ryr1 Search Results


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  • 95
    Thermo Fisher ryr
    Acute ablation of both cardiac Mfn1 and Mfn2 reduces the interaction between the mitochondria and SR. ( a ) 2D confocal images showing the points of interaction between mitochondria and the SR in myocytes isolated from WT and DKO hearts, as visualized by the red dots, the nucleus is visualized in blue. Magnified insets show the zone of a line-scan fluorescence profile analysis (white arrow), plotted in the right. ( b ) Whole-cell 3D rendering from multiple z-stack images, compiling the points of interaction between the mitochondria and the SR in myocytes isolated from WT and DKO hearts, as visualized in gold, with the nucleus visualized in blue. ( c ) Quantification of the whole-cell 3D interactions visualized between the mitochondria and the SR. Antibodies to <t>VDAC</t> and the ryanodine receptor <t>(Pan-RyR)</t> were used as to mark the mitochondria and SR, respectively, prior to the proximal ligation assay. N =80 cells from three heart isolations. Error bars indicate S.E.M. and statistical analysis was performed by an unpaired t -test. * P ≤0.05. ( d ) Representative immunocytochemistry images of myocytes isolated from WT and DKO hearts stained with antibodies for VDAC (green) and Pan-RyR (red). ( e ) Representative western blot image of VDAC and RyR expression in whole heart homogenate taken from WT and DKO mice
    Ryr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 179 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore rabbit anti ryr1
    Future subplate genes are expressed in RELN-negative preplate and subplate neurons. ( a ) Genes expressed in future subplate neurons categorized by function. ( b – h ) Double immunofluorescent staining for RELN (red) and marker proteins for future subplate candidate genes (green) in coronal cryostat sections of wild-type E12.5 ( b , c ) and E14.5 ( c – h ) brains. Hpca ( c , e ) and EAAC1 ( b , d ) are expressed in RELN-negative preplate cells and in subplate cell bodies. At E14.5, SRF ( f ) and <t>RyR1</t> ( h ) are expressed in RELN-positive marginal zone neurons, as well as in intermediate zone fibers originating in the subplate, whereas Sez6 -driven EGFP ( g ) is expressed in subplate and cortical plate neurons, but not in RELN-positive cells. Scale bars = 50 μm.
    Rabbit Anti Ryr1, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher ryr1
    Splicing patterns of Ca 2+ channels and transporters in control and DM1 myotubes at 10 (T10) and 15 (T15) days of differentiation. ( A ) <t>RyR1</t> alternative splicing of fetal ASII(−) and ASII(+) isoforms and schematic representation of the ASII splicing isoforms of RyR1. ( B ) Quantification of RyR ASII(−) as compared to the total transcript. ( C ) SERCA1 alternative splicing of fetal SERCA1b and adult SERCA1a isoforms and schematic representation of the SERCA1 splicing isoforms. ( D ) Quantification of fetal SERCA1b as compared to the total transcript. ( E ) Cav1.1 alternative splicing of fetal Cav1.1Δ29 and adult Cav1.1 isoforms and schematic representation of the Cav1.1 splicing isoforms. ( F ) Quantification of fetal Cav1.1 Δ29 as compared to the total transcript. PCR products were separated in 3% agarose gel by electrophoresis. Data are the mean ± SD. * p
    Ryr1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Developmental Studies Hybridoma Bank ryr1
    Effect of triadin on FKBP12 expression and its binding to <t>RyR1.</t> A , equivalent amounts of crude membrane homogenates from WT and triadin-null myotubes were loaded and immunoblotted for expression of RyR1, DHPR α 1S , CSQ-1, junctin, and FKBP12. Band intensity is expressed as fraction of the WT signal ( dotted line ). Numbers on the bars indicate the number of blots analyzed per protein. Data are presented as mean ± S.E. *, p
    Ryr1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Alomone Labs ryr1
    GST-RTN1 523 inhibits specific [ 3 H]ryanodine binding to rat brain synaptosomes. (A) Equilibrium [ 3 H]ryanodine binding to rat forebrain synaptosomal membrane preparations was carried out in binding buffer containing 10 nM [ 3 H]ryanodine at the indicated free calcium concentrations in control conditions ( closed circles ) and in the presence of 0.1 μM GST ( closed triangles ) or 0.1 μM GST-RTN1 523 ( open squares ). [Ca 2 + ] was maintained, in the range 0.01 μM–1 mM, by a combination of EGTA and CaCl 2 . Free Ca 2 + concentrations were calculated as described in material and methods. Data points shown are the mean ± S.E.M., from three separate experiments performed in triplicates. (B) [ 3 H]ryanodine binding in the presence of 0.1 μM GST or GST-RTN1 523 is presented as percent of control. No specific [ 3 H]ryanodine binding was observed at 0.01 μM Ca 2 + in the presence of GST or GST-RTN1 523 . Difference in [ 3 H]ryanodine binding was plotted as percent decrease in specific binding. Data points shown are the mean ± S.E.M., from three separate experiments (* p = 0.011 by Student's t -test). (C) <t>RyR2</t> evoked Ca 2 + oscillations in HEK293. Upper and lower left panels represent Fura-2 ratio time-courses of single cells expressing RyR2 together with mcherry, mcherry-RTN1A or EGFP-RTN4A. Cells were continuously perfused with buffer containing 0 mM Ca 2 + (nominal free), 1 mM Ca 2 + and 0 mM Ca 2 + + 10 mM caffeine as indicated by the bars at the top. Lower right panel shows a quantitative analysis performed by integration of the respective single peak areas referred to area under curve for estimation of the total amount of the cytosolic [Ca 2 + ] arising through RyR2 dependent Ca 2 + oscillations. The fraction of cells that showed both oscillations as well as a clear caffeine peak in comparison to those that lacked oscillations before a single caffeine peak are given in percentages in the graph. A two-sample t-test was carried out to test for significance as indicated by the p values at the bottom of the panel.
    Ryr1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ryr1  (Abcam)
    92
    Abcam ryr1
    T-tubule and sarcoplasmic reticulum abnormalities in DNM2-S619L larval muscle. (A–D) Electron micrographs of longitudinal sections through zebrafish muscle at 3 dpf. (A,C) Muscle from DNM2-WT larvae shows the typical sarcomere striations of vertebrate striated muscle. (B,D) Muscle from DNM2-S619L larvae demonstrates extensive swelling and vacuolization in the region of the SR and T-tubules. Scale bars: 1 μm. (E-H) Confocal micrographs of isolated myofibers subjected to immunofluorescence analysis. (E) Wild-type (WT) myofiber showing the expected pattern of <t>RyR1</t> staining. (F,G) RyR1 expression is irregular in DNM2-S619L myofibers, and is often found aggregated. (H) α-actinin staining was normal, indicating that other elements of the muscle structure in S619L myofibers are not disturbed.
    Ryr1, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Collaborative Drug Discovery Inc ryr1 gene
    Effect of the pY3016C mutation on <t>RyR1</t> protein expression on patient and control muscle biopsies. The western blot shows a dramatic decrease of the RyR1 protein and of the DHPRalpha 1.1 expression in the patient’s biopsy compared to control’s biopsy (P
    Ryr1 Gene, supplied by Collaborative Drug Discovery Inc, used in various techniques. Bioz Stars score: 88/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Upstate Biotechnology Inc ryr 1
    Immunofluorescence of control, β 1 -null and dysgenic, α 1S -null, myotubes with antibodies to β 1 and α 1S DHPR subunits and <t>RyR-1.</t> ( a and b ) Control myotubes labeled with either anti-β 1 ( a ) or anti-α 1S ( b ). ( c and d ) β 1 -Null myotubes labeled with either anti-β 1 ( c ) or anti-α 1S ( d ). ( e ) Dysgenic myotubes labeled with anti-β show a weak, but punctate pattern (arrows). ( f ) β 1 -Null myotubes labeled with anti-RyR-1 in a punctate pattern (arrows). The diffuse background staining in the myotube may represent RyR-1 that is not membrane localized. (Bar = 20 μM.)
    Ryr 1, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 88/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Collaborative Drug Discovery Inc ryr1 mutations
    <t>Cav1.1-RyR1</t> complex: molecular details . Shown is a reconstruction from cryo-electron microscopy of the mammalian RyR1 at 4.8 Å illustrating the different regions. This includes the large myoplasmic segment and its multiple cavities and processes, and the SR transmembranal segment, which forms the Ca 2+ conduction pathway (Electron microscopy data bank entry 6106, Zalk et al., 2015 ). The above properties allow the RyR1 to interact with multiple SR luminal and cytosolic proteins. Several 3-D structures of proteins interacting with RyR1, as well as their relative locations, are also shown. EMData bank PDB entry and references: calsequestrin-1 (Casq1) polymer (3UOM, Sanchez et al., 2012 ), FKBP-12 (1D6O, Burkhard et al., 2000 ), S100A1 (2K2F; Wright et al., 2008 ), calmodulin (CaM; 1CLL, Chattopadhyaya et al., 1992 ), Cav1.1α subunit (4MS2, Tang et al., 2014 ; Hu et al., 2015 ), and Cav1.1 β subunit (1T0J, Van Petegem et al., 2004 ; Hu et al., 2015 ); only two alpha and two beta subunits are shown. In the muscle fiber Ca v 1.1 channels are clustered in groups of four (or tetrads) in the T-tubules. Ca v 1.1 channels in the tetrad interact with the four subunits of the RyR1, one Cav1.1 per each RyR1 subunit. All structures were generated using UCSF Chimera (Pettersen et al., 2004 ).
    Ryr1 Mutations, supplied by Collaborative Drug Discovery Inc, used in various techniques. Bioz Stars score: 88/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore ryr1
    Confocal laser scanning microscopy demonstrates increased expression levels of IP3R3, <t>RYR1</t> and S100A6 in SH-SY5Y cells following CDDP or TOPO treatment ( A ) Representative fluorescence confocal images of SH-SY5Y cells that endogenously express IP3R1, IP3R3, RYR1, RYR3 and S100A6 as detected by Fluo-488 nm conjugated antibodies (green fluorescence) using images taken in similar experimental set ups following exposure to either 1 μM CDDP or 0.01 μM TOPO for 72 h. Cell nuclei are counterstained with DAPI (blue). scale bar = 20 μm. ( B ) Quantification of fluorescence intensity (protein expression) expressed as mean corrected total cell fluorescence (CTCF) ± standard deviation (SD). Data are derived from three independent biological experiments each. Statistical significance is relative to untreated cells and considered if p
    Ryr1, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti ryr
    Protein phosphorylation with Zn 2+ exposure. Western blots were examined for n = 4 rat hearts for each condition. Representative blots are shown for n = 2 in each condition. A : phosphorylated <t>RyR</t> <t>S2808</t> in rat LV was reduced after exposure to 50 μM
    Anti Ryr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher gene exp ryr1 mm01175211 m1
    mRNA levels of ryanodine receptors. ( A ) <t>Ryr1</t> , <t>Ryr2</t> and <t>Ryr3</t> expression in lysates prepared from whole mammary tissue (including luminal, basal and stromal cells) dissected from virgin or lactating animals (n = 4 mice). ( B ) Krt14 , Esr1 and Ryr1 levels in freshly sorted luminal and basal cells (n = 3 mice). Graphs show mean ± SEM; * P
    Gene Exp Ryr1 Mm01175211 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher gene exp ryr1 hs00166991 m1
    RyR gene expression and function are up-regulated after T cell activation. A , average normalized linearized <t>RyR1</t> ( left ), <t>RyR2</t> ( middle ), and <t>RyR3</t> ( right ) C q values in resting T cells ( R ; n = 9) and 5-day activated ( A ; n = 7) primary human T cells. B , anti-RyR
    Gene Exp Ryr1 Hs00166991 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc ryr1
    Pregnancy upregulates ryanodine receptors in uterine arteries. Ryanodine receptor type 1 <t>(RyR1),</t> type 2 <t>(RyR2)</t> and type 3 <t>(RyR3)</t> mRNA ( A ) and protein ( B ) abundance in uterine arteries of nonpregnant (UA NP ) and pregnant (UA P ) sheep were determined with real-time RT-PCR and Western blotting. Data are means ± SEM from 4–5 animals of each group. *P
    Ryr1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Collaborative Drug Discovery Inc ryr1 related ccd
    Pregnancy upregulates ryanodine receptors in uterine arteries. Ryanodine receptor type 1 <t>(RyR1),</t> type 2 <t>(RyR2)</t> and type 3 <t>(RyR3)</t> mRNA ( A ) and protein ( B ) abundance in uterine arteries of nonpregnant (UA NP ) and pregnant (UA P ) sheep were determined with real-time RT-PCR and Western blotting. Data are means ± SEM from 4–5 animals of each group. *P
    Ryr1 Related Ccd, supplied by Collaborative Drug Discovery Inc, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Alexis Inc ryr1
    BST detection of <t>SNO-RyR1</t> in human skeletal muscle SOL and VL biopsies. A. Upper panel , Immunoblot of BST streptavidin column eluate of SOL for RyR1. A. Lower panel , Immunoblot of BST streptavidin column eluate of VL for RyR1. Increased RyR1 immunoreactive band was detected in CTR End-only samples. B. Percent changes biotin-labeled RyR1 proteins from Pre and End bed rest biopsies. Bed rest without exercise promoted S -nitrosylation of RyR1 in disused SOL (CTR n =9, 18.6%, p ≤0.01) and VL (CTR n =9, 17.2%, p ≤0.01) as counteracted by both exercise countermeasures. (* significance).
    Ryr1, supplied by Alexis Inc, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Acute ablation of both cardiac Mfn1 and Mfn2 reduces the interaction between the mitochondria and SR. ( a ) 2D confocal images showing the points of interaction between mitochondria and the SR in myocytes isolated from WT and DKO hearts, as visualized by the red dots, the nucleus is visualized in blue. Magnified insets show the zone of a line-scan fluorescence profile analysis (white arrow), plotted in the right. ( b ) Whole-cell 3D rendering from multiple z-stack images, compiling the points of interaction between the mitochondria and the SR in myocytes isolated from WT and DKO hearts, as visualized in gold, with the nucleus visualized in blue. ( c ) Quantification of the whole-cell 3D interactions visualized between the mitochondria and the SR. Antibodies to VDAC and the ryanodine receptor (Pan-RyR) were used as to mark the mitochondria and SR, respectively, prior to the proximal ligation assay. N =80 cells from three heart isolations. Error bars indicate S.E.M. and statistical analysis was performed by an unpaired t -test. * P ≤0.05. ( d ) Representative immunocytochemistry images of myocytes isolated from WT and DKO hearts stained with antibodies for VDAC (green) and Pan-RyR (red). ( e ) Representative western blot image of VDAC and RyR expression in whole heart homogenate taken from WT and DKO mice

    Journal: Cell Death & Disease

    Article Title: Hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction

    doi: 10.1038/cddis.2016.139

    Figure Lengend Snippet: Acute ablation of both cardiac Mfn1 and Mfn2 reduces the interaction between the mitochondria and SR. ( a ) 2D confocal images showing the points of interaction between mitochondria and the SR in myocytes isolated from WT and DKO hearts, as visualized by the red dots, the nucleus is visualized in blue. Magnified insets show the zone of a line-scan fluorescence profile analysis (white arrow), plotted in the right. ( b ) Whole-cell 3D rendering from multiple z-stack images, compiling the points of interaction between the mitochondria and the SR in myocytes isolated from WT and DKO hearts, as visualized in gold, with the nucleus visualized in blue. ( c ) Quantification of the whole-cell 3D interactions visualized between the mitochondria and the SR. Antibodies to VDAC and the ryanodine receptor (Pan-RyR) were used as to mark the mitochondria and SR, respectively, prior to the proximal ligation assay. N =80 cells from three heart isolations. Error bars indicate S.E.M. and statistical analysis was performed by an unpaired t -test. * P ≤0.05. ( d ) Representative immunocytochemistry images of myocytes isolated from WT and DKO hearts stained with antibodies for VDAC (green) and Pan-RyR (red). ( e ) Representative western blot image of VDAC and RyR expression in whole heart homogenate taken from WT and DKO mice

    Article Snippet: Primary antibodies for VDAC (#4661, Cell Signalling) and the RyR (Pan-RyR, #MA3-925, Thermo Scientific, Paisley, UK) were incubated overnight (1:500).

    Techniques: Isolation, Fluorescence, Ligation, Immunocytochemistry, Staining, Western Blot, Expressing, Mouse Assay

    Future subplate genes are expressed in RELN-negative preplate and subplate neurons. ( a ) Genes expressed in future subplate neurons categorized by function. ( b – h ) Double immunofluorescent staining for RELN (red) and marker proteins for future subplate candidate genes (green) in coronal cryostat sections of wild-type E12.5 ( b , c ) and E14.5 ( c – h ) brains. Hpca ( c , e ) and EAAC1 ( b , d ) are expressed in RELN-negative preplate cells and in subplate cell bodies. At E14.5, SRF ( f ) and RyR1 ( h ) are expressed in RELN-positive marginal zone neurons, as well as in intermediate zone fibers originating in the subplate, whereas Sez6 -driven EGFP ( g ) is expressed in subplate and cortical plate neurons, but not in RELN-positive cells. Scale bars = 50 μm.

    Journal: Cerebral Cortex (New York, NY)

    Article Title: Gene Expression Profiling of Preplate Neurons Destined for the Subplate: Genes Involved in Transcription, Axon Extension, Neurotransmitter Regulation, Steroid Hormone Signaling, and Neuronal Survival

    doi: 10.1093/cercor/bhp034

    Figure Lengend Snippet: Future subplate genes are expressed in RELN-negative preplate and subplate neurons. ( a ) Genes expressed in future subplate neurons categorized by function. ( b – h ) Double immunofluorescent staining for RELN (red) and marker proteins for future subplate candidate genes (green) in coronal cryostat sections of wild-type E12.5 ( b , c ) and E14.5 ( c – h ) brains. Hpca ( c , e ) and EAAC1 ( b , d ) are expressed in RELN-negative preplate cells and in subplate cell bodies. At E14.5, SRF ( f ) and RyR1 ( h ) are expressed in RELN-positive marginal zone neurons, as well as in intermediate zone fibers originating in the subplate, whereas Sez6 -driven EGFP ( g ) is expressed in subplate and cortical plate neurons, but not in RELN-positive cells. Scale bars = 50 μm.

    Article Snippet: Primary antibodies used were: mouse anti-Cajal–Retzius cell marker reelin (RELN) (1:1000, MAB5364, Chemicon, Billerica, MA), rabbit anti-GFP (1:2000, Molecular Probes, Carlsbad, CA), rabbit anti-calretinin (1:2000, Swant, Bellinzona, Switzerland), rabbit anti-hippocalcin (1:2000, Abcam, Cambridge, MA), goat anti-EAAC1 (1:2000, Chemicon), rabbit anti-Ryr1 (1:1000, Chemicon), rabbit anti-serum response factor (SRF) (1:100, Abcam), rat anti-L1 (1:20, a gift from Dr J. Trotter), mouse IgM anti-TAG1 (1:2, gift from Dr Jane Dodd, Columbia University, NY).

    Techniques: Staining, Marker

    Splicing patterns of Ca 2+ channels and transporters in control and DM1 myotubes at 10 (T10) and 15 (T15) days of differentiation. ( A ) RyR1 alternative splicing of fetal ASII(−) and ASII(+) isoforms and schematic representation of the ASII splicing isoforms of RyR1. ( B ) Quantification of RyR ASII(−) as compared to the total transcript. ( C ) SERCA1 alternative splicing of fetal SERCA1b and adult SERCA1a isoforms and schematic representation of the SERCA1 splicing isoforms. ( D ) Quantification of fetal SERCA1b as compared to the total transcript. ( E ) Cav1.1 alternative splicing of fetal Cav1.1Δ29 and adult Cav1.1 isoforms and schematic representation of the Cav1.1 splicing isoforms. ( F ) Quantification of fetal Cav1.1 Δ29 as compared to the total transcript. PCR products were separated in 3% agarose gel by electrophoresis. Data are the mean ± SD. * p

    Journal: Genes

    Article Title: Altered Ca2+ Homeostasis and Endoplasmic Reticulum Stress in Myotonic Dystrophy Type 1 Muscle Cells

    doi: 10.3390/genes4020275

    Figure Lengend Snippet: Splicing patterns of Ca 2+ channels and transporters in control and DM1 myotubes at 10 (T10) and 15 (T15) days of differentiation. ( A ) RyR1 alternative splicing of fetal ASII(−) and ASII(+) isoforms and schematic representation of the ASII splicing isoforms of RyR1. ( B ) Quantification of RyR ASII(−) as compared to the total transcript. ( C ) SERCA1 alternative splicing of fetal SERCA1b and adult SERCA1a isoforms and schematic representation of the SERCA1 splicing isoforms. ( D ) Quantification of fetal SERCA1b as compared to the total transcript. ( E ) Cav1.1 alternative splicing of fetal Cav1.1Δ29 and adult Cav1.1 isoforms and schematic representation of the Cav1.1 splicing isoforms. ( F ) Quantification of fetal Cav1.1 Δ29 as compared to the total transcript. PCR products were separated in 3% agarose gel by electrophoresis. Data are the mean ± SD. * p

    Article Snippet: The splicing pattern of the SERCA1, RyR1 and Cav 1.1 was assessed in the parental adult muscles from the same patients, from whom the muscle cells were established in culture.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis

    Expression of Ca 2+ channels and transporters in control and DM1 myotubes at 10 (T10) and 15 (T15) days of differentiation. ( A ) Representative images of SERCA2 (green) and RyR1 (red), nuclei (blue-Dapi) immunofluorescence of T15 differentiated control and DM1 myotubes. Scale bar is 10 μm. ( B ) Representative Western blot analysis of RyR1, DHPR α1s (Cav1.1) and SERCA2 of control and DM1 myotubes at 10 and 15 days of differentiation. ( C – E ) Quantification of RyR1, DHPRα1s and SERCA2 protein levels normalized to β-actin. Data are the mean ± SD of two experiments. ** p

    Journal: Genes

    Article Title: Altered Ca2+ Homeostasis and Endoplasmic Reticulum Stress in Myotonic Dystrophy Type 1 Muscle Cells

    doi: 10.3390/genes4020275

    Figure Lengend Snippet: Expression of Ca 2+ channels and transporters in control and DM1 myotubes at 10 (T10) and 15 (T15) days of differentiation. ( A ) Representative images of SERCA2 (green) and RyR1 (red), nuclei (blue-Dapi) immunofluorescence of T15 differentiated control and DM1 myotubes. Scale bar is 10 μm. ( B ) Representative Western blot analysis of RyR1, DHPR α1s (Cav1.1) and SERCA2 of control and DM1 myotubes at 10 and 15 days of differentiation. ( C – E ) Quantification of RyR1, DHPRα1s and SERCA2 protein levels normalized to β-actin. Data are the mean ± SD of two experiments. ** p

    Article Snippet: The splicing pattern of the SERCA1, RyR1 and Cav 1.1 was assessed in the parental adult muscles from the same patients, from whom the muscle cells were established in culture.

    Techniques: Expressing, Immunofluorescence, Western Blot

    Effect of triadin on FKBP12 expression and its binding to RyR1. A , equivalent amounts of crude membrane homogenates from WT and triadin-null myotubes were loaded and immunoblotted for expression of RyR1, DHPR α 1S , CSQ-1, junctin, and FKBP12. Band intensity is expressed as fraction of the WT signal ( dotted line ). Numbers on the bars indicate the number of blots analyzed per protein. Data are presented as mean ± S.E. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Ablation of Skeletal Muscle Triadin Impairs FKBP12/RyR1 Channel Interactions Essential for Maintaining Resting Cytoplasmic Ca2+ *

    doi: 10.1074/jbc.M110.164525

    Figure Lengend Snippet: Effect of triadin on FKBP12 expression and its binding to RyR1. A , equivalent amounts of crude membrane homogenates from WT and triadin-null myotubes were loaded and immunoblotted for expression of RyR1, DHPR α 1S , CSQ-1, junctin, and FKBP12. Band intensity is expressed as fraction of the WT signal ( dotted line ). Numbers on the bars indicate the number of blots analyzed per protein. Data are presented as mean ± S.E. *, p

    Article Snippet: Triadin-null RyR1 channels exhibit more frequent and much longer lived subconductances within typical recordings as contrasted by the brief subconductance transitions typically observed when FKBP12 is dissociated from RyR1 in the presence of triadin.

    Techniques: Expressing, Binding Assay

    GST-RTN1 523 inhibits specific [ 3 H]ryanodine binding to rat brain synaptosomes. (A) Equilibrium [ 3 H]ryanodine binding to rat forebrain synaptosomal membrane preparations was carried out in binding buffer containing 10 nM [ 3 H]ryanodine at the indicated free calcium concentrations in control conditions ( closed circles ) and in the presence of 0.1 μM GST ( closed triangles ) or 0.1 μM GST-RTN1 523 ( open squares ). [Ca 2 + ] was maintained, in the range 0.01 μM–1 mM, by a combination of EGTA and CaCl 2 . Free Ca 2 + concentrations were calculated as described in material and methods. Data points shown are the mean ± S.E.M., from three separate experiments performed in triplicates. (B) [ 3 H]ryanodine binding in the presence of 0.1 μM GST or GST-RTN1 523 is presented as percent of control. No specific [ 3 H]ryanodine binding was observed at 0.01 μM Ca 2 + in the presence of GST or GST-RTN1 523 . Difference in [ 3 H]ryanodine binding was plotted as percent decrease in specific binding. Data points shown are the mean ± S.E.M., from three separate experiments (* p = 0.011 by Student's t -test). (C) RyR2 evoked Ca 2 + oscillations in HEK293. Upper and lower left panels represent Fura-2 ratio time-courses of single cells expressing RyR2 together with mcherry, mcherry-RTN1A or EGFP-RTN4A. Cells were continuously perfused with buffer containing 0 mM Ca 2 + (nominal free), 1 mM Ca 2 + and 0 mM Ca 2 + + 10 mM caffeine as indicated by the bars at the top. Lower right panel shows a quantitative analysis performed by integration of the respective single peak areas referred to area under curve for estimation of the total amount of the cytosolic [Ca 2 + ] arising through RyR2 dependent Ca 2 + oscillations. The fraction of cells that showed both oscillations as well as a clear caffeine peak in comparison to those that lacked oscillations before a single caffeine peak are given in percentages in the graph. A two-sample t-test was carried out to test for significance as indicated by the p values at the bottom of the panel.

    Journal: Biochimica et Biophysica Acta

    Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

    doi: 10.1016/j.bbamcr.2013.02.012

    Figure Lengend Snippet: GST-RTN1 523 inhibits specific [ 3 H]ryanodine binding to rat brain synaptosomes. (A) Equilibrium [ 3 H]ryanodine binding to rat forebrain synaptosomal membrane preparations was carried out in binding buffer containing 10 nM [ 3 H]ryanodine at the indicated free calcium concentrations in control conditions ( closed circles ) and in the presence of 0.1 μM GST ( closed triangles ) or 0.1 μM GST-RTN1 523 ( open squares ). [Ca 2 + ] was maintained, in the range 0.01 μM–1 mM, by a combination of EGTA and CaCl 2 . Free Ca 2 + concentrations were calculated as described in material and methods. Data points shown are the mean ± S.E.M., from three separate experiments performed in triplicates. (B) [ 3 H]ryanodine binding in the presence of 0.1 μM GST or GST-RTN1 523 is presented as percent of control. No specific [ 3 H]ryanodine binding was observed at 0.01 μM Ca 2 + in the presence of GST or GST-RTN1 523 . Difference in [ 3 H]ryanodine binding was plotted as percent decrease in specific binding. Data points shown are the mean ± S.E.M., from three separate experiments (* p = 0.011 by Student's t -test). (C) RyR2 evoked Ca 2 + oscillations in HEK293. Upper and lower left panels represent Fura-2 ratio time-courses of single cells expressing RyR2 together with mcherry, mcherry-RTN1A or EGFP-RTN4A. Cells were continuously perfused with buffer containing 0 mM Ca 2 + (nominal free), 1 mM Ca 2 + and 0 mM Ca 2 + + 10 mM caffeine as indicated by the bars at the top. Lower right panel shows a quantitative analysis performed by integration of the respective single peak areas referred to area under curve for estimation of the total amount of the cytosolic [Ca 2 + ] arising through RyR2 dependent Ca 2 + oscillations. The fraction of cells that showed both oscillations as well as a clear caffeine peak in comparison to those that lacked oscillations before a single caffeine peak are given in percentages in the graph. A two-sample t-test was carried out to test for significance as indicated by the p values at the bottom of the panel.

    Article Snippet: Nevertheless, this selectivity could reflect preferential binding of RTN1A to RyR2 in the absence of RyR1 such as in hippocampal mossy fibers (see below).

    Techniques: Binding Assay, Expressing

    Immunohistochemical distribution pattern of RTN1A and RyR2 in rat hippocampus and cerebellum. Representative staining patterns for RTN1A (A,C,D) and RyR2 (B,E) on sections of rat hippocampus (A,B) and cerebellar cortex (C,D,E). In the hippocampus, immunoreactivity for both proteins is found in granule cells (G), mossy fiber axons, and in stratum lucidum (SL). In the cerebellum, RTN1A-immunoreactivity was confined to Purkinje cell bodies (P), their dendrites in ML (C; arrow) and axons (D; arrowheads). (E) Confocal immunofluorescent image showing RyR2 staining in Purkinje cells. Unlike RTN1A, RyR2 was also found in granule cell layer (Gl). Scale bars: A and B, 500 μm; H, hilus; G, Granule cell layer; So, stratum oriens; Sr, stratum radiatum; Slm, stratum lacunosum molecular; Iml, inner molecular layer; M + Oml, Middle outer molecular layer; CA1-3, Cornu ammonis; SL, stratum lucidum; S, Subiculum; Pr, Presubiculum; Pa, Parasubiculum.

    Journal: Biochimica et Biophysica Acta

    Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

    doi: 10.1016/j.bbamcr.2013.02.012

    Figure Lengend Snippet: Immunohistochemical distribution pattern of RTN1A and RyR2 in rat hippocampus and cerebellum. Representative staining patterns for RTN1A (A,C,D) and RyR2 (B,E) on sections of rat hippocampus (A,B) and cerebellar cortex (C,D,E). In the hippocampus, immunoreactivity for both proteins is found in granule cells (G), mossy fiber axons, and in stratum lucidum (SL). In the cerebellum, RTN1A-immunoreactivity was confined to Purkinje cell bodies (P), their dendrites in ML (C; arrow) and axons (D; arrowheads). (E) Confocal immunofluorescent image showing RyR2 staining in Purkinje cells. Unlike RTN1A, RyR2 was also found in granule cell layer (Gl). Scale bars: A and B, 500 μm; H, hilus; G, Granule cell layer; So, stratum oriens; Sr, stratum radiatum; Slm, stratum lacunosum molecular; Iml, inner molecular layer; M + Oml, Middle outer molecular layer; CA1-3, Cornu ammonis; SL, stratum lucidum; S, Subiculum; Pr, Presubiculum; Pa, Parasubiculum.

    Article Snippet: Nevertheless, this selectivity could reflect preferential binding of RTN1A to RyR2 in the absence of RyR1 such as in hippocampal mossy fibers (see below).

    Techniques: Immunohistochemistry, Staining

    Identification of RyR2 as a binding partner of RTN1A. (A) Schematic representation of full length RTN1A and the GST-RTN1 523 construct ( right ). RHD: reticulon homology domain; TM1 and TM2: transmembrane domain 1 and 2. GST pull-downs were performed with GST-RTN1 523 using detergent-solubilized mouse brain proteins. GST or empty glutathione beads served as negative controls, whereas GST-NiR was tested to control for GST-RTN1 523 binding specificity. Arrow denotes protein band that was consistently pulled down with GST-RTN1 523 and from which RyR2 was identified by mass spectrometry. Note that this band is absent in the different control samples. Asterisks indicate the GST fusion proteins and their relative amounts used in the GST pull-down. The silver stained gel shown is representative of three independent experiments. (B) Upper panel , Western Blot of samples from (A) probed with RyR2 antibody. Note the double-band that is identified as RyR2. The higher molecular mass band corresponds to intact RyR2, while the lower band presumably represents a proteolytic degradation fragment of RyR2. The intensity of the RyR2 bands increase with increasing amounts of GST-RTN1 523 . Lower panel , shows Ponceau S staining of the pull-downs to assess the relative amounts of each GST fusion protein. Input lane shows one-twentieth of the amount used for pull-down. WB, Western blot. The Western blot shown is representative of three independent experiments.

    Journal: Biochimica et Biophysica Acta

    Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

    doi: 10.1016/j.bbamcr.2013.02.012

    Figure Lengend Snippet: Identification of RyR2 as a binding partner of RTN1A. (A) Schematic representation of full length RTN1A and the GST-RTN1 523 construct ( right ). RHD: reticulon homology domain; TM1 and TM2: transmembrane domain 1 and 2. GST pull-downs were performed with GST-RTN1 523 using detergent-solubilized mouse brain proteins. GST or empty glutathione beads served as negative controls, whereas GST-NiR was tested to control for GST-RTN1 523 binding specificity. Arrow denotes protein band that was consistently pulled down with GST-RTN1 523 and from which RyR2 was identified by mass spectrometry. Note that this band is absent in the different control samples. Asterisks indicate the GST fusion proteins and their relative amounts used in the GST pull-down. The silver stained gel shown is representative of three independent experiments. (B) Upper panel , Western Blot of samples from (A) probed with RyR2 antibody. Note the double-band that is identified as RyR2. The higher molecular mass band corresponds to intact RyR2, while the lower band presumably represents a proteolytic degradation fragment of RyR2. The intensity of the RyR2 bands increase with increasing amounts of GST-RTN1 523 . Lower panel , shows Ponceau S staining of the pull-downs to assess the relative amounts of each GST fusion protein. Input lane shows one-twentieth of the amount used for pull-down. WB, Western blot. The Western blot shown is representative of three independent experiments.

    Article Snippet: Nevertheless, this selectivity could reflect preferential binding of RTN1A to RyR2 in the absence of RyR1 such as in hippocampal mossy fibers (see below).

    Techniques: Binding Assay, Construct, Mass Spectrometry, Staining, Western Blot

    Immunocytochemical distribution of mCherry-RTN1 523 and RyRs in HEK293 cells. (A) HEK293 cells were transiently transfected with mCherry-RTN1 523 in the absence (a) or presence of untagged RyR2 (d–f) or RyR1 (g–i). Single-transfections of untagged RyR2 (b) and RyR1 (c) were carried out as a comparison. Cells were immunostained with anti-RyR2 (b,d–f) or anti-RyR1 (c,g-i) antibodies and visualized by confocal microscopy. Note that single-transfected mCherry-RTN1 523 is uniformly distributed in the cytosol (a), but altered to a more reticular staining pattern when co-expressed with RyR2 (d–f). mCherry-RTN1 523 remained uniformly distributed in cells cotransfected with RyR1 (g–i). Cells shown represent at least 20 representative cells per condition. (B) Immunofluorescence confocal microscopy analysis of RyR ER localization in HEK293 cells. HEK293 cells were single-transfected with untagged RyR1 cDNA or RyR2 cDNA, immunostained with the indicated antibodies and imaged using immunofluorescence microscopy to demonstrate RyR and calreticulin (ER-specific marker) colocalization. Cells were immunostained for RyR isoforms (green) and Calreticulin (red), respectively. Space bar: 10 μm. (C) Extent of colocalization between mCherry-RTN1 523 and RyR isoforms was quantified using Pearson correlation coefficient and determined through correlation analysis with Leica SP5 software from 10 different cells per group. (*** p = 0.003 by Student's t -test).

    Journal: Biochimica et Biophysica Acta

    Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

    doi: 10.1016/j.bbamcr.2013.02.012

    Figure Lengend Snippet: Immunocytochemical distribution of mCherry-RTN1 523 and RyRs in HEK293 cells. (A) HEK293 cells were transiently transfected with mCherry-RTN1 523 in the absence (a) or presence of untagged RyR2 (d–f) or RyR1 (g–i). Single-transfections of untagged RyR2 (b) and RyR1 (c) were carried out as a comparison. Cells were immunostained with anti-RyR2 (b,d–f) or anti-RyR1 (c,g-i) antibodies and visualized by confocal microscopy. Note that single-transfected mCherry-RTN1 523 is uniformly distributed in the cytosol (a), but altered to a more reticular staining pattern when co-expressed with RyR2 (d–f). mCherry-RTN1 523 remained uniformly distributed in cells cotransfected with RyR1 (g–i). Cells shown represent at least 20 representative cells per condition. (B) Immunofluorescence confocal microscopy analysis of RyR ER localization in HEK293 cells. HEK293 cells were single-transfected with untagged RyR1 cDNA or RyR2 cDNA, immunostained with the indicated antibodies and imaged using immunofluorescence microscopy to demonstrate RyR and calreticulin (ER-specific marker) colocalization. Cells were immunostained for RyR isoforms (green) and Calreticulin (red), respectively. Space bar: 10 μm. (C) Extent of colocalization between mCherry-RTN1 523 and RyR isoforms was quantified using Pearson correlation coefficient and determined through correlation analysis with Leica SP5 software from 10 different cells per group. (*** p = 0.003 by Student's t -test).

    Article Snippet: Nevertheless, this selectivity could reflect preferential binding of RTN1A to RyR2 in the absence of RyR1 such as in hippocampal mossy fibers (see below).

    Techniques: Transfection, Confocal Microscopy, Staining, Immunofluorescence, Microscopy, Marker, Software

    RTN1A associates preferentially with RyR2 channel in vivo . (A) Left panels , detergent-solubilized protein from HEK293 cells transiently transfected with untagged RyR2 plus RTN1A-myc was immunoprecipitated (IP) with rabbit polyclonal anti-RTN1A antibodies or control rabbit IgG. Immunoprecipitated proteins were detected on immunoblots with monoclonal anti-RyR2 or anti-RTN1A antibodies. Note that native HEK293 cells do not express endogenous levels of RTN1A or RyR2. Right panels , detergent-solubilized protein from HEK293 cells transiently transfected with untagged RyR2 plus RTN4A-myc was immunoprecipitated with rabbit polyclonal anti-RTN4 antibodies or control rabbit IgG. Immunoprecipitated proteins were detected on immunoblots with monoclonal anti-RyR2 or anti-RTN4A antibodies. Input lane shows one-eighth of the amount used for immunoprecipitation. WB, Western blot. (B) Detergent-solubilized protein from rat cerebellum was used for co-immunoprecipitations with rabbit anti-RTN1A, rabbit anti-RTN1A, or control rabbit IgG. Immunoprecipitated proteins were resolved by SDS-PAGE blotted on PVDF membranes and probed with monoclonal antibodies as indicated at the right. Input lane shows one-tenth of the amount used for immunoprecipitation. WB, Western blot. (C) Detergent-solubilized protein from rat cerebellum was used for co-immunoprecipitations with mouse anti-RyR2, mouse anti-RTN1A, mouse anti-RTN1A, or control mouse IgG. Immunoprecipitated proteins were resolved by SDS-PAGE, blotted on PVDF membranes and probed with antibodies as indicated at the right. Input lane shows one-tenth of the amount used for immunoprecipitation. WB, Western blot. (D) Detergent-solubilized protein from rat cerebellum was immunoprecipitated with mouse anti-RTN1A, mouse anti-RyR2, or control mouse IgG. Immunoprecipitated proteins were resolved by SDS-PAGE, blotted on PVDF membranes and probed with antibodies as indicated at the right. Note that RyR1 co-immunoprecipitates with mouse anti-RyR2, but not with mouse anti-RTN1A antibodies. Input lane shows one-fifth of the amount used for immunoprecipitation. WB, Western blot.

    Journal: Biochimica et Biophysica Acta

    Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

    doi: 10.1016/j.bbamcr.2013.02.012

    Figure Lengend Snippet: RTN1A associates preferentially with RyR2 channel in vivo . (A) Left panels , detergent-solubilized protein from HEK293 cells transiently transfected with untagged RyR2 plus RTN1A-myc was immunoprecipitated (IP) with rabbit polyclonal anti-RTN1A antibodies or control rabbit IgG. Immunoprecipitated proteins were detected on immunoblots with monoclonal anti-RyR2 or anti-RTN1A antibodies. Note that native HEK293 cells do not express endogenous levels of RTN1A or RyR2. Right panels , detergent-solubilized protein from HEK293 cells transiently transfected with untagged RyR2 plus RTN4A-myc was immunoprecipitated with rabbit polyclonal anti-RTN4 antibodies or control rabbit IgG. Immunoprecipitated proteins were detected on immunoblots with monoclonal anti-RyR2 or anti-RTN4A antibodies. Input lane shows one-eighth of the amount used for immunoprecipitation. WB, Western blot. (B) Detergent-solubilized protein from rat cerebellum was used for co-immunoprecipitations with rabbit anti-RTN1A, rabbit anti-RTN1A, or control rabbit IgG. Immunoprecipitated proteins were resolved by SDS-PAGE blotted on PVDF membranes and probed with monoclonal antibodies as indicated at the right. Input lane shows one-tenth of the amount used for immunoprecipitation. WB, Western blot. (C) Detergent-solubilized protein from rat cerebellum was used for co-immunoprecipitations with mouse anti-RyR2, mouse anti-RTN1A, mouse anti-RTN1A, or control mouse IgG. Immunoprecipitated proteins were resolved by SDS-PAGE, blotted on PVDF membranes and probed with antibodies as indicated at the right. Input lane shows one-tenth of the amount used for immunoprecipitation. WB, Western blot. (D) Detergent-solubilized protein from rat cerebellum was immunoprecipitated with mouse anti-RTN1A, mouse anti-RyR2, or control mouse IgG. Immunoprecipitated proteins were resolved by SDS-PAGE, blotted on PVDF membranes and probed with antibodies as indicated at the right. Note that RyR1 co-immunoprecipitates with mouse anti-RyR2, but not with mouse anti-RTN1A antibodies. Input lane shows one-fifth of the amount used for immunoprecipitation. WB, Western blot.

    Article Snippet: Nevertheless, this selectivity could reflect preferential binding of RTN1A to RyR2 in the absence of RyR1 such as in hippocampal mossy fibers (see below).

    Techniques: In Vivo, Transfection, Immunoprecipitation, Western Blot, SDS Page

    Identification of the RyR2 binding domain. (A) Schematic representation of rat RTN1A protein and RTN1A fragments used to construct GST fusion proteins for the pull-down experiments. HHD: high homology domain; LNT: long N-terminal fragment. (B) GST-RTN1 fragments were used as baits in pull-down experiments using detergent-solubilized mouse brain proteins. GST, GST-NiR or empty glutathione beads served as negative controls. Binding of RyR2 was subsequently detected by immunoblot ( upper panel ). Ponceau S staining of the pull-downs shows the relative amounts of each GST fusion protein ( lower panel ). Input lane shows one-tenth of the amount used for immunoprecipitation. WB, Western blot. Results are representative of three independent experiments.

    Journal: Biochimica et Biophysica Acta

    Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

    doi: 10.1016/j.bbamcr.2013.02.012

    Figure Lengend Snippet: Identification of the RyR2 binding domain. (A) Schematic representation of rat RTN1A protein and RTN1A fragments used to construct GST fusion proteins for the pull-down experiments. HHD: high homology domain; LNT: long N-terminal fragment. (B) GST-RTN1 fragments were used as baits in pull-down experiments using detergent-solubilized mouse brain proteins. GST, GST-NiR or empty glutathione beads served as negative controls. Binding of RyR2 was subsequently detected by immunoblot ( upper panel ). Ponceau S staining of the pull-downs shows the relative amounts of each GST fusion protein ( lower panel ). Input lane shows one-tenth of the amount used for immunoprecipitation. WB, Western blot. Results are representative of three independent experiments.

    Article Snippet: Nevertheless, this selectivity could reflect preferential binding of RTN1A to RyR2 in the absence of RyR1 such as in hippocampal mossy fibers (see below).

    Techniques: Binding Assay, Construct, Staining, Immunoprecipitation, Western Blot

    T-tubule and sarcoplasmic reticulum abnormalities in DNM2-S619L larval muscle. (A–D) Electron micrographs of longitudinal sections through zebrafish muscle at 3 dpf. (A,C) Muscle from DNM2-WT larvae shows the typical sarcomere striations of vertebrate striated muscle. (B,D) Muscle from DNM2-S619L larvae demonstrates extensive swelling and vacuolization in the region of the SR and T-tubules. Scale bars: 1 μm. (E-H) Confocal micrographs of isolated myofibers subjected to immunofluorescence analysis. (E) Wild-type (WT) myofiber showing the expected pattern of RyR1 staining. (F,G) RyR1 expression is irregular in DNM2-S619L myofibers, and is often found aggregated. (H) α-actinin staining was normal, indicating that other elements of the muscle structure in S619L myofibers are not disturbed.

    Journal: Disease Models & Mechanisms

    Article Title: The myopathy-causing mutation DNM2-S619L leads to defective tubulation in vitro and in developing zebrafish

    doi: 10.1242/dmm.012286

    Figure Lengend Snippet: T-tubule and sarcoplasmic reticulum abnormalities in DNM2-S619L larval muscle. (A–D) Electron micrographs of longitudinal sections through zebrafish muscle at 3 dpf. (A,C) Muscle from DNM2-WT larvae shows the typical sarcomere striations of vertebrate striated muscle. (B,D) Muscle from DNM2-S619L larvae demonstrates extensive swelling and vacuolization in the region of the SR and T-tubules. Scale bars: 1 μm. (E-H) Confocal micrographs of isolated myofibers subjected to immunofluorescence analysis. (E) Wild-type (WT) myofiber showing the expected pattern of RyR1 staining. (F,G) RyR1 expression is irregular in DNM2-S619L myofibers, and is often found aggregated. (H) α-actinin staining was normal, indicating that other elements of the muscle structure in S619L myofibers are not disturbed.

    Article Snippet: Immunostaining was done as previously reporting using antibodies to a-actinin (Sigma), RyR1 (Developmental Hybridoma Bank) and DHPR (Abcam) ( ; ).

    Techniques: Isolation, Immunofluorescence, Staining, Expressing

    Effect of the pY3016C mutation on RyR1 protein expression on patient and control muscle biopsies. The western blot shows a dramatic decrease of the RyR1 protein and of the DHPRalpha 1.1 expression in the patient’s biopsy compared to control’s biopsy (P

    Journal: PLoS ONE

    Article Title: Variable Myopathic Presentation in a Single Family with Novel Skeletal RYR1 Mutation

    doi: 10.1371/journal.pone.0069296

    Figure Lengend Snippet: Effect of the pY3016C mutation on RyR1 protein expression on patient and control muscle biopsies. The western blot shows a dramatic decrease of the RyR1 protein and of the DHPRalpha 1.1 expression in the patient’s biopsy compared to control’s biopsy (P

    Article Snippet: Indeed, mutations in the RYR1 gene can lead to several clinical phenotypes: Malignant hyperthermia susceptibility including King-Denborough syndrome (MHS, pharmacogenetic disorder of skeletal muscle, MIM#145600), Central Core Disease (CCD, MIM#117000), as well as some forms of Multi minicore disease (MmD, MIM#255320), Centronuclear myopathy (CNM, MIM# 160150), congenital fiber type disproportion (CFTD, MIM#255310) and other rare atypical myopathies (see for review).

    Techniques: Mutagenesis, Expressing, Western Blot

    Analysis of RYR1 at the DNA level in patients and controls. ( A ) Sequence of the RYR1 gene revealed a homozygous A to G nucleotide substitution leading to an amino acid change (p.Y3016C) within exon 60 in patient (arrow). ( B ) Analysis of the mutation in family members and control. Left panel: PCR amplification products of RYR1 from exon -60 to intron-60 using primers specific to the mutated allele (MUT Primers, product size = 210 bp). Right panel: PCR amplification products of RYR1 from exon-60 to intron – 60 using primers specific to wild type allele (WT Primers, product size = 210 bp). This test confirms the cosegregation of the RYR1 mutation with the phenotype and haplotypes in the family. C: control. Affected individuals are underlined.

    Journal: PLoS ONE

    Article Title: Variable Myopathic Presentation in a Single Family with Novel Skeletal RYR1 Mutation

    doi: 10.1371/journal.pone.0069296

    Figure Lengend Snippet: Analysis of RYR1 at the DNA level in patients and controls. ( A ) Sequence of the RYR1 gene revealed a homozygous A to G nucleotide substitution leading to an amino acid change (p.Y3016C) within exon 60 in patient (arrow). ( B ) Analysis of the mutation in family members and control. Left panel: PCR amplification products of RYR1 from exon -60 to intron-60 using primers specific to the mutated allele (MUT Primers, product size = 210 bp). Right panel: PCR amplification products of RYR1 from exon-60 to intron – 60 using primers specific to wild type allele (WT Primers, product size = 210 bp). This test confirms the cosegregation of the RYR1 mutation with the phenotype and haplotypes in the family. C: control. Affected individuals are underlined.

    Article Snippet: Indeed, mutations in the RYR1 gene can lead to several clinical phenotypes: Malignant hyperthermia susceptibility including King-Denborough syndrome (MHS, pharmacogenetic disorder of skeletal muscle, MIM#145600), Central Core Disease (CCD, MIM#117000), as well as some forms of Multi minicore disease (MmD, MIM#255320), Centronuclear myopathy (CNM, MIM# 160150), congenital fiber type disproportion (CFTD, MIM#255310) and other rare atypical myopathies (see for review).

    Techniques: Sequencing, Mutagenesis, Polymerase Chain Reaction, Amplification

    Immunofluorescence of control, β 1 -null and dysgenic, α 1S -null, myotubes with antibodies to β 1 and α 1S DHPR subunits and RyR-1. ( a and b ) Control myotubes labeled with either anti-β 1 ( a ) or anti-α 1S ( b ). ( c and d ) β 1 -Null myotubes labeled with either anti-β 1 ( c ) or anti-α 1S ( d ). ( e ) Dysgenic myotubes labeled with anti-β show a weak, but punctate pattern (arrows). ( f ) β 1 -Null myotubes labeled with anti-RyR-1 in a punctate pattern (arrows). The diffuse background staining in the myotube may represent RyR-1 that is not membrane localized. (Bar = 20 μM.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Absence of the ? subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the ?1 subunit and eliminates excitation-contraction coupling

    doi:

    Figure Lengend Snippet: Immunofluorescence of control, β 1 -null and dysgenic, α 1S -null, myotubes with antibodies to β 1 and α 1S DHPR subunits and RyR-1. ( a and b ) Control myotubes labeled with either anti-β 1 ( a ) or anti-α 1S ( b ). ( c and d ) β 1 -Null myotubes labeled with either anti-β 1 ( c ) or anti-α 1S ( d ). ( e ) Dysgenic myotubes labeled with anti-β show a weak, but punctate pattern (arrows). ( f ) β 1 -Null myotubes labeled with anti-RyR-1 in a punctate pattern (arrows). The diffuse background staining in the myotube may represent RyR-1 that is not membrane localized. (Bar = 20 μM.)

    Article Snippet: Abbreviations: T tubule, transverse tubule; SR, sarcoplasmic reticulum; DHPR, dihydropyridine receptor; RyR-1, ryanodine receptor; E–C, excitation–contraction; ES cells, embryonic stem cells; E n , embryonic day n .

    Techniques: Immunofluorescence, Labeling, Staining

    Cav1.1-RyR1 complex: molecular details . Shown is a reconstruction from cryo-electron microscopy of the mammalian RyR1 at 4.8 Å illustrating the different regions. This includes the large myoplasmic segment and its multiple cavities and processes, and the SR transmembranal segment, which forms the Ca 2+ conduction pathway (Electron microscopy data bank entry 6106, Zalk et al., 2015 ). The above properties allow the RyR1 to interact with multiple SR luminal and cytosolic proteins. Several 3-D structures of proteins interacting with RyR1, as well as their relative locations, are also shown. EMData bank PDB entry and references: calsequestrin-1 (Casq1) polymer (3UOM, Sanchez et al., 2012 ), FKBP-12 (1D6O, Burkhard et al., 2000 ), S100A1 (2K2F; Wright et al., 2008 ), calmodulin (CaM; 1CLL, Chattopadhyaya et al., 1992 ), Cav1.1α subunit (4MS2, Tang et al., 2014 ; Hu et al., 2015 ), and Cav1.1 β subunit (1T0J, Van Petegem et al., 2004 ; Hu et al., 2015 ); only two alpha and two beta subunits are shown. In the muscle fiber Ca v 1.1 channels are clustered in groups of four (or tetrads) in the T-tubules. Ca v 1.1 channels in the tetrad interact with the four subunits of the RyR1, one Cav1.1 per each RyR1 subunit. All structures were generated using UCSF Chimera (Pettersen et al., 2004 ).

    Journal: Frontiers in Physiology

    Article Title: Critical Role of Intracellular RyR1 Calcium Release Channels in Skeletal Muscle Function and Disease

    doi: 10.3389/fphys.2015.00420

    Figure Lengend Snippet: Cav1.1-RyR1 complex: molecular details . Shown is a reconstruction from cryo-electron microscopy of the mammalian RyR1 at 4.8 Å illustrating the different regions. This includes the large myoplasmic segment and its multiple cavities and processes, and the SR transmembranal segment, which forms the Ca 2+ conduction pathway (Electron microscopy data bank entry 6106, Zalk et al., 2015 ). The above properties allow the RyR1 to interact with multiple SR luminal and cytosolic proteins. Several 3-D structures of proteins interacting with RyR1, as well as their relative locations, are also shown. EMData bank PDB entry and references: calsequestrin-1 (Casq1) polymer (3UOM, Sanchez et al., 2012 ), FKBP-12 (1D6O, Burkhard et al., 2000 ), S100A1 (2K2F; Wright et al., 2008 ), calmodulin (CaM; 1CLL, Chattopadhyaya et al., 1992 ), Cav1.1α subunit (4MS2, Tang et al., 2014 ; Hu et al., 2015 ), and Cav1.1 β subunit (1T0J, Van Petegem et al., 2004 ; Hu et al., 2015 ); only two alpha and two beta subunits are shown. In the muscle fiber Ca v 1.1 channels are clustered in groups of four (or tetrads) in the T-tubules. Ca v 1.1 channels in the tetrad interact with the four subunits of the RyR1, one Cav1.1 per each RyR1 subunit. All structures were generated using UCSF Chimera (Pettersen et al., 2004 ).

    Article Snippet: In the second category, RyR1 mutations cause leaky channels leading to Ca2+ dysregulation and depletion of Ca2+ from SR, and result in central core disease (CCD).

    Techniques: Electron Microscopy, Chick Chorioallantoic Membrane Assay, Generated

    Muscle structure and function: from the myofiber to RyR1 Ca 2+ release complex . (A) Morphology of skeletal muscle fibers. Top Panel: Transmitted light image of isolated muscle fibers from mouse flexor digitorum brevis via enzymatic dissociation; image was acquired using low magnification. Lower Panel: A higher magnification image of the fiber segment enclosed by a dashed rectangle. Note the characteristic striated pattern of muscle fibers, which results from highly organized array between sarcolemma, sarcoplasmic reticulum (SR), contractile elements and cytoarchitecture of the fibers. (B) Structure of the triad. In most cases the myoplasmic side of the junctional SR is closely apposed either to T-tubule or surface membrane, forming different junctions called dyads, triads, or peripheral coupling. The cartoon depicts a longitudinal section of the T-tubule axis, but a cross-section of the triad, a specialized array formed by the T-tubule and two segments of the terminal junctional SR (aka the terminal cisternae). The T-tubules are invaginations of the sarcolemma that propagate the action potential and possess the Ca v 1.1 voltage sensors that initiate the early steps of ECC. The voltage dependent Ca 2+ channels, Cav1.1 (blue, aka DHPR), are positioned in both the T-tubule and sarcolemma. The SR Ca 2+ release channel, RyR1 (brown), is located on the junctional domain of the SR surface, facing the T-tubules, and is also known as the junctional SR face membrane. Some RyRs may be present in adjacent parajunctional SR domains (orange). (C) Detailed architecture of the Cav1.1-RyR1 complex shown in (B) . About half of the total RyR1s do not associate with Cav1.1, resulting in an alternating pattern of “free” and Cav1.1-associated RyR1s. Note: In addition to Cav1.1 and RyR1, many other proteins form part of the T-tubule- junctional SR complex (e.g., FKPB12, triadin, junctin, Casq1) and are not shown here. Panels (B,C) are based on references: (Franzini-Armstrong and Porter, 1964 ; Franzini-Armstrong and Nunzi, 1983 ; Block et al., 1988 ; Franzini-Armstrong and Jorgensen, 1994 ; Franzini-Armstrong and Kish, 1995 ; Franzini-Armstrong and Protasi, 1997 ).

    Journal: Frontiers in Physiology

    Article Title: Critical Role of Intracellular RyR1 Calcium Release Channels in Skeletal Muscle Function and Disease

    doi: 10.3389/fphys.2015.00420

    Figure Lengend Snippet: Muscle structure and function: from the myofiber to RyR1 Ca 2+ release complex . (A) Morphology of skeletal muscle fibers. Top Panel: Transmitted light image of isolated muscle fibers from mouse flexor digitorum brevis via enzymatic dissociation; image was acquired using low magnification. Lower Panel: A higher magnification image of the fiber segment enclosed by a dashed rectangle. Note the characteristic striated pattern of muscle fibers, which results from highly organized array between sarcolemma, sarcoplasmic reticulum (SR), contractile elements and cytoarchitecture of the fibers. (B) Structure of the triad. In most cases the myoplasmic side of the junctional SR is closely apposed either to T-tubule or surface membrane, forming different junctions called dyads, triads, or peripheral coupling. The cartoon depicts a longitudinal section of the T-tubule axis, but a cross-section of the triad, a specialized array formed by the T-tubule and two segments of the terminal junctional SR (aka the terminal cisternae). The T-tubules are invaginations of the sarcolemma that propagate the action potential and possess the Ca v 1.1 voltage sensors that initiate the early steps of ECC. The voltage dependent Ca 2+ channels, Cav1.1 (blue, aka DHPR), are positioned in both the T-tubule and sarcolemma. The SR Ca 2+ release channel, RyR1 (brown), is located on the junctional domain of the SR surface, facing the T-tubules, and is also known as the junctional SR face membrane. Some RyRs may be present in adjacent parajunctional SR domains (orange). (C) Detailed architecture of the Cav1.1-RyR1 complex shown in (B) . About half of the total RyR1s do not associate with Cav1.1, resulting in an alternating pattern of “free” and Cav1.1-associated RyR1s. Note: In addition to Cav1.1 and RyR1, many other proteins form part of the T-tubule- junctional SR complex (e.g., FKPB12, triadin, junctin, Casq1) and are not shown here. Panels (B,C) are based on references: (Franzini-Armstrong and Porter, 1964 ; Franzini-Armstrong and Nunzi, 1983 ; Block et al., 1988 ; Franzini-Armstrong and Jorgensen, 1994 ; Franzini-Armstrong and Kish, 1995 ; Franzini-Armstrong and Protasi, 1997 ).

    Article Snippet: In the second category, RyR1 mutations cause leaky channels leading to Ca2+ dysregulation and depletion of Ca2+ from SR, and result in central core disease (CCD).

    Techniques: Isolation, Blocking Assay

    Impact of the NEB/RYR1 mutations. (A) Normal sized cDNA amplicons for exons 44 to 47 from patient ARX30 with the heterozygous c.5574C > G nonsense mutation (exon 45) and from patient ARX33 with the heterozygous c.5783_5784delAT deletion (exon 46). The splice mutation c.8160+1G > A (intron 58, ARX33) resulted in a shorter NEB cDNA amplicon (exons 57–60) compared to the control. The splice mutation c.19101+5G > A (intron 122, ARX30) involved a weak cDNA amplicon (exons 120–124) of normal size and a strong amplicon of smaller size. (B) The NEB c.8160+1G > A splice mutation (ARX33) causes a complete skipping of the in-frame exon 58. The c.5783_5784delAT mutation was not seen in the cDNA, indicating mRNA degradation by nonsense-mediated mRNA decay (NMD) of the allele containing this deletion. The NEB cDNA amplicon of patient ARX30 (exons 122–126) did not contain the in-frame exon 122. The c.5574C > G mutation in exon 46 was not seen by cDNA sequencing, suggesting NMD of the allele harboring this nonsense mutation. (C) Western blot of a deltoid muscle extract revealed a strong reduction of the RYR1 protein level in patient AHE6 compared to a healthy age-matched control. Desmin was used for normalization.

    Journal: PLoS ONE

    Article Title: An Integrated Diagnosis Strategy for Congenital Myopathies

    doi: 10.1371/journal.pone.0067527

    Figure Lengend Snippet: Impact of the NEB/RYR1 mutations. (A) Normal sized cDNA amplicons for exons 44 to 47 from patient ARX30 with the heterozygous c.5574C > G nonsense mutation (exon 45) and from patient ARX33 with the heterozygous c.5783_5784delAT deletion (exon 46). The splice mutation c.8160+1G > A (intron 58, ARX33) resulted in a shorter NEB cDNA amplicon (exons 57–60) compared to the control. The splice mutation c.19101+5G > A (intron 122, ARX30) involved a weak cDNA amplicon (exons 120–124) of normal size and a strong amplicon of smaller size. (B) The NEB c.8160+1G > A splice mutation (ARX33) causes a complete skipping of the in-frame exon 58. The c.5783_5784delAT mutation was not seen in the cDNA, indicating mRNA degradation by nonsense-mediated mRNA decay (NMD) of the allele containing this deletion. The NEB cDNA amplicon of patient ARX30 (exons 122–126) did not contain the in-frame exon 122. The c.5574C > G mutation in exon 46 was not seen by cDNA sequencing, suggesting NMD of the allele harboring this nonsense mutation. (C) Western blot of a deltoid muscle extract revealed a strong reduction of the RYR1 protein level in patient AHE6 compared to a healthy age-matched control. Desmin was used for normalization.

    Article Snippet: Mutations in RYR1 have been associated with different neuromuscular phenotypes including central core disease (CCD, MIM# 117000) , , multiminicore disease (MmD, MIM# 255320) , congenital myopathy with central or internalized nuclei , , congenital fiber-type disproportion (CFTD, MIM# 255310) , foetal akinesia , benign Samaritan congenital myopathy , and malignant hyperthermia susceptibility (MH, MIM# 145600) .

    Techniques: Mutagenesis, Amplification, Sequencing, Western Blot

    Pedigrees and RYR1/NEB mutations in six families with different muscle disorders. Patient ARX30 harbors the NEB c.5574C > G mutation on the maternal and the NEB c.19101+5G > A mutation on the paternal allele. Patient ARX33 harbors the NEB c.8160+1G > A mutation on the maternal and the NEB c.5783_5784delAT mutation on the paternal allele. Patients AKY21 and IM26 carry the heterozygous c.3223C > T, c.7025A > G and c.7645-7650dupGCGCTG mutations in RYR1 . The c.3223C > T mutation was found on the maternal allele, the father’s DNA was not available. AHY58 harbors two heterozygous RYR1 mutations: c.8953C > T on the paternal allele and c.9758T > C on the maternal allele. In patients AGT66 and AGT67 we identified the heterozygous RYR1 mutations c.325C > T on the maternal and c.8140_8141delTA on the paternal allele. Patient AHE6 from a consanguineous family was found to harbor the homozygous RYR1 c.8888T > C mutation, both parents were heterozygous.

    Journal: PLoS ONE

    Article Title: An Integrated Diagnosis Strategy for Congenital Myopathies

    doi: 10.1371/journal.pone.0067527

    Figure Lengend Snippet: Pedigrees and RYR1/NEB mutations in six families with different muscle disorders. Patient ARX30 harbors the NEB c.5574C > G mutation on the maternal and the NEB c.19101+5G > A mutation on the paternal allele. Patient ARX33 harbors the NEB c.8160+1G > A mutation on the maternal and the NEB c.5783_5784delAT mutation on the paternal allele. Patients AKY21 and IM26 carry the heterozygous c.3223C > T, c.7025A > G and c.7645-7650dupGCGCTG mutations in RYR1 . The c.3223C > T mutation was found on the maternal allele, the father’s DNA was not available. AHY58 harbors two heterozygous RYR1 mutations: c.8953C > T on the paternal allele and c.9758T > C on the maternal allele. In patients AGT66 and AGT67 we identified the heterozygous RYR1 mutations c.325C > T on the maternal and c.8140_8141delTA on the paternal allele. Patient AHE6 from a consanguineous family was found to harbor the homozygous RYR1 c.8888T > C mutation, both parents were heterozygous.

    Article Snippet: Mutations in RYR1 have been associated with different neuromuscular phenotypes including central core disease (CCD, MIM# 117000) , , multiminicore disease (MmD, MIM# 255320) , congenital myopathy with central or internalized nuclei , , congenital fiber-type disproportion (CFTD, MIM# 255310) , foetal akinesia , benign Samaritan congenital myopathy , and malignant hyperthermia susceptibility (MH, MIM# 145600) .

    Techniques: Mutagenesis

    Confocal laser scanning microscopy demonstrates increased expression levels of IP3R3, RYR1 and S100A6 in SH-SY5Y cells following CDDP or TOPO treatment ( A ) Representative fluorescence confocal images of SH-SY5Y cells that endogenously express IP3R1, IP3R3, RYR1, RYR3 and S100A6 as detected by Fluo-488 nm conjugated antibodies (green fluorescence) using images taken in similar experimental set ups following exposure to either 1 μM CDDP or 0.01 μM TOPO for 72 h. Cell nuclei are counterstained with DAPI (blue). scale bar = 20 μm. ( B ) Quantification of fluorescence intensity (protein expression) expressed as mean corrected total cell fluorescence (CTCF) ± standard deviation (SD). Data are derived from three independent biological experiments each. Statistical significance is relative to untreated cells and considered if p

    Journal: Oncotarget

    Article Title: Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma

    doi: 10.18632/oncotarget.15283

    Figure Lengend Snippet: Confocal laser scanning microscopy demonstrates increased expression levels of IP3R3, RYR1 and S100A6 in SH-SY5Y cells following CDDP or TOPO treatment ( A ) Representative fluorescence confocal images of SH-SY5Y cells that endogenously express IP3R1, IP3R3, RYR1, RYR3 and S100A6 as detected by Fluo-488 nm conjugated antibodies (green fluorescence) using images taken in similar experimental set ups following exposure to either 1 μM CDDP or 0.01 μM TOPO for 72 h. Cell nuclei are counterstained with DAPI (blue). scale bar = 20 μm. ( B ) Quantification of fluorescence intensity (protein expression) expressed as mean corrected total cell fluorescence (CTCF) ± standard deviation (SD). Data are derived from three independent biological experiments each. Statistical significance is relative to untreated cells and considered if p

    Article Snippet: Monoclonal primary antibodies; IP3R1 (Ms, Santa Cruz), IP3R3 (Rb, Santa Cruz), RYR1 (Rb, Millipore), RYR3 (Rb, Millipore) and S100A6 (Rb, Ab Cam) detected protein expression in SH-SY5Y and IMR-32 cells fluorescently stained with alexa-488 conjugated secondary Ab (Life Technologies).

    Techniques: Confocal Laser Scanning Microscopy, Expressing, Fluorescence, Standard Deviation, Derivative Assay, IF-P

    Treatment with CDDP or TOPO induces alterations in the mRNA expression of key [Ca 2+ ] i modulators in neuroblastoma cells Changes in mRNA expression of ITPR1, ITPR3, RYR1, RYR3 , S100A6 and COX2 were assessed via qRT-PCR in SH-SY5Y cells following 12 h, 24 h, 48 h or 72 h exposure to ( A ) 0.1 μM–10 μM CDDP or ( B ) 0.001 μM–1 μM TOPO. The expression pattern in IMR-32 cells was also assessed following exposure to 0.1 μM–10 μM CDDP ( C ). Data are expressed as fold-change (RQ), relative to the untreated control and are normalized to expression of ARF-1 mRNA as reference. Data are derived from three independent biological experiments each. Statistical significance is relative to the untreated control and considered if p

    Journal: Oncotarget

    Article Title: Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma

    doi: 10.18632/oncotarget.15283

    Figure Lengend Snippet: Treatment with CDDP or TOPO induces alterations in the mRNA expression of key [Ca 2+ ] i modulators in neuroblastoma cells Changes in mRNA expression of ITPR1, ITPR3, RYR1, RYR3 , S100A6 and COX2 were assessed via qRT-PCR in SH-SY5Y cells following 12 h, 24 h, 48 h or 72 h exposure to ( A ) 0.1 μM–10 μM CDDP or ( B ) 0.001 μM–1 μM TOPO. The expression pattern in IMR-32 cells was also assessed following exposure to 0.1 μM–10 μM CDDP ( C ). Data are expressed as fold-change (RQ), relative to the untreated control and are normalized to expression of ARF-1 mRNA as reference. Data are derived from three independent biological experiments each. Statistical significance is relative to the untreated control and considered if p

    Article Snippet: Monoclonal primary antibodies; IP3R1 (Ms, Santa Cruz), IP3R3 (Rb, Santa Cruz), RYR1 (Rb, Millipore), RYR3 (Rb, Millipore) and S100A6 (Rb, Ab Cam) detected protein expression in SH-SY5Y and IMR-32 cells fluorescently stained with alexa-488 conjugated secondary Ab (Life Technologies).

    Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, IF-P

    Changes in the expression of IP3R3, RYR3 and S100A6 at the protein level in SH-SY5Y cells following exposure to CDDP or TOPO SH-SY5Y cells were treated with either ( A ) CDDP (1 μM/10 μM) or ( B ) TOPO (0.01 μM/0.1 μM) for 72 hours and then harvested, fixed, permeabilized and incubated with primary antibodies specific for IP3R1, IP3R3, RYR1, RYR3 or S100A6 followed by incubation with a fluorescently conjugated (Alexa-488 nm) secondary antibody. The percentage of positive cells is individually presented for each protein or together for comparative analysis (expressed as a fold-change, relative to the untreated control). Data are derived from three independent biological experiments each. Statistical significance was calculated relative to untreated cells and considered if p

    Journal: Oncotarget

    Article Title: Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma

    doi: 10.18632/oncotarget.15283

    Figure Lengend Snippet: Changes in the expression of IP3R3, RYR3 and S100A6 at the protein level in SH-SY5Y cells following exposure to CDDP or TOPO SH-SY5Y cells were treated with either ( A ) CDDP (1 μM/10 μM) or ( B ) TOPO (0.01 μM/0.1 μM) for 72 hours and then harvested, fixed, permeabilized and incubated with primary antibodies specific for IP3R1, IP3R3, RYR1, RYR3 or S100A6 followed by incubation with a fluorescently conjugated (Alexa-488 nm) secondary antibody. The percentage of positive cells is individually presented for each protein or together for comparative analysis (expressed as a fold-change, relative to the untreated control). Data are derived from three independent biological experiments each. Statistical significance was calculated relative to untreated cells and considered if p

    Article Snippet: Monoclonal primary antibodies; IP3R1 (Ms, Santa Cruz), IP3R3 (Rb, Santa Cruz), RYR1 (Rb, Millipore), RYR3 (Rb, Millipore) and S100A6 (Rb, Ab Cam) detected protein expression in SH-SY5Y and IMR-32 cells fluorescently stained with alexa-488 conjugated secondary Ab (Life Technologies).

    Techniques: Expressing, Incubation, Derivative Assay, IF-P

    Protein phosphorylation with Zn 2+ exposure. Western blots were examined for n = 4 rat hearts for each condition. Representative blots are shown for n = 2 in each condition. A : phosphorylated RyR S2808 in rat LV was reduced after exposure to 50 μM

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Identifying cellular mechanisms of zinc-induced relaxation in isolated cardiomyocytes

    doi: 10.1152/ajpheart.00025.2013

    Figure Lengend Snippet: Protein phosphorylation with Zn 2+ exposure. Western blots were examined for n = 4 rat hearts for each condition. Representative blots are shown for n = 2 in each condition. A : phosphorylated RyR S2808 in rat LV was reduced after exposure to 50 μM

    Article Snippet: We used the following antibodies: anti-RyR (Thermo Scientific MA3–916); anti-phospho-RyR2 S2808 (Badrilla A010–30); anti-PLB (Thermo Scientific 2D12); anti-phospho-PLB-S16,T17 (Cell Signaling no. 8496); anti-phospho-TnI-S23,24 (Cell Signaling no. 4004) and anti-phospho-RLC-S20 (Abcam ab2480).

    Techniques: Western Blot

    Western Blots. ( A ) Western blots probed with anti FKBP12 (two blots displayed, the bottom blot shows a visible band in control lane) and the proteins selected as loading controls, calsequestrin and RyR. ( B and C ) An average of the western blots demonstrated that FKBP12 and FKBP12.6 purified peptides produced a significant increase in FKBPs association with RyR2 in treated cells. ( D ) Western blots probed with anti p-S2814, a phosphorylation site on RyR2, and the loading control, calsequestrin (CSQ). ( E ) An average of the blots demonstrated that the phosphorylation cocktail produced a significant increase in phosphorylation of S2814. Band densities were measured using ImageJ and were expressed as mean ± SEM. n = 7 blots of 4 hearts in B and C. n = 4 blots of 4 hearts in E. Data were analyzed using student t-test. *p

    Journal: eLife

    Article Title: Cardiac ryanodine receptor distribution is dynamic and changed by auxiliary proteins and post-translational modification

    doi: 10.7554/eLife.51602

    Figure Lengend Snippet: Western Blots. ( A ) Western blots probed with anti FKBP12 (two blots displayed, the bottom blot shows a visible band in control lane) and the proteins selected as loading controls, calsequestrin and RyR. ( B and C ) An average of the western blots demonstrated that FKBP12 and FKBP12.6 purified peptides produced a significant increase in FKBPs association with RyR2 in treated cells. ( D ) Western blots probed with anti p-S2814, a phosphorylation site on RyR2, and the loading control, calsequestrin (CSQ). ( E ) An average of the blots demonstrated that the phosphorylation cocktail produced a significant increase in phosphorylation of S2814. Band densities were measured using ImageJ and were expressed as mean ± SEM. n = 7 blots of 4 hearts in B and C. n = 4 blots of 4 hearts in E. Data were analyzed using student t-test. *p

    Article Snippet: We used the following antibodies: Anti-FKBP12 (ab2918; Abcam, Cambridge, UK) to detect both FKBP 12 and FKBP12.6, anti Calsequestrin (ab3516; Abcam) and anti-RyR2 (clone C3-33, MA3-916; Invitrogen, Carlsbad CA) were used as loading controls.

    Techniques: Western Blot, Purification, Produced

    Localization, Distribution and NND Measurements of RyR2 on the Surface of the jSR in a Permeabilized Rat Ventricular Myocyte Exposed to the Phosphorylation Cocktail. Orthogonal planes within the tomogram are outlined in different colors; XY in red, YZ in green and XZ in blue. ( A ) A single XY plane extracted from the dual-tilt tomogram. Single arrow points to the jSR, and the double arrow points to the t-tubule. ( B ) YZ view. (C) (i) An enlarged area of the junction in A. ( C ) (ii) Multiple XZ planes, at 0.5 nm intervals, were positioned within the cleft to bisect the ryanodine receptors. The planes paralleled, as nearly as possible, the jSR membrane; two of which (planes 2 and 8) are highlighted in blue. ( C ) (iii) Demonstration of how the tilt in X changed the appearance of the tetramers in Cii8. ( D ) Identification of RyR2 in Cii8. Each required a different rotation to identify the tetramers’ corners (red arrows). ( E ) Final distribution and orientation of all of the tetramers. The tetramers identified in Di and Dii are highlighted. ( F ) Histogram of the NND of 61 tetramers in four junctions.

    Journal: eLife

    Article Title: Cardiac ryanodine receptor distribution is dynamic and changed by auxiliary proteins and post-translational modification

    doi: 10.7554/eLife.51602

    Figure Lengend Snippet: Localization, Distribution and NND Measurements of RyR2 on the Surface of the jSR in a Permeabilized Rat Ventricular Myocyte Exposed to the Phosphorylation Cocktail. Orthogonal planes within the tomogram are outlined in different colors; XY in red, YZ in green and XZ in blue. ( A ) A single XY plane extracted from the dual-tilt tomogram. Single arrow points to the jSR, and the double arrow points to the t-tubule. ( B ) YZ view. (C) (i) An enlarged area of the junction in A. ( C ) (ii) Multiple XZ planes, at 0.5 nm intervals, were positioned within the cleft to bisect the ryanodine receptors. The planes paralleled, as nearly as possible, the jSR membrane; two of which (planes 2 and 8) are highlighted in blue. ( C ) (iii) Demonstration of how the tilt in X changed the appearance of the tetramers in Cii8. ( D ) Identification of RyR2 in Cii8. Each required a different rotation to identify the tetramers’ corners (red arrows). ( E ) Final distribution and orientation of all of the tetramers. The tetramers identified in Di and Dii are highlighted. ( F ) Histogram of the NND of 61 tetramers in four junctions.

    Article Snippet: We used the following antibodies: Anti-FKBP12 (ab2918; Abcam, Cambridge, UK) to detect both FKBP 12 and FKBP12.6, anti Calsequestrin (ab3516; Abcam) and anti-RyR2 (clone C3-33, MA3-916; Invitrogen, Carlsbad CA) were used as loading controls.

    Techniques:

    Localization and Distribution of 3 RyR2 Tetramers Identified in Figure 2 (a,b and c) on the Surface of the jSR. ( a ) (i) The RyR2 shown in Figure 2D , (D12); the red arrows point at the identified RyR throughout the panel. (ii) Tilt rotation in X (blue lines), centred on the identified tetramer. (iii) The tetramer changed appearance as a function of tilt angle. iv, a red box, 27 nm ( Van Petegem, 2015 ), was placed on the tetramer when its four corners (red arrows) could be clearly identified. Placement and orientation of tetramers in ( b and c ) are similar to a.

    Journal: eLife

    Article Title: Cardiac ryanodine receptor distribution is dynamic and changed by auxiliary proteins and post-translational modification

    doi: 10.7554/eLife.51602

    Figure Lengend Snippet: Localization and Distribution of 3 RyR2 Tetramers Identified in Figure 2 (a,b and c) on the Surface of the jSR. ( a ) (i) The RyR2 shown in Figure 2D , (D12); the red arrows point at the identified RyR throughout the panel. (ii) Tilt rotation in X (blue lines), centred on the identified tetramer. (iii) The tetramer changed appearance as a function of tilt angle. iv, a red box, 27 nm ( Van Petegem, 2015 ), was placed on the tetramer when its four corners (red arrows) could be clearly identified. Placement and orientation of tetramers in ( b and c ) are similar to a.

    Article Snippet: We used the following antibodies: Anti-FKBP12 (ab2918; Abcam, Cambridge, UK) to detect both FKBP 12 and FKBP12.6, anti Calsequestrin (ab3516; Abcam) and anti-RyR2 (clone C3-33, MA3-916; Invitrogen, Carlsbad CA) were used as loading controls.

    Techniques:

    Spontaneous RyR2-mediated Ca 2+ release in cardiac myocytes with loss of SPEG ( A–B ) Confocal microscopy line scans revealing increased number of Ca 2+ sparks in MCM-Speg fl/fl mice 8 weeks post tamoxifen. ( C ) Bar graphs showing quantification of Ca 2+ spark frequency (CaSpF), ( D ) Ca 2+ spark amplitude, and ( E ) full-width of half max (FWHM). n , number of sparks (mice). * P

    Journal: Circulation research

    Article Title: ‘Striated Muscle Preferentially Expressed Protein Kinase’ (SPEG) Is Essential for Cardiac Function by Regulating Junctional Membrane Complex Activity

    doi: 10.1161/CIRCRESAHA.116.309977

    Figure Lengend Snippet: Spontaneous RyR2-mediated Ca 2+ release in cardiac myocytes with loss of SPEG ( A–B ) Confocal microscopy line scans revealing increased number of Ca 2+ sparks in MCM-Speg fl/fl mice 8 weeks post tamoxifen. ( C ) Bar graphs showing quantification of Ca 2+ spark frequency (CaSpF), ( D ) Ca 2+ spark amplitude, and ( E ) full-width of half max (FWHM). n , number of sparks (mice). * P

    Article Snippet: Briefly, RyR2 was immunoprecipitated from WT mouse hearts using RyR2 monoclonal antibody (MA3–916, Thermo Fisher Scientific, Waltham, MA) and JPH2 was immunoprecipitated from JPH2 overexpressing hearts using monoclonal HA antibody (Sigma-Aldrich, St. Louis, MO).

    Techniques: Confocal Microscopy, Mouse Assay

    SPEG directly binds to RyR2 and JPH2 within JMCs ( A–B ) Unbiased proteomics analyses of putative RyR2 and JPH2 binding partners based on adult mouse heart co-immunoprecipitation of RyR2 and JPH2, respectively, coupled to mass spectrometry (IP-MS). Specific binding partners (red dots) were defined based on cut-off ratios of 3 and 4, respectively, of the MS intensity of each protein that came down with RyR2 or JPH2 IP vs. control IgG IP (y-axis) and RyR2 or JPH2 IP vs. beads only IP (x-axis). ( C ) Schematic overview of SPEG protein structure with key functional domains marked, and SPEG fragments used for binding-site analysis. ( D ) Western blots of reciprocal SPEG co-IP experiment from mouse heart showing that both RyR2 and JPH2 but not SERCA2a pulled down with SPEG. ( E ) RyR2, Myc-tagged SPEGβ N-terminal peptide, and FLAG-tagged SPEGα were co-expressed in HEK293 cells. Only SPEGβ N-terminal peptide was shown to bind the RyR2 pull-down. ( F ) JPH2 was co-expressed with Myc-tagged SPEGβ N-terminal peptide and FLAG-tagged SPEGα; only FLAG-tagged SPEGα was shown to bind the JPH2 pull-down.

    Journal: Circulation research

    Article Title: ‘Striated Muscle Preferentially Expressed Protein Kinase’ (SPEG) Is Essential for Cardiac Function by Regulating Junctional Membrane Complex Activity

    doi: 10.1161/CIRCRESAHA.116.309977

    Figure Lengend Snippet: SPEG directly binds to RyR2 and JPH2 within JMCs ( A–B ) Unbiased proteomics analyses of putative RyR2 and JPH2 binding partners based on adult mouse heart co-immunoprecipitation of RyR2 and JPH2, respectively, coupled to mass spectrometry (IP-MS). Specific binding partners (red dots) were defined based on cut-off ratios of 3 and 4, respectively, of the MS intensity of each protein that came down with RyR2 or JPH2 IP vs. control IgG IP (y-axis) and RyR2 or JPH2 IP vs. beads only IP (x-axis). ( C ) Schematic overview of SPEG protein structure with key functional domains marked, and SPEG fragments used for binding-site analysis. ( D ) Western blots of reciprocal SPEG co-IP experiment from mouse heart showing that both RyR2 and JPH2 but not SERCA2a pulled down with SPEG. ( E ) RyR2, Myc-tagged SPEGβ N-terminal peptide, and FLAG-tagged SPEGα were co-expressed in HEK293 cells. Only SPEGβ N-terminal peptide was shown to bind the RyR2 pull-down. ( F ) JPH2 was co-expressed with Myc-tagged SPEGβ N-terminal peptide and FLAG-tagged SPEGα; only FLAG-tagged SPEGα was shown to bind the JPH2 pull-down.

    Article Snippet: Briefly, RyR2 was immunoprecipitated from WT mouse hearts using RyR2 monoclonal antibody (MA3–916, Thermo Fisher Scientific, Waltham, MA) and JPH2 was immunoprecipitated from JPH2 overexpressing hearts using monoclonal HA antibody (Sigma-Aldrich, St. Louis, MO).

    Techniques: Binding Assay, Immunoprecipitation, Mass Spectrometry, Functional Assay, Western Blot, Co-Immunoprecipitation Assay

    RyR co-localisation with JPH2 is altered in human HF. Deconvolved confocal micrographs showing RyR (red) and JPH2 (green) dual labelling in ( a ) a trabecula and ( b ) ventricle wall sample from failing hearts. ( c ) Co-localisation analysis reveals the extent of RyR co-localised with JPH2 and JPH2 co-localised with RyR in the trabeculae and ventricle wall from failing human hearts. ( d ) Tissue labelling density revealed no differences for RyR or JPH2. ( e ) A positive relationship is observed between JPH2 tissue density and peak stress development (normalised to MCSA) in trabeculae from the failing human heart (p = 0.041). ( f ) JPH2 tissue labelling density forms a strong, positive relationship with the percentage of RyR clusters associated with t-tubules (tt) in failing human trabeculae (p = 0.0014). Co-localisation analysis: Trabeculae: n = 21 images, 14 trabeculae, 5 hearts; failing wall n = 22 images, 5 hearts. Density analysis: Trabeculae: n = 14 images, 14 trabeculae, 5 hearts; failing wall n = 14 images, 5 hearts. Data displayed as mean ± SEM.

    Journal: Scientific Reports

    Article Title: Highly variable contractile performance correlates with myocyte content in trabeculae from failing human hearts

    doi: 10.1038/s41598-018-21199-y

    Figure Lengend Snippet: RyR co-localisation with JPH2 is altered in human HF. Deconvolved confocal micrographs showing RyR (red) and JPH2 (green) dual labelling in ( a ) a trabecula and ( b ) ventricle wall sample from failing hearts. ( c ) Co-localisation analysis reveals the extent of RyR co-localised with JPH2 and JPH2 co-localised with RyR in the trabeculae and ventricle wall from failing human hearts. ( d ) Tissue labelling density revealed no differences for RyR or JPH2. ( e ) A positive relationship is observed between JPH2 tissue density and peak stress development (normalised to MCSA) in trabeculae from the failing human heart (p = 0.041). ( f ) JPH2 tissue labelling density forms a strong, positive relationship with the percentage of RyR clusters associated with t-tubules (tt) in failing human trabeculae (p = 0.0014). Co-localisation analysis: Trabeculae: n = 21 images, 14 trabeculae, 5 hearts; failing wall n = 22 images, 5 hearts. Density analysis: Trabeculae: n = 14 images, 14 trabeculae, 5 hearts; failing wall n = 14 images, 5 hearts. Data displayed as mean ± SEM.

    Article Snippet: Immunolabelling was performed using rabbit anti-JPH2 (1:100, custom polyclonal, as previously detailed) , combined with either mouse anti-RyR (1:100, ma3-916, Thermo) or mouse anti-α-tubulin (1:200, , Abcam) as a microtubule marker , or rabbit anti-collagen I (1:200, ab292, Abcam) or anti-collagen VI (1:100, ab6588, Abcam) with mouse anti-collagen III (1:200, ab6310, Abcam) with the primary antibodies incubated overnight at 4 °C.

    Techniques:

    iPSC can differentiate into functional CMs. ( a ) Reverse transcription-polymerase chain reaction (RT-PCR) for the expression of cardiac-specific genes in control- (WT) and CPVT-iPSC-derived beating explants (beating EBs); undifferentiated iPSCs and FHs were used as negative and positive controls, respectively. HGPRT : housekeeping gene; CACNA1C : calcium channel, voltage-dependent, α 1A subunit; SCN5A : sodium channel, voltage-gated, type V, alpha subunit; KCNQ1 : potassium, voltage-gated channel, KQT-like subfamily, member 1; MHCα : myosin heavy-chain α ; MHC β : myosin heavy chain β . ( b ) Western blot of WT- and CPVT-IPSC-derived beating explants for RyR2. β -Actin was used as the loading control, and human FH was used as a positive control. ( c ) RyR2 expression quantification in WT- and CPVT- beating explants, calculated as densitometry RyR2/ β -actin ratio (the diagram represents the mean of four independent experiments). ( d ) Immunostaining of CPVT-iPSC-derived CMs for α -actinin and RyR2. Nuclei stained in DAPI. Scale bar=20 μ m. ( e ) Representative traces of spontaneous APs recorded in iPSC harvested from the healthy donor (WT-iPSC-CMs, left) and the patient carrying the CPVT mutation (CPVT-iPSC-CMs, right). A DAD is indicated by the arrow. ( f ) The main AP features measured in both iPSC-derived lines: overshoot, amplitude, MDP, maximal upstroke velocity, maximal repolarization velocity and AP duration at 30%, 50% or 90% of repolarization (respectively APD 30 , APD 50 and APD 90 ). WT-iPSC-CMs, n =26; CPVT-iPSC-CMs, n =35. Values are mean±MSE

    Journal: Cell Death & Disease

    Article Title: CaMKII inhibition rectifies arrhythmic phenotype in a patient-specific model of catecholaminergic polymorphic ventricular tachycardia

    doi: 10.1038/cddis.2013.369

    Figure Lengend Snippet: iPSC can differentiate into functional CMs. ( a ) Reverse transcription-polymerase chain reaction (RT-PCR) for the expression of cardiac-specific genes in control- (WT) and CPVT-iPSC-derived beating explants (beating EBs); undifferentiated iPSCs and FHs were used as negative and positive controls, respectively. HGPRT : housekeeping gene; CACNA1C : calcium channel, voltage-dependent, α 1A subunit; SCN5A : sodium channel, voltage-gated, type V, alpha subunit; KCNQ1 : potassium, voltage-gated channel, KQT-like subfamily, member 1; MHCα : myosin heavy-chain α ; MHC β : myosin heavy chain β . ( b ) Western blot of WT- and CPVT-IPSC-derived beating explants for RyR2. β -Actin was used as the loading control, and human FH was used as a positive control. ( c ) RyR2 expression quantification in WT- and CPVT- beating explants, calculated as densitometry RyR2/ β -actin ratio (the diagram represents the mean of four independent experiments). ( d ) Immunostaining of CPVT-iPSC-derived CMs for α -actinin and RyR2. Nuclei stained in DAPI. Scale bar=20 μ m. ( e ) Representative traces of spontaneous APs recorded in iPSC harvested from the healthy donor (WT-iPSC-CMs, left) and the patient carrying the CPVT mutation (CPVT-iPSC-CMs, right). A DAD is indicated by the arrow. ( f ) The main AP features measured in both iPSC-derived lines: overshoot, amplitude, MDP, maximal upstroke velocity, maximal repolarization velocity and AP duration at 30%, 50% or 90% of repolarization (respectively APD 30 , APD 50 and APD 90 ). WT-iPSC-CMs, n =26; CPVT-iPSC-CMs, n =35. Values are mean±MSE

    Article Snippet: Monoclonal anti-RyR2 (1 : 1000; Thermo Fisher, Waltham, MA, USA) and polyclonal anti-β Actin (1 : 2000; Santa Cruz Biotechnologies, Dallas, TX, USA) antibodies were used for detection.

    Techniques: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Western Blot, Positive Control, Immunostaining, Staining, Mutagenesis

    Generation of iPSC from a CPVT patient skin biopsy. ( A ) Pedigree of the RyR2-He +/− CPVT kindred modeled in this study. Proband (II-2) is indicated by an arrow. Filled symbols indicate clinically and genetically affected subjects. Half-black symbols indicate genetically affected individuals, and upper half-black symbols indicate sudden cardiac death cases. Square=male; circle=female. ( B ) Example of bidirectional ventricular tachycardia recorded off-therapy in the proband (paper speed 25 mm/s). ( C ) Representative images of dermal fibroblasts derived from the CPVT patient (a) and of an iPSC colony derived from the patient's fibroblasts (b) showing alkaline phosphatase activity (c) and positivity for the pluripotency markers OCT4 (d), TRA1-60 (e) and SSEA4 (f). Scale bars=100 μ m. ( D ) Sequencing analysis confirming that the CPVT-iPSC line (He) carried the specific G-to-C mutation on one allele of the RyR2 gene, whereas control-iPSC (WT) did not show any genetic alteration. ( E ) iPSC lines maintained a normal karyotype after expansion

    Journal: Cell Death & Disease

    Article Title: CaMKII inhibition rectifies arrhythmic phenotype in a patient-specific model of catecholaminergic polymorphic ventricular tachycardia

    doi: 10.1038/cddis.2013.369

    Figure Lengend Snippet: Generation of iPSC from a CPVT patient skin biopsy. ( A ) Pedigree of the RyR2-He +/− CPVT kindred modeled in this study. Proband (II-2) is indicated by an arrow. Filled symbols indicate clinically and genetically affected subjects. Half-black symbols indicate genetically affected individuals, and upper half-black symbols indicate sudden cardiac death cases. Square=male; circle=female. ( B ) Example of bidirectional ventricular tachycardia recorded off-therapy in the proband (paper speed 25 mm/s). ( C ) Representative images of dermal fibroblasts derived from the CPVT patient (a) and of an iPSC colony derived from the patient's fibroblasts (b) showing alkaline phosphatase activity (c) and positivity for the pluripotency markers OCT4 (d), TRA1-60 (e) and SSEA4 (f). Scale bars=100 μ m. ( D ) Sequencing analysis confirming that the CPVT-iPSC line (He) carried the specific G-to-C mutation on one allele of the RyR2 gene, whereas control-iPSC (WT) did not show any genetic alteration. ( E ) iPSC lines maintained a normal karyotype after expansion

    Article Snippet: Monoclonal anti-RyR2 (1 : 1000; Thermo Fisher, Waltham, MA, USA) and polyclonal anti-β Actin (1 : 2000; Santa Cruz Biotechnologies, Dallas, TX, USA) antibodies were used for detection.

    Techniques: Derivative Assay, Activity Assay, Sequencing, Mutagenesis

    AβOs injections decrease RyR2 immunolabeling. (A) Representative confocal microscopy 3-D projections (20 slices, 1.5 μM each) of CA1, CA3, and DG hippocampal regions isolated from food restricted rats exposed to the memory training protocol, fixed after the sixth training session and labeled with a RyR2 antibody (green) and DAPI-nuclear staining (blue). Scale bar: 20 μM. (B) Quantification of total RyR2 fluorescence normalized by total DAPI nuclear staining. Values are expressed as mean ± SE ( n = 6). Statistical analysis was determined by two-tailed unpaired Student’s t -test; ∗ p

    Journal: Frontiers in Aging Neuroscience

    Article Title: N-Acetylcysteine Prevents the Spatial Memory Deficits and the Redox-Dependent RyR2 Decrease Displayed by an Alzheimer’s Disease Rat Model

    doi: 10.3389/fnagi.2018.00399

    Figure Lengend Snippet: AβOs injections decrease RyR2 immunolabeling. (A) Representative confocal microscopy 3-D projections (20 slices, 1.5 μM each) of CA1, CA3, and DG hippocampal regions isolated from food restricted rats exposed to the memory training protocol, fixed after the sixth training session and labeled with a RyR2 antibody (green) and DAPI-nuclear staining (blue). Scale bar: 20 μM. (B) Quantification of total RyR2 fluorescence normalized by total DAPI nuclear staining. Values are expressed as mean ± SE ( n = 6). Statistical analysis was determined by two-tailed unpaired Student’s t -test; ∗ p

    Article Snippet: Primary antibodies: mouse monoclonal anti-RyR2 (MA3-916) was from Thermo Fisher Scientific (Waltham, MA, United States); rabbit monoclonal anti-RyR3 (AB9082) and rabbit polyclonal anti-IP3 receptor type-1 (IP3 R1) (AB5882) antibodies were from former Merck-Millipore (Darmstadt, Germany); rabbit polyclonal anti-ACSL4 (SAB2100035) and anti β-actin (A5316) were from Sigma-Aldrich (St. Louis, MI, United States); anti-VDAC (sc-390996), anti-Calnexin (sc-6465) and anti-COX4 (sc-69359) were from Santa Cruz Biotechnology (Dallas, TX, United States); rabbit monoclonal anti-β-amyloid (H31L21) was from Life Technologies (Waltham, MA, United States); rabbit c-Fos polyclonal antibody Ab-5 was from Oncogene (San Diego, CA, United States); Anti-Arc Polyclonal rabbit affinity purified antibody was from Synaptic System (Göttingen, Germany); ERK1/2 and phospho-ERK1/2 antibodies were from Cell Signaling Technologies (Danvers, MA, United States).

    Techniques: Immunolabeling, Confocal Microscopy, Isolation, Labeling, Staining, Fluorescence, Two Tailed Test

    NAC feeding prevents the RyR2 downregulation induced by AβOs, without modifying RyR3 protein levels. The hippocampus of water deprived, spatial memory trained rats belonging to the following groups was removed and homog enized 1 h after the last training session. The four groups were: vehicle-fed rats injected with saline (control), vehicle-fed rats injected with AβOs (AβOs), NAC-fed rats injected with AβOs (NAC/AβOs), and NAC-fed rats injected with saline (NAC/Saline). (A) . Representative Western blot and relative quantification of RyR2 protein bands normalized to β-actin and expressed as fold of control. (B) Representative Western blot and relative quantification of RyR3 protein bands normalized to β-actin and expressed as fold of control. Values are expressed as mean ± SE ( n = 3, for each group). Statistical analysis was determined by one-way ANOVA followed by Holm-Sidak post hoc test; ∗ p

    Journal: Frontiers in Aging Neuroscience

    Article Title: N-Acetylcysteine Prevents the Spatial Memory Deficits and the Redox-Dependent RyR2 Decrease Displayed by an Alzheimer’s Disease Rat Model

    doi: 10.3389/fnagi.2018.00399

    Figure Lengend Snippet: NAC feeding prevents the RyR2 downregulation induced by AβOs, without modifying RyR3 protein levels. The hippocampus of water deprived, spatial memory trained rats belonging to the following groups was removed and homog enized 1 h after the last training session. The four groups were: vehicle-fed rats injected with saline (control), vehicle-fed rats injected with AβOs (AβOs), NAC-fed rats injected with AβOs (NAC/AβOs), and NAC-fed rats injected with saline (NAC/Saline). (A) . Representative Western blot and relative quantification of RyR2 protein bands normalized to β-actin and expressed as fold of control. (B) Representative Western blot and relative quantification of RyR3 protein bands normalized to β-actin and expressed as fold of control. Values are expressed as mean ± SE ( n = 3, for each group). Statistical analysis was determined by one-way ANOVA followed by Holm-Sidak post hoc test; ∗ p

    Article Snippet: Primary antibodies: mouse monoclonal anti-RyR2 (MA3-916) was from Thermo Fisher Scientific (Waltham, MA, United States); rabbit monoclonal anti-RyR3 (AB9082) and rabbit polyclonal anti-IP3 receptor type-1 (IP3 R1) (AB5882) antibodies were from former Merck-Millipore (Darmstadt, Germany); rabbit polyclonal anti-ACSL4 (SAB2100035) and anti β-actin (A5316) were from Sigma-Aldrich (St. Louis, MI, United States); anti-VDAC (sc-390996), anti-Calnexin (sc-6465) and anti-COX4 (sc-69359) were from Santa Cruz Biotechnology (Dallas, TX, United States); rabbit monoclonal anti-β-amyloid (H31L21) was from Life Technologies (Waltham, MA, United States); rabbit c-Fos polyclonal antibody Ab-5 was from Oncogene (San Diego, CA, United States); Anti-Arc Polyclonal rabbit affinity purified antibody was from Synaptic System (Göttingen, Germany); ERK1/2 and phospho-ERK1/2 antibodies were from Cell Signaling Technologies (Danvers, MA, United States).

    Techniques: Injection, Western Blot

    Hippocampal-derived MAM fractions contain RyR2, which is enriched in the MAM fraction from AβOs-injected rats. Western blot images of MAM fractions isolated from rat hippocampus (see Materials and Methods). The ER proteins analyzed were Calnexin (CNX), IP 3 R1, and RyR2. The inner and outer mitochondrial membrane proteins analyzed were COX1 and VDAC1, respectively; the characteristic MAM protein was ACSL-4. C, control, saline-injected; AβOs, AβOs-injected. The column at right indicates the ratios between band densities of each MAM protein (AβOs/control) normalized to b-tubulin protein levels.

    Journal: Frontiers in Aging Neuroscience

    Article Title: N-Acetylcysteine Prevents the Spatial Memory Deficits and the Redox-Dependent RyR2 Decrease Displayed by an Alzheimer’s Disease Rat Model

    doi: 10.3389/fnagi.2018.00399

    Figure Lengend Snippet: Hippocampal-derived MAM fractions contain RyR2, which is enriched in the MAM fraction from AβOs-injected rats. Western blot images of MAM fractions isolated from rat hippocampus (see Materials and Methods). The ER proteins analyzed were Calnexin (CNX), IP 3 R1, and RyR2. The inner and outer mitochondrial membrane proteins analyzed were COX1 and VDAC1, respectively; the characteristic MAM protein was ACSL-4. C, control, saline-injected; AβOs, AβOs-injected. The column at right indicates the ratios between band densities of each MAM protein (AβOs/control) normalized to b-tubulin protein levels.

    Article Snippet: Primary antibodies: mouse monoclonal anti-RyR2 (MA3-916) was from Thermo Fisher Scientific (Waltham, MA, United States); rabbit monoclonal anti-RyR3 (AB9082) and rabbit polyclonal anti-IP3 receptor type-1 (IP3 R1) (AB5882) antibodies were from former Merck-Millipore (Darmstadt, Germany); rabbit polyclonal anti-ACSL4 (SAB2100035) and anti β-actin (A5316) were from Sigma-Aldrich (St. Louis, MI, United States); anti-VDAC (sc-390996), anti-Calnexin (sc-6465) and anti-COX4 (sc-69359) were from Santa Cruz Biotechnology (Dallas, TX, United States); rabbit monoclonal anti-β-amyloid (H31L21) was from Life Technologies (Waltham, MA, United States); rabbit c-Fos polyclonal antibody Ab-5 was from Oncogene (San Diego, CA, United States); Anti-Arc Polyclonal rabbit affinity purified antibody was from Synaptic System (Göttingen, Germany); ERK1/2 and phospho-ERK1/2 antibodies were from Cell Signaling Technologies (Danvers, MA, United States).

    Techniques: Derivative Assay, Injection, Western Blot, Isolation

    AβOs-injections do not modify RyR3 immunolabeling. (A) Representative confocal microscopy 3-D projections (20 slices, 1.5 μM each) of CA1, CA3, and DG hippocampal regions from food restricted trained rats, fixed after the sixth training session and labeled with a RyR3 antibody (green) and DAPI staining for the nucleus (blue). Scale bar 20 μM. (B) Quantification of total fluorescence of RyR3 normalized by the total fluorescence of the DAPI nuclear staining. Statistical analysis was determined by two-tailed unpaired Student’s t -test.

    Journal: Frontiers in Aging Neuroscience

    Article Title: N-Acetylcysteine Prevents the Spatial Memory Deficits and the Redox-Dependent RyR2 Decrease Displayed by an Alzheimer’s Disease Rat Model

    doi: 10.3389/fnagi.2018.00399

    Figure Lengend Snippet: AβOs-injections do not modify RyR3 immunolabeling. (A) Representative confocal microscopy 3-D projections (20 slices, 1.5 μM each) of CA1, CA3, and DG hippocampal regions from food restricted trained rats, fixed after the sixth training session and labeled with a RyR3 antibody (green) and DAPI staining for the nucleus (blue). Scale bar 20 μM. (B) Quantification of total fluorescence of RyR3 normalized by the total fluorescence of the DAPI nuclear staining. Statistical analysis was determined by two-tailed unpaired Student’s t -test.

    Article Snippet: Primary antibodies: mouse monoclonal anti-RyR2 (MA3-916) was from Thermo Fisher Scientific (Waltham, MA, United States); rabbit monoclonal anti-RyR3 (AB9082) and rabbit polyclonal anti-IP3 receptor type-1 (IP3 R1) (AB5882) antibodies were from former Merck-Millipore (Darmstadt, Germany); rabbit polyclonal anti-ACSL4 (SAB2100035) and anti β-actin (A5316) were from Sigma-Aldrich (St. Louis, MI, United States); anti-VDAC (sc-390996), anti-Calnexin (sc-6465) and anti-COX4 (sc-69359) were from Santa Cruz Biotechnology (Dallas, TX, United States); rabbit monoclonal anti-β-amyloid (H31L21) was from Life Technologies (Waltham, MA, United States); rabbit c-Fos polyclonal antibody Ab-5 was from Oncogene (San Diego, CA, United States); Anti-Arc Polyclonal rabbit affinity purified antibody was from Synaptic System (Göttingen, Germany); ERK1/2 and phospho-ERK1/2 antibodies were from Cell Signaling Technologies (Danvers, MA, United States).

    Techniques: Immunolabeling, Confocal Microscopy, Labeling, Staining, Fluorescence, Two Tailed Test

    Representative images of isolated CM of PV ( A ), LV ( B ) and LA ( C ) with fluorescent RyR2 (green) and Ca v 1.2 (red) double immunostaining recorded with confocal microscopy. For each cell, RyR2 and Ca v 1.2 fluorescence intensity profiles along the white line were traced in a graph and the histograms represent the distribution of the distances between each RyR2 cluster and its nearest Ca v 1.2 cluster calculated across the entire cell slice. Aa and B illustrate a high level of RyR2-Ca v 1.2 co-localisation. Ab and Ca illustrate CM where RyR2 and Ca v 1.2 are only partially co-localised. Ac and Cb show CM where RyR2 and Ca v 1.2 are co-localised only at the sarcolemmal membrane.

    Journal: Scientific Reports

    Article Title: Structural heterogeneity of the rat pulmonary vein myocardium: consequences on intracellular calcium dynamics and arrhythmogenic potential

    doi: 10.1038/s41598-018-21671-9

    Figure Lengend Snippet: Representative images of isolated CM of PV ( A ), LV ( B ) and LA ( C ) with fluorescent RyR2 (green) and Ca v 1.2 (red) double immunostaining recorded with confocal microscopy. For each cell, RyR2 and Ca v 1.2 fluorescence intensity profiles along the white line were traced in a graph and the histograms represent the distribution of the distances between each RyR2 cluster and its nearest Ca v 1.2 cluster calculated across the entire cell slice. Aa and B illustrate a high level of RyR2-Ca v 1.2 co-localisation. Ab and Ca illustrate CM where RyR2 and Ca v 1.2 are only partially co-localised. Ac and Cb show CM where RyR2 and Ca v 1.2 are co-localised only at the sarcolemmal membrane.

    Article Snippet: Saturation with 4% BSA for 40 min was followed by double-labelling with primary antibodies against RyR2 (mouse monoclonal, Pierce) and Cav 1.2 (rabbit polyclonal, Calbiochem) overnight at 4 °C.

    Techniques: Isolation, Double Immunostaining, Confocal Microscopy, Fluorescence

    Expression of ryanodine and dihydropyridine receptors in WT and C3KO fibers. a Cross-sections of FDB muscles from WT and C3KO mice (HE). The arrows indicate non-peripheral nuclei. b Western blot analysis of CAPN3 ( upper row ), RyR1 ( middle row ), and αDHPR ( bottom row ) concentration in WT and C3KO muscles. Arrowhead indicates the CAPN3 band, which is absent in the C3KO muscles. c Quantitative comparison of the expression of αDHPR and RyR1. The bars are the means of densitometry measurements of triplicate data in ( b )

    Journal: Skeletal Muscle

    Article Title: Attenuated Ca2+ release in a mouse model of limb girdle muscular dystrophy 2A

    doi: 10.1186/s13395-016-0081-y

    Figure Lengend Snippet: Expression of ryanodine and dihydropyridine receptors in WT and C3KO fibers. a Cross-sections of FDB muscles from WT and C3KO mice (HE). The arrows indicate non-peripheral nuclei. b Western blot analysis of CAPN3 ( upper row ), RyR1 ( middle row ), and αDHPR ( bottom row ) concentration in WT and C3KO muscles. Arrowhead indicates the CAPN3 band, which is absent in the C3KO muscles. c Quantitative comparison of the expression of αDHPR and RyR1. The bars are the means of densitometry measurements of triplicate data in ( b )

    Article Snippet: The following antibodies were used for Western blot analyses: anti-calpain3 12A2 (Novocastra), anti-RyR1 (Thermo), anti-vinculin (Sigma), and anti-DHPRα (Thermo).

    Techniques: Expressing, Mouse Assay, Western Blot, Concentration Assay

    mRNA levels of ryanodine receptors. ( A ) Ryr1 , Ryr2 and Ryr3 expression in lysates prepared from whole mammary tissue (including luminal, basal and stromal cells) dissected from virgin or lactating animals (n = 4 mice). ( B ) Krt14 , Esr1 and Ryr1 levels in freshly sorted luminal and basal cells (n = 3 mice). Graphs show mean ± SEM; * P

    Journal: bioRxiv

    Article Title: Multiscale activity imaging in mammary gland reveals how oxytocin enables lactation

    doi: 10.1101/657510

    Figure Lengend Snippet: mRNA levels of ryanodine receptors. ( A ) Ryr1 , Ryr2 and Ryr3 expression in lysates prepared from whole mammary tissue (including luminal, basal and stromal cells) dissected from virgin or lactating animals (n = 4 mice). ( B ) Krt14 , Esr1 and Ryr1 levels in freshly sorted luminal and basal cells (n = 3 mice). Graphs show mean ± SEM; * P

    Article Snippet: The following Gene Expression Assays were used in this study: Ryr1 (Mm01175211_m1), Ryr2 (Mm00465877_m1), Ryr3 (Mm01328395_m1), Krt14 (Mm00516876_m1) and Esr1 (Mm00433149_m1).

    Techniques: Expressing, Mouse Assay

    RyR gene expression and function are up-regulated after T cell activation. A , average normalized linearized RyR1 ( left ), RyR2 ( middle ), and RyR3 ( right ) C q values in resting T cells ( R ; n = 9) and 5-day activated ( A ; n = 7) primary human T cells. B , anti-RyR

    Journal: The Journal of Biological Chemistry

    Article Title: Bidirectional Coupling between Ryanodine Receptors and Ca2+ Release-activated Ca2+ (CRAC) Channel Machinery Sustains Store-operated Ca2+

    doi: 10.1074/jbc.M112.398974

    Figure Lengend Snippet: RyR gene expression and function are up-regulated after T cell activation. A , average normalized linearized RyR1 ( left ), RyR2 ( middle ), and RyR3 ( right ) C q values in resting T cells ( R ; n = 9) and 5-day activated ( A ; n = 7) primary human T cells. B , anti-RyR

    Article Snippet: Human RT-PCR gene expression assays used for RyR1 (Hs00166991_m1), RyR2 (Hs00892902_m1), RyR3 (Hs01050911_m1), β2 -microglobulin ( B2M ; Hs99999907_m1), and ribosomal protein L13a ( RPL13a ; Hs01926559_g1) were obtained from Applied Biosystems.

    Techniques: Expressing, Activation Assay

    RyR co-localize with STIM1 puncta following store depletion in HEK293 cells. Confocal images of HEK293 cells co-overexpressing Cherry-STIM1 and RyR2 S437 -YFP recorded in red and green channels , respectively, prior to store depletion ( top panels ) and 10

    Journal: The Journal of Biological Chemistry

    Article Title: Bidirectional Coupling between Ryanodine Receptors and Ca2+ Release-activated Ca2+ (CRAC) Channel Machinery Sustains Store-operated Ca2+

    doi: 10.1074/jbc.M112.398974

    Figure Lengend Snippet: RyR co-localize with STIM1 puncta following store depletion in HEK293 cells. Confocal images of HEK293 cells co-overexpressing Cherry-STIM1 and RyR2 S437 -YFP recorded in red and green channels , respectively, prior to store depletion ( top panels ) and 10

    Article Snippet: Human RT-PCR gene expression assays used for RyR1 (Hs00166991_m1), RyR2 (Hs00892902_m1), RyR3 (Hs01050911_m1), β2 -microglobulin ( B2M ; Hs99999907_m1), and ribosomal protein L13a ( RPL13a ; Hs01926559_g1) were obtained from Applied Biosystems.

    Techniques:

    Pregnancy upregulates ryanodine receptors in uterine arteries. Ryanodine receptor type 1 (RyR1), type 2 (RyR2) and type 3 (RyR3) mRNA ( A ) and protein ( B ) abundance in uterine arteries of nonpregnant (UA NP ) and pregnant (UA P ) sheep were determined with real-time RT-PCR and Western blotting. Data are means ± SEM from 4–5 animals of each group. *P

    Journal: Hypertension (Dallas, Tex. : 1979)

    Article Title: Pregnancy increases Ca2+ sparks/STOCs and reduces uterine arterial myogenic tone

    doi: 10.1161/HYPERTENSIONAHA.118.12484

    Figure Lengend Snippet: Pregnancy upregulates ryanodine receptors in uterine arteries. Ryanodine receptor type 1 (RyR1), type 2 (RyR2) and type 3 (RyR3) mRNA ( A ) and protein ( B ) abundance in uterine arteries of nonpregnant (UA NP ) and pregnant (UA P ) sheep were determined with real-time RT-PCR and Western blotting. Data are means ± SEM from 4–5 animals of each group. *P

    Article Snippet: After blocking nonspecific binding sites by dry milk, membranes were incubated with primary antibodies (1:1000 dilution) against RyR1 (8153, Cell Signaling, Danvers, MA), RyR2 (AB9080, EMD Millipore, Billerica, MA) or RyR3 (AB9082, EMD Millipore).

    Techniques: Quantitative RT-PCR, Western Blot

    BST detection of SNO-RyR1 in human skeletal muscle SOL and VL biopsies. A. Upper panel , Immunoblot of BST streptavidin column eluate of SOL for RyR1. A. Lower panel , Immunoblot of BST streptavidin column eluate of VL for RyR1. Increased RyR1 immunoreactive band was detected in CTR End-only samples. B. Percent changes biotin-labeled RyR1 proteins from Pre and End bed rest biopsies. Bed rest without exercise promoted S -nitrosylation of RyR1 in disused SOL (CTR n =9, 18.6%, p ≤0.01) and VL (CTR n =9, 17.2%, p ≤0.01) as counteracted by both exercise countermeasures. (* significance).

    Journal: Redox Biology

    Article Title: Nitrosative stress in human skeletal muscle attenuated by exercise countermeasure after chronic disuse

    doi: 10.1016/j.redox.2013.10.006

    Figure Lengend Snippet: BST detection of SNO-RyR1 in human skeletal muscle SOL and VL biopsies. A. Upper panel , Immunoblot of BST streptavidin column eluate of SOL for RyR1. A. Lower panel , Immunoblot of BST streptavidin column eluate of VL for RyR1. Increased RyR1 immunoreactive band was detected in CTR End-only samples. B. Percent changes biotin-labeled RyR1 proteins from Pre and End bed rest biopsies. Bed rest without exercise promoted S -nitrosylation of RyR1 in disused SOL (CTR n =9, 18.6%, p ≤0.01) and VL (CTR n =9, 17.2%, p ≤0.01) as counteracted by both exercise countermeasures. (* significance).

    Article Snippet: Like RyR1, however, moderate SNO-SERCA levels were found in all Pre bed rest samples ( A and B, upper panel).

    Techniques: Labeling