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: 93/100, 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 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal ryr2 - by Bioz Stars, 2024-07
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    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

    rabbit anti ryanodine receptor 2 ryr2  (Alomone Labs)


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    Alomone Labs rabbit anti ryanodine receptor 2 ryr2
    Rabbit Anti Ryanodine Receptor 2 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/rabbit anti ryanodine receptor 2 ryr2/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    rabbit anti ryanodine receptor 2 ryr2  (Alomone Labs)


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    Alomone Labs rabbit anti ryanodine receptor 2 ryr2
    Rabbit Anti Ryanodine Receptor 2 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/rabbit anti ryanodine receptor 2 ryr2/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti ryanodine receptor 2 ryr2 - by Bioz Stars, 2024-07
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    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: 93/100, 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 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal ryr2 - by Bioz Stars, 2024-07
    93/100 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

    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: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti ryr2/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti ryr2 - by Bioz Stars, 2024-07
    93/100 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 ryr2  (Alomone Labs)


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    Alomone Labs rabbit polyclonal ryr2
    Pregnancy increased co-localization of <t>RyR1/RyR2</t> with BKCa channel in uterine arteries. (A) Representative confocal immunofluorescence images from five replicates show the co-localization of RyRs and BKCa channels in uterine arteries. Uterine arteries of non-pregnant (NUA) and pregnant (PUA) animals were stained with antibodies against the β1 subunit of BKCa channel (BKCa β1, red) and <t>RyR1,</t> <t>RyR2,</t> or <t>RyR3</t> (green). Merged images show the co-localization of RyR1 or RyR2 with BKCa β1 (in yellow). The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (B) PLA assay to confirm the co-localization of RyR1/RyR2 and BKCa β1 in uterine arteries. (C) Quantification of the PLA signals. Images from five independent replicates were analysed. The nuclear region was stained with DAPI and shown in blue. Scale bar: 50 µm. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, PUA vs. NUA.
    Rabbit Polyclonal Ryr2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal ryr2/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal ryr2 - by Bioz Stars, 2024-07
    93/100 stars

    Images

    1) Product Images from "Ryanodine receptor subtypes regulate Ca 2+ sparks/spontaneous transient outward currents and myogenic tone of uterine arteries in pregnancy"

    Article Title: Ryanodine receptor subtypes regulate Ca 2+ sparks/spontaneous transient outward currents and myogenic tone of uterine arteries in pregnancy

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvaa089

    Pregnancy increased co-localization of RyR1/RyR2 with BKCa channel in uterine arteries. (A) Representative confocal immunofluorescence images from five replicates show the co-localization of RyRs and BKCa channels in uterine arteries. Uterine arteries of non-pregnant (NUA) and pregnant (PUA) animals were stained with antibodies against the β1 subunit of BKCa channel (BKCa β1, red) and RyR1, RyR2, or RyR3 (green). Merged images show the co-localization of RyR1 or RyR2 with BKCa β1 (in yellow). The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (B) PLA assay to confirm the co-localization of RyR1/RyR2 and BKCa β1 in uterine arteries. (C) Quantification of the PLA signals. Images from five independent replicates were analysed. The nuclear region was stained with DAPI and shown in blue. Scale bar: 50 µm. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, PUA vs. NUA.
    Figure Legend Snippet: Pregnancy increased co-localization of RyR1/RyR2 with BKCa channel in uterine arteries. (A) Representative confocal immunofluorescence images from five replicates show the co-localization of RyRs and BKCa channels in uterine arteries. Uterine arteries of non-pregnant (NUA) and pregnant (PUA) animals were stained with antibodies against the β1 subunit of BKCa channel (BKCa β1, red) and RyR1, RyR2, or RyR3 (green). Merged images show the co-localization of RyR1 or RyR2 with BKCa β1 (in yellow). The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (B) PLA assay to confirm the co-localization of RyR1/RyR2 and BKCa β1 in uterine arteries. (C) Quantification of the PLA signals. Images from five independent replicates were analysed. The nuclear region was stained with DAPI and shown in blue. Scale bar: 50 µm. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, PUA vs. NUA.

    Techniques Used: Immunofluorescence, Staining

    Knockdown of RyR1/RyR2 reduced the interaction of BKCa channel α and β1 subunits in uterine arteries. (A) Representative immunoblots from five replicates show co-immunoprecipitation of BKCa channel α and β1 subunits in uterine arteries of non-pregnant (NUA) and pregnant (PUA) sheep. Uterine arteries were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively, for 48 h. IgG was used as a control to show antibody specificity. (B) Representative confocal immunofluorescence images from five replicates show the co-localization of BKCa channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNAs or RyR siRNAs treatments. The arteries were stained with antibodies against α (green) and β1 (red) subunits. Merged images show in yellow. The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (C) Representative immunoblots from five replicates show co-immunoprecipitation of BKCa channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNA or RyR siRNAs treatments. β-Actin blots showing equal total protein lysates (input).
    Figure Legend Snippet: Knockdown of RyR1/RyR2 reduced the interaction of BKCa channel α and β1 subunits in uterine arteries. (A) Representative immunoblots from five replicates show co-immunoprecipitation of BKCa channel α and β1 subunits in uterine arteries of non-pregnant (NUA) and pregnant (PUA) sheep. Uterine arteries were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively, for 48 h. IgG was used as a control to show antibody specificity. (B) Representative confocal immunofluorescence images from five replicates show the co-localization of BKCa channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNAs or RyR siRNAs treatments. The arteries were stained with antibodies against α (green) and β1 (red) subunits. Merged images show in yellow. The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (C) Representative immunoblots from five replicates show co-immunoprecipitation of BKCa channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNA or RyR siRNAs treatments. β-Actin blots showing equal total protein lysates (input).

    Techniques Used: Western Blot, Immunoprecipitation, Immunofluorescence, Staining

    Knockdown of RyR1/RyR2 decreased Ca2+ sparks in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively. Ca2+ sparks in uterine arteries were measured 48 h later. (A) Representative line-scan images of Fluo-4AM loaded uterine arteries showing Ca2+ sparks recorded before and after the sequential application of 30 mmol/L K+ (30 K) following siRNA treatments. (B) Percentage of Ca2+ spark-firing vascular smooth muscle cells. (C) Ca2+ spark frequency. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, RyR siRNAs vs. control siRNA.
    Figure Legend Snippet: Knockdown of RyR1/RyR2 decreased Ca2+ sparks in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively. Ca2+ sparks in uterine arteries were measured 48 h later. (A) Representative line-scan images of Fluo-4AM loaded uterine arteries showing Ca2+ sparks recorded before and after the sequential application of 30 mmol/L K+ (30 K) following siRNA treatments. (B) Percentage of Ca2+ spark-firing vascular smooth muscle cells. (C) Ca2+ spark frequency. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, RyR siRNAs vs. control siRNA.

    Techniques Used:

    Knockdown of RyR1/RyR2 suppressed STOCs in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNAs or siRNAs for RyR1, RyR2, and RyR3, respectively. STOCs were measured in uterine artery vascular smooth muscle cells 48 hours later. (A) Representative traces showing STOCs at holding potentials of 10 mV in uterine artery vascular smooth muscle cells. (B) STOC frequency. (C) STOC amplitude. Data are means ± SEM from five animals of each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, RyR siRNAs vs. control siRNA.
    Figure Legend Snippet: Knockdown of RyR1/RyR2 suppressed STOCs in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNAs or siRNAs for RyR1, RyR2, and RyR3, respectively. STOCs were measured in uterine artery vascular smooth muscle cells 48 hours later. (A) Representative traces showing STOCs at holding potentials of 10 mV in uterine artery vascular smooth muscle cells. (B) STOC frequency. (C) STOC amplitude. Data are means ± SEM from five animals of each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, RyR siRNAs vs. control siRNA.

    Techniques Used:

    Knockdown of RyR1/RyR2 increased myogenic tone in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNAs or siRNAs for RyR1, RyR2, and RyR3, respectively. Pressure-dependent myogenic tone was measured in uterine arteries 48 h later. (A–C) Changes of lumen diameters of uterine arteries in response to increases in intravascular pressure. (D) Data summary showing the percentage myogenic tone in RyR siRNA-treated uterine arteries. Data are means ± SEM from five animals of each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, RyR siRNAs vs. untreated control or control siRNA.
    Figure Legend Snippet: Knockdown of RyR1/RyR2 increased myogenic tone in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNAs or siRNAs for RyR1, RyR2, and RyR3, respectively. Pressure-dependent myogenic tone was measured in uterine arteries 48 h later. (A–C) Changes of lumen diameters of uterine arteries in response to increases in intravascular pressure. (D) Data summary showing the percentage myogenic tone in RyR siRNA-treated uterine arteries. Data are means ± SEM from five animals of each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, RyR siRNAs vs. untreated control or control siRNA.

    Techniques Used:

    Knockdown of RyR1/RyR2 downregulated the expression of BKCa channel β1 subunit in uterine arteries of pregnant animals. Uterine arteries of pregnant sheep were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively. Protein abundance of BKCa channel α and β1 subunits was measured by western blot in uterine arteries 48 h after the treatment. (A) RyR1 siRNAs treatment. (B) RyR2 siRNAs treatment. (C) RyR3 siRNAs treatment. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, RyR siRNAs vs. control siRNA.
    Figure Legend Snippet: Knockdown of RyR1/RyR2 downregulated the expression of BKCa channel β1 subunit in uterine arteries of pregnant animals. Uterine arteries of pregnant sheep were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively. Protein abundance of BKCa channel α and β1 subunits was measured by western blot in uterine arteries 48 h after the treatment. (A) RyR1 siRNAs treatment. (B) RyR2 siRNAs treatment. (C) RyR3 siRNAs treatment. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, RyR siRNAs vs. control siRNA.

    Techniques Used: Expressing, Western Blot

    rabbit polyclonal ryr2  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit polyclonal ryr2
    Pregnancy increased co-localization of <t>RyR1/RyR2</t> with BKCa channel in uterine arteries. (A) Representative confocal immunofluorescence images from five replicates show the co-localization of RyRs and BKCa channels in uterine arteries. Uterine arteries of non-pregnant (NUA) and pregnant (PUA) animals were stained with antibodies against the β1 subunit of BKCa channel (BKCa β1, red) and <t>RyR1,</t> <t>RyR2,</t> or <t>RyR3</t> (green). Merged images show the co-localization of RyR1 or RyR2 with BKCa β1 (in yellow). The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (B) PLA assay to confirm the co-localization of RyR1/RyR2 and BKCa β1 in uterine arteries. (C) Quantification of the PLA signals. Images from five independent replicates were analysed. The nuclear region was stained with DAPI and shown in blue. Scale bar: 50 µm. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, PUA vs. NUA.
    Rabbit Polyclonal Ryr2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal ryr2/product/Alomone Labs
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    Images

    1) Product Images from "Ryanodine receptor subtypes regulate Ca 2+ sparks/spontaneous transient outward currents and myogenic tone of uterine arteries in pregnancy"

    Article Title: Ryanodine receptor subtypes regulate Ca 2+ sparks/spontaneous transient outward currents and myogenic tone of uterine arteries in pregnancy

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvaa089

    Pregnancy increased co-localization of RyR1/RyR2 with BKCa channel in uterine arteries. (A) Representative confocal immunofluorescence images from five replicates show the co-localization of RyRs and BKCa channels in uterine arteries. Uterine arteries of non-pregnant (NUA) and pregnant (PUA) animals were stained with antibodies against the β1 subunit of BKCa channel (BKCa β1, red) and RyR1, RyR2, or RyR3 (green). Merged images show the co-localization of RyR1 or RyR2 with BKCa β1 (in yellow). The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (B) PLA assay to confirm the co-localization of RyR1/RyR2 and BKCa β1 in uterine arteries. (C) Quantification of the PLA signals. Images from five independent replicates were analysed. The nuclear region was stained with DAPI and shown in blue. Scale bar: 50 µm. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, PUA vs. NUA.
    Figure Legend Snippet: Pregnancy increased co-localization of RyR1/RyR2 with BKCa channel in uterine arteries. (A) Representative confocal immunofluorescence images from five replicates show the co-localization of RyRs and BKCa channels in uterine arteries. Uterine arteries of non-pregnant (NUA) and pregnant (PUA) animals were stained with antibodies against the β1 subunit of BKCa channel (BKCa β1, red) and RyR1, RyR2, or RyR3 (green). Merged images show the co-localization of RyR1 or RyR2 with BKCa β1 (in yellow). The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (B) PLA assay to confirm the co-localization of RyR1/RyR2 and BKCa β1 in uterine arteries. (C) Quantification of the PLA signals. Images from five independent replicates were analysed. The nuclear region was stained with DAPI and shown in blue. Scale bar: 50 µm. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, PUA vs. NUA.

    Techniques Used: Immunofluorescence, Staining

    Knockdown of RyR1/RyR2 reduced the interaction of BKCa channel α and β1 subunits in uterine arteries. (A) Representative immunoblots from five replicates show co-immunoprecipitation of BKCa channel α and β1 subunits in uterine arteries of non-pregnant (NUA) and pregnant (PUA) sheep. Uterine arteries were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively, for 48 h. IgG was used as a control to show antibody specificity. (B) Representative confocal immunofluorescence images from five replicates show the co-localization of BKCa channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNAs or RyR siRNAs treatments. The arteries were stained with antibodies against α (green) and β1 (red) subunits. Merged images show in yellow. The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (C) Representative immunoblots from five replicates show co-immunoprecipitation of BKCa channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNA or RyR siRNAs treatments. β-Actin blots showing equal total protein lysates (input).
    Figure Legend Snippet: Knockdown of RyR1/RyR2 reduced the interaction of BKCa channel α and β1 subunits in uterine arteries. (A) Representative immunoblots from five replicates show co-immunoprecipitation of BKCa channel α and β1 subunits in uterine arteries of non-pregnant (NUA) and pregnant (PUA) sheep. Uterine arteries were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively, for 48 h. IgG was used as a control to show antibody specificity. (B) Representative confocal immunofluorescence images from five replicates show the co-localization of BKCa channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNAs or RyR siRNAs treatments. The arteries were stained with antibodies against α (green) and β1 (red) subunits. Merged images show in yellow. The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (C) Representative immunoblots from five replicates show co-immunoprecipitation of BKCa channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNA or RyR siRNAs treatments. β-Actin blots showing equal total protein lysates (input).

    Techniques Used: Western Blot, Immunoprecipitation, Immunofluorescence, Staining

    Knockdown of RyR1/RyR2 decreased Ca2+ sparks in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively. Ca2+ sparks in uterine arteries were measured 48 h later. (A) Representative line-scan images of Fluo-4AM loaded uterine arteries showing Ca2+ sparks recorded before and after the sequential application of 30 mmol/L K+ (30 K) following siRNA treatments. (B) Percentage of Ca2+ spark-firing vascular smooth muscle cells. (C) Ca2+ spark frequency. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, RyR siRNAs vs. control siRNA.
    Figure Legend Snippet: Knockdown of RyR1/RyR2 decreased Ca2+ sparks in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively. Ca2+ sparks in uterine arteries were measured 48 h later. (A) Representative line-scan images of Fluo-4AM loaded uterine arteries showing Ca2+ sparks recorded before and after the sequential application of 30 mmol/L K+ (30 K) following siRNA treatments. (B) Percentage of Ca2+ spark-firing vascular smooth muscle cells. (C) Ca2+ spark frequency. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, RyR siRNAs vs. control siRNA.

    Techniques Used:

    Knockdown of RyR1/RyR2 suppressed STOCs in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNAs or siRNAs for RyR1, RyR2, and RyR3, respectively. STOCs were measured in uterine artery vascular smooth muscle cells 48 hours later. (A) Representative traces showing STOCs at holding potentials of 10 mV in uterine artery vascular smooth muscle cells. (B) STOC frequency. (C) STOC amplitude. Data are means ± SEM from five animals of each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, RyR siRNAs vs. control siRNA.
    Figure Legend Snippet: Knockdown of RyR1/RyR2 suppressed STOCs in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNAs or siRNAs for RyR1, RyR2, and RyR3, respectively. STOCs were measured in uterine artery vascular smooth muscle cells 48 hours later. (A) Representative traces showing STOCs at holding potentials of 10 mV in uterine artery vascular smooth muscle cells. (B) STOC frequency. (C) STOC amplitude. Data are means ± SEM from five animals of each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, RyR siRNAs vs. control siRNA.

    Techniques Used:

    Knockdown of RyR1/RyR2 increased myogenic tone in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNAs or siRNAs for RyR1, RyR2, and RyR3, respectively. Pressure-dependent myogenic tone was measured in uterine arteries 48 h later. (A–C) Changes of lumen diameters of uterine arteries in response to increases in intravascular pressure. (D) Data summary showing the percentage myogenic tone in RyR siRNA-treated uterine arteries. Data are means ± SEM from five animals of each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, RyR siRNAs vs. untreated control or control siRNA.
    Figure Legend Snippet: Knockdown of RyR1/RyR2 increased myogenic tone in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNAs or siRNAs for RyR1, RyR2, and RyR3, respectively. Pressure-dependent myogenic tone was measured in uterine arteries 48 h later. (A–C) Changes of lumen diameters of uterine arteries in response to increases in intravascular pressure. (D) Data summary showing the percentage myogenic tone in RyR siRNA-treated uterine arteries. Data are means ± SEM from five animals of each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, RyR siRNAs vs. untreated control or control siRNA.

    Techniques Used:

    Knockdown of RyR1/RyR2 downregulated the expression of BKCa channel β1 subunit in uterine arteries of pregnant animals. Uterine arteries of pregnant sheep were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively. Protein abundance of BKCa channel α and β1 subunits was measured by western blot in uterine arteries 48 h after the treatment. (A) RyR1 siRNAs treatment. (B) RyR2 siRNAs treatment. (C) RyR3 siRNAs treatment. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, RyR siRNAs vs. control siRNA.
    Figure Legend Snippet: Knockdown of RyR1/RyR2 downregulated the expression of BKCa channel β1 subunit in uterine arteries of pregnant animals. Uterine arteries of pregnant sheep were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively. Protein abundance of BKCa channel α and β1 subunits was measured by western blot in uterine arteries 48 h after the treatment. (A) RyR1 siRNAs treatment. (B) RyR2 siRNAs treatment. (C) RyR3 siRNAs treatment. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, RyR siRNAs vs. control siRNA.

    Techniques Used: Expressing, Western Blot

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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: 93/100, 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
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    86
    Alomone Labs rabbit anti ryanodine receptor 2 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 Anti Ryanodine Receptor 2 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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    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: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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, 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

    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