rabbit anti mglur1a b  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti mglur1a b
    mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with <t>mGluR1a</t> or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.
    Rabbit Anti Mglur1a B, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti mglur1a b/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti mglur1a b - by Bioz Stars, 2024-07
    93/100 stars

    Images

    1) Product Images from "Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons"

    Article Title: Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0705-12.2012

    mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with mGluR1a or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.
    Figure Legend Snippet: mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with mGluR1a or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.

    Techniques Used: Silver Staining, Mass Spectrometry

    TRPC3 associates with mGluR–GluRδ2–PKCγ in cerebellum. Triton X-100-solubilized postnuclear membrane fractions from either wild-type or mutant mice were immunoprecipitated and blotted as indicated. A, mGluR1 coimmunoprecipitated with TRPC3 from either wild-type (A1) or GluRδ2 (A2) mutant cerebella. B1, GluRδ2 coimmunoprecipitated with TRPC3. B2, In ho-4J mice, a truncated GluRδ2 is expressed at very low levels. C, PKCγ associates with mGluR1 or TRPC3 in the presence (C1) or absence (C2) of GluRδ2. D, TRPC3 and GluRδ2 interact in the presence (D1) or absence (D2) of mGluR1. E, PKCγ associates with GluRδ2 and TRPC3, which is not affected by the presence or absence of mGluR1. F, GluK2/3 did not interact with mGluR1, GluRδ2, or TRPC3.
    Figure Legend Snippet: TRPC3 associates with mGluR–GluRδ2–PKCγ in cerebellum. Triton X-100-solubilized postnuclear membrane fractions from either wild-type or mutant mice were immunoprecipitated and blotted as indicated. A, mGluR1 coimmunoprecipitated with TRPC3 from either wild-type (A1) or GluRδ2 (A2) mutant cerebella. B1, GluRδ2 coimmunoprecipitated with TRPC3. B2, In ho-4J mice, a truncated GluRδ2 is expressed at very low levels. C, PKCγ associates with mGluR1 or TRPC3 in the presence (C1) or absence (C2) of GluRδ2. D, TRPC3 and GluRδ2 interact in the presence (D1) or absence (D2) of mGluR1. E, PKCγ associates with GluRδ2 and TRPC3, which is not affected by the presence or absence of mGluR1. F, GluK2/3 did not interact with mGluR1, GluRδ2, or TRPC3.

    Techniques Used: Mutagenesis, Immunoprecipitation

    Neither GluRδ2 nor mGluR1 mutation affects cerebellar levels of interacting proteins. A, GluRδ2 mutation results in increased levels of GluA2 but does not affect levels of mGluR1, TRPC3, or other synaptic proteins. B, Mutation of GluR1 does not affect levels of any of the proteins analyzed. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ or mGluR1+/+ (t test). n = 4 for each sample. *p < 0.05.
    Figure Legend Snippet: Neither GluRδ2 nor mGluR1 mutation affects cerebellar levels of interacting proteins. A, GluRδ2 mutation results in increased levels of GluA2 but does not affect levels of mGluR1, TRPC3, or other synaptic proteins. B, Mutation of GluR1 does not affect levels of any of the proteins analyzed. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ or mGluR1+/+ (t test). n = 4 for each sample. *p < 0.05.

    Techniques Used: Mutagenesis

    GluRδ2 or mGluR1 mutation does not affect immunofluorescent distribution of the interacting proteins. A–C, Immunofluorescent double labeling of sagittal cerebellar sections; the bottom panels are magnified images of the molecular layer. Specificity of staining is confirmed by elimination of immunosignal in corresponding mutant mice. A, Punctate GluRδ2 staining partially colocalizes with mGluR1 in molecular layer of wild type. The molecular layer staining patterns for GluRδ2 and mGluR1 were not dramatically altered in the mGluR1-KO or GluRδ2ho-4J/ho-4J, respectively. B, PKCγ shows strong labeling of Purkinje cell bodies, dendrites, and neuropil. The colabeling of mGluR1 and PKCγ in the molecular layer was not dramatically altered in the GluRδ2ho-4J/ho-4J, and the PKCγ distribution was not changed in the mGlu1-KO. C, mGluR1 and TRPC3 partially colocalize in wild-type mouse. No obvious difference in the staining patterns was detected in either GluRδ2ho-4J/ho-4J or mGluR1-KO. ML, Molecular layer; PC, Purkinje cell.
    Figure Legend Snippet: GluRδ2 or mGluR1 mutation does not affect immunofluorescent distribution of the interacting proteins. A–C, Immunofluorescent double labeling of sagittal cerebellar sections; the bottom panels are magnified images of the molecular layer. Specificity of staining is confirmed by elimination of immunosignal in corresponding mutant mice. A, Punctate GluRδ2 staining partially colocalizes with mGluR1 in molecular layer of wild type. The molecular layer staining patterns for GluRδ2 and mGluR1 were not dramatically altered in the mGluR1-KO or GluRδ2ho-4J/ho-4J, respectively. B, PKCγ shows strong labeling of Purkinje cell bodies, dendrites, and neuropil. The colabeling of mGluR1 and PKCγ in the molecular layer was not dramatically altered in the GluRδ2ho-4J/ho-4J, and the PKCγ distribution was not changed in the mGlu1-KO. C, mGluR1 and TRPC3 partially colocalize in wild-type mouse. No obvious difference in the staining patterns was detected in either GluRδ2ho-4J/ho-4J or mGluR1-KO. ML, Molecular layer; PC, Purkinje cell.

    Techniques Used: Mutagenesis, Labeling, Staining

    In GluRδ2ho-4J/ho-4J, TRPC3 partially redistributes to the Triton X-100-soluble fraction. A, Synaptosomal (Syp) and PSD fractions of mouse cerebella from wild-type, GluRδ2ho-4J/ho-4J, and mGluR1-KO were prepared and immunoblotted. mGluR1, GluRδ2, PKCγ, and TRPC3 are detected in the synaptosomal and PSD fractions. The blotting profiles of PSD-95 and synaptophysin validate the subcellular fractionations. B, Cerebellar homogenates were treated with 2.5% Triton X-100, followed by ultracentrifugation to yield supernatant (Sup) and pellet (Ppt). C, A greater percentage of TRPC3 was detected in Triton X-100-soluble fraction in GluRδ2ho-4J/ho-4J than in wild type (GluRδ2+/+). Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for each sample. *p < 0.05.
    Figure Legend Snippet: In GluRδ2ho-4J/ho-4J, TRPC3 partially redistributes to the Triton X-100-soluble fraction. A, Synaptosomal (Syp) and PSD fractions of mouse cerebella from wild-type, GluRδ2ho-4J/ho-4J, and mGluR1-KO were prepared and immunoblotted. mGluR1, GluRδ2, PKCγ, and TRPC3 are detected in the synaptosomal and PSD fractions. The blotting profiles of PSD-95 and synaptophysin validate the subcellular fractionations. B, Cerebellar homogenates were treated with 2.5% Triton X-100, followed by ultracentrifugation to yield supernatant (Sup) and pellet (Ppt). C, A greater percentage of TRPC3 was detected in Triton X-100-soluble fraction in GluRδ2ho-4J/ho-4J than in wild type (GluRδ2+/+). Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for each sample. *p < 0.05.

    Techniques Used:

    Effects of mGluR1 and GluRδ2 mutation on surface expression of interacting proteins. Mouse cerebellar slices were treated with a membrane-impermeable biotinylation reagent to mark cell-surface proteins. Total, internal, and surface proteins were resolved by SDS-PAGE and subjected to immunoblotting. GluRδ2 mutation increased surface expression of TRPC3 and mGluR1, whereas other components were unchanged. mGluR1b is the shorter splice variant of mGluR1. β3-Tubulin and/or synaptophysin served as internal protein controls. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for mGluR1, mGluR1b, TRPC3, and GluK2/3. n = 3 for GluA2, synaptophysin, and β3-tubulin. *p < 0.05.
    Figure Legend Snippet: Effects of mGluR1 and GluRδ2 mutation on surface expression of interacting proteins. Mouse cerebellar slices were treated with a membrane-impermeable biotinylation reagent to mark cell-surface proteins. Total, internal, and surface proteins were resolved by SDS-PAGE and subjected to immunoblotting. GluRδ2 mutation increased surface expression of TRPC3 and mGluR1, whereas other components were unchanged. mGluR1b is the shorter splice variant of mGluR1. β3-Tubulin and/or synaptophysin served as internal protein controls. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for mGluR1, mGluR1b, TRPC3, and GluK2/3. n = 3 for GluA2, synaptophysin, and β3-tubulin. *p < 0.05.

    Techniques Used: Mutagenesis, Expressing, SDS Page, Western Blot, Variant Assay

    GluRδ2ho-4J/ho-4J alters the time course of the mGluR1-dependent sEPSC at PF–PC synapses. A, Typical traces of sEPSCs recorded from wild-type or GluRδ2ho-4J/ho-4J mice. Application of the mGluR1 antagonist CPCCOEt blocks sEPSCs. Inset shows corresponding fEPSCs, which are blocked by CNQX. B, GluRδ2ho-4J/ho-4J shows a similar fEPSC input–output relationship as wild type. C, Stimulation intensity for fEPSCs and sEPSCs was set to evoke similar fEPSC amplitudes (∼500 pA). D, The decay constant of fEPSCs was not affected by GluRδ2 mutation. E, The amplitude of sEPSCs was not changed significantly by GluRδ2 mutation. F, The integrated area of the CPCCOEt-sensitive sEPSC was not affected by GluRδ2 mutation. G, The onset of the sEPSC was significantly slowed in GluRδ2ho-4J/ho-4J. The average duration between the stimulus and the peak of the sEPSC was calculated. H, The average FWHM for the CPCCOEt-sensitive sEPSC was calculated. I, FWHM of sEPSC was normalized by the decay constant of fEPSC. The kinetics of sEPSCs was specifically slowed in GluRδ2ho-4J/ho-4J mice. Error bars indicate SEM. C–F, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 11). G–I, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 7). The waveforms of sEPSC in 4 of 11 samples recorded from GluRδ2ho-4J/ho-4J Purkinje cells were too small to evaluate, so we omitted these samples from G–I. J, K, NMDA receptors are not involved in the slower synaptic responses at PF–PC synapses in GluRδ2ho-4J/ho-4J. J, Typical sEPSC traces from wild-type and GluRδ2ho-4J/ho-4J in the presence or absence of 100 μm AP-5. All responses were evoked by five pulses at 100 Hz in the presence of 20 μm bicuculline and 40 μm CNQX. K, Quantified AP-5-sensitive charge transfer. Statistical significance with respect to GluRδ2+/+ (t test). **p < 0.01, ***p < 0.001.
    Figure Legend Snippet: GluRδ2ho-4J/ho-4J alters the time course of the mGluR1-dependent sEPSC at PF–PC synapses. A, Typical traces of sEPSCs recorded from wild-type or GluRδ2ho-4J/ho-4J mice. Application of the mGluR1 antagonist CPCCOEt blocks sEPSCs. Inset shows corresponding fEPSCs, which are blocked by CNQX. B, GluRδ2ho-4J/ho-4J shows a similar fEPSC input–output relationship as wild type. C, Stimulation intensity for fEPSCs and sEPSCs was set to evoke similar fEPSC amplitudes (∼500 pA). D, The decay constant of fEPSCs was not affected by GluRδ2 mutation. E, The amplitude of sEPSCs was not changed significantly by GluRδ2 mutation. F, The integrated area of the CPCCOEt-sensitive sEPSC was not affected by GluRδ2 mutation. G, The onset of the sEPSC was significantly slowed in GluRδ2ho-4J/ho-4J. The average duration between the stimulus and the peak of the sEPSC was calculated. H, The average FWHM for the CPCCOEt-sensitive sEPSC was calculated. I, FWHM of sEPSC was normalized by the decay constant of fEPSC. The kinetics of sEPSCs was specifically slowed in GluRδ2ho-4J/ho-4J mice. Error bars indicate SEM. C–F, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 11). G–I, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 7). The waveforms of sEPSC in 4 of 11 samples recorded from GluRδ2ho-4J/ho-4J Purkinje cells were too small to evaluate, so we omitted these samples from G–I. J, K, NMDA receptors are not involved in the slower synaptic responses at PF–PC synapses in GluRδ2ho-4J/ho-4J. J, Typical sEPSC traces from wild-type and GluRδ2ho-4J/ho-4J in the presence or absence of 100 μm AP-5. All responses were evoked by five pulses at 100 Hz in the presence of 20 μm bicuculline and 40 μm CNQX. K, Quantified AP-5-sensitive charge transfer. Statistical significance with respect to GluRδ2+/+ (t test). **p < 0.01, ***p < 0.001.

    Techniques Used: Mutagenesis

    rabbit anti mglur1a b  (Alomone Labs)


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  • 93

    Structured Review

    Alomone Labs rabbit anti mglur1a b
    mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with <t>mGluR1a</t> or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.
    Rabbit Anti Mglur1a B, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti mglur1a b/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti mglur1a b - by Bioz Stars, 2024-07
    93/100 stars

    Images

    1) Product Images from "Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons"

    Article Title: Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0705-12.2012

    mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with mGluR1a or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.
    Figure Legend Snippet: mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with mGluR1a or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.

    Techniques Used: Silver Staining, Mass Spectrometry

    TRPC3 associates with mGluR–GluRδ2–PKCγ in cerebellum. Triton X-100-solubilized postnuclear membrane fractions from either wild-type or mutant mice were immunoprecipitated and blotted as indicated. A, mGluR1 coimmunoprecipitated with TRPC3 from either wild-type (A1) or GluRδ2 (A2) mutant cerebella. B1, GluRδ2 coimmunoprecipitated with TRPC3. B2, In ho-4J mice, a truncated GluRδ2 is expressed at very low levels. C, PKCγ associates with mGluR1 or TRPC3 in the presence (C1) or absence (C2) of GluRδ2. D, TRPC3 and GluRδ2 interact in the presence (D1) or absence (D2) of mGluR1. E, PKCγ associates with GluRδ2 and TRPC3, which is not affected by the presence or absence of mGluR1. F, GluK2/3 did not interact with mGluR1, GluRδ2, or TRPC3.
    Figure Legend Snippet: TRPC3 associates with mGluR–GluRδ2–PKCγ in cerebellum. Triton X-100-solubilized postnuclear membrane fractions from either wild-type or mutant mice were immunoprecipitated and blotted as indicated. A, mGluR1 coimmunoprecipitated with TRPC3 from either wild-type (A1) or GluRδ2 (A2) mutant cerebella. B1, GluRδ2 coimmunoprecipitated with TRPC3. B2, In ho-4J mice, a truncated GluRδ2 is expressed at very low levels. C, PKCγ associates with mGluR1 or TRPC3 in the presence (C1) or absence (C2) of GluRδ2. D, TRPC3 and GluRδ2 interact in the presence (D1) or absence (D2) of mGluR1. E, PKCγ associates with GluRδ2 and TRPC3, which is not affected by the presence or absence of mGluR1. F, GluK2/3 did not interact with mGluR1, GluRδ2, or TRPC3.

    Techniques Used: Mutagenesis, Immunoprecipitation

    Neither GluRδ2 nor mGluR1 mutation affects cerebellar levels of interacting proteins. A, GluRδ2 mutation results in increased levels of GluA2 but does not affect levels of mGluR1, TRPC3, or other synaptic proteins. B, Mutation of GluR1 does not affect levels of any of the proteins analyzed. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ or mGluR1+/+ (t test). n = 4 for each sample. *p < 0.05.
    Figure Legend Snippet: Neither GluRδ2 nor mGluR1 mutation affects cerebellar levels of interacting proteins. A, GluRδ2 mutation results in increased levels of GluA2 but does not affect levels of mGluR1, TRPC3, or other synaptic proteins. B, Mutation of GluR1 does not affect levels of any of the proteins analyzed. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ or mGluR1+/+ (t test). n = 4 for each sample. *p < 0.05.

    Techniques Used: Mutagenesis

    GluRδ2 or mGluR1 mutation does not affect immunofluorescent distribution of the interacting proteins. A–C, Immunofluorescent double labeling of sagittal cerebellar sections; the bottom panels are magnified images of the molecular layer. Specificity of staining is confirmed by elimination of immunosignal in corresponding mutant mice. A, Punctate GluRδ2 staining partially colocalizes with mGluR1 in molecular layer of wild type. The molecular layer staining patterns for GluRδ2 and mGluR1 were not dramatically altered in the mGluR1-KO or GluRδ2ho-4J/ho-4J, respectively. B, PKCγ shows strong labeling of Purkinje cell bodies, dendrites, and neuropil. The colabeling of mGluR1 and PKCγ in the molecular layer was not dramatically altered in the GluRδ2ho-4J/ho-4J, and the PKCγ distribution was not changed in the mGlu1-KO. C, mGluR1 and TRPC3 partially colocalize in wild-type mouse. No obvious difference in the staining patterns was detected in either GluRδ2ho-4J/ho-4J or mGluR1-KO. ML, Molecular layer; PC, Purkinje cell.
    Figure Legend Snippet: GluRδ2 or mGluR1 mutation does not affect immunofluorescent distribution of the interacting proteins. A–C, Immunofluorescent double labeling of sagittal cerebellar sections; the bottom panels are magnified images of the molecular layer. Specificity of staining is confirmed by elimination of immunosignal in corresponding mutant mice. A, Punctate GluRδ2 staining partially colocalizes with mGluR1 in molecular layer of wild type. The molecular layer staining patterns for GluRδ2 and mGluR1 were not dramatically altered in the mGluR1-KO or GluRδ2ho-4J/ho-4J, respectively. B, PKCγ shows strong labeling of Purkinje cell bodies, dendrites, and neuropil. The colabeling of mGluR1 and PKCγ in the molecular layer was not dramatically altered in the GluRδ2ho-4J/ho-4J, and the PKCγ distribution was not changed in the mGlu1-KO. C, mGluR1 and TRPC3 partially colocalize in wild-type mouse. No obvious difference in the staining patterns was detected in either GluRδ2ho-4J/ho-4J or mGluR1-KO. ML, Molecular layer; PC, Purkinje cell.

    Techniques Used: Mutagenesis, Labeling, Staining

    In GluRδ2ho-4J/ho-4J, TRPC3 partially redistributes to the Triton X-100-soluble fraction. A, Synaptosomal (Syp) and PSD fractions of mouse cerebella from wild-type, GluRδ2ho-4J/ho-4J, and mGluR1-KO were prepared and immunoblotted. mGluR1, GluRδ2, PKCγ, and TRPC3 are detected in the synaptosomal and PSD fractions. The blotting profiles of PSD-95 and synaptophysin validate the subcellular fractionations. B, Cerebellar homogenates were treated with 2.5% Triton X-100, followed by ultracentrifugation to yield supernatant (Sup) and pellet (Ppt). C, A greater percentage of TRPC3 was detected in Triton X-100-soluble fraction in GluRδ2ho-4J/ho-4J than in wild type (GluRδ2+/+). Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for each sample. *p < 0.05.
    Figure Legend Snippet: In GluRδ2ho-4J/ho-4J, TRPC3 partially redistributes to the Triton X-100-soluble fraction. A, Synaptosomal (Syp) and PSD fractions of mouse cerebella from wild-type, GluRδ2ho-4J/ho-4J, and mGluR1-KO were prepared and immunoblotted. mGluR1, GluRδ2, PKCγ, and TRPC3 are detected in the synaptosomal and PSD fractions. The blotting profiles of PSD-95 and synaptophysin validate the subcellular fractionations. B, Cerebellar homogenates were treated with 2.5% Triton X-100, followed by ultracentrifugation to yield supernatant (Sup) and pellet (Ppt). C, A greater percentage of TRPC3 was detected in Triton X-100-soluble fraction in GluRδ2ho-4J/ho-4J than in wild type (GluRδ2+/+). Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for each sample. *p < 0.05.

    Techniques Used:

    Effects of mGluR1 and GluRδ2 mutation on surface expression of interacting proteins. Mouse cerebellar slices were treated with a membrane-impermeable biotinylation reagent to mark cell-surface proteins. Total, internal, and surface proteins were resolved by SDS-PAGE and subjected to immunoblotting. GluRδ2 mutation increased surface expression of TRPC3 and mGluR1, whereas other components were unchanged. mGluR1b is the shorter splice variant of mGluR1. β3-Tubulin and/or synaptophysin served as internal protein controls. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for mGluR1, mGluR1b, TRPC3, and GluK2/3. n = 3 for GluA2, synaptophysin, and β3-tubulin. *p < 0.05.
    Figure Legend Snippet: Effects of mGluR1 and GluRδ2 mutation on surface expression of interacting proteins. Mouse cerebellar slices were treated with a membrane-impermeable biotinylation reagent to mark cell-surface proteins. Total, internal, and surface proteins were resolved by SDS-PAGE and subjected to immunoblotting. GluRδ2 mutation increased surface expression of TRPC3 and mGluR1, whereas other components were unchanged. mGluR1b is the shorter splice variant of mGluR1. β3-Tubulin and/or synaptophysin served as internal protein controls. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for mGluR1, mGluR1b, TRPC3, and GluK2/3. n = 3 for GluA2, synaptophysin, and β3-tubulin. *p < 0.05.

    Techniques Used: Mutagenesis, Expressing, SDS Page, Western Blot, Variant Assay

    GluRδ2ho-4J/ho-4J alters the time course of the mGluR1-dependent sEPSC at PF–PC synapses. A, Typical traces of sEPSCs recorded from wild-type or GluRδ2ho-4J/ho-4J mice. Application of the mGluR1 antagonist CPCCOEt blocks sEPSCs. Inset shows corresponding fEPSCs, which are blocked by CNQX. B, GluRδ2ho-4J/ho-4J shows a similar fEPSC input–output relationship as wild type. C, Stimulation intensity for fEPSCs and sEPSCs was set to evoke similar fEPSC amplitudes (∼500 pA). D, The decay constant of fEPSCs was not affected by GluRδ2 mutation. E, The amplitude of sEPSCs was not changed significantly by GluRδ2 mutation. F, The integrated area of the CPCCOEt-sensitive sEPSC was not affected by GluRδ2 mutation. G, The onset of the sEPSC was significantly slowed in GluRδ2ho-4J/ho-4J. The average duration between the stimulus and the peak of the sEPSC was calculated. H, The average FWHM for the CPCCOEt-sensitive sEPSC was calculated. I, FWHM of sEPSC was normalized by the decay constant of fEPSC. The kinetics of sEPSCs was specifically slowed in GluRδ2ho-4J/ho-4J mice. Error bars indicate SEM. C–F, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 11). G–I, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 7). The waveforms of sEPSC in 4 of 11 samples recorded from GluRδ2ho-4J/ho-4J Purkinje cells were too small to evaluate, so we omitted these samples from G–I. J, K, NMDA receptors are not involved in the slower synaptic responses at PF–PC synapses in GluRδ2ho-4J/ho-4J. J, Typical sEPSC traces from wild-type and GluRδ2ho-4J/ho-4J in the presence or absence of 100 μm AP-5. All responses were evoked by five pulses at 100 Hz in the presence of 20 μm bicuculline and 40 μm CNQX. K, Quantified AP-5-sensitive charge transfer. Statistical significance with respect to GluRδ2+/+ (t test). **p < 0.01, ***p < 0.001.
    Figure Legend Snippet: GluRδ2ho-4J/ho-4J alters the time course of the mGluR1-dependent sEPSC at PF–PC synapses. A, Typical traces of sEPSCs recorded from wild-type or GluRδ2ho-4J/ho-4J mice. Application of the mGluR1 antagonist CPCCOEt blocks sEPSCs. Inset shows corresponding fEPSCs, which are blocked by CNQX. B, GluRδ2ho-4J/ho-4J shows a similar fEPSC input–output relationship as wild type. C, Stimulation intensity for fEPSCs and sEPSCs was set to evoke similar fEPSC amplitudes (∼500 pA). D, The decay constant of fEPSCs was not affected by GluRδ2 mutation. E, The amplitude of sEPSCs was not changed significantly by GluRδ2 mutation. F, The integrated area of the CPCCOEt-sensitive sEPSC was not affected by GluRδ2 mutation. G, The onset of the sEPSC was significantly slowed in GluRδ2ho-4J/ho-4J. The average duration between the stimulus and the peak of the sEPSC was calculated. H, The average FWHM for the CPCCOEt-sensitive sEPSC was calculated. I, FWHM of sEPSC was normalized by the decay constant of fEPSC. The kinetics of sEPSCs was specifically slowed in GluRδ2ho-4J/ho-4J mice. Error bars indicate SEM. C–F, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 11). G–I, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 7). The waveforms of sEPSC in 4 of 11 samples recorded from GluRδ2ho-4J/ho-4J Purkinje cells were too small to evaluate, so we omitted these samples from G–I. J, K, NMDA receptors are not involved in the slower synaptic responses at PF–PC synapses in GluRδ2ho-4J/ho-4J. J, Typical sEPSC traces from wild-type and GluRδ2ho-4J/ho-4J in the presence or absence of 100 μm AP-5. All responses were evoked by five pulses at 100 Hz in the presence of 20 μm bicuculline and 40 μm CNQX. K, Quantified AP-5-sensitive charge transfer. Statistical significance with respect to GluRδ2+/+ (t test). **p < 0.01, ***p < 0.001.

    Techniques Used: Mutagenesis

    rabbit anti mglur1a b  (Alomone Labs)


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    Alomone Labs rabbit anti mglur1a b
    mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with <t>mGluR1a</t> or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.
    Rabbit Anti Mglur1a B, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons"

    Article Title: Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0705-12.2012

    mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with mGluR1a or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.
    Figure Legend Snippet: mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with mGluR1a or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.

    Techniques Used: Silver Staining, Mass Spectrometry

    TRPC3 associates with mGluR–GluRδ2–PKCγ in cerebellum. Triton X-100-solubilized postnuclear membrane fractions from either wild-type or mutant mice were immunoprecipitated and blotted as indicated. A, mGluR1 coimmunoprecipitated with TRPC3 from either wild-type (A1) or GluRδ2 (A2) mutant cerebella. B1, GluRδ2 coimmunoprecipitated with TRPC3. B2, In ho-4J mice, a truncated GluRδ2 is expressed at very low levels. C, PKCγ associates with mGluR1 or TRPC3 in the presence (C1) or absence (C2) of GluRδ2. D, TRPC3 and GluRδ2 interact in the presence (D1) or absence (D2) of mGluR1. E, PKCγ associates with GluRδ2 and TRPC3, which is not affected by the presence or absence of mGluR1. F, GluK2/3 did not interact with mGluR1, GluRδ2, or TRPC3.
    Figure Legend Snippet: TRPC3 associates with mGluR–GluRδ2–PKCγ in cerebellum. Triton X-100-solubilized postnuclear membrane fractions from either wild-type or mutant mice were immunoprecipitated and blotted as indicated. A, mGluR1 coimmunoprecipitated with TRPC3 from either wild-type (A1) or GluRδ2 (A2) mutant cerebella. B1, GluRδ2 coimmunoprecipitated with TRPC3. B2, In ho-4J mice, a truncated GluRδ2 is expressed at very low levels. C, PKCγ associates with mGluR1 or TRPC3 in the presence (C1) or absence (C2) of GluRδ2. D, TRPC3 and GluRδ2 interact in the presence (D1) or absence (D2) of mGluR1. E, PKCγ associates with GluRδ2 and TRPC3, which is not affected by the presence or absence of mGluR1. F, GluK2/3 did not interact with mGluR1, GluRδ2, or TRPC3.

    Techniques Used: Mutagenesis, Immunoprecipitation

    Neither GluRδ2 nor mGluR1 mutation affects cerebellar levels of interacting proteins. A, GluRδ2 mutation results in increased levels of GluA2 but does not affect levels of mGluR1, TRPC3, or other synaptic proteins. B, Mutation of GluR1 does not affect levels of any of the proteins analyzed. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ or mGluR1+/+ (t test). n = 4 for each sample. *p < 0.05.
    Figure Legend Snippet: Neither GluRδ2 nor mGluR1 mutation affects cerebellar levels of interacting proteins. A, GluRδ2 mutation results in increased levels of GluA2 but does not affect levels of mGluR1, TRPC3, or other synaptic proteins. B, Mutation of GluR1 does not affect levels of any of the proteins analyzed. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ or mGluR1+/+ (t test). n = 4 for each sample. *p < 0.05.

    Techniques Used: Mutagenesis

    GluRδ2 or mGluR1 mutation does not affect immunofluorescent distribution of the interacting proteins. A–C, Immunofluorescent double labeling of sagittal cerebellar sections; the bottom panels are magnified images of the molecular layer. Specificity of staining is confirmed by elimination of immunosignal in corresponding mutant mice. A, Punctate GluRδ2 staining partially colocalizes with mGluR1 in molecular layer of wild type. The molecular layer staining patterns for GluRδ2 and mGluR1 were not dramatically altered in the mGluR1-KO or GluRδ2ho-4J/ho-4J, respectively. B, PKCγ shows strong labeling of Purkinje cell bodies, dendrites, and neuropil. The colabeling of mGluR1 and PKCγ in the molecular layer was not dramatically altered in the GluRδ2ho-4J/ho-4J, and the PKCγ distribution was not changed in the mGlu1-KO. C, mGluR1 and TRPC3 partially colocalize in wild-type mouse. No obvious difference in the staining patterns was detected in either GluRδ2ho-4J/ho-4J or mGluR1-KO. ML, Molecular layer; PC, Purkinje cell.
    Figure Legend Snippet: GluRδ2 or mGluR1 mutation does not affect immunofluorescent distribution of the interacting proteins. A–C, Immunofluorescent double labeling of sagittal cerebellar sections; the bottom panels are magnified images of the molecular layer. Specificity of staining is confirmed by elimination of immunosignal in corresponding mutant mice. A, Punctate GluRδ2 staining partially colocalizes with mGluR1 in molecular layer of wild type. The molecular layer staining patterns for GluRδ2 and mGluR1 were not dramatically altered in the mGluR1-KO or GluRδ2ho-4J/ho-4J, respectively. B, PKCγ shows strong labeling of Purkinje cell bodies, dendrites, and neuropil. The colabeling of mGluR1 and PKCγ in the molecular layer was not dramatically altered in the GluRδ2ho-4J/ho-4J, and the PKCγ distribution was not changed in the mGlu1-KO. C, mGluR1 and TRPC3 partially colocalize in wild-type mouse. No obvious difference in the staining patterns was detected in either GluRδ2ho-4J/ho-4J or mGluR1-KO. ML, Molecular layer; PC, Purkinje cell.

    Techniques Used: Mutagenesis, Labeling, Staining

    In GluRδ2ho-4J/ho-4J, TRPC3 partially redistributes to the Triton X-100-soluble fraction. A, Synaptosomal (Syp) and PSD fractions of mouse cerebella from wild-type, GluRδ2ho-4J/ho-4J, and mGluR1-KO were prepared and immunoblotted. mGluR1, GluRδ2, PKCγ, and TRPC3 are detected in the synaptosomal and PSD fractions. The blotting profiles of PSD-95 and synaptophysin validate the subcellular fractionations. B, Cerebellar homogenates were treated with 2.5% Triton X-100, followed by ultracentrifugation to yield supernatant (Sup) and pellet (Ppt). C, A greater percentage of TRPC3 was detected in Triton X-100-soluble fraction in GluRδ2ho-4J/ho-4J than in wild type (GluRδ2+/+). Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for each sample. *p < 0.05.
    Figure Legend Snippet: In GluRδ2ho-4J/ho-4J, TRPC3 partially redistributes to the Triton X-100-soluble fraction. A, Synaptosomal (Syp) and PSD fractions of mouse cerebella from wild-type, GluRδ2ho-4J/ho-4J, and mGluR1-KO were prepared and immunoblotted. mGluR1, GluRδ2, PKCγ, and TRPC3 are detected in the synaptosomal and PSD fractions. The blotting profiles of PSD-95 and synaptophysin validate the subcellular fractionations. B, Cerebellar homogenates were treated with 2.5% Triton X-100, followed by ultracentrifugation to yield supernatant (Sup) and pellet (Ppt). C, A greater percentage of TRPC3 was detected in Triton X-100-soluble fraction in GluRδ2ho-4J/ho-4J than in wild type (GluRδ2+/+). Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for each sample. *p < 0.05.

    Techniques Used:

    Effects of mGluR1 and GluRδ2 mutation on surface expression of interacting proteins. Mouse cerebellar slices were treated with a membrane-impermeable biotinylation reagent to mark cell-surface proteins. Total, internal, and surface proteins were resolved by SDS-PAGE and subjected to immunoblotting. GluRδ2 mutation increased surface expression of TRPC3 and mGluR1, whereas other components were unchanged. mGluR1b is the shorter splice variant of mGluR1. β3-Tubulin and/or synaptophysin served as internal protein controls. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for mGluR1, mGluR1b, TRPC3, and GluK2/3. n = 3 for GluA2, synaptophysin, and β3-tubulin. *p < 0.05.
    Figure Legend Snippet: Effects of mGluR1 and GluRδ2 mutation on surface expression of interacting proteins. Mouse cerebellar slices were treated with a membrane-impermeable biotinylation reagent to mark cell-surface proteins. Total, internal, and surface proteins were resolved by SDS-PAGE and subjected to immunoblotting. GluRδ2 mutation increased surface expression of TRPC3 and mGluR1, whereas other components were unchanged. mGluR1b is the shorter splice variant of mGluR1. β3-Tubulin and/or synaptophysin served as internal protein controls. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for mGluR1, mGluR1b, TRPC3, and GluK2/3. n = 3 for GluA2, synaptophysin, and β3-tubulin. *p < 0.05.

    Techniques Used: Mutagenesis, Expressing, SDS Page, Western Blot, Variant Assay

    GluRδ2ho-4J/ho-4J alters the time course of the mGluR1-dependent sEPSC at PF–PC synapses. A, Typical traces of sEPSCs recorded from wild-type or GluRδ2ho-4J/ho-4J mice. Application of the mGluR1 antagonist CPCCOEt blocks sEPSCs. Inset shows corresponding fEPSCs, which are blocked by CNQX. B, GluRδ2ho-4J/ho-4J shows a similar fEPSC input–output relationship as wild type. C, Stimulation intensity for fEPSCs and sEPSCs was set to evoke similar fEPSC amplitudes (∼500 pA). D, The decay constant of fEPSCs was not affected by GluRδ2 mutation. E, The amplitude of sEPSCs was not changed significantly by GluRδ2 mutation. F, The integrated area of the CPCCOEt-sensitive sEPSC was not affected by GluRδ2 mutation. G, The onset of the sEPSC was significantly slowed in GluRδ2ho-4J/ho-4J. The average duration between the stimulus and the peak of the sEPSC was calculated. H, The average FWHM for the CPCCOEt-sensitive sEPSC was calculated. I, FWHM of sEPSC was normalized by the decay constant of fEPSC. The kinetics of sEPSCs was specifically slowed in GluRδ2ho-4J/ho-4J mice. Error bars indicate SEM. C–F, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 11). G–I, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 7). The waveforms of sEPSC in 4 of 11 samples recorded from GluRδ2ho-4J/ho-4J Purkinje cells were too small to evaluate, so we omitted these samples from G–I. J, K, NMDA receptors are not involved in the slower synaptic responses at PF–PC synapses in GluRδ2ho-4J/ho-4J. J, Typical sEPSC traces from wild-type and GluRδ2ho-4J/ho-4J in the presence or absence of 100 μm AP-5. All responses were evoked by five pulses at 100 Hz in the presence of 20 μm bicuculline and 40 μm CNQX. K, Quantified AP-5-sensitive charge transfer. Statistical significance with respect to GluRδ2+/+ (t test). **p < 0.01, ***p < 0.001.
    Figure Legend Snippet: GluRδ2ho-4J/ho-4J alters the time course of the mGluR1-dependent sEPSC at PF–PC synapses. A, Typical traces of sEPSCs recorded from wild-type or GluRδ2ho-4J/ho-4J mice. Application of the mGluR1 antagonist CPCCOEt blocks sEPSCs. Inset shows corresponding fEPSCs, which are blocked by CNQX. B, GluRδ2ho-4J/ho-4J shows a similar fEPSC input–output relationship as wild type. C, Stimulation intensity for fEPSCs and sEPSCs was set to evoke similar fEPSC amplitudes (∼500 pA). D, The decay constant of fEPSCs was not affected by GluRδ2 mutation. E, The amplitude of sEPSCs was not changed significantly by GluRδ2 mutation. F, The integrated area of the CPCCOEt-sensitive sEPSC was not affected by GluRδ2 mutation. G, The onset of the sEPSC was significantly slowed in GluRδ2ho-4J/ho-4J. The average duration between the stimulus and the peak of the sEPSC was calculated. H, The average FWHM for the CPCCOEt-sensitive sEPSC was calculated. I, FWHM of sEPSC was normalized by the decay constant of fEPSC. The kinetics of sEPSCs was specifically slowed in GluRδ2ho-4J/ho-4J mice. Error bars indicate SEM. C–F, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 11). G–I, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 7). The waveforms of sEPSC in 4 of 11 samples recorded from GluRδ2ho-4J/ho-4J Purkinje cells were too small to evaluate, so we omitted these samples from G–I. J, K, NMDA receptors are not involved in the slower synaptic responses at PF–PC synapses in GluRδ2ho-4J/ho-4J. J, Typical sEPSC traces from wild-type and GluRδ2ho-4J/ho-4J in the presence or absence of 100 μm AP-5. All responses were evoked by five pulses at 100 Hz in the presence of 20 μm bicuculline and 40 μm CNQX. K, Quantified AP-5-sensitive charge transfer. Statistical significance with respect to GluRδ2+/+ (t test). **p < 0.01, ***p < 0.001.

    Techniques Used: Mutagenesis

    rabbit anti mglur1a b  (Alomone Labs)


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    Alomone Labs rabbit anti mglur1a b
    mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with <t>mGluR1a</t> or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.
    Rabbit Anti Mglur1a B, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti mglur1a b/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti mglur1a b - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons"

    Article Title: Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0705-12.2012

    mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with mGluR1a or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.
    Figure Legend Snippet: mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with mGluR1a or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.

    Techniques Used: Silver Staining, Mass Spectrometry

    TRPC3 associates with mGluR–GluRδ2–PKCγ in cerebellum. Triton X-100-solubilized postnuclear membrane fractions from either wild-type or mutant mice were immunoprecipitated and blotted as indicated. A, mGluR1 coimmunoprecipitated with TRPC3 from either wild-type (A1) or GluRδ2 (A2) mutant cerebella. B1, GluRδ2 coimmunoprecipitated with TRPC3. B2, In ho-4J mice, a truncated GluRδ2 is expressed at very low levels. C, PKCγ associates with mGluR1 or TRPC3 in the presence (C1) or absence (C2) of GluRδ2. D, TRPC3 and GluRδ2 interact in the presence (D1) or absence (D2) of mGluR1. E, PKCγ associates with GluRδ2 and TRPC3, which is not affected by the presence or absence of mGluR1. F, GluK2/3 did not interact with mGluR1, GluRδ2, or TRPC3.
    Figure Legend Snippet: TRPC3 associates with mGluR–GluRδ2–PKCγ in cerebellum. Triton X-100-solubilized postnuclear membrane fractions from either wild-type or mutant mice were immunoprecipitated and blotted as indicated. A, mGluR1 coimmunoprecipitated with TRPC3 from either wild-type (A1) or GluRδ2 (A2) mutant cerebella. B1, GluRδ2 coimmunoprecipitated with TRPC3. B2, In ho-4J mice, a truncated GluRδ2 is expressed at very low levels. C, PKCγ associates with mGluR1 or TRPC3 in the presence (C1) or absence (C2) of GluRδ2. D, TRPC3 and GluRδ2 interact in the presence (D1) or absence (D2) of mGluR1. E, PKCγ associates with GluRδ2 and TRPC3, which is not affected by the presence or absence of mGluR1. F, GluK2/3 did not interact with mGluR1, GluRδ2, or TRPC3.

    Techniques Used: Mutagenesis, Immunoprecipitation

    Neither GluRδ2 nor mGluR1 mutation affects cerebellar levels of interacting proteins. A, GluRδ2 mutation results in increased levels of GluA2 but does not affect levels of mGluR1, TRPC3, or other synaptic proteins. B, Mutation of GluR1 does not affect levels of any of the proteins analyzed. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ or mGluR1+/+ (t test). n = 4 for each sample. *p < 0.05.
    Figure Legend Snippet: Neither GluRδ2 nor mGluR1 mutation affects cerebellar levels of interacting proteins. A, GluRδ2 mutation results in increased levels of GluA2 but does not affect levels of mGluR1, TRPC3, or other synaptic proteins. B, Mutation of GluR1 does not affect levels of any of the proteins analyzed. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ or mGluR1+/+ (t test). n = 4 for each sample. *p < 0.05.

    Techniques Used: Mutagenesis

    GluRδ2 or mGluR1 mutation does not affect immunofluorescent distribution of the interacting proteins. A–C, Immunofluorescent double labeling of sagittal cerebellar sections; the bottom panels are magnified images of the molecular layer. Specificity of staining is confirmed by elimination of immunosignal in corresponding mutant mice. A, Punctate GluRδ2 staining partially colocalizes with mGluR1 in molecular layer of wild type. The molecular layer staining patterns for GluRδ2 and mGluR1 were not dramatically altered in the mGluR1-KO or GluRδ2ho-4J/ho-4J, respectively. B, PKCγ shows strong labeling of Purkinje cell bodies, dendrites, and neuropil. The colabeling of mGluR1 and PKCγ in the molecular layer was not dramatically altered in the GluRδ2ho-4J/ho-4J, and the PKCγ distribution was not changed in the mGlu1-KO. C, mGluR1 and TRPC3 partially colocalize in wild-type mouse. No obvious difference in the staining patterns was detected in either GluRδ2ho-4J/ho-4J or mGluR1-KO. ML, Molecular layer; PC, Purkinje cell.
    Figure Legend Snippet: GluRδ2 or mGluR1 mutation does not affect immunofluorescent distribution of the interacting proteins. A–C, Immunofluorescent double labeling of sagittal cerebellar sections; the bottom panels are magnified images of the molecular layer. Specificity of staining is confirmed by elimination of immunosignal in corresponding mutant mice. A, Punctate GluRδ2 staining partially colocalizes with mGluR1 in molecular layer of wild type. The molecular layer staining patterns for GluRδ2 and mGluR1 were not dramatically altered in the mGluR1-KO or GluRδ2ho-4J/ho-4J, respectively. B, PKCγ shows strong labeling of Purkinje cell bodies, dendrites, and neuropil. The colabeling of mGluR1 and PKCγ in the molecular layer was not dramatically altered in the GluRδ2ho-4J/ho-4J, and the PKCγ distribution was not changed in the mGlu1-KO. C, mGluR1 and TRPC3 partially colocalize in wild-type mouse. No obvious difference in the staining patterns was detected in either GluRδ2ho-4J/ho-4J or mGluR1-KO. ML, Molecular layer; PC, Purkinje cell.

    Techniques Used: Mutagenesis, Labeling, Staining

    In GluRδ2ho-4J/ho-4J, TRPC3 partially redistributes to the Triton X-100-soluble fraction. A, Synaptosomal (Syp) and PSD fractions of mouse cerebella from wild-type, GluRδ2ho-4J/ho-4J, and mGluR1-KO were prepared and immunoblotted. mGluR1, GluRδ2, PKCγ, and TRPC3 are detected in the synaptosomal and PSD fractions. The blotting profiles of PSD-95 and synaptophysin validate the subcellular fractionations. B, Cerebellar homogenates were treated with 2.5% Triton X-100, followed by ultracentrifugation to yield supernatant (Sup) and pellet (Ppt). C, A greater percentage of TRPC3 was detected in Triton X-100-soluble fraction in GluRδ2ho-4J/ho-4J than in wild type (GluRδ2+/+). Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for each sample. *p < 0.05.
    Figure Legend Snippet: In GluRδ2ho-4J/ho-4J, TRPC3 partially redistributes to the Triton X-100-soluble fraction. A, Synaptosomal (Syp) and PSD fractions of mouse cerebella from wild-type, GluRδ2ho-4J/ho-4J, and mGluR1-KO were prepared and immunoblotted. mGluR1, GluRδ2, PKCγ, and TRPC3 are detected in the synaptosomal and PSD fractions. The blotting profiles of PSD-95 and synaptophysin validate the subcellular fractionations. B, Cerebellar homogenates were treated with 2.5% Triton X-100, followed by ultracentrifugation to yield supernatant (Sup) and pellet (Ppt). C, A greater percentage of TRPC3 was detected in Triton X-100-soluble fraction in GluRδ2ho-4J/ho-4J than in wild type (GluRδ2+/+). Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for each sample. *p < 0.05.

    Techniques Used:

    Effects of mGluR1 and GluRδ2 mutation on surface expression of interacting proteins. Mouse cerebellar slices were treated with a membrane-impermeable biotinylation reagent to mark cell-surface proteins. Total, internal, and surface proteins were resolved by SDS-PAGE and subjected to immunoblotting. GluRδ2 mutation increased surface expression of TRPC3 and mGluR1, whereas other components were unchanged. mGluR1b is the shorter splice variant of mGluR1. β3-Tubulin and/or synaptophysin served as internal protein controls. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for mGluR1, mGluR1b, TRPC3, and GluK2/3. n = 3 for GluA2, synaptophysin, and β3-tubulin. *p < 0.05.
    Figure Legend Snippet: Effects of mGluR1 and GluRδ2 mutation on surface expression of interacting proteins. Mouse cerebellar slices were treated with a membrane-impermeable biotinylation reagent to mark cell-surface proteins. Total, internal, and surface proteins were resolved by SDS-PAGE and subjected to immunoblotting. GluRδ2 mutation increased surface expression of TRPC3 and mGluR1, whereas other components were unchanged. mGluR1b is the shorter splice variant of mGluR1. β3-Tubulin and/or synaptophysin served as internal protein controls. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for mGluR1, mGluR1b, TRPC3, and GluK2/3. n = 3 for GluA2, synaptophysin, and β3-tubulin. *p < 0.05.

    Techniques Used: Mutagenesis, Expressing, SDS Page, Western Blot, Variant Assay

    GluRδ2ho-4J/ho-4J alters the time course of the mGluR1-dependent sEPSC at PF–PC synapses. A, Typical traces of sEPSCs recorded from wild-type or GluRδ2ho-4J/ho-4J mice. Application of the mGluR1 antagonist CPCCOEt blocks sEPSCs. Inset shows corresponding fEPSCs, which are blocked by CNQX. B, GluRδ2ho-4J/ho-4J shows a similar fEPSC input–output relationship as wild type. C, Stimulation intensity for fEPSCs and sEPSCs was set to evoke similar fEPSC amplitudes (∼500 pA). D, The decay constant of fEPSCs was not affected by GluRδ2 mutation. E, The amplitude of sEPSCs was not changed significantly by GluRδ2 mutation. F, The integrated area of the CPCCOEt-sensitive sEPSC was not affected by GluRδ2 mutation. G, The onset of the sEPSC was significantly slowed in GluRδ2ho-4J/ho-4J. The average duration between the stimulus and the peak of the sEPSC was calculated. H, The average FWHM for the CPCCOEt-sensitive sEPSC was calculated. I, FWHM of sEPSC was normalized by the decay constant of fEPSC. The kinetics of sEPSCs was specifically slowed in GluRδ2ho-4J/ho-4J mice. Error bars indicate SEM. C–F, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 11). G–I, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 7). The waveforms of sEPSC in 4 of 11 samples recorded from GluRδ2ho-4J/ho-4J Purkinje cells were too small to evaluate, so we omitted these samples from G–I. J, K, NMDA receptors are not involved in the slower synaptic responses at PF–PC synapses in GluRδ2ho-4J/ho-4J. J, Typical sEPSC traces from wild-type and GluRδ2ho-4J/ho-4J in the presence or absence of 100 μm AP-5. All responses were evoked by five pulses at 100 Hz in the presence of 20 μm bicuculline and 40 μm CNQX. K, Quantified AP-5-sensitive charge transfer. Statistical significance with respect to GluRδ2+/+ (t test). **p < 0.01, ***p < 0.001.
    Figure Legend Snippet: GluRδ2ho-4J/ho-4J alters the time course of the mGluR1-dependent sEPSC at PF–PC synapses. A, Typical traces of sEPSCs recorded from wild-type or GluRδ2ho-4J/ho-4J mice. Application of the mGluR1 antagonist CPCCOEt blocks sEPSCs. Inset shows corresponding fEPSCs, which are blocked by CNQX. B, GluRδ2ho-4J/ho-4J shows a similar fEPSC input–output relationship as wild type. C, Stimulation intensity for fEPSCs and sEPSCs was set to evoke similar fEPSC amplitudes (∼500 pA). D, The decay constant of fEPSCs was not affected by GluRδ2 mutation. E, The amplitude of sEPSCs was not changed significantly by GluRδ2 mutation. F, The integrated area of the CPCCOEt-sensitive sEPSC was not affected by GluRδ2 mutation. G, The onset of the sEPSC was significantly slowed in GluRδ2ho-4J/ho-4J. The average duration between the stimulus and the peak of the sEPSC was calculated. H, The average FWHM for the CPCCOEt-sensitive sEPSC was calculated. I, FWHM of sEPSC was normalized by the decay constant of fEPSC. The kinetics of sEPSCs was specifically slowed in GluRδ2ho-4J/ho-4J mice. Error bars indicate SEM. C–F, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 11). G–I, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 7). The waveforms of sEPSC in 4 of 11 samples recorded from GluRδ2ho-4J/ho-4J Purkinje cells were too small to evaluate, so we omitted these samples from G–I. J, K, NMDA receptors are not involved in the slower synaptic responses at PF–PC synapses in GluRδ2ho-4J/ho-4J. J, Typical sEPSC traces from wild-type and GluRδ2ho-4J/ho-4J in the presence or absence of 100 μm AP-5. All responses were evoked by five pulses at 100 Hz in the presence of 20 μm bicuculline and 40 μm CNQX. K, Quantified AP-5-sensitive charge transfer. Statistical significance with respect to GluRδ2+/+ (t test). **p < 0.01, ***p < 0.001.

    Techniques Used: Mutagenesis

    rabbit anti mglur1a b  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti mglur1a b
    mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with <t>mGluR1a</t> or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.
    Rabbit Anti Mglur1a B, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons"

    Article Title: Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0705-12.2012

    mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with mGluR1a or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.
    Figure Legend Snippet: mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with mGluR1a or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.

    Techniques Used: Silver Staining, Mass Spectrometry

    TRPC3 associates with mGluR–GluRδ2–PKCγ in cerebellum. Triton X-100-solubilized postnuclear membrane fractions from either wild-type or mutant mice were immunoprecipitated and blotted as indicated. A, mGluR1 coimmunoprecipitated with TRPC3 from either wild-type (A1) or GluRδ2 (A2) mutant cerebella. B1, GluRδ2 coimmunoprecipitated with TRPC3. B2, In ho-4J mice, a truncated GluRδ2 is expressed at very low levels. C, PKCγ associates with mGluR1 or TRPC3 in the presence (C1) or absence (C2) of GluRδ2. D, TRPC3 and GluRδ2 interact in the presence (D1) or absence (D2) of mGluR1. E, PKCγ associates with GluRδ2 and TRPC3, which is not affected by the presence or absence of mGluR1. F, GluK2/3 did not interact with mGluR1, GluRδ2, or TRPC3.
    Figure Legend Snippet: TRPC3 associates with mGluR–GluRδ2–PKCγ in cerebellum. Triton X-100-solubilized postnuclear membrane fractions from either wild-type or mutant mice were immunoprecipitated and blotted as indicated. A, mGluR1 coimmunoprecipitated with TRPC3 from either wild-type (A1) or GluRδ2 (A2) mutant cerebella. B1, GluRδ2 coimmunoprecipitated with TRPC3. B2, In ho-4J mice, a truncated GluRδ2 is expressed at very low levels. C, PKCγ associates with mGluR1 or TRPC3 in the presence (C1) or absence (C2) of GluRδ2. D, TRPC3 and GluRδ2 interact in the presence (D1) or absence (D2) of mGluR1. E, PKCγ associates with GluRδ2 and TRPC3, which is not affected by the presence or absence of mGluR1. F, GluK2/3 did not interact with mGluR1, GluRδ2, or TRPC3.

    Techniques Used: Mutagenesis, Immunoprecipitation

    Neither GluRδ2 nor mGluR1 mutation affects cerebellar levels of interacting proteins. A, GluRδ2 mutation results in increased levels of GluA2 but does not affect levels of mGluR1, TRPC3, or other synaptic proteins. B, Mutation of GluR1 does not affect levels of any of the proteins analyzed. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ or mGluR1+/+ (t test). n = 4 for each sample. *p < 0.05.
    Figure Legend Snippet: Neither GluRδ2 nor mGluR1 mutation affects cerebellar levels of interacting proteins. A, GluRδ2 mutation results in increased levels of GluA2 but does not affect levels of mGluR1, TRPC3, or other synaptic proteins. B, Mutation of GluR1 does not affect levels of any of the proteins analyzed. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ or mGluR1+/+ (t test). n = 4 for each sample. *p < 0.05.

    Techniques Used: Mutagenesis

    GluRδ2 or mGluR1 mutation does not affect immunofluorescent distribution of the interacting proteins. A–C, Immunofluorescent double labeling of sagittal cerebellar sections; the bottom panels are magnified images of the molecular layer. Specificity of staining is confirmed by elimination of immunosignal in corresponding mutant mice. A, Punctate GluRδ2 staining partially colocalizes with mGluR1 in molecular layer of wild type. The molecular layer staining patterns for GluRδ2 and mGluR1 were not dramatically altered in the mGluR1-KO or GluRδ2ho-4J/ho-4J, respectively. B, PKCγ shows strong labeling of Purkinje cell bodies, dendrites, and neuropil. The colabeling of mGluR1 and PKCγ in the molecular layer was not dramatically altered in the GluRδ2ho-4J/ho-4J, and the PKCγ distribution was not changed in the mGlu1-KO. C, mGluR1 and TRPC3 partially colocalize in wild-type mouse. No obvious difference in the staining patterns was detected in either GluRδ2ho-4J/ho-4J or mGluR1-KO. ML, Molecular layer; PC, Purkinje cell.
    Figure Legend Snippet: GluRδ2 or mGluR1 mutation does not affect immunofluorescent distribution of the interacting proteins. A–C, Immunofluorescent double labeling of sagittal cerebellar sections; the bottom panels are magnified images of the molecular layer. Specificity of staining is confirmed by elimination of immunosignal in corresponding mutant mice. A, Punctate GluRδ2 staining partially colocalizes with mGluR1 in molecular layer of wild type. The molecular layer staining patterns for GluRδ2 and mGluR1 were not dramatically altered in the mGluR1-KO or GluRδ2ho-4J/ho-4J, respectively. B, PKCγ shows strong labeling of Purkinje cell bodies, dendrites, and neuropil. The colabeling of mGluR1 and PKCγ in the molecular layer was not dramatically altered in the GluRδ2ho-4J/ho-4J, and the PKCγ distribution was not changed in the mGlu1-KO. C, mGluR1 and TRPC3 partially colocalize in wild-type mouse. No obvious difference in the staining patterns was detected in either GluRδ2ho-4J/ho-4J or mGluR1-KO. ML, Molecular layer; PC, Purkinje cell.

    Techniques Used: Mutagenesis, Labeling, Staining

    In GluRδ2ho-4J/ho-4J, TRPC3 partially redistributes to the Triton X-100-soluble fraction. A, Synaptosomal (Syp) and PSD fractions of mouse cerebella from wild-type, GluRδ2ho-4J/ho-4J, and mGluR1-KO were prepared and immunoblotted. mGluR1, GluRδ2, PKCγ, and TRPC3 are detected in the synaptosomal and PSD fractions. The blotting profiles of PSD-95 and synaptophysin validate the subcellular fractionations. B, Cerebellar homogenates were treated with 2.5% Triton X-100, followed by ultracentrifugation to yield supernatant (Sup) and pellet (Ppt). C, A greater percentage of TRPC3 was detected in Triton X-100-soluble fraction in GluRδ2ho-4J/ho-4J than in wild type (GluRδ2+/+). Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for each sample. *p < 0.05.
    Figure Legend Snippet: In GluRδ2ho-4J/ho-4J, TRPC3 partially redistributes to the Triton X-100-soluble fraction. A, Synaptosomal (Syp) and PSD fractions of mouse cerebella from wild-type, GluRδ2ho-4J/ho-4J, and mGluR1-KO were prepared and immunoblotted. mGluR1, GluRδ2, PKCγ, and TRPC3 are detected in the synaptosomal and PSD fractions. The blotting profiles of PSD-95 and synaptophysin validate the subcellular fractionations. B, Cerebellar homogenates were treated with 2.5% Triton X-100, followed by ultracentrifugation to yield supernatant (Sup) and pellet (Ppt). C, A greater percentage of TRPC3 was detected in Triton X-100-soluble fraction in GluRδ2ho-4J/ho-4J than in wild type (GluRδ2+/+). Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for each sample. *p < 0.05.

    Techniques Used:

    Effects of mGluR1 and GluRδ2 mutation on surface expression of interacting proteins. Mouse cerebellar slices were treated with a membrane-impermeable biotinylation reagent to mark cell-surface proteins. Total, internal, and surface proteins were resolved by SDS-PAGE and subjected to immunoblotting. GluRδ2 mutation increased surface expression of TRPC3 and mGluR1, whereas other components were unchanged. mGluR1b is the shorter splice variant of mGluR1. β3-Tubulin and/or synaptophysin served as internal protein controls. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for mGluR1, mGluR1b, TRPC3, and GluK2/3. n = 3 for GluA2, synaptophysin, and β3-tubulin. *p < 0.05.
    Figure Legend Snippet: Effects of mGluR1 and GluRδ2 mutation on surface expression of interacting proteins. Mouse cerebellar slices were treated with a membrane-impermeable biotinylation reagent to mark cell-surface proteins. Total, internal, and surface proteins were resolved by SDS-PAGE and subjected to immunoblotting. GluRδ2 mutation increased surface expression of TRPC3 and mGluR1, whereas other components were unchanged. mGluR1b is the shorter splice variant of mGluR1. β3-Tubulin and/or synaptophysin served as internal protein controls. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for mGluR1, mGluR1b, TRPC3, and GluK2/3. n = 3 for GluA2, synaptophysin, and β3-tubulin. *p < 0.05.

    Techniques Used: Mutagenesis, Expressing, SDS Page, Western Blot, Variant Assay

    GluRδ2ho-4J/ho-4J alters the time course of the mGluR1-dependent sEPSC at PF–PC synapses. A, Typical traces of sEPSCs recorded from wild-type or GluRδ2ho-4J/ho-4J mice. Application of the mGluR1 antagonist CPCCOEt blocks sEPSCs. Inset shows corresponding fEPSCs, which are blocked by CNQX. B, GluRδ2ho-4J/ho-4J shows a similar fEPSC input–output relationship as wild type. C, Stimulation intensity for fEPSCs and sEPSCs was set to evoke similar fEPSC amplitudes (∼500 pA). D, The decay constant of fEPSCs was not affected by GluRδ2 mutation. E, The amplitude of sEPSCs was not changed significantly by GluRδ2 mutation. F, The integrated area of the CPCCOEt-sensitive sEPSC was not affected by GluRδ2 mutation. G, The onset of the sEPSC was significantly slowed in GluRδ2ho-4J/ho-4J. The average duration between the stimulus and the peak of the sEPSC was calculated. H, The average FWHM for the CPCCOEt-sensitive sEPSC was calculated. I, FWHM of sEPSC was normalized by the decay constant of fEPSC. The kinetics of sEPSCs was specifically slowed in GluRδ2ho-4J/ho-4J mice. Error bars indicate SEM. C–F, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 11). G–I, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 7). The waveforms of sEPSC in 4 of 11 samples recorded from GluRδ2ho-4J/ho-4J Purkinje cells were too small to evaluate, so we omitted these samples from G–I. J, K, NMDA receptors are not involved in the slower synaptic responses at PF–PC synapses in GluRδ2ho-4J/ho-4J. J, Typical sEPSC traces from wild-type and GluRδ2ho-4J/ho-4J in the presence or absence of 100 μm AP-5. All responses were evoked by five pulses at 100 Hz in the presence of 20 μm bicuculline and 40 μm CNQX. K, Quantified AP-5-sensitive charge transfer. Statistical significance with respect to GluRδ2+/+ (t test). **p < 0.01, ***p < 0.001.
    Figure Legend Snippet: GluRδ2ho-4J/ho-4J alters the time course of the mGluR1-dependent sEPSC at PF–PC synapses. A, Typical traces of sEPSCs recorded from wild-type or GluRδ2ho-4J/ho-4J mice. Application of the mGluR1 antagonist CPCCOEt blocks sEPSCs. Inset shows corresponding fEPSCs, which are blocked by CNQX. B, GluRδ2ho-4J/ho-4J shows a similar fEPSC input–output relationship as wild type. C, Stimulation intensity for fEPSCs and sEPSCs was set to evoke similar fEPSC amplitudes (∼500 pA). D, The decay constant of fEPSCs was not affected by GluRδ2 mutation. E, The amplitude of sEPSCs was not changed significantly by GluRδ2 mutation. F, The integrated area of the CPCCOEt-sensitive sEPSC was not affected by GluRδ2 mutation. G, The onset of the sEPSC was significantly slowed in GluRδ2ho-4J/ho-4J. The average duration between the stimulus and the peak of the sEPSC was calculated. H, The average FWHM for the CPCCOEt-sensitive sEPSC was calculated. I, FWHM of sEPSC was normalized by the decay constant of fEPSC. The kinetics of sEPSCs was specifically slowed in GluRδ2ho-4J/ho-4J mice. Error bars indicate SEM. C–F, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 11). G–I, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 7). The waveforms of sEPSC in 4 of 11 samples recorded from GluRδ2ho-4J/ho-4J Purkinje cells were too small to evaluate, so we omitted these samples from G–I. J, K, NMDA receptors are not involved in the slower synaptic responses at PF–PC synapses in GluRδ2ho-4J/ho-4J. J, Typical sEPSC traces from wild-type and GluRδ2ho-4J/ho-4J in the presence or absence of 100 μm AP-5. All responses were evoked by five pulses at 100 Hz in the presence of 20 μm bicuculline and 40 μm CNQX. K, Quantified AP-5-sensitive charge transfer. Statistical significance with respect to GluRδ2+/+ (t test). **p < 0.01, ***p < 0.001.

    Techniques Used: Mutagenesis

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    Alomone Labs rabbit anti mglur1a b
    mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with <t>mGluR1a</t> or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.
    Rabbit Anti Mglur1a B, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti mglur1a b/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
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    mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with mGluR1a or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.

    Journal: The Journal of Neuroscience

    Article Title: Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

    doi: 10.1523/JNEUROSCI.0705-12.2012

    Figure Lengend Snippet: mGluR1, GluRδ2, and PKCγ form protein complexes in cerebellum. A, B, Silver staining of proteins coimmunoprecipitated with mGluR1a or anti-GluRδ2. Each protein lane was excised, divided into six pieces, and analyzed by mass spectroscopy. Proteins identified by their tryptic fragments are indicated. Contaminating IgG, keratin, and trypsin were omitted. C, PKCγ coimmunoprecipitated with mGluR1 or GluRδ2. D, GluRδ2 coimmunoprecipitated with mGluR1 or PKCγ. E, mGluR1a coimmunoprecipitated with GluRδ2 or PKCγ. F, Other metabotropic and ionotropic glutamate receptors did not coimmunoprecipitate with mGluR1, GluRδ2, or PKCγ, confirming the specificity of the IP. G, mGluR1 and GluRδ2 did not associate with other PKC subtypes; H, PKCγ did not associate with mGluR1 or GluRδ2 in cerebral cortex.

    Article Snippet: We used following antibodies: rabbit anti-mGluR1a (07-617; Millipore), mouse anti-mGluR1a (clone G209-488; BD Biosciences), rabbit anti-mGluR1a/b (AGC-006; Alamone Labs), anti-GluRδ2 (AB-1514; Millipore; GluRδ2-Rb-Af500-1; Frontier Institute), anti-PKCγ (sc-211; Santa Cruz Biotechnology), anti-PKCα (sc-208; Santa Cruz Biotechnology), anti-PKCβII (sc-210; Santa Cruz Biotechnology), anti-mGluR2/3 (AB1553; Millipore), anti-GluA2 (MAB-397; Millipore), anti-GluN1 (clone 54.1; BD Biosciences Pharmingen), and anti-TRPC3 (ACC-016; Alamone Labs).

    Techniques: Silver Staining, Mass Spectrometry

    TRPC3 associates with mGluR–GluRδ2–PKCγ in cerebellum. Triton X-100-solubilized postnuclear membrane fractions from either wild-type or mutant mice were immunoprecipitated and blotted as indicated. A, mGluR1 coimmunoprecipitated with TRPC3 from either wild-type (A1) or GluRδ2 (A2) mutant cerebella. B1, GluRδ2 coimmunoprecipitated with TRPC3. B2, In ho-4J mice, a truncated GluRδ2 is expressed at very low levels. C, PKCγ associates with mGluR1 or TRPC3 in the presence (C1) or absence (C2) of GluRδ2. D, TRPC3 and GluRδ2 interact in the presence (D1) or absence (D2) of mGluR1. E, PKCγ associates with GluRδ2 and TRPC3, which is not affected by the presence or absence of mGluR1. F, GluK2/3 did not interact with mGluR1, GluRδ2, or TRPC3.

    Journal: The Journal of Neuroscience

    Article Title: Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

    doi: 10.1523/JNEUROSCI.0705-12.2012

    Figure Lengend Snippet: TRPC3 associates with mGluR–GluRδ2–PKCγ in cerebellum. Triton X-100-solubilized postnuclear membrane fractions from either wild-type or mutant mice were immunoprecipitated and blotted as indicated. A, mGluR1 coimmunoprecipitated with TRPC3 from either wild-type (A1) or GluRδ2 (A2) mutant cerebella. B1, GluRδ2 coimmunoprecipitated with TRPC3. B2, In ho-4J mice, a truncated GluRδ2 is expressed at very low levels. C, PKCγ associates with mGluR1 or TRPC3 in the presence (C1) or absence (C2) of GluRδ2. D, TRPC3 and GluRδ2 interact in the presence (D1) or absence (D2) of mGluR1. E, PKCγ associates with GluRδ2 and TRPC3, which is not affected by the presence or absence of mGluR1. F, GluK2/3 did not interact with mGluR1, GluRδ2, or TRPC3.

    Article Snippet: We used following antibodies: rabbit anti-mGluR1a (07-617; Millipore), mouse anti-mGluR1a (clone G209-488; BD Biosciences), rabbit anti-mGluR1a/b (AGC-006; Alamone Labs), anti-GluRδ2 (AB-1514; Millipore; GluRδ2-Rb-Af500-1; Frontier Institute), anti-PKCγ (sc-211; Santa Cruz Biotechnology), anti-PKCα (sc-208; Santa Cruz Biotechnology), anti-PKCβII (sc-210; Santa Cruz Biotechnology), anti-mGluR2/3 (AB1553; Millipore), anti-GluA2 (MAB-397; Millipore), anti-GluN1 (clone 54.1; BD Biosciences Pharmingen), and anti-TRPC3 (ACC-016; Alamone Labs).

    Techniques: Mutagenesis, Immunoprecipitation

    Neither GluRδ2 nor mGluR1 mutation affects cerebellar levels of interacting proteins. A, GluRδ2 mutation results in increased levels of GluA2 but does not affect levels of mGluR1, TRPC3, or other synaptic proteins. B, Mutation of GluR1 does not affect levels of any of the proteins analyzed. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ or mGluR1+/+ (t test). n = 4 for each sample. *p < 0.05.

    Journal: The Journal of Neuroscience

    Article Title: Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

    doi: 10.1523/JNEUROSCI.0705-12.2012

    Figure Lengend Snippet: Neither GluRδ2 nor mGluR1 mutation affects cerebellar levels of interacting proteins. A, GluRδ2 mutation results in increased levels of GluA2 but does not affect levels of mGluR1, TRPC3, or other synaptic proteins. B, Mutation of GluR1 does not affect levels of any of the proteins analyzed. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ or mGluR1+/+ (t test). n = 4 for each sample. *p < 0.05.

    Article Snippet: We used following antibodies: rabbit anti-mGluR1a (07-617; Millipore), mouse anti-mGluR1a (clone G209-488; BD Biosciences), rabbit anti-mGluR1a/b (AGC-006; Alamone Labs), anti-GluRδ2 (AB-1514; Millipore; GluRδ2-Rb-Af500-1; Frontier Institute), anti-PKCγ (sc-211; Santa Cruz Biotechnology), anti-PKCα (sc-208; Santa Cruz Biotechnology), anti-PKCβII (sc-210; Santa Cruz Biotechnology), anti-mGluR2/3 (AB1553; Millipore), anti-GluA2 (MAB-397; Millipore), anti-GluN1 (clone 54.1; BD Biosciences Pharmingen), and anti-TRPC3 (ACC-016; Alamone Labs).

    Techniques: Mutagenesis

    GluRδ2 or mGluR1 mutation does not affect immunofluorescent distribution of the interacting proteins. A–C, Immunofluorescent double labeling of sagittal cerebellar sections; the bottom panels are magnified images of the molecular layer. Specificity of staining is confirmed by elimination of immunosignal in corresponding mutant mice. A, Punctate GluRδ2 staining partially colocalizes with mGluR1 in molecular layer of wild type. The molecular layer staining patterns for GluRδ2 and mGluR1 were not dramatically altered in the mGluR1-KO or GluRδ2ho-4J/ho-4J, respectively. B, PKCγ shows strong labeling of Purkinje cell bodies, dendrites, and neuropil. The colabeling of mGluR1 and PKCγ in the molecular layer was not dramatically altered in the GluRδ2ho-4J/ho-4J, and the PKCγ distribution was not changed in the mGlu1-KO. C, mGluR1 and TRPC3 partially colocalize in wild-type mouse. No obvious difference in the staining patterns was detected in either GluRδ2ho-4J/ho-4J or mGluR1-KO. ML, Molecular layer; PC, Purkinje cell.

    Journal: The Journal of Neuroscience

    Article Title: Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

    doi: 10.1523/JNEUROSCI.0705-12.2012

    Figure Lengend Snippet: GluRδ2 or mGluR1 mutation does not affect immunofluorescent distribution of the interacting proteins. A–C, Immunofluorescent double labeling of sagittal cerebellar sections; the bottom panels are magnified images of the molecular layer. Specificity of staining is confirmed by elimination of immunosignal in corresponding mutant mice. A, Punctate GluRδ2 staining partially colocalizes with mGluR1 in molecular layer of wild type. The molecular layer staining patterns for GluRδ2 and mGluR1 were not dramatically altered in the mGluR1-KO or GluRδ2ho-4J/ho-4J, respectively. B, PKCγ shows strong labeling of Purkinje cell bodies, dendrites, and neuropil. The colabeling of mGluR1 and PKCγ in the molecular layer was not dramatically altered in the GluRδ2ho-4J/ho-4J, and the PKCγ distribution was not changed in the mGlu1-KO. C, mGluR1 and TRPC3 partially colocalize in wild-type mouse. No obvious difference in the staining patterns was detected in either GluRδ2ho-4J/ho-4J or mGluR1-KO. ML, Molecular layer; PC, Purkinje cell.

    Article Snippet: We used following antibodies: rabbit anti-mGluR1a (07-617; Millipore), mouse anti-mGluR1a (clone G209-488; BD Biosciences), rabbit anti-mGluR1a/b (AGC-006; Alamone Labs), anti-GluRδ2 (AB-1514; Millipore; GluRδ2-Rb-Af500-1; Frontier Institute), anti-PKCγ (sc-211; Santa Cruz Biotechnology), anti-PKCα (sc-208; Santa Cruz Biotechnology), anti-PKCβII (sc-210; Santa Cruz Biotechnology), anti-mGluR2/3 (AB1553; Millipore), anti-GluA2 (MAB-397; Millipore), anti-GluN1 (clone 54.1; BD Biosciences Pharmingen), and anti-TRPC3 (ACC-016; Alamone Labs).

    Techniques: Mutagenesis, Labeling, Staining

    In GluRδ2ho-4J/ho-4J, TRPC3 partially redistributes to the Triton X-100-soluble fraction. A, Synaptosomal (Syp) and PSD fractions of mouse cerebella from wild-type, GluRδ2ho-4J/ho-4J, and mGluR1-KO were prepared and immunoblotted. mGluR1, GluRδ2, PKCγ, and TRPC3 are detected in the synaptosomal and PSD fractions. The blotting profiles of PSD-95 and synaptophysin validate the subcellular fractionations. B, Cerebellar homogenates were treated with 2.5% Triton X-100, followed by ultracentrifugation to yield supernatant (Sup) and pellet (Ppt). C, A greater percentage of TRPC3 was detected in Triton X-100-soluble fraction in GluRδ2ho-4J/ho-4J than in wild type (GluRδ2+/+). Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for each sample. *p < 0.05.

    Journal: The Journal of Neuroscience

    Article Title: Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

    doi: 10.1523/JNEUROSCI.0705-12.2012

    Figure Lengend Snippet: In GluRδ2ho-4J/ho-4J, TRPC3 partially redistributes to the Triton X-100-soluble fraction. A, Synaptosomal (Syp) and PSD fractions of mouse cerebella from wild-type, GluRδ2ho-4J/ho-4J, and mGluR1-KO were prepared and immunoblotted. mGluR1, GluRδ2, PKCγ, and TRPC3 are detected in the synaptosomal and PSD fractions. The blotting profiles of PSD-95 and synaptophysin validate the subcellular fractionations. B, Cerebellar homogenates were treated with 2.5% Triton X-100, followed by ultracentrifugation to yield supernatant (Sup) and pellet (Ppt). C, A greater percentage of TRPC3 was detected in Triton X-100-soluble fraction in GluRδ2ho-4J/ho-4J than in wild type (GluRδ2+/+). Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for each sample. *p < 0.05.

    Article Snippet: We used following antibodies: rabbit anti-mGluR1a (07-617; Millipore), mouse anti-mGluR1a (clone G209-488; BD Biosciences), rabbit anti-mGluR1a/b (AGC-006; Alamone Labs), anti-GluRδ2 (AB-1514; Millipore; GluRδ2-Rb-Af500-1; Frontier Institute), anti-PKCγ (sc-211; Santa Cruz Biotechnology), anti-PKCα (sc-208; Santa Cruz Biotechnology), anti-PKCβII (sc-210; Santa Cruz Biotechnology), anti-mGluR2/3 (AB1553; Millipore), anti-GluA2 (MAB-397; Millipore), anti-GluN1 (clone 54.1; BD Biosciences Pharmingen), and anti-TRPC3 (ACC-016; Alamone Labs).

    Techniques:

    Effects of mGluR1 and GluRδ2 mutation on surface expression of interacting proteins. Mouse cerebellar slices were treated with a membrane-impermeable biotinylation reagent to mark cell-surface proteins. Total, internal, and surface proteins were resolved by SDS-PAGE and subjected to immunoblotting. GluRδ2 mutation increased surface expression of TRPC3 and mGluR1, whereas other components were unchanged. mGluR1b is the shorter splice variant of mGluR1. β3-Tubulin and/or synaptophysin served as internal protein controls. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for mGluR1, mGluR1b, TRPC3, and GluK2/3. n = 3 for GluA2, synaptophysin, and β3-tubulin. *p < 0.05.

    Journal: The Journal of Neuroscience

    Article Title: Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

    doi: 10.1523/JNEUROSCI.0705-12.2012

    Figure Lengend Snippet: Effects of mGluR1 and GluRδ2 mutation on surface expression of interacting proteins. Mouse cerebellar slices were treated with a membrane-impermeable biotinylation reagent to mark cell-surface proteins. Total, internal, and surface proteins were resolved by SDS-PAGE and subjected to immunoblotting. GluRδ2 mutation increased surface expression of TRPC3 and mGluR1, whereas other components were unchanged. mGluR1b is the shorter splice variant of mGluR1. β3-Tubulin and/or synaptophysin served as internal protein controls. Error bars indicate SEM. Statistical significance with respect to GluRδ2+/+ (t test). n = 4 for mGluR1, mGluR1b, TRPC3, and GluK2/3. n = 3 for GluA2, synaptophysin, and β3-tubulin. *p < 0.05.

    Article Snippet: We used following antibodies: rabbit anti-mGluR1a (07-617; Millipore), mouse anti-mGluR1a (clone G209-488; BD Biosciences), rabbit anti-mGluR1a/b (AGC-006; Alamone Labs), anti-GluRδ2 (AB-1514; Millipore; GluRδ2-Rb-Af500-1; Frontier Institute), anti-PKCγ (sc-211; Santa Cruz Biotechnology), anti-PKCα (sc-208; Santa Cruz Biotechnology), anti-PKCβII (sc-210; Santa Cruz Biotechnology), anti-mGluR2/3 (AB1553; Millipore), anti-GluA2 (MAB-397; Millipore), anti-GluN1 (clone 54.1; BD Biosciences Pharmingen), and anti-TRPC3 (ACC-016; Alamone Labs).

    Techniques: Mutagenesis, Expressing, SDS Page, Western Blot, Variant Assay

    GluRδ2ho-4J/ho-4J alters the time course of the mGluR1-dependent sEPSC at PF–PC synapses. A, Typical traces of sEPSCs recorded from wild-type or GluRδ2ho-4J/ho-4J mice. Application of the mGluR1 antagonist CPCCOEt blocks sEPSCs. Inset shows corresponding fEPSCs, which are blocked by CNQX. B, GluRδ2ho-4J/ho-4J shows a similar fEPSC input–output relationship as wild type. C, Stimulation intensity for fEPSCs and sEPSCs was set to evoke similar fEPSC amplitudes (∼500 pA). D, The decay constant of fEPSCs was not affected by GluRδ2 mutation. E, The amplitude of sEPSCs was not changed significantly by GluRδ2 mutation. F, The integrated area of the CPCCOEt-sensitive sEPSC was not affected by GluRδ2 mutation. G, The onset of the sEPSC was significantly slowed in GluRδ2ho-4J/ho-4J. The average duration between the stimulus and the peak of the sEPSC was calculated. H, The average FWHM for the CPCCOEt-sensitive sEPSC was calculated. I, FWHM of sEPSC was normalized by the decay constant of fEPSC. The kinetics of sEPSCs was specifically slowed in GluRδ2ho-4J/ho-4J mice. Error bars indicate SEM. C–F, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 11). G–I, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 7). The waveforms of sEPSC in 4 of 11 samples recorded from GluRδ2ho-4J/ho-4J Purkinje cells were too small to evaluate, so we omitted these samples from G–I. J, K, NMDA receptors are not involved in the slower synaptic responses at PF–PC synapses in GluRδ2ho-4J/ho-4J. J, Typical sEPSC traces from wild-type and GluRδ2ho-4J/ho-4J in the presence or absence of 100 μm AP-5. All responses were evoked by five pulses at 100 Hz in the presence of 20 μm bicuculline and 40 μm CNQX. K, Quantified AP-5-sensitive charge transfer. Statistical significance with respect to GluRδ2+/+ (t test). **p < 0.01, ***p < 0.001.

    Journal: The Journal of Neuroscience

    Article Title: Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

    doi: 10.1523/JNEUROSCI.0705-12.2012

    Figure Lengend Snippet: GluRδ2ho-4J/ho-4J alters the time course of the mGluR1-dependent sEPSC at PF–PC synapses. A, Typical traces of sEPSCs recorded from wild-type or GluRδ2ho-4J/ho-4J mice. Application of the mGluR1 antagonist CPCCOEt blocks sEPSCs. Inset shows corresponding fEPSCs, which are blocked by CNQX. B, GluRδ2ho-4J/ho-4J shows a similar fEPSC input–output relationship as wild type. C, Stimulation intensity for fEPSCs and sEPSCs was set to evoke similar fEPSC amplitudes (∼500 pA). D, The decay constant of fEPSCs was not affected by GluRδ2 mutation. E, The amplitude of sEPSCs was not changed significantly by GluRδ2 mutation. F, The integrated area of the CPCCOEt-sensitive sEPSC was not affected by GluRδ2 mutation. G, The onset of the sEPSC was significantly slowed in GluRδ2ho-4J/ho-4J. The average duration between the stimulus and the peak of the sEPSC was calculated. H, The average FWHM for the CPCCOEt-sensitive sEPSC was calculated. I, FWHM of sEPSC was normalized by the decay constant of fEPSC. The kinetics of sEPSCs was specifically slowed in GluRδ2ho-4J/ho-4J mice. Error bars indicate SEM. C–F, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 11). G–I, GluRδ2+/+ (n = 18) and GluRδ2ho-4J/ho-4J (n = 7). The waveforms of sEPSC in 4 of 11 samples recorded from GluRδ2ho-4J/ho-4J Purkinje cells were too small to evaluate, so we omitted these samples from G–I. J, K, NMDA receptors are not involved in the slower synaptic responses at PF–PC synapses in GluRδ2ho-4J/ho-4J. J, Typical sEPSC traces from wild-type and GluRδ2ho-4J/ho-4J in the presence or absence of 100 μm AP-5. All responses were evoked by five pulses at 100 Hz in the presence of 20 μm bicuculline and 40 μm CNQX. K, Quantified AP-5-sensitive charge transfer. Statistical significance with respect to GluRδ2+/+ (t test). **p < 0.01, ***p < 0.001.

    Article Snippet: We used following antibodies: rabbit anti-mGluR1a (07-617; Millipore), mouse anti-mGluR1a (clone G209-488; BD Biosciences), rabbit anti-mGluR1a/b (AGC-006; Alamone Labs), anti-GluRδ2 (AB-1514; Millipore; GluRδ2-Rb-Af500-1; Frontier Institute), anti-PKCγ (sc-211; Santa Cruz Biotechnology), anti-PKCα (sc-208; Santa Cruz Biotechnology), anti-PKCβII (sc-210; Santa Cruz Biotechnology), anti-mGluR2/3 (AB1553; Millipore), anti-GluA2 (MAB-397; Millipore), anti-GluN1 (clone 54.1; BD Biosciences Pharmingen), and anti-TRPC3 (ACC-016; Alamone Labs).

    Techniques: Mutagenesis