anti cav1 3  (Alomone Labs)


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    Structured Review

    Alomone Labs anti cav1 3
    Graphic representation of the results. The activity-dependent signaling between the nerve terminals that are in competition through several metabotropic receptors can result in the modulation of the downstream effector kinases, specifically cPKCβI, nPKCε, and PKA. Changes in kinases activity can allow the coordinate phosphorylation of the L-type <t>CaV1.3</t> and P/Q-type VGCC. The high calcium entry through these operative channels present in immature nerve endings can result in their final loss. Also, muscle CaV1.1 and contractile activity can contribute to the synapse elimination. A component of this mechanism may be mediated by a retrograde influence from the postsynaptic site, via the BDNF-TrkB pathway, on the presynaptic calcium channels
    Anti Cav1 3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development"

    Article Title: Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development

    Journal: Molecular Neurobiology

    doi: 10.1007/s12035-022-02818-2

    Graphic representation of the results. The activity-dependent signaling between the nerve terminals that are in competition through several metabotropic receptors can result in the modulation of the downstream effector kinases, specifically cPKCβI, nPKCε, and PKA. Changes in kinases activity can allow the coordinate phosphorylation of the L-type CaV1.3 and P/Q-type VGCC. The high calcium entry through these operative channels present in immature nerve endings can result in their final loss. Also, muscle CaV1.1 and contractile activity can contribute to the synapse elimination. A component of this mechanism may be mediated by a retrograde influence from the postsynaptic site, via the BDNF-TrkB pathway, on the presynaptic calcium channels
    Figure Legend Snippet: Graphic representation of the results. The activity-dependent signaling between the nerve terminals that are in competition through several metabotropic receptors can result in the modulation of the downstream effector kinases, specifically cPKCβI, nPKCε, and PKA. Changes in kinases activity can allow the coordinate phosphorylation of the L-type CaV1.3 and P/Q-type VGCC. The high calcium entry through these operative channels present in immature nerve endings can result in their final loss. Also, muscle CaV1.1 and contractile activity can contribute to the synapse elimination. A component of this mechanism may be mediated by a retrograde influence from the postsynaptic site, via the BDNF-TrkB pathway, on the presynaptic calcium channels

    Techniques Used: Activity Assay

    2) Product Images from "Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development"

    Article Title: Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development

    Journal: Molecular Neurobiology

    doi: 10.1007/s12035-022-02818-2

    Graphic representation of the results. The activity-dependent signaling between the nerve terminals that are in competition through several metabotropic receptors can result in the modulation of the downstream effector kinases, specifically cPKCβI, nPKCε, and PKA. Changes in kinases activity can allow the coordinate phosphorylation of the L-type CaV1.3 and P/Q-type VGCC. The high calcium entry through these operative channels present in immature nerve endings can result in their final loss. Also, muscle CaV1.1 and contractile activity can contribute to the synapse elimination. A component of this mechanism may be mediated by a retrograde influence from the postsynaptic site, via the BDNF-TrkB pathway, on the presynaptic calcium channels
    Figure Legend Snippet: Graphic representation of the results. The activity-dependent signaling between the nerve terminals that are in competition through several metabotropic receptors can result in the modulation of the downstream effector kinases, specifically cPKCβI, nPKCε, and PKA. Changes in kinases activity can allow the coordinate phosphorylation of the L-type CaV1.3 and P/Q-type VGCC. The high calcium entry through these operative channels present in immature nerve endings can result in their final loss. Also, muscle CaV1.1 and contractile activity can contribute to the synapse elimination. A component of this mechanism may be mediated by a retrograde influence from the postsynaptic site, via the BDNF-TrkB pathway, on the presynaptic calcium channels

    Techniques Used: Activity Assay

    3) Product Images from "Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development"

    Article Title: Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development

    Journal: Molecular Neurobiology

    doi: 10.1007/s12035-022-02818-2

    Graphic representation of the results. The activity-dependent signaling between the nerve terminals that are in competition through several metabotropic receptors can result in the modulation of the downstream effector kinases, specifically cPKCβI, nPKCε, and PKA. Changes in kinases activity can allow the coordinate phosphorylation of the L-type CaV1.3 and P/Q-type VGCC. The high calcium entry through these operative channels present in immature nerve endings can result in their final loss. Also, muscle CaV1.1 and contractile activity can contribute to the synapse elimination. A component of this mechanism may be mediated by a retrograde influence from the postsynaptic site, via the BDNF-TrkB pathway, on the presynaptic calcium channels
    Figure Legend Snippet: Graphic representation of the results. The activity-dependent signaling between the nerve terminals that are in competition through several metabotropic receptors can result in the modulation of the downstream effector kinases, specifically cPKCβI, nPKCε, and PKA. Changes in kinases activity can allow the coordinate phosphorylation of the L-type CaV1.3 and P/Q-type VGCC. The high calcium entry through these operative channels present in immature nerve endings can result in their final loss. Also, muscle CaV1.1 and contractile activity can contribute to the synapse elimination. A component of this mechanism may be mediated by a retrograde influence from the postsynaptic site, via the BDNF-TrkB pathway, on the presynaptic calcium channels

    Techniques Used: Activity Assay

    4) Product Images from "L-Type Cav1.3 Calcium Channels Are Required for Beta-Adrenergic Triggered Automaticity in Dormant Mouse Sinoatrial Pacemaker Cells"

    Article Title: L-Type Cav1.3 Calcium Channels Are Required for Beta-Adrenergic Triggered Automaticity in Dormant Mouse Sinoatrial Pacemaker Cells

    Journal: Cells

    doi: 10.3390/cells11071114

    Dormant SANC express reduced I Cav1.3 and I f . Representative traces and I-V curves of I f (( A ), n = 10 dormant and n = 8 firing) and Nife-sensitive current ( I Cav1.3 ) (( B ), n = 12 dormant and n = 9 firing) in dormant (blue) and firing (red) SANC. ( C ) Nife-sensitive current ( I Cav1.3 ) and Nife-insensitive current ( I Cav1.2 ) densities at 0 mV in dormant and firing Ca v 1.2 DHP−/− SANC. Averaged SR Ca 2+ load ( D ), time constant of caffeine-induced Ca 2+ transient ( E ), number of LCRs ( F ) and diastolic Ca 2+ ( G ), in dormant (blue, n = 9) and firing (red, n = 15) SANC. ns: non-significant, * p
    Figure Legend Snippet: Dormant SANC express reduced I Cav1.3 and I f . Representative traces and I-V curves of I f (( A ), n = 10 dormant and n = 8 firing) and Nife-sensitive current ( I Cav1.3 ) (( B ), n = 12 dormant and n = 9 firing) in dormant (blue) and firing (red) SANC. ( C ) Nife-sensitive current ( I Cav1.3 ) and Nife-insensitive current ( I Cav1.2 ) densities at 0 mV in dormant and firing Ca v 1.2 DHP−/− SANC. Averaged SR Ca 2+ load ( D ), time constant of caffeine-induced Ca 2+ transient ( E ), number of LCRs ( F ) and diastolic Ca 2+ ( G ), in dormant (blue, n = 9) and firing (red, n = 15) SANC. ns: non-significant, * p

    Techniques Used:

    5) Product Images from "TRPC7 regulates the electrophysiological functions of embryonic stem cell-derived cardiomyocytes"

    Article Title: TRPC7 regulates the electrophysiological functions of embryonic stem cell-derived cardiomyocytes

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-021-02308-7

    Knockdown or overexpression of TRPC7 did not alter the expression of several important ion channels/pump in NRVMs. a – g Western blots showing the expression of a TRPC7, b HCN4, c Cav1.3, d IP3R1, e Cav3.1, f Cav3.2, g SERCA in NRVMs infected with different adenoviruses to knockdown or overexpress TRPC7. h – n Bar charts showing the quantification of each protein from a – g . To eliminate the loading bias, intensity of each target protein was normalized to that of its corresponding β-tubulin. TRPC7 was successfully knocked down or overexpressed in NRVMs but the change of TRPC7 expression did not alter the expression of HCN4, Cav1.3, IP3R1, Cav3.1, Cav3.2, and SERCA. Data were presented as mean ± SEM ( n = 4). * P
    Figure Legend Snippet: Knockdown or overexpression of TRPC7 did not alter the expression of several important ion channels/pump in NRVMs. a – g Western blots showing the expression of a TRPC7, b HCN4, c Cav1.3, d IP3R1, e Cav3.1, f Cav3.2, g SERCA in NRVMs infected with different adenoviruses to knockdown or overexpress TRPC7. h – n Bar charts showing the quantification of each protein from a – g . To eliminate the loading bias, intensity of each target protein was normalized to that of its corresponding β-tubulin. TRPC7 was successfully knocked down or overexpressed in NRVMs but the change of TRPC7 expression did not alter the expression of HCN4, Cav1.3, IP3R1, Cav3.1, Cav3.2, and SERCA. Data were presented as mean ± SEM ( n = 4). * P

    Techniques Used: Over Expression, Expressing, Western Blot, Infection

    6) Product Images from "Biophysical classification of a CACNA1D de novo mutation as a high-risk mutation for a severe neurodevelopmental disorder"

    Article Title: Biophysical classification of a CACNA1D de novo mutation as a high-risk mutation for a severe neurodevelopmental disorder

    Journal: Molecular Autism

    doi: 10.1186/s13229-019-0310-4

    Mutation S652L increases intracellular Ca 2+ during simulated action potential firing. a Upper left: Shape of single action potential waveform (APW) mimicked by the following voltage steps: HP: − 80 mV, − 80 to − 60 mV for 2.5 ms, − 60 to + 20 mV in 1 ms, + 20 to − 70 mV in 1.5 ms, − 70 to − 60 mV in 5 ms, − 60 mV for 90 ms. The corresponding I Ca of WT L and S652L L are shown below. Right: Representative current responses of WT L and S652L L during 30 s of stimulation with APW-like stimuli at a frequency of 10 Hz. b Peak I Ca of S652L L Cav1.3 channels decayed faster than WT L during stimulation. Statistics: unpaired student´s t-test ([mean ± SEM]; WT L , 14.94 ± 2.19, n = 20; S652L L , 30.94 ± 2.85***, n = 21; *** p
    Figure Legend Snippet: Mutation S652L increases intracellular Ca 2+ during simulated action potential firing. a Upper left: Shape of single action potential waveform (APW) mimicked by the following voltage steps: HP: − 80 mV, − 80 to − 60 mV for 2.5 ms, − 60 to + 20 mV in 1 ms, + 20 to − 70 mV in 1.5 ms, − 70 to − 60 mV in 5 ms, − 60 mV for 90 ms. The corresponding I Ca of WT L and S652L L are shown below. Right: Representative current responses of WT L and S652L L during 30 s of stimulation with APW-like stimuli at a frequency of 10 Hz. b Peak I Ca of S652L L Cav1.3 channels decayed faster than WT L during stimulation. Statistics: unpaired student´s t-test ([mean ± SEM]; WT L , 14.94 ± 2.19, n = 20; S652L L , 30.94 ± 2.85***, n = 21; *** p

    Techniques Used: Mutagenesis, Mass Spectrometry

    Molecular modeling of Cav1.3 WT α 1 -subunits, mutations S652L and S652W. Top: Top view and side view of the Cav1.3 α 1 -subunit structure. The region involving the inter-domain interactions (IIS4-S5–IS4-IS5) affected by the mutation is highlighted (left). Bottom: a WT inter-domain interaction of S652 in repeat II and S256 in the S4-S5 linker in repeat I. b Weaker hydrophobic interactions of the mutated residue L652 with the hydrophobic cloud in the S4-S5 linker of repeat I. c Stabilizing effect of the W652 mutation; the tryptophan residue can form an intra-domain hydrogen bond with the backbone of K648 and due to its aromatic character an inter-domain pi-H interaction with S256.
    Figure Legend Snippet: Molecular modeling of Cav1.3 WT α 1 -subunits, mutations S652L and S652W. Top: Top view and side view of the Cav1.3 α 1 -subunit structure. The region involving the inter-domain interactions (IIS4-S5–IS4-IS5) affected by the mutation is highlighted (left). Bottom: a WT inter-domain interaction of S652 in repeat II and S256 in the S4-S5 linker in repeat I. b Weaker hydrophobic interactions of the mutated residue L652 with the hydrophobic cloud in the S4-S5 linker of repeat I. c Stabilizing effect of the W652 mutation; the tryptophan residue can form an intra-domain hydrogen bond with the backbone of K648 and due to its aromatic character an inter-domain pi-H interaction with S256.

    Techniques Used: Mutagenesis

    Mutation S652L induces severe gating changes. a , b Current-voltage relationship ( I Ca ; mean ± SEM) of WT and mutant C-terminal long (WT L , S652L L , A) and short (WT S , S652L S , B) Cav1.3 splice variants recorded in parallel on the same day using 50-ms depolarizing square pulses to various test potentials from a holding potential (HP) of -89 mV. Inset: Representative I Ca traces upon depolarization to the potential of maximal inward current ( V max ). Statistics: two-way ANOVA followed by Bonferroni post hoc test, * p
    Figure Legend Snippet: Mutation S652L induces severe gating changes. a , b Current-voltage relationship ( I Ca ; mean ± SEM) of WT and mutant C-terminal long (WT L , S652L L , A) and short (WT S , S652L S , B) Cav1.3 splice variants recorded in parallel on the same day using 50-ms depolarizing square pulses to various test potentials from a holding potential (HP) of -89 mV. Inset: Representative I Ca traces upon depolarization to the potential of maximal inward current ( V max ). Statistics: two-way ANOVA followed by Bonferroni post hoc test, * p

    Techniques Used: Mutagenesis, Mass Spectrometry

    7) Product Images from "RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells"

    Article Title: RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells

    Journal: Pflugers Archiv

    doi: 10.1007/s00424-019-02338-4

    Modulation of Cav1.3 L /α2δ1/β2a Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8
    Figure Legend Snippet: Modulation of Cav1.3 L /α2δ1/β2a Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8

    Techniques Used: Expressing

    Modulation of Cav1.3 42A /α2δ1/β3 Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8
    Figure Legend Snippet: Modulation of Cav1.3 42A /α2δ1/β3 Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8

    Techniques Used: Expressing

    Modulation of Cav1.3 L /α2δ1/β3 Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. a Schematic illustration of measured LTCC complexes, from left to right: control (Cav1.3 L /α2δ1/β3); plus RIM2α; plus RBP2; plus RIM2α/RBP2. Data in panels b and c are shown for each recording condition. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). b I Ba inactivation time course during a 5-s long depolarization to the V max ( y -axis labels as in the left panel). Traces were normalized to the I Ba peak and are shown as mean ± SEM for the indicated number of recordings. c Voltage dependence of I Ba steady-state activation and inactivation ( y -axis label as in the left panel). For parameters and statistics, see panel d and Table 1 . d Statistics of two activation parameters ( V 0.5,act and activation threshold) are shown. e Bar graphs showing the remaining I Ba after 250, 500, 1000, or 5000 ms. Data shown as mean ± SEM. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparison post hoc test as indicated in the graph: versus control (Cav1.3 without RIM2α and/or RBP2): *** p
    Figure Legend Snippet: Modulation of Cav1.3 L /α2δ1/β3 Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. a Schematic illustration of measured LTCC complexes, from left to right: control (Cav1.3 L /α2δ1/β3); plus RIM2α; plus RBP2; plus RIM2α/RBP2. Data in panels b and c are shown for each recording condition. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). b I Ba inactivation time course during a 5-s long depolarization to the V max ( y -axis labels as in the left panel). Traces were normalized to the I Ba peak and are shown as mean ± SEM for the indicated number of recordings. c Voltage dependence of I Ba steady-state activation and inactivation ( y -axis label as in the left panel). For parameters and statistics, see panel d and Table 1 . d Statistics of two activation parameters ( V 0.5,act and activation threshold) are shown. e Bar graphs showing the remaining I Ba after 250, 500, 1000, or 5000 ms. Data shown as mean ± SEM. Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparison post hoc test as indicated in the graph: versus control (Cav1.3 without RIM2α and/or RBP2): *** p

    Techniques Used: Expressing, Activation Assay

    Comparison of RIM2α/RBP2-stabilized Cav1.3 L I Ba inactivation (β3 and β2a; tsA-201 cells) with I Ba VDI measured in IHCs. Mean I Ba (15 mM) traces of Cav1.3 L /α2δ1 with β3 (black), β3/RIM2α/RBP2 (dark red), or β2a/RIM2α/RBP2 (red) during the first 2 s of a depolarization to V max . For comparison, we recorded I Ba in mature mouse IHCs measured as recently described [ 53 ] in mature mouse IHCs (mean I Ba trace from 5 individual recordings; gray; 10 mM Ba 2+ ). Circles indicate the remaining Ba 2+ current at the indicated time points recorded from IHCs taken from previously published papers: [ 33 ] (dark blue; 10 mM Ba 2+ , mouse P20); [ 42 ] (turquoise; 5 mM Ba 2+ , mouse P40–70); [ 9 ] (purple; 5 mM Ba 2+ , mouse 2–4 weeks); [ 24 ] (yellow; 5 mM Ba 2+ , gerbil P50); [ 34 ] (green; 20 mM Ba 2+ , chicken 1–21 days). Turquoise and purple circles are overlapping and are therefore shown together as half-filled circle
    Figure Legend Snippet: Comparison of RIM2α/RBP2-stabilized Cav1.3 L I Ba inactivation (β3 and β2a; tsA-201 cells) with I Ba VDI measured in IHCs. Mean I Ba (15 mM) traces of Cav1.3 L /α2δ1 with β3 (black), β3/RIM2α/RBP2 (dark red), or β2a/RIM2α/RBP2 (red) during the first 2 s of a depolarization to V max . For comparison, we recorded I Ba in mature mouse IHCs measured as recently described [ 53 ] in mature mouse IHCs (mean I Ba trace from 5 individual recordings; gray; 10 mM Ba 2+ ). Circles indicate the remaining Ba 2+ current at the indicated time points recorded from IHCs taken from previously published papers: [ 33 ] (dark blue; 10 mM Ba 2+ , mouse P20); [ 42 ] (turquoise; 5 mM Ba 2+ , mouse P40–70); [ 9 ] (purple; 5 mM Ba 2+ , mouse 2–4 weeks); [ 24 ] (yellow; 5 mM Ba 2+ , gerbil P50); [ 34 ] (green; 20 mM Ba 2+ , chicken 1–21 days). Turquoise and purple circles are overlapping and are therefore shown together as half-filled circle

    Techniques Used:

    Modulation of Cav1.3 L /α2δ1/β2e Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8
    Figure Legend Snippet: Modulation of Cav1.3 L /α2δ1/β2e Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8

    Techniques Used: Expressing

    Modulation of VDI by β3 and different β2 subunit splic e variants (15 mM Ba 2+ ). a , b Left panels: mean (± SEM) I Ba traces for Cav1.3 L /α2δ1 ( a ) or Cav1.3 42A /α2δ1 ( b ) co-expressed with either β3 (black/gray), β2a (red), or β2e (purple). The number of individual recordings is indicated in parentheses. VDI was quantified using 15 mM Ba 2+ as charge carrier and calculated as residual I Ba at the indicated predefined time points (bar graphs). Statistical significance was determined using one-way ANOVA with Bonferroni post hoc test ( a ) or unpaired Student’s t test ( b ): *** p
    Figure Legend Snippet: Modulation of VDI by β3 and different β2 subunit splic e variants (15 mM Ba 2+ ). a , b Left panels: mean (± SEM) I Ba traces for Cav1.3 L /α2δ1 ( a ) or Cav1.3 42A /α2δ1 ( b ) co-expressed with either β3 (black/gray), β2a (red), or β2e (purple). The number of individual recordings is indicated in parentheses. VDI was quantified using 15 mM Ba 2+ as charge carrier and calculated as residual I Ba at the indicated predefined time points (bar graphs). Statistical significance was determined using one-way ANOVA with Bonferroni post hoc test ( a ) or unpaired Student’s t test ( b ): *** p

    Techniques Used:

    Interaction of RBP2 with Cav1.3 channels. RIMs and RBPs are multidomain proteins [ 41 , 46 ]. All RIM isoforms (RIM1α and 1β; RIM2α, 2β, and 2γ; RIM3γ and RIM4γ) bind via their C 2 B domain to the auxiliary β subunit of the Ca 2+ channel complex. Disruption of the SH3 or GK domain in the β subunit prevents the interaction with RIM [ 28 ]. All three RBP isoforms contain three SH3 domains and two (RBP3) or three (RBP1 and 2) FN3 domains [ 41 ]. The second SH3 domain of RBP binds to the proline-rich region (PXXP) present only in RIMα or β isoforms, located between the two C 2 domains. The other SH3 domains, marked by “x,” in turn can interact with a proline-rich region (PXXP) localized in the full-length Cav1.3 C terminus [ 23 ]. Note that incorporation of alternative exons 42A and 43S leads to short C-terminal splice variants (Cav1.3 42A or Cav1.3 43S , respectively; C-terminal ends indicated by orange dots) lacking the PXXP interaction site. AID, α-interaction domain; FN3, fibronectin 3 domain; GK, guanylate-kinase like domain; PXXP, proline-rich region; SH3, SRC homology 3 domain; Zn 2+ , zinc finger domain. Note that RIM may also interact via its C 2 B domain with the C terminus of Cav1.3, but the interaction site is unknown [ 49 ]
    Figure Legend Snippet: Interaction of RBP2 with Cav1.3 channels. RIMs and RBPs are multidomain proteins [ 41 , 46 ]. All RIM isoforms (RIM1α and 1β; RIM2α, 2β, and 2γ; RIM3γ and RIM4γ) bind via their C 2 B domain to the auxiliary β subunit of the Ca 2+ channel complex. Disruption of the SH3 or GK domain in the β subunit prevents the interaction with RIM [ 28 ]. All three RBP isoforms contain three SH3 domains and two (RBP3) or three (RBP1 and 2) FN3 domains [ 41 ]. The second SH3 domain of RBP binds to the proline-rich region (PXXP) present only in RIMα or β isoforms, located between the two C 2 domains. The other SH3 domains, marked by “x,” in turn can interact with a proline-rich region (PXXP) localized in the full-length Cav1.3 C terminus [ 23 ]. Note that incorporation of alternative exons 42A and 43S leads to short C-terminal splice variants (Cav1.3 42A or Cav1.3 43S , respectively; C-terminal ends indicated by orange dots) lacking the PXXP interaction site. AID, α-interaction domain; FN3, fibronectin 3 domain; GK, guanylate-kinase like domain; PXXP, proline-rich region; SH3, SRC homology 3 domain; Zn 2+ , zinc finger domain. Note that RIM may also interact via its C 2 B domain with the C terminus of Cav1.3, but the interaction site is unknown [ 49 ]

    Techniques Used:

    RIM, RBP, and Cav1.3 α1 subunit expression in IHCs. Control experiments in IHC preparations revealed the expected transcripts of long (containing exon 43) and short C-terminal splice variants (containing exon 43S) of Cav1.3 α1 subunits (top left). RIM2α was reliably detected in IHCs (4 out of 4 independent preparations) before (P6) and after hearing onset (after P12) (top right). RBP1 was the only isoform, which could not be detected in IHCs at any tested developmental stage (cDNA preparations from 5 different mice at different postnatal days, not shown). RBP2 transcripts (bottom left) were found only in 1 out of 5 different samples before hearing onset but were consistently detected in mature IHCs (8 out of 9 separate preparations). RBP3 transcripts (bottom right) were identified before as well as after hearing onset (6 out of 6 and 8 out of 10 independent samples, respectively). Brain samples from adult mice and reactions without template (“ctrl”) were used as positive and negative controls, respectively. Representative PCRs from > 3 independent experiments are shown
    Figure Legend Snippet: RIM, RBP, and Cav1.3 α1 subunit expression in IHCs. Control experiments in IHC preparations revealed the expected transcripts of long (containing exon 43) and short C-terminal splice variants (containing exon 43S) of Cav1.3 α1 subunits (top left). RIM2α was reliably detected in IHCs (4 out of 4 independent preparations) before (P6) and after hearing onset (after P12) (top right). RBP1 was the only isoform, which could not be detected in IHCs at any tested developmental stage (cDNA preparations from 5 different mice at different postnatal days, not shown). RBP2 transcripts (bottom left) were found only in 1 out of 5 different samples before hearing onset but were consistently detected in mature IHCs (8 out of 9 separate preparations). RBP3 transcripts (bottom right) were identified before as well as after hearing onset (6 out of 6 and 8 out of 10 independent samples, respectively). Brain samples from adult mice and reactions without template (“ctrl”) were used as positive and negative controls, respectively. Representative PCRs from > 3 independent experiments are shown

    Techniques Used: Expressing, Immunohistochemistry, Mouse Assay

    Modulation of Cav1.3 42A /α2δ1/β2a Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8
    Figure Legend Snippet: Modulation of Cav1.3 42A /α2δ1/β2a Ba 2+ currents (15 mM) by co-expression of RIM2α and/or RBP2. Color code: control (black), plus RIM2α (blue), plus RBP2 (green), and plus RIM2α/RBP2 (red). Experimental conditions and statistical analysis are as described in Fig. 8

    Techniques Used: Expressing

    RBP2 interaction with Cav1.3 C-terminal splice variants. a Schematic representation of the Cav1.3 C-terminal GST-fusion proteins: GST-Cav1.3 42 C-term (GST-42), GST-Cav1.3 42A C-term (GST-42A), and GST-Cav1.3 43S C-term (GST-43S) including the binding position for the anti-Cav1.3α1 2022–2138 antibody (anti-42) in the full-length C terminus. Numbers indicate the amino acid position in the Cav1.3 protein (GenBank™ accession number NM_000720). b GST pull-down of whole-cell extracts prepared from HEK293 cells transfected with HA-RBP2 with the indicated Cav1.3 C termini coupled to GST; 1 of 4 similar experiments is illustrated. Bound HA-RBP2 was visualized by western blotting using anti-HA. Anti-GAPDH staining served as a negative control. Input—0.5, 0.25, and 0.1% of the lysate. GST, GST-RIIβ, and GST-max p14 were control peptides not binding to HA-RBP2. Migration of molecular mass markers is indicated. c Left: Ponceau staining of GST-fusion proteins. Arrows indicate the migration of the full-length construct. Despite the partial degradation of GST-fusion proteins GST-42 and GST-42A, we observed selective protein–protein interactions between GST-42 and RBP2. Right: Immunoblot from panel b was stripped and the presence of GST-Cav1.3 42 C-term was verified by immunoblotting using anti-Cav1.3α1 2022–2138 antibody directed against an epitope present only in the long C-terminal splice variant as illustrated in panel a . d Confirmation of HA-RBP2 interaction with the long Cav1.3 C terminus by co-immunoprecipitation of HA-rRBP2 expressed in tsA-201 cells with YFP-tagged long Cav1.3 C terminus (YFP-Cav1.3 42 C-term; YFP-42). Top: Verification of the presence of YFP-Cav1.3 42 C-term by immunoblotting using an YFP antibody. Bottom: Specific immunoprecipitation of RBP2 by Cav1.3 42 C-term (detection by anti-RBP2-1318). Input control—1 and 0.5% of the lysate. Mock: untransfected control
    Figure Legend Snippet: RBP2 interaction with Cav1.3 C-terminal splice variants. a Schematic representation of the Cav1.3 C-terminal GST-fusion proteins: GST-Cav1.3 42 C-term (GST-42), GST-Cav1.3 42A C-term (GST-42A), and GST-Cav1.3 43S C-term (GST-43S) including the binding position for the anti-Cav1.3α1 2022–2138 antibody (anti-42) in the full-length C terminus. Numbers indicate the amino acid position in the Cav1.3 protein (GenBank™ accession number NM_000720). b GST pull-down of whole-cell extracts prepared from HEK293 cells transfected with HA-RBP2 with the indicated Cav1.3 C termini coupled to GST; 1 of 4 similar experiments is illustrated. Bound HA-RBP2 was visualized by western blotting using anti-HA. Anti-GAPDH staining served as a negative control. Input—0.5, 0.25, and 0.1% of the lysate. GST, GST-RIIβ, and GST-max p14 were control peptides not binding to HA-RBP2. Migration of molecular mass markers is indicated. c Left: Ponceau staining of GST-fusion proteins. Arrows indicate the migration of the full-length construct. Despite the partial degradation of GST-fusion proteins GST-42 and GST-42A, we observed selective protein–protein interactions between GST-42 and RBP2. Right: Immunoblot from panel b was stripped and the presence of GST-Cav1.3 42 C-term was verified by immunoblotting using anti-Cav1.3α1 2022–2138 antibody directed against an epitope present only in the long C-terminal splice variant as illustrated in panel a . d Confirmation of HA-RBP2 interaction with the long Cav1.3 C terminus by co-immunoprecipitation of HA-rRBP2 expressed in tsA-201 cells with YFP-tagged long Cav1.3 C terminus (YFP-Cav1.3 42 C-term; YFP-42). Top: Verification of the presence of YFP-Cav1.3 42 C-term by immunoblotting using an YFP antibody. Bottom: Specific immunoprecipitation of RBP2 by Cav1.3 42 C-term (detection by anti-RBP2-1318). Input control—1 and 0.5% of the lysate. Mock: untransfected control

    Techniques Used: Binding Assay, Transfection, Western Blot, Staining, Negative Control, Migration, Construct, Variant Assay, Immunoprecipitation

    RBP2 co-localization with Cav1.3 at ribbon synapses in mouse IHCs. a – h Maximum intensity projection (MIP) of confocal stacks of whole-mount organs of Corti with stretches of 7–8 IHCs. a – d IHCs from the apical cochlear turn of a 4-week-old NMRI mouse co-immunolabeled for Cav1.3 and RBP2 demonstrate that almost every Cav1.3 cluster co-localized with RBP2 at the basolateral pole of the IHCs ( a ), which is shown in more detail in the enlargements of the box in a ( b – d ). e – h IHCs from the apical cochlear turn of a 4-week-old NMRI mouse co-immunolabeled for the ribbon synapse marker CtBP2 and RBP2 show that almost every ribbon co-localized with RBP2 at the basolateral pole ( e ), which is shown in more detail in the enlargements of the box in e ( f – h ). Nuclei stained in blue with DAPI are shown only in the merged images. The dotted lines in a and e outline the basolateral pole of one IHC in each specimen. Scale bars: a , e , 10 μm; d , h , 5 μm
    Figure Legend Snippet: RBP2 co-localization with Cav1.3 at ribbon synapses in mouse IHCs. a – h Maximum intensity projection (MIP) of confocal stacks of whole-mount organs of Corti with stretches of 7–8 IHCs. a – d IHCs from the apical cochlear turn of a 4-week-old NMRI mouse co-immunolabeled for Cav1.3 and RBP2 demonstrate that almost every Cav1.3 cluster co-localized with RBP2 at the basolateral pole of the IHCs ( a ), which is shown in more detail in the enlargements of the box in a ( b – d ). e – h IHCs from the apical cochlear turn of a 4-week-old NMRI mouse co-immunolabeled for the ribbon synapse marker CtBP2 and RBP2 show that almost every ribbon co-localized with RBP2 at the basolateral pole ( e ), which is shown in more detail in the enlargements of the box in e ( f – h ). Nuclei stained in blue with DAPI are shown only in the merged images. The dotted lines in a and e outline the basolateral pole of one IHC in each specimen. Scale bars: a , e , 10 μm; d , h , 5 μm

    Techniques Used: Immunolabeling, Marker, Staining, Immunohistochemistry

    8) Product Images from "Down-regulation of Cav1.3 in auditory pathway promotes age-related hearing loss by enhancing calcium-mediated oxidative stress in male mice"

    Article Title: Down-regulation of Cav1.3 in auditory pathway promotes age-related hearing loss by enhancing calcium-mediated oxidative stress in male mice

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.102203

    Age-related expression of Cav1.3 in auditory pathway. ( A ) The immunofluorescence of CaV1.3 in the auditory cortex (green, magnification, ×400). ( B ) the quantitative analysis of CaV1.3 expression in the auditory cortex. ( C ) the western-blotting analysis of CaV1.3 expression in auditory cortex (top), the bottom panel is the quantitative analysis. ( D ) the mRNA expression of CaV1.3 in auditory cortex, inferior colliculus and cochlear nucleus. ( E ) CaV1.3 expression in auditory cortex was analyzed by flow cytometry, the right panel is the quantitative analysis. ( F ) Cav1.3 expression in neurons of auditory cortex, the neuron cells were gated as NeuN+, the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Age-related expression of Cav1.3 in auditory pathway. ( A ) The immunofluorescence of CaV1.3 in the auditory cortex (green, magnification, ×400). ( B ) the quantitative analysis of CaV1.3 expression in the auditory cortex. ( C ) the western-blotting analysis of CaV1.3 expression in auditory cortex (top), the bottom panel is the quantitative analysis. ( D ) the mRNA expression of CaV1.3 in auditory cortex, inferior colliculus and cochlear nucleus. ( E ) CaV1.3 expression in auditory cortex was analyzed by flow cytometry, the right panel is the quantitative analysis. ( F ) Cav1.3 expression in neurons of auditory cortex, the neuron cells were gated as NeuN+, the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P

    Techniques Used: Expressing, Immunofluorescence, Western Blot, Flow Cytometry

    Age-related Cav1.3 expression in cochlea. ( A , B ) immunofluorescence of CaV1.3(green) and Myo7a (red) in the organ of Corti (left) and spiral ganglion (right) (magnification, ×400), nuclei was visualized by DAPI (blue). ( C ) the immunofluorescent staining for CaV1.3 (green) in the whole cochlear basilar membrane. ( D ) quantitative analysis of CaV1.3 expression in hair cells, spiral ganglion and cochlea basilar membrane.
    Figure Legend Snippet: Age-related Cav1.3 expression in cochlea. ( A , B ) immunofluorescence of CaV1.3(green) and Myo7a (red) in the organ of Corti (left) and spiral ganglion (right) (magnification, ×400), nuclei was visualized by DAPI (blue). ( C ) the immunofluorescent staining for CaV1.3 (green) in the whole cochlear basilar membrane. ( D ) quantitative analysis of CaV1.3 expression in hair cells, spiral ganglion and cochlea basilar membrane.

    Techniques Used: Expressing, Immunofluorescence, Staining

    Hair cells were vulnerable to ROS injury after Cav1.3 was knocked out. ( A ) the effect of CaV1.3 knock out in HEI-OC1 was analyzed by flow cytometry. ( B ) membrane potential (top) and non-linear capacitance (NLC) (bottom) studies in WT HEI-OC1 and CaV1.3 KO HEI-OC1 cells (n=5). ( C ) western-blotting analysis of CaV1.3 and p53 expression in control and senescence HEI-OC1 cells induced by D-galactose (D-Gal) or hydrogen peroxide (H 2 O 2 ), the bottom panel is the quantitative analysis. ( D ) β-Galactosidase staining (top) and C12FDG staining (bottom) of control and senescent HEI-OC1 cells induced by H 2 O 2 . ( E ) flow cytometry analysis of CaV1.3 in control and H 2 O 2 induced HEI-OC1 cells. ( F ) C12FDG staining (top) and β-Galactosidase staining (bottom) of NC (negative control) and KO (CaV1.3 knock out) HEI-OC1 cells after H 2 O 2 induction. ( G, H ) CFSE staining and red dot staining of NC and KO HEI-OC1 cells with or without H 2 O 2 induction. ( I ) LDH assay of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). ( J ) caspase-3/7-AAD staining of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Hair cells were vulnerable to ROS injury after Cav1.3 was knocked out. ( A ) the effect of CaV1.3 knock out in HEI-OC1 was analyzed by flow cytometry. ( B ) membrane potential (top) and non-linear capacitance (NLC) (bottom) studies in WT HEI-OC1 and CaV1.3 KO HEI-OC1 cells (n=5). ( C ) western-blotting analysis of CaV1.3 and p53 expression in control and senescence HEI-OC1 cells induced by D-galactose (D-Gal) or hydrogen peroxide (H 2 O 2 ), the bottom panel is the quantitative analysis. ( D ) β-Galactosidase staining (top) and C12FDG staining (bottom) of control and senescent HEI-OC1 cells induced by H 2 O 2 . ( E ) flow cytometry analysis of CaV1.3 in control and H 2 O 2 induced HEI-OC1 cells. ( F ) C12FDG staining (top) and β-Galactosidase staining (bottom) of NC (negative control) and KO (CaV1.3 knock out) HEI-OC1 cells after H 2 O 2 induction. ( G, H ) CFSE staining and red dot staining of NC and KO HEI-OC1 cells with or without H 2 O 2 induction. ( I ) LDH assay of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). ( J ) caspase-3/7-AAD staining of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). Error bars represent mean ± s.d.; *P

    Techniques Used: Knock-Out, Flow Cytometry, Western Blot, Expressing, Staining, Negative Control, Lactate Dehydrogenase Assay

    Cav1.3 knock out decrease intra cellular calcium and subsequently result in reduction of complex I derived ROS inactivation. ( A ) intra-cellular ROS detection by flow cytometry (n=3). ( B – E ) the intra cellular calcium, intra cellular ROS, caspase-3/7-AAD staining and C12FDG staining of NC and KO HEI-OC1 cells with or without Ionmycin (n=3). ( F ) immunofluorescence of mitoSOX (red) and mitotrackor (green) in NC and KO HEI-OC1 cells, nuclei was visualized by DAPI (magnification, ×400, scal bar: 50μm), the right panels are the quantitative analysis of mitoSOX (top) and mitoTrackor (bottom). ( G ) the intra cellular ROS of KO HEI-OC1 cells with or without gradient Retenone (Ret) and Antimycin A (AMA)(n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Cav1.3 knock out decrease intra cellular calcium and subsequently result in reduction of complex I derived ROS inactivation. ( A ) intra-cellular ROS detection by flow cytometry (n=3). ( B – E ) the intra cellular calcium, intra cellular ROS, caspase-3/7-AAD staining and C12FDG staining of NC and KO HEI-OC1 cells with or without Ionmycin (n=3). ( F ) immunofluorescence of mitoSOX (red) and mitotrackor (green) in NC and KO HEI-OC1 cells, nuclei was visualized by DAPI (magnification, ×400, scal bar: 50μm), the right panels are the quantitative analysis of mitoSOX (top) and mitoTrackor (bottom). ( G ) the intra cellular ROS of KO HEI-OC1 cells with or without gradient Retenone (Ret) and Antimycin A (AMA)(n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P

    Techniques Used: Knock-Out, Derivative Assay, Flow Cytometry, Staining, Immunofluorescence

    Cav1.3 knock down aggravated the loss of hair cells after senescence induction and resulted in hearing impairment. ( A ) immunofluorescence of CaV1.3 (green) and mCherry (red) in the organ of Corti (left) and spiral ganglion (right) of control and CaV1.3 knock down AAV group, nuclei was visualized by DAPI (magnification, ×400, scale bar: 50μm), the right panels are the quantitative analysis of CaV1.3 expression in organ of corti (top) and spiral ganglion (bottom). ( B ) auditory brainstem response (ABR) (top) and the whole cochlear basilar membrane after DAPI staining (bottom) of NC, CaV1.3 knock down, NC+D-Gal and CaV1.3 knock down+D-Gal group (n=6). ( C ) phalloidine staining for control and negative control AAV or Cav1.3 knock down AAV infected OC segment explants with H 2 O 2 treatment (n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Cav1.3 knock down aggravated the loss of hair cells after senescence induction and resulted in hearing impairment. ( A ) immunofluorescence of CaV1.3 (green) and mCherry (red) in the organ of Corti (left) and spiral ganglion (right) of control and CaV1.3 knock down AAV group, nuclei was visualized by DAPI (magnification, ×400, scale bar: 50μm), the right panels are the quantitative analysis of CaV1.3 expression in organ of corti (top) and spiral ganglion (bottom). ( B ) auditory brainstem response (ABR) (top) and the whole cochlear basilar membrane after DAPI staining (bottom) of NC, CaV1.3 knock down, NC+D-Gal and CaV1.3 knock down+D-Gal group (n=6). ( C ) phalloidine staining for control and negative control AAV or Cav1.3 knock down AAV infected OC segment explants with H 2 O 2 treatment (n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P

    Techniques Used: Immunofluorescence, Expressing, Staining, Negative Control, Infection

    9) Product Images from "Down-regulation of Cav1.3 in auditory pathway promotes age-related hearing loss by enhancing calcium-mediated oxidative stress in male mice"

    Article Title: Down-regulation of Cav1.3 in auditory pathway promotes age-related hearing loss by enhancing calcium-mediated oxidative stress in male mice

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.102203

    Age-related expression of Cav1.3 in auditory pathway. ( A ) The immunofluorescence of CaV1.3 in the auditory cortex (green, magnification, ×400). ( B ) the quantitative analysis of CaV1.3 expression in the auditory cortex. ( C ) the western-blotting analysis of CaV1.3 expression in auditory cortex (top), the bottom panel is the quantitative analysis. ( D ) the mRNA expression of CaV1.3 in auditory cortex, inferior colliculus and cochlear nucleus. ( E ) CaV1.3 expression in auditory cortex was analyzed by flow cytometry, the right panel is the quantitative analysis. ( F ) Cav1.3 expression in neurons of auditory cortex, the neuron cells were gated as NeuN+, the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Age-related expression of Cav1.3 in auditory pathway. ( A ) The immunofluorescence of CaV1.3 in the auditory cortex (green, magnification, ×400). ( B ) the quantitative analysis of CaV1.3 expression in the auditory cortex. ( C ) the western-blotting analysis of CaV1.3 expression in auditory cortex (top), the bottom panel is the quantitative analysis. ( D ) the mRNA expression of CaV1.3 in auditory cortex, inferior colliculus and cochlear nucleus. ( E ) CaV1.3 expression in auditory cortex was analyzed by flow cytometry, the right panel is the quantitative analysis. ( F ) Cav1.3 expression in neurons of auditory cortex, the neuron cells were gated as NeuN+, the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P

    Techniques Used: Expressing, Immunofluorescence, Western Blot, Flow Cytometry

    Age-related Cav1.3 expression in cochlea. ( A , B ) immunofluorescence of CaV1.3(green) and Myo7a (red) in the organ of Corti (left) and spiral ganglion (right) (magnification, ×400), nuclei was visualized by DAPI (blue). ( C ) the immunofluorescent staining for CaV1.3 (green) in the whole cochlear basilar membrane. ( D ) quantitative analysis of CaV1.3 expression in hair cells, spiral ganglion and cochlea basilar membrane.
    Figure Legend Snippet: Age-related Cav1.3 expression in cochlea. ( A , B ) immunofluorescence of CaV1.3(green) and Myo7a (red) in the organ of Corti (left) and spiral ganglion (right) (magnification, ×400), nuclei was visualized by DAPI (blue). ( C ) the immunofluorescent staining for CaV1.3 (green) in the whole cochlear basilar membrane. ( D ) quantitative analysis of CaV1.3 expression in hair cells, spiral ganglion and cochlea basilar membrane.

    Techniques Used: Expressing, Immunofluorescence, Staining

    Hair cells were vulnerable to ROS injury after Cav1.3 was knocked out. ( A ) the effect of CaV1.3 knock out in HEI-OC1 was analyzed by flow cytometry. ( B ) membrane potential (top) and non-linear capacitance (NLC) (bottom) studies in WT HEI-OC1 and CaV1.3 KO HEI-OC1 cells (n=5). ( C ) western-blotting analysis of CaV1.3 and p53 expression in control and senescence HEI-OC1 cells induced by D-galactose (D-Gal) or hydrogen peroxide (H 2 O 2 ), the bottom panel is the quantitative analysis. ( D ) β-Galactosidase staining (top) and C12FDG staining (bottom) of control and senescent HEI-OC1 cells induced by H 2 O 2 . ( E ) flow cytometry analysis of CaV1.3 in control and H 2 O 2 induced HEI-OC1 cells. ( F ) C12FDG staining (top) and β-Galactosidase staining (bottom) of NC (negative control) and KO (CaV1.3 knock out) HEI-OC1 cells after H 2 O 2 induction. ( G, H ) CFSE staining and red dot staining of NC and KO HEI-OC1 cells with or without H 2 O 2 induction. ( I ) LDH assay of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). ( J ) caspase-3/7-AAD staining of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Hair cells were vulnerable to ROS injury after Cav1.3 was knocked out. ( A ) the effect of CaV1.3 knock out in HEI-OC1 was analyzed by flow cytometry. ( B ) membrane potential (top) and non-linear capacitance (NLC) (bottom) studies in WT HEI-OC1 and CaV1.3 KO HEI-OC1 cells (n=5). ( C ) western-blotting analysis of CaV1.3 and p53 expression in control and senescence HEI-OC1 cells induced by D-galactose (D-Gal) or hydrogen peroxide (H 2 O 2 ), the bottom panel is the quantitative analysis. ( D ) β-Galactosidase staining (top) and C12FDG staining (bottom) of control and senescent HEI-OC1 cells induced by H 2 O 2 . ( E ) flow cytometry analysis of CaV1.3 in control and H 2 O 2 induced HEI-OC1 cells. ( F ) C12FDG staining (top) and β-Galactosidase staining (bottom) of NC (negative control) and KO (CaV1.3 knock out) HEI-OC1 cells after H 2 O 2 induction. ( G, H ) CFSE staining and red dot staining of NC and KO HEI-OC1 cells with or without H 2 O 2 induction. ( I ) LDH assay of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). ( J ) caspase-3/7-AAD staining of NC and KO HEI-OC1 cells after H 2 O 2 induction (n=3). Error bars represent mean ± s.d.; *P

    Techniques Used: Knock-Out, Flow Cytometry, Western Blot, Expressing, Staining, Negative Control, Lactate Dehydrogenase Assay

    Cav1.3 knock out decrease intra cellular calcium and subsequently result in reduction of complex I derived ROS inactivation. ( A ) intra-cellular ROS detection by flow cytometry (n=3). ( B – E ) the intra cellular calcium, intra cellular ROS, caspase-3/7-AAD staining and C12FDG staining of NC and KO HEI-OC1 cells with or without Ionmycin (n=3). ( F ) immunofluorescence of mitoSOX (red) and mitotrackor (green) in NC and KO HEI-OC1 cells, nuclei was visualized by DAPI (magnification, ×400, scal bar: 50μm), the right panels are the quantitative analysis of mitoSOX (top) and mitoTrackor (bottom). ( G ) the intra cellular ROS of KO HEI-OC1 cells with or without gradient Retenone (Ret) and Antimycin A (AMA)(n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Cav1.3 knock out decrease intra cellular calcium and subsequently result in reduction of complex I derived ROS inactivation. ( A ) intra-cellular ROS detection by flow cytometry (n=3). ( B – E ) the intra cellular calcium, intra cellular ROS, caspase-3/7-AAD staining and C12FDG staining of NC and KO HEI-OC1 cells with or without Ionmycin (n=3). ( F ) immunofluorescence of mitoSOX (red) and mitotrackor (green) in NC and KO HEI-OC1 cells, nuclei was visualized by DAPI (magnification, ×400, scal bar: 50μm), the right panels are the quantitative analysis of mitoSOX (top) and mitoTrackor (bottom). ( G ) the intra cellular ROS of KO HEI-OC1 cells with or without gradient Retenone (Ret) and Antimycin A (AMA)(n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P

    Techniques Used: Knock-Out, Derivative Assay, Flow Cytometry, Staining, Immunofluorescence

    Cav1.3 knock down aggravated the loss of hair cells after senescence induction and resulted in hearing impairment. ( A ) immunofluorescence of CaV1.3 (green) and mCherry (red) in the organ of Corti (left) and spiral ganglion (right) of control and CaV1.3 knock down AAV group, nuclei was visualized by DAPI (magnification, ×400, scale bar: 50μm), the right panels are the quantitative analysis of CaV1.3 expression in organ of corti (top) and spiral ganglion (bottom). ( B ) auditory brainstem response (ABR) (top) and the whole cochlear basilar membrane after DAPI staining (bottom) of NC, CaV1.3 knock down, NC+D-Gal and CaV1.3 knock down+D-Gal group (n=6). ( C ) phalloidine staining for control and negative control AAV or Cav1.3 knock down AAV infected OC segment explants with H 2 O 2 treatment (n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P
    Figure Legend Snippet: Cav1.3 knock down aggravated the loss of hair cells after senescence induction and resulted in hearing impairment. ( A ) immunofluorescence of CaV1.3 (green) and mCherry (red) in the organ of Corti (left) and spiral ganglion (right) of control and CaV1.3 knock down AAV group, nuclei was visualized by DAPI (magnification, ×400, scale bar: 50μm), the right panels are the quantitative analysis of CaV1.3 expression in organ of corti (top) and spiral ganglion (bottom). ( B ) auditory brainstem response (ABR) (top) and the whole cochlear basilar membrane after DAPI staining (bottom) of NC, CaV1.3 knock down, NC+D-Gal and CaV1.3 knock down+D-Gal group (n=6). ( C ) phalloidine staining for control and negative control AAV or Cav1.3 knock down AAV infected OC segment explants with H 2 O 2 treatment (n=3), the right panel is the quantitative analysis. Error bars represent mean ± s.d.; *P

    Techniques Used: Immunofluorescence, Expressing, Staining, Negative Control, Infection

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    Alomone Labs anti cav1 3
    Graphic representation of the results. The activity-dependent signaling between the nerve terminals that are in competition through several metabotropic receptors can result in the modulation of the downstream effector kinases, specifically cPKCβI, nPKCε, and PKA. Changes in kinases activity can allow the coordinate phosphorylation of the L-type <t>CaV1.3</t> and P/Q-type VGCC. The high calcium entry through these operative channels present in immature nerve endings can result in their final loss. Also, muscle CaV1.1 and contractile activity can contribute to the synapse elimination. A component of this mechanism may be mediated by a retrograde influence from the postsynaptic site, via the BDNF-TrkB pathway, on the presynaptic calcium channels
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    Graphic representation of the results. The activity-dependent signaling between the nerve terminals that are in competition through several metabotropic receptors can result in the modulation of the downstream effector kinases, specifically cPKCβI, nPKCε, and PKA. Changes in kinases activity can allow the coordinate phosphorylation of the L-type CaV1.3 and P/Q-type VGCC. The high calcium entry through these operative channels present in immature nerve endings can result in their final loss. Also, muscle CaV1.1 and contractile activity can contribute to the synapse elimination. A component of this mechanism may be mediated by a retrograde influence from the postsynaptic site, via the BDNF-TrkB pathway, on the presynaptic calcium channels

    Journal: Molecular Neurobiology

    Article Title: Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development

    doi: 10.1007/s12035-022-02818-2

    Figure Lengend Snippet: Graphic representation of the results. The activity-dependent signaling between the nerve terminals that are in competition through several metabotropic receptors can result in the modulation of the downstream effector kinases, specifically cPKCβI, nPKCε, and PKA. Changes in kinases activity can allow the coordinate phosphorylation of the L-type CaV1.3 and P/Q-type VGCC. The high calcium entry through these operative channels present in immature nerve endings can result in their final loss. Also, muscle CaV1.1 and contractile activity can contribute to the synapse elimination. A component of this mechanism may be mediated by a retrograde influence from the postsynaptic site, via the BDNF-TrkB pathway, on the presynaptic calcium channels

    Article Snippet: Primary antibodies were incubated at 4 °C overnight (rabbit anti-P/Q-type calcium channel (1:1000; ACC-001, Alomone; Jerusalem, Israel); rabbit anti-α1D L-type calcium channel (CaV1.3, 1:500; ACC-005, Alomone, Jerusalem, Israel); rabbit anti-N-type calcium channel (1:500; ACC-002, Alomone); rabbit anti-Munc18-1 (1:1000; ≠ 13414, Cell Signalling Technology; Massachusetts, USA), and rabbit anti-PKCε (1:1000; ≠ 2683, Cell Signalling Technology; Massachusetts, USA)).

    Techniques: Activity Assay