polyclonal rabbit anti trpm8 extracellular  (Alomone Labs)

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

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

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, Immunocytochemistry, Western Blot, Staining, Quantitation Assay, MANN-WHITNEY

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.

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: MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Immunocytochemistry of mTRPM8 in transfected HEK-293 cells. ( A – F left) Confocal images of mTRPM8-EYFP transiently expressed in HEK-293 cells. EYFP (green), TRPM8 antibody (red) and Hoechst staining (blue). Merge images correspond to the overlap of the three fluorescent signals plus the bright field image. Scale bar: 30 µm. ( A – F right) Box plots represent the specificity ratio (SR) for each dilution of the respective antibody. Each dot corresponds to an individual EYFP+ cell. Box contains the 25th to 75th percentiles. Whiskers mark the 5th and 95th percentiles. The line inside the box denotes the median and the black dot represents the mean. (*** p < 0.001, Mann–Whitney test). ( G ) Bar histogram summarizing the SR mean ± SEM of each TRPM8 antibody and the controls without primary antibody (αRabbit or αMouse secondary antibodies alone). Dashed line indicates SR = 1. (*** p < 0.001, Kruskal–Wallis followed by Dunn’s post hoc test vs. αRabbit or αMouse). For each antibody and dilution, n > 270 cells; 4 fields from 2 independent transfections.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Immunocytochemistry, Transfection, Staining, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Western blot analysis for TRPM8 antibodies specificity. ( A – F ) TRPM8 immunoblots using ( A ) Alomone ( B ) ECM1, ( C ) Origene1, ( D ) ECM2, ( E ) ECM3 or ( F ) Origene2 antibodies. (−) lanes: untransfected HEK-293 cells, (+) lanes: HEK-293 cells transfected with mTRPM8-EYFP. Left: Immunoblot with each TRPM8 antibody. Right-top: EYFP immunoblotting on the same membrane. Right-bottom: GAPDH loading control. Black arrowheads indicate mTRPM8-EYFP bands revealed with antiTRPM8 antibody. Green arrowheads indicate mTRPM8-EYFP bands revealed with anti-GFP antibody. Red arrowheads indicate GAPDH bands. All blots were repeated at least 3 times to exclude a technical artefact when no anti-TRPM8 signal was observed. For each replicate, the same lysate was used for all antibodies.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Western Blot, Transfection, Membrane, Control

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Calcium imaging in DRG-cultured cells from Trpm8 BAC -EYFP + mice. ( A ) Representative traces of calcium transients evoked by mild cold (∼18–20 °C), WS-12 (10 µM) and KCl (30 mM) in EYFP+ and EYFP− neurons. Traces corresponding to individual cells are shown in grey. Colored traces represent averages of the respective individual traces. ( B ) Proportion of EYFP+ and EYFP− cells responding to cold and WS-12. ( C ) Representative immunocytochemistry performed after a calcium recording. Upper- and bottom-left: confocal images of the immunofluorescence. Green: EYFP. Red: TRPM8 (ECM1 antibody). Grey: βIII-Tubulin. Bottom-right: transmitted light and EYFP fluorescence from the same live cells before fixation. n = 432 cells from 2 mice. Scale bar: 50 µm.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Imaging, Cell Culture, Immunocytochemistry, Immunofluorescence, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Immunocytochemistry of endogenous TRPM8 in cultured DRG cells from the Trpm8 BAC -EYFP + mouse. ( A – F left) Confocal images of TRPM8+ sensory neurons. EYFP (green), TRPM8 antibody (red) and βIII-Tubulin (grey). Scale bar: 50 µm. ( A – F right) Box plots represent specificity ratio (SR) for each dilution of the respective antibody. Each dot corresponds to an individual EYFP+ cell. Box contains the 25th to 75th percentiles. Whiskers mark the 5th and 95th percentiles. The line inside the box denotes the median and the black dot represents the mean (* p < 0.05, ** p < 0.01, Mann–Whitney test). ( G ) Bar histogram summarizing the SR mean ± SEM of each TRPM8 antibody and the controls without primary antibody (αRabbit or αMouse secondary antibodies alone). Dashed line indicates SR = 1 (* p < 0.05, ** p < 0.01, Kruskal–Wallis followed by Dunn’s post hoc test vs. αRabbit or αMouse). n = at least 20 cells; 4 pictures from 2 mice for each antibody and dilution.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Immunocytochemistry, Cell Culture, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Immunohistochemistry of endogenous TRPM8 in DRG slices from the Trpm8 BAC -EYFP + mouse. ( A – F left) Confocal images of TRPM8+ sensory neurons. EYFP (green), TRPM8 antibody (red) and βIII-Tubulin (grey). Scale bar: 50 µm. ( A – F right) Box plots represent specificity ratio (SR) for each dilution of the respective antibody. Each dot corresponds to an individual EYFP+ cell. Box contains the 25th to 75th percentiles. Whiskers mark the 5th and 95th percentiles. The line inside the box denotes the median and the black dot represents the mean. (Differences among dilutions were not significant, Mann–Whitney test). ( G ) Bar histogram summarizing the SR mean ± SEM of each TRPM8 antibody and the controls without primary antibody (αRabbit or αMouse secondary antibodies alone). Dashed line indicates SR = 1. (* p < 0.05, ** p < 0.01, Kruskal–Wallis followed by Dunn´s post hoc test vs. αRabbit or αMouse). n = at least 18 cells; 4 images from 2 mice for each antibody and dilution.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Immunohistochemistry, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Immunofluorescence of endogenous TRPM8 in DRG cells and slices from the TRPM8 KO mouse. ( A , C ) Immunocytochemistry. ( B , D ) Immunohistochemistry. ( A – D left) Confocal images of TRPM8 KO ( Trpm8 EGFPf ; B6;129S1(FVB)- Trpm8 tm1Apat /J) sensory neurons. EGFP (green), TRPM8 antibody (red) and βIII-Tubulin (grey). Scale bar: 50 µm. ( A – D right) Box plots represent specificity ratio (SR) for each dilution of the respective antibody. Each dot corresponds to an individual EGFP+ cell. Box contains the 25th to 75th percentiles. Whiskers mark the 5th and 95th percentiles. The line inside the box denotes the median and the black dot represents the mean (** p < 0.01, Mann–Whitney test). ( E,F ) Bar histograms summarizing the SR mean ± SEM of each TRPM8 antibody in the KO and the reporter ( Trpm8 BAC -EYFP + , named M8-EYFP in the figure) mouse in ICC ( E ) and IHC ( F ). Dashed line indicates mean SR for the control without primary antibody (** p < 0.01, Mann–Whitney test). n = at least 22 cells; 4 pictures from 2 mice for each antibody and dilution.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Immunofluorescence, Immunocytochemistry, Immunohistochemistry, MANN-WHITNEY, Control

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Antibody performance with the different techniques used in this study. − poor, + regular, ++ good, +++ excellent.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Isolation

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

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.

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:

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.

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: Patch Clamp, Expressing, Variant Assay

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.

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

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.

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

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.

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: Patch Clamp, Expressing, Variant Assay, Transferring

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

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.

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: Blocking Assay, Activation Assay, Positive Control

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.

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: Concentration Assay, Activation Assay, Positive Control

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.

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: Activation Assay

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.

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: Concentration Assay, Activation Assay, Positive Control

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.

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: Concentration Assay, Activation Assay, Positive Control

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.

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