ryr2  (Alomone Labs)


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    Structured Review

    Alomone Labs ryr2
    Super-resolution imaging demonstrates nanoscale colocalization of TRPML1 and <t>RyR2</t> in native SMCs. A–C) Representative super-resolution localization maps of native, contractile cerebral artery SMCs co-immunolabeled for Lamp-1 and TRPML1 (A), Lamp-1 and RyR2 (B), or TRPML1 and RyR2 (C). Scale bars = 3 μm. Representative of n= 8–10 cells isolated from N = 3 animals. The second column of images shows a magnified view of the region enclosed in the white boxes. Scale bars = 1 μm. Insets show magnified views of the indicated regions of interest. Scale bars = 0.1 μm. D and E) Histograms showing the distribution of the surface areas of individual protein clusters for TRPML1 (D) and RyR2 (E) (TRPML1, n = 6143 clusters; RyR2, n = 35432 clusters). F) TRPML1 and RyR2 protein cluster density (n = 19 cells from N = 6 animals/group; *P ≤ 0.05) G) Histogram showing the area distribution of Lamp-1–positive LELs (n = 216 ovoids). H) Nearest neighbor analysis showing the distance between the center of RyR2 protein clusters and the edge of Lamp-1–positive LELs (n = 1409 RyR2 protein clusters). I) Object-based analysis comparing the fraction of TRPML1 and RyR2 co-localizing clusters with the fraction of clusters that co-localize in a simulated random distribution of RyR2 protein clusters (TRPML1-RyR2, 1.51 ± 0.12 %; Random, 0.44 ± 0.07 %; n = 10 cells from 3 animals; *P ≤ 0.05). All data are shown as mean ± SEM.
    Ryr2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 2 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 2 article reviews
    Price from $9.99 to $1999.99
    ryr2 - by Bioz Stars, 2022-12
    94/100 stars

    Images

    1) Product Images from "TRPML1 Channels Initiate Ca2+ Sparks in Vascular Smooth Muscle Cells"

    Article Title: TRPML1 Channels Initiate Ca2+ Sparks in Vascular Smooth Muscle Cells

    Journal: Science signaling

    doi: 10.1126/scisignal.aba1015

    Super-resolution imaging demonstrates nanoscale colocalization of TRPML1 and RyR2 in native SMCs. A–C) Representative super-resolution localization maps of native, contractile cerebral artery SMCs co-immunolabeled for Lamp-1 and TRPML1 (A), Lamp-1 and RyR2 (B), or TRPML1 and RyR2 (C). Scale bars = 3 μm. Representative of n= 8–10 cells isolated from N = 3 animals. The second column of images shows a magnified view of the region enclosed in the white boxes. Scale bars = 1 μm. Insets show magnified views of the indicated regions of interest. Scale bars = 0.1 μm. D and E) Histograms showing the distribution of the surface areas of individual protein clusters for TRPML1 (D) and RyR2 (E) (TRPML1, n = 6143 clusters; RyR2, n = 35432 clusters). F) TRPML1 and RyR2 protein cluster density (n = 19 cells from N = 6 animals/group; *P ≤ 0.05) G) Histogram showing the area distribution of Lamp-1–positive LELs (n = 216 ovoids). H) Nearest neighbor analysis showing the distance between the center of RyR2 protein clusters and the edge of Lamp-1–positive LELs (n = 1409 RyR2 protein clusters). I) Object-based analysis comparing the fraction of TRPML1 and RyR2 co-localizing clusters with the fraction of clusters that co-localize in a simulated random distribution of RyR2 protein clusters (TRPML1-RyR2, 1.51 ± 0.12 %; Random, 0.44 ± 0.07 %; n = 10 cells from 3 animals; *P ≤ 0.05). All data are shown as mean ± SEM.
    Figure Legend Snippet: Super-resolution imaging demonstrates nanoscale colocalization of TRPML1 and RyR2 in native SMCs. A–C) Representative super-resolution localization maps of native, contractile cerebral artery SMCs co-immunolabeled for Lamp-1 and TRPML1 (A), Lamp-1 and RyR2 (B), or TRPML1 and RyR2 (C). Scale bars = 3 μm. Representative of n= 8–10 cells isolated from N = 3 animals. The second column of images shows a magnified view of the region enclosed in the white boxes. Scale bars = 1 μm. Insets show magnified views of the indicated regions of interest. Scale bars = 0.1 μm. D and E) Histograms showing the distribution of the surface areas of individual protein clusters for TRPML1 (D) and RyR2 (E) (TRPML1, n = 6143 clusters; RyR2, n = 35432 clusters). F) TRPML1 and RyR2 protein cluster density (n = 19 cells from N = 6 animals/group; *P ≤ 0.05) G) Histogram showing the area distribution of Lamp-1–positive LELs (n = 216 ovoids). H) Nearest neighbor analysis showing the distance between the center of RyR2 protein clusters and the edge of Lamp-1–positive LELs (n = 1409 RyR2 protein clusters). I) Object-based analysis comparing the fraction of TRPML1 and RyR2 co-localizing clusters with the fraction of clusters that co-localize in a simulated random distribution of RyR2 protein clusters (TRPML1-RyR2, 1.51 ± 0.12 %; Random, 0.44 ± 0.07 %; n = 10 cells from 3 animals; *P ≤ 0.05). All data are shown as mean ± SEM.

    Techniques Used: Imaging, Immunolabeling, Isolation

    2) Product Images from "Simultaneous recordings of action potentials and calcium transients from human induced pluripotent stem cell derived cardiomyocytes"

    Article Title: Simultaneous recordings of action potentials and calcium transients from human induced pluripotent stem cell derived cardiomyocytes

    Journal: Biology Open

    doi: 10.1242/bio.035030

    Representative immunofluorescence images of hiPSC-CMs showing cardiac proteins. (Upper) 50 slices were combined from confocal immunofluorescence images of nucleus (DAPI, blue), cardiac troponin T (cTnT, green) and cardiac ryanodine receptors (RyR2, red). Final panel shows the former three merged. (Lower) 40 slices were combined from confocal immunoflurescence images of nucleus (DAPI, blue), cTnT (green), L-type calcium channels (Ca V 1.2, red). Final panel shows the former three merged. Scale bars: 10 µM.
    Figure Legend Snippet: Representative immunofluorescence images of hiPSC-CMs showing cardiac proteins. (Upper) 50 slices were combined from confocal immunofluorescence images of nucleus (DAPI, blue), cardiac troponin T (cTnT, green) and cardiac ryanodine receptors (RyR2, red). Final panel shows the former three merged. (Lower) 40 slices were combined from confocal immunoflurescence images of nucleus (DAPI, blue), cTnT (green), L-type calcium channels (Ca V 1.2, red). Final panel shows the former three merged. Scale bars: 10 µM.

    Techniques Used: Immunofluorescence

    3) Product Images from "Simultaneous recordings of action potentials and calcium transients from human induced pluripotent stem cell derived cardiomyocytes"

    Article Title: Simultaneous recordings of action potentials and calcium transients from human induced pluripotent stem cell derived cardiomyocytes

    Journal: Biology Open

    doi: 10.1242/bio.035030

    Representative immunofluorescence images of hiPSC-CMs showing cardiac proteins. (Upper) 50 slices were combined from confocal immunofluorescence images of nucleus (DAPI, blue), cardiac troponin T (cTnT, green) and cardiac ryanodine receptors (RyR2, red). Final panel shows the former three merged. (Lower) 40 slices were combined from confocal immunoflurescence images of nucleus (DAPI, blue), cTnT (green), L-type calcium channels (Ca V 1.2, red). Final panel shows the former three merged. Scale bars: 10 µM.
    Figure Legend Snippet: Representative immunofluorescence images of hiPSC-CMs showing cardiac proteins. (Upper) 50 slices were combined from confocal immunofluorescence images of nucleus (DAPI, blue), cardiac troponin T (cTnT, green) and cardiac ryanodine receptors (RyR2, red). Final panel shows the former three merged. (Lower) 40 slices were combined from confocal immunoflurescence images of nucleus (DAPI, blue), cTnT (green), L-type calcium channels (Ca V 1.2, red). Final panel shows the former three merged. Scale bars: 10 µM.

    Techniques Used: Immunofluorescence

    4) Product Images from "Simultaneous recordings of action potentials and calcium transients from human induced pluripotent stem cell derived cardiomyocytes"

    Article Title: Simultaneous recordings of action potentials and calcium transients from human induced pluripotent stem cell derived cardiomyocytes

    Journal: Biology Open

    doi: 10.1242/bio.035030

    Representative immunofluorescence images of hiPSC-CMs showing cardiac proteins. (Upper) 50 slices were combined from confocal immunofluorescence images of nucleus (DAPI, blue), cardiac troponin T (cTnT, green) and cardiac ryanodine receptors (RyR2, red). Final panel shows the former three merged. (Lower) 40 slices were combined from confocal immunoflurescence images of nucleus (DAPI, blue), cTnT (green), L-type calcium channels (Ca V 1.2, red). Final panel shows the former three merged. Scale bars: 10 µM.
    Figure Legend Snippet: Representative immunofluorescence images of hiPSC-CMs showing cardiac proteins. (Upper) 50 slices were combined from confocal immunofluorescence images of nucleus (DAPI, blue), cardiac troponin T (cTnT, green) and cardiac ryanodine receptors (RyR2, red). Final panel shows the former three merged. (Lower) 40 slices were combined from confocal immunoflurescence images of nucleus (DAPI, blue), cTnT (green), L-type calcium channels (Ca V 1.2, red). Final panel shows the former three merged. Scale bars: 10 µM.

    Techniques Used: Immunofluorescence

    5) Product Images from "Upregulation of the CaV 1.1-ryanodine receptor complex in a rat model of critical illness myopathy"

    Article Title: Upregulation of the CaV 1.1-ryanodine receptor complex in a rat model of critical illness myopathy

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    doi: 10.1152/ajpregu.00032.2011

    Fibers with elevated RYR1 contain fast myosin. Shown is a field from a tibialis anterior muscle double labeled for myosin II and RYR1. All 3 fibers in the field are positive for fast myosin. In the lower center of the field is a small fiber that stains
    Figure Legend Snippet: Fibers with elevated RYR1 contain fast myosin. Shown is a field from a tibialis anterior muscle double labeled for myosin II and RYR1. All 3 fibers in the field are positive for fast myosin. In the lower center of the field is a small fiber that stains

    Techniques Used: Labeling

    Marked elevation of RYR1 in a subset of fibers. A : RYR1 staining in 2 fibers from the tibialis anterior muscle in a control rat. The staining is well organized into parallel stripes running perpendicular to the length of the fiber. B : RYR1 staining in
    Figure Legend Snippet: Marked elevation of RYR1 in a subset of fibers. A : RYR1 staining in 2 fibers from the tibialis anterior muscle in a control rat. The staining is well organized into parallel stripes running perpendicular to the length of the fiber. B : RYR1 staining in

    Techniques Used: Staining

    Expression of the ryanodine receptor (RYR) increases in critical illness myopathy (CIM).  A : skeletal muscle membranes prepared from individual control (Con) or CIM animals were analyzed in Western blot analysis using a monoclonal antibody to RYR. There
    Figure Legend Snippet: Expression of the ryanodine receptor (RYR) increases in critical illness myopathy (CIM). A : skeletal muscle membranes prepared from individual control (Con) or CIM animals were analyzed in Western blot analysis using a monoclonal antibody to RYR. There

    Techniques Used: Expressing, Western Blot

    Voltage-gated calcium channel type 1.1 (Ca V 1.1) and RYR1 are upregulated in the same fibers in CIM. Two fields from an individual tibialis anterior muscle double labeled for Ca V 1.1 and RYR1 are shown. In the field at the top , a severely atrophied muscle
    Figure Legend Snippet: Voltage-gated calcium channel type 1.1 (Ca V 1.1) and RYR1 are upregulated in the same fibers in CIM. Two fields from an individual tibialis anterior muscle double labeled for Ca V 1.1 and RYR1 are shown. In the field at the top , a severely atrophied muscle

    Techniques Used: Labeling

    Calpain II is elevated in fibers with elevated RYR1. Shown is a field from a tibialis anterior muscle double labeled for calpain II and RYR1. In the field are two atrophied fibers that have elevated levels of both calpain II and RYR1. The normal fibers
    Figure Legend Snippet: Calpain II is elevated in fibers with elevated RYR1. Shown is a field from a tibialis anterior muscle double labeled for calpain II and RYR1. In the field are two atrophied fibers that have elevated levels of both calpain II and RYR1. The normal fibers

    Techniques Used: Labeling

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    Alomone Labs anti ryanodine receptor 2 antibody
    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 <t>antibodies</t> 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
    Anti Ryanodine Receptor 2 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ryanodine receptor 2 antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ryanodine receptor 2 antibody - by Bioz Stars, 2022-12
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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

    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

    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: Mouse Assay, Injection, Staining, Western Blot

    CCN5 enhances exercise performance of mdx/utrn (±) mice. (A) Experimental scheme for panel (B) . Mice were injected with AAV.9-Con or CCN5 into the tail vein, and treadmill tests were performed 8 weeks later. Warming up exercises were serially performed at 4 m/min for 2 min and 8 m/min for 2 min. Main exercise was performed at 12 m/min until the mice were exhausted. (B) Total running distance was measured by calculating running time and velocity. (C) Comparison of grip strength tests was performed after AAV.9-Con or CCN5 injection. (D) Proteins from skeletal muscle tissue were immunoblotted with antibodies against CCN5, utrophin, α-SMA and GAPDH. (E) Protein bands on western blots were scanned and plotted. n = 6. ** p

    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 enhances exercise performance of mdx/utrn (±) mice. (A) Experimental scheme for panel (B) . Mice were injected with AAV.9-Con or CCN5 into the tail vein, and treadmill tests were performed 8 weeks later. Warming up exercises were serially performed at 4 m/min for 2 min and 8 m/min for 2 min. Main exercise was performed at 12 m/min until the mice were exhausted. (B) Total running distance was measured by calculating running time and velocity. (C) Comparison of grip strength tests was performed after AAV.9-Con or CCN5 injection. (D) Proteins from skeletal muscle tissue were immunoblotted with antibodies against CCN5, utrophin, α-SMA and GAPDH. (E) Protein bands on western blots were scanned and plotted. n = 6. ** p

    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: Mouse Assay, Injection, Western Blot

    CCN5 directly regulates utrophin expression. (A) Proteins obtained from cardiac tissues were immunoblotted with antibodies against CCN5, dystrophin, utrophin and GAPDH. (B) Protein bands on western blots were scanned and plotted. (C) Relative mRNA expression level of utrophin in hearts. (D) Utrophin (UTRN) promoter only, UTRN promoter + GABPα, and UTRN promoter + CCN5 were transfected to 293T cells. Luciferase activity was measured 72 h later. Luciferase activity was analyzed by GloMax Microplate Reader. n = 6 for western blots. n = 4 for luciferase assay. * p

    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 directly regulates utrophin expression. (A) Proteins obtained from cardiac tissues were immunoblotted with antibodies against CCN5, dystrophin, utrophin and GAPDH. (B) Protein bands on western blots were scanned and plotted. (C) Relative mRNA expression level of utrophin in hearts. (D) Utrophin (UTRN) promoter only, UTRN promoter + GABPα, and UTRN promoter + CCN5 were transfected to 293T cells. Luciferase activity was measured 72 h later. Luciferase activity was analyzed by GloMax Microplate Reader. n = 6 for western blots. n = 4 for luciferase assay. * p

    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: Expressing, Western Blot, Transfection, Luciferase, Activity Assay

    Orai1 blockade by Synta66 does not affect STOC amplitude or frequency in cerebral artery SMCs. ( A ) mRNA expression levels (transcript copies/ng of RNA) of Kcnma1 (BKα1), Kcnmb1 (BKβ1), and Ryr2 (RyR2) in cerebral arteries from control and Stim1 -smKO mice. Data are presented as means ± SEM (n = 3 mice in each group, unpaired t -test). ns, not significant. ( B ) Representative trace of spontaneous transient outward currents (STOCs) recorded from a vascular smooth muscle cell (VSMC) isolated from a control mouse before and after the selective Orai1 blocker Synta66 (10 µM) was applied. ( C, D ) Summary data showing STOC amplitude ( C ) and frequency ( D ) in the absence (control) and presence of Synta66 (10 µM) (n = 7 cells from four animals, unpaired t -test). ns, not significant. Individual data points and analysis summaries for datasets shown in Figure 7—figure supplement 1 .

    Journal: eLife

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

    doi: 10.7554/eLife.70278

    Figure Lengend Snippet: Orai1 blockade by Synta66 does not affect STOC amplitude or frequency in cerebral artery SMCs. ( A ) mRNA expression levels (transcript copies/ng of RNA) of Kcnma1 (BKα1), Kcnmb1 (BKβ1), and Ryr2 (RyR2) in cerebral arteries from control and Stim1 -smKO mice. Data are presented as means ± SEM (n = 3 mice in each group, unpaired t -test). ns, not significant. ( B ) Representative trace of spontaneous transient outward currents (STOCs) recorded from a vascular smooth muscle cell (VSMC) isolated from a control mouse before and after the selective Orai1 blocker Synta66 (10 µM) was applied. ( C, D ) Summary data showing STOC amplitude ( C ) and frequency ( D ) in the absence (control) and presence of Synta66 (10 µM) (n = 7 cells from four animals, unpaired t -test). ns, not significant. Individual data points and analysis summaries for datasets shown in Figure 7—figure supplement 1 .

    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-IP3R- (ab5804), Abcam, Cambridge, UK) diluted in PBS containing 20% SEABLOCK, 1% BSA, and 0.05% Triton X-100.

    Techniques: Expressing, Mouse Assay, Isolation

    Stim1 knockout decreases colocalization of BK and RyR2 protein clusters. ( A ) Epifluorescence-mode superresolution localization maps of freshly isolated vascular smooth muscle cells (VSMCs) from control and Stim1- smKO mice immunolabeled for BK (red) and RyR2 (green). Colocalized BK and RyR2 clusters were identified by object-based analysis (OBA) and mapped (cyan). Scale bar: 3 µm. Panels to the right show enlarged areas of the original superresolution maps indicated by the white boxes. Scale bar: 500 nm. ( B ) Summary data showing the density (clusters per unit area), frequency distribution of sizes, and mean size of BK channel clusters. ( C ) Summary data showing the density, frequency distribution of sizes, and mean size of RyR2 clusters. ( D ) Summary data showing the density, frequency distribution of sizes, and mean size of colocalizing BK and RyR2 clusters, identified using OBA. For density data, n = 20 cells from three mice for controls and n = 18 cells from three mice for Stim1- smKO mice. For frequency distribution and mean cluster size data: control, n = 44,340 BK channel clusters, n = 15,193 RyR2 clusters, and n = 1054 colocalizing clusters; Stim1-smKO: n = 30,552 BK channel clusters, n = 9702 RyR2 clusters, and n = 547 colocalizing clusters (*p

    Journal: eLife

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

    doi: 10.7554/eLife.70278

    Figure Lengend Snippet: Stim1 knockout decreases colocalization of BK and RyR2 protein clusters. ( A ) Epifluorescence-mode superresolution localization maps of freshly isolated vascular smooth muscle cells (VSMCs) from control and Stim1- smKO mice immunolabeled for BK (red) and RyR2 (green). Colocalized BK and RyR2 clusters were identified by object-based analysis (OBA) and mapped (cyan). Scale bar: 3 µm. Panels to the right show enlarged areas of the original superresolution maps indicated by the white boxes. Scale bar: 500 nm. ( B ) Summary data showing the density (clusters per unit area), frequency distribution of sizes, and mean size of BK channel clusters. ( C ) Summary data showing the density, frequency distribution of sizes, and mean size of RyR2 clusters. ( D ) Summary data showing the density, frequency distribution of sizes, and mean size of colocalizing BK and RyR2 clusters, identified using OBA. For density data, n = 20 cells from three mice for controls and n = 18 cells from three mice for Stim1- smKO mice. For frequency distribution and mean cluster size data: control, n = 44,340 BK channel clusters, n = 15,193 RyR2 clusters, and n = 1054 colocalizing clusters; Stim1-smKO: n = 30,552 BK channel clusters, n = 9702 RyR2 clusters, and n = 547 colocalizing clusters (*p

    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-IP3R- (ab5804), Abcam, Cambridge, UK) diluted in PBS containing 20% SEABLOCK, 1% BSA, and 0.05% Triton X-100.

    Techniques: Knock-Out, Isolation, Mouse Assay, Immunolabeling

    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 

    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 

    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