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: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Command and Compensation in a Neuromodulatory Decision Network

doi: 10.1523/JNEUROSCI.3707-11.2012

Figure Lengend Snippet: A, CNS wholemounts from newly eclosed flies expressing either TRPM8 alone (left), or TRPM8 plus an RNAi construct directed against the bursicon subunit pburs (right) in CCAP-expressing neurons, were labeled with an anti-pburs antibody. pburs-RNAi expression substantially reduces anti-pburs immunostaining in both groups of bursicon-expressing neurons, the BSEG (arrowheads) and the BAG (brackets). Scale bar, 50 μm. B, Bar graphs showing that flies expressing pburs-RNAi did not expand their wings (top). Unlike control flies not expressing pburs-RNAi, approximately 30% of flies in which pburs levels are knocked down exhibit expansional behavior (middle), but only after environmental delays in the time till abdominal flexion (bottom), even when the CCAP-expressing neurons are stimulated (ΔTemp). C, Burs-Gal4 drives UAS-TRPM8 expression in both subsets of bursicon-expressing neurons, the BAG and the BSEG, as revealed by double-labeling with anti-burs (magenta) and anti-TRPM8 (green) immunolabeling. Scale bar, 50 μm. D, Box plots show that under adverse conditions, a 15 min temperature shift to 18°C from 25°C (ΔTemp) rapidly induces wing expansion in Burs-Gal4>TRPM8 flies (+) compared with control animals either of the same genotype and not subjected to temperature shift (-) or lacking the Burs-Gal4 driver (Con). The no temperature shift control plot includes two animals that tonically flexed their abdomens, but did not fully expand their wings. As noted in Materials and Methods, this sometimes happens after prolonged delays. In these cases, “Expansional Delay” indicates the time until termination of abdominal flexion. *** and NS, indicate p<0.001 and p>0.05 as evaluated by t-test. Number of animals in each group is in parentheses above each plot.

Article Snippet: Immunohistochemistry and immunoblotting Whole mounts of the central nervous system from either males or females were prepared, stained, and analyzed by confocal microscopy as described previously ( Luan et al., 2006b ) using the following primary antibodies: rabbit anti-burs (AKA, anti-bursicon α-subunit; 1: 5000), rat anti-pburs (AKA, anti-bursicon β-subunit; kind gift of Aaron Hsueh, used at 1: 500 ), goat anti-TRPM8 (Everest Biotech, Oxfordshire, UK, used at 1:2000).

Techniques: Expressing, Construct, Labeling, Immunostaining, Immunolabeling

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Command and Compensation in a Neuromodulatory Decision Network

doi: 10.1523/JNEUROSCI.3707-11.2012

Figure Lengend Snippet: A, Box plots showing the time required to complete expansional behaviors (i.e. Expansional Delay) for flies expressing TRPM8 in the indicated cell group (Driver) with or without stimulation (ΔTemp). (-)DBD, controls lacking the BursGalDBD-U6A1 hemidriver. As noted in the legend of Fig. 1, some flies that attempted expansion after prolonged delays failed to expand their wings, in which case the expansional delay represents the time they took to complete abdominal flexion. The number of flies tested in each group is indicated above the graph, as are the results of t-test comparisons between groups. NS, p>0.05; ***, p<0.001. B, Western blots showing blood titers of bursicon (i.e. anti-burs immunostaining) resulting from stimulation (ΔTemp) of the BAG (top) or BSEG (bottom). PC, burs positive control indicates release during normal expansion. Volumes of blood loaded per sample are indicated. C, Wing morphology (arrows) and tanning (arrowheads) of flies expressing TRPM8 in either the BAG or BSEG evaluated 3 h after eclosion. D, Abdominal nerves from BSEG>TRPM8 animals with (right) or without (left) BSEG stimulation immunostained for bursicon using anti-burs antibodies. Scale bars, 50 μm. E, Box plot showing quantified burs-IR data from abdominal nerves of the indicated number of animals of each kind shown in D, demonstrating release of bursicon from these nerves in response to BSEG stimulation. mpi; mean pixel intensity (background-subtracted). **, p<0.01 by t-test.

Article Snippet: Immunohistochemistry and immunoblotting Whole mounts of the central nervous system from either males or females were prepared, stained, and analyzed by confocal microscopy as described previously ( Luan et al., 2006b ) using the following primary antibodies: rabbit anti-burs (AKA, anti-bursicon α-subunit; 1: 5000), rat anti-pburs (AKA, anti-bursicon β-subunit; kind gift of Aaron Hsueh, used at 1: 500 ), goat anti-TRPM8 (Everest Biotech, Oxfordshire, UK, used at 1:2000).

Techniques: Expressing, Western Blot, Immunostaining, Positive Control

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Command and Compensation in a Neuromodulatory Decision Network

doi: 10.1523/JNEUROSCI.3707-11.2012

Figure Lengend Snippet: Summary of Manipulations and Results

Article Snippet: Immunohistochemistry and immunoblotting Whole mounts of the central nervous system from either males or females were prepared, stained, and analyzed by confocal microscopy as described previously ( Luan et al., 2006b ) using the following primary antibodies: rabbit anti-burs (AKA, anti-bursicon α-subunit; 1: 5000), rat anti-pburs (AKA, anti-bursicon β-subunit; kind gift of Aaron Hsueh, used at 1: 500 ), goat anti-TRPM8 (Everest Biotech, Oxfordshire, UK, used at 1:2000).

Techniques: Activation 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: 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