antibodies to the Extracellular Pore Loop of TRPM8 Act as Antagonists of Channel Activation

doi: 10.1371/journal.pone.0107151

Figure Lengend Snippet: Concentration dependent antagonism of icilin induced activation of the human (A), rat (B) or mouse (C) TRPM8 channels by ACC-049, control IgG and M8-B measured by 45 calcium uptake. Note the right shifted concentration response of ACC-049 on human TRPM8 (A) compared to rat (B) or mouse (C) TRPM8, while the small molecule antagonist positive control M8-B exhibited comparable responses on TRPM8 channels of all species tested (A–C). Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). Icilin induced 45 Ca 2+ uptake was considered as 100 percent and wells with only assay buffer plus 45 Ca 2+ were set as zero percent.

Techniques: Concentration Assay, Activation Assay, Positive Control

Concentration dependent antagonism of menthol induced activation of the human (A), rat (B) or mouse (C) TRPM8 channels by ACC-049, control IgG and M8-B measured by 45 calcium uptake. Human TRPM8 channel activation was blocked by ACC-049 in a concentration dependent manner (A), but there was no antagonistic effect of ACC-049 on either rat (B) or mouse (C) TRPM8 channels activated by menthol. The small molecule antagonist positive control M8-B exhibited comparable responses on TRPM8 channels of all species tested (A–C). Values are means of triplicate measures and expressed as percent of control (POC). Menthol induced 45 Ca 2+ uptake was considered as 100 percent and wells with only assay buffer plus 45 Ca 2+ were set as zero percent.

Journal: PLoS ONE

Article Title: Antibodies to the Extracellular Pore Loop of TRPM8 Act as Antagonists of Channel Activation

doi: 10.1371/journal.pone.0107151

Figure Lengend Snippet: Concentration dependent antagonism of menthol induced activation of the human (A), rat (B) or mouse (C) TRPM8 channels by ACC-049, control IgG and M8-B measured by 45 calcium uptake. Human TRPM8 channel activation was blocked by ACC-049 in a concentration dependent manner (A), but there was no antagonistic effect of ACC-049 on either rat (B) or mouse (C) TRPM8 channels activated by menthol. The small molecule antagonist positive control M8-B exhibited comparable responses on TRPM8 channels of all species tested (A–C). Values are means of triplicate measures and expressed as percent of control (POC). Menthol induced 45 Ca 2+ uptake was considered as 100 percent and wells with only assay buffer plus 45 Ca 2+ were set as zero percent.

Techniques: Concentration Assay, Activation Assay, Positive Control

Antagonism of icilin induced activation of human TRPM8 recombinantly expressed by CHO cells (A) and rat DRG neurons (B) by additional poly- and monoclonal antibodies generated against the third extracellular pore loop. a . Alomone ACC-049. b . MyBiosource MBS609041. c . Creative Diagnostics CABT37242RH. d. Thermo Scientific OST00133W. e . Antibodies Online ABIN351226. f . Lifespan Biosciences LS-B6668. g . Enzo Lifesciences BML-SA664. h . M8-B. i . 1 µM icilin. j . 1 µM icilin + peptide (SDVD GTTYDFAHC). k . Buffer. A. Note the complete block of TRPM8 channel activation by ACC-049 ( a ), MyBiosource ( b ) and Enzo Lifesciences ( g ) antibodies at the single concentration tested. Small molecule positive control M8-B also completely blocked TRPM8 channel activation ( h ). B. Five out of seven antibodies tested ( a, b, e, f, g ) block icilin activation of rat DRG neurons by 70–80%, two antibodies ( c, d ) are ineffective. Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). 45 Ca 2+ uptake of CHO-TRPM8 cells activated with 1 µM icilin and antigen peptide ( j ) was considered as 100 percent and wells with only assay buffer plus 45 Ca 2+ were set as zero percent.

Journal: PLoS ONE

Article Title: Antibodies to the Extracellular Pore Loop of TRPM8 Act as Antagonists of Channel Activation

doi: 10.1371/journal.pone.0107151

Figure Lengend Snippet: Antagonism of icilin induced activation of human TRPM8 recombinantly expressed by CHO cells (A) and rat DRG neurons (B) by additional poly- and monoclonal antibodies generated against the third extracellular pore loop. a . Alomone ACC-049. b . MyBiosource MBS609041. c . Creative Diagnostics CABT37242RH. d. Thermo Scientific OST00133W. e . Antibodies Online ABIN351226. f . Lifespan Biosciences LS-B6668. g . Enzo Lifesciences BML-SA664. h . M8-B. i . 1 µM icilin. j . 1 µM icilin + peptide (SDVD GTTYDFAHC). k . Buffer. A. Note the complete block of TRPM8 channel activation by ACC-049 ( a ), MyBiosource ( b ) and Enzo Lifesciences ( g ) antibodies at the single concentration tested. Small molecule positive control M8-B also completely blocked TRPM8 channel activation ( h ). B. Five out of seven antibodies tested ( a, b, e, f, g ) block icilin activation of rat DRG neurons by 70–80%, two antibodies ( c, d ) are ineffective. Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). 45 Ca 2+ uptake of CHO-TRPM8 cells activated with 1 µM icilin and antigen peptide ( j ) was considered as 100 percent and wells with only assay buffer plus 45 Ca 2+ were set as zero percent.

Techniques: Activation Assay, Generated, Blocking Assay, Concentration Assay, Positive Control

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