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: 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: 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 , Origene2 , OriGene , TA811228 , Mouse , Primary , Human 1–300. 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: Antibody performance with the different techniques used in this study. − poor, + regular, ++ good, +++ excellent.

Article Snippet: TRPM8 , Origene2 , OriGene , TA811228 , Mouse , Primary , Human 1–300. 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 , Origene2 , OriGene , TA811228 , Mouse , Primary , Human 1–300. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Isolation

Journal: Food & function

Article Title: The mechanism of ginger and its processed products in the treatment of estradiol valerate coupled with oxytocin-induced dysmenorrhea in mice via regulating the TRP ion channel-mediated ERK 1/2 /NF-κB signaling pathway.

doi: 10.1039/d2fo01845d

Figure Lengend Snippet: Fig. 4 RZ and RZPPs can significantly affect TRPV1, TRPM8 and ERK1/2/NF-κB signaling pathway protein expression levels in the uteri and DRG of PD mice. The expression levels in the uteri (A) and in the DRG (B) of PD mice were detected by western blotting, and their expression was normalized relative to β-actin. Each band was presented as a representative figure, and a histogram was calculated from the band density value of at least three independent experiments. β-Actin was used as an internal control. Data are presented as mean ± SD for each group (n = 3). ##P < 0.01, ###P < 0.001 vs. normal group. Normal: blank, control: model, IBF: ibuprofen, RZL: low-dose RZ, RZM: medium-dose RZ, RZH: high-dose RZ, BGL: low-dose BG, BGM: medium-dose BG, BGH: high-dose BG, GCL: low-dose GC, GCM: medium-dose GC, GCH: high-dose GC. *P < 0.05, **P < 0.01 vs. control group. +P < 0.05, ++P < 0.01 vs. RZL group. ΔP < 0.05 vs. BGL group.

Article Snippet: The membrane was blocked at room temperature for 2 h, followed by overnight incubation with primary antibodies, including TRPV1, TRPM8, CaMKII, p-ERK1/2/ERK1/2, p-NF-κB (p65)/NFκB (p65), p-IκBα/IκBα, COX-2, and β-actin (ABclonal Technology Co. Ltd, Wuhan, China) at 4 °C.

Techniques: Expressing, Western Blot, Control

Journal: Food & function

Article Title: The mechanism of ginger and its processed products in the treatment of estradiol valerate coupled with oxytocin-induced dysmenorrhea in mice via regulating the TRP ion channel-mediated ERK 1/2 /NF-κB signaling pathway.

doi: 10.1039/d2fo01845d

Figure Lengend Snippet: Fig. 5 Immunohistochemistry analysis. The TRPV1 (A), TRPM8 (B) and COX-2 (C) expressions were detected by immunohistochemical analysis (400×). Normal: blank, control: model, IBF: ibuprofen, RZL: low-dose RZ, RZM: medium-dose RZ, RZH: high-dose RZ, BGL: low-dose BG, BGM: medium-dose BG, BGH: high-dose BG, GCL: low-dose GC, GCM: medium-dose GC, GCH: high-dose GC. ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. normal group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group. +P < 0.05 vs. RZL group.

Article Snippet: The membrane was blocked at room temperature for 2 h, followed by overnight incubation with primary antibodies, including TRPV1, TRPM8, CaMKII, p-ERK1/2/ERK1/2, p-NF-κB (p65)/NFκB (p65), p-IκBα/IκBα, COX-2, and β-actin (ABclonal Technology Co. Ltd, Wuhan, China) at 4 °C.

Techniques: Immunohistochemistry, Immunohistochemical staining, 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: 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℃ 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℃.

Techniques: Expressing, Injection, Western Blot

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℃ 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℃.

Techniques: Double Immunofluorescence Staining, Injection, Marker

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Grancalcin Accelerates Wound Healing by Improving Angiogenesis in Diabetes.

doi: 10.1002/advs.202305856

Figure Lengend Snippet: Figure 5. TRPM8 is a receptor of GCA in endothelial cells. A) TRPM8 protein binding to GCA was identified by LC-MS/MS analysis. B) TRPM8 expression in HUVECs was determined by Western blotting after incubation with normal or high glucose. Immunoprecipitation (IP) analysis of Flag-TRPM8 C) and Myc-GCA D) binding. GCA and siRNA-Trpm8 treatments induced functional changes in HUVECs (n = 3), including migration, tube formation, and expression of various angiogenic factors. Live cell images E) were captured 12 h after the wound-healing assay (scale bars, 100 μm), and the wound-

Article Snippet: [58] The membranes were probed with specific antibodies against GCA (PA577127, 1:1000, Invitrogen), TRPM8 (DF7966, 1:2000, Affinity), ERK (4695T, 1:2000, Cell Signaling Technology), p-ERK (4370S, 1:2000, Cell Signaling Technology), HIF-1α (3281T, 1:2000, Cell Signaling Technology), and VEGF (50661S, 1:2000, Cell Signaling Technology), and GAPDH (5174, 1:2000, Cell Signaling Technology), and subsequently reprobed with the appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Protein Binding, Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, Incubation, Immunoprecipitation, Binding Assay, Functional Assay, Migration, Wound Healing Assay

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