cav3 1  (Alomone Labs)


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    Alomone Labs cav3 1
    NmbR mediates the Nmb-induced I T increase. A, western blot analysis of NmbR in mouse TGs. A prominent band was also observed in the spinal cord of mice. Blots depicted are representative of three independent experiments. B, double immunostaining of NmbR with calcitonin gene-related peptide (CGRP), isolectin B4 (IB 4 ), or neurofilament 200 (NF200) in mouse TG sections. Arrows in white indicate the co-localization. Scale bar, 30 µm. C, time course ( left panel ) and summary data ( right panel ) show the effects of 100 nM Nmb on I T in TG neurons pretreated with BIM23042 (0.5 µM) ( n = 7). Bath application of 0.5 µM BIM23042 alone did not affect I T ( n = 6). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus Nmb, two-tailed t test. D, double immunostaining of NmbR with <t>Cav3.2</t> in TG neurons of naïve mice. Arrows in white show the colocalization. Scale bars, 30 µm. Insets indicate the percentage of double-labeled neurons in NmbR + or Cav3.2 + neurons. E, total contact number of orofacial operant test with mechanical stimulation was comparable between normal saline (NS)- and CFA-treated mice. BL, baseline. F, total contact time was significantly decreased from 1 to 7 d after CFA injection, compared to NS group. * P < 0.05, ** P < 0.01 versus NS, two-way ANOVA followed by Bonferroni post hoc test. G, mechanical hypersensitivity induced by CFA injection. Decreased escape threshold was observed at the 12th h after CFA injection and lasted to ~7 d ( n = 9 mice per group). * p < 0.05 and ** p < 0.01 versus normal saline (NS) at the corresponding time point, two-way ANOVA followed by Bonferroni post hoc test. H, representative immunoblots show that Nmb expression was increased in the local inflamed tissue of mice at 2 d after CFA. Blots depicted are representative of three independent experiments. # p < 0.05 versus NS, two-tailed t test. I, abundance of NmbR protein in mouse TGs at 2 d after NS- or CFA- treatment. Blots depicted are representative of three independent experiments. ## p < 0.01 versus NS, two-tailed t test.
    Cav3 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cav3 1 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity"

    Article Title: Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity

    Journal: Theranostics

    doi: 10.7150/thno.62255

    NmbR mediates the Nmb-induced I T increase. A, western blot analysis of NmbR in mouse TGs. A prominent band was also observed in the spinal cord of mice. Blots depicted are representative of three independent experiments. B, double immunostaining of NmbR with calcitonin gene-related peptide (CGRP), isolectin B4 (IB 4 ), or neurofilament 200 (NF200) in mouse TG sections. Arrows in white indicate the co-localization. Scale bar, 30 µm. C, time course ( left panel ) and summary data ( right panel ) show the effects of 100 nM Nmb on I T in TG neurons pretreated with BIM23042 (0.5 µM) ( n = 7). Bath application of 0.5 µM BIM23042 alone did not affect I T ( n = 6). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus Nmb, two-tailed t test. D, double immunostaining of NmbR with Cav3.2 in TG neurons of naïve mice. Arrows in white show the colocalization. Scale bars, 30 µm. Insets indicate the percentage of double-labeled neurons in NmbR + or Cav3.2 + neurons. E, total contact number of orofacial operant test with mechanical stimulation was comparable between normal saline (NS)- and CFA-treated mice. BL, baseline. F, total contact time was significantly decreased from 1 to 7 d after CFA injection, compared to NS group. * P < 0.05, ** P < 0.01 versus NS, two-way ANOVA followed by Bonferroni post hoc test. G, mechanical hypersensitivity induced by CFA injection. Decreased escape threshold was observed at the 12th h after CFA injection and lasted to ~7 d ( n = 9 mice per group). * p < 0.05 and ** p < 0.01 versus normal saline (NS) at the corresponding time point, two-way ANOVA followed by Bonferroni post hoc test. H, representative immunoblots show that Nmb expression was increased in the local inflamed tissue of mice at 2 d after CFA. Blots depicted are representative of three independent experiments. # p < 0.05 versus NS, two-tailed t test. I, abundance of NmbR protein in mouse TGs at 2 d after NS- or CFA- treatment. Blots depicted are representative of three independent experiments. ## p < 0.01 versus NS, two-tailed t test.
    Figure Legend Snippet: NmbR mediates the Nmb-induced I T increase. A, western blot analysis of NmbR in mouse TGs. A prominent band was also observed in the spinal cord of mice. Blots depicted are representative of three independent experiments. B, double immunostaining of NmbR with calcitonin gene-related peptide (CGRP), isolectin B4 (IB 4 ), or neurofilament 200 (NF200) in mouse TG sections. Arrows in white indicate the co-localization. Scale bar, 30 µm. C, time course ( left panel ) and summary data ( right panel ) show the effects of 100 nM Nmb on I T in TG neurons pretreated with BIM23042 (0.5 µM) ( n = 7). Bath application of 0.5 µM BIM23042 alone did not affect I T ( n = 6). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus Nmb, two-tailed t test. D, double immunostaining of NmbR with Cav3.2 in TG neurons of naïve mice. Arrows in white show the colocalization. Scale bars, 30 µm. Insets indicate the percentage of double-labeled neurons in NmbR + or Cav3.2 + neurons. E, total contact number of orofacial operant test with mechanical stimulation was comparable between normal saline (NS)- and CFA-treated mice. BL, baseline. F, total contact time was significantly decreased from 1 to 7 d after CFA injection, compared to NS group. * P < 0.05, ** P < 0.01 versus NS, two-way ANOVA followed by Bonferroni post hoc test. G, mechanical hypersensitivity induced by CFA injection. Decreased escape threshold was observed at the 12th h after CFA injection and lasted to ~7 d ( n = 9 mice per group). * p < 0.05 and ** p < 0.01 versus normal saline (NS) at the corresponding time point, two-way ANOVA followed by Bonferroni post hoc test. H, representative immunoblots show that Nmb expression was increased in the local inflamed tissue of mice at 2 d after CFA. Blots depicted are representative of three independent experiments. # p < 0.05 versus NS, two-tailed t test. I, abundance of NmbR protein in mouse TGs at 2 d after NS- or CFA- treatment. Blots depicted are representative of three independent experiments. ## p < 0.01 versus NS, two-tailed t test.

    Techniques Used: Western Blot, Double Immunostaining, Two Tailed Test, Labeling, Injection, Expressing

    The G βγ -dependent AMPK/PKA pathway mediates the NmbR response. A, time course of I T changes induced by 100 nM Nmb in TG neurons dialyzed with QEHA (10 μM). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. B, summary data indicate the effect of 100 nM Nmb on I T in TG neurons dialyzed with QEHA (10 μM, n = 6) or SKEE (10 μM, n = 7). ** p < 0.01 versus control, two-tailed t test. C, protein expression of G β in cells transduced with G β shRNA (G β -shRNA) or negative control shRNA (NC-shRNA). Blots depicted are representative of three independent experiments. # p < 0.05 versus NC-shRNA, two-tailed t test. D, summary data show the effect of Nmb (100 nM) on I T in TG neurons transduced with NC-shRNA ( n = 9) or G β -shRNA ( n = 8). * p < 0.05 versus NC-shRNA + control, one-way ANOVA followed by Bonferroni post hoc test. E, time course ( left panel ) and summary data ( right panel ) show the effect of Nmb (100 nM) on I T in cells pre-incubated with KT-5720 (1 μM, n = 7). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. ** p < 0.01 versus control, two-tailed t test. F, exemplary current traces and summary of results show the effect of forskolin (10 µM) on I T in TG neurons in the absence ( n = 7) or presence of KT-5720 (1 μM, n = 6). ** p < 0.01 versus control, two-tailed t test. G, representative immunoblot and summary data show that treatment of TG cells with 100 nM Nmb did not affect the protein expression level of Cav3.2. GAPDH was used as the loading control. H, pretreatment of TG cells with 0.5 µM BIM23042, but not 1 μM KT-5720, attenuates the increased expression level of phosphorylated AMPK ( p -AMPK) induced by 100 nM Nmb. GAPDH was used as the loading control. Blots depicted are representative of three independent experiments. * p < 0.05 versus control, two-tailed t test. I, summary data show the effect of 100 nM Nmb on PKA activity in TG cells pretreated with 0.5 µM BIM23042 or 10 µM compound C. All experiments were performed in triplicate with similar results. # p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. J, time course ( left panel ) and summary of results ( right panel ) indicate the effect of 100 nM Nmb on I T in TG neurons pretreated with compound C (10 µM, n = 7). Inset s show the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus control, two-tailed t test.
    Figure Legend Snippet: The G βγ -dependent AMPK/PKA pathway mediates the NmbR response. A, time course of I T changes induced by 100 nM Nmb in TG neurons dialyzed with QEHA (10 μM). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. B, summary data indicate the effect of 100 nM Nmb on I T in TG neurons dialyzed with QEHA (10 μM, n = 6) or SKEE (10 μM, n = 7). ** p < 0.01 versus control, two-tailed t test. C, protein expression of G β in cells transduced with G β shRNA (G β -shRNA) or negative control shRNA (NC-shRNA). Blots depicted are representative of three independent experiments. # p < 0.05 versus NC-shRNA, two-tailed t test. D, summary data show the effect of Nmb (100 nM) on I T in TG neurons transduced with NC-shRNA ( n = 9) or G β -shRNA ( n = 8). * p < 0.05 versus NC-shRNA + control, one-way ANOVA followed by Bonferroni post hoc test. E, time course ( left panel ) and summary data ( right panel ) show the effect of Nmb (100 nM) on I T in cells pre-incubated with KT-5720 (1 μM, n = 7). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. ** p < 0.01 versus control, two-tailed t test. F, exemplary current traces and summary of results show the effect of forskolin (10 µM) on I T in TG neurons in the absence ( n = 7) or presence of KT-5720 (1 μM, n = 6). ** p < 0.01 versus control, two-tailed t test. G, representative immunoblot and summary data show that treatment of TG cells with 100 nM Nmb did not affect the protein expression level of Cav3.2. GAPDH was used as the loading control. H, pretreatment of TG cells with 0.5 µM BIM23042, but not 1 μM KT-5720, attenuates the increased expression level of phosphorylated AMPK ( p -AMPK) induced by 100 nM Nmb. GAPDH was used as the loading control. Blots depicted are representative of three independent experiments. * p < 0.05 versus control, two-tailed t test. I, summary data show the effect of 100 nM Nmb on PKA activity in TG cells pretreated with 0.5 µM BIM23042 or 10 µM compound C. All experiments were performed in triplicate with similar results. # p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. J, time course ( left panel ) and summary of results ( right panel ) indicate the effect of 100 nM Nmb on I T in TG neurons pretreated with compound C (10 µM, n = 7). Inset s show the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus control, two-tailed t test.

    Techniques Used: Two Tailed Test, Expressing, Transduction, shRNA, Negative Control, Incubation, Western Blot, Activity Assay

    Activation of NmbR stimulates recombinant Cav3.2 channels heterologously expressed in HEK293 cells. A, western blot analysis of NmbR in HEK293 cells transiently transfected with NmbR cDNA. Blots depicted are representative of three independent experiments. B, membrane localization of NmbR in transfected HEK293 cells. Alphabets a through c in the diagram indicate the differential interference contrast (DIC, a ), the EGFP fluorescent signals of NmbR ( b ), and the merged image ( c ), respectively. C, exemplary current traces show the effect of Nmb (100 nM) on Cav3.1 (α1G), Cav3.2 (α1H) and Cav3.3 (α1I) channel currents. Currents were elicited by a 100 ms depolarizing step pulse from the holding potential of -110 mV to -30 mV. D, summary data show the effect of 100 nM Nmb on Cav3.1 ( n = 10), Cav3.2 ( n = 9) and Cav3.3 ( n = 7) channel currents. ** p < 0.01 versus control, two-tailed t test. E, dose-dependent effects of Nmb on Cav3.2 channel currents. Solid line represents the sigmoidal dose-response fits. Numbers in parentheses denote n cells tested at each concentration. F, summary data show the effect of Nmb (100 nM) on Cav3.2 channel currents in cells pre-incubated with 10 µM compound C ( n = 8), in cells pretreated with 1 µM KT-5720 ( n = 9), and in cells dialyzed with 10 μM PKC 19-36 ( n = 9), respectively. ** p < 0.01 and *** p < 0.001 versus control, two-tailed t test.
    Figure Legend Snippet: Activation of NmbR stimulates recombinant Cav3.2 channels heterologously expressed in HEK293 cells. A, western blot analysis of NmbR in HEK293 cells transiently transfected with NmbR cDNA. Blots depicted are representative of three independent experiments. B, membrane localization of NmbR in transfected HEK293 cells. Alphabets a through c in the diagram indicate the differential interference contrast (DIC, a ), the EGFP fluorescent signals of NmbR ( b ), and the merged image ( c ), respectively. C, exemplary current traces show the effect of Nmb (100 nM) on Cav3.1 (α1G), Cav3.2 (α1H) and Cav3.3 (α1I) channel currents. Currents were elicited by a 100 ms depolarizing step pulse from the holding potential of -110 mV to -30 mV. D, summary data show the effect of 100 nM Nmb on Cav3.1 ( n = 10), Cav3.2 ( n = 9) and Cav3.3 ( n = 7) channel currents. ** p < 0.01 versus control, two-tailed t test. E, dose-dependent effects of Nmb on Cav3.2 channel currents. Solid line represents the sigmoidal dose-response fits. Numbers in parentheses denote n cells tested at each concentration. F, summary data show the effect of Nmb (100 nM) on Cav3.2 channel currents in cells pre-incubated with 10 µM compound C ( n = 8), in cells pretreated with 1 µM KT-5720 ( n = 9), and in cells dialyzed with 10 μM PKC 19-36 ( n = 9), respectively. ** p < 0.01 and *** p < 0.001 versus control, two-tailed t test.

    Techniques Used: Activation Assay, Recombinant, Western Blot, Transfection, Two Tailed Test, Concentration Assay, Incubation

    Involvement of peripheral NmbR in mechanical pain hypersensitivity. A, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces mechanical hypersensitivity ( n = 6 - 8 mice per group). * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. BL, baseline. B, the total contact time assessed by orofacial operant test was significantly decreased 1 h after intra-TG injection of 1 nmol Nmb, compared to vehicle group. * p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. C, pretreatment of BIM23042 (5 nmol), KT-5720 (2 nmol), Z941 (2 nmol) or TTA-P2 (1 nmol) significantly attenuates 1 nmol Nmb-induced mechanical hypersensitivity ( n = 8 - 10 mice per group). * p < 0.05 versus vehicle at 1 h, + p < 0.05 versus Z941 + vehicle, # p < 0.05 versus TTA-P2 + vehicle, one-way ANOVA followed by Bonferroni post hoc test. D, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces heat hypersensitivity. * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. E, intra-TG administration of BIM23042 (5 nmol) prevented 1 nmol Nmb-induced heat hypersensitivity (n = 6 - 8 mice per group). * p < 0.05 versus vehicle at 1 h, one-way ANOVA followed by Bonferroni post hoc test. F, rofecoxib (1 mg/kg administrated subcutaneously) reduced the mechanical allodynia produced by CFA. Behavioral analysis was performed at 2 d after CFA injection, and the effect of rofecoxib was assessed at 1 h after administration (n = 7 - 9 mice per group). ** p < 0.01 versus basline (BL) in vehicle group, ## p < 0.01 versus basline (BL) in rofecoxib group, ++ p < 0.01 versus vehicle at 1 h. G, intra-TG injection of BIM23042 (5 nmol) 2 d after CFA injection significantly suppresses the mechanical hypersensitivity in CFA-treated group ( n = 7 - 9 mice per group). * p < 0.05 and ** p < 0.01 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. H-I, intra-TG administration of Cav3.2 siRNA resulted in a significant decrease of Cav3.2 protein abundance in mouse TGs ( H ), while the expression level of Cav3.1 or Cav3.3 ( I ) remained unchanged. The blots shown are representative of three independent experiments. # p < 0.05 versus NC-siRNA, two-tailed t test. J, siRNA knock-down of Cav3.2 attenuates the BIM23042-induced alleviation of mechanical allodynia in CFA-treated mice ( n = 8 - 10 mice per group). * p < 0.01 and ** p < 0.01 versus NC-siRNA in CFA-treated mice; ++ p < 0.01 versus CFA 2 d; # p < 0.05 versus 0 h point in NC-siRNA-treated mice, two-way ANOVA followed by Bonferroni post hoc test. The black arrow indicates the intra-TG injection of 5 nmol BIM23042.
    Figure Legend Snippet: Involvement of peripheral NmbR in mechanical pain hypersensitivity. A, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces mechanical hypersensitivity ( n = 6 - 8 mice per group). * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. BL, baseline. B, the total contact time assessed by orofacial operant test was significantly decreased 1 h after intra-TG injection of 1 nmol Nmb, compared to vehicle group. * p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. C, pretreatment of BIM23042 (5 nmol), KT-5720 (2 nmol), Z941 (2 nmol) or TTA-P2 (1 nmol) significantly attenuates 1 nmol Nmb-induced mechanical hypersensitivity ( n = 8 - 10 mice per group). * p < 0.05 versus vehicle at 1 h, + p < 0.05 versus Z941 + vehicle, # p < 0.05 versus TTA-P2 + vehicle, one-way ANOVA followed by Bonferroni post hoc test. D, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces heat hypersensitivity. * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. E, intra-TG administration of BIM23042 (5 nmol) prevented 1 nmol Nmb-induced heat hypersensitivity (n = 6 - 8 mice per group). * p < 0.05 versus vehicle at 1 h, one-way ANOVA followed by Bonferroni post hoc test. F, rofecoxib (1 mg/kg administrated subcutaneously) reduced the mechanical allodynia produced by CFA. Behavioral analysis was performed at 2 d after CFA injection, and the effect of rofecoxib was assessed at 1 h after administration (n = 7 - 9 mice per group). ** p < 0.01 versus basline (BL) in vehicle group, ## p < 0.01 versus basline (BL) in rofecoxib group, ++ p < 0.01 versus vehicle at 1 h. G, intra-TG injection of BIM23042 (5 nmol) 2 d after CFA injection significantly suppresses the mechanical hypersensitivity in CFA-treated group ( n = 7 - 9 mice per group). * p < 0.05 and ** p < 0.01 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. H-I, intra-TG administration of Cav3.2 siRNA resulted in a significant decrease of Cav3.2 protein abundance in mouse TGs ( H ), while the expression level of Cav3.1 or Cav3.3 ( I ) remained unchanged. The blots shown are representative of three independent experiments. # p < 0.05 versus NC-siRNA, two-tailed t test. J, siRNA knock-down of Cav3.2 attenuates the BIM23042-induced alleviation of mechanical allodynia in CFA-treated mice ( n = 8 - 10 mice per group). * p < 0.01 and ** p < 0.01 versus NC-siRNA in CFA-treated mice; ++ p < 0.01 versus CFA 2 d; # p < 0.05 versus 0 h point in NC-siRNA-treated mice, two-way ANOVA followed by Bonferroni post hoc test. The black arrow indicates the intra-TG injection of 5 nmol BIM23042.

    Techniques Used: Injection, Produced, Expressing, Two Tailed Test

    Proposed mechanisms of NmbR signalling on Cav3.2 channels in TG neurons. The binding of Nmb triggers NmbR to activate G q protein and releases the G βγ subunits. The released G βγ dimer then stimulates AMPK activity, induces the activation of PKA and subsequently phosphorylates Cav3.2 channels to increase I T . Neither PLC/PKC nor direct binding of G βγ to Cav3.2 channels is required for T-type channel response mediated by NmbR.
    Figure Legend Snippet: Proposed mechanisms of NmbR signalling on Cav3.2 channels in TG neurons. The binding of Nmb triggers NmbR to activate G q protein and releases the G βγ subunits. The released G βγ dimer then stimulates AMPK activity, induces the activation of PKA and subsequently phosphorylates Cav3.2 channels to increase I T . Neither PLC/PKC nor direct binding of G βγ to Cav3.2 channels is required for T-type channel response mediated by NmbR.

    Techniques Used: Binding Assay, Activity Assay, Activation Assay

    cav3 1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
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    Structured Review

    Alomone Labs cav3 1
    NmbR mediates the Nmb-induced I T increase. A, western blot analysis of NmbR in mouse TGs. A prominent band was also observed in the spinal cord of mice. Blots depicted are representative of three independent experiments. B, double immunostaining of NmbR with calcitonin gene-related peptide (CGRP), isolectin B4 (IB 4 ), or neurofilament 200 (NF200) in mouse TG sections. Arrows in white indicate the co-localization. Scale bar, 30 µm. C, time course ( left panel ) and summary data ( right panel ) show the effects of 100 nM Nmb on I T in TG neurons pretreated with BIM23042 (0.5 µM) ( n = 7). Bath application of 0.5 µM BIM23042 alone did not affect I T ( n = 6). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus Nmb, two-tailed t test. D, double immunostaining of NmbR with <t>Cav3.2</t> in TG neurons of naïve mice. Arrows in white show the colocalization. Scale bars, 30 µm. Insets indicate the percentage of double-labeled neurons in NmbR + or Cav3.2 + neurons. E, total contact number of orofacial operant test with mechanical stimulation was comparable between normal saline (NS)- and CFA-treated mice. BL, baseline. F, total contact time was significantly decreased from 1 to 7 d after CFA injection, compared to NS group. * P < 0.05, ** P < 0.01 versus NS, two-way ANOVA followed by Bonferroni post hoc test. G, mechanical hypersensitivity induced by CFA injection. Decreased escape threshold was observed at the 12th h after CFA injection and lasted to ~7 d ( n = 9 mice per group). * p < 0.05 and ** p < 0.01 versus normal saline (NS) at the corresponding time point, two-way ANOVA followed by Bonferroni post hoc test. H, representative immunoblots show that Nmb expression was increased in the local inflamed tissue of mice at 2 d after CFA. Blots depicted are representative of three independent experiments. # p < 0.05 versus NS, two-tailed t test. I, abundance of NmbR protein in mouse TGs at 2 d after NS- or CFA- treatment. Blots depicted are representative of three independent experiments. ## p < 0.01 versus NS, two-tailed t test.
    Cav3 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cav3 1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cav3 1 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity"

    Article Title: Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity

    Journal: Theranostics

    doi: 10.7150/thno.62255

    NmbR mediates the Nmb-induced I T increase. A, western blot analysis of NmbR in mouse TGs. A prominent band was also observed in the spinal cord of mice. Blots depicted are representative of three independent experiments. B, double immunostaining of NmbR with calcitonin gene-related peptide (CGRP), isolectin B4 (IB 4 ), or neurofilament 200 (NF200) in mouse TG sections. Arrows in white indicate the co-localization. Scale bar, 30 µm. C, time course ( left panel ) and summary data ( right panel ) show the effects of 100 nM Nmb on I T in TG neurons pretreated with BIM23042 (0.5 µM) ( n = 7). Bath application of 0.5 µM BIM23042 alone did not affect I T ( n = 6). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus Nmb, two-tailed t test. D, double immunostaining of NmbR with Cav3.2 in TG neurons of naïve mice. Arrows in white show the colocalization. Scale bars, 30 µm. Insets indicate the percentage of double-labeled neurons in NmbR + or Cav3.2 + neurons. E, total contact number of orofacial operant test with mechanical stimulation was comparable between normal saline (NS)- and CFA-treated mice. BL, baseline. F, total contact time was significantly decreased from 1 to 7 d after CFA injection, compared to NS group. * P < 0.05, ** P < 0.01 versus NS, two-way ANOVA followed by Bonferroni post hoc test. G, mechanical hypersensitivity induced by CFA injection. Decreased escape threshold was observed at the 12th h after CFA injection and lasted to ~7 d ( n = 9 mice per group). * p < 0.05 and ** p < 0.01 versus normal saline (NS) at the corresponding time point, two-way ANOVA followed by Bonferroni post hoc test. H, representative immunoblots show that Nmb expression was increased in the local inflamed tissue of mice at 2 d after CFA. Blots depicted are representative of three independent experiments. # p < 0.05 versus NS, two-tailed t test. I, abundance of NmbR protein in mouse TGs at 2 d after NS- or CFA- treatment. Blots depicted are representative of three independent experiments. ## p < 0.01 versus NS, two-tailed t test.
    Figure Legend Snippet: NmbR mediates the Nmb-induced I T increase. A, western blot analysis of NmbR in mouse TGs. A prominent band was also observed in the spinal cord of mice. Blots depicted are representative of three independent experiments. B, double immunostaining of NmbR with calcitonin gene-related peptide (CGRP), isolectin B4 (IB 4 ), or neurofilament 200 (NF200) in mouse TG sections. Arrows in white indicate the co-localization. Scale bar, 30 µm. C, time course ( left panel ) and summary data ( right panel ) show the effects of 100 nM Nmb on I T in TG neurons pretreated with BIM23042 (0.5 µM) ( n = 7). Bath application of 0.5 µM BIM23042 alone did not affect I T ( n = 6). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus Nmb, two-tailed t test. D, double immunostaining of NmbR with Cav3.2 in TG neurons of naïve mice. Arrows in white show the colocalization. Scale bars, 30 µm. Insets indicate the percentage of double-labeled neurons in NmbR + or Cav3.2 + neurons. E, total contact number of orofacial operant test with mechanical stimulation was comparable between normal saline (NS)- and CFA-treated mice. BL, baseline. F, total contact time was significantly decreased from 1 to 7 d after CFA injection, compared to NS group. * P < 0.05, ** P < 0.01 versus NS, two-way ANOVA followed by Bonferroni post hoc test. G, mechanical hypersensitivity induced by CFA injection. Decreased escape threshold was observed at the 12th h after CFA injection and lasted to ~7 d ( n = 9 mice per group). * p < 0.05 and ** p < 0.01 versus normal saline (NS) at the corresponding time point, two-way ANOVA followed by Bonferroni post hoc test. H, representative immunoblots show that Nmb expression was increased in the local inflamed tissue of mice at 2 d after CFA. Blots depicted are representative of three independent experiments. # p < 0.05 versus NS, two-tailed t test. I, abundance of NmbR protein in mouse TGs at 2 d after NS- or CFA- treatment. Blots depicted are representative of three independent experiments. ## p < 0.01 versus NS, two-tailed t test.

    Techniques Used: Western Blot, Double Immunostaining, Two Tailed Test, Labeling, Injection, Expressing

    The G βγ -dependent AMPK/PKA pathway mediates the NmbR response. A, time course of I T changes induced by 100 nM Nmb in TG neurons dialyzed with QEHA (10 μM). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. B, summary data indicate the effect of 100 nM Nmb on I T in TG neurons dialyzed with QEHA (10 μM, n = 6) or SKEE (10 μM, n = 7). ** p < 0.01 versus control, two-tailed t test. C, protein expression of G β in cells transduced with G β shRNA (G β -shRNA) or negative control shRNA (NC-shRNA). Blots depicted are representative of three independent experiments. # p < 0.05 versus NC-shRNA, two-tailed t test. D, summary data show the effect of Nmb (100 nM) on I T in TG neurons transduced with NC-shRNA ( n = 9) or G β -shRNA ( n = 8). * p < 0.05 versus NC-shRNA + control, one-way ANOVA followed by Bonferroni post hoc test. E, time course ( left panel ) and summary data ( right panel ) show the effect of Nmb (100 nM) on I T in cells pre-incubated with KT-5720 (1 μM, n = 7). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. ** p < 0.01 versus control, two-tailed t test. F, exemplary current traces and summary of results show the effect of forskolin (10 µM) on I T in TG neurons in the absence ( n = 7) or presence of KT-5720 (1 μM, n = 6). ** p < 0.01 versus control, two-tailed t test. G, representative immunoblot and summary data show that treatment of TG cells with 100 nM Nmb did not affect the protein expression level of Cav3.2. GAPDH was used as the loading control. H, pretreatment of TG cells with 0.5 µM BIM23042, but not 1 μM KT-5720, attenuates the increased expression level of phosphorylated AMPK ( p -AMPK) induced by 100 nM Nmb. GAPDH was used as the loading control. Blots depicted are representative of three independent experiments. * p < 0.05 versus control, two-tailed t test. I, summary data show the effect of 100 nM Nmb on PKA activity in TG cells pretreated with 0.5 µM BIM23042 or 10 µM compound C. All experiments were performed in triplicate with similar results. # p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. J, time course ( left panel ) and summary of results ( right panel ) indicate the effect of 100 nM Nmb on I T in TG neurons pretreated with compound C (10 µM, n = 7). Inset s show the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus control, two-tailed t test.
    Figure Legend Snippet: The G βγ -dependent AMPK/PKA pathway mediates the NmbR response. A, time course of I T changes induced by 100 nM Nmb in TG neurons dialyzed with QEHA (10 μM). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. B, summary data indicate the effect of 100 nM Nmb on I T in TG neurons dialyzed with QEHA (10 μM, n = 6) or SKEE (10 μM, n = 7). ** p < 0.01 versus control, two-tailed t test. C, protein expression of G β in cells transduced with G β shRNA (G β -shRNA) or negative control shRNA (NC-shRNA). Blots depicted are representative of three independent experiments. # p < 0.05 versus NC-shRNA, two-tailed t test. D, summary data show the effect of Nmb (100 nM) on I T in TG neurons transduced with NC-shRNA ( n = 9) or G β -shRNA ( n = 8). * p < 0.05 versus NC-shRNA + control, one-way ANOVA followed by Bonferroni post hoc test. E, time course ( left panel ) and summary data ( right panel ) show the effect of Nmb (100 nM) on I T in cells pre-incubated with KT-5720 (1 μM, n = 7). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. ** p < 0.01 versus control, two-tailed t test. F, exemplary current traces and summary of results show the effect of forskolin (10 µM) on I T in TG neurons in the absence ( n = 7) or presence of KT-5720 (1 μM, n = 6). ** p < 0.01 versus control, two-tailed t test. G, representative immunoblot and summary data show that treatment of TG cells with 100 nM Nmb did not affect the protein expression level of Cav3.2. GAPDH was used as the loading control. H, pretreatment of TG cells with 0.5 µM BIM23042, but not 1 μM KT-5720, attenuates the increased expression level of phosphorylated AMPK ( p -AMPK) induced by 100 nM Nmb. GAPDH was used as the loading control. Blots depicted are representative of three independent experiments. * p < 0.05 versus control, two-tailed t test. I, summary data show the effect of 100 nM Nmb on PKA activity in TG cells pretreated with 0.5 µM BIM23042 or 10 µM compound C. All experiments were performed in triplicate with similar results. # p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. J, time course ( left panel ) and summary of results ( right panel ) indicate the effect of 100 nM Nmb on I T in TG neurons pretreated with compound C (10 µM, n = 7). Inset s show the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus control, two-tailed t test.

    Techniques Used: Two Tailed Test, Expressing, Transduction, shRNA, Negative Control, Incubation, Western Blot, Activity Assay

    Activation of NmbR stimulates recombinant Cav3.2 channels heterologously expressed in HEK293 cells. A, western blot analysis of NmbR in HEK293 cells transiently transfected with NmbR cDNA. Blots depicted are representative of three independent experiments. B, membrane localization of NmbR in transfected HEK293 cells. Alphabets a through c in the diagram indicate the differential interference contrast (DIC, a ), the EGFP fluorescent signals of NmbR ( b ), and the merged image ( c ), respectively. C, exemplary current traces show the effect of Nmb (100 nM) on Cav3.1 (α1G), Cav3.2 (α1H) and Cav3.3 (α1I) channel currents. Currents were elicited by a 100 ms depolarizing step pulse from the holding potential of -110 mV to -30 mV. D, summary data show the effect of 100 nM Nmb on Cav3.1 ( n = 10), Cav3.2 ( n = 9) and Cav3.3 ( n = 7) channel currents. ** p < 0.01 versus control, two-tailed t test. E, dose-dependent effects of Nmb on Cav3.2 channel currents. Solid line represents the sigmoidal dose-response fits. Numbers in parentheses denote n cells tested at each concentration. F, summary data show the effect of Nmb (100 nM) on Cav3.2 channel currents in cells pre-incubated with 10 µM compound C ( n = 8), in cells pretreated with 1 µM KT-5720 ( n = 9), and in cells dialyzed with 10 μM PKC 19-36 ( n = 9), respectively. ** p < 0.01 and *** p < 0.001 versus control, two-tailed t test.
    Figure Legend Snippet: Activation of NmbR stimulates recombinant Cav3.2 channels heterologously expressed in HEK293 cells. A, western blot analysis of NmbR in HEK293 cells transiently transfected with NmbR cDNA. Blots depicted are representative of three independent experiments. B, membrane localization of NmbR in transfected HEK293 cells. Alphabets a through c in the diagram indicate the differential interference contrast (DIC, a ), the EGFP fluorescent signals of NmbR ( b ), and the merged image ( c ), respectively. C, exemplary current traces show the effect of Nmb (100 nM) on Cav3.1 (α1G), Cav3.2 (α1H) and Cav3.3 (α1I) channel currents. Currents were elicited by a 100 ms depolarizing step pulse from the holding potential of -110 mV to -30 mV. D, summary data show the effect of 100 nM Nmb on Cav3.1 ( n = 10), Cav3.2 ( n = 9) and Cav3.3 ( n = 7) channel currents. ** p < 0.01 versus control, two-tailed t test. E, dose-dependent effects of Nmb on Cav3.2 channel currents. Solid line represents the sigmoidal dose-response fits. Numbers in parentheses denote n cells tested at each concentration. F, summary data show the effect of Nmb (100 nM) on Cav3.2 channel currents in cells pre-incubated with 10 µM compound C ( n = 8), in cells pretreated with 1 µM KT-5720 ( n = 9), and in cells dialyzed with 10 μM PKC 19-36 ( n = 9), respectively. ** p < 0.01 and *** p < 0.001 versus control, two-tailed t test.

    Techniques Used: Activation Assay, Recombinant, Western Blot, Transfection, Two Tailed Test, Concentration Assay, Incubation

    Involvement of peripheral NmbR in mechanical pain hypersensitivity. A, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces mechanical hypersensitivity ( n = 6 - 8 mice per group). * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. BL, baseline. B, the total contact time assessed by orofacial operant test was significantly decreased 1 h after intra-TG injection of 1 nmol Nmb, compared to vehicle group. * p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. C, pretreatment of BIM23042 (5 nmol), KT-5720 (2 nmol), Z941 (2 nmol) or TTA-P2 (1 nmol) significantly attenuates 1 nmol Nmb-induced mechanical hypersensitivity ( n = 8 - 10 mice per group). * p < 0.05 versus vehicle at 1 h, + p < 0.05 versus Z941 + vehicle, # p < 0.05 versus TTA-P2 + vehicle, one-way ANOVA followed by Bonferroni post hoc test. D, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces heat hypersensitivity. * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. E, intra-TG administration of BIM23042 (5 nmol) prevented 1 nmol Nmb-induced heat hypersensitivity (n = 6 - 8 mice per group). * p < 0.05 versus vehicle at 1 h, one-way ANOVA followed by Bonferroni post hoc test. F, rofecoxib (1 mg/kg administrated subcutaneously) reduced the mechanical allodynia produced by CFA. Behavioral analysis was performed at 2 d after CFA injection, and the effect of rofecoxib was assessed at 1 h after administration (n = 7 - 9 mice per group). ** p < 0.01 versus basline (BL) in vehicle group, ## p < 0.01 versus basline (BL) in rofecoxib group, ++ p < 0.01 versus vehicle at 1 h. G, intra-TG injection of BIM23042 (5 nmol) 2 d after CFA injection significantly suppresses the mechanical hypersensitivity in CFA-treated group ( n = 7 - 9 mice per group). * p < 0.05 and ** p < 0.01 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. H-I, intra-TG administration of Cav3.2 siRNA resulted in a significant decrease of Cav3.2 protein abundance in mouse TGs ( H ), while the expression level of Cav3.1 or Cav3.3 ( I ) remained unchanged. The blots shown are representative of three independent experiments. # p < 0.05 versus NC-siRNA, two-tailed t test. J, siRNA knock-down of Cav3.2 attenuates the BIM23042-induced alleviation of mechanical allodynia in CFA-treated mice ( n = 8 - 10 mice per group). * p < 0.01 and ** p < 0.01 versus NC-siRNA in CFA-treated mice; ++ p < 0.01 versus CFA 2 d; # p < 0.05 versus 0 h point in NC-siRNA-treated mice, two-way ANOVA followed by Bonferroni post hoc test. The black arrow indicates the intra-TG injection of 5 nmol BIM23042.
    Figure Legend Snippet: Involvement of peripheral NmbR in mechanical pain hypersensitivity. A, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces mechanical hypersensitivity ( n = 6 - 8 mice per group). * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. BL, baseline. B, the total contact time assessed by orofacial operant test was significantly decreased 1 h after intra-TG injection of 1 nmol Nmb, compared to vehicle group. * p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. C, pretreatment of BIM23042 (5 nmol), KT-5720 (2 nmol), Z941 (2 nmol) or TTA-P2 (1 nmol) significantly attenuates 1 nmol Nmb-induced mechanical hypersensitivity ( n = 8 - 10 mice per group). * p < 0.05 versus vehicle at 1 h, + p < 0.05 versus Z941 + vehicle, # p < 0.05 versus TTA-P2 + vehicle, one-way ANOVA followed by Bonferroni post hoc test. D, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces heat hypersensitivity. * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. E, intra-TG administration of BIM23042 (5 nmol) prevented 1 nmol Nmb-induced heat hypersensitivity (n = 6 - 8 mice per group). * p < 0.05 versus vehicle at 1 h, one-way ANOVA followed by Bonferroni post hoc test. F, rofecoxib (1 mg/kg administrated subcutaneously) reduced the mechanical allodynia produced by CFA. Behavioral analysis was performed at 2 d after CFA injection, and the effect of rofecoxib was assessed at 1 h after administration (n = 7 - 9 mice per group). ** p < 0.01 versus basline (BL) in vehicle group, ## p < 0.01 versus basline (BL) in rofecoxib group, ++ p < 0.01 versus vehicle at 1 h. G, intra-TG injection of BIM23042 (5 nmol) 2 d after CFA injection significantly suppresses the mechanical hypersensitivity in CFA-treated group ( n = 7 - 9 mice per group). * p < 0.05 and ** p < 0.01 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. H-I, intra-TG administration of Cav3.2 siRNA resulted in a significant decrease of Cav3.2 protein abundance in mouse TGs ( H ), while the expression level of Cav3.1 or Cav3.3 ( I ) remained unchanged. The blots shown are representative of three independent experiments. # p < 0.05 versus NC-siRNA, two-tailed t test. J, siRNA knock-down of Cav3.2 attenuates the BIM23042-induced alleviation of mechanical allodynia in CFA-treated mice ( n = 8 - 10 mice per group). * p < 0.01 and ** p < 0.01 versus NC-siRNA in CFA-treated mice; ++ p < 0.01 versus CFA 2 d; # p < 0.05 versus 0 h point in NC-siRNA-treated mice, two-way ANOVA followed by Bonferroni post hoc test. The black arrow indicates the intra-TG injection of 5 nmol BIM23042.

    Techniques Used: Injection, Produced, Expressing, Two Tailed Test

    Proposed mechanisms of NmbR signalling on Cav3.2 channels in TG neurons. The binding of Nmb triggers NmbR to activate G q protein and releases the G βγ subunits. The released G βγ dimer then stimulates AMPK activity, induces the activation of PKA and subsequently phosphorylates Cav3.2 channels to increase I T . Neither PLC/PKC nor direct binding of G βγ to Cav3.2 channels is required for T-type channel response mediated by NmbR.
    Figure Legend Snippet: Proposed mechanisms of NmbR signalling on Cav3.2 channels in TG neurons. The binding of Nmb triggers NmbR to activate G q protein and releases the G βγ subunits. The released G βγ dimer then stimulates AMPK activity, induces the activation of PKA and subsequently phosphorylates Cav3.2 channels to increase I T . Neither PLC/PKC nor direct binding of G βγ to Cav3.2 channels is required for T-type channel response mediated by NmbR.

    Techniques Used: Binding Assay, Activity Assay, Activation Assay

    anti cav3 1 antibody  (Alomone Labs)


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    Alomone Labs anti cav3 1 antibody
    Ca currents in wt and <t>Cav3.2</t> KO ventricular cardiomyocytes. ( A ) Original traces of Ca currents of a typical wt and Cav3.2 KO myocyte elicited by the pulse protocol displayed on top. ( B ) Current density–voltage relationships of wt (n = 20) and Cav3.2 KO (n = 20) myocytes. Data are expressed as means ± SEM. The current density–voltage relations hardly revealed any T-type Ca channel activity (expected to be maximal at −45 mV) but did reveal significant L-type Ca channel activity (peaking at +5 mV), which was similar in wt and Cav3.2 KO myocytes ( p = 0.67 at +5 mV, unpaired Student’s t -test). Capacitance values of the examined myocytes were 184 ± 13 pF for wt and 169 ± 11 pF for Cav3.2 KO ( p = 0.37). ( C ) The effect of the external application of 100 nM isoprenaline (ISO) on ICaT in a wt myocyte. From a holding potential of −95 mV, depolarizing voltage steps to −45 mV were applied every 3 s to elicit the currents. The arrows indicate the beginning and end of ISO application. Current peaks were finally plotted against time. ( D ) ICaT peaks before, during application after the steady-state was reached, and after the washout of ISO in wt (top) and Cav3.2 KO (bottom) myocytes. There was no significant difference between wt and Cav3.2 KO under control conditions ( p = 0.81, unpaired Student’s t -test) or in the presence of ISO ( p = 0.54). Data are expressed as means ± SEM. Each data point represents a single cell. ** p < 0.01; *** p < 0.001; paired Student’s t -test.
    Anti Cav3 1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice"

    Article Title: Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice

    Journal: Membranes

    doi: 10.3390/membranes12060566

    Ca currents in wt and Cav3.2 KO ventricular cardiomyocytes. ( A ) Original traces of Ca currents of a typical wt and Cav3.2 KO myocyte elicited by the pulse protocol displayed on top. ( B ) Current density–voltage relationships of wt (n = 20) and Cav3.2 KO (n = 20) myocytes. Data are expressed as means ± SEM. The current density–voltage relations hardly revealed any T-type Ca channel activity (expected to be maximal at −45 mV) but did reveal significant L-type Ca channel activity (peaking at +5 mV), which was similar in wt and Cav3.2 KO myocytes ( p = 0.67 at +5 mV, unpaired Student’s t -test). Capacitance values of the examined myocytes were 184 ± 13 pF for wt and 169 ± 11 pF for Cav3.2 KO ( p = 0.37). ( C ) The effect of the external application of 100 nM isoprenaline (ISO) on ICaT in a wt myocyte. From a holding potential of −95 mV, depolarizing voltage steps to −45 mV were applied every 3 s to elicit the currents. The arrows indicate the beginning and end of ISO application. Current peaks were finally plotted against time. ( D ) ICaT peaks before, during application after the steady-state was reached, and after the washout of ISO in wt (top) and Cav3.2 KO (bottom) myocytes. There was no significant difference between wt and Cav3.2 KO under control conditions ( p = 0.81, unpaired Student’s t -test) or in the presence of ISO ( p = 0.54). Data are expressed as means ± SEM. Each data point represents a single cell. ** p < 0.01; *** p < 0.001; paired Student’s t -test.
    Figure Legend Snippet: Ca currents in wt and Cav3.2 KO ventricular cardiomyocytes. ( A ) Original traces of Ca currents of a typical wt and Cav3.2 KO myocyte elicited by the pulse protocol displayed on top. ( B ) Current density–voltage relationships of wt (n = 20) and Cav3.2 KO (n = 20) myocytes. Data are expressed as means ± SEM. The current density–voltage relations hardly revealed any T-type Ca channel activity (expected to be maximal at −45 mV) but did reveal significant L-type Ca channel activity (peaking at +5 mV), which was similar in wt and Cav3.2 KO myocytes ( p = 0.67 at +5 mV, unpaired Student’s t -test). Capacitance values of the examined myocytes were 184 ± 13 pF for wt and 169 ± 11 pF for Cav3.2 KO ( p = 0.37). ( C ) The effect of the external application of 100 nM isoprenaline (ISO) on ICaT in a wt myocyte. From a holding potential of −95 mV, depolarizing voltage steps to −45 mV were applied every 3 s to elicit the currents. The arrows indicate the beginning and end of ISO application. Current peaks were finally plotted against time. ( D ) ICaT peaks before, during application after the steady-state was reached, and after the washout of ISO in wt (top) and Cav3.2 KO (bottom) myocytes. There was no significant difference between wt and Cav3.2 KO under control conditions ( p = 0.81, unpaired Student’s t -test) or in the presence of ISO ( p = 0.54). Data are expressed as means ± SEM. Each data point represents a single cell. ** p < 0.01; *** p < 0.001; paired Student’s t -test.

    Techniques Used: Activity Assay

    Immunostaining of T-type Ca channels in ventricular cardiomyocytes isolated from adult wt mice. Cav3.1 ( top ) channel expression and localization was detected using a selective anti-Cav3.1 antibody (anti-CACNA1G, #ACC-021, source: rabbit, Alomone Labs, 1:500). Cav3.2 ( bottom ) was detected with the selective anti-Cav3.2 antibodies (anti-CACNA1H, #ACC-025, source: rabbit, Alomone Labs, 1:1000; left and right cell; or anti-Cav3.2 polyclonal, #PA5-106771, source: rabbit, Invitrogen, 1:200; middle cell). Secondary antibody: Alexa Fluor 488, #A11008, goat anti-rabbit, Invitrogen, 1:500.
    Figure Legend Snippet: Immunostaining of T-type Ca channels in ventricular cardiomyocytes isolated from adult wt mice. Cav3.1 ( top ) channel expression and localization was detected using a selective anti-Cav3.1 antibody (anti-CACNA1G, #ACC-021, source: rabbit, Alomone Labs, 1:500). Cav3.2 ( bottom ) was detected with the selective anti-Cav3.2 antibodies (anti-CACNA1H, #ACC-025, source: rabbit, Alomone Labs, 1:1000; left and right cell; or anti-Cav3.2 polyclonal, #PA5-106771, source: rabbit, Invitrogen, 1:200; middle cell). Secondary antibody: Alexa Fluor 488, #A11008, goat anti-rabbit, Invitrogen, 1:500.

    Techniques Used: Immunostaining, Isolation, Expressing

    Electrically induced and caffeine-induced intracellular Ca transients in wt and Cav3.2 KO ventricular cardiomyocytes. ( A ) Left: Representative time courses of Fluo-4 fluorescence reporting rises in cytosolic Ca concentration during electrical field stimulation at 0.1 Hz frequency in a single wt and Cav3.2 KO myocyte. The orange lines represent fits of the data with a single exponential function. Right: Comparison of mean Ca peak fluorescence relative to baseline (F/F0) between wt and Cav3.2 KO myocytes. Each data point represents a single cell, and values are expressed as means ± SEM (n= 68 for wt and 59 for Cav3.2 KO myocytes). * p < 0.05 (0.02); unpaired Student’s t -test. The Ca transients decayed with time constants of 0.30 ± 0.02 s in wt and 0.25 ± 0.01 s in Cav3.2 KO myocytes, which were significantly different ( p = 0.04). ( B ) Comparison of mean Ca peak fluorescence (F/F0) before and after the application of 100 nM ISO in wt (n = 20) and Cav3.2 KO (n = 21) myocytes. **** p < 0.0001 (paired Student’s t -test). There was no significant difference between wt and Cav3.2 KO in the presence of ISO ( p = 0.37; unpaired Student’s t -test). The Ca transients under ISO decayed with time constants of 0.14 ± 0.02 s in wt and 0.11 ± 0.02 s in Cav3.2 KO myocytes (not significantly different, p = 0.19). ( C ) Comparison of mean Ca peak fluorescence elicited by an application of 20 mM caffeine, relative to baseline (F/F0), between wt (n = 26) and Cav3.2 KO (n = 35) myocytes. ns—not significant (unpaired Student’s t -test, p = 0.42).
    Figure Legend Snippet: Electrically induced and caffeine-induced intracellular Ca transients in wt and Cav3.2 KO ventricular cardiomyocytes. ( A ) Left: Representative time courses of Fluo-4 fluorescence reporting rises in cytosolic Ca concentration during electrical field stimulation at 0.1 Hz frequency in a single wt and Cav3.2 KO myocyte. The orange lines represent fits of the data with a single exponential function. Right: Comparison of mean Ca peak fluorescence relative to baseline (F/F0) between wt and Cav3.2 KO myocytes. Each data point represents a single cell, and values are expressed as means ± SEM (n= 68 for wt and 59 for Cav3.2 KO myocytes). * p < 0.05 (0.02); unpaired Student’s t -test. The Ca transients decayed with time constants of 0.30 ± 0.02 s in wt and 0.25 ± 0.01 s in Cav3.2 KO myocytes, which were significantly different ( p = 0.04). ( B ) Comparison of mean Ca peak fluorescence (F/F0) before and after the application of 100 nM ISO in wt (n = 20) and Cav3.2 KO (n = 21) myocytes. **** p < 0.0001 (paired Student’s t -test). There was no significant difference between wt and Cav3.2 KO in the presence of ISO ( p = 0.37; unpaired Student’s t -test). The Ca transients under ISO decayed with time constants of 0.14 ± 0.02 s in wt and 0.11 ± 0.02 s in Cav3.2 KO myocytes (not significantly different, p = 0.19). ( C ) Comparison of mean Ca peak fluorescence elicited by an application of 20 mM caffeine, relative to baseline (F/F0), between wt (n = 26) and Cav3.2 KO (n = 35) myocytes. ns—not significant (unpaired Student’s t -test, p = 0.42).

    Techniques Used: Fluorescence, Concentration Assay

    acc 021 rrid ab 2039779  (Alomone Labs)


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    Alomone Labs acc 021 rrid ab 2039779
    Acc 021 Rrid Ab 2039779, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti cav3 1  (Alomone Labs)


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    Alomone Labs anti cav3 1
    ( A and B ) Confocal images of EP13 transplanted ZsGreen-labeled ChCs transfected with LacZ sgRNAs (A, left) or <t>Cav3.3</t> sgRNAs (A, right) as well as EP13 transplanted GFP-labeled ChCs originating from Cav3.3 germline KO mice (B). Single optical sections indicating synaptic cartridges apposed to AnkG-labeled AISs are shown in the middle panels. The areas in white boxes with arrowheads are enlarged and overlaid with the AnkG channel. Bottom panels indicate reconstructed axonal arbors in the area defined in . ( C ) The number of branch points in LacZ sgRNA –transfected ChCs ( n = 5 cells from three mice), Cav3.3 sgRNA– transfected homozygous KO ChCs ( n = 5 cells from three mice), transplanted ChCs originating from Cav3.3 homozygous KO mice ( n = 6 cells from three mice), and transplanted ChCs originating from Cav3.3 heterozygous KO mice ( n = 5 cells from three mice). One-way ANOVA, * P < 0.05, *** P < 0.001, F = 14.3, df = 20. ( D ) Confocal images of P13 endogenous RFP-labeled ChCs in WT mice (left) and Cav3.3 heterozygous KO mouse (right). ( E ) The number of branch points in endogenous ChCs in WT mice ( n = 5 cells from three mice) and Cav3.3 heterozygous KO mice ( n = 6 cells from three mice). t test (two-tailed), ** P < 0.01, t = 5.80, df = 9. Scale bars, 10 μm.
    Anti Cav3 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Neuromodulatory control of inhibitory network arborization in the developing postnatal neocortex"

    Article Title: Neuromodulatory control of inhibitory network arborization in the developing postnatal neocortex

    Journal: Science Advances

    doi: 10.1126/sciadv.abe7192

    ( A and B ) Confocal images of EP13 transplanted ZsGreen-labeled ChCs transfected with LacZ sgRNAs (A, left) or Cav3.3 sgRNAs (A, right) as well as EP13 transplanted GFP-labeled ChCs originating from Cav3.3 germline KO mice (B). Single optical sections indicating synaptic cartridges apposed to AnkG-labeled AISs are shown in the middle panels. The areas in white boxes with arrowheads are enlarged and overlaid with the AnkG channel. Bottom panels indicate reconstructed axonal arbors in the area defined in . ( C ) The number of branch points in LacZ sgRNA –transfected ChCs ( n = 5 cells from three mice), Cav3.3 sgRNA– transfected homozygous KO ChCs ( n = 5 cells from three mice), transplanted ChCs originating from Cav3.3 homozygous KO mice ( n = 6 cells from three mice), and transplanted ChCs originating from Cav3.3 heterozygous KO mice ( n = 5 cells from three mice). One-way ANOVA, * P < 0.05, *** P < 0.001, F = 14.3, df = 20. ( D ) Confocal images of P13 endogenous RFP-labeled ChCs in WT mice (left) and Cav3.3 heterozygous KO mouse (right). ( E ) The number of branch points in endogenous ChCs in WT mice ( n = 5 cells from three mice) and Cav3.3 heterozygous KO mice ( n = 6 cells from three mice). t test (two-tailed), ** P < 0.01, t = 5.80, df = 9. Scale bars, 10 μm.
    Figure Legend Snippet: ( A and B ) Confocal images of EP13 transplanted ZsGreen-labeled ChCs transfected with LacZ sgRNAs (A, left) or Cav3.3 sgRNAs (A, right) as well as EP13 transplanted GFP-labeled ChCs originating from Cav3.3 germline KO mice (B). Single optical sections indicating synaptic cartridges apposed to AnkG-labeled AISs are shown in the middle panels. The areas in white boxes with arrowheads are enlarged and overlaid with the AnkG channel. Bottom panels indicate reconstructed axonal arbors in the area defined in . ( C ) The number of branch points in LacZ sgRNA –transfected ChCs ( n = 5 cells from three mice), Cav3.3 sgRNA– transfected homozygous KO ChCs ( n = 5 cells from three mice), transplanted ChCs originating from Cav3.3 homozygous KO mice ( n = 6 cells from three mice), and transplanted ChCs originating from Cav3.3 heterozygous KO mice ( n = 5 cells from three mice). One-way ANOVA, * P < 0.05, *** P < 0.001, F = 14.3, df = 20. ( D ) Confocal images of P13 endogenous RFP-labeled ChCs in WT mice (left) and Cav3.3 heterozygous KO mouse (right). ( E ) The number of branch points in endogenous ChCs in WT mice ( n = 5 cells from three mice) and Cav3.3 heterozygous KO mice ( n = 6 cells from three mice). t test (two-tailed), ** P < 0.01, t = 5.80, df = 9. Scale bars, 10 μm.

    Techniques Used: Labeling, Transfection, Two Tailed Test

    cav3 1  (Alomone Labs)


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    Alomone Labs cav3 1
    Cav3 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cav3 1  (Alomone Labs)


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    voltage dependent t type calcium channel subunits cav3 1  (Alomone Labs)


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    Alomone Labs voltage dependent t type calcium channel subunits cav3 1
    Summary of primary antibodies and dilutions for the immunolabeling
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    1) Product Images from "Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei"

    Article Title: Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei

    Journal: Brain Structure & Function

    doi: 10.1007/s00429-021-02315-7

    Summary of primary antibodies and dilutions for the immunolabeling
    Figure Legend Snippet: Summary of primary antibodies and dilutions for the immunolabeling

    Techniques Used: Marker

    Qualitative summary of immunohistochemical results ( − no labeling, + weak, + + moderate, + + + strong labeling)
    Figure Legend Snippet: Qualitative summary of immunohistochemical results ( − no labeling, + weak, + + moderate, + + + strong labeling)

    Techniques Used: Immunohistochemical staining, Labeling

    Low-voltage activated calcium channel family (Cav3) subunits in the abducens and trochlear nucleus. a Consecutive coronal paraffin sections through the abducens nucleus (nVI) ( a ) depicting the immunolabeling for Cav3.1 (first panel), Cav3.2 (third panel) and Cav3.3 (fourth panel) subunits with combined immunostaining for ChAT (brown) and ACAN (black) (second panel) as reference. b, c Close-up of Cav subunit expression in nVI MIF (red arrowheads) and SIF motoneurons (MNs) (green arrows) and INTs (blue arrow). d Consecutive coronal paraffin sections through the trochlear nucleus (nVI) depicting the immunolabeling for Cav3.1 (first panel), Cav3.2 (second panel) and Cav3.3 (fourth panel) subunits with combined immunostaining for ChAT (brown) and ACAN (black) (third panel) as reference. Thin dashed lines in d indicate the border of nIV and thick dashed lines indicate the boundary between the MIF and SIF MNs. e Close-up of Cav subunit expression in MIF (red arrowheads) and SIF MNs (green arrow) on different sections as those illustrated in d . Note the weak Cav3.1 expression along the membrane of some SIF MNs (left column, black star). Red dashed lines indicate the tentative position of the border delineating the dorsal cap of nIV. f Cerebellar Purkinje cells located on the same consecutive sections as nVI as controls for immunopositivity of the different Cav subunits. Scale bar indicates 100 μm in a, d, f and 50 μm in b, c, e
    Figure Legend Snippet: Low-voltage activated calcium channel family (Cav3) subunits in the abducens and trochlear nucleus. a Consecutive coronal paraffin sections through the abducens nucleus (nVI) ( a ) depicting the immunolabeling for Cav3.1 (first panel), Cav3.2 (third panel) and Cav3.3 (fourth panel) subunits with combined immunostaining for ChAT (brown) and ACAN (black) (second panel) as reference. b, c Close-up of Cav subunit expression in nVI MIF (red arrowheads) and SIF motoneurons (MNs) (green arrows) and INTs (blue arrow). d Consecutive coronal paraffin sections through the trochlear nucleus (nVI) depicting the immunolabeling for Cav3.1 (first panel), Cav3.2 (second panel) and Cav3.3 (fourth panel) subunits with combined immunostaining for ChAT (brown) and ACAN (black) (third panel) as reference. Thin dashed lines in d indicate the border of nIV and thick dashed lines indicate the boundary between the MIF and SIF MNs. e Close-up of Cav subunit expression in MIF (red arrowheads) and SIF MNs (green arrow) on different sections as those illustrated in d . Note the weak Cav3.1 expression along the membrane of some SIF MNs (left column, black star). Red dashed lines indicate the tentative position of the border delineating the dorsal cap of nIV. f Cerebellar Purkinje cells located on the same consecutive sections as nVI as controls for immunopositivity of the different Cav subunits. Scale bar indicates 100 μm in a, d, f and 50 μm in b, c, e

    Techniques Used: Immunolabeling, Immunostaining, Expressing

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    Alomone Labs cav3 1
    NmbR mediates the Nmb-induced I T increase. A, western blot analysis of NmbR in mouse TGs. A prominent band was also observed in the spinal cord of mice. Blots depicted are representative of three independent experiments. B, double immunostaining of NmbR with calcitonin gene-related peptide (CGRP), isolectin B4 (IB 4 ), or neurofilament 200 (NF200) in mouse TG sections. Arrows in white indicate the co-localization. Scale bar, 30 µm. C, time course ( left panel ) and summary data ( right panel ) show the effects of 100 nM Nmb on I T in TG neurons pretreated with BIM23042 (0.5 µM) ( n = 7). Bath application of 0.5 µM BIM23042 alone did not affect I T ( n = 6). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus Nmb, two-tailed t test. D, double immunostaining of NmbR with <t>Cav3.2</t> in TG neurons of naïve mice. Arrows in white show the colocalization. Scale bars, 30 µm. Insets indicate the percentage of double-labeled neurons in NmbR + or Cav3.2 + neurons. E, total contact number of orofacial operant test with mechanical stimulation was comparable between normal saline (NS)- and CFA-treated mice. BL, baseline. F, total contact time was significantly decreased from 1 to 7 d after CFA injection, compared to NS group. * P < 0.05, ** P < 0.01 versus NS, two-way ANOVA followed by Bonferroni post hoc test. G, mechanical hypersensitivity induced by CFA injection. Decreased escape threshold was observed at the 12th h after CFA injection and lasted to ~7 d ( n = 9 mice per group). * p < 0.05 and ** p < 0.01 versus normal saline (NS) at the corresponding time point, two-way ANOVA followed by Bonferroni post hoc test. H, representative immunoblots show that Nmb expression was increased in the local inflamed tissue of mice at 2 d after CFA. Blots depicted are representative of three independent experiments. # p < 0.05 versus NS, two-tailed t test. I, abundance of NmbR protein in mouse TGs at 2 d after NS- or CFA- treatment. Blots depicted are representative of three independent experiments. ## p < 0.01 versus NS, two-tailed t test.
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    Alomone Labs anti cav3 1 antibody
    Ca currents in wt and <t>Cav3.2</t> KO ventricular cardiomyocytes. ( A ) Original traces of Ca currents of a typical wt and Cav3.2 KO myocyte elicited by the pulse protocol displayed on top. ( B ) Current density–voltage relationships of wt (n = 20) and Cav3.2 KO (n = 20) myocytes. Data are expressed as means ± SEM. The current density–voltage relations hardly revealed any T-type Ca channel activity (expected to be maximal at −45 mV) but did reveal significant L-type Ca channel activity (peaking at +5 mV), which was similar in wt and Cav3.2 KO myocytes ( p = 0.67 at +5 mV, unpaired Student’s t -test). Capacitance values of the examined myocytes were 184 ± 13 pF for wt and 169 ± 11 pF for Cav3.2 KO ( p = 0.37). ( C ) The effect of the external application of 100 nM isoprenaline (ISO) on ICaT in a wt myocyte. From a holding potential of −95 mV, depolarizing voltage steps to −45 mV were applied every 3 s to elicit the currents. The arrows indicate the beginning and end of ISO application. Current peaks were finally plotted against time. ( D ) ICaT peaks before, during application after the steady-state was reached, and after the washout of ISO in wt (top) and Cav3.2 KO (bottom) myocytes. There was no significant difference between wt and Cav3.2 KO under control conditions ( p = 0.81, unpaired Student’s t -test) or in the presence of ISO ( p = 0.54). Data are expressed as means ± SEM. Each data point represents a single cell. ** p < 0.01; *** p < 0.001; paired Student’s t -test.
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    Alomone Labs acc 021 rrid ab 2039779
    Ca currents in wt and <t>Cav3.2</t> KO ventricular cardiomyocytes. ( A ) Original traces of Ca currents of a typical wt and Cav3.2 KO myocyte elicited by the pulse protocol displayed on top. ( B ) Current density–voltage relationships of wt (n = 20) and Cav3.2 KO (n = 20) myocytes. Data are expressed as means ± SEM. The current density–voltage relations hardly revealed any T-type Ca channel activity (expected to be maximal at −45 mV) but did reveal significant L-type Ca channel activity (peaking at +5 mV), which was similar in wt and Cav3.2 KO myocytes ( p = 0.67 at +5 mV, unpaired Student’s t -test). Capacitance values of the examined myocytes were 184 ± 13 pF for wt and 169 ± 11 pF for Cav3.2 KO ( p = 0.37). ( C ) The effect of the external application of 100 nM isoprenaline (ISO) on ICaT in a wt myocyte. From a holding potential of −95 mV, depolarizing voltage steps to −45 mV were applied every 3 s to elicit the currents. The arrows indicate the beginning and end of ISO application. Current peaks were finally plotted against time. ( D ) ICaT peaks before, during application after the steady-state was reached, and after the washout of ISO in wt (top) and Cav3.2 KO (bottom) myocytes. There was no significant difference between wt and Cav3.2 KO under control conditions ( p = 0.81, unpaired Student’s t -test) or in the presence of ISO ( p = 0.54). Data are expressed as means ± SEM. Each data point represents a single cell. ** p < 0.01; *** p < 0.001; paired Student’s t -test.
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    Alomone Labs anti cav3 1
    ( A and B ) Confocal images of EP13 transplanted ZsGreen-labeled ChCs transfected with LacZ sgRNAs (A, left) or <t>Cav3.3</t> sgRNAs (A, right) as well as EP13 transplanted GFP-labeled ChCs originating from Cav3.3 germline KO mice (B). Single optical sections indicating synaptic cartridges apposed to AnkG-labeled AISs are shown in the middle panels. The areas in white boxes with arrowheads are enlarged and overlaid with the AnkG channel. Bottom panels indicate reconstructed axonal arbors in the area defined in . ( C ) The number of branch points in LacZ sgRNA –transfected ChCs ( n = 5 cells from three mice), Cav3.3 sgRNA– transfected homozygous KO ChCs ( n = 5 cells from three mice), transplanted ChCs originating from Cav3.3 homozygous KO mice ( n = 6 cells from three mice), and transplanted ChCs originating from Cav3.3 heterozygous KO mice ( n = 5 cells from three mice). One-way ANOVA, * P < 0.05, *** P < 0.001, F = 14.3, df = 20. ( D ) Confocal images of P13 endogenous RFP-labeled ChCs in WT mice (left) and Cav3.3 heterozygous KO mouse (right). ( E ) The number of branch points in endogenous ChCs in WT mice ( n = 5 cells from three mice) and Cav3.3 heterozygous KO mice ( n = 6 cells from three mice). t test (two-tailed), ** P < 0.01, t = 5.80, df = 9. Scale bars, 10 μm.
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    Alomone Labs voltage dependent t type calcium channel subunits cav3 1
    Summary of primary antibodies and dilutions for the immunolabeling
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    NmbR mediates the Nmb-induced I T increase. A, western blot analysis of NmbR in mouse TGs. A prominent band was also observed in the spinal cord of mice. Blots depicted are representative of three independent experiments. B, double immunostaining of NmbR with calcitonin gene-related peptide (CGRP), isolectin B4 (IB 4 ), or neurofilament 200 (NF200) in mouse TG sections. Arrows in white indicate the co-localization. Scale bar, 30 µm. C, time course ( left panel ) and summary data ( right panel ) show the effects of 100 nM Nmb on I T in TG neurons pretreated with BIM23042 (0.5 µM) ( n = 7). Bath application of 0.5 µM BIM23042 alone did not affect I T ( n = 6). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus Nmb, two-tailed t test. D, double immunostaining of NmbR with Cav3.2 in TG neurons of naïve mice. Arrows in white show the colocalization. Scale bars, 30 µm. Insets indicate the percentage of double-labeled neurons in NmbR + or Cav3.2 + neurons. E, total contact number of orofacial operant test with mechanical stimulation was comparable between normal saline (NS)- and CFA-treated mice. BL, baseline. F, total contact time was significantly decreased from 1 to 7 d after CFA injection, compared to NS group. * P < 0.05, ** P < 0.01 versus NS, two-way ANOVA followed by Bonferroni post hoc test. G, mechanical hypersensitivity induced by CFA injection. Decreased escape threshold was observed at the 12th h after CFA injection and lasted to ~7 d ( n = 9 mice per group). * p < 0.05 and ** p < 0.01 versus normal saline (NS) at the corresponding time point, two-way ANOVA followed by Bonferroni post hoc test. H, representative immunoblots show that Nmb expression was increased in the local inflamed tissue of mice at 2 d after CFA. Blots depicted are representative of three independent experiments. # p < 0.05 versus NS, two-tailed t test. I, abundance of NmbR protein in mouse TGs at 2 d after NS- or CFA- treatment. Blots depicted are representative of three independent experiments. ## p < 0.01 versus NS, two-tailed t test.

    Journal: Theranostics

    Article Title: Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity

    doi: 10.7150/thno.62255

    Figure Lengend Snippet: NmbR mediates the Nmb-induced I T increase. A, western blot analysis of NmbR in mouse TGs. A prominent band was also observed in the spinal cord of mice. Blots depicted are representative of three independent experiments. B, double immunostaining of NmbR with calcitonin gene-related peptide (CGRP), isolectin B4 (IB 4 ), or neurofilament 200 (NF200) in mouse TG sections. Arrows in white indicate the co-localization. Scale bar, 30 µm. C, time course ( left panel ) and summary data ( right panel ) show the effects of 100 nM Nmb on I T in TG neurons pretreated with BIM23042 (0.5 µM) ( n = 7). Bath application of 0.5 µM BIM23042 alone did not affect I T ( n = 6). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus Nmb, two-tailed t test. D, double immunostaining of NmbR with Cav3.2 in TG neurons of naïve mice. Arrows in white show the colocalization. Scale bars, 30 µm. Insets indicate the percentage of double-labeled neurons in NmbR + or Cav3.2 + neurons. E, total contact number of orofacial operant test with mechanical stimulation was comparable between normal saline (NS)- and CFA-treated mice. BL, baseline. F, total contact time was significantly decreased from 1 to 7 d after CFA injection, compared to NS group. * P < 0.05, ** P < 0.01 versus NS, two-way ANOVA followed by Bonferroni post hoc test. G, mechanical hypersensitivity induced by CFA injection. Decreased escape threshold was observed at the 12th h after CFA injection and lasted to ~7 d ( n = 9 mice per group). * p < 0.05 and ** p < 0.01 versus normal saline (NS) at the corresponding time point, two-way ANOVA followed by Bonferroni post hoc test. H, representative immunoblots show that Nmb expression was increased in the local inflamed tissue of mice at 2 d after CFA. Blots depicted are representative of three independent experiments. # p < 0.05 versus NS, two-tailed t test. I, abundance of NmbR protein in mouse TGs at 2 d after NS- or CFA- treatment. Blots depicted are representative of three independent experiments. ## p < 0.01 versus NS, two-tailed t test.

    Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and probed with antibodies against Nmb (rabbit, 1:1000, Abcam, Cat. No. ab191499), Cav3.1 (rabbit, 1:1000, Alomone, Cat. No. ACC-021), Cav3.2 (mouse, 1:1000, Novus, Cat. No. NBP1-22444), Cav3.3 (rabbit, 1:1000, Alomone, Cat. No. ACC-009), NmbR (rabbit, 1:500, Sigma, Cat. No. SAB4502914), phospho-AMPKα1 (rabbit, 1:500, Abcam, Cat. No. ab92701), AMPKα1 (rabbit, 1:600, Abcam, Cat. No. ab110036), Gαq/11 (mouse, 1:600, Santa Cruz Biotechnology, Cat. No. SC-365906), and Gβ (mouse, 1:500, Santa Cruz Biotechnology, Cat. No. SC-515822).

    Techniques: Western Blot, Double Immunostaining, Two Tailed Test, Labeling, Injection, Expressing

    The G βγ -dependent AMPK/PKA pathway mediates the NmbR response. A, time course of I T changes induced by 100 nM Nmb in TG neurons dialyzed with QEHA (10 μM). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. B, summary data indicate the effect of 100 nM Nmb on I T in TG neurons dialyzed with QEHA (10 μM, n = 6) or SKEE (10 μM, n = 7). ** p < 0.01 versus control, two-tailed t test. C, protein expression of G β in cells transduced with G β shRNA (G β -shRNA) or negative control shRNA (NC-shRNA). Blots depicted are representative of three independent experiments. # p < 0.05 versus NC-shRNA, two-tailed t test. D, summary data show the effect of Nmb (100 nM) on I T in TG neurons transduced with NC-shRNA ( n = 9) or G β -shRNA ( n = 8). * p < 0.05 versus NC-shRNA + control, one-way ANOVA followed by Bonferroni post hoc test. E, time course ( left panel ) and summary data ( right panel ) show the effect of Nmb (100 nM) on I T in cells pre-incubated with KT-5720 (1 μM, n = 7). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. ** p < 0.01 versus control, two-tailed t test. F, exemplary current traces and summary of results show the effect of forskolin (10 µM) on I T in TG neurons in the absence ( n = 7) or presence of KT-5720 (1 μM, n = 6). ** p < 0.01 versus control, two-tailed t test. G, representative immunoblot and summary data show that treatment of TG cells with 100 nM Nmb did not affect the protein expression level of Cav3.2. GAPDH was used as the loading control. H, pretreatment of TG cells with 0.5 µM BIM23042, but not 1 μM KT-5720, attenuates the increased expression level of phosphorylated AMPK ( p -AMPK) induced by 100 nM Nmb. GAPDH was used as the loading control. Blots depicted are representative of three independent experiments. * p < 0.05 versus control, two-tailed t test. I, summary data show the effect of 100 nM Nmb on PKA activity in TG cells pretreated with 0.5 µM BIM23042 or 10 µM compound C. All experiments were performed in triplicate with similar results. # p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. J, time course ( left panel ) and summary of results ( right panel ) indicate the effect of 100 nM Nmb on I T in TG neurons pretreated with compound C (10 µM, n = 7). Inset s show the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus control, two-tailed t test.

    Journal: Theranostics

    Article Title: Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity

    doi: 10.7150/thno.62255

    Figure Lengend Snippet: The G βγ -dependent AMPK/PKA pathway mediates the NmbR response. A, time course of I T changes induced by 100 nM Nmb in TG neurons dialyzed with QEHA (10 μM). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. B, summary data indicate the effect of 100 nM Nmb on I T in TG neurons dialyzed with QEHA (10 μM, n = 6) or SKEE (10 μM, n = 7). ** p < 0.01 versus control, two-tailed t test. C, protein expression of G β in cells transduced with G β shRNA (G β -shRNA) or negative control shRNA (NC-shRNA). Blots depicted are representative of three independent experiments. # p < 0.05 versus NC-shRNA, two-tailed t test. D, summary data show the effect of Nmb (100 nM) on I T in TG neurons transduced with NC-shRNA ( n = 9) or G β -shRNA ( n = 8). * p < 0.05 versus NC-shRNA + control, one-way ANOVA followed by Bonferroni post hoc test. E, time course ( left panel ) and summary data ( right panel ) show the effect of Nmb (100 nM) on I T in cells pre-incubated with KT-5720 (1 μM, n = 7). Inset s indicate the exemplary current traces. The Arabic numbers represent points used for representative traces. ** p < 0.01 versus control, two-tailed t test. F, exemplary current traces and summary of results show the effect of forskolin (10 µM) on I T in TG neurons in the absence ( n = 7) or presence of KT-5720 (1 μM, n = 6). ** p < 0.01 versus control, two-tailed t test. G, representative immunoblot and summary data show that treatment of TG cells with 100 nM Nmb did not affect the protein expression level of Cav3.2. GAPDH was used as the loading control. H, pretreatment of TG cells with 0.5 µM BIM23042, but not 1 μM KT-5720, attenuates the increased expression level of phosphorylated AMPK ( p -AMPK) induced by 100 nM Nmb. GAPDH was used as the loading control. Blots depicted are representative of three independent experiments. * p < 0.05 versus control, two-tailed t test. I, summary data show the effect of 100 nM Nmb on PKA activity in TG cells pretreated with 0.5 µM BIM23042 or 10 µM compound C. All experiments were performed in triplicate with similar results. # p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. J, time course ( left panel ) and summary of results ( right panel ) indicate the effect of 100 nM Nmb on I T in TG neurons pretreated with compound C (10 µM, n = 7). Inset s show the exemplary current traces. The Arabic numbers represent points used for representative traces. *** p < 0.001 versus control, two-tailed t test.

    Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and probed with antibodies against Nmb (rabbit, 1:1000, Abcam, Cat. No. ab191499), Cav3.1 (rabbit, 1:1000, Alomone, Cat. No. ACC-021), Cav3.2 (mouse, 1:1000, Novus, Cat. No. NBP1-22444), Cav3.3 (rabbit, 1:1000, Alomone, Cat. No. ACC-009), NmbR (rabbit, 1:500, Sigma, Cat. No. SAB4502914), phospho-AMPKα1 (rabbit, 1:500, Abcam, Cat. No. ab92701), AMPKα1 (rabbit, 1:600, Abcam, Cat. No. ab110036), Gαq/11 (mouse, 1:600, Santa Cruz Biotechnology, Cat. No. SC-365906), and Gβ (mouse, 1:500, Santa Cruz Biotechnology, Cat. No. SC-515822).

    Techniques: Two Tailed Test, Expressing, Transduction, shRNA, Negative Control, Incubation, Western Blot, Activity Assay

    Activation of NmbR stimulates recombinant Cav3.2 channels heterologously expressed in HEK293 cells. A, western blot analysis of NmbR in HEK293 cells transiently transfected with NmbR cDNA. Blots depicted are representative of three independent experiments. B, membrane localization of NmbR in transfected HEK293 cells. Alphabets a through c in the diagram indicate the differential interference contrast (DIC, a ), the EGFP fluorescent signals of NmbR ( b ), and the merged image ( c ), respectively. C, exemplary current traces show the effect of Nmb (100 nM) on Cav3.1 (α1G), Cav3.2 (α1H) and Cav3.3 (α1I) channel currents. Currents were elicited by a 100 ms depolarizing step pulse from the holding potential of -110 mV to -30 mV. D, summary data show the effect of 100 nM Nmb on Cav3.1 ( n = 10), Cav3.2 ( n = 9) and Cav3.3 ( n = 7) channel currents. ** p < 0.01 versus control, two-tailed t test. E, dose-dependent effects of Nmb on Cav3.2 channel currents. Solid line represents the sigmoidal dose-response fits. Numbers in parentheses denote n cells tested at each concentration. F, summary data show the effect of Nmb (100 nM) on Cav3.2 channel currents in cells pre-incubated with 10 µM compound C ( n = 8), in cells pretreated with 1 µM KT-5720 ( n = 9), and in cells dialyzed with 10 μM PKC 19-36 ( n = 9), respectively. ** p < 0.01 and *** p < 0.001 versus control, two-tailed t test.

    Journal: Theranostics

    Article Title: Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity

    doi: 10.7150/thno.62255

    Figure Lengend Snippet: Activation of NmbR stimulates recombinant Cav3.2 channels heterologously expressed in HEK293 cells. A, western blot analysis of NmbR in HEK293 cells transiently transfected with NmbR cDNA. Blots depicted are representative of three independent experiments. B, membrane localization of NmbR in transfected HEK293 cells. Alphabets a through c in the diagram indicate the differential interference contrast (DIC, a ), the EGFP fluorescent signals of NmbR ( b ), and the merged image ( c ), respectively. C, exemplary current traces show the effect of Nmb (100 nM) on Cav3.1 (α1G), Cav3.2 (α1H) and Cav3.3 (α1I) channel currents. Currents were elicited by a 100 ms depolarizing step pulse from the holding potential of -110 mV to -30 mV. D, summary data show the effect of 100 nM Nmb on Cav3.1 ( n = 10), Cav3.2 ( n = 9) and Cav3.3 ( n = 7) channel currents. ** p < 0.01 versus control, two-tailed t test. E, dose-dependent effects of Nmb on Cav3.2 channel currents. Solid line represents the sigmoidal dose-response fits. Numbers in parentheses denote n cells tested at each concentration. F, summary data show the effect of Nmb (100 nM) on Cav3.2 channel currents in cells pre-incubated with 10 µM compound C ( n = 8), in cells pretreated with 1 µM KT-5720 ( n = 9), and in cells dialyzed with 10 μM PKC 19-36 ( n = 9), respectively. ** p < 0.01 and *** p < 0.001 versus control, two-tailed t test.

    Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and probed with antibodies against Nmb (rabbit, 1:1000, Abcam, Cat. No. ab191499), Cav3.1 (rabbit, 1:1000, Alomone, Cat. No. ACC-021), Cav3.2 (mouse, 1:1000, Novus, Cat. No. NBP1-22444), Cav3.3 (rabbit, 1:1000, Alomone, Cat. No. ACC-009), NmbR (rabbit, 1:500, Sigma, Cat. No. SAB4502914), phospho-AMPKα1 (rabbit, 1:500, Abcam, Cat. No. ab92701), AMPKα1 (rabbit, 1:600, Abcam, Cat. No. ab110036), Gαq/11 (mouse, 1:600, Santa Cruz Biotechnology, Cat. No. SC-365906), and Gβ (mouse, 1:500, Santa Cruz Biotechnology, Cat. No. SC-515822).

    Techniques: Activation Assay, Recombinant, Western Blot, Transfection, Two Tailed Test, Concentration Assay, Incubation

    Involvement of peripheral NmbR in mechanical pain hypersensitivity. A, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces mechanical hypersensitivity ( n = 6 - 8 mice per group). * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. BL, baseline. B, the total contact time assessed by orofacial operant test was significantly decreased 1 h after intra-TG injection of 1 nmol Nmb, compared to vehicle group. * p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. C, pretreatment of BIM23042 (5 nmol), KT-5720 (2 nmol), Z941 (2 nmol) or TTA-P2 (1 nmol) significantly attenuates 1 nmol Nmb-induced mechanical hypersensitivity ( n = 8 - 10 mice per group). * p < 0.05 versus vehicle at 1 h, + p < 0.05 versus Z941 + vehicle, # p < 0.05 versus TTA-P2 + vehicle, one-way ANOVA followed by Bonferroni post hoc test. D, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces heat hypersensitivity. * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. E, intra-TG administration of BIM23042 (5 nmol) prevented 1 nmol Nmb-induced heat hypersensitivity (n = 6 - 8 mice per group). * p < 0.05 versus vehicle at 1 h, one-way ANOVA followed by Bonferroni post hoc test. F, rofecoxib (1 mg/kg administrated subcutaneously) reduced the mechanical allodynia produced by CFA. Behavioral analysis was performed at 2 d after CFA injection, and the effect of rofecoxib was assessed at 1 h after administration (n = 7 - 9 mice per group). ** p < 0.01 versus basline (BL) in vehicle group, ## p < 0.01 versus basline (BL) in rofecoxib group, ++ p < 0.01 versus vehicle at 1 h. G, intra-TG injection of BIM23042 (5 nmol) 2 d after CFA injection significantly suppresses the mechanical hypersensitivity in CFA-treated group ( n = 7 - 9 mice per group). * p < 0.05 and ** p < 0.01 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. H-I, intra-TG administration of Cav3.2 siRNA resulted in a significant decrease of Cav3.2 protein abundance in mouse TGs ( H ), while the expression level of Cav3.1 or Cav3.3 ( I ) remained unchanged. The blots shown are representative of three independent experiments. # p < 0.05 versus NC-siRNA, two-tailed t test. J, siRNA knock-down of Cav3.2 attenuates the BIM23042-induced alleviation of mechanical allodynia in CFA-treated mice ( n = 8 - 10 mice per group). * p < 0.01 and ** p < 0.01 versus NC-siRNA in CFA-treated mice; ++ p < 0.01 versus CFA 2 d; # p < 0.05 versus 0 h point in NC-siRNA-treated mice, two-way ANOVA followed by Bonferroni post hoc test. The black arrow indicates the intra-TG injection of 5 nmol BIM23042.

    Journal: Theranostics

    Article Title: Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity

    doi: 10.7150/thno.62255

    Figure Lengend Snippet: Involvement of peripheral NmbR in mechanical pain hypersensitivity. A, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces mechanical hypersensitivity ( n = 6 - 8 mice per group). * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. BL, baseline. B, the total contact time assessed by orofacial operant test was significantly decreased 1 h after intra-TG injection of 1 nmol Nmb, compared to vehicle group. * p < 0.05 versus vehicle, one-way ANOVA followed by Bonferroni post hoc test. C, pretreatment of BIM23042 (5 nmol), KT-5720 (2 nmol), Z941 (2 nmol) or TTA-P2 (1 nmol) significantly attenuates 1 nmol Nmb-induced mechanical hypersensitivity ( n = 8 - 10 mice per group). * p < 0.05 versus vehicle at 1 h, + p < 0.05 versus Z941 + vehicle, # p < 0.05 versus TTA-P2 + vehicle, one-way ANOVA followed by Bonferroni post hoc test. D, intra-TG injection of 1 nmol or 5 nmol of Nmb significantly induces heat hypersensitivity. * p < 0.05 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. E, intra-TG administration of BIM23042 (5 nmol) prevented 1 nmol Nmb-induced heat hypersensitivity (n = 6 - 8 mice per group). * p < 0.05 versus vehicle at 1 h, one-way ANOVA followed by Bonferroni post hoc test. F, rofecoxib (1 mg/kg administrated subcutaneously) reduced the mechanical allodynia produced by CFA. Behavioral analysis was performed at 2 d after CFA injection, and the effect of rofecoxib was assessed at 1 h after administration (n = 7 - 9 mice per group). ** p < 0.01 versus basline (BL) in vehicle group, ## p < 0.01 versus basline (BL) in rofecoxib group, ++ p < 0.01 versus vehicle at 1 h. G, intra-TG injection of BIM23042 (5 nmol) 2 d after CFA injection significantly suppresses the mechanical hypersensitivity in CFA-treated group ( n = 7 - 9 mice per group). * p < 0.05 and ** p < 0.01 versus vehicle, two-way ANOVA followed by Bonferroni post hoc test. H-I, intra-TG administration of Cav3.2 siRNA resulted in a significant decrease of Cav3.2 protein abundance in mouse TGs ( H ), while the expression level of Cav3.1 or Cav3.3 ( I ) remained unchanged. The blots shown are representative of three independent experiments. # p < 0.05 versus NC-siRNA, two-tailed t test. J, siRNA knock-down of Cav3.2 attenuates the BIM23042-induced alleviation of mechanical allodynia in CFA-treated mice ( n = 8 - 10 mice per group). * p < 0.01 and ** p < 0.01 versus NC-siRNA in CFA-treated mice; ++ p < 0.01 versus CFA 2 d; # p < 0.05 versus 0 h point in NC-siRNA-treated mice, two-way ANOVA followed by Bonferroni post hoc test. The black arrow indicates the intra-TG injection of 5 nmol BIM23042.

    Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and probed with antibodies against Nmb (rabbit, 1:1000, Abcam, Cat. No. ab191499), Cav3.1 (rabbit, 1:1000, Alomone, Cat. No. ACC-021), Cav3.2 (mouse, 1:1000, Novus, Cat. No. NBP1-22444), Cav3.3 (rabbit, 1:1000, Alomone, Cat. No. ACC-009), NmbR (rabbit, 1:500, Sigma, Cat. No. SAB4502914), phospho-AMPKα1 (rabbit, 1:500, Abcam, Cat. No. ab92701), AMPKα1 (rabbit, 1:600, Abcam, Cat. No. ab110036), Gαq/11 (mouse, 1:600, Santa Cruz Biotechnology, Cat. No. SC-365906), and Gβ (mouse, 1:500, Santa Cruz Biotechnology, Cat. No. SC-515822).

    Techniques: Injection, Produced, Expressing, Two Tailed Test

    Proposed mechanisms of NmbR signalling on Cav3.2 channels in TG neurons. The binding of Nmb triggers NmbR to activate G q protein and releases the G βγ subunits. The released G βγ dimer then stimulates AMPK activity, induces the activation of PKA and subsequently phosphorylates Cav3.2 channels to increase I T . Neither PLC/PKC nor direct binding of G βγ to Cav3.2 channels is required for T-type channel response mediated by NmbR.

    Journal: Theranostics

    Article Title: Neuromedin B receptor stimulation of Cav3.2 T-type Ca 2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity

    doi: 10.7150/thno.62255

    Figure Lengend Snippet: Proposed mechanisms of NmbR signalling on Cav3.2 channels in TG neurons. The binding of Nmb triggers NmbR to activate G q protein and releases the G βγ subunits. The released G βγ dimer then stimulates AMPK activity, induces the activation of PKA and subsequently phosphorylates Cav3.2 channels to increase I T . Neither PLC/PKC nor direct binding of G βγ to Cav3.2 channels is required for T-type channel response mediated by NmbR.

    Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and probed with antibodies against Nmb (rabbit, 1:1000, Abcam, Cat. No. ab191499), Cav3.1 (rabbit, 1:1000, Alomone, Cat. No. ACC-021), Cav3.2 (mouse, 1:1000, Novus, Cat. No. NBP1-22444), Cav3.3 (rabbit, 1:1000, Alomone, Cat. No. ACC-009), NmbR (rabbit, 1:500, Sigma, Cat. No. SAB4502914), phospho-AMPKα1 (rabbit, 1:500, Abcam, Cat. No. ab92701), AMPKα1 (rabbit, 1:600, Abcam, Cat. No. ab110036), Gαq/11 (mouse, 1:600, Santa Cruz Biotechnology, Cat. No. SC-365906), and Gβ (mouse, 1:500, Santa Cruz Biotechnology, Cat. No. SC-515822).

    Techniques: Binding Assay, Activity Assay, Activation Assay

    Ca currents in wt and Cav3.2 KO ventricular cardiomyocytes. ( A ) Original traces of Ca currents of a typical wt and Cav3.2 KO myocyte elicited by the pulse protocol displayed on top. ( B ) Current density–voltage relationships of wt (n = 20) and Cav3.2 KO (n = 20) myocytes. Data are expressed as means ± SEM. The current density–voltage relations hardly revealed any T-type Ca channel activity (expected to be maximal at −45 mV) but did reveal significant L-type Ca channel activity (peaking at +5 mV), which was similar in wt and Cav3.2 KO myocytes ( p = 0.67 at +5 mV, unpaired Student’s t -test). Capacitance values of the examined myocytes were 184 ± 13 pF for wt and 169 ± 11 pF for Cav3.2 KO ( p = 0.37). ( C ) The effect of the external application of 100 nM isoprenaline (ISO) on ICaT in a wt myocyte. From a holding potential of −95 mV, depolarizing voltage steps to −45 mV were applied every 3 s to elicit the currents. The arrows indicate the beginning and end of ISO application. Current peaks were finally plotted against time. ( D ) ICaT peaks before, during application after the steady-state was reached, and after the washout of ISO in wt (top) and Cav3.2 KO (bottom) myocytes. There was no significant difference between wt and Cav3.2 KO under control conditions ( p = 0.81, unpaired Student’s t -test) or in the presence of ISO ( p = 0.54). Data are expressed as means ± SEM. Each data point represents a single cell. ** p < 0.01; *** p < 0.001; paired Student’s t -test.

    Journal: Membranes

    Article Title: Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice

    doi: 10.3390/membranes12060566

    Figure Lengend Snippet: Ca currents in wt and Cav3.2 KO ventricular cardiomyocytes. ( A ) Original traces of Ca currents of a typical wt and Cav3.2 KO myocyte elicited by the pulse protocol displayed on top. ( B ) Current density–voltage relationships of wt (n = 20) and Cav3.2 KO (n = 20) myocytes. Data are expressed as means ± SEM. The current density–voltage relations hardly revealed any T-type Ca channel activity (expected to be maximal at −45 mV) but did reveal significant L-type Ca channel activity (peaking at +5 mV), which was similar in wt and Cav3.2 KO myocytes ( p = 0.67 at +5 mV, unpaired Student’s t -test). Capacitance values of the examined myocytes were 184 ± 13 pF for wt and 169 ± 11 pF for Cav3.2 KO ( p = 0.37). ( C ) The effect of the external application of 100 nM isoprenaline (ISO) on ICaT in a wt myocyte. From a holding potential of −95 mV, depolarizing voltage steps to −45 mV were applied every 3 s to elicit the currents. The arrows indicate the beginning and end of ISO application. Current peaks were finally plotted against time. ( D ) ICaT peaks before, during application after the steady-state was reached, and after the washout of ISO in wt (top) and Cav3.2 KO (bottom) myocytes. There was no significant difference between wt and Cav3.2 KO under control conditions ( p = 0.81, unpaired Student’s t -test) or in the presence of ISO ( p = 0.54). Data are expressed as means ± SEM. Each data point represents a single cell. ** p < 0.01; *** p < 0.001; paired Student’s t -test.

    Article Snippet: This was followed by blocking with 10% goat serum (Sigma-Aldrich, Vienna, Austria) and 0.01% azide (Sigma-Aldrich, Vienna, Austria) in PBS for 2 h. Subsequently, the cells were incubated with a selective anti-Cav3.1 antibody (anti-CACNA1G, #ACC-021, source: rabbit, Alomone Labs, Jerusalem, Israel, 1:500) or with one of two selective anti-Cav3.2 antibodies (anti-CACNA1H, #ACC-025, source: rabbit, Alomone Labs, Jerusalem, Israel, 1:1000; anti-Cav3.2 polyclonal antibody, #PA5-106771, source: rabbit, Invitrogen, Paisley, England, 1:200) at 4 °C overnight.

    Techniques: Activity Assay

    Immunostaining of T-type Ca channels in ventricular cardiomyocytes isolated from adult wt mice. Cav3.1 ( top ) channel expression and localization was detected using a selective anti-Cav3.1 antibody (anti-CACNA1G, #ACC-021, source: rabbit, Alomone Labs, 1:500). Cav3.2 ( bottom ) was detected with the selective anti-Cav3.2 antibodies (anti-CACNA1H, #ACC-025, source: rabbit, Alomone Labs, 1:1000; left and right cell; or anti-Cav3.2 polyclonal, #PA5-106771, source: rabbit, Invitrogen, 1:200; middle cell). Secondary antibody: Alexa Fluor 488, #A11008, goat anti-rabbit, Invitrogen, 1:500.

    Journal: Membranes

    Article Title: Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice

    doi: 10.3390/membranes12060566

    Figure Lengend Snippet: Immunostaining of T-type Ca channels in ventricular cardiomyocytes isolated from adult wt mice. Cav3.1 ( top ) channel expression and localization was detected using a selective anti-Cav3.1 antibody (anti-CACNA1G, #ACC-021, source: rabbit, Alomone Labs, 1:500). Cav3.2 ( bottom ) was detected with the selective anti-Cav3.2 antibodies (anti-CACNA1H, #ACC-025, source: rabbit, Alomone Labs, 1:1000; left and right cell; or anti-Cav3.2 polyclonal, #PA5-106771, source: rabbit, Invitrogen, 1:200; middle cell). Secondary antibody: Alexa Fluor 488, #A11008, goat anti-rabbit, Invitrogen, 1:500.

    Article Snippet: This was followed by blocking with 10% goat serum (Sigma-Aldrich, Vienna, Austria) and 0.01% azide (Sigma-Aldrich, Vienna, Austria) in PBS for 2 h. Subsequently, the cells were incubated with a selective anti-Cav3.1 antibody (anti-CACNA1G, #ACC-021, source: rabbit, Alomone Labs, Jerusalem, Israel, 1:500) or with one of two selective anti-Cav3.2 antibodies (anti-CACNA1H, #ACC-025, source: rabbit, Alomone Labs, Jerusalem, Israel, 1:1000; anti-Cav3.2 polyclonal antibody, #PA5-106771, source: rabbit, Invitrogen, Paisley, England, 1:200) at 4 °C overnight.

    Techniques: Immunostaining, Isolation, Expressing

    Electrically induced and caffeine-induced intracellular Ca transients in wt and Cav3.2 KO ventricular cardiomyocytes. ( A ) Left: Representative time courses of Fluo-4 fluorescence reporting rises in cytosolic Ca concentration during electrical field stimulation at 0.1 Hz frequency in a single wt and Cav3.2 KO myocyte. The orange lines represent fits of the data with a single exponential function. Right: Comparison of mean Ca peak fluorescence relative to baseline (F/F0) between wt and Cav3.2 KO myocytes. Each data point represents a single cell, and values are expressed as means ± SEM (n= 68 for wt and 59 for Cav3.2 KO myocytes). * p < 0.05 (0.02); unpaired Student’s t -test. The Ca transients decayed with time constants of 0.30 ± 0.02 s in wt and 0.25 ± 0.01 s in Cav3.2 KO myocytes, which were significantly different ( p = 0.04). ( B ) Comparison of mean Ca peak fluorescence (F/F0) before and after the application of 100 nM ISO in wt (n = 20) and Cav3.2 KO (n = 21) myocytes. **** p < 0.0001 (paired Student’s t -test). There was no significant difference between wt and Cav3.2 KO in the presence of ISO ( p = 0.37; unpaired Student’s t -test). The Ca transients under ISO decayed with time constants of 0.14 ± 0.02 s in wt and 0.11 ± 0.02 s in Cav3.2 KO myocytes (not significantly different, p = 0.19). ( C ) Comparison of mean Ca peak fluorescence elicited by an application of 20 mM caffeine, relative to baseline (F/F0), between wt (n = 26) and Cav3.2 KO (n = 35) myocytes. ns—not significant (unpaired Student’s t -test, p = 0.42).

    Journal: Membranes

    Article Title: Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice

    doi: 10.3390/membranes12060566

    Figure Lengend Snippet: Electrically induced and caffeine-induced intracellular Ca transients in wt and Cav3.2 KO ventricular cardiomyocytes. ( A ) Left: Representative time courses of Fluo-4 fluorescence reporting rises in cytosolic Ca concentration during electrical field stimulation at 0.1 Hz frequency in a single wt and Cav3.2 KO myocyte. The orange lines represent fits of the data with a single exponential function. Right: Comparison of mean Ca peak fluorescence relative to baseline (F/F0) between wt and Cav3.2 KO myocytes. Each data point represents a single cell, and values are expressed as means ± SEM (n= 68 for wt and 59 for Cav3.2 KO myocytes). * p < 0.05 (0.02); unpaired Student’s t -test. The Ca transients decayed with time constants of 0.30 ± 0.02 s in wt and 0.25 ± 0.01 s in Cav3.2 KO myocytes, which were significantly different ( p = 0.04). ( B ) Comparison of mean Ca peak fluorescence (F/F0) before and after the application of 100 nM ISO in wt (n = 20) and Cav3.2 KO (n = 21) myocytes. **** p < 0.0001 (paired Student’s t -test). There was no significant difference between wt and Cav3.2 KO in the presence of ISO ( p = 0.37; unpaired Student’s t -test). The Ca transients under ISO decayed with time constants of 0.14 ± 0.02 s in wt and 0.11 ± 0.02 s in Cav3.2 KO myocytes (not significantly different, p = 0.19). ( C ) Comparison of mean Ca peak fluorescence elicited by an application of 20 mM caffeine, relative to baseline (F/F0), between wt (n = 26) and Cav3.2 KO (n = 35) myocytes. ns—not significant (unpaired Student’s t -test, p = 0.42).

    Article Snippet: This was followed by blocking with 10% goat serum (Sigma-Aldrich, Vienna, Austria) and 0.01% azide (Sigma-Aldrich, Vienna, Austria) in PBS for 2 h. Subsequently, the cells were incubated with a selective anti-Cav3.1 antibody (anti-CACNA1G, #ACC-021, source: rabbit, Alomone Labs, Jerusalem, Israel, 1:500) or with one of two selective anti-Cav3.2 antibodies (anti-CACNA1H, #ACC-025, source: rabbit, Alomone Labs, Jerusalem, Israel, 1:1000; anti-Cav3.2 polyclonal antibody, #PA5-106771, source: rabbit, Invitrogen, Paisley, England, 1:200) at 4 °C overnight.

    Techniques: Fluorescence, Concentration Assay

    ( A and B ) Confocal images of EP13 transplanted ZsGreen-labeled ChCs transfected with LacZ sgRNAs (A, left) or Cav3.3 sgRNAs (A, right) as well as EP13 transplanted GFP-labeled ChCs originating from Cav3.3 germline KO mice (B). Single optical sections indicating synaptic cartridges apposed to AnkG-labeled AISs are shown in the middle panels. The areas in white boxes with arrowheads are enlarged and overlaid with the AnkG channel. Bottom panels indicate reconstructed axonal arbors in the area defined in . ( C ) The number of branch points in LacZ sgRNA –transfected ChCs ( n = 5 cells from three mice), Cav3.3 sgRNA– transfected homozygous KO ChCs ( n = 5 cells from three mice), transplanted ChCs originating from Cav3.3 homozygous KO mice ( n = 6 cells from three mice), and transplanted ChCs originating from Cav3.3 heterozygous KO mice ( n = 5 cells from three mice). One-way ANOVA, * P < 0.05, *** P < 0.001, F = 14.3, df = 20. ( D ) Confocal images of P13 endogenous RFP-labeled ChCs in WT mice (left) and Cav3.3 heterozygous KO mouse (right). ( E ) The number of branch points in endogenous ChCs in WT mice ( n = 5 cells from three mice) and Cav3.3 heterozygous KO mice ( n = 6 cells from three mice). t test (two-tailed), ** P < 0.01, t = 5.80, df = 9. Scale bars, 10 μm.

    Journal: Science Advances

    Article Title: Neuromodulatory control of inhibitory network arborization in the developing postnatal neocortex

    doi: 10.1126/sciadv.abe7192

    Figure Lengend Snippet: ( A and B ) Confocal images of EP13 transplanted ZsGreen-labeled ChCs transfected with LacZ sgRNAs (A, left) or Cav3.3 sgRNAs (A, right) as well as EP13 transplanted GFP-labeled ChCs originating from Cav3.3 germline KO mice (B). Single optical sections indicating synaptic cartridges apposed to AnkG-labeled AISs are shown in the middle panels. The areas in white boxes with arrowheads are enlarged and overlaid with the AnkG channel. Bottom panels indicate reconstructed axonal arbors in the area defined in . ( C ) The number of branch points in LacZ sgRNA –transfected ChCs ( n = 5 cells from three mice), Cav3.3 sgRNA– transfected homozygous KO ChCs ( n = 5 cells from three mice), transplanted ChCs originating from Cav3.3 homozygous KO mice ( n = 6 cells from three mice), and transplanted ChCs originating from Cav3.3 heterozygous KO mice ( n = 5 cells from three mice). One-way ANOVA, * P < 0.05, *** P < 0.001, F = 14.3, df = 20. ( D ) Confocal images of P13 endogenous RFP-labeled ChCs in WT mice (left) and Cav3.3 heterozygous KO mouse (right). ( E ) The number of branch points in endogenous ChCs in WT mice ( n = 5 cells from three mice) and Cav3.3 heterozygous KO mice ( n = 6 cells from three mice). t test (two-tailed), ** P < 0.01, t = 5.80, df = 9. Scale bars, 10 μm.

    Article Snippet: The following primary antibodies were used: goat anti-ChAT (1:200; Millipore, catalog no. AB144P), rabbit anti-Cav3.1 (1:100; Alomone Labs, catalog no.ACC-021), rabbit anti-Cav3.2 (1:100; Alomone Labs, catalog no. ACC-025), rabbit anti-Cav3.3 (1:100; Alomone Labs, catalog no. ACC-009), guinea pig polyclonal anti-PV (1:2000; Swant, PVG-213), rat anti-hemagglutinin (1:500; Roche, catalog no.11-867-423-001), chicken anti-GFP (1:800; Abcam, catalog no.ab13970), mouse anti-AnkG (1:500; UC Davis/NIH Neuromab, catalog no. clone N106/36 75-146), rabbit anti-ZsGreen (1:500; Clontech, catalog no. 632474), and rabbit anti-RFP (1:800; Rockland, catalog no. 600-401-379).

    Techniques: Labeling, Transfection, Two Tailed Test

    Summary of primary antibodies and dilutions for the immunolabeling

    Journal: Brain Structure & Function

    Article Title: Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei

    doi: 10.1007/s00429-021-02315-7

    Figure Lengend Snippet: Summary of primary antibodies and dilutions for the immunolabeling

    Article Snippet: Voltage-dependent T-type calcium channel subunits Cav3.1 (CACNA1G, α1G; Cat #: ACC-021; RRID: AB_2039779), Cav3.2 (CACNA1H, α1H; Cat #: ACC-025; RRID: AB_2039781) and Cav3.3 (CACNA1I, α1H; Cat #: ACC-009; RRID: AB_2039783) were detected with polyclonal rabbit antibodies from (Alomone Labs, Jerusalem, ISRAEL).

    Techniques: Marker

    Qualitative summary of immunohistochemical results ( − no labeling, + weak, + + moderate, + + + strong labeling)

    Journal: Brain Structure & Function

    Article Title: Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei

    doi: 10.1007/s00429-021-02315-7

    Figure Lengend Snippet: Qualitative summary of immunohistochemical results ( − no labeling, + weak, + + moderate, + + + strong labeling)

    Article Snippet: Voltage-dependent T-type calcium channel subunits Cav3.1 (CACNA1G, α1G; Cat #: ACC-021; RRID: AB_2039779), Cav3.2 (CACNA1H, α1H; Cat #: ACC-025; RRID: AB_2039781) and Cav3.3 (CACNA1I, α1H; Cat #: ACC-009; RRID: AB_2039783) were detected with polyclonal rabbit antibodies from (Alomone Labs, Jerusalem, ISRAEL).

    Techniques: Immunohistochemical staining, Labeling

    Low-voltage activated calcium channel family (Cav3) subunits in the abducens and trochlear nucleus. a Consecutive coronal paraffin sections through the abducens nucleus (nVI) ( a ) depicting the immunolabeling for Cav3.1 (first panel), Cav3.2 (third panel) and Cav3.3 (fourth panel) subunits with combined immunostaining for ChAT (brown) and ACAN (black) (second panel) as reference. b, c Close-up of Cav subunit expression in nVI MIF (red arrowheads) and SIF motoneurons (MNs) (green arrows) and INTs (blue arrow). d Consecutive coronal paraffin sections through the trochlear nucleus (nVI) depicting the immunolabeling for Cav3.1 (first panel), Cav3.2 (second panel) and Cav3.3 (fourth panel) subunits with combined immunostaining for ChAT (brown) and ACAN (black) (third panel) as reference. Thin dashed lines in d indicate the border of nIV and thick dashed lines indicate the boundary between the MIF and SIF MNs. e Close-up of Cav subunit expression in MIF (red arrowheads) and SIF MNs (green arrow) on different sections as those illustrated in d . Note the weak Cav3.1 expression along the membrane of some SIF MNs (left column, black star). Red dashed lines indicate the tentative position of the border delineating the dorsal cap of nIV. f Cerebellar Purkinje cells located on the same consecutive sections as nVI as controls for immunopositivity of the different Cav subunits. Scale bar indicates 100 μm in a, d, f and 50 μm in b, c, e

    Journal: Brain Structure & Function

    Article Title: Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei

    doi: 10.1007/s00429-021-02315-7

    Figure Lengend Snippet: Low-voltage activated calcium channel family (Cav3) subunits in the abducens and trochlear nucleus. a Consecutive coronal paraffin sections through the abducens nucleus (nVI) ( a ) depicting the immunolabeling for Cav3.1 (first panel), Cav3.2 (third panel) and Cav3.3 (fourth panel) subunits with combined immunostaining for ChAT (brown) and ACAN (black) (second panel) as reference. b, c Close-up of Cav subunit expression in nVI MIF (red arrowheads) and SIF motoneurons (MNs) (green arrows) and INTs (blue arrow). d Consecutive coronal paraffin sections through the trochlear nucleus (nVI) depicting the immunolabeling for Cav3.1 (first panel), Cav3.2 (second panel) and Cav3.3 (fourth panel) subunits with combined immunostaining for ChAT (brown) and ACAN (black) (third panel) as reference. Thin dashed lines in d indicate the border of nIV and thick dashed lines indicate the boundary between the MIF and SIF MNs. e Close-up of Cav subunit expression in MIF (red arrowheads) and SIF MNs (green arrow) on different sections as those illustrated in d . Note the weak Cav3.1 expression along the membrane of some SIF MNs (left column, black star). Red dashed lines indicate the tentative position of the border delineating the dorsal cap of nIV. f Cerebellar Purkinje cells located on the same consecutive sections as nVI as controls for immunopositivity of the different Cav subunits. Scale bar indicates 100 μm in a, d, f and 50 μm in b, c, e

    Article Snippet: Voltage-dependent T-type calcium channel subunits Cav3.1 (CACNA1G, α1G; Cat #: ACC-021; RRID: AB_2039779), Cav3.2 (CACNA1H, α1H; Cat #: ACC-025; RRID: AB_2039781) and Cav3.3 (CACNA1I, α1H; Cat #: ACC-009; RRID: AB_2039783) were detected with polyclonal rabbit antibodies from (Alomone Labs, Jerusalem, ISRAEL).

    Techniques: Immunolabeling, Immunostaining, Expressing