anti cav 2 3  (Alomone Labs)


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    Alomone Labs anti cav 2 3
    Anti Cav 2 3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cav 2 3/product/Alomone Labs
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    anti cav 2 3 - by Bioz Stars, 2022-08
    93/100 stars

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    Alomone Labs anti cav2 3 cacna1e antibody
    KCTDs directly bind <t>Cav2.3</t> in vitro and KCTD8 enhances currents through <t>Cav2.3</t> A Co-immunoprecipitation from total cell lysates of HEK293 cells transfected with Flag-tagged KCTDs and Cav2.3. Immunoprecipitation of Cav2.3 co-precipitated KCTD8 and KCTD12b, but not KCTD12. Input lanes (bottom) indicate expression of the tagged proteins in the cell lysates. B Whole-cell recordings from HEK293 cells stably expressing Cav2.3. Ba 2+ current densities measured in response to a single depolarizing voltage step from −80 to 10 mV were significantly increased in KCTD8 co-transfected cells. * P
    Anti Cav2 3 Cacna1e Antibody, 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/anti cav2 3 cacna1e antibody/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti cav2 3 cacna1e antibody - by Bioz Stars, 2022-08
    93/100 stars
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    KCTDs directly bind Cav2.3 in vitro and KCTD8 enhances currents through Cav2.3 A Co-immunoprecipitation from total cell lysates of HEK293 cells transfected with Flag-tagged KCTDs and Cav2.3. Immunoprecipitation of Cav2.3 co-precipitated KCTD8 and KCTD12b, but not KCTD12. Input lanes (bottom) indicate expression of the tagged proteins in the cell lysates. B Whole-cell recordings from HEK293 cells stably expressing Cav2.3. Ba 2+ current densities measured in response to a single depolarizing voltage step from −80 to 10 mV were significantly increased in KCTD8 co-transfected cells. * P

    Journal: bioRxiv

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.1101/2020.04.16.045112

    Figure Lengend Snippet: KCTDs directly bind Cav2.3 in vitro and KCTD8 enhances currents through Cav2.3 A Co-immunoprecipitation from total cell lysates of HEK293 cells transfected with Flag-tagged KCTDs and Cav2.3. Immunoprecipitation of Cav2.3 co-precipitated KCTD8 and KCTD12b, but not KCTD12. Input lanes (bottom) indicate expression of the tagged proteins in the cell lysates. B Whole-cell recordings from HEK293 cells stably expressing Cav2.3. Ba 2+ current densities measured in response to a single depolarizing voltage step from −80 to 10 mV were significantly increased in KCTD8 co-transfected cells. * P

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 h at 4 °C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: In Vitro, Immunoprecipitation, Transfection, Expressing, Stable Transfection

    Co-localization of Cav2.3 with GBR and KCTDs in the active zone of medial habenula terminals A Active zones double-labeled for Cav2.3 and either GABA B1 (left), KCTD8 (middle) or KCTD12b (right) in IPN replicas. Top row images are from presynaptic terminals in the rostral IPN, bottom row images are from presynaptic terminals in the lateral IPN. Scale bar: 100 nm. B Quantification of active zone immunolabeling in the rostral and lateral IPN. With the exception of the absence of KCTD12b in lateral IPN terminals, absolute particle numbers per active zone (left graph) and particle densities (middle graph) are comparable between MHb terminals in the rostral and lateral IPN. Right graph: Over 97% of active zones positive for Cav2.3 labeling also show labeling for one of the other molecules (GABA B1 , KCTD8 or KCTD12b), suggesting co-localization of all presynaptic molecules inside the same active zone. Numbers inside the bars indicate the number of replicas used for each quantification. C Nearest neighbor distance (NND) for all presynaptic molecules in MHb terminals inside the rostral and lateral IPN based on the real (black line) and simulated random distribution (blue line). Smaller NND values in real distributions compared to simulation suggest clustering of all presynaptic molecules. P values calculated via Kolmogorov-Smirnov test.

    Journal: bioRxiv

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.1101/2020.04.16.045112

    Figure Lengend Snippet: Co-localization of Cav2.3 with GBR and KCTDs in the active zone of medial habenula terminals A Active zones double-labeled for Cav2.3 and either GABA B1 (left), KCTD8 (middle) or KCTD12b (right) in IPN replicas. Top row images are from presynaptic terminals in the rostral IPN, bottom row images are from presynaptic terminals in the lateral IPN. Scale bar: 100 nm. B Quantification of active zone immunolabeling in the rostral and lateral IPN. With the exception of the absence of KCTD12b in lateral IPN terminals, absolute particle numbers per active zone (left graph) and particle densities (middle graph) are comparable between MHb terminals in the rostral and lateral IPN. Right graph: Over 97% of active zones positive for Cav2.3 labeling also show labeling for one of the other molecules (GABA B1 , KCTD8 or KCTD12b), suggesting co-localization of all presynaptic molecules inside the same active zone. Numbers inside the bars indicate the number of replicas used for each quantification. C Nearest neighbor distance (NND) for all presynaptic molecules in MHb terminals inside the rostral and lateral IPN based on the real (black line) and simulated random distribution (blue line). Smaller NND values in real distributions compared to simulation suggest clustering of all presynaptic molecules. P values calculated via Kolmogorov-Smirnov test.

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 h at 4 °C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Labeling, Immunolabeling

    Absence of KCTD12b leads to a compensatory increase of KCTD8 inside the active zone of ventral MHb terminals A Example images of active zones containing Cav2.3 and either GABA B1 (left panels) or KCTD8 (right panels) in replicas of WT (upper row) and KCTD12b KO IPN tissue (lower row). Scale bars: 100 nm B Quantification of relative densities for Cav2.3, KCTD8 and GABA B1 in active zones located in the rostral IPN of WT and KCTD12b KO mice. Densities were normalized to the average density in MHb terminals inside the lateral IPN of the same replica. The number inside the bars indicate the number of replicas used for quantification. ** indicate P

    Journal: bioRxiv

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.1101/2020.04.16.045112

    Figure Lengend Snippet: Absence of KCTD12b leads to a compensatory increase of KCTD8 inside the active zone of ventral MHb terminals A Example images of active zones containing Cav2.3 and either GABA B1 (left panels) or KCTD8 (right panels) in replicas of WT (upper row) and KCTD12b KO IPN tissue (lower row). Scale bars: 100 nm B Quantification of relative densities for Cav2.3, KCTD8 and GABA B1 in active zones located in the rostral IPN of WT and KCTD12b KO mice. Densities were normalized to the average density in MHb terminals inside the lateral IPN of the same replica. The number inside the bars indicate the number of replicas used for quantification. ** indicate P

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 h at 4 °C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Mouse Assay

    Expression and function of Cav2.3 and GABA B receptors at two parallel MHb-IPN pathways A Schematic drawing of the two MHb-IPN pathways. In red: the dorsal part of the MHb projects to the lateral subnuclei of the IPN. In blue: the ventral part of the MHb projects to the rostral/central subnuclei of the IPN. B Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. C Pharmacological inhibition of Cav2.3 with SNX-482 in whole-cell recordings of rostral IPN neurons. Left: example traces before and after the application of SNX-482; middle: example time course of EPSC amplitude reduction by SNX-482; right: averaged time course of relative EPSC amplitude reduction by SNX-482. EPSC amplitudes were reduced by 83% on average (n=9 cells/9 mice). D In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n=8 cells/4 mice). E Confocal image of GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. F In whole-cell recordings of rostral IPN neurons, activation of GBRs by baclofen (1 µM) produced a potentiation of electrically evoked EPSC amplitudes. Left: example EPSC traces before (black) and during the application of baclofen (red) and after washout of baclofen (blue); middle: example time course of EPSC amplitudes in one cell; right: averaged time course of relative EPSC amplitude change after baclofen (n=13 cells/9 mice). G Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n=10 cells/5 mice). Scale bars in panels ( B ) and ( E ) are 100 µm. Averaged data is presented as mean ± SEM.

    Journal: bioRxiv

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.1101/2020.04.16.045112

    Figure Lengend Snippet: Expression and function of Cav2.3 and GABA B receptors at two parallel MHb-IPN pathways A Schematic drawing of the two MHb-IPN pathways. In red: the dorsal part of the MHb projects to the lateral subnuclei of the IPN. In blue: the ventral part of the MHb projects to the rostral/central subnuclei of the IPN. B Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. C Pharmacological inhibition of Cav2.3 with SNX-482 in whole-cell recordings of rostral IPN neurons. Left: example traces before and after the application of SNX-482; middle: example time course of EPSC amplitude reduction by SNX-482; right: averaged time course of relative EPSC amplitude reduction by SNX-482. EPSC amplitudes were reduced by 83% on average (n=9 cells/9 mice). D In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n=8 cells/4 mice). E Confocal image of GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. F In whole-cell recordings of rostral IPN neurons, activation of GBRs by baclofen (1 µM) produced a potentiation of electrically evoked EPSC amplitudes. Left: example EPSC traces before (black) and during the application of baclofen (red) and after washout of baclofen (blue); middle: example time course of EPSC amplitudes in one cell; right: averaged time course of relative EPSC amplitude change after baclofen (n=13 cells/9 mice). G Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n=10 cells/5 mice). Scale bars in panels ( B ) and ( E ) are 100 µm. Averaged data is presented as mean ± SEM.

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 h at 4 °C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing, Immunofluorescence, Inhibition, Mouse Assay, Activation Assay, Produced

    SDS-digested freeze-fracture replica labeling confirms Cav2.3 in the active zone of medial habenula terminals in the IPN A Example image of a grid-glued replica containing the whole IPN. White line indicates demarcation of rostral/central and lateral subnuclei. Scale bar: 20 µm B Example image of a presynaptic P face and a postsynaptic E face of a habenular synapse in the rostral IPN that was double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold). Scale bar: 100 nm. C Example image of a similar synaptic profile double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold) in the rostral IPN of a Cav2.3 KO mouse. Scale bar: 100 nm. D Left panel: double labeling of a WT carbon-only replica with antibodies against Cav2.3 (5 nm gold) and a mixture of active zone proteins (2 nm gold), including RIM1/2, CAST and neurexin. Right panel: the same image with additional coloring of 2 nm (red) and 5 nm (blue) particles and demarcation of the active zone area based on active zone marker labeling. Scale bars: 100 nm. F Left panel: quantification of Cav2.3 labeling densities in the presynaptic P face in WT and Cav2.3 KO mice. *** indicates P

    Journal: bioRxiv

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.1101/2020.04.16.045112

    Figure Lengend Snippet: SDS-digested freeze-fracture replica labeling confirms Cav2.3 in the active zone of medial habenula terminals in the IPN A Example image of a grid-glued replica containing the whole IPN. White line indicates demarcation of rostral/central and lateral subnuclei. Scale bar: 20 µm B Example image of a presynaptic P face and a postsynaptic E face of a habenular synapse in the rostral IPN that was double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold). Scale bar: 100 nm. C Example image of a similar synaptic profile double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold) in the rostral IPN of a Cav2.3 KO mouse. Scale bar: 100 nm. D Left panel: double labeling of a WT carbon-only replica with antibodies against Cav2.3 (5 nm gold) and a mixture of active zone proteins (2 nm gold), including RIM1/2, CAST and neurexin. Right panel: the same image with additional coloring of 2 nm (red) and 5 nm (blue) particles and demarcation of the active zone area based on active zone marker labeling. Scale bars: 100 nm. F Left panel: quantification of Cav2.3 labeling densities in the presynaptic P face in WT and Cav2.3 KO mice. *** indicates P

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 h at 4 °C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Labeling, Marker, Mouse Assay

    Quantification of the sub-synaptic localization of presynaptic Cav2.3, GBRs and KCTDs along both MHb-IPN pathways Transmission electron microscopy images of 70 nm−thick sections following pre-embedding immunolabeled IPN slices for Cav2.3 ( A ), GABA B1 ( B ), KCTD8 ( C ), KCTD12 ( D ) and KCTD12b ( E ) from synapses in the rostral (left images) and lateral (right image) IPN subnuclei. Scale bars: 200 nm. Graph on the right displays quantification of relative and absolute silver-enhanced gold particle densities in the active zone and at distances of 50 − 200 nm from the edge of the active zone (50 nm bins). F Absolute labeling densities are summarized for synapses in the rostral (left panel) and lateral IPN (right panel). Note absence of KCTD12 and KCTD12b particles in presynaptic terminals inside the lateral IPN subnuclei. KCTD12 was not included in panel F because of predominantly postsynaptic localization inside the rostral IPN. Data was pooled from two animals, showing no significant difference in gold particle distribution patterns with Kolmogorov-Smirnov test (see Supplementary Figure S1).

    Journal: bioRxiv

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.1101/2020.04.16.045112

    Figure Lengend Snippet: Quantification of the sub-synaptic localization of presynaptic Cav2.3, GBRs and KCTDs along both MHb-IPN pathways Transmission electron microscopy images of 70 nm−thick sections following pre-embedding immunolabeled IPN slices for Cav2.3 ( A ), GABA B1 ( B ), KCTD8 ( C ), KCTD12 ( D ) and KCTD12b ( E ) from synapses in the rostral (left images) and lateral (right image) IPN subnuclei. Scale bars: 200 nm. Graph on the right displays quantification of relative and absolute silver-enhanced gold particle densities in the active zone and at distances of 50 − 200 nm from the edge of the active zone (50 nm bins). F Absolute labeling densities are summarized for synapses in the rostral (left panel) and lateral IPN (right panel). Note absence of KCTD12 and KCTD12b particles in presynaptic terminals inside the lateral IPN subnuclei. KCTD12 was not included in panel F because of predominantly postsynaptic localization inside the rostral IPN. Data was pooled from two animals, showing no significant difference in gold particle distribution patterns with Kolmogorov-Smirnov test (see Supplementary Figure S1).

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 h at 4 °C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Transmission Assay, Electron Microscopy, Immunolabeling, Labeling

    Quantification of KCTDs at the plasma and total membrane in HEK cells co-expressing Cav2.3, auxiliary β3 and α2δ1 subunits. Fold increase of KCTD signal at the plasma membrane (PM) compared to the averaged KCTD signal in total membrane (TM). Quantification of KCTDs at the plasma and total membrane in HEK cells co-expressing Cav2.3, auxiliary β3 and α2δ1 subunits.

    Journal: eLife

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: Quantification of KCTDs at the plasma and total membrane in HEK cells co-expressing Cav2.3, auxiliary β3 and α2δ1 subunits. Fold increase of KCTD signal at the plasma membrane (PM) compared to the averaged KCTD signal in total membrane (TM). Quantification of KCTDs at the plasma and total membrane in HEK cells co-expressing Cav2.3, auxiliary β3 and α2δ1 subunits.

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 hr at 4°C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing

    Co-localization of Cav2.3 with GBR and KCTDs in the active zone of medial habenula terminals. ( A ) Active zones double labeled for Cav2.3 and either GABA B1 (left), KCTD8 (middle), or KCTD12b (right) in IPN replicas. Top row images are from presynaptic terminals in the rostral IPN; bottom row images are from presynaptic terminals in the lateral IPN. Scale bar: 100 nm. ( B ) Quantification of active zone immunolabeling in the rostral and lateral IPN. With the exception of the absence of KCTD12b in lateral IPN terminals, absolute particle numbers per active zone (left graph) and particle densities (middle graph) are comparable between MHb terminals in the rostral and lateral IPN. Right graph: Over 97% of active zones positive for Cav2.3 labeling also show labeling for one of the other molecules (GABA B1 , KCTD8, or KCTD12b), suggesting co-localization of all presynaptic molecules inside the same active zone. Numbers inside the bars indicate the number of replicas used for each quantification. ( C ) Nearest-neighbor distance (NND) for all presynaptic molecules in MHb terminals inside the rostral and lateral IPN based on the real (black line) and simulated random distribution (blue line). Smaller NND values in real distributions compared to simulation suggest clustering of all presynaptic molecules. p-values calculated via Kolmogorov–Smirnov test. Co-localization of Cav2.3 with GBR and KCTDs in the active zone of medial habenula terminals.

    Journal: eLife

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: Co-localization of Cav2.3 with GBR and KCTDs in the active zone of medial habenula terminals. ( A ) Active zones double labeled for Cav2.3 and either GABA B1 (left), KCTD8 (middle), or KCTD12b (right) in IPN replicas. Top row images are from presynaptic terminals in the rostral IPN; bottom row images are from presynaptic terminals in the lateral IPN. Scale bar: 100 nm. ( B ) Quantification of active zone immunolabeling in the rostral and lateral IPN. With the exception of the absence of KCTD12b in lateral IPN terminals, absolute particle numbers per active zone (left graph) and particle densities (middle graph) are comparable between MHb terminals in the rostral and lateral IPN. Right graph: Over 97% of active zones positive for Cav2.3 labeling also show labeling for one of the other molecules (GABA B1 , KCTD8, or KCTD12b), suggesting co-localization of all presynaptic molecules inside the same active zone. Numbers inside the bars indicate the number of replicas used for each quantification. ( C ) Nearest-neighbor distance (NND) for all presynaptic molecules in MHb terminals inside the rostral and lateral IPN based on the real (black line) and simulated random distribution (blue line). Smaller NND values in real distributions compared to simulation suggest clustering of all presynaptic molecules. p-values calculated via Kolmogorov–Smirnov test. Co-localization of Cav2.3 with GBR and KCTDs in the active zone of medial habenula terminals.

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 hr at 4°C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Labeling, Immunolabeling

    Allen Brain Atlas images showing lack of expression of Cav2.1 ( http://mouse.brain-map.org/gene/show/12071 ) and Cav2.2 ( http://mouse.brain-map.org/gene/show/12072 ) mRNA in MHb neurons, whereas Cav2.3 shows strong expression in MHb neurons ( http://mouse.brain-map.org/gene/show/12075 ).

    Journal: eLife

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: Allen Brain Atlas images showing lack of expression of Cav2.1 ( http://mouse.brain-map.org/gene/show/12071 ) and Cav2.2 ( http://mouse.brain-map.org/gene/show/12072 ) mRNA in MHb neurons, whereas Cav2.3 shows strong expression in MHb neurons ( http://mouse.brain-map.org/gene/show/12075 ).

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 hr at 4°C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing

    SDS-digested freeze-fracture replica labeling confirms Cav2.3 in the active zone of medial habenula terminals in the IPN. ( A ) Example image of a grid-glued replica containing the whole IPN. White dashed line indicates demarcation of rostral/central and lateral subnuclei. Scale bar: 20 µm. ( B ) Example image of a presynaptic P-face and a postsynaptic E-face of a habenular synapse in the rostral IPN that was double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold). Scale bar: 100 nm. ( C ) Example image of a similar synaptic profile double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold) in the rostral IPN of a Cav2.3 KO mouse. Scale bar: 100 nm. ( D ) Left: double labeling of a WT carbon-only replica with antibodies against Cav2.3 (5 nm gold) and a mixture of active zone proteins (2 nm gold), including RIM1/2, CAST, and neurexin. Right: the same image with additional coloring of 2 nm (red) and 5 nm (blue) particles and demarcation of the active zone area based on active zone-marker labeling. Scale bars: 100 nm. ( F ) Left: quantification of Cav2.3 labeling densities in the presynaptic P-face in WT and Cav2.3 KO mice. ***p

    Journal: eLife

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: SDS-digested freeze-fracture replica labeling confirms Cav2.3 in the active zone of medial habenula terminals in the IPN. ( A ) Example image of a grid-glued replica containing the whole IPN. White dashed line indicates demarcation of rostral/central and lateral subnuclei. Scale bar: 20 µm. ( B ) Example image of a presynaptic P-face and a postsynaptic E-face of a habenular synapse in the rostral IPN that was double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold). Scale bar: 100 nm. ( C ) Example image of a similar synaptic profile double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold) in the rostral IPN of a Cav2.3 KO mouse. Scale bar: 100 nm. ( D ) Left: double labeling of a WT carbon-only replica with antibodies against Cav2.3 (5 nm gold) and a mixture of active zone proteins (2 nm gold), including RIM1/2, CAST, and neurexin. Right: the same image with additional coloring of 2 nm (red) and 5 nm (blue) particles and demarcation of the active zone area based on active zone-marker labeling. Scale bars: 100 nm. ( F ) Left: quantification of Cav2.3 labeling densities in the presynaptic P-face in WT and Cav2.3 KO mice. ***p

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 hr at 4°C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Labeling, Marker, Mouse Assay

    Comparison of distribution of silver-enhanced immunogold particles outside the active zone for Cav2.3, KCTD8, KCTD12, and KCTD12b in MHb terminals inside the rostral and lateral IPN. Distribution of particles in samples from two mice was not significantly different. p-values above graph resulted from Kolmogorov–Smirnov test. M1 = mouse 1, M2 = mouse 2, n indicates the total number of particles analyzed. Comparison of distribution of silver-enhanced immunogold particles outside the active zone for Cav2.3, KCTD8, KCTD12, and KCTD12b in MHb terminals inside the rostral and lateral IPN.

    Journal: eLife

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: Comparison of distribution of silver-enhanced immunogold particles outside the active zone for Cav2.3, KCTD8, KCTD12, and KCTD12b in MHb terminals inside the rostral and lateral IPN. Distribution of particles in samples from two mice was not significantly different. p-values above graph resulted from Kolmogorov–Smirnov test. M1 = mouse 1, M2 = mouse 2, n indicates the total number of particles analyzed. Comparison of distribution of silver-enhanced immunogold particles outside the active zone for Cav2.3, KCTD8, KCTD12, and KCTD12b in MHb terminals inside the rostral and lateral IPN.

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 hr at 4°C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Mouse Assay

    Absence of KCTD12b leads to a compensatory increase of KCTD8 inside the active zone of ventral MHb terminals. ( A ) Example images of active zones containing Cav2.3 and either GABA B1 (left) or KCTD8 (right) in replicas of WT (upper row) and KCTD12b KO IPN tissue (lower row). Scale bars: 100 nm. ( B ) Quantification of relative densities for Cav2.3, KCTD8, and GABA B1 in active zones located in the rostral IPN of WT and KCTD12b KO mice. Densities were normalized to the average density in MHb terminals inside the lateral IPN of the same replica. The number inside the bars indicates the number of replicas used for quantification. **p

    Journal: eLife

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: Absence of KCTD12b leads to a compensatory increase of KCTD8 inside the active zone of ventral MHb terminals. ( A ) Example images of active zones containing Cav2.3 and either GABA B1 (left) or KCTD8 (right) in replicas of WT (upper row) and KCTD12b KO IPN tissue (lower row). Scale bars: 100 nm. ( B ) Quantification of relative densities for Cav2.3, KCTD8, and GABA B1 in active zones located in the rostral IPN of WT and KCTD12b KO mice. Densities were normalized to the average density in MHb terminals inside the lateral IPN of the same replica. The number inside the bars indicates the number of replicas used for quantification. **p

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 hr at 4°C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Mouse Assay

    Analyses of expression of the Cav2.3 protein in neuronal and nonneuronal cells or in cell membrane of the cells in dorsal root ganglia in vivo. Representative images to demonstrate expression of Cav2.3 in mouse DRG and colabeling with PGP9.5-positive neuronal cells (A), GFAP-positive satellite cells (B) and with HCN1 (hyperpolarization-activated cyclic nucleotide-gated) channel in the cell membrane (C). Observed colocalization is highlighted with white arrows. Quantification of coexpression of each neuronal subtype with the Cav2.3 expressing neurons is shown in panel D. Scale bars represent 50 µm in all panels. Tissue samples from 3 independent mice were analyzed.

    Journal: Pain

    Article Title: miR-34c-5p functions as pronociceptive microRNA in cancer pain by targeting Cav2.3 containing calcium channels

    doi: 10.1097/j.pain.0000000000000971

    Figure Lengend Snippet: Analyses of expression of the Cav2.3 protein in neuronal and nonneuronal cells or in cell membrane of the cells in dorsal root ganglia in vivo. Representative images to demonstrate expression of Cav2.3 in mouse DRG and colabeling with PGP9.5-positive neuronal cells (A), GFAP-positive satellite cells (B) and with HCN1 (hyperpolarization-activated cyclic nucleotide-gated) channel in the cell membrane (C). Observed colocalization is highlighted with white arrows. Quantification of coexpression of each neuronal subtype with the Cav2.3 expressing neurons is shown in panel D. Scale bars represent 50 µm in all panels. Tissue samples from 3 independent mice were analyzed.

    Article Snippet: Primary antibodies used for IF are Guinea pig anti-PGP9.5 (1:100 dilution, 14104, Neuromics, Edina, MN), Guinea pig anti-HCN1 (1:100, Alomone Labs, AGP203) rabbit anti-Cav2.3 antibody (1:80, Alomone Labs, ACC-006), Biotinylated-Isolectin B4 (1:100; B-1205, Vector, Burlingame, CA), Guinea pig Substance P (1:150; Neuromics GP14103), Anti-GFAP (1:500; NeuroMab clone N206A/8, UC Davis, Davis, CA) and Chicken anti-NF200 (1:500; Neuromics CH23015).

    Techniques: Expressing, In Vivo, Mouse Assay

    Analyses of cellular expression of the Cav2.3 protein in dorsal root ganglia in vivo. Representative images to demonstrate expression of Cav2.3 in mouse DRG and colabeling with isolectin‐B4‐binding (IB4) nonpeptidergic nociceptors (A), substance P‐positive peptidergic nociceptors (B) and NF200-positive large diameter sensory neurons (C) in mouse DRG. Observed colocalization is highlighted with white arrows. Quantification of coexpression of each neuronal subtype with the Cav2.3 expressing neurons is shown in panel D. Scale bars represent 50 μm in all panels. Tissue samples from 3 independent mice were analyzed.

    Journal: Pain

    Article Title: miR-34c-5p functions as pronociceptive microRNA in cancer pain by targeting Cav2.3 containing calcium channels

    doi: 10.1097/j.pain.0000000000000971

    Figure Lengend Snippet: Analyses of cellular expression of the Cav2.3 protein in dorsal root ganglia in vivo. Representative images to demonstrate expression of Cav2.3 in mouse DRG and colabeling with isolectin‐B4‐binding (IB4) nonpeptidergic nociceptors (A), substance P‐positive peptidergic nociceptors (B) and NF200-positive large diameter sensory neurons (C) in mouse DRG. Observed colocalization is highlighted with white arrows. Quantification of coexpression of each neuronal subtype with the Cav2.3 expressing neurons is shown in panel D. Scale bars represent 50 μm in all panels. Tissue samples from 3 independent mice were analyzed.

    Article Snippet: Primary antibodies used for IF are Guinea pig anti-PGP9.5 (1:100 dilution, 14104, Neuromics, Edina, MN), Guinea pig anti-HCN1 (1:100, Alomone Labs, AGP203) rabbit anti-Cav2.3 antibody (1:80, Alomone Labs, ACC-006), Biotinylated-Isolectin B4 (1:100; B-1205, Vector, Burlingame, CA), Guinea pig Substance P (1:150; Neuromics GP14103), Anti-GFAP (1:500; NeuroMab clone N206A/8, UC Davis, Davis, CA) and Chicken anti-NF200 (1:500; Neuromics CH23015).

    Techniques: Expressing, In Vivo, Binding Assay, Mouse Assay

    Analyses of cellular expression of miR-34c-5p in dorsal root ganglia in vivo. Representative images to demonstrate fluorescent in situ hybridization (FISH) analysis of miR-34c-5p expression in mouse DRG with specific or negative control probes (A) and immunofluorescence analysis of colabeling with its mRNA target Cav2.3 (A), isolectin‐B4‐binding (IB4) nonpeptidergic nociceptors (B), substance P‐positive peptidergic nociceptors (C) and NF200-positive large diameter sensory neurons (D) in mouse DRG. Cell nuclei were counterstained with DAPI. Quantification of coexpression of each neuronal subtype with the miR-34c-5p expressing neurons is shown in panel E. Scale bars represent 50 µm in all panels. Tissue samples from 3 independent mice were analyzed.

    Journal: Pain

    Article Title: miR-34c-5p functions as pronociceptive microRNA in cancer pain by targeting Cav2.3 containing calcium channels

    doi: 10.1097/j.pain.0000000000000971

    Figure Lengend Snippet: Analyses of cellular expression of miR-34c-5p in dorsal root ganglia in vivo. Representative images to demonstrate fluorescent in situ hybridization (FISH) analysis of miR-34c-5p expression in mouse DRG with specific or negative control probes (A) and immunofluorescence analysis of colabeling with its mRNA target Cav2.3 (A), isolectin‐B4‐binding (IB4) nonpeptidergic nociceptors (B), substance P‐positive peptidergic nociceptors (C) and NF200-positive large diameter sensory neurons (D) in mouse DRG. Cell nuclei were counterstained with DAPI. Quantification of coexpression of each neuronal subtype with the miR-34c-5p expressing neurons is shown in panel E. Scale bars represent 50 µm in all panels. Tissue samples from 3 independent mice were analyzed.

    Article Snippet: Primary antibodies used for IF are Guinea pig anti-PGP9.5 (1:100 dilution, 14104, Neuromics, Edina, MN), Guinea pig anti-HCN1 (1:100, Alomone Labs, AGP203) rabbit anti-Cav2.3 antibody (1:80, Alomone Labs, ACC-006), Biotinylated-Isolectin B4 (1:100; B-1205, Vector, Burlingame, CA), Guinea pig Substance P (1:150; Neuromics GP14103), Anti-GFAP (1:500; NeuroMab clone N206A/8, UC Davis, Davis, CA) and Chicken anti-NF200 (1:500; Neuromics CH23015).

    Techniques: Expressing, In Vivo, In Situ Hybridization, Fluorescence In Situ Hybridization, Negative Control, Immunofluorescence, Binding Assay, Mouse Assay

    Analyses of prioritized putative targets for miR-3c-5p in sensory neurons isolated from tumor-bearing mice. (A) qRTPCR analyses demonstrating the change in the expression of miR-34c-5p and 4 of its putative targets in cultured sensory neurons in the presence of miR-34c-5p specific mimic as compared to nontargeting mimic transfected controls. (B) qRTPCR analyses demonstrating the change in the expression of miR-34c-5p and 4 of its putative targets in cultured sensory neurons in the presence of miR-34c-5p specific inhibitor as compared to scrambled nontargeting inhibitor-transfected controls. (C) qRTPCR analyses demonstrating the change in the expression of miR-34c-5p and 4 of its putative targets in the DRGs isolated from tumor-bearing mice as compared to sham controls. In panels A-C, *denotes P ≤ 0.05 as compared to control group, analysis of variance followed by post hoc Fischer test from at least 3 biological replicate experiments. The dotted line represents expression levels in the control group, bars above the line represent upregulation and below the line represent downregulation of tested transcripts. (D) Representation of miR-34c-5p binding sites in the 3′UTR of Cav2.3 and strategy for the cloning of 3′UTR sequence into the dual luciferase vector. (E) Luciferase reporter assay in HEK293 cells demonstrating changes in the translation of the Cav2.3 gene in the presence of intact or mutated binding sites for miR-34c-5p and following induction of miR‐34c‐5p expression via specific mimic. (F) Representative western blot analysis images and their quantification for Cacna1e or β-tubulin protein expression in the lysates of cultured DRG neuronal cells following transfection with control (non-targeting mimic) or miR-34c-5p specific mimic. *Denotes P ≤ 0.05 as compared to nontargeting mimic group, analysis of variance followed by post hoc Fischer test, n = 3 independent experiments.

    Journal: Pain

    Article Title: miR-34c-5p functions as pronociceptive microRNA in cancer pain by targeting Cav2.3 containing calcium channels

    doi: 10.1097/j.pain.0000000000000971

    Figure Lengend Snippet: Analyses of prioritized putative targets for miR-3c-5p in sensory neurons isolated from tumor-bearing mice. (A) qRTPCR analyses demonstrating the change in the expression of miR-34c-5p and 4 of its putative targets in cultured sensory neurons in the presence of miR-34c-5p specific mimic as compared to nontargeting mimic transfected controls. (B) qRTPCR analyses demonstrating the change in the expression of miR-34c-5p and 4 of its putative targets in cultured sensory neurons in the presence of miR-34c-5p specific inhibitor as compared to scrambled nontargeting inhibitor-transfected controls. (C) qRTPCR analyses demonstrating the change in the expression of miR-34c-5p and 4 of its putative targets in the DRGs isolated from tumor-bearing mice as compared to sham controls. In panels A-C, *denotes P ≤ 0.05 as compared to control group, analysis of variance followed by post hoc Fischer test from at least 3 biological replicate experiments. The dotted line represents expression levels in the control group, bars above the line represent upregulation and below the line represent downregulation of tested transcripts. (D) Representation of miR-34c-5p binding sites in the 3′UTR of Cav2.3 and strategy for the cloning of 3′UTR sequence into the dual luciferase vector. (E) Luciferase reporter assay in HEK293 cells demonstrating changes in the translation of the Cav2.3 gene in the presence of intact or mutated binding sites for miR-34c-5p and following induction of miR‐34c‐5p expression via specific mimic. (F) Representative western blot analysis images and their quantification for Cacna1e or β-tubulin protein expression in the lysates of cultured DRG neuronal cells following transfection with control (non-targeting mimic) or miR-34c-5p specific mimic. *Denotes P ≤ 0.05 as compared to nontargeting mimic group, analysis of variance followed by post hoc Fischer test, n = 3 independent experiments.

    Article Snippet: Primary antibodies used for IF are Guinea pig anti-PGP9.5 (1:100 dilution, 14104, Neuromics, Edina, MN), Guinea pig anti-HCN1 (1:100, Alomone Labs, AGP203) rabbit anti-Cav2.3 antibody (1:80, Alomone Labs, ACC-006), Biotinylated-Isolectin B4 (1:100; B-1205, Vector, Burlingame, CA), Guinea pig Substance P (1:150; Neuromics GP14103), Anti-GFAP (1:500; NeuroMab clone N206A/8, UC Davis, Davis, CA) and Chicken anti-NF200 (1:500; Neuromics CH23015).

    Techniques: Isolation, Mouse Assay, Expressing, Cell Culture, Transfection, Binding Assay, Clone Assay, Sequencing, Luciferase, Plasmid Preparation, Reporter Assay, Western Blot

    Analyses of functional contribution of Cav2.3 and miR-34c-5p in the mediation of mechanical sensitivity in wild-type mice. (A) Change in the frequency of paw withdrawal to the plantar application of graded von Frey filament forces of different strength in ipsilateral paws following intraganglionic injection of AAVs carrying either scrambled shRNA (AAV-Scr-shRNA) or shRNA directed against coding sequence of Cav2.3 (AAV-Cav2.3-shRNA), measured before and at 3W after viral injections. (B) Mechanical response thresholds calculated as von Frey filament strength required to achieve 60% withdrawal frequency in ipsilateral paws following intraganglionic injection of AAV-Scr-shRNA or AAV-Cav2.3-shRNA, measured before and at 3W following viral injection. (C) Representative western blot analysis images and their quantification for Cacna1e or β-tubulin protein expression in the DRG tissue lysates following intraganglionic injection of AAV-Scr-shRNA or AAV-Cav2.3-shRNA. (D) Change in the frequency of paw withdrawal to the plantar application of graded von Frey filament forces of different strength in ipsilateral paws following intraganglionic injection of lentivirus carrying either nontargeting miRNA mimic (Lenti-nontargeting-mimic) miR-34c-5p specific mimic (Lenti-miR-34c-mimic), measured before and at 4W after viral injections. (E) Mechanical response thresholds calculated as von Frey filament strength required to achieve 60% withdrawal frequency in ipsilateral paws following intraganglionic injection of Lenti-nontargeting-mimic or Lenti-miR-34c-mimic, measured before and at 3W following viral injection. (F) Representative western blot analysis images and their quantification for Cacna1e or β-tubulin protein expression in the DRG tissue lysates following intraganglionic injection of Lenti-nontargeting-mimic or Lenti-miR-34c-mimic. In panels A, B, D, and E, * P ≤ 0.05 as compared to basal readings and †denotes P ≤ 0.05 as compared to a corresponding data point in other 3 groups, 2-way analysis of variance of repeated measures followed by Bonferroni multiple comparisons post hoc test, n = 8 mice per group. In panels C and F, * P ≤ 0.05 as compared to control group, analysis of variance followed by post hoc Fischer test, n = 5 independent experiments.

    Journal: Pain

    Article Title: miR-34c-5p functions as pronociceptive microRNA in cancer pain by targeting Cav2.3 containing calcium channels

    doi: 10.1097/j.pain.0000000000000971

    Figure Lengend Snippet: Analyses of functional contribution of Cav2.3 and miR-34c-5p in the mediation of mechanical sensitivity in wild-type mice. (A) Change in the frequency of paw withdrawal to the plantar application of graded von Frey filament forces of different strength in ipsilateral paws following intraganglionic injection of AAVs carrying either scrambled shRNA (AAV-Scr-shRNA) or shRNA directed against coding sequence of Cav2.3 (AAV-Cav2.3-shRNA), measured before and at 3W after viral injections. (B) Mechanical response thresholds calculated as von Frey filament strength required to achieve 60% withdrawal frequency in ipsilateral paws following intraganglionic injection of AAV-Scr-shRNA or AAV-Cav2.3-shRNA, measured before and at 3W following viral injection. (C) Representative western blot analysis images and their quantification for Cacna1e or β-tubulin protein expression in the DRG tissue lysates following intraganglionic injection of AAV-Scr-shRNA or AAV-Cav2.3-shRNA. (D) Change in the frequency of paw withdrawal to the plantar application of graded von Frey filament forces of different strength in ipsilateral paws following intraganglionic injection of lentivirus carrying either nontargeting miRNA mimic (Lenti-nontargeting-mimic) miR-34c-5p specific mimic (Lenti-miR-34c-mimic), measured before and at 4W after viral injections. (E) Mechanical response thresholds calculated as von Frey filament strength required to achieve 60% withdrawal frequency in ipsilateral paws following intraganglionic injection of Lenti-nontargeting-mimic or Lenti-miR-34c-mimic, measured before and at 3W following viral injection. (F) Representative western blot analysis images and their quantification for Cacna1e or β-tubulin protein expression in the DRG tissue lysates following intraganglionic injection of Lenti-nontargeting-mimic or Lenti-miR-34c-mimic. In panels A, B, D, and E, * P ≤ 0.05 as compared to basal readings and †denotes P ≤ 0.05 as compared to a corresponding data point in other 3 groups, 2-way analysis of variance of repeated measures followed by Bonferroni multiple comparisons post hoc test, n = 8 mice per group. In panels C and F, * P ≤ 0.05 as compared to control group, analysis of variance followed by post hoc Fischer test, n = 5 independent experiments.

    Article Snippet: Primary antibodies used for IF are Guinea pig anti-PGP9.5 (1:100 dilution, 14104, Neuromics, Edina, MN), Guinea pig anti-HCN1 (1:100, Alomone Labs, AGP203) rabbit anti-Cav2.3 antibody (1:80, Alomone Labs, ACC-006), Biotinylated-Isolectin B4 (1:100; B-1205, Vector, Burlingame, CA), Guinea pig Substance P (1:150; Neuromics GP14103), Anti-GFAP (1:500; NeuroMab clone N206A/8, UC Davis, Davis, CA) and Chicken anti-NF200 (1:500; Neuromics CH23015).

    Techniques: Functional Assay, Mouse Assay, Injection, shRNA, Sequencing, Western Blot, Expressing