ryr2  (Alomone Labs)


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    Alomone Labs ryr2
    The effect of hypoxia and Rycal treatment on <t>RyR2</t> and SERCA2a gene expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a gene expression. n = 6, * P < .050 for comparison with 1% O 2 , # P < .050 for comparison with Vehicle, $ P < .050 for comparison with 0.3 mM (all 1-way ANOVA with Tukey post hoc test).
    Ryr2, 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/ryr2/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ryr2 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes"

    Article Title: Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes

    Journal: Anatolian Journal of Cardiology

    doi: 10.5152/AnatolJCardiol.2022.1223

    The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a gene expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a gene expression. n = 6, * P < .050 for comparison with 1% O 2 , # P < .050 for comparison with Vehicle, $ P < .050 for comparison with 0.3 mM (all 1-way ANOVA with Tukey post hoc test).
    Figure Legend Snippet: The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a gene expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a gene expression. n = 6, * P < .050 for comparison with 1% O 2 , # P < .050 for comparison with Vehicle, $ P < .050 for comparison with 0.3 mM (all 1-way ANOVA with Tukey post hoc test).

    Techniques Used: Expressing

    The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a protein expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a protein expression. n = 6. No statistically significant comparison.
    Figure Legend Snippet: The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a protein expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a protein expression. n = 6. No statistically significant comparison.

    Techniques Used: Expressing

    mouse monoclonal antibody igg1 anti ryr2  (Alomone Labs)


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    Alomone Labs mouse monoclonal antibody igg1 anti ryr2
    Integrin ligand ff_RGD induced changes in the Ca 2+ cycling ultrastructure. L-type calcium channel (LTCC) subunit CaV1.2 (green) and <t>ryanodine</t> <t>receptor</t> (RyR) 2 (red) were labelled and orthoview z-stack formed with wheat germ agglutinin (WGA) (white) and Hoechst (blue) staining ( A , B ) control and ( C , D ) ff_RGD-treated cardiomyocytes (day 40–46 of differentiation). Scale bar for A and 6 = 20 μm, B and D = 50 μm. Merged images used to calculate the Mander’s correlation coefficient for ( E ) LTCC overlapping RyR background pixels (M1) (* p = 0.0363), ( F ) RyR overlapping LTCC background pixels (M2) (ns p = 0.1143), and ( G ) Pearson’s correlation coefficient between LTCC and RyR (** p = 0.001) illustrated as means ± SEM ( n = 39 control, n = 18 ff_RGD cells from ten batches).
    Mouse Monoclonal Antibody Igg1 Anti Ryr2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal antibody igg1 anti ryr2/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal antibody igg1 anti ryr2 - by Bioz Stars, 2023-01
    86/100 stars

    Images

    1) Product Images from "Integrins Increase Sarcoplasmic Reticulum Activity for Excitation—Contraction Coupling in Human Stem Cell-Derived Cardiomyocytes"

    Article Title: Integrins Increase Sarcoplasmic Reticulum Activity for Excitation—Contraction Coupling in Human Stem Cell-Derived Cardiomyocytes

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms231810940

    Integrin ligand ff_RGD induced changes in the Ca 2+ cycling ultrastructure. L-type calcium channel (LTCC) subunit CaV1.2 (green) and ryanodine receptor (RyR) 2 (red) were labelled and orthoview z-stack formed with wheat germ agglutinin (WGA) (white) and Hoechst (blue) staining ( A , B ) control and ( C , D ) ff_RGD-treated cardiomyocytes (day 40–46 of differentiation). Scale bar for A and 6 = 20 μm, B and D = 50 μm. Merged images used to calculate the Mander’s correlation coefficient for ( E ) LTCC overlapping RyR background pixels (M1) (* p = 0.0363), ( F ) RyR overlapping LTCC background pixels (M2) (ns p = 0.1143), and ( G ) Pearson’s correlation coefficient between LTCC and RyR (** p = 0.001) illustrated as means ± SEM ( n = 39 control, n = 18 ff_RGD cells from ten batches).
    Figure Legend Snippet: Integrin ligand ff_RGD induced changes in the Ca 2+ cycling ultrastructure. L-type calcium channel (LTCC) subunit CaV1.2 (green) and ryanodine receptor (RyR) 2 (red) were labelled and orthoview z-stack formed with wheat germ agglutinin (WGA) (white) and Hoechst (blue) staining ( A , B ) control and ( C , D ) ff_RGD-treated cardiomyocytes (day 40–46 of differentiation). Scale bar for A and 6 = 20 μm, B and D = 50 μm. Merged images used to calculate the Mander’s correlation coefficient for ( E ) LTCC overlapping RyR background pixels (M1) (* p = 0.0363), ( F ) RyR overlapping LTCC background pixels (M2) (ns p = 0.1143), and ( G ) Pearson’s correlation coefficient between LTCC and RyR (** p = 0.001) illustrated as means ± SEM ( n = 39 control, n = 18 ff_RGD cells from ten batches).

    Techniques Used: Staining

    anti pser2808 ryr2  (Alomone Labs)


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    Alomone Labs anti pser2808 ryr2
    Effects of macitentan on the calcium handling in LA in SHR. (a) Representative immunoblots showing expression of α1C, NCX1, CSQ and <t>RyR2</t> from SHR treated with MAC or DOX versus SHR-CTL (b-g) Quantification of immunoblots (N = 6–9 per group) (h) Representative immunoblots showing expression of SERCA2a, CaMKIIδ and PLB. Actin controls for pS16, pT17 and pS10 are not shown (i-m) Quantification of immunoblots (N = 6–9); # P < 0.05. Note that some blots/bands of housekeeping proteins used for normalization are identical (e.g. in (a) GAPDH blot/bands shown below CSQ and GAPDH blot/bands shown below RyR) as they are derived from the same membrane.
    Anti Pser2808 Ryr2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti pser2808 ryr2/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti pser2808 ryr2 - by Bioz Stars, 2023-01
    86/100 stars

    Images

    1) Product Images from "Anti-inflammatory effects of endothelin receptor blockade in left atrial tissue of spontaneously hypertensive rats"

    Article Title: Anti-inflammatory effects of endothelin receptor blockade in left atrial tissue of spontaneously hypertensive rats

    Journal: International Journal of Cardiology. Heart & Vasculature

    doi: 10.1016/j.ijcha.2022.101088

    Effects of macitentan on the calcium handling in LA in SHR. (a) Representative immunoblots showing expression of α1C, NCX1, CSQ and RyR2 from SHR treated with MAC or DOX versus SHR-CTL (b-g) Quantification of immunoblots (N = 6–9 per group) (h) Representative immunoblots showing expression of SERCA2a, CaMKIIδ and PLB. Actin controls for pS16, pT17 and pS10 are not shown (i-m) Quantification of immunoblots (N = 6–9); # P < 0.05. Note that some blots/bands of housekeeping proteins used for normalization are identical (e.g. in (a) GAPDH blot/bands shown below CSQ and GAPDH blot/bands shown below RyR) as they are derived from the same membrane.
    Figure Legend Snippet: Effects of macitentan on the calcium handling in LA in SHR. (a) Representative immunoblots showing expression of α1C, NCX1, CSQ and RyR2 from SHR treated with MAC or DOX versus SHR-CTL (b-g) Quantification of immunoblots (N = 6–9 per group) (h) Representative immunoblots showing expression of SERCA2a, CaMKIIδ and PLB. Actin controls for pS16, pT17 and pS10 are not shown (i-m) Quantification of immunoblots (N = 6–9); # P < 0.05. Note that some blots/bands of housekeeping proteins used for normalization are identical (e.g. in (a) GAPDH blot/bands shown below CSQ and GAPDH blot/bands shown below RyR) as they are derived from the same membrane.

    Techniques Used: Western Blot, Expressing, Derivative Assay

    ryr2  (Alomone Labs)


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    Alomone Labs ryr2
    The effect of hypoxia and Rycal treatment on <t>RyR2</t> and SERCA2a gene expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a gene expression. n = 6, * P < .050 for comparison with 1% O 2 , # P < .050 for comparison with Vehicle, $ P < .050 for comparison with 0.3 mM (all 1-way ANOVA with Tukey post hoc test).
    Ryr2, 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/ryr2/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ryr2 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes"

    Article Title: Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes

    Journal: Anatolian Journal of Cardiology

    doi: 10.5152/AnatolJCardiol.2022.1223

    The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a gene expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a gene expression. n = 6, * P < .050 for comparison with 1% O 2 , # P < .050 for comparison with Vehicle, $ P < .050 for comparison with 0.3 mM (all 1-way ANOVA with Tukey post hoc test).
    Figure Legend Snippet: The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a gene expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a gene expression. n = 6, * P < .050 for comparison with 1% O 2 , # P < .050 for comparison with Vehicle, $ P < .050 for comparison with 0.3 mM (all 1-way ANOVA with Tukey post hoc test).

    Techniques Used: Expressing

    The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a protein expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a protein expression. n = 6. No statistically significant comparison.
    Figure Legend Snippet: The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a protein expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a protein expression. n = 6. No statistically significant comparison.

    Techniques Used: Expressing

    rabbit polyclonal ryr2  (Alomone Labs)


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    Alomone Labs rabbit polyclonal ryr2
    CCN5 prevents cardiac fibrosis in mdx/utrn (±) mice. (A) Experimental scheme for panels (B–E) . mdx/utrn (±) mice were injected with AAV.9-Con or CCN5 into the tail vein, and hearts were harvested for experiments 8 weeks later. Age-matched WT mice are shown in comparison. (B) Hearts were sectioned and stained with trichrome. Blue areas indicate fibrotic tissue and red areas indicate normal tissue. (C) The ratio of fibrotic area over total tissue of the stained hearts was plotted. (D) Proteins obtained from cardiac tissue were immunoblotted with antibodies against CCN5, α-SMA, collagen I, SERCA2a, <t>RyR2,</t> NCX1, phosphorylated phospholamban (p-PLN), t-PLN and α-tubulin. (E) Protein bands on western blots were scanned and plotted. n = 6. * p < 0.05 and ** p < 0.01.
    Rabbit Polyclonal Ryr2, 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/rabbit polyclonal ryr2/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal ryr2 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Matricellular Protein CCN5 Gene Transfer Ameliorates Cardiac and Skeletal Dysfunction in mdx/utrn (±) Haploinsufficient Mice by Reducing Fibrosis and Upregulating Utrophin Expression"

    Article Title: Matricellular Protein CCN5 Gene Transfer Ameliorates Cardiac and Skeletal Dysfunction in mdx/utrn (±) Haploinsufficient Mice by Reducing Fibrosis and Upregulating Utrophin Expression

    Journal: Frontiers in Cardiovascular Medicine

    doi: 10.3389/fcvm.2022.763544

    CCN5 prevents cardiac fibrosis in mdx/utrn (±) mice. (A) Experimental scheme for panels (B–E) . mdx/utrn (±) mice were injected with AAV.9-Con or CCN5 into the tail vein, and hearts were harvested for experiments 8 weeks later. Age-matched WT mice are shown in comparison. (B) Hearts were sectioned and stained with trichrome. Blue areas indicate fibrotic tissue and red areas indicate normal tissue. (C) The ratio of fibrotic area over total tissue of the stained hearts was plotted. (D) Proteins obtained from cardiac tissue were immunoblotted with antibodies against CCN5, α-SMA, collagen I, SERCA2a, RyR2, NCX1, phosphorylated phospholamban (p-PLN), t-PLN and α-tubulin. (E) Protein bands on western blots were scanned and plotted. n = 6. * p < 0.05 and ** p < 0.01.
    Figure Legend Snippet: CCN5 prevents cardiac fibrosis in mdx/utrn (±) mice. (A) Experimental scheme for panels (B–E) . mdx/utrn (±) mice were injected with AAV.9-Con or CCN5 into the tail vein, and hearts were harvested for experiments 8 weeks later. Age-matched WT mice are shown in comparison. (B) Hearts were sectioned and stained with trichrome. Blue areas indicate fibrotic tissue and red areas indicate normal tissue. (C) The ratio of fibrotic area over total tissue of the stained hearts was plotted. (D) Proteins obtained from cardiac tissue were immunoblotted with antibodies against CCN5, α-SMA, collagen I, SERCA2a, RyR2, NCX1, phosphorylated phospholamban (p-PLN), t-PLN and α-tubulin. (E) Protein bands on western blots were scanned and plotted. n = 6. * p < 0.05 and ** p < 0.01.

    Techniques Used: Injection, Staining, Western Blot

    anti ryr2  (Alomone Labs)


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    Alomone Labs anti ryr2

    Anti Ryr2, 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 ryr2/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ryr2 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells"

    Article Title: STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells

    Journal: eLife

    doi: 10.7554/eLife.70278


    Figure Legend Snippet:

    Techniques Used: Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Software

    anti ryanodine receptor 2 ryr2  (Alomone Labs)


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    Alomone Labs anti ryanodine receptor 2 ryr2
    Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine <t>receptor</t> <t>2</t> <t>[RyR2]),</t> MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.
    Anti Ryanodine Receptor 2 Ryr2, 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 ryanodine receptor 2 ryr2/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ryanodine receptor 2 ryr2 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Deciphering cellular signals in adult mouse sinoatrial node cells"

    Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

    Journal: iScience

    doi: 10.1016/j.isci.2021.103693

    Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.
    Figure Legend Snippet: Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.

    Techniques Used: Derivative Assay, Sequencing, Expressing, Fluorescence, Marker, Concentration Assay, Generated, Two Tailed Test, MANN-WHITNEY


    Figure Legend Snippet:

    Techniques Used: Recombinant, Software

    rabbit polyclonal anti ryr2  (Alomone Labs)


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    Alomone Labs rabbit polyclonal anti ryr2
    Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 <t>[RyR2]),</t> MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.
    Rabbit Polyclonal Anti Ryr2, 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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    Images

    1) Product Images from "Deciphering cellular signals in adult mouse sinoatrial node cells"

    Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

    Journal: iScience

    doi: 10.1016/j.isci.2021.103693

    Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.
    Figure Legend Snippet: Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.

    Techniques Used: Derivative Assay, Sequencing, Expressing, Fluorescence, Marker, Concentration Assay, Generated, Two Tailed Test, MANN-WHITNEY


    Figure Legend Snippet:

    Techniques Used: Recombinant, Software

    ryr2  (Alomone Labs)


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    Alomone Labs ryr2
    Ca2+ handling protein expression or phosphorylation at ZT4 and ZT14 in adult hearts. A-B. Phosphorylation of PLB at serine16 was essentially non-detectable following 5min perfusion (A) and 1hr perfusion (to match optical mapping data collection timepoint, B). Band labeled ‘Iso’ in (B) was a single heart perfused with isoproterenol for a positive control for pPLB. C-D. PLB phosphorylation was higher at the Thr17 site at ZT4 vs. ZT14 following 5min perfusion (C). However, phosphorylation levels were reduced and were not different between ZT4 and ZT14 following 1hr of perfusion (D). Band labeled ‘Iso’ in (D) was a single heart perfused with isoproterenol for a positive control for pPLB. E-G. Total PLB (E), SERCA2a (F), and <t>RyR2</t> (G) expression. α-tubulin was used as loading control. ZT4 n=4; ZT14 n=4; except RyR (G) where ZT4 n=3; ZT14 n=3. *p<0.05.
    Ryr2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Aging Disrupts Normal Time-of-day Variation in Cardiac Electrophysiology"

    Article Title: Aging Disrupts Normal Time-of-day Variation in Cardiac Electrophysiology

    Journal: Circulation. Arrhythmia and electrophysiology

    doi: 10.1161/CIRCEP.119.008093

    Ca2+ handling protein expression or phosphorylation at ZT4 and ZT14 in adult hearts. A-B. Phosphorylation of PLB at serine16 was essentially non-detectable following 5min perfusion (A) and 1hr perfusion (to match optical mapping data collection timepoint, B). Band labeled ‘Iso’ in (B) was a single heart perfused with isoproterenol for a positive control for pPLB. C-D. PLB phosphorylation was higher at the Thr17 site at ZT4 vs. ZT14 following 5min perfusion (C). However, phosphorylation levels were reduced and were not different between ZT4 and ZT14 following 1hr of perfusion (D). Band labeled ‘Iso’ in (D) was a single heart perfused with isoproterenol for a positive control for pPLB. E-G. Total PLB (E), SERCA2a (F), and RyR2 (G) expression. α-tubulin was used as loading control. ZT4 n=4; ZT14 n=4; except RyR (G) where ZT4 n=3; ZT14 n=3. *p<0.05.
    Figure Legend Snippet: Ca2+ handling protein expression or phosphorylation at ZT4 and ZT14 in adult hearts. A-B. Phosphorylation of PLB at serine16 was essentially non-detectable following 5min perfusion (A) and 1hr perfusion (to match optical mapping data collection timepoint, B). Band labeled ‘Iso’ in (B) was a single heart perfused with isoproterenol for a positive control for pPLB. C-D. PLB phosphorylation was higher at the Thr17 site at ZT4 vs. ZT14 following 5min perfusion (C). However, phosphorylation levels were reduced and were not different between ZT4 and ZT14 following 1hr of perfusion (D). Band labeled ‘Iso’ in (D) was a single heart perfused with isoproterenol for a positive control for pPLB. E-G. Total PLB (E), SERCA2a (F), and RyR2 (G) expression. α-tubulin was used as loading control. ZT4 n=4; ZT14 n=4; except RyR (G) where ZT4 n=3; ZT14 n=3. *p<0.05.

    Techniques Used: Expressing, Labeling, Positive Control

    ryr2  (Alomone Labs)


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    Alomone Labs ryr2
    Ca2+ handling protein expression or phosphorylation at ZT4 and ZT14 in adult hearts. A-B. Phosphorylation of PLB at serine16 was essentially non-detectable following 5min perfusion (A) and 1hr perfusion (to match optical mapping data collection timepoint, B). Band labeled ‘Iso’ in (B) was a single heart perfused with isoproterenol for a positive control for pPLB. C-D. PLB phosphorylation was higher at the Thr17 site at ZT4 vs. ZT14 following 5min perfusion (C). However, phosphorylation levels were reduced and were not different between ZT4 and ZT14 following 1hr of perfusion (D). Band labeled ‘Iso’ in (D) was a single heart perfused with isoproterenol for a positive control for pPLB. E-G. Total PLB (E), SERCA2a (F), and <t>RyR2</t> (G) expression. α-tubulin was used as loading control. ZT4 n=4; ZT14 n=4; except RyR (G) where ZT4 n=3; ZT14 n=3. *p<0.05.
    Ryr2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Aging Disrupts Normal Time-of-day Variation in Cardiac Electrophysiology"

    Article Title: Aging Disrupts Normal Time-of-day Variation in Cardiac Electrophysiology

    Journal: Circulation. Arrhythmia and electrophysiology

    doi: 10.1161/CIRCEP.119.008093

    Ca2+ handling protein expression or phosphorylation at ZT4 and ZT14 in adult hearts. A-B. Phosphorylation of PLB at serine16 was essentially non-detectable following 5min perfusion (A) and 1hr perfusion (to match optical mapping data collection timepoint, B). Band labeled ‘Iso’ in (B) was a single heart perfused with isoproterenol for a positive control for pPLB. C-D. PLB phosphorylation was higher at the Thr17 site at ZT4 vs. ZT14 following 5min perfusion (C). However, phosphorylation levels were reduced and were not different between ZT4 and ZT14 following 1hr of perfusion (D). Band labeled ‘Iso’ in (D) was a single heart perfused with isoproterenol for a positive control for pPLB. E-G. Total PLB (E), SERCA2a (F), and RyR2 (G) expression. α-tubulin was used as loading control. ZT4 n=4; ZT14 n=4; except RyR (G) where ZT4 n=3; ZT14 n=3. *p<0.05.
    Figure Legend Snippet: Ca2+ handling protein expression or phosphorylation at ZT4 and ZT14 in adult hearts. A-B. Phosphorylation of PLB at serine16 was essentially non-detectable following 5min perfusion (A) and 1hr perfusion (to match optical mapping data collection timepoint, B). Band labeled ‘Iso’ in (B) was a single heart perfused with isoproterenol for a positive control for pPLB. C-D. PLB phosphorylation was higher at the Thr17 site at ZT4 vs. ZT14 following 5min perfusion (C). However, phosphorylation levels were reduced and were not different between ZT4 and ZT14 following 1hr of perfusion (D). Band labeled ‘Iso’ in (D) was a single heart perfused with isoproterenol for a positive control for pPLB. E-G. Total PLB (E), SERCA2a (F), and RyR2 (G) expression. α-tubulin was used as loading control. ZT4 n=4; ZT14 n=4; except RyR (G) where ZT4 n=3; ZT14 n=3. *p<0.05.

    Techniques Used: Expressing, Labeling, Positive Control

    antibody ryr1  (Alomone Labs)


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    Alomone Labs antibody ryr1
    Flow cytometry detects <t>RYR1</t> in freshly isolated LMCs. Scatter plots of LMC suspensions plotted as side scatter (SSC-W) vs. fluorescent signals. Black horizontal lines denote the gate set. Fluorescent signal appearing above gate indicates positive staining. (A) Negative control (Neg) showing background FITC fluorescence of cell population with no antibody staining. Another LMC suspension was used to identify (B, left ) the LMC population stained by α-SMA-FITC ( red box ) and (B, right ) the gate set for non-specific staining of Alexa-Fluor 647 conjugated secondary antibody without primary antibodies within the α-SMA-FITC gated region. (C) Positive detection of RYR1 above the Alexa-Fluor 674 gate in LMCs isolated from rat mesenteric LVs. Data representative of five isolations each from male and female rats ( n = 10).
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    Images

    1) Product Images from "Dantrolene Prevents the Lymphostasis Caused by Doxorubicin in the Rat Mesenteric Circulation"

    Article Title: Dantrolene Prevents the Lymphostasis Caused by Doxorubicin in the Rat Mesenteric Circulation

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2021.727526

    Flow cytometry detects RYR1 in freshly isolated LMCs. Scatter plots of LMC suspensions plotted as side scatter (SSC-W) vs. fluorescent signals. Black horizontal lines denote the gate set. Fluorescent signal appearing above gate indicates positive staining. (A) Negative control (Neg) showing background FITC fluorescence of cell population with no antibody staining. Another LMC suspension was used to identify (B, left ) the LMC population stained by α-SMA-FITC ( red box ) and (B, right ) the gate set for non-specific staining of Alexa-Fluor 647 conjugated secondary antibody without primary antibodies within the α-SMA-FITC gated region. (C) Positive detection of RYR1 above the Alexa-Fluor 674 gate in LMCs isolated from rat mesenteric LVs. Data representative of five isolations each from male and female rats ( n = 10).
    Figure Legend Snippet: Flow cytometry detects RYR1 in freshly isolated LMCs. Scatter plots of LMC suspensions plotted as side scatter (SSC-W) vs. fluorescent signals. Black horizontal lines denote the gate set. Fluorescent signal appearing above gate indicates positive staining. (A) Negative control (Neg) showing background FITC fluorescence of cell population with no antibody staining. Another LMC suspension was used to identify (B, left ) the LMC population stained by α-SMA-FITC ( red box ) and (B, right ) the gate set for non-specific staining of Alexa-Fluor 647 conjugated secondary antibody without primary antibodies within the α-SMA-FITC gated region. (C) Positive detection of RYR1 above the Alexa-Fluor 674 gate in LMCs isolated from rat mesenteric LVs. Data representative of five isolations each from male and female rats ( n = 10).

    Techniques Used: Flow Cytometry, Isolation, Staining, Negative Control, Fluorescence

    RYR1 antibody selectivity. (A) Western blot shows positive detection of RYR1 (∼565 kD) in rat skeletal muscle lysate and no detection in rat heart or brain lysate. (B,C) Contour plots of RYR1 antibody testing. (B) Red population represents positive detection of RYR1 subtype in male LMCs. Grey population represents the LMC suspension containing no primary antibody. (C) No detection in male LMCs after co-incubation with competing peptide (CP; 1:50 dilution) for the RYR1 antibody. Data representative of three isolations for each sex ( n = 6).
    Figure Legend Snippet: RYR1 antibody selectivity. (A) Western blot shows positive detection of RYR1 (∼565 kD) in rat skeletal muscle lysate and no detection in rat heart or brain lysate. (B,C) Contour plots of RYR1 antibody testing. (B) Red population represents positive detection of RYR1 subtype in male LMCs. Grey population represents the LMC suspension containing no primary antibody. (C) No detection in male LMCs after co-incubation with competing peptide (CP; 1:50 dilution) for the RYR1 antibody. Data representative of three isolations for each sex ( n = 6).

    Techniques Used: Western Blot, Incubation

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    Alomone Labs ryr2
    The effect of hypoxia and Rycal treatment on <t>RyR2</t> and SERCA2a gene expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a gene expression. n = 6, * P < .050 for comparison with 1% O 2 , # P < .050 for comparison with Vehicle, $ P < .050 for comparison with 0.3 mM (all 1-way ANOVA with Tukey post hoc test).
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    Integrin ligand ff_RGD induced changes in the Ca 2+ cycling ultrastructure. L-type calcium channel (LTCC) subunit CaV1.2 (green) and <t>ryanodine</t> <t>receptor</t> (RyR) 2 (red) were labelled and orthoview z-stack formed with wheat germ agglutinin (WGA) (white) and Hoechst (blue) staining ( A , B ) control and ( C , D ) ff_RGD-treated cardiomyocytes (day 40–46 of differentiation). Scale bar for A and 6 = 20 μm, B and D = 50 μm. Merged images used to calculate the Mander’s correlation coefficient for ( E ) LTCC overlapping RyR background pixels (M1) (* p = 0.0363), ( F ) RyR overlapping LTCC background pixels (M2) (ns p = 0.1143), and ( G ) Pearson’s correlation coefficient between LTCC and RyR (** p = 0.001) illustrated as means ± SEM ( n = 39 control, n = 18 ff_RGD cells from ten batches).
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    Alomone Labs anti pser2808 ryr2
    Effects of macitentan on the calcium handling in LA in SHR. (a) Representative immunoblots showing expression of α1C, NCX1, CSQ and <t>RyR2</t> from SHR treated with MAC or DOX versus SHR-CTL (b-g) Quantification of immunoblots (N = 6–9 per group) (h) Representative immunoblots showing expression of SERCA2a, CaMKIIδ and PLB. Actin controls for pS16, pT17 and pS10 are not shown (i-m) Quantification of immunoblots (N = 6–9); # P < 0.05. Note that some blots/bands of housekeeping proteins used for normalization are identical (e.g. in (a) GAPDH blot/bands shown below CSQ and GAPDH blot/bands shown below RyR) as they are derived from the same membrane.
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    CCN5 prevents cardiac fibrosis in mdx/utrn (±) mice. (A) Experimental scheme for panels (B–E) . mdx/utrn (±) mice were injected with AAV.9-Con or CCN5 into the tail vein, and hearts were harvested for experiments 8 weeks later. Age-matched WT mice are shown in comparison. (B) Hearts were sectioned and stained with trichrome. Blue areas indicate fibrotic tissue and red areas indicate normal tissue. (C) The ratio of fibrotic area over total tissue of the stained hearts was plotted. (D) Proteins obtained from cardiac tissue were immunoblotted with antibodies against CCN5, α-SMA, collagen I, SERCA2a, <t>RyR2,</t> NCX1, phosphorylated phospholamban (p-PLN), t-PLN and α-tubulin. (E) Protein bands on western blots were scanned and plotted. n = 6. * p < 0.05 and ** p < 0.01.
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    Anti Ryr2, 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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    Alomone Labs anti ryanodine receptor 2 ryr2
    Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine <t>receptor</t> <t>2</t> <t>[RyR2]),</t> MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.
    Anti Ryanodine Receptor 2 Ryr2, 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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    Alomone Labs rabbit polyclonal anti ryr2
    Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 <t>[RyR2]),</t> MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.
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    Alomone Labs antibody ryr1
    Flow cytometry detects <t>RYR1</t> in freshly isolated LMCs. Scatter plots of LMC suspensions plotted as side scatter (SSC-W) vs. fluorescent signals. Black horizontal lines denote the gate set. Fluorescent signal appearing above gate indicates positive staining. (A) Negative control (Neg) showing background FITC fluorescence of cell population with no antibody staining. Another LMC suspension was used to identify (B, left ) the LMC population stained by α-SMA-FITC ( red box ) and (B, right ) the gate set for non-specific staining of Alexa-Fluor 647 conjugated secondary antibody without primary antibodies within the α-SMA-FITC gated region. (C) Positive detection of RYR1 above the Alexa-Fluor 674 gate in LMCs isolated from rat mesenteric LVs. Data representative of five isolations each from male and female rats ( n = 10).
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    Image Search Results


    The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a gene expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a gene expression. n = 6, * P < .050 for comparison with 1% O 2 , # P < .050 for comparison with Vehicle, $ P < .050 for comparison with 0.3 mM (all 1-way ANOVA with Tukey post hoc test).

    Journal: Anatolian Journal of Cardiology

    Article Title: Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes

    doi: 10.5152/AnatolJCardiol.2022.1223

    Figure Lengend Snippet: The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a gene expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a gene expression. n = 6, * P < .050 for comparison with 1% O 2 , # P < .050 for comparison with Vehicle, $ P < .050 for comparison with 0.3 mM (all 1-way ANOVA with Tukey post hoc test).

    Article Snippet: After blocking, the membranes were washed using Tris-buffered saline with Tween (TBS-T) and then incubated with primary antibody SERCA2a (A010-23S, Badrilla, Leeds, UK, dilution 1:1000), RyR2 (ARR-002, Alomone Labs, Jerusalem, Israel, dilution 1:1000) and β-tubulin (ab6046, Abcam, Cambridge, UK, dilution 1:1000) overnight.

    Techniques: Expressing

    The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a protein expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a protein expression. n = 6. No statistically significant comparison.

    Journal: Anatolian Journal of Cardiology

    Article Title: Hypoxia-Induced Sarcoplasmic Reticulum Ca 2+ Leak Is Reversed by Ryanodine Receptor Stabilizer JTV-519 in HL-1 Cardiomyocytes

    doi: 10.5152/AnatolJCardiol.2022.1223

    Figure Lengend Snippet: The effect of hypoxia and Rycal treatment on RyR2 and SERCA2a protein expression. The effect of 7-day hypoxic and normoxic exposure with Rycals/Vehicle (DMSO) treatment on RyR2 and SERCA2a protein expression. n = 6. No statistically significant comparison.

    Article Snippet: After blocking, the membranes were washed using Tris-buffered saline with Tween (TBS-T) and then incubated with primary antibody SERCA2a (A010-23S, Badrilla, Leeds, UK, dilution 1:1000), RyR2 (ARR-002, Alomone Labs, Jerusalem, Israel, dilution 1:1000) and β-tubulin (ab6046, Abcam, Cambridge, UK, dilution 1:1000) overnight.

    Techniques: Expressing

    Integrin ligand ff_RGD induced changes in the Ca 2+ cycling ultrastructure. L-type calcium channel (LTCC) subunit CaV1.2 (green) and ryanodine receptor (RyR) 2 (red) were labelled and orthoview z-stack formed with wheat germ agglutinin (WGA) (white) and Hoechst (blue) staining ( A , B ) control and ( C , D ) ff_RGD-treated cardiomyocytes (day 40–46 of differentiation). Scale bar for A and 6 = 20 μm, B and D = 50 μm. Merged images used to calculate the Mander’s correlation coefficient for ( E ) LTCC overlapping RyR background pixels (M1) (* p = 0.0363), ( F ) RyR overlapping LTCC background pixels (M2) (ns p = 0.1143), and ( G ) Pearson’s correlation coefficient between LTCC and RyR (** p = 0.001) illustrated as means ± SEM ( n = 39 control, n = 18 ff_RGD cells from ten batches).

    Journal: International Journal of Molecular Sciences

    Article Title: Integrins Increase Sarcoplasmic Reticulum Activity for Excitation—Contraction Coupling in Human Stem Cell-Derived Cardiomyocytes

    doi: 10.3390/ijms231810940

    Figure Lengend Snippet: Integrin ligand ff_RGD induced changes in the Ca 2+ cycling ultrastructure. L-type calcium channel (LTCC) subunit CaV1.2 (green) and ryanodine receptor (RyR) 2 (red) were labelled and orthoview z-stack formed with wheat germ agglutinin (WGA) (white) and Hoechst (blue) staining ( A , B ) control and ( C , D ) ff_RGD-treated cardiomyocytes (day 40–46 of differentiation). Scale bar for A and 6 = 20 μm, B and D = 50 μm. Merged images used to calculate the Mander’s correlation coefficient for ( E ) LTCC overlapping RyR background pixels (M1) (* p = 0.0363), ( F ) RyR overlapping LTCC background pixels (M2) (ns p = 0.1143), and ( G ) Pearson’s correlation coefficient between LTCC and RyR (** p = 0.001) illustrated as means ± SEM ( n = 39 control, n = 18 ff_RGD cells from ten batches).

    Article Snippet: In the co-localisation immunostaining, preparations were stained with mouse monoclonal antibody IgG1 anti-RyR2 in a 1/500 dilution in PBS and guinea pig polyclonal antibody (Alomone Labs, Jerusalem, Israel) anti-1.2 CaV in 1/250 dilution in PBS.

    Techniques: Staining

    Effects of macitentan on the calcium handling in LA in SHR. (a) Representative immunoblots showing expression of α1C, NCX1, CSQ and RyR2 from SHR treated with MAC or DOX versus SHR-CTL (b-g) Quantification of immunoblots (N = 6–9 per group) (h) Representative immunoblots showing expression of SERCA2a, CaMKIIδ and PLB. Actin controls for pS16, pT17 and pS10 are not shown (i-m) Quantification of immunoblots (N = 6–9); # P < 0.05. Note that some blots/bands of housekeeping proteins used for normalization are identical (e.g. in (a) GAPDH blot/bands shown below CSQ and GAPDH blot/bands shown below RyR) as they are derived from the same membrane.

    Journal: International Journal of Cardiology. Heart & Vasculature

    Article Title: Anti-inflammatory effects of endothelin receptor blockade in left atrial tissue of spontaneously hypertensive rats

    doi: 10.1016/j.ijcha.2022.101088

    Figure Lengend Snippet: Effects of macitentan on the calcium handling in LA in SHR. (a) Representative immunoblots showing expression of α1C, NCX1, CSQ and RyR2 from SHR treated with MAC or DOX versus SHR-CTL (b-g) Quantification of immunoblots (N = 6–9 per group) (h) Representative immunoblots showing expression of SERCA2a, CaMKIIδ and PLB. Actin controls for pS16, pT17 and pS10 are not shown (i-m) Quantification of immunoblots (N = 6–9); # P < 0.05. Note that some blots/bands of housekeeping proteins used for normalization are identical (e.g. in (a) GAPDH blot/bands shown below CSQ and GAPDH blot/bands shown below RyR) as they are derived from the same membrane.

    Article Snippet: Quality of transfer was probed using Ponceau S. Primary antibodies used for detection of protein expression and phosphorylation were 1) on 4–20% gradient gels (BioRad, Germany): anti-CaMKII (50 kD, Badrilla, A010-55AP, 1:5,000), anti-CSQ (55 kD, Thermo Scientific, PA1-913, 1:2,500), anti-GAPDH (34 kD, Calbiochem, CB1001, 1:50,000), anti-IP 3 R2 (313 kD, Abcam, ab77838, 1:1,000), anti-NCX1 (120 kD, Thermo Scientific, MA1-4672, 1:1,000), anti-SERCA2a (100 kD, Badrilla, A010-20, 1:5,000), anti-RyR (565 kD, Thermo Scientific, MA3-916, 1:5,000), anti-pSer2808-RyR2 (Badrilla, A010-30, 1:5,000); 2) on 8% glycine gels: anti-Cav1.2a, i.e. cardiac type α1C subunit of the L-type Ca channel (250 kD, Alomone Labs, ACC-013, 1:200); and 3) on 14% tricine gels: anti-actin (44 kD, clone C4, MP Biomedicals #69100, 1:50,000), anti-PLB (25 kD, Badrilla, A010-14, 1:5,000), anti-Ser10-PLB (Badrilla, A010-10AP, 1:1,000), anti-pSer16-PLB (Badrilla, A010-12, 1:5,000), anti-pThr17-PLB (Badrilla, A010-13, 1:5,000).

    Techniques: Western Blot, Expressing, Derivative Assay

    CCN5 prevents cardiac fibrosis in mdx/utrn (±) mice. (A) Experimental scheme for panels (B–E) . mdx/utrn (±) mice were injected with AAV.9-Con or CCN5 into the tail vein, and hearts were harvested for experiments 8 weeks later. Age-matched WT mice are shown in comparison. (B) Hearts were sectioned and stained with trichrome. Blue areas indicate fibrotic tissue and red areas indicate normal tissue. (C) The ratio of fibrotic area over total tissue of the stained hearts was plotted. (D) Proteins obtained from cardiac tissue were immunoblotted with antibodies against CCN5, α-SMA, collagen I, SERCA2a, RyR2, NCX1, phosphorylated phospholamban (p-PLN), t-PLN and α-tubulin. (E) Protein bands on western blots were scanned and plotted. n = 6. * p < 0.05 and ** p < 0.01.

    Journal: Frontiers in Cardiovascular Medicine

    Article Title: Matricellular Protein CCN5 Gene Transfer Ameliorates Cardiac and Skeletal Dysfunction in mdx/utrn (±) Haploinsufficient Mice by Reducing Fibrosis and Upregulating Utrophin Expression

    doi: 10.3389/fcvm.2022.763544

    Figure Lengend Snippet: CCN5 prevents cardiac fibrosis in mdx/utrn (±) mice. (A) Experimental scheme for panels (B–E) . mdx/utrn (±) mice were injected with AAV.9-Con or CCN5 into the tail vein, and hearts were harvested for experiments 8 weeks later. Age-matched WT mice are shown in comparison. (B) Hearts were sectioned and stained with trichrome. Blue areas indicate fibrotic tissue and red areas indicate normal tissue. (C) The ratio of fibrotic area over total tissue of the stained hearts was plotted. (D) Proteins obtained from cardiac tissue were immunoblotted with antibodies against CCN5, α-SMA, collagen I, SERCA2a, RyR2, NCX1, phosphorylated phospholamban (p-PLN), t-PLN and α-tubulin. (E) Protein bands on western blots were scanned and plotted. n = 6. * p < 0.05 and ** p < 0.01.

    Article Snippet: Transferred blots were blocked with 5% non-fat skim milk and incubated with antibodies against mouse monoclonal CCN5 (1:1,000, Sigma, #WH0008839M9), mouse monoclonal α-SMA (1:1,000, Sigma-Aldrich, #A5228), goat polyclonal collagen I (1:1,000, Abcam, ab34710), rabbit polyclonal SERCA2a (1:1,000, a custom antibody from 21st Century Biochemicals), rabbit polyclonal RyR2 (1:1,000, Alomone Lab, ARR-002), rabbit monoclonal NCX1 (1:1,000, Abcam, EPR12739), rabbit polyclonal p-PLN (1:1,000, Badrilla, A010-12AP), mouse monoclonal t-PLN (1:1,000, Badrilla, A010-14), and mouse monoclonal α-tubulin (1:3,000, Santa Cruz, #sc-8035) for 12–16 h at 4°C.

    Techniques: Injection, Staining, Western Blot

    Journal: eLife

    Article Title: STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells

    doi: 10.7554/eLife.70278

    Figure Lengend Snippet:

    Article Snippet: Cells were then blocked with 50% SEABLOCK blocking buffer (Thermo Fisher Scientific) for 2 hr and incubated overnight at 4°C with primary antibody (anti-STIM1- [4916], Cell Signaling Technologies, Danvers, MA; anti-STIM1 [610954], BD Biosciences, Franklin Lakes, NJ; anti-BKα1- [APC-021], Alomone Labs, Jerusalem, Israel; anti-RyR2- [MA3-916], Thermo Fisher Scientific; anti-TRPM4- (ABIN572220); https://antibodies-online.com , Limerick, PA; anti-IP 3 R- (ab5804), Abcam, Cambridge, UK) diluted in PBS containing 20% SEABLOCK, 1% BSA, and 0.05% Triton X-100.

    Techniques: Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Software

    Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.

    Journal: iScience

    Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

    doi: 10.1016/j.isci.2021.103693

    Figure Lengend Snippet: Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.

    Article Snippet: For experiments in , cells infected with the different ICU3 biosensors were incubated with an anti-caveolin 3 antibody (1:200, Thermo-Fisher Scientific PA1-066) to label the PM, an anti-ryanodine receptor 2 (RyR2) antibody (1:200, Alomone Labs ARR-002) to label the SR, an Alexa Fluor 647 Phalloidin (1.3 U; Thermo-Fisher Scientific A22287) to label MF, or DAPI from the ProLong Diamond Antifade Mountant (Thermo-Fisher Scientific) to label the nucleus.

    Techniques: Derivative Assay, Sequencing, Expressing, Fluorescence, Marker, Concentration Assay, Generated, Two Tailed Test, MANN-WHITNEY

    Journal: iScience

    Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

    doi: 10.1016/j.isci.2021.103693

    Figure Lengend Snippet:

    Article Snippet: For experiments in , cells infected with the different ICU3 biosensors were incubated with an anti-caveolin 3 antibody (1:200, Thermo-Fisher Scientific PA1-066) to label the PM, an anti-ryanodine receptor 2 (RyR2) antibody (1:200, Alomone Labs ARR-002) to label the SR, an Alexa Fluor 647 Phalloidin (1.3 U; Thermo-Fisher Scientific A22287) to label MF, or DAPI from the ProLong Diamond Antifade Mountant (Thermo-Fisher Scientific) to label the nucleus.

    Techniques: Recombinant, Software

    Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.

    Journal: iScience

    Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

    doi: 10.1016/j.isci.2021.103693

    Figure Lengend Snippet: Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.

    Article Snippet: Rabbit polyclonal anti RyR2 , Alomone , Cat# ARR-002; RRID: AB_2040184.

    Techniques: Derivative Assay, Sequencing, Expressing, Fluorescence, Marker, Concentration Assay, Generated, Two Tailed Test, MANN-WHITNEY

    Journal: iScience

    Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

    doi: 10.1016/j.isci.2021.103693

    Figure Lengend Snippet:

    Article Snippet: Rabbit polyclonal anti RyR2 , Alomone , Cat# ARR-002; RRID: AB_2040184.

    Techniques: Recombinant, Software

    Flow cytometry detects RYR1 in freshly isolated LMCs. Scatter plots of LMC suspensions plotted as side scatter (SSC-W) vs. fluorescent signals. Black horizontal lines denote the gate set. Fluorescent signal appearing above gate indicates positive staining. (A) Negative control (Neg) showing background FITC fluorescence of cell population with no antibody staining. Another LMC suspension was used to identify (B, left ) the LMC population stained by α-SMA-FITC ( red box ) and (B, right ) the gate set for non-specific staining of Alexa-Fluor 647 conjugated secondary antibody without primary antibodies within the α-SMA-FITC gated region. (C) Positive detection of RYR1 above the Alexa-Fluor 674 gate in LMCs isolated from rat mesenteric LVs. Data representative of five isolations each from male and female rats ( n = 10).

    Journal: Frontiers in Pharmacology

    Article Title: Dantrolene Prevents the Lymphostasis Caused by Doxorubicin in the Rat Mesenteric Circulation

    doi: 10.3389/fphar.2021.727526

    Figure Lengend Snippet: Flow cytometry detects RYR1 in freshly isolated LMCs. Scatter plots of LMC suspensions plotted as side scatter (SSC-W) vs. fluorescent signals. Black horizontal lines denote the gate set. Fluorescent signal appearing above gate indicates positive staining. (A) Negative control (Neg) showing background FITC fluorescence of cell population with no antibody staining. Another LMC suspension was used to identify (B, left ) the LMC population stained by α-SMA-FITC ( red box ) and (B, right ) the gate set for non-specific staining of Alexa-Fluor 647 conjugated secondary antibody without primary antibodies within the α-SMA-FITC gated region. (C) Positive detection of RYR1 above the Alexa-Fluor 674 gate in LMCs isolated from rat mesenteric LVs. Data representative of five isolations each from male and female rats ( n = 10).

    Article Snippet: The membrane was first incubated in tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% non-fat dry milk to reduce non-specific antibody binding, then incubated overnight at 4°C with primary antibody RYR1 (1:1,000, Alomone Labs, Jerusalem, Israel) prepared in TBS containing 0.05% Tween-20 and 5% non-fat dry milk.

    Techniques: Flow Cytometry, Isolation, Staining, Negative Control, Fluorescence

    RYR1 antibody selectivity. (A) Western blot shows positive detection of RYR1 (∼565 kD) in rat skeletal muscle lysate and no detection in rat heart or brain lysate. (B,C) Contour plots of RYR1 antibody testing. (B) Red population represents positive detection of RYR1 subtype in male LMCs. Grey population represents the LMC suspension containing no primary antibody. (C) No detection in male LMCs after co-incubation with competing peptide (CP; 1:50 dilution) for the RYR1 antibody. Data representative of three isolations for each sex ( n = 6).

    Journal: Frontiers in Pharmacology

    Article Title: Dantrolene Prevents the Lymphostasis Caused by Doxorubicin in the Rat Mesenteric Circulation

    doi: 10.3389/fphar.2021.727526

    Figure Lengend Snippet: RYR1 antibody selectivity. (A) Western blot shows positive detection of RYR1 (∼565 kD) in rat skeletal muscle lysate and no detection in rat heart or brain lysate. (B,C) Contour plots of RYR1 antibody testing. (B) Red population represents positive detection of RYR1 subtype in male LMCs. Grey population represents the LMC suspension containing no primary antibody. (C) No detection in male LMCs after co-incubation with competing peptide (CP; 1:50 dilution) for the RYR1 antibody. Data representative of three isolations for each sex ( n = 6).

    Article Snippet: The membrane was first incubated in tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% non-fat dry milk to reduce non-specific antibody binding, then incubated overnight at 4°C with primary antibody RYR1 (1:1,000, Alomone Labs, Jerusalem, Israel) prepared in TBS containing 0.05% Tween-20 and 5% non-fat dry milk.

    Techniques: Western Blot, Incubation