apc 027  (Alomone Labs)


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    Alomone Labs apc 027
    Apc 027, 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
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    apc 027 - by Bioz Stars, 2023-01
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    apc 027  (Alomone Labs)


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  • 94

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    Alomone Labs apc 027
    Apc 027, 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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    kir3 4  (Alomone Labs)


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    Alomone Labs kir3 4
    Kir3 4, 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 girk4  (Alomone Labs)


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    Alomone Labs anti girk4
    Heart rate recorded in sedentary (left panel) and trained (right panel) WT (A) and <t>Girk4</t> –/– mice (B) at different days during 5 min sham-training (WT and Girk4 –/– sedentary, see methods) and training (WT and Girk4 –/– trained) protocol. (C) Histogram of the average values of the slopes of the regression line between time and heart rate. Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. (D) Representative examples of ECG traces and averaged heart rate recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. Tertiapin-Q (Tert, 5 mg/kg) effect in WT (E) and Girk4 –/– (F) sedentary (empty bar) and trained (filled bars) mice. Statistics: two-way analysis of variance followed by Sidak multiple comparisons test. (G) Close-up of ECG traces and averaged PR interval recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, ### p < 0.001, #### p < 0.0001. Error bars indicate s.e.m. WTS: WT sedentary; WTT: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.
    Anti Girk4, 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/anti girk4/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti girk4 - by Bioz Stars, 2023-01
    94/100 stars

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    1) Product Images from "Genetic Ablation of G Protein-Gated Inwardly Rectifying K + Channels Prevents Training-Induced Sinus Bradycardia"

    Article Title: Genetic Ablation of G Protein-Gated Inwardly Rectifying K + Channels Prevents Training-Induced Sinus Bradycardia

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2020.519382

    Heart rate recorded in sedentary (left panel) and trained (right panel) WT (A) and Girk4 –/– mice (B) at different days during 5 min sham-training (WT and Girk4 –/– sedentary, see methods) and training (WT and Girk4 –/– trained) protocol. (C) Histogram of the average values of the slopes of the regression line between time and heart rate. Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. (D) Representative examples of ECG traces and averaged heart rate recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. Tertiapin-Q (Tert, 5 mg/kg) effect in WT (E) and Girk4 –/– (F) sedentary (empty bar) and trained (filled bars) mice. Statistics: two-way analysis of variance followed by Sidak multiple comparisons test. (G) Close-up of ECG traces and averaged PR interval recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, ### p < 0.001, #### p < 0.0001. Error bars indicate s.e.m. WTS: WT sedentary; WTT: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.
    Figure Legend Snippet: Heart rate recorded in sedentary (left panel) and trained (right panel) WT (A) and Girk4 –/– mice (B) at different days during 5 min sham-training (WT and Girk4 –/– sedentary, see methods) and training (WT and Girk4 –/– trained) protocol. (C) Histogram of the average values of the slopes of the regression line between time and heart rate. Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. (D) Representative examples of ECG traces and averaged heart rate recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. Tertiapin-Q (Tert, 5 mg/kg) effect in WT (E) and Girk4 –/– (F) sedentary (empty bar) and trained (filled bars) mice. Statistics: two-way analysis of variance followed by Sidak multiple comparisons test. (G) Close-up of ECG traces and averaged PR interval recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, ### p < 0.001, #### p < 0.0001. Error bars indicate s.e.m. WTS: WT sedentary; WTT: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.

    Techniques Used:

    Standard deviation of intervals between two consecutive heart beats (SDNN) calculated in 5-min stable ECG periods at day 0 and at day 28 in WT (A) and Girk4 –/– (B) sedentary (open bars) and trained (filled bars) animals. Power spectral density (PSD) of heart rate variability determined by Fast Fourier Transformation analysis of 5-min of stable ECG segments at day 0 and at day 28 in WT (C) and Girk4 –/– (D) sedentary (open bars) and trained (filled bars) animals. Heart rate (non-invasive ECG recordings) measured in sedentary (open bars, left panel) and trained (filled bars, right panel) WT mice before (day 0) and after (day 28) training period in control condition (ANS +, E ) or following intraperitoneal injection of atropine (0.5 mg/kg) and propranolol (5 mg/kg) to inhibit the input of the autonomic nervous system (ANS-, F ). (G,H) same as (E,F) but in Girk4 –/– animals. Statistics: unpaired Student’s t -test. ** p < 0.01, **** p < 0.0001. Error bars indicate s.e.m.
    Figure Legend Snippet: Standard deviation of intervals between two consecutive heart beats (SDNN) calculated in 5-min stable ECG periods at day 0 and at day 28 in WT (A) and Girk4 –/– (B) sedentary (open bars) and trained (filled bars) animals. Power spectral density (PSD) of heart rate variability determined by Fast Fourier Transformation analysis of 5-min of stable ECG segments at day 0 and at day 28 in WT (C) and Girk4 –/– (D) sedentary (open bars) and trained (filled bars) animals. Heart rate (non-invasive ECG recordings) measured in sedentary (open bars, left panel) and trained (filled bars, right panel) WT mice before (day 0) and after (day 28) training period in control condition (ANS +, E ) or following intraperitoneal injection of atropine (0.5 mg/kg) and propranolol (5 mg/kg) to inhibit the input of the autonomic nervous system (ANS-, F ). (G,H) same as (E,F) but in Girk4 –/– animals. Statistics: unpaired Student’s t -test. ** p < 0.01, **** p < 0.0001. Error bars indicate s.e.m.

    Techniques Used: Standard Deviation, Transformation Assay, Injection

    Action potential recordings of SAN myocytes isolated from WT sedentary (WT S), WT trained (WT T), Girk4 –/– sedentary ( Girk4 –/– S) and Girk4 –/– trained ( Girk4 –/– T) in Tyrode’s solution at the end of training, or sham-training protocol (day 28) (A) . The dotted line indicates the 0 mV. Rate of spontaneous action potentials (B) and slope of the linear part of the diastolic depolarization (SLDD, C ) recorded at day 28 in SAN myocytes in Tyrode’s solution ( n = 6 WT S, n = 10 WT T, n = 7 Girk4 –/– S and n = 8 Girk4 –/– T). Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. * p < 0.05, **** p < 0.0001. Error bars indicate s.e.m.
    Figure Legend Snippet: Action potential recordings of SAN myocytes isolated from WT sedentary (WT S), WT trained (WT T), Girk4 –/– sedentary ( Girk4 –/– S) and Girk4 –/– trained ( Girk4 –/– T) in Tyrode’s solution at the end of training, or sham-training protocol (day 28) (A) . The dotted line indicates the 0 mV. Rate of spontaneous action potentials (B) and slope of the linear part of the diastolic depolarization (SLDD, C ) recorded at day 28 in SAN myocytes in Tyrode’s solution ( n = 6 WT S, n = 10 WT T, n = 7 Girk4 –/– S and n = 8 Girk4 –/– T). Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. * p < 0.05, **** p < 0.0001. Error bars indicate s.e.m.

    Techniques Used: Isolation

    Representative traces of I f recordings (A) and averaged current-to-voltage (I-V) curve (B) in sedentary (open black circles, n = 15) and trained (filled black circles, n = 23) WT SAN myocytes. I f recordings (C) and I-V curve (D) in SAN myocytes from sedentary (open blue circles, n = 12) and trained (filled blue circles, n = 18) isolated Girk4 –/– SAN pacemaker cells. The voltage-clamp protocol used for all the recordings is shown at the bottom of panel (C) . Statistical significance was tested at each voltage using the unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001. ( E , left panel) Steady state I f activation curves in isolated SAN cells from sedentary (dotted line) and trained (continuous line), WT (black) and Girk4 –/– (blue) mice. Representative ramp current trace recorded in a WT sedentary SAN cell and corresponding voltage protocol are shown under the curves. ( E , right panel) Histograms representing averaged half-activation voltages (V 1/2 ) values for I f recorded in SAN cells from WT (black bars) and Girk4 –/– (blue bars) sedentary (open bars) and trained (filled bars) animals. Data have been collected at day 28 (end of training, or sham-training, protocol). Statistics: one-way analysis of variance. Error bars indicate s.e.m.. WT S: WT sedentary; WT T: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.
    Figure Legend Snippet: Representative traces of I f recordings (A) and averaged current-to-voltage (I-V) curve (B) in sedentary (open black circles, n = 15) and trained (filled black circles, n = 23) WT SAN myocytes. I f recordings (C) and I-V curve (D) in SAN myocytes from sedentary (open blue circles, n = 12) and trained (filled blue circles, n = 18) isolated Girk4 –/– SAN pacemaker cells. The voltage-clamp protocol used for all the recordings is shown at the bottom of panel (C) . Statistical significance was tested at each voltage using the unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001. ( E , left panel) Steady state I f activation curves in isolated SAN cells from sedentary (dotted line) and trained (continuous line), WT (black) and Girk4 –/– (blue) mice. Representative ramp current trace recorded in a WT sedentary SAN cell and corresponding voltage protocol are shown under the curves. ( E , right panel) Histograms representing averaged half-activation voltages (V 1/2 ) values for I f recorded in SAN cells from WT (black bars) and Girk4 –/– (blue bars) sedentary (open bars) and trained (filled bars) animals. Data have been collected at day 28 (end of training, or sham-training, protocol). Statistics: one-way analysis of variance. Error bars indicate s.e.m.. WT S: WT sedentary; WT T: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.

    Techniques Used: Isolation, Activation Assay

    Representative Ca 2+ current traces of the L-type Ca 2+ current ( I CaL ) measured from a holding potential (HP) of −55 mV (A) and current-to-voltage (I-V) relationships (B) from sedentary (open black circles, n = 14) and trained (filled black circles, n = 13) WT SAN myocytes. Traces of I CaL (C) and I-V curves (D) from sedentary (open blue circles, n = 15) compared with trained (filled blue circles, n = 22) Girk4 –/– SAN myocytes. Data, collected at day 28 were fitted with a modified Boltzmann equation. Voltage protocol is shown in panel C (bottom). (E) Sample traces of I Ca ( I CaT + I CaL ) recorded from a HP of −80 mV in WT and Girk4 –/– SAN myocytes. (F) Sample I Ca traces for same myocytes as in (E) , but after switching to HP = −55 to inactivate I CaT . (G) Net peak I CaT I-V curves measured following subtraction of traces recorded from HP = −55 mV from traces obtained at HP = −80 mV in WT (left panel) and in Girk4 –/– (right panel) SAN myocytes from sedentary and trained mice. Statistical significance was tested at each voltage using the unpaired Student’s t -test. * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars indicate s.e.m. WT S: WT sedentary; WT T: WT trained; Girk4 –/– S: Girk4 –/– sedentary, and Girk4 –/– T: Girk4 –/– trained.
    Figure Legend Snippet: Representative Ca 2+ current traces of the L-type Ca 2+ current ( I CaL ) measured from a holding potential (HP) of −55 mV (A) and current-to-voltage (I-V) relationships (B) from sedentary (open black circles, n = 14) and trained (filled black circles, n = 13) WT SAN myocytes. Traces of I CaL (C) and I-V curves (D) from sedentary (open blue circles, n = 15) compared with trained (filled blue circles, n = 22) Girk4 –/– SAN myocytes. Data, collected at day 28 were fitted with a modified Boltzmann equation. Voltage protocol is shown in panel C (bottom). (E) Sample traces of I Ca ( I CaT + I CaL ) recorded from a HP of −80 mV in WT and Girk4 –/– SAN myocytes. (F) Sample I Ca traces for same myocytes as in (E) , but after switching to HP = −55 to inactivate I CaT . (G) Net peak I CaT I-V curves measured following subtraction of traces recorded from HP = −55 mV from traces obtained at HP = −80 mV in WT (left panel) and in Girk4 –/– (right panel) SAN myocytes from sedentary and trained mice. Statistical significance was tested at each voltage using the unpaired Student’s t -test. * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars indicate s.e.m. WT S: WT sedentary; WT T: WT trained; Girk4 –/– S: Girk4 –/– sedentary, and Girk4 –/– T: Girk4 –/– trained.

    Techniques Used: Modification

    Expression of HCN4 mRNA (A) normalized to expression of Tbp in SAN biopsies of from WT sedentary ( n = 8), WT trained ( n = 8), Girk4 –/– sedentary ( n = 7), and Girk4 –/– trained ( n = 6) mice. Statistics: two-way analysis of variance with Sidak’s multiple comparisons test. Representative HCN4 western blot ( B , left panel) with corresponding stain-free total protein gel used for quantification (lower left panel). ( B , right panel) Protein expression determined by western blot in individual SAN biopsies isolated from WT sedentary ( n = 4), WT trained ( n = 4) sedentary Girk4 –/– mice ( n = 5) and trained Girk4 –/– mice ( n = 4). Statistics: Students t -test. SAN Expression of miR-423-5p (C) in WT sedentary ( n = 8), WT trained ( n = 7) sedentary Girk4 –/– mice ( n = 8) and trained Girk4 –/– mice ( n = 7). (D) Fold change in expression of selected miRs in the SAN of WT trained ( n = 8) and Girk4 –/– trained mice ( n = 7) relative to respective WT sedentary ( n = 8) and Girk4 –/– sedentary mice ( n = 7). Statistics: one-way analysis of variance with Tukey’s multiple comparisons test. * p < 0.05. miR expression in (C,D) was normalized to expression of Snord61 and Snord95.
    Figure Legend Snippet: Expression of HCN4 mRNA (A) normalized to expression of Tbp in SAN biopsies of from WT sedentary ( n = 8), WT trained ( n = 8), Girk4 –/– sedentary ( n = 7), and Girk4 –/– trained ( n = 6) mice. Statistics: two-way analysis of variance with Sidak’s multiple comparisons test. Representative HCN4 western blot ( B , left panel) with corresponding stain-free total protein gel used for quantification (lower left panel). ( B , right panel) Protein expression determined by western blot in individual SAN biopsies isolated from WT sedentary ( n = 4), WT trained ( n = 4) sedentary Girk4 –/– mice ( n = 5) and trained Girk4 –/– mice ( n = 4). Statistics: Students t -test. SAN Expression of miR-423-5p (C) in WT sedentary ( n = 8), WT trained ( n = 7) sedentary Girk4 –/– mice ( n = 8) and trained Girk4 –/– mice ( n = 7). (D) Fold change in expression of selected miRs in the SAN of WT trained ( n = 8) and Girk4 –/– trained mice ( n = 7) relative to respective WT sedentary ( n = 8) and Girk4 –/– sedentary mice ( n = 7). Statistics: one-way analysis of variance with Tukey’s multiple comparisons test. * p < 0.05. miR expression in (C,D) was normalized to expression of Snord61 and Snord95.

    Techniques Used: Expressing, Western Blot, Staining, Isolation

    mRNA expression of L-type calcium channel subunits Ca v 1.2, Ca v 1.3, Ca v 3.1, and Ca v 3.2 (A) (normalized to expression of Tbp) in SAN biopsies from from WT sedentary ( n = 10), WT trained ( n = 10), Girk4 –/– sedentary ( n = 9) and Girk4 –/– trained ( n = 10) mice. Protein expression determined by western blot using antibodies directed against Ca v 1.2 (B) and Ca v 1.3 (C) in individual sinus node biopsies isolated from sedentary WT ( n = 6), trained WT ( n = 4) sedentary Girk4 –/– mice ( n = 6) and trained Girk4 –/– mice ( n = 6). Representative western blots with corresponding stain-free total protein blot used for quantification shown in lower panel. Statistics: Students t -test. * p < 0.05.
    Figure Legend Snippet: mRNA expression of L-type calcium channel subunits Ca v 1.2, Ca v 1.3, Ca v 3.1, and Ca v 3.2 (A) (normalized to expression of Tbp) in SAN biopsies from from WT sedentary ( n = 10), WT trained ( n = 10), Girk4 –/– sedentary ( n = 9) and Girk4 –/– trained ( n = 10) mice. Protein expression determined by western blot using antibodies directed against Ca v 1.2 (B) and Ca v 1.3 (C) in individual sinus node biopsies isolated from sedentary WT ( n = 6), trained WT ( n = 4) sedentary Girk4 –/– mice ( n = 6) and trained Girk4 –/– mice ( n = 6). Representative western blots with corresponding stain-free total protein blot used for quantification shown in lower panel. Statistics: Students t -test. * p < 0.05.

    Techniques Used: Expressing, Western Blot, Isolation, Staining

    Numerical simulation of current traces for I f (A) , I CaT (B) , and I CaL (C) calculated using current density and activation parameters recorded in WT sedentary (WT S), WT trained (WT T), Girk4 –/– sedentary ( Girk4 –/– S) and Girk4 –/– trained ( Girk4 –/– T) at day 28 of experimental protocol. (D) Corresponding predicted I-V curves of I f (left panel), I CaT (central panel) and I CaL (right panel), calculated at the peak of current density from simulations in (A–C) . In I-V curves, open circles show predicted current densities of WT sedentary SAN cells and black circles densities of WT trained SAN cells. (E) Comparison between predicted pacemaker activities simulated for control (sedentary) condition (left panel), or including training-dependent changes of I f , I CaT and I CaL . Abbreviations: CL, pacemaker activity cycle length; DI, diastolic interval.
    Figure Legend Snippet: Numerical simulation of current traces for I f (A) , I CaT (B) , and I CaL (C) calculated using current density and activation parameters recorded in WT sedentary (WT S), WT trained (WT T), Girk4 –/– sedentary ( Girk4 –/– S) and Girk4 –/– trained ( Girk4 –/– T) at day 28 of experimental protocol. (D) Corresponding predicted I-V curves of I f (left panel), I CaT (central panel) and I CaL (right panel), calculated at the peak of current density from simulations in (A–C) . In I-V curves, open circles show predicted current densities of WT sedentary SAN cells and black circles densities of WT trained SAN cells. (E) Comparison between predicted pacemaker activities simulated for control (sedentary) condition (left panel), or including training-dependent changes of I f , I CaT and I CaL . Abbreviations: CL, pacemaker activity cycle length; DI, diastolic interval.

    Techniques Used: Activation Assay, Activity Assay

    kir3 4  (Alomone Labs)


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    Alomone Labs kir3 4
    Kir3 4, 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
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    kir3 4  (Alomone Labs)


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    Alomone Labs kir3 4
    Kir3 4, 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
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    kir3 4 girk4  (Alomone Labs)


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    Alomone Labs kir3 4 girk4
    Kir3 4 Girk4, 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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    kir3 4  (Alomone Labs)


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    Alomone Labs kir3 4
    Kir3 4, 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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    apc  (Alomone Labs)


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    Alomone Labs apc
    Apc, 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 girk4  (Alomone Labs)


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    Alomone Labs anti girk4
    (A) Alignment of <t>GIRK4</t> with GIRK1, GIRK2, and GIRK3 shows that R52, E246, and G247R are conserved amino acids. (B) Crystal structure of GIRK2 marked with highlighted positions of the mutations; R52 – red, E246 – blue, and G247 – orange (numbering corresponds to GIRK4). (C) Example of the recording protocol in GIRK1/4 WT injected oocytes without (gray) or with (black) coinjected Gβγ. Horizontal bars show the corresponding external solutions. Voltage ramps were applied in HK24 and HK24 + 2.5 mM Ba 2+ solutions. (D) Normalized I basal (left) and I βγ (right). Currents were measured at V m =-80 mV. (E) Representative I-V relationships of currents of GIRK1/4 (I basal ; left) and GIRK1/4 + Gβγ (Iβγ ; right). Net GIRK currents were obtained by subtraction of I-V curve in Ba 2+ (“Ba 2+ -subtraction” procedure). (F) Representative net I-V relationships of GIRK1/4 and mutants; I basal (left) and I βγ (right).
    Anti Girk4, 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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    94/100 stars

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    1) Product Images from "A revised mechanism of action of hyperaldosteronism-linked mutations in cytosolic domains of GIRK4 (KCNJ5)"

    Article Title: A revised mechanism of action of hyperaldosteronism-linked mutations in cytosolic domains of GIRK4 (KCNJ5)

    Journal: bioRxiv

    doi: 10.1101/866202

    (A) Alignment of GIRK4 with GIRK1, GIRK2, and GIRK3 shows that R52, E246, and G247R are conserved amino acids. (B) Crystal structure of GIRK2 marked with highlighted positions of the mutations; R52 – red, E246 – blue, and G247 – orange (numbering corresponds to GIRK4). (C) Example of the recording protocol in GIRK1/4 WT injected oocytes without (gray) or with (black) coinjected Gβγ. Horizontal bars show the corresponding external solutions. Voltage ramps were applied in HK24 and HK24 + 2.5 mM Ba 2+ solutions. (D) Normalized I basal (left) and I βγ (right). Currents were measured at V m =-80 mV. (E) Representative I-V relationships of currents of GIRK1/4 (I basal ; left) and GIRK1/4 + Gβγ (Iβγ ; right). Net GIRK currents were obtained by subtraction of I-V curve in Ba 2+ (“Ba 2+ -subtraction” procedure). (F) Representative net I-V relationships of GIRK1/4 and mutants; I basal (left) and I βγ (right).
    Figure Legend Snippet: (A) Alignment of GIRK4 with GIRK1, GIRK2, and GIRK3 shows that R52, E246, and G247R are conserved amino acids. (B) Crystal structure of GIRK2 marked with highlighted positions of the mutations; R52 – red, E246 – blue, and G247 – orange (numbering corresponds to GIRK4). (C) Example of the recording protocol in GIRK1/4 WT injected oocytes without (gray) or with (black) coinjected Gβγ. Horizontal bars show the corresponding external solutions. Voltage ramps were applied in HK24 and HK24 + 2.5 mM Ba 2+ solutions. (D) Normalized I basal (left) and I βγ (right). Currents were measured at V m =-80 mV. (E) Representative I-V relationships of currents of GIRK1/4 (I basal ; left) and GIRK1/4 + Gβγ (Iβγ ; right). Net GIRK currents were obtained by subtraction of I-V curve in Ba 2+ (“Ba 2+ -subtraction” procedure). (F) Representative net I-V relationships of GIRK1/4 and mutants; I basal (left) and I βγ (right).

    Techniques Used: Injection

    A separate experiment was performed for each mutant. (A) Representative confocal images of YFP-GIRK4 expressing oocytes, with or without Gβγ. (B) YFP-GIRK4 expression in the PM; except for YFP-GIRK4 G247R +Gβγ, all mutants express much less than YFP-GIRK4 WT . (C) I βγ of YFP-GIRK4 homotetramers. All mutants were not active as homotetramers. (D) Representative I-V relationships of YFP-GIRK4 (WT and mutants) coexpressed with Gβγ.
    Figure Legend Snippet: A separate experiment was performed for each mutant. (A) Representative confocal images of YFP-GIRK4 expressing oocytes, with or without Gβγ. (B) YFP-GIRK4 expression in the PM; except for YFP-GIRK4 G247R +Gβγ, all mutants express much less than YFP-GIRK4 WT . (C) I βγ of YFP-GIRK4 homotetramers. All mutants were not active as homotetramers. (D) Representative I-V relationships of YFP-GIRK4 (WT and mutants) coexpressed with Gβγ.

    Techniques Used: Mutagenesis, Expressing

    (A) Representative confocal images of GIRK1/YFP-GIRK4 expressing oocytes, with or without Gβγ. (B) GIRK1/YFP-GIRK4 expression in PM; mutated channels express less well than GIRK1/YFP-GIRK4 WT . A separate experiment was performed for each mutant. (C) Currents of GIRK1/YFP-GIRK4 WT and mutated channels. (D) Raw data from FRET experiments, showing E app as a function of donor/acceptor molar ratio. Each symbol corresponds to E app from one oocyte, from 4 experiments. (E) Binned analysis of E app shows that, at all donor/acceptor molar ratios, the interaction of CFP-Gβγ with GIRK1/YFP-GIRK4 R52H and GIRK1/YFP-GIRK4 E246K was impaired, whereas the interaction of CFP-Gβγ with GIRK1/YFP-GIRK4 G247R was more similar to GIRK1/YFP-GIRK4 WT . Molar ratios >2.5 were not included in the analysis because of insufficient data points forGIRK1/YFP-GIRK4 WT .
    Figure Legend Snippet: (A) Representative confocal images of GIRK1/YFP-GIRK4 expressing oocytes, with or without Gβγ. (B) GIRK1/YFP-GIRK4 expression in PM; mutated channels express less well than GIRK1/YFP-GIRK4 WT . A separate experiment was performed for each mutant. (C) Currents of GIRK1/YFP-GIRK4 WT and mutated channels. (D) Raw data from FRET experiments, showing E app as a function of donor/acceptor molar ratio. Each symbol corresponds to E app from one oocyte, from 4 experiments. (E) Binned analysis of E app shows that, at all donor/acceptor molar ratios, the interaction of CFP-Gβγ with GIRK1/YFP-GIRK4 R52H and GIRK1/YFP-GIRK4 E246K was impaired, whereas the interaction of CFP-Gβγ with GIRK1/YFP-GIRK4 G247R was more similar to GIRK1/YFP-GIRK4 WT . Molar ratios >2.5 were not included in the analysis because of insufficient data points forGIRK1/YFP-GIRK4 WT .

    Techniques Used: Expressing, Mutagenesis

    (A-C) Currents in oocytes injected with RNAs of YFP-GIRK4 mutants, measured at −80 mV, were indistinguishable from currents recorded in uninjected oocytes. (D-H) Representative I-V relationships of uninjected oocytes (D) and homotetrameric YFP-GIRK4WT (E), YFP-GIRK4R52H (F), YFP-GIRK4E246K (G), YFP-GIRK4G247R (H).
    Figure Legend Snippet: (A-C) Currents in oocytes injected with RNAs of YFP-GIRK4 mutants, measured at −80 mV, were indistinguishable from currents recorded in uninjected oocytes. (D-H) Representative I-V relationships of uninjected oocytes (D) and homotetrameric YFP-GIRK4WT (E), YFP-GIRK4R52H (F), YFP-GIRK4E246K (G), YFP-GIRK4G247R (H).

    Techniques Used: Injection

    (A) Normalization of current to expression of YFP-GIRK4 homotetramers indicates an impaired gating of the mutated channels. The normalization procedure included the division of mean value of current on mean value of channel expression in each experiment, giving one value for each experiment. Therefore, no statistical analysis could be performed. (B) Normalization of current to expression of GIRK1/YFP-GIRK4 heterotetramers indicates an impaired expression and gating of GIRK1/YFP-GIRK4R52H heterotetramers; on the other hand, E246K and G247R mutations appear to have the same open probability suggesting unimpaired gating.
    Figure Legend Snippet: (A) Normalization of current to expression of YFP-GIRK4 homotetramers indicates an impaired gating of the mutated channels. The normalization procedure included the division of mean value of current on mean value of channel expression in each experiment, giving one value for each experiment. Therefore, no statistical analysis could be performed. (B) Normalization of current to expression of GIRK1/YFP-GIRK4 heterotetramers indicates an impaired expression and gating of GIRK1/YFP-GIRK4R52H heterotetramers; on the other hand, E246K and G247R mutations appear to have the same open probability suggesting unimpaired gating.

    Techniques Used: Expressing

    (A) Western blots of HAC15 cells with GAPDH antibody (both panels) and GIRK1 antibody (left) and GIRK4 antibody (right) show that HAC15 cells express GIRK1 and GIRK4 channels. Oocytes expressing GIRK1, GIRK4, and YFP-GIRK4 were used as positive controls, naïve oocytes as negative control, and GAPDH as loading control. (B) Examples (left) and summary (right) of currents in HAC15 cells expressing GFP-GIRK1/GIRK4 WT , GFP-GIRK1/GIRK4 R52H , GFP-GIRK1/GIRK4 E246K , and GFP-GIRK1/GIRK4 G247R . Channels were expressed with D 2 dopamine receptor and Gβγ (I βγ ), thus the small I DA .
    Figure Legend Snippet: (A) Western blots of HAC15 cells with GAPDH antibody (both panels) and GIRK1 antibody (left) and GIRK4 antibody (right) show that HAC15 cells express GIRK1 and GIRK4 channels. Oocytes expressing GIRK1, GIRK4, and YFP-GIRK4 were used as positive controls, naïve oocytes as negative control, and GAPDH as loading control. (B) Examples (left) and summary (right) of currents in HAC15 cells expressing GFP-GIRK1/GIRK4 WT , GFP-GIRK1/GIRK4 R52H , GFP-GIRK1/GIRK4 E246K , and GFP-GIRK1/GIRK4 G247R . Channels were expressed with D 2 dopamine receptor and Gβγ (I βγ ), thus the small I DA .

    Techniques Used: Western Blot, Expressing, Negative Control

    Oocytes expressed GIRK4 mutants and GIRK4 WT , with or without Gβγ. Cells were exposed to 4 concentrations of VU0529331 (1, 5, 20, and 60 μM). Only GIRK4 WT and GIRK4 G247R mutant responded to VU0529331. (A) I basal of GIRK4 WT and GIRK4 G247R . GIRK4 WT and GIRK4 G247R react similarly to VU0529331. (B) I βγ of GIRK4 WT and GIRK4 G247R . GIRK4 WT had larger current at 0 μM VU0529931, while GIRK4 G247R did not show any response to concentrations lower than 20 μM VU0529331. (C) Normalized I basal (I/I control ). GIRK4 WT and GIRK4 G247R were activated similarly by VU0529331. (D) I/I control of I βγ show that GIRK4 G247R was activated by VU0529331 stronger than GIRK4 WT , in relative terms. (E) I-V relationship of GIRK4 WT (top) and GIRK4 G247R (bottom) without Gβγ. (F) I-V relationship of GIRK4 WT (top) and GIRK4 G247R (bottom) with Gβγ.
    Figure Legend Snippet: Oocytes expressed GIRK4 mutants and GIRK4 WT , with or without Gβγ. Cells were exposed to 4 concentrations of VU0529331 (1, 5, 20, and 60 μM). Only GIRK4 WT and GIRK4 G247R mutant responded to VU0529331. (A) I basal of GIRK4 WT and GIRK4 G247R . GIRK4 WT and GIRK4 G247R react similarly to VU0529331. (B) I βγ of GIRK4 WT and GIRK4 G247R . GIRK4 WT had larger current at 0 μM VU0529931, while GIRK4 G247R did not show any response to concentrations lower than 20 μM VU0529331. (C) Normalized I basal (I/I control ). GIRK4 WT and GIRK4 G247R were activated similarly by VU0529331. (D) I/I control of I βγ show that GIRK4 G247R was activated by VU0529331 stronger than GIRK4 WT , in relative terms. (E) I-V relationship of GIRK4 WT (top) and GIRK4 G247R (bottom) without Gβγ. (F) I-V relationship of GIRK4 WT (top) and GIRK4 G247R (bottom) with Gβγ.

    Techniques Used: Mutagenesis

    girk4  (Alomone Labs)


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    Alomone Labs girk4
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    Alomone Labs anti girk4
    Heart rate recorded in sedentary (left panel) and trained (right panel) WT (A) and <t>Girk4</t> –/– mice (B) at different days during 5 min sham-training (WT and Girk4 –/– sedentary, see methods) and training (WT and Girk4 –/– trained) protocol. (C) Histogram of the average values of the slopes of the regression line between time and heart rate. Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. (D) Representative examples of ECG traces and averaged heart rate recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. Tertiapin-Q (Tert, 5 mg/kg) effect in WT (E) and Girk4 –/– (F) sedentary (empty bar) and trained (filled bars) mice. Statistics: two-way analysis of variance followed by Sidak multiple comparisons test. (G) Close-up of ECG traces and averaged PR interval recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, ### p < 0.001, #### p < 0.0001. Error bars indicate s.e.m. WTS: WT sedentary; WTT: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.
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    Alomone Labs kir3 4 girk4
    Heart rate recorded in sedentary (left panel) and trained (right panel) WT (A) and <t>Girk4</t> –/– mice (B) at different days during 5 min sham-training (WT and Girk4 –/– sedentary, see methods) and training (WT and Girk4 –/– trained) protocol. (C) Histogram of the average values of the slopes of the regression line between time and heart rate. Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. (D) Representative examples of ECG traces and averaged heart rate recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. Tertiapin-Q (Tert, 5 mg/kg) effect in WT (E) and Girk4 –/– (F) sedentary (empty bar) and trained (filled bars) mice. Statistics: two-way analysis of variance followed by Sidak multiple comparisons test. (G) Close-up of ECG traces and averaged PR interval recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, ### p < 0.001, #### p < 0.0001. Error bars indicate s.e.m. WTS: WT sedentary; WTT: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.
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    Heart rate recorded in sedentary (left panel) and trained (right panel) WT (A) and <t>Girk4</t> –/– mice (B) at different days during 5 min sham-training (WT and Girk4 –/– sedentary, see methods) and training (WT and Girk4 –/– trained) protocol. (C) Histogram of the average values of the slopes of the regression line between time and heart rate. Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. (D) Representative examples of ECG traces and averaged heart rate recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. Tertiapin-Q (Tert, 5 mg/kg) effect in WT (E) and Girk4 –/– (F) sedentary (empty bar) and trained (filled bars) mice. Statistics: two-way analysis of variance followed by Sidak multiple comparisons test. (G) Close-up of ECG traces and averaged PR interval recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, ### p < 0.001, #### p < 0.0001. Error bars indicate s.e.m. WTS: WT sedentary; WTT: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.
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    Heart rate recorded in sedentary (left panel) and trained (right panel) WT (A) and <t>Girk4</t> –/– mice (B) at different days during 5 min sham-training (WT and Girk4 –/– sedentary, see methods) and training (WT and Girk4 –/– trained) protocol. (C) Histogram of the average values of the slopes of the regression line between time and heart rate. Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. (D) Representative examples of ECG traces and averaged heart rate recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. Tertiapin-Q (Tert, 5 mg/kg) effect in WT (E) and Girk4 –/– (F) sedentary (empty bar) and trained (filled bars) mice. Statistics: two-way analysis of variance followed by Sidak multiple comparisons test. (G) Close-up of ECG traces and averaged PR interval recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, ### p < 0.001, #### p < 0.0001. Error bars indicate s.e.m. WTS: WT sedentary; WTT: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.
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    Heart rate recorded in sedentary (left panel) and trained (right panel) WT (A) and Girk4 –/– mice (B) at different days during 5 min sham-training (WT and Girk4 –/– sedentary, see methods) and training (WT and Girk4 –/– trained) protocol. (C) Histogram of the average values of the slopes of the regression line between time and heart rate. Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. (D) Representative examples of ECG traces and averaged heart rate recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. Tertiapin-Q (Tert, 5 mg/kg) effect in WT (E) and Girk4 –/– (F) sedentary (empty bar) and trained (filled bars) mice. Statistics: two-way analysis of variance followed by Sidak multiple comparisons test. (G) Close-up of ECG traces and averaged PR interval recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, ### p < 0.001, #### p < 0.0001. Error bars indicate s.e.m. WTS: WT sedentary; WTT: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.

    Journal: Frontiers in Physiology

    Article Title: Genetic Ablation of G Protein-Gated Inwardly Rectifying K + Channels Prevents Training-Induced Sinus Bradycardia

    doi: 10.3389/fphys.2020.519382

    Figure Lengend Snippet: Heart rate recorded in sedentary (left panel) and trained (right panel) WT (A) and Girk4 –/– mice (B) at different days during 5 min sham-training (WT and Girk4 –/– sedentary, see methods) and training (WT and Girk4 –/– trained) protocol. (C) Histogram of the average values of the slopes of the regression line between time and heart rate. Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. (D) Representative examples of ECG traces and averaged heart rate recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. Tertiapin-Q (Tert, 5 mg/kg) effect in WT (E) and Girk4 –/– (F) sedentary (empty bar) and trained (filled bars) mice. Statistics: two-way analysis of variance followed by Sidak multiple comparisons test. (G) Close-up of ECG traces and averaged PR interval recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4 –/– mice (right panel). Statistics: unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, ### p < 0.001, #### p < 0.0001. Error bars indicate s.e.m. WTS: WT sedentary; WTT: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.

    Article Snippet: Rabbit polyclonal anti-HCN4 (Alomone Labs, APC-052, Lot #APC052AN2802), anti-Ca v 1.2 (ACC-003, Lot #ACC003AN6802), anti-GIRK1 (APC-005, Lot #APC005AN1125) and anti-GIRK4 (APC-027, Lot #APC027AN0725), were used at 1:200. anti-Ca v 1.3 ( ) was used at 1/2000.

    Techniques:

    Standard deviation of intervals between two consecutive heart beats (SDNN) calculated in 5-min stable ECG periods at day 0 and at day 28 in WT (A) and Girk4 –/– (B) sedentary (open bars) and trained (filled bars) animals. Power spectral density (PSD) of heart rate variability determined by Fast Fourier Transformation analysis of 5-min of stable ECG segments at day 0 and at day 28 in WT (C) and Girk4 –/– (D) sedentary (open bars) and trained (filled bars) animals. Heart rate (non-invasive ECG recordings) measured in sedentary (open bars, left panel) and trained (filled bars, right panel) WT mice before (day 0) and after (day 28) training period in control condition (ANS +, E ) or following intraperitoneal injection of atropine (0.5 mg/kg) and propranolol (5 mg/kg) to inhibit the input of the autonomic nervous system (ANS-, F ). (G,H) same as (E,F) but in Girk4 –/– animals. Statistics: unpaired Student’s t -test. ** p < 0.01, **** p < 0.0001. Error bars indicate s.e.m.

    Journal: Frontiers in Physiology

    Article Title: Genetic Ablation of G Protein-Gated Inwardly Rectifying K + Channels Prevents Training-Induced Sinus Bradycardia

    doi: 10.3389/fphys.2020.519382

    Figure Lengend Snippet: Standard deviation of intervals between two consecutive heart beats (SDNN) calculated in 5-min stable ECG periods at day 0 and at day 28 in WT (A) and Girk4 –/– (B) sedentary (open bars) and trained (filled bars) animals. Power spectral density (PSD) of heart rate variability determined by Fast Fourier Transformation analysis of 5-min of stable ECG segments at day 0 and at day 28 in WT (C) and Girk4 –/– (D) sedentary (open bars) and trained (filled bars) animals. Heart rate (non-invasive ECG recordings) measured in sedentary (open bars, left panel) and trained (filled bars, right panel) WT mice before (day 0) and after (day 28) training period in control condition (ANS +, E ) or following intraperitoneal injection of atropine (0.5 mg/kg) and propranolol (5 mg/kg) to inhibit the input of the autonomic nervous system (ANS-, F ). (G,H) same as (E,F) but in Girk4 –/– animals. Statistics: unpaired Student’s t -test. ** p < 0.01, **** p < 0.0001. Error bars indicate s.e.m.

    Article Snippet: Rabbit polyclonal anti-HCN4 (Alomone Labs, APC-052, Lot #APC052AN2802), anti-Ca v 1.2 (ACC-003, Lot #ACC003AN6802), anti-GIRK1 (APC-005, Lot #APC005AN1125) and anti-GIRK4 (APC-027, Lot #APC027AN0725), were used at 1:200. anti-Ca v 1.3 ( ) was used at 1/2000.

    Techniques: Standard Deviation, Transformation Assay, Injection

    Action potential recordings of SAN myocytes isolated from WT sedentary (WT S), WT trained (WT T), Girk4 –/– sedentary ( Girk4 –/– S) and Girk4 –/– trained ( Girk4 –/– T) in Tyrode’s solution at the end of training, or sham-training protocol (day 28) (A) . The dotted line indicates the 0 mV. Rate of spontaneous action potentials (B) and slope of the linear part of the diastolic depolarization (SLDD, C ) recorded at day 28 in SAN myocytes in Tyrode’s solution ( n = 6 WT S, n = 10 WT T, n = 7 Girk4 –/– S and n = 8 Girk4 –/– T). Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. * p < 0.05, **** p < 0.0001. Error bars indicate s.e.m.

    Journal: Frontiers in Physiology

    Article Title: Genetic Ablation of G Protein-Gated Inwardly Rectifying K + Channels Prevents Training-Induced Sinus Bradycardia

    doi: 10.3389/fphys.2020.519382

    Figure Lengend Snippet: Action potential recordings of SAN myocytes isolated from WT sedentary (WT S), WT trained (WT T), Girk4 –/– sedentary ( Girk4 –/– S) and Girk4 –/– trained ( Girk4 –/– T) in Tyrode’s solution at the end of training, or sham-training protocol (day 28) (A) . The dotted line indicates the 0 mV. Rate of spontaneous action potentials (B) and slope of the linear part of the diastolic depolarization (SLDD, C ) recorded at day 28 in SAN myocytes in Tyrode’s solution ( n = 6 WT S, n = 10 WT T, n = 7 Girk4 –/– S and n = 8 Girk4 –/– T). Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. * p < 0.05, **** p < 0.0001. Error bars indicate s.e.m.

    Article Snippet: Rabbit polyclonal anti-HCN4 (Alomone Labs, APC-052, Lot #APC052AN2802), anti-Ca v 1.2 (ACC-003, Lot #ACC003AN6802), anti-GIRK1 (APC-005, Lot #APC005AN1125) and anti-GIRK4 (APC-027, Lot #APC027AN0725), were used at 1:200. anti-Ca v 1.3 ( ) was used at 1/2000.

    Techniques: Isolation

    Representative traces of I f recordings (A) and averaged current-to-voltage (I-V) curve (B) in sedentary (open black circles, n = 15) and trained (filled black circles, n = 23) WT SAN myocytes. I f recordings (C) and I-V curve (D) in SAN myocytes from sedentary (open blue circles, n = 12) and trained (filled blue circles, n = 18) isolated Girk4 –/– SAN pacemaker cells. The voltage-clamp protocol used for all the recordings is shown at the bottom of panel (C) . Statistical significance was tested at each voltage using the unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001. ( E , left panel) Steady state I f activation curves in isolated SAN cells from sedentary (dotted line) and trained (continuous line), WT (black) and Girk4 –/– (blue) mice. Representative ramp current trace recorded in a WT sedentary SAN cell and corresponding voltage protocol are shown under the curves. ( E , right panel) Histograms representing averaged half-activation voltages (V 1/2 ) values for I f recorded in SAN cells from WT (black bars) and Girk4 –/– (blue bars) sedentary (open bars) and trained (filled bars) animals. Data have been collected at day 28 (end of training, or sham-training, protocol). Statistics: one-way analysis of variance. Error bars indicate s.e.m.. WT S: WT sedentary; WT T: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.

    Journal: Frontiers in Physiology

    Article Title: Genetic Ablation of G Protein-Gated Inwardly Rectifying K + Channels Prevents Training-Induced Sinus Bradycardia

    doi: 10.3389/fphys.2020.519382

    Figure Lengend Snippet: Representative traces of I f recordings (A) and averaged current-to-voltage (I-V) curve (B) in sedentary (open black circles, n = 15) and trained (filled black circles, n = 23) WT SAN myocytes. I f recordings (C) and I-V curve (D) in SAN myocytes from sedentary (open blue circles, n = 12) and trained (filled blue circles, n = 18) isolated Girk4 –/– SAN pacemaker cells. The voltage-clamp protocol used for all the recordings is shown at the bottom of panel (C) . Statistical significance was tested at each voltage using the unpaired Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001. ( E , left panel) Steady state I f activation curves in isolated SAN cells from sedentary (dotted line) and trained (continuous line), WT (black) and Girk4 –/– (blue) mice. Representative ramp current trace recorded in a WT sedentary SAN cell and corresponding voltage protocol are shown under the curves. ( E , right panel) Histograms representing averaged half-activation voltages (V 1/2 ) values for I f recorded in SAN cells from WT (black bars) and Girk4 –/– (blue bars) sedentary (open bars) and trained (filled bars) animals. Data have been collected at day 28 (end of training, or sham-training, protocol). Statistics: one-way analysis of variance. Error bars indicate s.e.m.. WT S: WT sedentary; WT T: WT trained; Girk4 –/– S: Girk4 –/– sedentary and Girk4 –/– T: Girk4 –/– trained.

    Article Snippet: Rabbit polyclonal anti-HCN4 (Alomone Labs, APC-052, Lot #APC052AN2802), anti-Ca v 1.2 (ACC-003, Lot #ACC003AN6802), anti-GIRK1 (APC-005, Lot #APC005AN1125) and anti-GIRK4 (APC-027, Lot #APC027AN0725), were used at 1:200. anti-Ca v 1.3 ( ) was used at 1/2000.

    Techniques: Isolation, Activation Assay

    Representative Ca 2+ current traces of the L-type Ca 2+ current ( I CaL ) measured from a holding potential (HP) of −55 mV (A) and current-to-voltage (I-V) relationships (B) from sedentary (open black circles, n = 14) and trained (filled black circles, n = 13) WT SAN myocytes. Traces of I CaL (C) and I-V curves (D) from sedentary (open blue circles, n = 15) compared with trained (filled blue circles, n = 22) Girk4 –/– SAN myocytes. Data, collected at day 28 were fitted with a modified Boltzmann equation. Voltage protocol is shown in panel C (bottom). (E) Sample traces of I Ca ( I CaT + I CaL ) recorded from a HP of −80 mV in WT and Girk4 –/– SAN myocytes. (F) Sample I Ca traces for same myocytes as in (E) , but after switching to HP = −55 to inactivate I CaT . (G) Net peak I CaT I-V curves measured following subtraction of traces recorded from HP = −55 mV from traces obtained at HP = −80 mV in WT (left panel) and in Girk4 –/– (right panel) SAN myocytes from sedentary and trained mice. Statistical significance was tested at each voltage using the unpaired Student’s t -test. * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars indicate s.e.m. WT S: WT sedentary; WT T: WT trained; Girk4 –/– S: Girk4 –/– sedentary, and Girk4 –/– T: Girk4 –/– trained.

    Journal: Frontiers in Physiology

    Article Title: Genetic Ablation of G Protein-Gated Inwardly Rectifying K + Channels Prevents Training-Induced Sinus Bradycardia

    doi: 10.3389/fphys.2020.519382

    Figure Lengend Snippet: Representative Ca 2+ current traces of the L-type Ca 2+ current ( I CaL ) measured from a holding potential (HP) of −55 mV (A) and current-to-voltage (I-V) relationships (B) from sedentary (open black circles, n = 14) and trained (filled black circles, n = 13) WT SAN myocytes. Traces of I CaL (C) and I-V curves (D) from sedentary (open blue circles, n = 15) compared with trained (filled blue circles, n = 22) Girk4 –/– SAN myocytes. Data, collected at day 28 were fitted with a modified Boltzmann equation. Voltage protocol is shown in panel C (bottom). (E) Sample traces of I Ca ( I CaT + I CaL ) recorded from a HP of −80 mV in WT and Girk4 –/– SAN myocytes. (F) Sample I Ca traces for same myocytes as in (E) , but after switching to HP = −55 to inactivate I CaT . (G) Net peak I CaT I-V curves measured following subtraction of traces recorded from HP = −55 mV from traces obtained at HP = −80 mV in WT (left panel) and in Girk4 –/– (right panel) SAN myocytes from sedentary and trained mice. Statistical significance was tested at each voltage using the unpaired Student’s t -test. * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars indicate s.e.m. WT S: WT sedentary; WT T: WT trained; Girk4 –/– S: Girk4 –/– sedentary, and Girk4 –/– T: Girk4 –/– trained.

    Article Snippet: Rabbit polyclonal anti-HCN4 (Alomone Labs, APC-052, Lot #APC052AN2802), anti-Ca v 1.2 (ACC-003, Lot #ACC003AN6802), anti-GIRK1 (APC-005, Lot #APC005AN1125) and anti-GIRK4 (APC-027, Lot #APC027AN0725), were used at 1:200. anti-Ca v 1.3 ( ) was used at 1/2000.

    Techniques: Modification

    Expression of HCN4 mRNA (A) normalized to expression of Tbp in SAN biopsies of from WT sedentary ( n = 8), WT trained ( n = 8), Girk4 –/– sedentary ( n = 7), and Girk4 –/– trained ( n = 6) mice. Statistics: two-way analysis of variance with Sidak’s multiple comparisons test. Representative HCN4 western blot ( B , left panel) with corresponding stain-free total protein gel used for quantification (lower left panel). ( B , right panel) Protein expression determined by western blot in individual SAN biopsies isolated from WT sedentary ( n = 4), WT trained ( n = 4) sedentary Girk4 –/– mice ( n = 5) and trained Girk4 –/– mice ( n = 4). Statistics: Students t -test. SAN Expression of miR-423-5p (C) in WT sedentary ( n = 8), WT trained ( n = 7) sedentary Girk4 –/– mice ( n = 8) and trained Girk4 –/– mice ( n = 7). (D) Fold change in expression of selected miRs in the SAN of WT trained ( n = 8) and Girk4 –/– trained mice ( n = 7) relative to respective WT sedentary ( n = 8) and Girk4 –/– sedentary mice ( n = 7). Statistics: one-way analysis of variance with Tukey’s multiple comparisons test. * p < 0.05. miR expression in (C,D) was normalized to expression of Snord61 and Snord95.

    Journal: Frontiers in Physiology

    Article Title: Genetic Ablation of G Protein-Gated Inwardly Rectifying K + Channels Prevents Training-Induced Sinus Bradycardia

    doi: 10.3389/fphys.2020.519382

    Figure Lengend Snippet: Expression of HCN4 mRNA (A) normalized to expression of Tbp in SAN biopsies of from WT sedentary ( n = 8), WT trained ( n = 8), Girk4 –/– sedentary ( n = 7), and Girk4 –/– trained ( n = 6) mice. Statistics: two-way analysis of variance with Sidak’s multiple comparisons test. Representative HCN4 western blot ( B , left panel) with corresponding stain-free total protein gel used for quantification (lower left panel). ( B , right panel) Protein expression determined by western blot in individual SAN biopsies isolated from WT sedentary ( n = 4), WT trained ( n = 4) sedentary Girk4 –/– mice ( n = 5) and trained Girk4 –/– mice ( n = 4). Statistics: Students t -test. SAN Expression of miR-423-5p (C) in WT sedentary ( n = 8), WT trained ( n = 7) sedentary Girk4 –/– mice ( n = 8) and trained Girk4 –/– mice ( n = 7). (D) Fold change in expression of selected miRs in the SAN of WT trained ( n = 8) and Girk4 –/– trained mice ( n = 7) relative to respective WT sedentary ( n = 8) and Girk4 –/– sedentary mice ( n = 7). Statistics: one-way analysis of variance with Tukey’s multiple comparisons test. * p < 0.05. miR expression in (C,D) was normalized to expression of Snord61 and Snord95.

    Article Snippet: Rabbit polyclonal anti-HCN4 (Alomone Labs, APC-052, Lot #APC052AN2802), anti-Ca v 1.2 (ACC-003, Lot #ACC003AN6802), anti-GIRK1 (APC-005, Lot #APC005AN1125) and anti-GIRK4 (APC-027, Lot #APC027AN0725), were used at 1:200. anti-Ca v 1.3 ( ) was used at 1/2000.

    Techniques: Expressing, Western Blot, Staining, Isolation

    mRNA expression of L-type calcium channel subunits Ca v 1.2, Ca v 1.3, Ca v 3.1, and Ca v 3.2 (A) (normalized to expression of Tbp) in SAN biopsies from from WT sedentary ( n = 10), WT trained ( n = 10), Girk4 –/– sedentary ( n = 9) and Girk4 –/– trained ( n = 10) mice. Protein expression determined by western blot using antibodies directed against Ca v 1.2 (B) and Ca v 1.3 (C) in individual sinus node biopsies isolated from sedentary WT ( n = 6), trained WT ( n = 4) sedentary Girk4 –/– mice ( n = 6) and trained Girk4 –/– mice ( n = 6). Representative western blots with corresponding stain-free total protein blot used for quantification shown in lower panel. Statistics: Students t -test. * p < 0.05.

    Journal: Frontiers in Physiology

    Article Title: Genetic Ablation of G Protein-Gated Inwardly Rectifying K + Channels Prevents Training-Induced Sinus Bradycardia

    doi: 10.3389/fphys.2020.519382

    Figure Lengend Snippet: mRNA expression of L-type calcium channel subunits Ca v 1.2, Ca v 1.3, Ca v 3.1, and Ca v 3.2 (A) (normalized to expression of Tbp) in SAN biopsies from from WT sedentary ( n = 10), WT trained ( n = 10), Girk4 –/– sedentary ( n = 9) and Girk4 –/– trained ( n = 10) mice. Protein expression determined by western blot using antibodies directed against Ca v 1.2 (B) and Ca v 1.3 (C) in individual sinus node biopsies isolated from sedentary WT ( n = 6), trained WT ( n = 4) sedentary Girk4 –/– mice ( n = 6) and trained Girk4 –/– mice ( n = 6). Representative western blots with corresponding stain-free total protein blot used for quantification shown in lower panel. Statistics: Students t -test. * p < 0.05.

    Article Snippet: Rabbit polyclonal anti-HCN4 (Alomone Labs, APC-052, Lot #APC052AN2802), anti-Ca v 1.2 (ACC-003, Lot #ACC003AN6802), anti-GIRK1 (APC-005, Lot #APC005AN1125) and anti-GIRK4 (APC-027, Lot #APC027AN0725), were used at 1:200. anti-Ca v 1.3 ( ) was used at 1/2000.

    Techniques: Expressing, Western Blot, Isolation, Staining

    Numerical simulation of current traces for I f (A) , I CaT (B) , and I CaL (C) calculated using current density and activation parameters recorded in WT sedentary (WT S), WT trained (WT T), Girk4 –/– sedentary ( Girk4 –/– S) and Girk4 –/– trained ( Girk4 –/– T) at day 28 of experimental protocol. (D) Corresponding predicted I-V curves of I f (left panel), I CaT (central panel) and I CaL (right panel), calculated at the peak of current density from simulations in (A–C) . In I-V curves, open circles show predicted current densities of WT sedentary SAN cells and black circles densities of WT trained SAN cells. (E) Comparison between predicted pacemaker activities simulated for control (sedentary) condition (left panel), or including training-dependent changes of I f , I CaT and I CaL . Abbreviations: CL, pacemaker activity cycle length; DI, diastolic interval.

    Journal: Frontiers in Physiology

    Article Title: Genetic Ablation of G Protein-Gated Inwardly Rectifying K + Channels Prevents Training-Induced Sinus Bradycardia

    doi: 10.3389/fphys.2020.519382

    Figure Lengend Snippet: Numerical simulation of current traces for I f (A) , I CaT (B) , and I CaL (C) calculated using current density and activation parameters recorded in WT sedentary (WT S), WT trained (WT T), Girk4 –/– sedentary ( Girk4 –/– S) and Girk4 –/– trained ( Girk4 –/– T) at day 28 of experimental protocol. (D) Corresponding predicted I-V curves of I f (left panel), I CaT (central panel) and I CaL (right panel), calculated at the peak of current density from simulations in (A–C) . In I-V curves, open circles show predicted current densities of WT sedentary SAN cells and black circles densities of WT trained SAN cells. (E) Comparison between predicted pacemaker activities simulated for control (sedentary) condition (left panel), or including training-dependent changes of I f , I CaT and I CaL . Abbreviations: CL, pacemaker activity cycle length; DI, diastolic interval.

    Article Snippet: Rabbit polyclonal anti-HCN4 (Alomone Labs, APC-052, Lot #APC052AN2802), anti-Ca v 1.2 (ACC-003, Lot #ACC003AN6802), anti-GIRK1 (APC-005, Lot #APC005AN1125) and anti-GIRK4 (APC-027, Lot #APC027AN0725), were used at 1:200. anti-Ca v 1.3 ( ) was used at 1/2000.

    Techniques: Activation Assay, Activity Assay