Journal: Experimental Animals

Article Title: Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats

doi: 10.1538/expanim.17-0028

Figure Lengend Snippet: Sequences of primers used for real-time RT-PCR assays

Article Snippet: After rinsing with PBS, the sections were incubated with an anti-TRPM8 antibody (1:500, rabbit polyclonal, Novus Biologicals, Littleton, CO, USA) and anti-PGP9.5 antibody (1:500, mouse polyclonal, Abcam, Tokyo, Japan) overnight at 4°C.

Techniques: Quantitative RT-PCR

Journal: Experimental Animals

Article Title: Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats

doi: 10.1538/expanim.17-0028

Figure Lengend Snippet: Relative expression levels of TRPM8 mRNA. NON-OPE: non-operated rats. OVX: ovariectomized rats. OVX + E2: ovariectomized rats treated for 28 days with 17β-estradiol. The OVX + E2 group showed a trend for decreased expression of TRPM8 channel mRNA in lumbar skin in comparison with the OVX group, although the difference between the two groups did not reach statistical significance ( P =0.0873>0.05).

Article Snippet: After rinsing with PBS, the sections were incubated with an anti-TRPM8 antibody (1:500, rabbit polyclonal, Novus Biologicals, Littleton, CO, USA) and anti-PGP9.5 antibody (1:500, mouse polyclonal, Abcam, Tokyo, Japan) overnight at 4°C.

Techniques: Expressing, Comparison

Journal: Experimental Animals

Article Title: Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats

doi: 10.1538/expanim.17-0028

Figure Lengend Snippet: TRPM8 protein levels. NON-OPE: non-operated rats. OVX: ovariectomized rats. OVX + E2: ovariectomized rats treated for 28 days with 17β-estradiol. There was no statistical difference between mean TRPM8 protein levels in lumbar skin of the OVX and OVX + E2 groups ( P =0.3095>0.05).

Article Snippet: After rinsing with PBS, the sections were incubated with an anti-TRPM8 antibody (1:500, rabbit polyclonal, Novus Biologicals, Littleton, CO, USA) and anti-PGP9.5 antibody (1:500, mouse polyclonal, Abcam, Tokyo, Japan) overnight at 4°C.

Techniques:

Journal: Experimental Animals

Article Title: Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats

doi: 10.1538/expanim.17-0028

Figure Lengend Snippet: Immunohistochemistry. A. NON-OPE: non-operated rats. B. OVX: ovariectomized rats. C. OVX + E2: ovariectomized rats treated for 28 days with 17β-estradiol. TRPM8 channels and PGP9.5-positive nerve fibers in lumbar skin of NON-OPE, OVX, and OVX + E2 rats were visualized by immunohistochemistry. TRPM8 channels were stained red, and PGP9.5-positive nerve fibers were stained green. Cell nuclei are shown in blue. PGP9.5-positive nerve fibers were scattered in a line at the external side of the epidermal tissues (arrows). Scale bar, 10 µ m. Expression of TRPM8 in the OVX rat was greater than in the NON-OPE rat (A, B), whereas expression of TRPM8 in the OVX + E2 rat was lesser than in the OVX rat (B, C).

Article Snippet: After rinsing with PBS, the sections were incubated with an anti-TRPM8 antibody (1:500, rabbit polyclonal, Novus Biologicals, Littleton, CO, USA) and anti-PGP9.5 antibody (1:500, mouse polyclonal, Abcam, Tokyo, Japan) overnight at 4°C.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Poultry Science

Article Title: Differential expressions of hypothalamic thermosensitive TRP ion channels may underlie the posthatching ontogeny of brain cooling capacity in broiler chickens

doi: 10.1016/j.psj.2023.102782

Figure Lengend Snippet: PCR primers of relevant genes used in this study.

Article Snippet: The membrane was then incubated with specific primary antibodies (1:1,000 dilution) overnight at 4°C: TRPV1 (#AF8250, rabbit polyclonal antibody from Beyotime), TRPV2 (#bs-10297R, rabbit polyclonal antibody from Beijing Biosynthesis Biotechnology Co., Ltd., China), TRPV3 (#BA2875-2, rabbit polyclonal antibody from Boster Biological Technology Co., Ltd., Wuhan, Hubei, China), TRPV4 (#AF8253, rabbit polyclonal antibody from Beyotime), TRPA1 (#AF8241, rabbit polyclonal antibody from Beyotime), TRPM8 (#PB0882, rabbit polyclonal antibody from Boster), and GAPDH (#AF0006, mouse monoclonal antibody from Beyotime).

Techniques: Sequencing

Journal: Scientific Reports

Article Title: DF2726A, a new IL-8 signalling inhibitor, is able to counteract chemotherapy-induced neuropathic pain

doi: 10.1038/s41598-019-48231-z

Figure Lengend Snippet: Western blotting analyisis for microglia marker Iba-1 and for astrocyte GFAP in control, OXA and (Oxa) + DF2726A rats. In the bottom, TRPA1 and TRPM8 receptors in in control, OXA and (Oxa) + DF2726A rats, evalutated by western blotting. Administration of DF2726A (Oxa + DF, light grey triangles) was able to significantly reduce reactive microglia and astrocytes as well as to reduce pain receptors. ***P < 0.001, **P < 0.01 and *P < 0.05 vs respective CTR group; +++ P < 0.001; ++ P < 0.01 and + P < 0.05 vs the respective Oxa + Saline group. 2-way repeated-measures ANOVA and Bonferroni test. Data are expressed as mean ± SEM; n 10 per group. ANOVA, analysis of variance.

Article Snippet: The filter was then blocked with 1 × PBS and 3% non-fat dried milk for 40 min at room temperature and probed with anti-Glial Fibrillary Acidic Protein (GFAP) antibody (diluition 1.1000; cat.no.Z0334, Dako), anti-ionized calcium-binding adapter molecule 1 (Iba-1) antibody (dilution 1:1000; cat. no.019-19741, Wako), anti-transient receptor potential ankyrin 1 (TRPA1) antibody (diluition 1:1000; cat. no. NB110-40763, Novus Biologicals) and anti- Transient receptor potential M8 (TRPM8) antibody (diluition 1:10100; cat. no. NBP1-97311, Novus Biologicals) in 1 × PBS, 3% non-fat dried milk, and 0.1% Tween 20 at 4 °C overnight.

Techniques: Western Blot, Marker, Control, Saline

Journal: The Korean Journal of Pain

Article Title: Effects of Nefopam on Streptozotocin-Induced Diabetic Neuropathic Pain in Rats

doi: 10.3344/kjp.2014.27.4.326

Figure Lengend Snippet: Effect of a nefopam pretreatment on the transient receptor potential ankyrin 1 (TRPA1) and melastatin 8 (TRPM8) protein expression levels in the dorsal root ganglion (DRG) of streptozotocin (STZ)-induced diabetic rats. Pretreatment with nefopam (30 mg/kg) was performed intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). To evaluate the expressions of TRPA1 (A) and TRPM8 (B) proteins located in the L4-L6 DRG, a Western blot analysis was performed 4 weeks after the final drug administration. The specific signals for TRPA1 and TRPM8 were quantified and plotted (lower panel). β-actin was used as an internal loading control. STZ injections did not significantly affect the basal level of the TRPA1 protein in the DRG, which was not altered by the nefopam pretreatment. The TRPM8 protein level in the DRG significantly increased 4 weeks after the STZ injection, which was reduced by the nefopam pretreatment. *** P < 0.001 compared to the control group. ††† P < 0.001 compared to the STZ group.

Article Snippet: After blocking with 5% goat serum, 0.3% Triton X-100, and 1% bovine serum albumin in phosphate-buffered saline (PBS-T), double-label immunofluorescence was conducted by incubating the paraffin sections overnight at 4°C with rabbit polyclonal antibodies against TRPM8 (1:1000, Abcam), with the IgG fraction of the mouse polyclonal antibody against the N52 clone of neurofilament 200 (NF200, 1:200; Sigma-Aldrich), followed by incubation with Alexa Flour 555 goat anti-rabbit IgG (red; 1:500; Invitrogen, Carlsbad, CA, USA) and Alexa Flour 488 goat anti-mouse IgG (green; 1:500; Invitrogen) for 2 hours at 20°C.

Techniques: Expressing, Injection, Western Blot, Control

Journal: The Korean Journal of Pain

Article Title: Effects of Nefopam on Streptozotocin-Induced Diabetic Neuropathic Pain in Rats

doi: 10.3344/kjp.2014.27.4.326

Figure Lengend Snippet: Typical double immunofluorescence staining of transient receptor potential melastatin 8 (TRPM8) and neurofilament 200 (NF200) in the rat dorsal root ganglion (DRG) of streptozotocin (STZ)-induced diabetic rats. Pretreatment with nefopam (30 mg/kg) was performed intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). Four weeks after the final drug treatment, L5 DRG samples were immunostained with TRPM8 and neurofilament 200 (NF200), a marker of myelinated neurons. The colocalization of TRPM8 was visualized in a merged image.

Article Snippet: After blocking with 5% goat serum, 0.3% Triton X-100, and 1% bovine serum albumin in phosphate-buffered saline (PBS-T), double-label immunofluorescence was conducted by incubating the paraffin sections overnight at 4°C with rabbit polyclonal antibodies against TRPM8 (1:1000, Abcam), with the IgG fraction of the mouse polyclonal antibody against the N52 clone of neurofilament 200 (NF200, 1:200; Sigma-Aldrich), followed by incubation with Alexa Flour 555 goat anti-rabbit IgG (red; 1:500; Invitrogen, Carlsbad, CA, USA) and Alexa Flour 488 goat anti-mouse IgG (green; 1:500; Invitrogen) for 2 hours at 20°C.

Techniques: Double Immunofluorescence Staining, Injection, Marker

Journal: Cell Death & Disease

Article Title: Rewiring melanoma cell fate: TRPM8 modulators trigger apoptosis and boost NK cell cytotoxicity

doi: 10.1038/s41419-026-08469-8

Figure Lengend Snippet: A TRPM8 gene expression in melanoma tissues (red, T; n = 461 ) vs. normal skin (green, N; n = 558 ) from The Genomic Data Commons (GDC) Cancer Genome Atlas (TCGA) database. Data are presented as log2 TPM; p < 0.001 by unpaired t -test. B Representative westen blot showing TRPM8 protein expression in the indicated cell lines. Tubulin was used as a loading control. Immunofluorescence analysis of TRPM8 localization in WM266-4 ( C ) and AMM16 cells ( D ). Cells were stained with anti-TRPM8 antibody (blu), Endoplasmic reticulum (ER)-tracker (green) and Plasma Membrane-tracker (red). Merged images indicate TRPM8 distribution in both plasma membrane and ER compartments. Scale bar: 10 µm. Viability of AMM16 ( E ) and WM266-4 ( F ) cells untreated or treated with compounds 3, 5, 4, 6 and 9 at the concentrations indicated in the legends above. Absorbance values from WST-1 assays after 24, 48 and 72 h are reported. Data are expressed as mean ± standard deviations (SDs) from n independent experiments. * p < 0.05 for the indicated time points vs. the corresponding untreated control.

Article Snippet: The following antibodies were used in WB and Co-IP analyzes: the mouse monoclonal: anti-IκBα (L35A5) (#4814, Cell Signaling); anti-p53 (DO-1) (sc-126, Santa Cruz); anti-IKKα (3G12) (#11930, Cell Signaling); anti-α-tubulin (#E-AB-20036, Elabsciences); anti-GAPDH (#E-AB-20079, Elabsciences); the rabbit polyclonal: anti-phospho-ATM (Ser1981) (D25E5) (#13050, Cell Signaling); anti-phospho-p53 (Ser15) (#AF1043, R&D Systems); anti-phospho-AKT (Ser473) (#9271, Cell Signaling); anti-AKT (#9272, Cell Signaling); anti-phospho-GSK-3α/β (Ser21/9) (#9331, Cell Signaling); anti-caspase-3 (#9662, Cell Signaling); anti-PARP cleavage site (214/215) (#AB3565, Millipore); anti-PARP (#06-557, Upstate); anti-TRPM8 (#NBP1-97311, Novus Biologicals); anti-PMAIP1 (NOXA) (#A9801, Abclonal); the recombinant rabbit monoclonal anti-PUMA (#A3752, Abclonal); anti-ULBP1 (#A21161, Abclonal); anti-IKKβ (D30C6) (#8943, Cell Signaling); anti-phospho-IKKα/β (Ser176/180) (#2697, Cell Signaling); anti-phospho-NF-κB p65 (Ser536) (93H1) (#3033, Cell Signaling); anti-PI3K p85 (#06-195; Millipore, Burlington, Massachusetts, USA); anti-PI3K p110 δ (#AB1678; Abcam, Cambridge, UK).

Techniques: Gene Expression, Expressing, Control, Immunofluorescence, Staining, Clinical Proteomics, Membrane

Journal: Cell Death & Disease

Article Title: Rewiring melanoma cell fate: TRPM8 modulators trigger apoptosis and boost NK cell cytotoxicity

doi: 10.1038/s41419-026-08469-8

Figure Lengend Snippet: A Western blot analysis of WM266-4 cell lysates collected after 60 or 120 min of treatment with TRPM8 modulators, using the indicated antibodies. B WM266-4 cells were untreated (-) or treated for 120 min with TRPM8 antagonists (4 and 9, used at 10 µM). Lysate proteins were immune-precipitated using the anti-TRPM8 (anti-TRPM8) or control (ctrl IgG) antibodies. WB analysis using antibodies against the indicated proteins was done to reveal co-immunoprecipitated proteins. Western blot analysis of cleaved caspase-3 and cleaved PARP in AMM16 ( C ) and WM266-4 ( D ) cells treated with TRPM8 modulators at the indicated concentrations and hours. The α-tubulin was used as loading control. Graphs in the lower part of the figure represent the densitometric analysis of the cleaved PARP/tubulin ratio obtained in three different experiments ( n = 3 ).

Article Snippet: The following antibodies were used in WB and Co-IP analyzes: the mouse monoclonal: anti-IκBα (L35A5) (#4814, Cell Signaling); anti-p53 (DO-1) (sc-126, Santa Cruz); anti-IKKα (3G12) (#11930, Cell Signaling); anti-α-tubulin (#E-AB-20036, Elabsciences); anti-GAPDH (#E-AB-20079, Elabsciences); the rabbit polyclonal: anti-phospho-ATM (Ser1981) (D25E5) (#13050, Cell Signaling); anti-phospho-p53 (Ser15) (#AF1043, R&D Systems); anti-phospho-AKT (Ser473) (#9271, Cell Signaling); anti-AKT (#9272, Cell Signaling); anti-phospho-GSK-3α/β (Ser21/9) (#9331, Cell Signaling); anti-caspase-3 (#9662, Cell Signaling); anti-PARP cleavage site (214/215) (#AB3565, Millipore); anti-PARP (#06-557, Upstate); anti-TRPM8 (#NBP1-97311, Novus Biologicals); anti-PMAIP1 (NOXA) (#A9801, Abclonal); the recombinant rabbit monoclonal anti-PUMA (#A3752, Abclonal); anti-ULBP1 (#A21161, Abclonal); anti-IKKβ (D30C6) (#8943, Cell Signaling); anti-phospho-IKKα/β (Ser176/180) (#2697, Cell Signaling); anti-phospho-NF-κB p65 (Ser536) (93H1) (#3033, Cell Signaling); anti-PI3K p85 (#06-195; Millipore, Burlington, Massachusetts, USA); anti-PI3K p110 δ (#AB1678; Abcam, Cambridge, UK).

Techniques: Western Blot, Control, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: TRPM8-independent Menthol-induced Ca2+ Release from Endoplasmic Reticulum and Golgi

doi: 10.1074/jbc.m605213200

Figure Lengend Snippet: FIGURE 1. Menthol-induced intracellular Ca2 release. A, time course of TRPM8 current activation in Ca2-free extracellular solution upon application of 1 mM menthol at 33 °C in HEK293 cells overexpressing TRPM8. The upper panel shows the in and outward current at 90 and 90 mV, whereas the lower panel displays the increase in [Ca2]i. B, current-voltage relations obtained at the time points indicated in panel A.

Article Snippet: Anti-TRPM8 Antibody Generation and Immunodetection— For immunodetection, two different anti-TRPM8 antibodies were used: we compared commercially available antiTRPM8 (Novus) to a newly generated anti-TRPM8 antibody designed in our laboratory.

Techniques: Activation Assay

Journal: Journal of Biological Chemistry

Article Title: TRPM8-independent Menthol-induced Ca2+ Release from Endoplasmic Reticulum and Golgi

doi: 10.1074/jbc.m605213200

Figure Lengend Snippet: FIGURE 2. Influence of temperature on the menthol-induced [Ca2]i release. A, typical time course of [Ca2]i for non-transfected HEK293 cells at 33 °C when applying 1 mM menthol in the absence of extracellular Ca2. A rise in [Ca2]i was only observed during the first menthol application. The inset shows the chemical structure of menthol. B, the left panel shows the averaged [Ca2]i increase upon application of 1 mM menthol for non-transfected cells compared with TRPM8-transfected cells at 33 °C. The right panel compares the time needed to reach half-maximal rise upon menthol application. C, a typical experiment showing that TRPM8- transfected cells in Ca2-containing solution only display a rise in [Ca2]i upon exposure to low menthol concentrations at low temperatures. The upper panel shows the temperature course, the lower panel displays the [Ca2]i increase upon application of 10 M menthol. D, the same as in C, but for untransfected cells exposed to 1 mM of menthol. A rise in [Ca2]i is favored by high temperatures. E, dose-response curves at 23 and 33 °C, respectively, for the [Ca2]i increase upon application of different concentrations of menthol on TRPM8-trans- fected cells. F, same as in E but for non-transfected cells.

Article Snippet: Anti-TRPM8 Antibody Generation and Immunodetection— For immunodetection, two different anti-TRPM8 antibodies were used: we compared commercially available antiTRPM8 (Novus) to a newly generated anti-TRPM8 antibody designed in our laboratory.

Techniques: Transfection

Journal: Journal of Biological Chemistry

Article Title: TRPM8-independent Menthol-induced Ca2+ Release from Endoplasmic Reticulum and Golgi

doi: 10.1074/jbc.m605213200

Figure Lengend Snippet: FIGURE 4. Lack of endogenous TRPM8 expression in non-transfected HEK293 cells. Agarose gel electro- phoresis of mRNA products obtained after amplification of 3 different base pair sequences specific for TRPM8 (see Table 1) with reverse transcriptase-PCR. Evaluation of the constituvely expressed actin gene was included as a quality control for the cDNA.

Article Snippet: Anti-TRPM8 Antibody Generation and Immunodetection— For immunodetection, two different anti-TRPM8 antibodies were used: we compared commercially available antiTRPM8 (Novus) to a newly generated anti-TRPM8 antibody designed in our laboratory.

Techniques: Expressing, Transfection, Agarose Gel Electrophoresis, Amplification, Reverse Transcription, Control

Journal: Journal of Biological Chemistry

Article Title: TRPM8-independent Menthol-induced Ca2+ Release from Endoplasmic Reticulum and Golgi

doi: 10.1074/jbc.m605213200

Figure Lengend Snippet: FIGURE 6. Comparison of intracellular Ca2 release induced by different TRPM8 agonists. A, typical time course of [Ca2]i for non-transfected HEK293 cells at 33 °C when applying 3 mM eucalyptol in the absence of extracellularCa2.Theinsetshowsthechemicalstructureofeucalyptol.B,sameasinAbutwith20Micilin.The inset shows the chemical structure of icilin. C, same as in A but with 3 mM linalool. D, same as in A but with 3 mM geraniol. E, dose-response curves at 23 and 33 °C for the [Ca2]i increase upon application of different concen- trations of linalool in non-transfected HEK293 cells. The inset shows the chemical structure of linalool. F, same as in E but for geraniol. The inset shows the chemical structure of geraniol.

Article Snippet: Anti-TRPM8 Antibody Generation and Immunodetection— For immunodetection, two different anti-TRPM8 antibodies were used: we compared commercially available antiTRPM8 (Novus) to a newly generated anti-TRPM8 antibody designed in our laboratory.

Techniques: Comparison, Transfection