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    Alomone Labs 1 ebio anticonvulsant effects 1 ebio
    1 Ebio Anticonvulsant Effects 1 Ebio, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs nimodipine
    Gonadotropin-releasing hormone (GnRH)-induced ERK1/2 phosphorylation is Gα q/11 and protein kinase C (PKC) dependent, and calcium independent in homologous LβT2 cells. ( A ) LβT2 cells were pretreated with 10 µM FR900359 (Gα q/11 inhibitor) or vehicle (dimethyl sulfoxide, DMSO) for 1 hr, and then treated with vehicle (water) or 10 nM GnRH for 5 or 15 min. Whole cell protein lysates were collected and subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting with phospho- (top) or total (bottom) ERK1/2 antibodies. Blots from one of two replicate experiments are shown. ( B ) Data from independent duplicate experiments exemplified in panel A were quantified by normalizing the densitometry for the pERK1/2 bands to the total ERK1/2 bands. Data are presented as fold phospho-ERK1/2 relative to the control condition. Bar height reflects the group mean. Data were analyzed by two-way analysis of variance (ANOVA), followed by Sidak’s post hoc comparison tests. Bars with different letters differed significantly. Vehicle vs. FR900359: 0 min GnRH p > 0.9999; 5 min GnRH p = 0.0106; 15 min GnRH p = 0.0085. ( C ) LβT2 cells were pretreated for 20 min with vehicle (dimethyl sulfoxide, DMSO) or 5 µM Gö6983 for 20 min followed by treatment with vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. ( D ) Data from three independent experiments in exemplified panel C were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post hoc comparison tests. Bars with different letters differed significantly. Control (vehicle) vs. GnRH (vehicle) p = 0.0048; Control (Gö6983) vs. GnRH (Gö6983) p = 0.3470. Control (vehicle) vs. Control (Gö6983) p = 0.9472; GnRH (vehicle) vs. GnRH (Gö6983) p = 0.0389. ( E ) LβT2 cells were pretreated for 20 min with vehicle (dimethyl sulfoxide, DMSO), 20 µM BAPTA-AM, 5 µM or 10 µM <t>nimodipine,</t> or 10 µM U0126 for 20 min followed by vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. ( F ) Data from three independent experiments in panel E were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post hoc comparison tests. Bars with different letters differed significantly. Control vs. GnRH: Vehicle p = 0.0103; BAPTA-AM p = 0.0229; Nimodipine (5 µM) p = 0.0377; Nimodipine (10 µM) p = 0.0350; U1026 p > 0.9999. Source data for Figure 7—figure supplement 1A . Source data for Figure 7—figure supplement 1C . Source data for Figure 7—figure supplement 1E .
    Nimodipine, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs 1 ebio anticonvulsant effects 1 ebio
    Gonadotropin-releasing hormone (GnRH)-induced ERK1/2 phosphorylation is Gα q/11 and protein kinase C (PKC) dependent, and calcium independent in homologous LβT2 cells. ( A ) LβT2 cells were pretreated with 10 µM FR900359 (Gα q/11 inhibitor) or vehicle (dimethyl sulfoxide, DMSO) for 1 hr, and then treated with vehicle (water) or 10 nM GnRH for 5 or 15 min. Whole cell protein lysates were collected and subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting with phospho- (top) or total (bottom) ERK1/2 antibodies. Blots from one of two replicate experiments are shown. ( B ) Data from independent duplicate experiments exemplified in panel A were quantified by normalizing the densitometry for the pERK1/2 bands to the total ERK1/2 bands. Data are presented as fold phospho-ERK1/2 relative to the control condition. Bar height reflects the group mean. Data were analyzed by two-way analysis of variance (ANOVA), followed by Sidak’s post hoc comparison tests. Bars with different letters differed significantly. Vehicle vs. FR900359: 0 min GnRH p > 0.9999; 5 min GnRH p = 0.0106; 15 min GnRH p = 0.0085. ( C ) LβT2 cells were pretreated for 20 min with vehicle (dimethyl sulfoxide, DMSO) or 5 µM Gö6983 for 20 min followed by treatment with vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. ( D ) Data from three independent experiments in exemplified panel C were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post hoc comparison tests. Bars with different letters differed significantly. Control (vehicle) vs. GnRH (vehicle) p = 0.0048; Control (Gö6983) vs. GnRH (Gö6983) p = 0.3470. Control (vehicle) vs. Control (Gö6983) p = 0.9472; GnRH (vehicle) vs. GnRH (Gö6983) p = 0.0389. ( E ) LβT2 cells were pretreated for 20 min with vehicle (dimethyl sulfoxide, DMSO), 20 µM BAPTA-AM, 5 µM or 10 µM <t>nimodipine,</t> or 10 µM U0126 for 20 min followed by vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. ( F ) Data from three independent experiments in panel E were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post hoc comparison tests. Bars with different letters differed significantly. Control vs. GnRH: Vehicle p = 0.0103; BAPTA-AM p = 0.0229; Nimodipine (5 µM) p = 0.0377; Nimodipine (10 µM) p = 0.0350; U1026 p > 0.9999. Source data for Figure 7—figure supplement 1A . Source data for Figure 7—figure supplement 1C . Source data for Figure 7—figure supplement 1E .
    1 Ebio Anticonvulsant Effects 1 Ebio, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    Gonadotropin-releasing hormone (GnRH)-induced ERK1/2 phosphorylation is Gα q/11 and protein kinase C (PKC) dependent, and calcium independent in homologous LβT2 cells. ( A ) LβT2 cells were pretreated with 10 µM FR900359 (Gα q/11 inhibitor) or vehicle (dimethyl sulfoxide, DMSO) for 1 hr, and then treated with vehicle (water) or 10 nM GnRH for 5 or 15 min. Whole cell protein lysates were collected and subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting with phospho- (top) or total (bottom) ERK1/2 antibodies. Blots from one of two replicate experiments are shown. ( B ) Data from independent duplicate experiments exemplified in panel A were quantified by normalizing the densitometry for the pERK1/2 bands to the total ERK1/2 bands. Data are presented as fold phospho-ERK1/2 relative to the control condition. Bar height reflects the group mean. Data were analyzed by two-way analysis of variance (ANOVA), followed by Sidak’s post hoc comparison tests. Bars with different letters differed significantly. Vehicle vs. FR900359: 0 min GnRH p > 0.9999; 5 min GnRH p = 0.0106; 15 min GnRH p = 0.0085. ( C ) LβT2 cells were pretreated for 20 min with vehicle (dimethyl sulfoxide, DMSO) or 5 µM Gö6983 for 20 min followed by treatment with vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. ( D ) Data from three independent experiments in exemplified panel C were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post hoc comparison tests. Bars with different letters differed significantly. Control (vehicle) vs. GnRH (vehicle) p = 0.0048; Control (Gö6983) vs. GnRH (Gö6983) p = 0.3470. Control (vehicle) vs. Control (Gö6983) p = 0.9472; GnRH (vehicle) vs. GnRH (Gö6983) p = 0.0389. ( E ) LβT2 cells were pretreated for 20 min with vehicle (dimethyl sulfoxide, DMSO), 20 µM BAPTA-AM, 5 µM or 10 µM nimodipine, or 10 µM U0126 for 20 min followed by vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. ( F ) Data from three independent experiments in panel E were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post hoc comparison tests. Bars with different letters differed significantly. Control vs. GnRH: Vehicle p = 0.0103; BAPTA-AM p = 0.0229; Nimodipine (5 µM) p = 0.0377; Nimodipine (10 µM) p = 0.0350; U1026 p > 0.9999. Source data for Figure 7—figure supplement 1A . Source data for Figure 7—figure supplement 1C . Source data for Figure 7—figure supplement 1E .

    Journal: eLife

    Article Title: Addition of a carboxy-terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

    doi: 10.7554/eLife.72937

    Figure Lengend Snippet: Gonadotropin-releasing hormone (GnRH)-induced ERK1/2 phosphorylation is Gα q/11 and protein kinase C (PKC) dependent, and calcium independent in homologous LβT2 cells. ( A ) LβT2 cells were pretreated with 10 µM FR900359 (Gα q/11 inhibitor) or vehicle (dimethyl sulfoxide, DMSO) for 1 hr, and then treated with vehicle (water) or 10 nM GnRH for 5 or 15 min. Whole cell protein lysates were collected and subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting with phospho- (top) or total (bottom) ERK1/2 antibodies. Blots from one of two replicate experiments are shown. ( B ) Data from independent duplicate experiments exemplified in panel A were quantified by normalizing the densitometry for the pERK1/2 bands to the total ERK1/2 bands. Data are presented as fold phospho-ERK1/2 relative to the control condition. Bar height reflects the group mean. Data were analyzed by two-way analysis of variance (ANOVA), followed by Sidak’s post hoc comparison tests. Bars with different letters differed significantly. Vehicle vs. FR900359: 0 min GnRH p > 0.9999; 5 min GnRH p = 0.0106; 15 min GnRH p = 0.0085. ( C ) LβT2 cells were pretreated for 20 min with vehicle (dimethyl sulfoxide, DMSO) or 5 µM Gö6983 for 20 min followed by treatment with vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. ( D ) Data from three independent experiments in exemplified panel C were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post hoc comparison tests. Bars with different letters differed significantly. Control (vehicle) vs. GnRH (vehicle) p = 0.0048; Control (Gö6983) vs. GnRH (Gö6983) p = 0.3470. Control (vehicle) vs. Control (Gö6983) p = 0.9472; GnRH (vehicle) vs. GnRH (Gö6983) p = 0.0389. ( E ) LβT2 cells were pretreated for 20 min with vehicle (dimethyl sulfoxide, DMSO), 20 µM BAPTA-AM, 5 µM or 10 µM nimodipine, or 10 µM U0126 for 20 min followed by vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. ( F ) Data from three independent experiments in panel E were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post hoc comparison tests. Bars with different letters differed significantly. Control vs. GnRH: Vehicle p = 0.0103; BAPTA-AM p = 0.0229; Nimodipine (5 µM) p = 0.0377; Nimodipine (10 µM) p = 0.0350; U1026 p > 0.9999. Source data for Figure 7—figure supplement 1A . Source data for Figure 7—figure supplement 1C . Source data for Figure 7—figure supplement 1E .

    Article Snippet: To evaluate the contribution of voltage-gated calcium channels, after 1 hr of recovery, tissue was incubated for 30 s with 20 μM nimodipine (ALOMONE LABS N-150 N150SM0250; Jerusalem, Israel) followed by a second application of 10 nM GnRH alone ( ) or in combination with 20 μM nimodipine ( ) for 30 s. Finally, to determine cell viability, high potassium solution (50 KCl mM, 120 NaCl mM, 10 HEPES mM, 2 CaCl2 mM, pH 7.4) was applied for 30 s. For each condition, the numbers of animals and cells analyzed are indicated in the figure legends.

    Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot

    Gonadotropin-releasing hormone (GnRH)-induced Fshb and Lhb expression does not depend on calcium entry via L-type channels in homologous LβT2 cells. ( A ) Relative Fshb and ( B ) Lhb expression in LβT2 cells treated with vehicle (dimethyl sulfoxide, DMSO) or 10 µM nimodipine for 20 min followed by treatment with water (vehicle) or high-frequency GnRH (10 nM) pulses. Gene expression was assessed by RT-qPCR and normalized to Rpl19 . Data reflect the means of two independent experiments. Data were analyzed with two-way analyses of variance (ANOVAs), followed by post hoc Tukey’s test for multiple comparisons. Bars with different letters differed significantly. Panel A: Control (vehicle) vs. GnRH (vehicle) p = 0.0178; Control (vehicle) vs. GnRH (Nimodipine) p = 0.0232; GnRH (vehicle) vs. GnRH (Nimodipine) p = 0.8545; Control (Nimodipine) vs. GnRH (Nimodipine) p = 0.0235. Panel B: Control (vehicle) vs. GnRH (vehicle) p = 0.0061; Control (vehicle) vs. GnRH (Nimodipine) p = 0.0043; GnRH (vehicle) vs. GnRH (Nimodipine) p = 0.8770; Control (Nimodipine) vs. GnRH (Nimodipine) p = 0.0178.

    Journal: eLife

    Article Title: Addition of a carboxy-terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

    doi: 10.7554/eLife.72937

    Figure Lengend Snippet: Gonadotropin-releasing hormone (GnRH)-induced Fshb and Lhb expression does not depend on calcium entry via L-type channels in homologous LβT2 cells. ( A ) Relative Fshb and ( B ) Lhb expression in LβT2 cells treated with vehicle (dimethyl sulfoxide, DMSO) or 10 µM nimodipine for 20 min followed by treatment with water (vehicle) or high-frequency GnRH (10 nM) pulses. Gene expression was assessed by RT-qPCR and normalized to Rpl19 . Data reflect the means of two independent experiments. Data were analyzed with two-way analyses of variance (ANOVAs), followed by post hoc Tukey’s test for multiple comparisons. Bars with different letters differed significantly. Panel A: Control (vehicle) vs. GnRH (vehicle) p = 0.0178; Control (vehicle) vs. GnRH (Nimodipine) p = 0.0232; GnRH (vehicle) vs. GnRH (Nimodipine) p = 0.8545; Control (Nimodipine) vs. GnRH (Nimodipine) p = 0.0235. Panel B: Control (vehicle) vs. GnRH (vehicle) p = 0.0061; Control (vehicle) vs. GnRH (Nimodipine) p = 0.0043; GnRH (vehicle) vs. GnRH (Nimodipine) p = 0.8770; Control (Nimodipine) vs. GnRH (Nimodipine) p = 0.0178.

    Article Snippet: To evaluate the contribution of voltage-gated calcium channels, after 1 hr of recovery, tissue was incubated for 30 s with 20 μM nimodipine (ALOMONE LABS N-150 N150SM0250; Jerusalem, Israel) followed by a second application of 10 nM GnRH alone ( ) or in combination with 20 μM nimodipine ( ) for 30 s. Finally, to determine cell viability, high potassium solution (50 KCl mM, 120 NaCl mM, 10 HEPES mM, 2 CaCl2 mM, pH 7.4) was applied for 30 s. For each condition, the numbers of animals and cells analyzed are indicated in the figure legends.

    Techniques: Expressing, Quantitative RT-PCR

    Nimodipine alters gonadotropin-releasing hormone (GnRH)-induced calcium responses in gonadotropes of wild-type and Ctail mice. The analysis in Figure 8—figure supplement 1 was repeated but with the L-type calcium channel blocker nimodipine (Nim) applied prior to and during the second GnRH pulse. Raster plots of GnRH-induced calcium responses in the absence (left) and presence of nimodipine (right) in gonadotropes from a representative adult male ( A ) wild-type and ( D ) Ctail mouse. The two stimuli were separated by 60 min wash with Krebs–Ringer. Comparisons of AUC from a ( B ) wild-type (275 cells; 11.5 ± 4.7 vs. 7.1 ± 2.8 a.u. for GnRH and GnRH/Nim, respectively; p

    Journal: eLife

    Article Title: Addition of a carboxy-terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

    doi: 10.7554/eLife.72937

    Figure Lengend Snippet: Nimodipine alters gonadotropin-releasing hormone (GnRH)-induced calcium responses in gonadotropes of wild-type and Ctail mice. The analysis in Figure 8—figure supplement 1 was repeated but with the L-type calcium channel blocker nimodipine (Nim) applied prior to and during the second GnRH pulse. Raster plots of GnRH-induced calcium responses in the absence (left) and presence of nimodipine (right) in gonadotropes from a representative adult male ( A ) wild-type and ( D ) Ctail mouse. The two stimuli were separated by 60 min wash with Krebs–Ringer. Comparisons of AUC from a ( B ) wild-type (275 cells; 11.5 ± 4.7 vs. 7.1 ± 2.8 a.u. for GnRH and GnRH/Nim, respectively; p

    Article Snippet: To evaluate the contribution of voltage-gated calcium channels, after 1 hr of recovery, tissue was incubated for 30 s with 20 μM nimodipine (ALOMONE LABS N-150 N150SM0250; Jerusalem, Israel) followed by a second application of 10 nM GnRH alone ( ) or in combination with 20 μM nimodipine ( ) for 30 s. Finally, to determine cell viability, high potassium solution (50 KCl mM, 120 NaCl mM, 10 HEPES mM, 2 CaCl2 mM, pH 7.4) was applied for 30 s. For each condition, the numbers of animals and cells analyzed are indicated in the figure legends.

    Techniques: Mouse Assay

    Nimodipine alters GnRH induced calcium responses in gonadotropes of wild-type and Ctail mice. The analysis in Figure 8 - figure supplement 1 was repeated but with the L-type calcium channel blocker nimodipine (Nim) applied prior to and during the second GnRH pulse. Raster plots of GnRH induced calcium responses in the absence (left) and presence of nimodipine (right) in gonadotropes from a representative adult male (A) wild-type and (D) Ctail mouse. The two stimuli were separated by 60 min wash with Krebs-Ringer. Comparisons of AUC from a (B) wild-type (275 cells; 11.5 ± 4.7 vs. 7.1 ± 2.8 a.u. for GnRH and GnRH/Nim, respectively; p

    Journal: bioRxiv

    Article Title: Addition of a carboxy terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

    doi: 10.1101/2021.09.13.460073

    Figure Lengend Snippet: Nimodipine alters GnRH induced calcium responses in gonadotropes of wild-type and Ctail mice. The analysis in Figure 8 - figure supplement 1 was repeated but with the L-type calcium channel blocker nimodipine (Nim) applied prior to and during the second GnRH pulse. Raster plots of GnRH induced calcium responses in the absence (left) and presence of nimodipine (right) in gonadotropes from a representative adult male (A) wild-type and (D) Ctail mouse. The two stimuli were separated by 60 min wash with Krebs-Ringer. Comparisons of AUC from a (B) wild-type (275 cells; 11.5 ± 4.7 vs. 7.1 ± 2.8 a.u. for GnRH and GnRH/Nim, respectively; p

    Article Snippet: To evaluate the contribution of voltage-gated calcium channels, after 1 h of recovery, tissue was incubated for 30 s with 20 μM nimodipine (ALOMONE LABS N-150 N150SM0250; Jerusalem, Israel) followed by a second application of 10 nM GnRH alone ( ) or in combination with 20 μM nimodipine ( ) for 30 s. Finally, to determine cell viability, high potassium solution (50 KCl mM, 120 NaCl mM, 10 HEPES mM, 2 CaCl2 mM, pH 7.4) was applied for 30 s. For each condition, the numbers of animals and cells analyzed are indicated in the figure legends.

    Techniques: Mouse Assay

    GnRH induced ERK1/2 phosphorylation is Gα q/11 - and PKC-dependent, and calcium-independent in homologous LβT2 cells. (A) LβT2 cells were pretreated with 10 µM FR900359 (Gα q/11 inhibitor) or vehicle (DMSO) for 1 h, and then treated with vehicle (water) or 10 nM GnRH for 5- or 15-min. Whole cell protein lysates were collected and subjected to SDS-PAGE and western blotting with phospho- (top) or total (bottom) ERK1/2 antibodies. Blots from one of two replicate experiments are shown. (B) Data from independent duplicate experiments exemplified in panel A were quantified by normalizing the densitometry for the pERK1/2 bands to the total ERK1/2 bands. Data are presented as fold phospho-ERK1/2 relative to the control condition. Bar height reflects the group mean. Data were analyzed by two-way ANOVA, followed by Sidak’s post-hoc comparison tests. Bars with different letters differed significantly. Vehicle vs. FR900359: 0 min GnRH p > 0.9999; 5 min GnRH p = 0.0106; 15 min GnRH p = 0.0085]. (C) LβT2 cells were pretreated for 20 min with vehicle (DMSO) or 5 µM Gö6983 for 20 min followed by treatment with vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. (D) Data from 3 independent experiments in exemplified panel C were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post-hoc comparison tests. Bars with different letters differed significantly. Control (vehicle) vs. GnRH (vehicle) p = 0.0048; Control (Gö6983) vs. GnRH (Gö6983) p = 0.3470. Control (vehicle) vs. Control (Gö6983) p = 0.9472; GnRH (vehicle) vs. GnRH (Gö6983) p = 0.0389. (E) LβT2 cells were pretreated for 20 min with vehicle (DMSO), 20 µM BAPTA-AM, 5 µM or 10 µM nimodipine, or 10 µM U0126 for 20 min followed by vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. (F) Data from 3 independent experiments in panel E were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post-hoc comparison tests. Bars with different letters differed significantly. Control vs. GnRH: Vehicle p = 0.0103; BAPTA-AM p = 0.0229; Nimodipine (5 µM) p = 0.0377; Nimodipine (10 µM) p = 0.0350; U1026 p > 0.9999.

    Journal: bioRxiv

    Article Title: Addition of a carboxy terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

    doi: 10.1101/2021.09.13.460073

    Figure Lengend Snippet: GnRH induced ERK1/2 phosphorylation is Gα q/11 - and PKC-dependent, and calcium-independent in homologous LβT2 cells. (A) LβT2 cells were pretreated with 10 µM FR900359 (Gα q/11 inhibitor) or vehicle (DMSO) for 1 h, and then treated with vehicle (water) or 10 nM GnRH for 5- or 15-min. Whole cell protein lysates were collected and subjected to SDS-PAGE and western blotting with phospho- (top) or total (bottom) ERK1/2 antibodies. Blots from one of two replicate experiments are shown. (B) Data from independent duplicate experiments exemplified in panel A were quantified by normalizing the densitometry for the pERK1/2 bands to the total ERK1/2 bands. Data are presented as fold phospho-ERK1/2 relative to the control condition. Bar height reflects the group mean. Data were analyzed by two-way ANOVA, followed by Sidak’s post-hoc comparison tests. Bars with different letters differed significantly. Vehicle vs. FR900359: 0 min GnRH p > 0.9999; 5 min GnRH p = 0.0106; 15 min GnRH p = 0.0085]. (C) LβT2 cells were pretreated for 20 min with vehicle (DMSO) or 5 µM Gö6983 for 20 min followed by treatment with vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. (D) Data from 3 independent experiments in exemplified panel C were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post-hoc comparison tests. Bars with different letters differed significantly. Control (vehicle) vs. GnRH (vehicle) p = 0.0048; Control (Gö6983) vs. GnRH (Gö6983) p = 0.3470. Control (vehicle) vs. Control (Gö6983) p = 0.9472; GnRH (vehicle) vs. GnRH (Gö6983) p = 0.0389. (E) LβT2 cells were pretreated for 20 min with vehicle (DMSO), 20 µM BAPTA-AM, 5 µM or 10 µM nimodipine, or 10 µM U0126 for 20 min followed by vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. (F) Data from 3 independent experiments in panel E were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post-hoc comparison tests. Bars with different letters differed significantly. Control vs. GnRH: Vehicle p = 0.0103; BAPTA-AM p = 0.0229; Nimodipine (5 µM) p = 0.0377; Nimodipine (10 µM) p = 0.0350; U1026 p > 0.9999.

    Article Snippet: To evaluate the contribution of voltage-gated calcium channels, after 1 h of recovery, tissue was incubated for 30 s with 20 μM nimodipine (ALOMONE LABS N-150 N150SM0250; Jerusalem, Israel) followed by a second application of 10 nM GnRH alone ( ) or in combination with 20 μM nimodipine ( ) for 30 s. Finally, to determine cell viability, high potassium solution (50 KCl mM, 120 NaCl mM, 10 HEPES mM, 2 CaCl2 mM, pH 7.4) was applied for 30 s. For each condition, the numbers of animals and cells analyzed are indicated in the figure legends.

    Techniques: SDS Page, Western Blot

    GnRH-induced Fshb and Lhb expression does not depend on calcium entry via L-type channels in homologous LβT2 cells. (A) Relative Fshb and (B) Lhb expression in LβT2 cells treated with vehicle (DMSO) or 10 µM nimodipine for 20 min followed by treatment with water (vehicle) or high frequency GnRH (10 nM) pulses. Gene expression was assessed by RT-qPCR and normalized to Rpl19 . Data reflect the means of 2 independent experiments. Data were analyzed with two-way ANOVAs, followed by post-hoc Tukey’s test for multiple comparisons. Bars with different letters differed significantly. Panel A: Control (vehicle) vs. GnRH (vehicle) p = 0.0178; Control (vehicle) vs. GnRH (Nimodipine) p = 0.0232; GnRH (vehicle) vs. GnRH (Nimodipine) p = 0.8545; Control (Nimodipine) vs. GnRH (Nimodipine) p = 0.0235. Panel B: Control (vehicle) vs. GnRH (vehicle) p = 0.0061; Control (vehicle) vs. GnRH (Nimodipine) p = 0.0043; GnRH (vehicle) vs. GnRH (Nimodipine) p = 0.8770; Control (Nimodipine) vs. GnRH (Nimodipine) p = 0.0178.

    Journal: bioRxiv

    Article Title: Addition of a carboxy terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

    doi: 10.1101/2021.09.13.460073

    Figure Lengend Snippet: GnRH-induced Fshb and Lhb expression does not depend on calcium entry via L-type channels in homologous LβT2 cells. (A) Relative Fshb and (B) Lhb expression in LβT2 cells treated with vehicle (DMSO) or 10 µM nimodipine for 20 min followed by treatment with water (vehicle) or high frequency GnRH (10 nM) pulses. Gene expression was assessed by RT-qPCR and normalized to Rpl19 . Data reflect the means of 2 independent experiments. Data were analyzed with two-way ANOVAs, followed by post-hoc Tukey’s test for multiple comparisons. Bars with different letters differed significantly. Panel A: Control (vehicle) vs. GnRH (vehicle) p = 0.0178; Control (vehicle) vs. GnRH (Nimodipine) p = 0.0232; GnRH (vehicle) vs. GnRH (Nimodipine) p = 0.8545; Control (Nimodipine) vs. GnRH (Nimodipine) p = 0.0235. Panel B: Control (vehicle) vs. GnRH (vehicle) p = 0.0061; Control (vehicle) vs. GnRH (Nimodipine) p = 0.0043; GnRH (vehicle) vs. GnRH (Nimodipine) p = 0.8770; Control (Nimodipine) vs. GnRH (Nimodipine) p = 0.0178.

    Article Snippet: To evaluate the contribution of voltage-gated calcium channels, after 1 h of recovery, tissue was incubated for 30 s with 20 μM nimodipine (ALOMONE LABS N-150 N150SM0250; Jerusalem, Israel) followed by a second application of 10 nM GnRH alone ( ) or in combination with 20 μM nimodipine ( ) for 30 s. Finally, to determine cell viability, high potassium solution (50 KCl mM, 120 NaCl mM, 10 HEPES mM, 2 CaCl2 mM, pH 7.4) was applied for 30 s. For each condition, the numbers of animals and cells analyzed are indicated in the figure legends.

    Techniques: Expressing, Quantitative RT-PCR

    Effects of Ca 2+ channel inhibitors on peak and trough Ca 2+ concentration. ( A and B ) Time course of the change in Ca 2+ concentration (ΔCa 2+ = Ca 2+ – average Ca 2+ from 2-min baseline) before and after the application of vehicle control (Veh) or Ca 2+ channel inhibitors at the peak ( A ) and trough ( B ). Data are mean ± SEM. ( C and D ) Plots of median, 25th and 75th percentile ( boxes ), and minimal and maximal ( whiskers ) changes in Ca 2+ concentration for individual slices quantified from 9 to 10 min after drugs were applied at the peak ( C ) and trough ( D ). Inhibition of L-type Ca 2+ channels with nimodipine (Nim, 10 μ M) or inhibition of N/P/Q/R/T-type Ca 2+ channels with VGC (a mixture of 3 μ M ConoGVIA, 200 nM AgaIVA, 30 μ M nickel, and 1 μ M TTA-P2) did not significantly affect peak or trough Ca 2+ levels compared to Veh. Inhibition of ryanodine receptors with dantrolene (Dan, 10 μ M), inhibition of SERCA-ATPase with cyclopiazonic acid (CPA, 10 μ M) and combined inhibition of voltage-gated Ca 2+ channels and ryanodine receptors with a cocktail containing Dan, Nim, and VGC (cocktail X) significantly decreased Ca 2+ at peak and trough. * p

    Journal: Biophysical reports

    Article Title: Comparative Ca2+ channel contributions to intracellular Ca2+ levels in the circadian clock

    doi: 10.1016/j.bpr.2021.100005

    Figure Lengend Snippet: Effects of Ca 2+ channel inhibitors on peak and trough Ca 2+ concentration. ( A and B ) Time course of the change in Ca 2+ concentration (ΔCa 2+ = Ca 2+ – average Ca 2+ from 2-min baseline) before and after the application of vehicle control (Veh) or Ca 2+ channel inhibitors at the peak ( A ) and trough ( B ). Data are mean ± SEM. ( C and D ) Plots of median, 25th and 75th percentile ( boxes ), and minimal and maximal ( whiskers ) changes in Ca 2+ concentration for individual slices quantified from 9 to 10 min after drugs were applied at the peak ( C ) and trough ( D ). Inhibition of L-type Ca 2+ channels with nimodipine (Nim, 10 μ M) or inhibition of N/P/Q/R/T-type Ca 2+ channels with VGC (a mixture of 3 μ M ConoGVIA, 200 nM AgaIVA, 30 μ M nickel, and 1 μ M TTA-P2) did not significantly affect peak or trough Ca 2+ levels compared to Veh. Inhibition of ryanodine receptors with dantrolene (Dan, 10 μ M), inhibition of SERCA-ATPase with cyclopiazonic acid (CPA, 10 μ M) and combined inhibition of voltage-gated Ca 2+ channels and ryanodine receptors with a cocktail containing Dan, Nim, and VGC (cocktail X) significantly decreased Ca 2+ at peak and trough. * p

    Article Snippet: Pharmacological reagents were used at final concentrations of 10 μ M nimodipine (Nim; N150; Alomone Labs, Jerusalem, Israel), 10 μ M dantrolene (Dan; D9175; Sigma-Aldrich), 30 μ M CPA (C-750; Alomone Labs), 3 μ M ω -conotoxin GVIA (ConoGVIA; C-300; Alomone Labs), 200 nM ω -agatoxin IVA (AgaIVA; STA-500; Alomone Labs), 30 μ M NiCl2 (Ni2+ ; 22387; Sigma-Aldrich), 10 μ M ionomycin (407951; Sigma-Aldrich), 10 μ M rotenone (R8875; Sigma-Aldrich), and 50 mM KCl (P9333; Sigma-Aldrich).

    Techniques: Concentration Assay, Inhibition