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: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Immunohistochemistry using anti-TRPM8 in pancreatic tissues. ( A ) Normal pancreatic tissues. ( B ) Chronic pancreatitis. ( C ) Pancreatic intra-epithelial neoplasm. ( D ) Intraductal papillary mucinous neoplasm.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Immunohistochemistry

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Immunohistochemical analysis of TRPM8 in malignant pancreatic tumors. ( A , B ) Adenocarcinoma. ( C , D ) Adenosquamous carcinoma. ( E , F ) Solid pseudo-papillary neoplasm ( G , H ) Acinar cell carcinoma. ( I , J ) Neuroendocrine tumor. ( A , C , E , G , I ) H and E, original magnification ×200. ( B , D , F , H , J ) Immunohistochemistry using anti-TRPM8 antibodies, original magnification ×400.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Immunohistochemical staining, Immunohistochemistry

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Expression of TRPM8 in various types of histopathology in pancreatic tumors with regard to the intensity of immunoreactivity and the percentage of positive cells. The total number of cases examined is 308. The values represent the number of specimens with the corresponding proportions in parentheses.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Expressing, Histopathology

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Expression levels of TRPM8 in pancreatic adenocarcinoma. Images in ( A , C , E ) (original magnification ×200) represent the H&E sections of the same tumor as the TRPM8 immunohistochemical staining in ( B , D , F ) (original magnification ×400), respectively. Expression levels of TRPM8: ( B ) no-to-low; ( D ) moderate; ( F ) high.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Anti-TRPM8 immunoreactivity in pancreatic adenocarcinoma positively correlates with primary tumor size and stages. The distribution of the IHC score for expression of TRPM8 is displayed against age/gender, histological grade, primary tumor (T), and stage. The total number of specimens of pancreatic adenocarcinoma examined is 280. Polyserial correlation between the primary tumor size (T) or the tumor stages and the log-transformed IHC score is 0.13 and 0.10, respectively.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Expressing, Transformation Assay

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: The percent coverage ± standard error for expression of TRPM8 in pancreatic adenocarcinoma with respect to the intensity and the tumor stage. The numbers in parentheses represent the patient counts. NA, not applicable.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Expressing

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Over-expression of TRPM8 protein in human pancreatic adenocarcinoma cell lines. ( A ) The protein levels of TRPM8 in H6c7, MIA PaCa-2, PANC-1, and BxPC-3 cells were determined using immunoblotting with anti-TRPM8 antibodies. The GAPDH protein levels were analyzed as internal controls. ( B ) The relative protein levels of TRPM8 are expressed as % (mean ± standard error) of that in H6c7.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Over Expression, Western Blot

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Short hairpin RNA-mediated silencing of TRPM8 impaired invasion of pancreatic adenocarcinoma cells. BxPC-3 and MIA PaCa-2 cells transfected with either anti- TRPM8 shRNA or NC shRNA were analyzed for cell invasion using the trans-well assay. Cell invasion is expressed % (mean ± standard error) of that in the cells transfected with control shRNA. Representative images of the invaded cells stained with crystal violet are shown at 200× magnification.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: shRNA, Transfection, Staining

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