monoclonal anti-ryanodine receptor ryr2 Search Results


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  • 99
    Thermo Fisher monoclonal anti ryanodine receptor ryr2
    Recordings of diastolic sarcoplasmic reticulum (SR) Ca 2+ leak after 1 Hz electrical stimulation in normal HEPES 1.8 mM Ca 2+ solution. A, Exemplary recordings show the protocol of quantification of SR Ca 2+ -leak by determination of diastolic Ca 2+ -levels in quiescent atrial cells with 0 Na + /0 Ca 2+ in the external perfusion solution compared to perfusion solution with 0 Na + /0 Ca 2+ +Tetracaine (TET) that inhibits the opening of the <t>ryanodine</t> receptor <t>(RyR2).</t> Recordings were followed by Caffeine (10 mM) induced Ca 2+ depletion of the SR to determine SR Ca 2+ storage B, Diastolic SR Ca 2+ -leak was significantly increased in Low Capacity Runner (LCR) rats compared to High Capacity Runner (HCR) rats. n = 5 animals, n = 4−6 cells from each animal. C, Western blot analyses of the ratio between phosphorylated Serine-2814/RyR2 display a significant higher expression in LCR rats (n = 4) compared to HCR rats (n = 3). D, Representative Western blots. Data are presented as mean±SD.
    Monoclonal Anti Ryanodine Receptor Ryr2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ryanodine receptor
    Predominant expression of IP 3 R type 1 in sub-PM SR encourages SPCU. (A) Ca 2+ stores visualized with fluo-3FF consisted of a sub-PM SR network and some central formation. (B) Immunolocalization of IP 3 R type 1 and <t>RyRs</t> using a double-staining protocol (see Section 2 ). Confocal image of Alexa Fluor 488 fluorescence (green), showing type 1 IP 3 R distribution (middle), and confocal image of Alexa Fluor 633 fluorescence (red), showing <t>RyR</t> distribution (right), are overlaid (left). (C) The fluo-4-loaded SMC was stimulated with 10 μM CCh and 5 mM caffeine (with 10-min interval). Two galleries of images (acquired at 42 Hz) highlight the difference in the initial phase of the responses. (D) The temporal profiles of the fluorescence at three regions outlined in red, green and magenta (left) are shown in corresponding colour.
    Ryanodine Receptor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam primary mouse monoclonal anti ryr2
    Predominant expression of IP 3 R type 1 in sub-PM SR encourages SPCU. (A) Ca 2+ stores visualized with fluo-3FF consisted of a sub-PM SR network and some central formation. (B) Immunolocalization of IP 3 R type 1 and <t>RyRs</t> using a double-staining protocol (see Section 2 ). Confocal image of Alexa Fluor 488 fluorescence (green), showing type 1 IP 3 R distribution (middle), and confocal image of Alexa Fluor 633 fluorescence (red), showing <t>RyR</t> distribution (right), are overlaid (left). (C) The fluo-4-loaded SMC was stimulated with 10 μM CCh and 5 mM caffeine (with 10-min interval). Two galleries of images (acquired at 42 Hz) highlight the difference in the initial phase of the responses. (D) The temporal profiles of the fluorescence at three regions outlined in red, green and magenta (left) are shown in corresponding colour.
    Primary Mouse Monoclonal Anti Ryr2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti ryanodine receptor 1 ryr1
    Neither contractile proteins nor excitation-contraction coupling proteins are altered in PH diaphragm muscles. A : Stain free images of myosin heavy chain (MyHC) and western blots of troponin (Tn) T. B : the expression levels of MyHC or TnT normalized to actin content. C : electrophoretically separated MyHC isoforms. D : percentage distribution of MyHC isoforms: I, slow myosin isoform; IIa, IId/x, and IIb, fast myosin isoforms. E : representative western blots of <t>ryanodine</t> receptor <t>(RyR1),</t> dihydropyridine receptor α2 subunit (DHPR), sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) 1, and SERCA2. F : the expression levels of RyR1, DHPR, SERCA1, or SERCA2 normalized to actin content. Control (CNT), white bars; AIA, black bars. Data represent mean ± SD for 4–8 rats in each group.
    Anti Ryanodine Receptor 1 Ryr1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher monoclonal anti ryanodine receptor
    Comparison of experimentally determined and computed calcium transients in Purkinje cells Top, representative recordings of I Ca measured at +10 mV from a Purkinje cell. I Ca measured at +10 mV was used to determine the time course of Ca 2+ movement. A and B , graphs showing time course of Ca 2+ movement and fluo-3 fluorescence signals obtained from a Purkinje cell in the absence and presence of <t>ryanodine.</t> Ryanodine decreased the fluorescence intensity but did not change the time course of Ca 2+ movement. C and D , time course of Ca 2+ movement and fluo-3 fluorescence signals obtained by mathematical modelling (see Results). No CICR mechanism was included in the formulation and all Ca 2+ movement was by simple diffusion. The time course of Ca 2+ movement in the model resembles the experimental data suggesting that the amplitude of the central transient is the result of simple diffusion from the cell periphery.
    Monoclonal Anti Ryanodine Receptor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam monoclonal antibody against total ryr1
    Western blotting analysis of <t>RyR1</t> phosphorylation at serine 2843 ( p RyR1 Ser2843 ) in human skeletal muscle tissue. A : Representative western blots of p RyR1 Ser2843 (left) and total RyR1 (right). Tissue was analyzed from muscle biopsies taken prior to exercise (B-pre), 25 min after completion of one set (B-I), five (B-V) and ten sets (B-X) from vastus lateralis of the loaded leg as well as 25–30 min after X set exercise from vastus lateralis of the non-exercised leg (B-0X). Total RyR1 was used as loading control. B : Bar graph of p RyR1 Ser2843 / total RyR1. a.u. = arbitrary units. Error bars are 1*SD of the mean.
    Monoclonal Antibody Against Total Ryr1, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti ryanodine receptor ryr monoclonal antibody
    Western blotting analysis of <t>RyR1</t> phosphorylation at serine 2843 ( p RyR1 Ser2843 ) in human skeletal muscle tissue. A : Representative western blots of p RyR1 Ser2843 (left) and total RyR1 (right). Tissue was analyzed from muscle biopsies taken prior to exercise (B-pre), 25 min after completion of one set (B-I), five (B-V) and ten sets (B-X) from vastus lateralis of the loaded leg as well as 25–30 min after X set exercise from vastus lateralis of the non-exercised leg (B-0X). Total RyR1 was used as loading control. B : Bar graph of p RyR1 Ser2843 / total RyR1. a.u. = arbitrary units. Error bars are 1*SD of the mean.
    Anti Ryanodine Receptor Ryr Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH ryanodine receptor
    Western blotting analysis of <t>RyR1</t> phosphorylation at serine 2843 ( p RyR1 Ser2843 ) in human skeletal muscle tissue. A : Representative western blots of p RyR1 Ser2843 (left) and total RyR1 (right). Tissue was analyzed from muscle biopsies taken prior to exercise (B-pre), 25 min after completion of one set (B-I), five (B-V) and ten sets (B-X) from vastus lateralis of the loaded leg as well as 25–30 min after X set exercise from vastus lateralis of the non-exercised leg (B-0X). Total RyR1 was used as loading control. B : Bar graph of p RyR1 Ser2843 / total RyR1. a.u. = arbitrary units. Error bars are 1*SD of the mean.
    Ryanodine Receptor, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novus Biologicals mouse monoclonal anti ryr1
    mRNA expression of calbindin D-28K ( Calb1 ), calretinin ( Calb2 ), tyrosine hydroxylase ( TH ), Zebrin II ( ZebrinII ), ryanodine receptor 1 ( <t>Ryr1</t> ), ryanodine receptor 2 ( Ryr2 ), and ryanodine receptor 3 ( Ryr3 ) genes in the cerebellum of wild-type and tottering-6j mice. The data are presented as means ± SEM. * P
    Mouse Monoclonal Anti Ryr1, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti ryanodine receptor monoclonal 34c antibody
    mRNA expression of calbindin D-28K ( Calb1 ), calretinin ( Calb2 ), tyrosine hydroxylase ( TH ), Zebrin II ( ZebrinII ), ryanodine receptor 1 ( <t>Ryr1</t> ), ryanodine receptor 2 ( Ryr2 ), and ryanodine receptor 3 ( Ryr3 ) genes in the cerebellum of wild-type and tottering-6j mice. The data are presented as means ± SEM. * P
    Anti Ryanodine Receptor Monoclonal 34c Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam serine 2808 phosphorylated ryr2 p ryr2
    mRNA expression of calbindin D-28K ( Calb1 ), calretinin ( Calb2 ), tyrosine hydroxylase ( TH ), Zebrin II ( ZebrinII ), ryanodine receptor 1 ( <t>Ryr1</t> ), ryanodine receptor 2 ( Ryr2 ), and ryanodine receptor 3 ( Ryr3 ) genes in the cerebellum of wild-type and tottering-6j mice. The data are presented as means ± SEM. * P
    Serine 2808 Phosphorylated Ryr2 P Ryr2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank anti ryr monoclonal antibody
    mRNA expression of calbindin D-28K ( Calb1 ), calretinin ( Calb2 ), tyrosine hydroxylase ( TH ), Zebrin II ( ZebrinII ), ryanodine receptor 1 ( <t>Ryr1</t> ), ryanodine receptor 2 ( Ryr2 ), and ryanodine receptor 3 ( Ryr3 ) genes in the cerebellum of wild-type and tottering-6j mice. The data are presented as means ± SEM. * P
    Anti Ryr Monoclonal Antibody, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ryanodine receptors expression primary antibody monoclonal rabbit anti ryr1
    Immunofluorescent visualization of calcium/sodium exchanger (NCX1) and <t>ryanodine</t> receptor <t>(Ryr1)</t> in normal (C2C12, C2C12-D) and malignant (RD, RD-D) cells after exposition to CaEP protocol (electric field intensity: 1000 V/cm and 0.5 mM Ca 2+ ) The left panel: CLSM images present changes in NCX1 (red) intracellular localization and signal intensity between untreated and CaEP cell lines: C2C12 ( A ), C2C12-D ( B ), RD ( C ), RD-D ( D ). The graph shows signal intensity for untreated cell lines (above) and 3 therapy conditions (below): Ca 2+ incubation, EP only and CaEP normalized to untreated cells. The right panel: confocal images present changes in RyR1 (green) signal intensity between untreated and CaEP cell lines: C2C12 ( E ), C2C12-D ( F ), RD ( G ), RD-D ( H ). Both NCX1 and RyR1 protein colocalized with F-actin and cellular nucleus. The graphs NCX1 (left) and RyR1 (right) show a signal intensity for untreated cell lines (above) and 3 therapy conditions (below): Ca 2+ incubation (0.5 mM), EP only (1000 V/cm) and CaEP (0.5 mM; 1000 V/cm) normalized to untreated cells. Fluorescent signal was detected 24 h after CaEP application; 20 μm; n = 3–5. The signal intensity was analyzed by ImageJ software.
    Ryanodine Receptors Expression Primary Antibody Monoclonal Rabbit Anti Ryr1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ryr1 monoclonal antibody
    Ryanodine receptor location. (A) representative image from a Sham left atrial myocyte. Lower image, higher magnification view of area indicated by dotted box. Arrows indicate intra-sarcomeric RyR at the cell periphery. (B) power spectrum of longitudinal scan along the cell. (C) representative image from an AoB left atrial myocyte. Lower image, higher magnification view of area indicated by dotted box. (D) power spectrum of longitudinal scan along the cell. Scale bars represent 5 μm. (E) sarcomere length. Data from 18/3 Sham cells/hearts and 18/3 AoB cells/hearts.
    Ryr1 Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem mouse monoclonal anti ryr
    Expression and triad targeting of Ca V 1.2 constructs. Dysgenic myotubes (Ca V 1.1 -/- ) were reconstituted with <t>GFP-Ca</t> V 1.2, GFP-Ca V 1.2-D4N, GFP-Ca V 1.2-ΔE33, or GFP-Ca V 1.2-ΔE33-D4N and double immunofluorescence labeled with anti-GFP and <t>anti-RyR.</t> All channel constructs are equally expressed and colocalized in clusters with the RyR1, indicative of their correct targeting into t-tubule/SR or plasma membrane/SR junctions. Scale bar, 10 μm.
    Mouse Monoclonal Anti Ryr, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 95/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank 34c anti ryr monoclonal antibody
    Expression and triad targeting of Ca V 1.2 constructs. Dysgenic myotubes (Ca V 1.1 -/- ) were reconstituted with <t>GFP-Ca</t> V 1.2, GFP-Ca V 1.2-D4N, GFP-Ca V 1.2-ΔE33, or GFP-Ca V 1.2-ΔE33-D4N and double immunofluorescence labeled with anti-GFP and <t>anti-RyR.</t> All channel constructs are equally expressed and colocalized in clusters with the RyR1, indicative of their correct targeting into t-tubule/SR or plasma membrane/SR junctions. Scale bar, 10 μm.
    34c Anti Ryr Monoclonal Antibody, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 85/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti ryr monoclonal antibody 34c
    Expression and triad targeting of Ca V 1.2 constructs. Dysgenic myotubes (Ca V 1.1 -/- ) were reconstituted with <t>GFP-Ca</t> V 1.2, GFP-Ca V 1.2-D4N, GFP-Ca V 1.2-ΔE33, or GFP-Ca V 1.2-ΔE33-D4N and double immunofluorescence labeled with anti-GFP and <t>anti-RyR.</t> All channel constructs are equally expressed and colocalized in clusters with the RyR1, indicative of their correct targeting into t-tubule/SR or plasma membrane/SR junctions. Scale bar, 10 μm.
    Anti Ryr Monoclonal Antibody 34c, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse monoclonal anti ryr
    Expression and triad targeting of Ca V 1.2 constructs. Dysgenic myotubes (Ca V 1.1 -/- ) were reconstituted with <t>GFP-Ca</t> V 1.2, GFP-Ca V 1.2-D4N, GFP-Ca V 1.2-ΔE33, or GFP-Ca V 1.2-ΔE33-D4N and double immunofluorescence labeled with anti-GFP and <t>anti-RyR.</t> All channel constructs are equally expressed and colocalized in clusters with the RyR1, indicative of their correct targeting into t-tubule/SR or plasma membrane/SR junctions. Scale bar, 10 μm.
    Mouse Monoclonal Anti Ryr, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1 ryr3
    Immunogold labeling shows i) preferential I band localization of STIM1 and ii) increased presence of Orai1 at the I band following exercise. Left) Representative immunogold EM images showing the localization of <t>RyR1</t> (top), STIM1 (center), and Orai1 (bottom) under control and exercised conditions. Right) Histograms of distances of immunogold particles from the Z line. The cyan line marks the position of the TT at the triad. Brackets with numeric values indicate the percentage of gold particles that fall within the triad-area vs. those that fall within the I-band area. Grey arrow points to the increased presence of Orai1 at the I-band following exercise. Sample size: control, 2 mice/4–8 fibers analyzed; exercised, 3 mice/3–8 fibers analyzed . *p
    Mouse Monoclonal Anti Ryr1 Ryr3, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ryr1 d4e1 rabbit mab
    Immunohistochemical analysis and subcellular localization of <t>RYR1</t> and of the α 1 subunit of the DHPR in single muscle fibers from WT and ryr3 −/− mice. (A) Left and central panels show the staining obtained using rabbit anti-RYR1 mAb <t>D4E1</t> (green in merged image) and mouse anti-Ca v 1.1 (red in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). (B) Left and central panels show the staining obtained using mouse anti-Ca v 1.1 (red in merged image) and rabbit anti-Ca v 1.2 (green in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). The bottom panels show staining of mouse EDL fibers, which are negative for Ca v 1.2 and were used as staining control. All images in B were acquired using the same settings for the laser intensities and acquisition parameters. Images were acquired using a Nikon A1 plus confocal microscope equipped with a Plan Apo 60× oil objective (numerical aperture, 1.4) and stained as described in Materials and methods. Orange pixels show areas of colocalization. Scale bars, 30 µm.
    Ryr1 D4e1 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 34c mouse anti ryr monoclonal antibody
    Immunohistochemical analysis and subcellular localization of <t>RYR1</t> and of the α 1 subunit of the DHPR in single muscle fibers from WT and ryr3 −/− mice. (A) Left and central panels show the staining obtained using rabbit anti-RYR1 mAb <t>D4E1</t> (green in merged image) and mouse anti-Ca v 1.1 (red in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). (B) Left and central panels show the staining obtained using mouse anti-Ca v 1.1 (red in merged image) and rabbit anti-Ca v 1.2 (green in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). The bottom panels show staining of mouse EDL fibers, which are negative for Ca v 1.2 and were used as staining control. All images in B were acquired using the same settings for the laser intensities and acquisition parameters. Images were acquired using a Nikon A1 plus confocal microscope equipped with a Plan Apo 60× oil objective (numerical aperture, 1.4) and stained as described in Materials and methods. Orange pixels show areas of colocalization. Scale bars, 30 µm.
    34c Mouse Anti Ryr Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc monoclonal anti ryr1 antibody
    Hypoxic increase in [Ca 2+ ] i and contraction are diminished in <t>RyR1</t> −/− and RyR1 +/− PASMCs. a Original recordings of hypoxic increase in [Ca 2+ ] i in an RyR1 +/+ and RyR1 −/− cell. Bar graph summarizes the effect of hypoxia
    Monoclonal Anti Ryr1 Antibody, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank mouse monoclonal anti ryr antibody
    Hypoxic increase in [Ca 2+ ] i and contraction are diminished in <t>RyR1</t> −/− and RyR1 +/− PASMCs. a Original recordings of hypoxic increase in [Ca 2+ ] i in an RyR1 +/+ and RyR1 −/− cell. Bar graph summarizes the effect of hypoxia
    Mouse Monoclonal Anti Ryr Antibody, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 87/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1 ryr3 34c
    Quantitative analysis in histologic sections of EDL fibers presenting structural damage following the ES protocol. A – C ) Histologic examination of EDL muscles after ES protocol allowed classification of fibers in 3 main classes: fibers with no apparent sign of damage ( A ); fibers losing striation (dashed oval; B ); and fibers with contractures (asterisks; C ). D ) Quantitative analysis showing the percentage of EDL fibers presenting the different features classified in A – C . Azu, azumolene; Y522S, <t>RYR1</t> Y522S/WT . Scale bar, 10 µm. * P
    Mouse Monoclonal Anti Ryr1 Ryr3 34c, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mouse monoclonal anti ryr antibody
    Quantitative analysis in histologic sections of EDL fibers presenting structural damage following the ES protocol. A – C ) Histologic examination of EDL muscles after ES protocol allowed classification of fibers in 3 main classes: fibers with no apparent sign of damage ( A ); fibers losing striation (dashed oval; B ); and fibers with contractures (asterisks; C ). D ) Quantitative analysis showing the percentage of EDL fibers presenting the different features classified in A – C . Azu, azumolene; Y522S, <t>RYR1</t> Y522S/WT . Scale bar, 10 µm. * P
    Mouse Monoclonal Anti Ryr Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse monoclonal anti ryr antibody
    Quantitative analysis in histologic sections of EDL fibers presenting structural damage following the ES protocol. A – C ) Histologic examination of EDL muscles after ES protocol allowed classification of fibers in 3 main classes: fibers with no apparent sign of damage ( A ); fibers losing striation (dashed oval; B ); and fibers with contractures (asterisks; C ). D ) Quantitative analysis showing the percentage of EDL fibers presenting the different features classified in A – C . Azu, azumolene; Y522S, <t>RYR1</t> Y522S/WT . Scale bar, 10 µm. * P
    Mouse Monoclonal Anti Ryr Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit monoclonal anti ryr1
    <t>ryr3</t> −/− EOM-derived myotubes show fewer calcium waves than WT EOM-derived myotubes. (A) WT (left) and ryr3 −/− (right) confocal image of myotubes loaded with Fluo-4. Spontaneous calcium waves can be seen as lines during image acquisition. Scale bars, 30 µm. (B) Representative Fluo-4 line scan traces of WT (top, black line) and ryr3 −/− (bottom, gray line) myotubes. a.u., arbitrary units. (C) Analysis of the frequency of the spontaneous Ca 2+ transient. Each point represents the number of transients per minute recorded in a single myotube. Each myotube was recorded for 30 s, and the frequency output is given as frequency per minute. Experiments were performed at room temperature. The horizontal black line represents the mean value. Empty circles, WT ( n = 95); gray circles, ryr3 −/− ( n = 22). (D) ΔF/F of the Fluo-4 transients. The number of transients analyzed was n = 9,319 and n = 1,368 for WT and ryr3 −/− , respectively. **, P
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    Abcam mab anti ryr2
    Distribution of <t>RyR2</t> signals in cytoplasm of SMB cells. Representative images double-immunofluorescently stained with the antibodies for RyR2 (green) and ER (red, A) or membrane (red, B) on the slices of SMB-S15 and SMB-PS cells.
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    Millipore monoclonal anti ryr2 antibody
    Immunoblot analysis of Ca 2+ -cycling proteins from WT and KO mice Cardiac protein samples were separated by SDS-PAGE and immunoblotting was performed with antibodies against various Ca 2+ -cycling proteins. a: Representative western blot results of SR Ca 2+ -cycling protein levels in WT and HRC-KO hearts. b: Quantification of SR protein levels in WT and HRC-KO hearts. <t>p-RyR2</t> indicates phosphorylated RyR2 at serine 2809 and 2814; PLN, phospholamban; CSQ, calsequestrin. Data are means ± SEM; n=6 hearts for WT or HRC KO mice. *P
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    Abcam mouse monoclonal anti ryr
    Immunoblot analysis of Ca 2+ -cycling proteins from WT and KO mice Cardiac protein samples were separated by SDS-PAGE and immunoblotting was performed with antibodies against various Ca 2+ -cycling proteins. a: Representative western blot results of SR Ca 2+ -cycling protein levels in WT and HRC-KO hearts. b: Quantification of SR protein levels in WT and HRC-KO hearts. <t>p-RyR2</t> indicates phosphorylated RyR2 at serine 2809 and 2814; PLN, phospholamban; CSQ, calsequestrin. Data are means ± SEM; n=6 hearts for WT or HRC KO mice. *P
    Mouse Monoclonal Anti Ryr, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Recordings of diastolic sarcoplasmic reticulum (SR) Ca 2+ leak after 1 Hz electrical stimulation in normal HEPES 1.8 mM Ca 2+ solution. A, Exemplary recordings show the protocol of quantification of SR Ca 2+ -leak by determination of diastolic Ca 2+ -levels in quiescent atrial cells with 0 Na + /0 Ca 2+ in the external perfusion solution compared to perfusion solution with 0 Na + /0 Ca 2+ +Tetracaine (TET) that inhibits the opening of the ryanodine receptor (RyR2). Recordings were followed by Caffeine (10 mM) induced Ca 2+ depletion of the SR to determine SR Ca 2+ storage B, Diastolic SR Ca 2+ -leak was significantly increased in Low Capacity Runner (LCR) rats compared to High Capacity Runner (HCR) rats. n = 5 animals, n = 4−6 cells from each animal. C, Western blot analyses of the ratio between phosphorylated Serine-2814/RyR2 display a significant higher expression in LCR rats (n = 4) compared to HCR rats (n = 3). D, Representative Western blots. Data are presented as mean±SD.

    Journal: PLoS ONE

    Article Title: Atrial Myocyte Function and Ca2+ Handling Is Associated with Inborn Aerobic Capacity

    doi: 10.1371/journal.pone.0076568

    Figure Lengend Snippet: Recordings of diastolic sarcoplasmic reticulum (SR) Ca 2+ leak after 1 Hz electrical stimulation in normal HEPES 1.8 mM Ca 2+ solution. A, Exemplary recordings show the protocol of quantification of SR Ca 2+ -leak by determination of diastolic Ca 2+ -levels in quiescent atrial cells with 0 Na + /0 Ca 2+ in the external perfusion solution compared to perfusion solution with 0 Na + /0 Ca 2+ +Tetracaine (TET) that inhibits the opening of the ryanodine receptor (RyR2). Recordings were followed by Caffeine (10 mM) induced Ca 2+ depletion of the SR to determine SR Ca 2+ storage B, Diastolic SR Ca 2+ -leak was significantly increased in Low Capacity Runner (LCR) rats compared to High Capacity Runner (HCR) rats. n = 5 animals, n = 4−6 cells from each animal. C, Western blot analyses of the ratio between phosphorylated Serine-2814/RyR2 display a significant higher expression in LCR rats (n = 4) compared to HCR rats (n = 3). D, Representative Western blots. Data are presented as mean±SD.

    Article Snippet: The membranes were blocked with Odyssey blocking buffer (LiCOR) prior to incubation with monoclonal anti-ryanodine receptor (RyR2) (1∶5,000; Thermo Fisher Scientific, Waltham, MA), polyclonal anti-pS2809-RyR2 (1∶1,000; Badrilla, Leeds, UK), and monoclonal anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶100,000; Millipore (Chemicon), Temecula, CA) antibodies overnight at 4°C.

    Techniques: Western Blot, Expressing

    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

    IP 3 R2 and RyR3 colocalized. Confocal images focused on OP processes show beaded pattern of expression IP 3 Rs and RyRs. Scale bars, 10 μm (for all panels). A , OPs soma and processes express RyR3 ( green ) and IP 3 R2 ( red ). Immunoreactivity for IP 3 R2 was strong throughout the cell, whereas RyR3 appeared to be excluded from the nuclear membrane area. Both receptors showed a patchy distribution along processes. B , A process from A enlarged to show frequent colocalization of IP 3 R2 and RyR3. C , Another process from A enlarged to show IP 3 R2 and RyR are not always colocalized. In this process, IP 3 R2 is expressed in small patches along the entire process. RyR expression is limited to larger patches that do not completely overlap with IP 3 R2.

    Journal: The Journal of Neuroscience

    Article Title: Sparks and Puffs in Oligodendrocyte Progenitors: Cross Talk between Ryanodine Receptors and Inositol Trisphosphate Receptors

    doi: 10.1523/JNEUROSCI.21-11-03860.2001

    Figure Lengend Snippet: IP 3 R2 and RyR3 colocalized. Confocal images focused on OP processes show beaded pattern of expression IP 3 Rs and RyRs. Scale bars, 10 μm (for all panels). A , OPs soma and processes express RyR3 ( green ) and IP 3 R2 ( red ). Immunoreactivity for IP 3 R2 was strong throughout the cell, whereas RyR3 appeared to be excluded from the nuclear membrane area. Both receptors showed a patchy distribution along processes. B , A process from A enlarged to show frequent colocalization of IP 3 R2 and RyR3. C , Another process from A enlarged to show IP 3 R2 and RyR are not always colocalized. In this process, IP 3 R2 is expressed in small patches along the entire process. RyR expression is limited to larger patches that do not completely overlap with IP 3 R2.

    Article Snippet: For dual RyR3–IP3 R2 staining, we used an anti-RyR3 antibody (1:100) raised in a goat and an anti-IP3 R2 antibody (1:100) raised in a rabbit (Affinity Bioreagents) diluted in 10% donkey serum, and FITC donkey anti-goat and RRX donkey anti-rabbit secondary antibodies (1:200; Jackson ImmunoResearch).

    Techniques: 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: Western blots were probed with anti-RyR1 (Thermo Scientific mAb 34C MA3-925), anti-Cav 1.1 (Santa Cruz sc-8160) and anti -Myosin (Millipore MAB1628) antibodies followed by the appropriate secondary conjugate and developed using the Super Signal chemiluminescence kit (Thermo scientific 34076) as previously described .

    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: Western blots were probed with anti-RyR1 (Thermo Scientific mAb 34C MA3-925), anti-Cav 1.1 (Santa Cruz sc-8160) and anti -Myosin (Millipore MAB1628) antibodies followed by the appropriate secondary conjugate and developed using the Super Signal chemiluminescence kit (Thermo scientific 34076) as previously described .

    Techniques: Sequencing, Mutagenesis, Polymerase Chain Reaction, Amplification

    Confocal images of CASQ1 and RYR1 co-localization in both myotube and muscle biopsy. Immunocytochemical staining of RYR1 and CASQ1 in patient ( upper panels ) and control myotubes (intermediate panels; scale bar = 10 μm). The lower panels show CASQ1 and RYR1 co-staining in vacuoles of fibers derived from a patient muscle biopsy (scale bar = 50 μm).

    Journal: PLoS ONE

    Article Title: A Calsequestrin-1 Mutation Associated with a Skeletal Muscle Disease Alters Sarcoplasmic Ca2+ Release

    doi: 10.1371/journal.pone.0155516

    Figure Lengend Snippet: Confocal images of CASQ1 and RYR1 co-localization in both myotube and muscle biopsy. Immunocytochemical staining of RYR1 and CASQ1 in patient ( upper panels ) and control myotubes (intermediate panels; scale bar = 10 μm). The lower panels show CASQ1 and RYR1 co-staining in vacuoles of fibers derived from a patient muscle biopsy (scale bar = 50 μm).

    Article Snippet: In patient muscle, CASQ1 was co-localized with RYR1 following incubation of sections with polyclonal anti-CASQ1 (Abcam; 1:500) plus monoclonal anti-RYR1 (Thermo Fischer Scientific, Waltham, MA USA; diluted 1:10), followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes Inc, Eugene, OR, USA), and successively by incubation with Alexa Fluor 546 goat anti-mouse IgG (Molecular Probes).

    Techniques: Staining, Derivative Assay

    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

    Visualization of the spatial distribution of SK2, RyR2, and Ca v 1.2 (AU: arbitrary units).

    Journal: Scientific Reports

    Article Title: Coupling of SK channels, L-type Ca2+ channels, and ryanodine receptors in cardiomyocytes

    doi: 10.1038/s41598-018-22843-3

    Figure Lengend Snippet: Visualization of the spatial distribution of SK2, RyR2, and Ca v 1.2 (AU: arbitrary units).

    Article Snippet: For double labeling experiments, cells were first treated with a primary antibody (anti-SK2 polyclonal antibody (Abcam 111939, Cambridge, MA, USA), which was tested in HEK 293 cells expressing human cardiac SK2 (Supplemental Fig. ); anti-Cav 1.2 monoclonal antibody (NeuroMab Cav1.2 N263/31, Davis, CA, USA) ; or anti-RyR2 monoclonal antibody (Thermo Fisher Scientific, Clone C3-33, Rockford, IL, USA)) , incubated overnight at 4 °C, followed by application of secondary antibodies sequentially with tetramethylrhodamine (TMR) donkey-anti-goat secondary antibody (Thermo Fisher Scientific) followed by Oregon Green 488 goat-anti-mouse (Thermo Fisher Scientific) for 1 hour incubation at RT.

    Techniques:

    Phosphorylation status of ryanodine receptors and SERCA. (A) Representative examples of ryanodine status immunolabeling in fresh and cultured cells after 48 h. (B) Representative examples of phospholamban phosphorylation and phospholamban immunolabeling in fresh and cultured cells after 48 h. Phosphorylation signal is normalized to the fresh level. BDM (2,3-Butanedione 2-monoxime), BLB (Blebbistatin).

    Journal: Cell calcium

    Article Title: Eliminating contraction during culture maintains global and local Ca2+ dynamics in cultured rabbit pacemaker cells

    doi: 10.1016/j.ceca.2018.12.008

    Figure Lengend Snippet: Phosphorylation status of ryanodine receptors and SERCA. (A) Representative examples of ryanodine status immunolabeling in fresh and cultured cells after 48 h. (B) Representative examples of phospholamban phosphorylation and phospholamban immunolabeling in fresh and cultured cells after 48 h. Phosphorylation signal is normalized to the fresh level. BDM (2,3-Butanedione 2-monoxime), BLB (Blebbistatin).

    Article Snippet: We employed either a monoclonal anti-RyR2 antibody (IgG1, clone C3–33, 1:100, ThermoFisher Scientific, Bothell, WA, U.S.), a polyclonal anti–HCN4 antibody (1:100, Alomone Labs), an anti-PLB antibody (1:200, Badrilla), a p-PLB PS-16 antibody (1:200, Badrilla) or a p-RyR2 (2809 site) antibody (1:200, Badrilla).

    Techniques: Immunolabeling, Cell Culture

    Structural changes consequent to PKP2 deletion. a STORM-acquired images of Cav1.2 ( green ) and RyR2 ( purple ) in a single myocyte. b Analysis of RyR2/Cav1.2 overlapping area in transverse ( left ) and longitudinal ( right ) clusters. n = 1335 and 1285 clusters from 30 control and 25 PKP2-cKO cardiomyocytes at 21 dpi, respectively. t -test, * p

    Journal: Nature Communications

    Article Title: Plakophilin-2 is required for transcription of genes that control calcium cycling and cardiac rhythm

    doi: 10.1038/s41467-017-00127-0

    Figure Lengend Snippet: Structural changes consequent to PKP2 deletion. a STORM-acquired images of Cav1.2 ( green ) and RyR2 ( purple ) in a single myocyte. b Analysis of RyR2/Cav1.2 overlapping area in transverse ( left ) and longitudinal ( right ) clusters. n = 1335 and 1285 clusters from 30 control and 25 PKP2-cKO cardiomyocytes at 21 dpi, respectively. t -test, * p

    Article Snippet: Antibodies The following primary antibodies were used: monoclonal mouse anti-PKP2 (K44262M; Biodesign International, Meridian Life Sciences), monoclonal mouse anti-AnkyrinB (N105-17; BioLegend), polyclonal rabbit anti-Cav1.2 (ACC-003; Alomone), polyclonal rabbit anti-CASQ2 (PA1-913; ThermoFisher), monoclonal mouse anti-RyR2 (MA3-916; ThermoFisher), polyclonal rabbit anti-RyR2 phospho serine-2808 and anti-RyR2 phospho serine-2814 (A010-30 and A010-31 respectively; Badrilla), monoclonal mouse anti-TriadinT32 (MA3-927; ThermoFisher), polyclonal rabbit anti-NCX (sc-32881, SantaCruz), polyclonal rabbit anti-SERCA2 (PA5-29380; ThermoFisher), monoclonal mouse anti-phospholamban (sc-393990; SantaCruz), polyclonal rabbit anti-phospholamban phospho serine-16 (sc-12963; SantaCruz) and phospho threonine-17 (sc-17024; SantaCruz), GAPDH (G109A; Fitzerald) monoclonal mouse anti-Cx43 (Clone 4E6.2, Millipore), polyclonal rabbit anti-β-catenin (C2206; Sigma), monoclonal mouse anti-JPH2 (sc-37086, SantaCruz), monoclonal mouse anti-Bin1 (Clone 99D; Sigma), polyclonal rabbit anti-PKC and anti-phospho PKC (#2056 and #9375; Cell Signaling), polyclonal rabbit anti-CaMKII (PA5-22168; ThermoFisher), monoclonal mouse anti-CaMKII phospho threonine-286 (MA1-047; ThermoFisher), polyclonal rabbit Desmocollin-2 (ab72792; Abcam), polyclonal rabbit anti-Nav1.5 (S0819; Sigma).

    Techniques:

    MCU IF distribution in the context of RyR2 and mitochondrial shape (mtHsp70) in adult mouse cardiomyocites visualized by the Zeiss LSM880 Airyscan super-resolution system. A , validation of two anti-MCU antibodies (Sigma and Cell Signaling as labeled on

    Journal: The Journal of Biological Chemistry

    Article Title: Strategic Positioning and Biased Activity of the Mitochondrial Calcium Uniporter in Cardiac Muscle *

    doi: 10.1074/jbc.M116.755496

    Figure Lengend Snippet: MCU IF distribution in the context of RyR2 and mitochondrial shape (mtHsp70) in adult mouse cardiomyocites visualized by the Zeiss LSM880 Airyscan super-resolution system. A , validation of two anti-MCU antibodies (Sigma and Cell Signaling as labeled on

    Article Snippet: Anti-HSP70 mouse monoclonal (MA3–028, WB 1:500, IF 1:50) and anti-RyR2 mouse monoclonal (MA3–916, WB 1:500, IF 1:50) were from Thermo Scientific.

    Techniques: Labeling

    MCU and RyR2 IF colocalization resolved by the Vutara 350 single-molecule super-resolution microscopy in adult cardiomyocytes. A , mouse adult cardiomyocites were labeled with anti-MCU (AF 647; red ) and anti-RyR2 (CF TM 568; red ). The left panels show point

    Journal: The Journal of Biological Chemistry

    Article Title: Strategic Positioning and Biased Activity of the Mitochondrial Calcium Uniporter in Cardiac Muscle *

    doi: 10.1074/jbc.M116.755496

    Figure Lengend Snippet: MCU and RyR2 IF colocalization resolved by the Vutara 350 single-molecule super-resolution microscopy in adult cardiomyocytes. A , mouse adult cardiomyocites were labeled with anti-MCU (AF 647; red ) and anti-RyR2 (CF TM 568; red ). The left panels show point

    Article Snippet: Anti-HSP70 mouse monoclonal (MA3–028, WB 1:500, IF 1:50) and anti-RyR2 mouse monoclonal (MA3–916, WB 1:500, IF 1:50) were from Thermo Scientific.

    Techniques: Microscopy, Labeling

    Mitochondrial content in SR and mitochondrial fractions differ in their protein profile and membrane (jSR) associations. A , anti-RyR2 IF (AF 488; green ) and MtTrRed (MtTr; red shades ) distribution in glass-mounted SR and mitochondrial fractions (as labeled).

    Journal: The Journal of Biological Chemistry

    Article Title: Strategic Positioning and Biased Activity of the Mitochondrial Calcium Uniporter in Cardiac Muscle *

    doi: 10.1074/jbc.M116.755496

    Figure Lengend Snippet: Mitochondrial content in SR and mitochondrial fractions differ in their protein profile and membrane (jSR) associations. A , anti-RyR2 IF (AF 488; green ) and MtTrRed (MtTr; red shades ) distribution in glass-mounted SR and mitochondrial fractions (as labeled).

    Article Snippet: Anti-HSP70 mouse monoclonal (MA3–028, WB 1:500, IF 1:50) and anti-RyR2 mouse monoclonal (MA3–916, WB 1:500, IF 1:50) were from Thermo Scientific.

    Techniques: Labeling

    Ca 2+ signaling activity promotes MCU recruitment to dyad (RyR2) areas. Colocalization of MCU and RyR2 fluorescence was determined in adult cardiomyocytes that were perfused with (1 m m ) or quasi-without (2 μ m ) Ca 2+ and field-stimulated for 10 min

    Journal: The Journal of Biological Chemistry

    Article Title: Strategic Positioning and Biased Activity of the Mitochondrial Calcium Uniporter in Cardiac Muscle *

    doi: 10.1074/jbc.M116.755496

    Figure Lengend Snippet: Ca 2+ signaling activity promotes MCU recruitment to dyad (RyR2) areas. Colocalization of MCU and RyR2 fluorescence was determined in adult cardiomyocytes that were perfused with (1 m m ) or quasi-without (2 μ m ) Ca 2+ and field-stimulated for 10 min

    Article Snippet: Anti-HSP70 mouse monoclonal (MA3–028, WB 1:500, IF 1:50) and anti-RyR2 mouse monoclonal (MA3–916, WB 1:500, IF 1:50) were from Thermo Scientific.

    Techniques: Activity Assay, Fluorescence

    Validation of MCU IF and colocalization between MCU and RyR2 in cardiac mitochondrial fractions. Shown is the distribution of anti-MCU IF (AF 647; red ), anti-RyR2 (AF 488; green ), and the mitochondrial matrix probe MtTrRed ( MtTr ) fluorescence ( purple

    Journal: The Journal of Biological Chemistry

    Article Title: Strategic Positioning and Biased Activity of the Mitochondrial Calcium Uniporter in Cardiac Muscle *

    doi: 10.1074/jbc.M116.755496

    Figure Lengend Snippet: Validation of MCU IF and colocalization between MCU and RyR2 in cardiac mitochondrial fractions. Shown is the distribution of anti-MCU IF (AF 647; red ), anti-RyR2 (AF 488; green ), and the mitochondrial matrix probe MtTrRed ( MtTr ) fluorescence ( purple

    Article Snippet: Anti-HSP70 mouse monoclonal (MA3–028, WB 1:500, IF 1:50) and anti-RyR2 mouse monoclonal (MA3–916, WB 1:500, IF 1:50) were from Thermo Scientific.

    Techniques: Fluorescence

    Immunohistochemistry of ryanodine receptor 2 (RyR2) expression in transverse sections of the heart + lungs of rats without arrhythmia. Expression of RyR2 in the (A) LV, (B) RV, (C) LAA and (D) LPV. Scale bar, 20 µm. (E) Masson's trichrome-stained transverse section of the whole heart, boxes represent the locations of A, B, C and D. Scale bar, 5,000 µm. LV, left ventricle; RV, right ventricle; LAA, left atrial appendage; LPV, left pulmonary vein.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Expression of connexin 43, ion channels and Ca2+-handling proteins in rat pulmonary vein cardiomyocytes

    doi: 10.3892/etm.2016.3766

    Figure Lengend Snippet: Immunohistochemistry of ryanodine receptor 2 (RyR2) expression in transverse sections of the heart + lungs of rats without arrhythmia. Expression of RyR2 in the (A) LV, (B) RV, (C) LAA and (D) LPV. Scale bar, 20 µm. (E) Masson's trichrome-stained transverse section of the whole heart, boxes represent the locations of A, B, C and D. Scale bar, 5,000 µm. LV, left ventricle; RV, right ventricle; LAA, left atrial appendage; LPV, left pulmonary vein.

    Article Snippet: Antibodies Eight primary antibodies were used in this study: Mouse anti-Cx43 (polyclonal; 1:200; cat. no. AB1727; Chemicon, Livingston, UK); rabbit anti-Cx43 (polyclonal; 1:200; cat. no. C6219; Sigma-Alrich, St. Louis, MO, USA); rabbit anti-HCN4 (polyclonal; 1:50; cat. no. APC-052; Alomone Labs, Jerusalem, Israel); mouse anti-caveolin-3 (Cav3) (monoclonal; 1:500; cat. no. 610420; BD Biosciences, Oxford, UK); mouse anti-RyR2 (clone C3-33; monoclonal; 1:100; cat. no. MA3-916; Thermo Fisher Scientific); mouse anti-SERCA1/2 (clone Y/1F4; monocolonal; 1:100; cat. no. A010-21AP; Badrilla, Ltd., Leeds, UK); rabbit anti-Nav 1.5, corresponding to amino acid residues 493–511 (polyclonal; 1:50; cat. no. ASC-005; Alomone Labs); and rabbit anti-Kir2.1, corresponding to amino acid residues 392–410 (polyclonal; 1:50; cat. no. APC-026; Alomone Labs).

    Techniques: Immunohistochemistry, Expressing, Staining

    Predominant expression of IP 3 R type 1 in sub-PM SR encourages SPCU. (A) Ca 2+ stores visualized with fluo-3FF consisted of a sub-PM SR network and some central formation. (B) Immunolocalization of IP 3 R type 1 and RyRs using a double-staining protocol (see Section 2 ). Confocal image of Alexa Fluor 488 fluorescence (green), showing type 1 IP 3 R distribution (middle), and confocal image of Alexa Fluor 633 fluorescence (red), showing RyR distribution (right), are overlaid (left). (C) The fluo-4-loaded SMC was stimulated with 10 μM CCh and 5 mM caffeine (with 10-min interval). Two galleries of images (acquired at 42 Hz) highlight the difference in the initial phase of the responses. (D) The temporal profiles of the fluorescence at three regions outlined in red, green and magenta (left) are shown in corresponding colour.

    Journal: Cell Calcium

    Article Title: Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

    doi: 10.1016/j.ceca.2007.04.012

    Figure Lengend Snippet: Predominant expression of IP 3 R type 1 in sub-PM SR encourages SPCU. (A) Ca 2+ stores visualized with fluo-3FF consisted of a sub-PM SR network and some central formation. (B) Immunolocalization of IP 3 R type 1 and RyRs using a double-staining protocol (see Section 2 ). Confocal image of Alexa Fluor 488 fluorescence (green), showing type 1 IP 3 R distribution (middle), and confocal image of Alexa Fluor 633 fluorescence (red), showing RyR distribution (right), are overlaid (left). (C) The fluo-4-loaded SMC was stimulated with 10 μM CCh and 5 mM caffeine (with 10-min interval). Two galleries of images (acquired at 42 Hz) highlight the difference in the initial phase of the responses. (D) The temporal profiles of the fluorescence at three regions outlined in red, green and magenta (left) are shown in corresponding colour.

    Article Snippet: RyRs were detected with a monoclonal anti-RyR antibody derived (Sigma–Aldrich) from the 34C hybridoma (produced by the fusion of P3X 63 Ag8.653 myeloma cells and spleen cells from Balb/c mice).

    Techniques: Expressing, Double Staining, Fluorescence

    Subcellular localization of myotubularin in skeletal muscle . (A) Detection of endogenous myotubularin by western blot. Left panel shows the presence of myotubularin in wild-type (WT) but not KO skeletal muscle and heart. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunoreactivity was used as an internal control. Right panel illustrates the level of expression of myotubularin 6 weeks after adeno-associated virus (AAV) injection; 75 and 7.5 µg of proteins were loaded for phosphate-buffered saline (PBS) (LTA) and AAV (RTA)-injected WT muscles, respectively. A lower exposure of myotubularin band in RTA (RTA*) is shown to illustrate the doublet. ( B ) Localization of endogenous myotubularin in skeletal muscle. Semithin cryosections (0.5 µm) of WT muscle were stained for MTM1 (myotubularin), α-actinin (Z-lines), ryanodine receptor (RYR) and triadin (both in triads). Occasional α-actinin-positive Z-lines appear yellow when oblique orientation of the sarcomeres in sections superimpose the Z-lines with the adjacent triadic regions. The bar represents 5 µm. ( C ) Localization of overexpressed myotubularin in skeletal muscle. Semithin Mtm1 -deficient (upper panels) and WT-AAV (middle and lower panels) muscle cryosections were stained for MTM1, SERCA1 (sarco-endoplasmic reticulum calcium ATPase 1) and α-actinin. Myotubularin antibody binds aspecifically to a region between I-bands in WT and KO myofibers, which probably corresponds to the M-band. Note myotubularin location at the sarcolemma (arrowheads) and around the Z-line (arrows) in WT-AAV muscle. The Z-line and I-band (endoplasmic reticulum) were labeled with antibodies against α-actinin and SERCA1, respectively. The bar represents 5 µm. ( D ) Subcellular localization of overexpressed myotubularin by immunoelectron microscopy. Triads (arrows) are labeled by r1947-coupled gold particles. Note the position of mitochondria (m, bar represents 200 nm). ( E ) Subcellular fractionation of skeletal muscle overexpressing myotubularin (upper panel) and wild-type muscle (lower). Proteins from total homogenate (HT), S1 (supernatant at 1000 g ), S2 (10 000 g ), S3 (100 000 g ) and the corresponding pellet fractions (P1, P2 and P3) were analyzed by immunoblotting. Myotubularin is distributed along all fractions. We used myosin heavy chain (MHC) and GAPDH as internal controls for cytosolic fractions. GRP78/BiP and caveolin 3 (CAV3) immunoreactivities are indicative of membrane fractions. Antibody r1947 was used in all illustrated experiments.

    Journal: Human Molecular Genetics

    Article Title: AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis

    doi: 10.1093/hmg/ddn112

    Figure Lengend Snippet: Subcellular localization of myotubularin in skeletal muscle . (A) Detection of endogenous myotubularin by western blot. Left panel shows the presence of myotubularin in wild-type (WT) but not KO skeletal muscle and heart. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunoreactivity was used as an internal control. Right panel illustrates the level of expression of myotubularin 6 weeks after adeno-associated virus (AAV) injection; 75 and 7.5 µg of proteins were loaded for phosphate-buffered saline (PBS) (LTA) and AAV (RTA)-injected WT muscles, respectively. A lower exposure of myotubularin band in RTA (RTA*) is shown to illustrate the doublet. ( B ) Localization of endogenous myotubularin in skeletal muscle. Semithin cryosections (0.5 µm) of WT muscle were stained for MTM1 (myotubularin), α-actinin (Z-lines), ryanodine receptor (RYR) and triadin (both in triads). Occasional α-actinin-positive Z-lines appear yellow when oblique orientation of the sarcomeres in sections superimpose the Z-lines with the adjacent triadic regions. The bar represents 5 µm. ( C ) Localization of overexpressed myotubularin in skeletal muscle. Semithin Mtm1 -deficient (upper panels) and WT-AAV (middle and lower panels) muscle cryosections were stained for MTM1, SERCA1 (sarco-endoplasmic reticulum calcium ATPase 1) and α-actinin. Myotubularin antibody binds aspecifically to a region between I-bands in WT and KO myofibers, which probably corresponds to the M-band. Note myotubularin location at the sarcolemma (arrowheads) and around the Z-line (arrows) in WT-AAV muscle. The Z-line and I-band (endoplasmic reticulum) were labeled with antibodies against α-actinin and SERCA1, respectively. The bar represents 5 µm. ( D ) Subcellular localization of overexpressed myotubularin by immunoelectron microscopy. Triads (arrows) are labeled by r1947-coupled gold particles. Note the position of mitochondria (m, bar represents 200 nm). ( E ) Subcellular fractionation of skeletal muscle overexpressing myotubularin (upper panel) and wild-type muscle (lower). Proteins from total homogenate (HT), S1 (supernatant at 1000 g ), S2 (10 000 g ), S3 (100 000 g ) and the corresponding pellet fractions (P1, P2 and P3) were analyzed by immunoblotting. Myotubularin is distributed along all fractions. We used myosin heavy chain (MHC) and GAPDH as internal controls for cytosolic fractions. GRP78/BiP and caveolin 3 (CAV3) immunoreactivities are indicative of membrane fractions. Antibody r1947 was used in all illustrated experiments.

    Article Snippet: Other antibodies used in this study are: monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase (MAB374, Chemicon), caveolin 3 (610420, BD Transduction Laboratories), pan-myosin (MF20, Developmental Studies Hybridoma Bank) and rabbit polyclonal anti-GRP78 (PA1-014, Affinity BioReagents) for immunoblot analysis, and monoclonal anti-ryanodine receptor (clone 34C, Sigma), anti-triadin (clone IIG12, Sigma), anti-SERCA1 (sarco-endoplasmic reticulum calcium ATPase 1) (MA3-911, Affinity BioReagents) and anti-α-actinin (EA-53, Sigma) for immunohistochemistry.

    Techniques: Western Blot, Expressing, Injection, Staining, Labeling, Immuno-Electron Microscopy, Fractionation

    Diagram of hypothetical model of the involvement of IP 3 - and ryanodine-sensitive Ca 2+ stores in the regulation of cytoplasmic [Ca 2+ ] in an individual varicosity Ca 2+ entry via voltage-sensitive Ca 2+ channels following membrane depolarization (Ψ) leads to direct elevation of cytosolic Ca 2+ level and to release (red arrow) of Ca 2+ from the ryanodine-sensitive Ca 2+ store (at left). Activation of bradykinin (BK) receptors leads to the formation of IP 3 via phospholipase C (PLC) and subsequent release (red arrow) of Ca 2+ from the IP 3 -sensitive Ca 2+ store (at right). Ca 2+ released (dashed black arrow) from the IP 3 -sensitive Ca 2+ store induces secondarily the release of Ca 2+ from the ryanodine-sensitive Ca 2+ store.

    Journal: The Journal of Physiology

    Article Title: Functional IP3- and ryanodine-sensitive calcium stores in presynaptic varicosities of NG108-15 (rodent neuroblastoma x glioma hybrid) cells

    doi: 10.1111/j.1469-7793.2000.00307.x

    Figure Lengend Snippet: Diagram of hypothetical model of the involvement of IP 3 - and ryanodine-sensitive Ca 2+ stores in the regulation of cytoplasmic [Ca 2+ ] in an individual varicosity Ca 2+ entry via voltage-sensitive Ca 2+ channels following membrane depolarization (Ψ) leads to direct elevation of cytosolic Ca 2+ level and to release (red arrow) of Ca 2+ from the ryanodine-sensitive Ca 2+ store (at left). Activation of bradykinin (BK) receptors leads to the formation of IP 3 via phospholipase C (PLC) and subsequent release (red arrow) of Ca 2+ from the IP 3 -sensitive Ca 2+ store (at right). Ca 2+ released (dashed black arrow) from the IP 3 -sensitive Ca 2+ store induces secondarily the release of Ca 2+ from the ryanodine-sensitive Ca 2+ store.

    Article Snippet: The following primary antibodies were used: anti-synaptophysin monoclonal antibody (1:100 dilution; Calbiochem), anti-syntaxin 1 polyclonal antibody (1:100 dilution; Alomone Laboratories), anti-IP3 receptor polyclonal antibody or anti-ryanodine receptor monoclonal antibody (1:100 dilution each; Chemicon).

    Techniques: Activation Assay, Planar Chromatography

    Schematic of RCMV r129 gene and mutants. (A) Schematic representation of the r129 gene and recombinant r129-His protein and mutants produced for this study. (B) r129-His protein and mutants expressed in E. coli and refolded as described in Materials and

    Journal: Journal of Virology

    Article Title: Cytomegalovirus CC Chemokine Promotes Immune Cell Migration

    doi: 10.1128/JVI.00452-12

    Figure Lengend Snippet: Schematic of RCMV r129 gene and mutants. (A) Schematic representation of the r129 gene and recombinant r129-His protein and mutants produced for this study. (B) r129-His protein and mutants expressed in E. coli and refolded as described in Materials and

    Article Snippet: BALB/c mice were subjected to peritoneal injection twice, 21 days apart, with 20 μg of r129-His emulsified with the Sigma adjuvant system (catalog no. S6322-1VL; Sigma).

    Techniques: Recombinant, Produced

    RCMV r129 mutants compete with r129-WT chemotactic stimulus. Shown are the results of transwell migration assays using rat macrophage cells stimulated with 10 mg/ml WT r129 protein and/or increasing concentrations of the mutant proteins F43A (A), C31A

    Journal: Journal of Virology

    Article Title: Cytomegalovirus CC Chemokine Promotes Immune Cell Migration

    doi: 10.1128/JVI.00452-12

    Figure Lengend Snippet: RCMV r129 mutants compete with r129-WT chemotactic stimulus. Shown are the results of transwell migration assays using rat macrophage cells stimulated with 10 mg/ml WT r129 protein and/or increasing concentrations of the mutant proteins F43A (A), C31A

    Article Snippet: BALB/c mice were subjected to peritoneal injection twice, 21 days apart, with 20 μg of r129-His emulsified with the Sigma adjuvant system (catalog no. S6322-1VL; Sigma).

    Techniques: Migration, Mutagenesis

    The RCMV r129 protein induces cellular migration. Transwell migration assays were performed with either a rat macrophage cell line (A) or primary rat peripheral blood lymphocytes (B) using various concentrations of the RCMV-r129 protein (black bars) or

    Journal: Journal of Virology

    Article Title: Cytomegalovirus CC Chemokine Promotes Immune Cell Migration

    doi: 10.1128/JVI.00452-12

    Figure Lengend Snippet: The RCMV r129 protein induces cellular migration. Transwell migration assays were performed with either a rat macrophage cell line (A) or primary rat peripheral blood lymphocytes (B) using various concentrations of the RCMV-r129 protein (black bars) or

    Article Snippet: BALB/c mice were subjected to peritoneal injection twice, 21 days apart, with 20 μg of r129-His emulsified with the Sigma adjuvant system (catalog no. S6322-1VL; Sigma).

    Techniques: Migration

    (A, C, and D) RCMV r129 is expressed with late viral expression kinetics. RCMV r129 mRNA was detected by quantitative RT-PCR using a gene-specific primer and probe set. RCMV-infected fibroblasts were harvested at 0 (mock infected), 8, 24, and 72 hpi.

    Journal: Journal of Virology

    Article Title: Cytomegalovirus CC Chemokine Promotes Immune Cell Migration

    doi: 10.1128/JVI.00452-12

    Figure Lengend Snippet: (A, C, and D) RCMV r129 is expressed with late viral expression kinetics. RCMV r129 mRNA was detected by quantitative RT-PCR using a gene-specific primer and probe set. RCMV-infected fibroblasts were harvested at 0 (mock infected), 8, 24, and 72 hpi.

    Article Snippet: BALB/c mice were subjected to peritoneal injection twice, 21 days apart, with 20 μg of r129-His emulsified with the Sigma adjuvant system (catalog no. S6322-1VL; Sigma).

    Techniques: Expressing, Quantitative RT-PCR, Infection

    RCMV r129 binds rat CC chemokine receptors. Vero cells were plated in 96-well plates and infected with adenoviruses expressing rat CC chemokine receptors. r129 was conjugated to IRDye 800CW, and 0.5 μg/ml of the ligand was added to Vero cells

    Journal: Journal of Virology

    Article Title: Cytomegalovirus CC Chemokine Promotes Immune Cell Migration

    doi: 10.1128/JVI.00452-12

    Figure Lengend Snippet: RCMV r129 binds rat CC chemokine receptors. Vero cells were plated in 96-well plates and infected with adenoviruses expressing rat CC chemokine receptors. r129 was conjugated to IRDye 800CW, and 0.5 μg/ml of the ligand was added to Vero cells

    Article Snippet: BALB/c mice were subjected to peritoneal injection twice, 21 days apart, with 20 μg of r129-His emulsified with the Sigma adjuvant system (catalog no. S6322-1VL; Sigma).

    Techniques: Infection, Expressing

    RCMV r129 induces the migration of CM/naïve CD4 T cells. Primary splenocytes were subfractionated into separate T cell populations using antibodies directed against CD4, CD8, and the activation marker CD62L. Results of the analyses of the sorted

    Journal: Journal of Virology

    Article Title: Cytomegalovirus CC Chemokine Promotes Immune Cell Migration

    doi: 10.1128/JVI.00452-12

    Figure Lengend Snippet: RCMV r129 induces the migration of CM/naïve CD4 T cells. Primary splenocytes were subfractionated into separate T cell populations using antibodies directed against CD4, CD8, and the activation marker CD62L. Results of the analyses of the sorted

    Article Snippet: BALB/c mice were subjected to peritoneal injection twice, 21 days apart, with 20 μg of r129-His emulsified with the Sigma adjuvant system (catalog no. S6322-1VL; Sigma).

    Techniques: Migration, Activation Assay, Marker

    In vitro migration of r129 mutants. Transwell migration assays were performed using rat alveolar macrophages (A), primary rat PBMC (B), primary rat splenocytes (C), and primary rat bone marrow lymphocytes (D) stimulated with 0.1 ng/ml of r129-His-WT,

    Journal: Journal of Virology

    Article Title: Cytomegalovirus CC Chemokine Promotes Immune Cell Migration

    doi: 10.1128/JVI.00452-12

    Figure Lengend Snippet: In vitro migration of r129 mutants. Transwell migration assays were performed using rat alveolar macrophages (A), primary rat PBMC (B), primary rat splenocytes (C), and primary rat bone marrow lymphocytes (D) stimulated with 0.1 ng/ml of r129-His-WT,

    Article Snippet: BALB/c mice were subjected to peritoneal injection twice, 21 days apart, with 20 μg of r129-His emulsified with the Sigma adjuvant system (catalog no. S6322-1VL; Sigma).

    Techniques: In Vitro, Migration

    RCMV r129 induces the migration of T cells. Primary splenocytes were subfractionated into T cell, B cell, and non-T cell and non-B cell populations using antibodies directed against CD3 and CD45R. Results of the analyses of the sorted cells are depicted

    Journal: Journal of Virology

    Article Title: Cytomegalovirus CC Chemokine Promotes Immune Cell Migration

    doi: 10.1128/JVI.00452-12

    Figure Lengend Snippet: RCMV r129 induces the migration of T cells. Primary splenocytes were subfractionated into T cell, B cell, and non-T cell and non-B cell populations using antibodies directed against CD3 and CD45R. Results of the analyses of the sorted cells are depicted

    Article Snippet: BALB/c mice were subjected to peritoneal injection twice, 21 days apart, with 20 μg of r129-His emulsified with the Sigma adjuvant system (catalog no. S6322-1VL; Sigma).

    Techniques: Migration

    US28 induces constitutively activates ß-catenin signaling. US28 is expressed and functional in NIH-3T3 cells. A, Whole cell binding of [ 125 I]-CCL5 on NIH-3T3 cells expressing wild-type (WT), mutant R 129 A or HA-tagged US28 is displaced by fractalkine (CX3CL1). B, US28-WT constitutively stimulates inositol phosphate (IPx) accumulation in NIH-3T3, while the non-G protein-coupling mutant US28-R 129 A shows no activation. C, Total cell extracts of NIH-3T3 cells stably expressing US28, the non G-protein-coupling mutant and empty plasmid control (mock) were analysed on Western blot with antibodies recognizing the non-phospho (active) ß-catenin, total ß-catenin and actin. D, NIH-3T3 cells stably expressing US28 and the non G-protein coupling US28 mutant R 129 A were transfected with the Tcf-Lef reportergene construct. Luciferase activity was measured 24 h after transfection. E, US28 dose-dependently induces Tcf-Lef transcriptional activation in HEK293T cells. The non-G protein-coupling mutant US28 R129A does not display activation of the reportergene at 25 ng DNA transfected (dark grey bar). Treatment of HEK293T cells transfected with 25 ng US28 DNA with inverse agonist VUF 6064 (10 µM) prevents activation of Tcf-Lef reortergene (light grey bar). F, HEK293T cells transfected with the human chemokine receptor CCR1 and the Tcf-Lef reportergene construct do not show Tcf-Lef activation nor after exposure to 100 nM CCL5 (RANTES). US28 expressing HEK293T cells display constitutive signaling to the Tcf-Lef reportergene, which is significantly enhanced by exposure to 100 nM CCL5 (RANTES).

    Journal: PLoS ONE

    Article Title: Constitutive ss-Catenin Signaling by the Viral Chemokine Receptor US28

    doi: 10.1371/journal.pone.0048935

    Figure Lengend Snippet: US28 induces constitutively activates ß-catenin signaling. US28 is expressed and functional in NIH-3T3 cells. A, Whole cell binding of [ 125 I]-CCL5 on NIH-3T3 cells expressing wild-type (WT), mutant R 129 A or HA-tagged US28 is displaced by fractalkine (CX3CL1). B, US28-WT constitutively stimulates inositol phosphate (IPx) accumulation in NIH-3T3, while the non-G protein-coupling mutant US28-R 129 A shows no activation. C, Total cell extracts of NIH-3T3 cells stably expressing US28, the non G-protein-coupling mutant and empty plasmid control (mock) were analysed on Western blot with antibodies recognizing the non-phospho (active) ß-catenin, total ß-catenin and actin. D, NIH-3T3 cells stably expressing US28 and the non G-protein coupling US28 mutant R 129 A were transfected with the Tcf-Lef reportergene construct. Luciferase activity was measured 24 h after transfection. E, US28 dose-dependently induces Tcf-Lef transcriptional activation in HEK293T cells. The non-G protein-coupling mutant US28 R129A does not display activation of the reportergene at 25 ng DNA transfected (dark grey bar). Treatment of HEK293T cells transfected with 25 ng US28 DNA with inverse agonist VUF 6064 (10 µM) prevents activation of Tcf-Lef reortergene (light grey bar). F, HEK293T cells transfected with the human chemokine receptor CCR1 and the Tcf-Lef reportergene construct do not show Tcf-Lef activation nor after exposure to 100 nM CCL5 (RANTES). US28 expressing HEK293T cells display constitutive signaling to the Tcf-Lef reportergene, which is significantly enhanced by exposure to 100 nM CCL5 (RANTES).

    Article Snippet: NIH-3T3-stable cell lines (Mock, US28, HA-US28 and US28-R129 A mutant) were kept under the selective pressure of neomycin (400 µg/ml) (Sigma) to ensure homogenous receptor expression.

    Techniques: Functional Assay, Binding Assay, Expressing, Mutagenesis, Activation Assay, Stable Transfection, Plasmid Preparation, Western Blot, Transfection, Construct, Luciferase, Activity Assay

    Classical Wnt/Frizzled/ß-catenin signaling is not involved in US28-mediated Tcf-Lef activation. A, Western blot analysis of total cell extracts of NIH-3T3 cells, stably expressing US28 or an empty plasmid (mock) which were treated with Wnt3a- (overnight, 200 ng/ml) and vehicle-treated mock cells. The blot was probed with antibodies recognizing the non-phosphorylated (active ß-catenin, total ß-catenin and actin. A representative blot is shown and normalized quantifications of (active) ß-catenin of independent experiments are shown below the blot. B, Western blot analysis of total cell extracts of NIH-3T3 cells stably expressing US28, the non G-protein coupling US28 mutant R 129 A or an empty plasmid (mock) and Wnt3a-treated mock cells. The blot was probed with antibodies recognizing Lrp6-phospho-ser 1490 and actin. A representative blot is shown and normalized quantifications of Lrp6-phospho-ser 1490 of independent experiments are shown below the blot. C, HEK293T cells co-transfected with the Tcf-Lef reporter gene construct and either US28-expressing or an empty control plasmid (mock) exposed to Wnt3a (overnight, 200 ng/ml). Luciferase activity was measured 24 hr after transfection and is displayed here as the percentage of the non-treated mock control that is set at 100%. D, HEK293T cells co- transfected with the Tcf-Lef reportergene and an US28-expressing construct or empty plasmid control (mock) were exposed to various concentrations (ON, 10–25 µM) of the COX2 inhibitor celecoxib (Cxb). Tcf-Lef reporter gene activation was measured 24 hr after transfection and is displayed here as the percentage of the mock control that is set at 100%.

    Journal: PLoS ONE

    Article Title: Constitutive ss-Catenin Signaling by the Viral Chemokine Receptor US28

    doi: 10.1371/journal.pone.0048935

    Figure Lengend Snippet: Classical Wnt/Frizzled/ß-catenin signaling is not involved in US28-mediated Tcf-Lef activation. A, Western blot analysis of total cell extracts of NIH-3T3 cells, stably expressing US28 or an empty plasmid (mock) which were treated with Wnt3a- (overnight, 200 ng/ml) and vehicle-treated mock cells. The blot was probed with antibodies recognizing the non-phosphorylated (active ß-catenin, total ß-catenin and actin. A representative blot is shown and normalized quantifications of (active) ß-catenin of independent experiments are shown below the blot. B, Western blot analysis of total cell extracts of NIH-3T3 cells stably expressing US28, the non G-protein coupling US28 mutant R 129 A or an empty plasmid (mock) and Wnt3a-treated mock cells. The blot was probed with antibodies recognizing Lrp6-phospho-ser 1490 and actin. A representative blot is shown and normalized quantifications of Lrp6-phospho-ser 1490 of independent experiments are shown below the blot. C, HEK293T cells co-transfected with the Tcf-Lef reporter gene construct and either US28-expressing or an empty control plasmid (mock) exposed to Wnt3a (overnight, 200 ng/ml). Luciferase activity was measured 24 hr after transfection and is displayed here as the percentage of the non-treated mock control that is set at 100%. D, HEK293T cells co- transfected with the Tcf-Lef reportergene and an US28-expressing construct or empty plasmid control (mock) were exposed to various concentrations (ON, 10–25 µM) of the COX2 inhibitor celecoxib (Cxb). Tcf-Lef reporter gene activation was measured 24 hr after transfection and is displayed here as the percentage of the mock control that is set at 100%.

    Article Snippet: NIH-3T3-stable cell lines (Mock, US28, HA-US28 and US28-R129 A mutant) were kept under the selective pressure of neomycin (400 µg/ml) (Sigma) to ensure homogenous receptor expression.

    Techniques: Activation Assay, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Transfection, Construct, Luciferase, Activity Assay

    Neither contractile proteins nor excitation-contraction coupling proteins are altered in PH diaphragm muscles. A : Stain free images of myosin heavy chain (MyHC) and western blots of troponin (Tn) T. B : the expression levels of MyHC or TnT normalized to actin content. C : electrophoretically separated MyHC isoforms. D : percentage distribution of MyHC isoforms: I, slow myosin isoform; IIa, IId/x, and IIb, fast myosin isoforms. E : representative western blots of ryanodine receptor (RyR1), dihydropyridine receptor α2 subunit (DHPR), sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) 1, and SERCA2. F : the expression levels of RyR1, DHPR, SERCA1, or SERCA2 normalized to actin content. Control (CNT), white bars; AIA, black bars. Data represent mean ± SD for 4–8 rats in each group.

    Journal: PLoS ONE

    Article Title: Superoxide dismutase/catalase mimetic EUK-134 prevents diaphragm muscle weakness in monocrotalin-induced pulmonary hypertension

    doi: 10.1371/journal.pone.0169146

    Figure Lengend Snippet: Neither contractile proteins nor excitation-contraction coupling proteins are altered in PH diaphragm muscles. A : Stain free images of myosin heavy chain (MyHC) and western blots of troponin (Tn) T. B : the expression levels of MyHC or TnT normalized to actin content. C : electrophoretically separated MyHC isoforms. D : percentage distribution of MyHC isoforms: I, slow myosin isoform; IIa, IId/x, and IIb, fast myosin isoforms. E : representative western blots of ryanodine receptor (RyR1), dihydropyridine receptor α2 subunit (DHPR), sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) 1, and SERCA2. F : the expression levels of RyR1, DHPR, SERCA1, or SERCA2 normalized to actin content. Control (CNT), white bars; AIA, black bars. Data represent mean ± SD for 4–8 rats in each group.

    Article Snippet: Immunoblots Immunoblots were performed using: anti-NADPH oxidase 2 catalytic subunit gp91phox (NOX2/gp91phox ) (Abcam); anti-neuronal, -endothelial, and -inducible nitric oxide synthases (nNOS, eNOS, and iNOS, respectively) (610308, 610296, 610328, respectively, BD Biosciences); anti-manganese SOD (SOD2) (06–984, Upstate); anti-catalase (C0979, Sigma); anti-tumor necrosis factor α (TNF-α) (11948, Cell Signaling); anti-high mobility group box 1 (HMGB1) (326059652, SHINO-TEST); anti-ryanodine receptor 1 (RyR1) (MA3-925, Thermo); anti-3-nitrotyrosine (3-NT) (ab52309, Abcam); anti-troponin T (TnT) (T6277, Sigma); anti-actin (A4700, Sigma); anti-dihydropyridine receptor α2 subunit (DHPR) (ab2864, Abcam); anti-sarcoplasmic reticulum (SR) Ca2+ -ATPase (SERCA) 1 (MA3-911, Thermo); anti-SERCA2 (MA3-919, Thermo).

    Techniques: Staining, Western Blot, Expressing

    Ryanodine receptor and TH colocalize in DA-IPC processes. Vertical sections mouse retina. a–c: A DA-IPC varicosity shows RyR-ir (a), TH-ir (b), and the colocalization of these two antibodies in the merged image (c). In (d) RyR and VMAT2 colocalize

    Journal:

    Article Title: Anatomical and Neurochemical Characterization of Dopaminergic Interplexiform Processes in Mouse and Rat Retinas

    doi: 10.1002/cne.21784

    Figure Lengend Snippet: Ryanodine receptor and TH colocalize in DA-IPC processes. Vertical sections mouse retina. a–c: A DA-IPC varicosity shows RyR-ir (a), TH-ir (b), and the colocalization of these two antibodies in the merged image (c). In (d) RyR and VMAT2 colocalize

    Article Snippet: The mouse monoclonal anti-ryanodine receptor (Affinity BioReagents, Golden, CO; MA3-925) was raised against partially purified chicken pectoral muscle ryanodine receptor.

    Techniques:

    Cellular distribution of RyR1 and Ca v 1.1 in differentiated orbicularis oculi–derived myotubes. Human myotubes were visualized with an A1R confocal microscope equipped with a CFI Apo TIRF 100× objective (1.49 NA) and stained as described in Materials and methods. Top panels show orbicularis oculi, and bottom panels show EOMs. (A and E) Anti-Ca v 1.1 (green). (B and F) Anti-RyR1 (red). (C and G) Merged image of anti-RyR1, anti-Ca v 1.1, and DAPI (blue); orange pixels show codistribution of RyR1 and Ca v 1.1. (D and H) Anti-Ca v 1.2 (green). Bars, 20 µm.

    Journal: The Journal of General Physiology

    Article Title: Functional characterization of orbicularis oculi and extraocular muscles

    doi: 10.1085/jgp.201511542

    Figure Lengend Snippet: Cellular distribution of RyR1 and Ca v 1.1 in differentiated orbicularis oculi–derived myotubes. Human myotubes were visualized with an A1R confocal microscope equipped with a CFI Apo TIRF 100× objective (1.49 NA) and stained as described in Materials and methods. Top panels show orbicularis oculi, and bottom panels show EOMs. (A and E) Anti-Ca v 1.1 (green). (B and F) Anti-RyR1 (red). (C and G) Merged image of anti-RyR1, anti-Ca v 1.1, and DAPI (blue); orange pixels show codistribution of RyR1 and Ca v 1.1. (D and H) Anti-Ca v 1.2 (green). Bars, 20 µm.

    Article Snippet: The following primary antibodies were used: mouse anti-RyR1 (MA3-925; Thermo Fisher Scientific), goat anti-Cav 1.1 (sc-8160; Santa Cruz Biotechnology, Inc.), rabbit anti-Cav 1.2 (sc-25686; Santa Cruz Biotechnology, Inc.), rabbit anti–calsequestrin 1 (C-0743; Sigma-Aldrich), anti–calsequestrin 2 (3516; Abcam), goat anti-SERCA1 (sc-8093; Santa Cruz Biotechnology, Inc.), mouse anti-sarcalumenin (SRL; MA3-932; Thermo Fisher Scientific), anti-dystrophin (ab-7164; Abcam), anti-utrophin (sc-15377; Santa Cruz Biotechnology, Inc.), and mouse anti–myosin heavy chain (05-716; EMD Millipore).

    Techniques: Derivative Assay, Microscopy, Staining

    Comparison of experimentally determined and computed calcium transients in Purkinje cells Top, representative recordings of I Ca measured at +10 mV from a Purkinje cell. I Ca measured at +10 mV was used to determine the time course of Ca 2+ movement. A and B , graphs showing time course of Ca 2+ movement and fluo-3 fluorescence signals obtained from a Purkinje cell in the absence and presence of ryanodine. Ryanodine decreased the fluorescence intensity but did not change the time course of Ca 2+ movement. C and D , time course of Ca 2+ movement and fluo-3 fluorescence signals obtained by mathematical modelling (see Results). No CICR mechanism was included in the formulation and all Ca 2+ movement was by simple diffusion. The time course of Ca 2+ movement in the model resembles the experimental data suggesting that the amplitude of the central transient is the result of simple diffusion from the cell periphery.

    Journal: The Journal of Physiology

    Article Title: Location of the initiation site of calcium transients and sparks in rabbit heart Purkinje cells

    doi: 10.1111/j.1469-7793.2001.0301i.x

    Figure Lengend Snippet: Comparison of experimentally determined and computed calcium transients in Purkinje cells Top, representative recordings of I Ca measured at +10 mV from a Purkinje cell. I Ca measured at +10 mV was used to determine the time course of Ca 2+ movement. A and B , graphs showing time course of Ca 2+ movement and fluo-3 fluorescence signals obtained from a Purkinje cell in the absence and presence of ryanodine. Ryanodine decreased the fluorescence intensity but did not change the time course of Ca 2+ movement. C and D , time course of Ca 2+ movement and fluo-3 fluorescence signals obtained by mathematical modelling (see Results). No CICR mechanism was included in the formulation and all Ca 2+ movement was by simple diffusion. The time course of Ca 2+ movement in the model resembles the experimental data suggesting that the amplitude of the central transient is the result of simple diffusion from the cell periphery.

    Article Snippet: After permeabilization, the fixed myocytes were washed with PBS and blocked with 10% goat serum for 30 min. Myocytes were then incubated with monoclonal anti-ryanodine receptor antibody (mouse anti-RyR2 antibody; Affinity Bioreagents, Inc.).

    Techniques: Fluorescence, Diffusion-based Assay

    Immunohistochemical localization of ryanodine receptors in Purkinje ( A ) and ventricular cells( B ) The ventricular cell shows prominent banding at the level of the t-tubules suggesting that ryanodine receptors were located at sites adjacent to the t-tubules ( B ). The Purkinje cell also exhibited bands of intense staining in the interior of the cell ( A ). This suggests that ryanodine receptors are located in the cell interior. The spacing between the bands of intensity measured in 4 Purkinje and 4 ventricular cells was found to be 1.95 and 1.98 μm, respectively.

    Journal: The Journal of Physiology

    Article Title: Location of the initiation site of calcium transients and sparks in rabbit heart Purkinje cells

    doi: 10.1111/j.1469-7793.2001.0301i.x

    Figure Lengend Snippet: Immunohistochemical localization of ryanodine receptors in Purkinje ( A ) and ventricular cells( B ) The ventricular cell shows prominent banding at the level of the t-tubules suggesting that ryanodine receptors were located at sites adjacent to the t-tubules ( B ). The Purkinje cell also exhibited bands of intense staining in the interior of the cell ( A ). This suggests that ryanodine receptors are located in the cell interior. The spacing between the bands of intensity measured in 4 Purkinje and 4 ventricular cells was found to be 1.95 and 1.98 μm, respectively.

    Article Snippet: After permeabilization, the fixed myocytes were washed with PBS and blocked with 10% goat serum for 30 min. Myocytes were then incubated with monoclonal anti-ryanodine receptor antibody (mouse anti-RyR2 antibody; Affinity Bioreagents, Inc.).

    Techniques: Immunohistochemistry, Staining

    Immunocytochemical staining of mouse heart. Identical thin sections of left ventricles were stained with monoclonal anti-AHNAK antibody ( Left Upper ), polyclonal anti-α-actinin antisera ( Left Lower ), or monoclonal anti-RyR ( Right Lower ). All three antibodies stained periodic structures in cardiomyocytes that contain the Z-band material, T-tubule membranes, and SR vesicles enriched with the RyR. ( Right Upper ) A heart section stained without a primary antibody is shown.

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

    Article Title: The AHNAKs are a class of giant propeller-like proteins that associate with calcium channel proteins of cardiomyocytes and other cells

    doi: 10.1073/pnas.0308619101

    Figure Lengend Snippet: Immunocytochemical staining of mouse heart. Identical thin sections of left ventricles were stained with monoclonal anti-AHNAK antibody ( Left Upper ), polyclonal anti-α-actinin antisera ( Left Lower ), or monoclonal anti-RyR ( Right Lower ). All three antibodies stained periodic structures in cardiomyocytes that contain the Z-band material, T-tubule membranes, and SR vesicles enriched with the RyR. ( Right Upper ) A heart section stained without a primary antibody is shown.

    Article Snippet: The sections were fixed with acetone for 10 min and blocked with 5% BSA/PBS for 1 h. The blocked samples were incubated with anti-AHNAK monoclonal antibody, or anti-RyR monoclonal antibody (Affinity Bioreagents), followed by incubation with rhodamine-conjugated anti-mouse IgG, or FITC-conjugated anti-mouse IgG.

    Techniques: Staining

    Sucrose gradient analysis of mouse heart homogenates. Low-speed supernatants of left ventricles were fractionated on continuous sucrose gradients (20–40%) and were analyzed by SDS/PAGE and immunoblotting with antibodies specific for RyR, DHP receptor, and AHNAK. Most of the AHNAK that was not sedimentable by low-speed centrifugation was found in vesicular fractions that comigrate with the DHP receptor, and presumably represent membrane vesicles derived from the T-tubule system and the sarcolemma. RyR immunoreactivity migrated as more dense membrane fractions derived from the SR. A small fraction of AHNAK cosedimented with the heavy vesicle fraction.

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

    Article Title: The AHNAKs are a class of giant propeller-like proteins that associate with calcium channel proteins of cardiomyocytes and other cells

    doi: 10.1073/pnas.0308619101

    Figure Lengend Snippet: Sucrose gradient analysis of mouse heart homogenates. Low-speed supernatants of left ventricles were fractionated on continuous sucrose gradients (20–40%) and were analyzed by SDS/PAGE and immunoblotting with antibodies specific for RyR, DHP receptor, and AHNAK. Most of the AHNAK that was not sedimentable by low-speed centrifugation was found in vesicular fractions that comigrate with the DHP receptor, and presumably represent membrane vesicles derived from the T-tubule system and the sarcolemma. RyR immunoreactivity migrated as more dense membrane fractions derived from the SR. A small fraction of AHNAK cosedimented with the heavy vesicle fraction.

    Article Snippet: The sections were fixed with acetone for 10 min and blocked with 5% BSA/PBS for 1 h. The blocked samples were incubated with anti-AHNAK monoclonal antibody, or anti-RyR monoclonal antibody (Affinity Bioreagents), followed by incubation with rhodamine-conjugated anti-mouse IgG, or FITC-conjugated anti-mouse IgG.

    Techniques: SDS Page, Centrifugation, Derivative Assay

    Western blotting analysis of RyR1 phosphorylation at serine 2843 ( p RyR1 Ser2843 ) in human skeletal muscle tissue. A : Representative western blots of p RyR1 Ser2843 (left) and total RyR1 (right). Tissue was analyzed from muscle biopsies taken prior to exercise (B-pre), 25 min after completion of one set (B-I), five (B-V) and ten sets (B-X) from vastus lateralis of the loaded leg as well as 25–30 min after X set exercise from vastus lateralis of the non-exercised leg (B-0X). Total RyR1 was used as loading control. B : Bar graph of p RyR1 Ser2843 / total RyR1. a.u. = arbitrary units. Error bars are 1*SD of the mean.

    Journal: PLoS ONE

    Article Title: Resistance exercise-induced muscle fatigue is not accompanied by increased phosphorylation of ryanodine receptor 1 at serine 2843

    doi: 10.1371/journal.pone.0199307

    Figure Lengend Snippet: Western blotting analysis of RyR1 phosphorylation at serine 2843 ( p RyR1 Ser2843 ) in human skeletal muscle tissue. A : Representative western blots of p RyR1 Ser2843 (left) and total RyR1 (right). Tissue was analyzed from muscle biopsies taken prior to exercise (B-pre), 25 min after completion of one set (B-I), five (B-V) and ten sets (B-X) from vastus lateralis of the loaded leg as well as 25–30 min after X set exercise from vastus lateralis of the non-exercised leg (B-0X). Total RyR1 was used as loading control. B : Bar graph of p RyR1 Ser2843 / total RyR1. a.u. = arbitrary units. Error bars are 1*SD of the mean.

    Article Snippet: Membranes were stripped with a commercially available stripping buffer (ThermoScientific, Rockford, USA) washed with TBST and re-incubated in the same way as described above, with primary monoclonal antibody against total RyR1 (host: mouse, diluted 1:5000 in 5% BSA, ab2868, Abcam, Cambridge, UK) and secondary goat anti mouse antibody (diluted 1:10.000 in 5% non-fat dry milk, ThermoScientific, Rockford, USA).

    Techniques: Western Blot

    Immunohistochemistry: RyR1 phosphorylation at serine 2843 ( p RyR1 Ser2843 ) in type I and type II human skeletal muscle fibers. A : Representative immunohistochemistry. At the top: p RyR1 Ser2843 ; below: fiber typing. Dark staining displays type 1 myofibers, unstained displays type 2 myofibers (MHC I = myosin heavy chain I). The p RyR1 Ser2843 was analyzed from muscle biopsies taken prior to exercise (B-pre), 25 min after completion of one set (B-I), five (B-V) and ten sets (B-X) from vastus lateralis of the loaded leg as well as 25–30 min after X set exercise from vastus lateralis of the non-exercised leg (B-0X). B : Immunohistochemical control. At the left: section where no primary antibody was used; at the right: consecutive section incubated with anti p RyR1 Ser2843 . C : Bar graph, p RyR1 Ser2843 . a.u. = arbitrary units. Error bars are 1*SD of the mean.

    Journal: PLoS ONE

    Article Title: Resistance exercise-induced muscle fatigue is not accompanied by increased phosphorylation of ryanodine receptor 1 at serine 2843

    doi: 10.1371/journal.pone.0199307

    Figure Lengend Snippet: Immunohistochemistry: RyR1 phosphorylation at serine 2843 ( p RyR1 Ser2843 ) in type I and type II human skeletal muscle fibers. A : Representative immunohistochemistry. At the top: p RyR1 Ser2843 ; below: fiber typing. Dark staining displays type 1 myofibers, unstained displays type 2 myofibers (MHC I = myosin heavy chain I). The p RyR1 Ser2843 was analyzed from muscle biopsies taken prior to exercise (B-pre), 25 min after completion of one set (B-I), five (B-V) and ten sets (B-X) from vastus lateralis of the loaded leg as well as 25–30 min after X set exercise from vastus lateralis of the non-exercised leg (B-0X). B : Immunohistochemical control. At the left: section where no primary antibody was used; at the right: consecutive section incubated with anti p RyR1 Ser2843 . C : Bar graph, p RyR1 Ser2843 . a.u. = arbitrary units. Error bars are 1*SD of the mean.

    Article Snippet: Membranes were stripped with a commercially available stripping buffer (ThermoScientific, Rockford, USA) washed with TBST and re-incubated in the same way as described above, with primary monoclonal antibody against total RyR1 (host: mouse, diluted 1:5000 in 5% BSA, ab2868, Abcam, Cambridge, UK) and secondary goat anti mouse antibody (diluted 1:10.000 in 5% non-fat dry milk, ThermoScientific, Rockford, USA).

    Techniques: Immunohistochemistry, Staining, Incubation

    HIIT exercise does not induce RyR1 fragmentation in elite endurance athletes. ( A ) Representative Western blots show no signs of RyR1 fragmentation after the cycling bouts in the elite athletes. Arrows indicate full-sized RyR1 (red arrow) and the location

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

    Article Title: Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

    doi: 10.1073/pnas.1507176112

    Figure Lengend Snippet: HIIT exercise does not induce RyR1 fragmentation in elite endurance athletes. ( A ) Representative Western blots show no signs of RyR1 fragmentation after the cycling bouts in the elite athletes. Arrows indicate full-sized RyR1 (red arrow) and the location

    Article Snippet: Similarly, a commercially available mouse monoclonal anti-RyR1 antibody (ab2868; Abcam) showed a shift from the full-length RyR1 monomer to a ∼375-kDa fragment 24 h after exercise ( ); note that the ab2868 antibody did not detect the smaller ∼80- and 60-kDa fragments, possibly because the cleavage sites then interfered with the binding site of this antibody.

    Techniques: Western Blot

    HIIT exercise induces extensive RyR1 fragmentation in recreationally active subjects. ( A ) Representative Western blot reveals decreased expression of full-sized RyR1 (red arrow) 24 h after exercise, which was accompanied by the appearance of fragments

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

    Article Title: Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

    doi: 10.1073/pnas.1507176112

    Figure Lengend Snippet: HIIT exercise induces extensive RyR1 fragmentation in recreationally active subjects. ( A ) Representative Western blot reveals decreased expression of full-sized RyR1 (red arrow) 24 h after exercise, which was accompanied by the appearance of fragments

    Article Snippet: Similarly, a commercially available mouse monoclonal anti-RyR1 antibody (ab2868; Abcam) showed a shift from the full-length RyR1 monomer to a ∼375-kDa fragment 24 h after exercise ( ); note that the ab2868 antibody did not detect the smaller ∼80- and 60-kDa fragments, possibly because the cleavage sites then interfered with the binding site of this antibody.

    Techniques: Western Blot, Expressing

    Simulated HIIT exercise causes a ROS-dependent RyR1 fragmentation in mouse muscle. ( A ) Representative Western blots of RyR1, MDA adducts on RyR1, DHPR, and actin obtained from single-digit FDB muscles 5 min after being fatigued by six bouts of 30-s simulated

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

    Article Title: Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

    doi: 10.1073/pnas.1507176112

    Figure Lengend Snippet: Simulated HIIT exercise causes a ROS-dependent RyR1 fragmentation in mouse muscle. ( A ) Representative Western blots of RyR1, MDA adducts on RyR1, DHPR, and actin obtained from single-digit FDB muscles 5 min after being fatigued by six bouts of 30-s simulated

    Article Snippet: Similarly, a commercially available mouse monoclonal anti-RyR1 antibody (ab2868; Abcam) showed a shift from the full-length RyR1 monomer to a ∼375-kDa fragment 24 h after exercise ( ); note that the ab2868 antibody did not detect the smaller ∼80- and 60-kDa fragments, possibly because the cleavage sites then interfered with the binding site of this antibody.

    Techniques: Western Blot, Multiple Displacement Amplification

    Comparison of DHPR-RyR1 coupling in dyspedic myotubes expressing either wild-type (WT) RyR1 or R2939K (RK). A: Representative voltage-gated Ca 2+ transients (upper traces) and L-type Ca 2+ . C: Average voltage dependence of SR Ca 2+ .

    Journal: Neuromuscular disorders : NMD

    Article Title: Multi-minicore disease and atypical periodic paralysis associated with novel mutations in the skeletal muscle ryanodine receptor (RYR1) gene

    doi: 10.1016/j.nmd.2009.12.005

    Figure Lengend Snippet: Comparison of DHPR-RyR1 coupling in dyspedic myotubes expressing either wild-type (WT) RyR1 or R2939K (RK). A: Representative voltage-gated Ca 2+ transients (upper traces) and L-type Ca 2+ . C: Average voltage dependence of SR Ca 2+ .

    Article Snippet: Immunohistochemistry was done by double staining with primary antibodies: mouse anti-RyR1 monoclonal antibody (1:500, Abcam) and goat anti-DHPR polyclonal antibody (1:1000, Santa Cruz).

    Techniques: Expressing

    Amino acid sequence alignment of the residues flanking the mutated arginine residue at position 2939 in RyR1. Residues conserved across different RyR isoforms and species are shaded. The substituted arginine residue is strictly conserved in all three RyR isoforms and across all species.

    Journal: Neuromuscular disorders : NMD

    Article Title: Multi-minicore disease and atypical periodic paralysis associated with novel mutations in the skeletal muscle ryanodine receptor (RYR1) gene

    doi: 10.1016/j.nmd.2009.12.005

    Figure Lengend Snippet: Amino acid sequence alignment of the residues flanking the mutated arginine residue at position 2939 in RyR1. Residues conserved across different RyR isoforms and species are shaded. The substituted arginine residue is strictly conserved in all three RyR isoforms and across all species.

    Article Snippet: Immunohistochemistry was done by double staining with primary antibodies: mouse anti-RyR1 monoclonal antibody (1:500, Abcam) and goat anti-DHPR polyclonal antibody (1:1000, Santa Cruz).

    Techniques: Sequencing

    Analysis of muscle biopsies. (A) Histochemical and electron microscopic analysis of transverse sections obtained from a biopsy of the patient’s quadriceps muscle. H E (a) and Gomori trichrome (c) stains show marked variability in fibre size with increased internal nucleation in the absence of excess connective or fatty tissue. ATPase stain pre-incubated at pH 9.4 (d) and cytochrome oxidase (COX) stain (b) are consistent with marked type 1 fiber predominance. Uneven COX staining reveals core-like structures in some fibres. Electron microscopy (e f) demonstrates variable degrees of sarcomeric disorganization, ranging from Z-band streaming to more advanced disarray of myofibrils. (B) Immunohistochemical distribution of DHPR and RyR1 protein in transverse sections obtained in the patient (a-c) and a normal control (d-f). In most fibres, reduced RyR1 immunofluorescence as well as the aggregation of DHPR in some fibres are observed in the patient compared to normal control (c f). (C) Western blot analysis of skeletal muscle tissue. Protein extracted from patient’s muscle biopsy shows a significant reduction of RyR1 expression (565kDa) compared to the control sample. Desmin immunoreactivity was used as a muscle-specific internal control. Semi-quantification by using densitometric analyses showed that in the patient there was only about 20% residual RyR1 protein expressed (result expressed as percentage of control).

    Journal: Neuromuscular disorders : NMD

    Article Title: Multi-minicore disease and atypical periodic paralysis associated with novel mutations in the skeletal muscle ryanodine receptor (RYR1) gene

    doi: 10.1016/j.nmd.2009.12.005

    Figure Lengend Snippet: Analysis of muscle biopsies. (A) Histochemical and electron microscopic analysis of transverse sections obtained from a biopsy of the patient’s quadriceps muscle. H E (a) and Gomori trichrome (c) stains show marked variability in fibre size with increased internal nucleation in the absence of excess connective or fatty tissue. ATPase stain pre-incubated at pH 9.4 (d) and cytochrome oxidase (COX) stain (b) are consistent with marked type 1 fiber predominance. Uneven COX staining reveals core-like structures in some fibres. Electron microscopy (e f) demonstrates variable degrees of sarcomeric disorganization, ranging from Z-band streaming to more advanced disarray of myofibrils. (B) Immunohistochemical distribution of DHPR and RyR1 protein in transverse sections obtained in the patient (a-c) and a normal control (d-f). In most fibres, reduced RyR1 immunofluorescence as well as the aggregation of DHPR in some fibres are observed in the patient compared to normal control (c f). (C) Western blot analysis of skeletal muscle tissue. Protein extracted from patient’s muscle biopsy shows a significant reduction of RyR1 expression (565kDa) compared to the control sample. Desmin immunoreactivity was used as a muscle-specific internal control. Semi-quantification by using densitometric analyses showed that in the patient there was only about 20% residual RyR1 protein expressed (result expressed as percentage of control).

    Article Snippet: Immunohistochemistry was done by double staining with primary antibodies: mouse anti-RyR1 monoclonal antibody (1:500, Abcam) and goat anti-DHPR polyclonal antibody (1:1000, Santa Cruz).

    Techniques: Staining, Incubation, Electron Microscopy, Immunohistochemistry, Immunofluorescence, Western Blot, Expressing

    Double staining sections were examined by confocal microscope at high magnification (40× objective). Compared to control biopsy, patient’s muscle fibre showed abnormal accumulation of DHPR protein and slight reduction of RyR1 antibody staining in the corresponding area (arrows).

    Journal: Neuromuscular disorders : NMD

    Article Title: Multi-minicore disease and atypical periodic paralysis associated with novel mutations in the skeletal muscle ryanodine receptor (RYR1) gene

    doi: 10.1016/j.nmd.2009.12.005

    Figure Lengend Snippet: Double staining sections were examined by confocal microscope at high magnification (40× objective). Compared to control biopsy, patient’s muscle fibre showed abnormal accumulation of DHPR protein and slight reduction of RyR1 antibody staining in the corresponding area (arrows).

    Article Snippet: Immunohistochemistry was done by double staining with primary antibodies: mouse anti-RyR1 monoclonal antibody (1:500, Abcam) and goat anti-DHPR polyclonal antibody (1:1000, Santa Cruz).

    Techniques: Double Staining, Microscopy, Staining

    Calcium (A) and caffeine (B) dependence of [ 3 H]ryanodine binding to recombinant WT RyR1 and the R2939K RyR1 mutant. Specific [ 3 H]ryanodine binding to crude membrane fractions containing WT or R2939K mutant RyR1 was determined as described in Methods. Data represent the mean of 3 independent experiments. There is no difference in Ca 2+ or caffeine dependence of [ 3 H] ryanodine binding to WT RyR1 (closed circles) and R2939K mutant RyR1 (closed triangles).

    Journal: Neuromuscular disorders : NMD

    Article Title: Multi-minicore disease and atypical periodic paralysis associated with novel mutations in the skeletal muscle ryanodine receptor (RYR1) gene

    doi: 10.1016/j.nmd.2009.12.005

    Figure Lengend Snippet: Calcium (A) and caffeine (B) dependence of [ 3 H]ryanodine binding to recombinant WT RyR1 and the R2939K RyR1 mutant. Specific [ 3 H]ryanodine binding to crude membrane fractions containing WT or R2939K mutant RyR1 was determined as described in Methods. Data represent the mean of 3 independent experiments. There is no difference in Ca 2+ or caffeine dependence of [ 3 H] ryanodine binding to WT RyR1 (closed circles) and R2939K mutant RyR1 (closed triangles).

    Article Snippet: Immunohistochemistry was done by double staining with primary antibodies: mouse anti-RyR1 monoclonal antibody (1:500, Abcam) and goat anti-DHPR polyclonal antibody (1:1000, Santa Cruz).

    Techniques: Binding Assay, Recombinant, Mutagenesis

    mRNA expression of calbindin D-28K ( Calb1 ), calretinin ( Calb2 ), tyrosine hydroxylase ( TH ), Zebrin II ( ZebrinII ), ryanodine receptor 1 ( Ryr1 ), ryanodine receptor 2 ( Ryr2 ), and ryanodine receptor 3 ( Ryr3 ) genes in the cerebellum of wild-type and tottering-6j mice. The data are presented as means ± SEM. * P

    Journal: Experimental Animals

    Article Title: Protein expression pattern in cerebellum of Cav2.1 mutant, tottering-6j mice

    doi: 10.1538/expanim.15-0120

    Figure Lengend Snippet: mRNA expression of calbindin D-28K ( Calb1 ), calretinin ( Calb2 ), tyrosine hydroxylase ( TH ), Zebrin II ( ZebrinII ), ryanodine receptor 1 ( Ryr1 ), ryanodine receptor 2 ( Ryr2 ), and ryanodine receptor 3 ( Ryr3 ) genes in the cerebellum of wild-type and tottering-6j mice. The data are presented as means ± SEM. * P

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-Cab1 (anti-Calbindin-D-28K Clone CB-955; 1:500; Sigma-Aldrich, St. Louis, MO, USA); rabbit monoclonal anti-Calb2 (1:100; Spring Bioscience, Pleasanton, CA, USA); rabbit monoclonal anti-TH (1:100; Sigma-Aldrich); rabbit polyclonal anti- ZebrinII (1:100; Novus Biologicals, Littleton, CO, USA); mouse monoclonal anti-Ryr1 (1:100; Novus Biologicals); rabbit polyclonal anti-Ryr2 (1:100; Novus Biologicals); and rabbit polyclonal anti-Ryr3 (1:100; Merck Millipore, Billerica, MA, USA).

    Techniques: Expressing, Mouse Assay

    Expression of Ryr3 in the cerebellum. Ryr3 expression was increased in tottering-6j mice compared with +/+ mice. The scale bar represents 50 µ m. M: molecular layer, P: Purkinje cell layer, Gr: granular layer.

    Journal: Experimental Animals

    Article Title: Protein expression pattern in cerebellum of Cav2.1 mutant, tottering-6j mice

    doi: 10.1538/expanim.15-0120

    Figure Lengend Snippet: Expression of Ryr3 in the cerebellum. Ryr3 expression was increased in tottering-6j mice compared with +/+ mice. The scale bar represents 50 µ m. M: molecular layer, P: Purkinje cell layer, Gr: granular layer.

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-Cab1 (anti-Calbindin-D-28K Clone CB-955; 1:500; Sigma-Aldrich, St. Louis, MO, USA); rabbit monoclonal anti-Calb2 (1:100; Spring Bioscience, Pleasanton, CA, USA); rabbit monoclonal anti-TH (1:100; Sigma-Aldrich); rabbit polyclonal anti- ZebrinII (1:100; Novus Biologicals, Littleton, CO, USA); mouse monoclonal anti-Ryr1 (1:100; Novus Biologicals); rabbit polyclonal anti-Ryr2 (1:100; Novus Biologicals); and rabbit polyclonal anti-Ryr3 (1:100; Merck Millipore, Billerica, MA, USA).

    Techniques: Expressing, Mouse Assay

    Immunofluorescent visualization of calcium/sodium exchanger (NCX1) and ryanodine receptor (Ryr1) in normal (C2C12, C2C12-D) and malignant (RD, RD-D) cells after exposition to CaEP protocol (electric field intensity: 1000 V/cm and 0.5 mM Ca 2+ ) The left panel: CLSM images present changes in NCX1 (red) intracellular localization and signal intensity between untreated and CaEP cell lines: C2C12 ( A ), C2C12-D ( B ), RD ( C ), RD-D ( D ). The graph shows signal intensity for untreated cell lines (above) and 3 therapy conditions (below): Ca 2+ incubation, EP only and CaEP normalized to untreated cells. The right panel: confocal images present changes in RyR1 (green) signal intensity between untreated and CaEP cell lines: C2C12 ( E ), C2C12-D ( F ), RD ( G ), RD-D ( H ). Both NCX1 and RyR1 protein colocalized with F-actin and cellular nucleus. The graphs NCX1 (left) and RyR1 (right) show a signal intensity for untreated cell lines (above) and 3 therapy conditions (below): Ca 2+ incubation (0.5 mM), EP only (1000 V/cm) and CaEP (0.5 mM; 1000 V/cm) normalized to untreated cells. Fluorescent signal was detected 24 h after CaEP application; 20 μm; n = 3–5. The signal intensity was analyzed by ImageJ software.

    Journal: Oncotarget

    Article Title: Calcium electroporation for treatment of sarcoma in preclinical studies

    doi: 10.18632/oncotarget.24352

    Figure Lengend Snippet: Immunofluorescent visualization of calcium/sodium exchanger (NCX1) and ryanodine receptor (Ryr1) in normal (C2C12, C2C12-D) and malignant (RD, RD-D) cells after exposition to CaEP protocol (electric field intensity: 1000 V/cm and 0.5 mM Ca 2+ ) The left panel: CLSM images present changes in NCX1 (red) intracellular localization and signal intensity between untreated and CaEP cell lines: C2C12 ( A ), C2C12-D ( B ), RD ( C ), RD-D ( D ). The graph shows signal intensity for untreated cell lines (above) and 3 therapy conditions (below): Ca 2+ incubation, EP only and CaEP normalized to untreated cells. The right panel: confocal images present changes in RyR1 (green) signal intensity between untreated and CaEP cell lines: C2C12 ( E ), C2C12-D ( F ), RD ( G ), RD-D ( H ). Both NCX1 and RyR1 protein colocalized with F-actin and cellular nucleus. The graphs NCX1 (left) and RyR1 (right) show a signal intensity for untreated cell lines (above) and 3 therapy conditions (below): Ca 2+ incubation (0.5 mM), EP only (1000 V/cm) and CaEP (0.5 mM; 1000 V/cm) normalized to untreated cells. Fluorescent signal was detected 24 h after CaEP application; 20 μm; n = 3–5. The signal intensity was analyzed by ImageJ software.

    Article Snippet: Then after 24 h cells were fixed in 4% formalin, permeabilized with 0.5% Triton X-100 in PBS for 5 min, raised in PBS 3 × 5 min, blocked with 1% Bovine Serum Albumin (BSA) in PBS for 1 h. These antibodies were used: 1) to show cytoskeleton structure – primary antibody monoclonal mouse anti-zyxin (overnight incubation at 4μ C; 1:500; Abcam) and secondary antibody Fluorescein (FITC)-conjugated AffiniPure Fragment Donkey Anti-Mouse IgG (for 60 min; at room temperature; 1:100; Jackson ImmunoResearch) mixed with Alexa 546-conjugated phalloidin (at a concentration of 2 μg/ml; Life Technologies); 2) to show ryanodine receptors expression – primary antibody monoclonal rabbit anti-RyR1 (overnight at 4μ C; 1:100, Cell Signaling) and secondary antibody Cy3-conjugated AffiniPure Donkey Anti-Rabbit IgG (for 60 min at the room temperature; 1;100; Jackson ImmunoResearch) mixed with Alexa 488-conjugated phalloidin (at a concentration of 2 μg/ml; Life Technologies); 3) to show sodium/calcium exchanger expression – primary antibody monoclonal mouse anti-NCX1 (overnight at 4μ C; 1:100, Abcam) and secondary antibody Fluorescein (FITC)-conjugated AffiniPure Fragment Donkey Anti-Mouse IgG (for 60 min; at the room temperature; 1:100; Jackson ImmunoResearch) mixed with Alexa 546-conjugated phalloidin (at a concentration of 2 μg/ml; Life Technologies).

    Techniques: Confocal Laser Scanning Microscopy, Incubation, Software

    Ryanodine receptor location. (A) representative image from a Sham left atrial myocyte. Lower image, higher magnification view of area indicated by dotted box. Arrows indicate intra-sarcomeric RyR at the cell periphery. (B) power spectrum of longitudinal scan along the cell. (C) representative image from an AoB left atrial myocyte. Lower image, higher magnification view of area indicated by dotted box. (D) power spectrum of longitudinal scan along the cell. Scale bars represent 5 μm. (E) sarcomere length. Data from 18/3 Sham cells/hearts and 18/3 AoB cells/hearts.

    Journal: PLoS ONE

    Article Title: Cellular Hypertrophy and Increased Susceptibility to Spontaneous Calcium-Release of Rat Left Atrial Myocytes Due to Elevated Afterload

    doi: 10.1371/journal.pone.0144309

    Figure Lengend Snippet: Ryanodine receptor location. (A) representative image from a Sham left atrial myocyte. Lower image, higher magnification view of area indicated by dotted box. Arrows indicate intra-sarcomeric RyR at the cell periphery. (B) power spectrum of longitudinal scan along the cell. (C) representative image from an AoB left atrial myocyte. Lower image, higher magnification view of area indicated by dotted box. (D) power spectrum of longitudinal scan along the cell. Scale bars represent 5 μm. (E) sarcomere length. Data from 18/3 Sham cells/hearts and 18/3 AoB cells/hearts.

    Article Snippet: Blots were probed with anti-ryanodine receptor 2 (anti-RyR2; MA3916, Thermo Scientific, UK), S2808-specific anti-phospho-RyR2 (2808 AP, Badrilla, UK), S2814-specific anti-phospho-RyR2 (A010-31AP, Badrilla, UK), anti-α1c LTCC subunit (ACC-003; Alomone, Israel), anti-SR Ca2+ ATPase (anti-SERCA2; MA3-919, Thermo Scientific, UK), anti-phospholamban (anti-PLB; A010-14, Badrilla, UK), S16-specific anti-phosphorylated PLB (A010-12AP, Badrilla, UK), anti-Na+ /Ca2+ exchanger (anti-NCX1; R3F1, Swannt, Switzerland) or anti-GAPDH (G9545; Sigma, UK) and protein bands visualized using relevant peroxidase-conjugated secondary antibodies, chemiluminescence and autoradiography.

    Techniques:

    Expression and triad targeting of Ca V 1.2 constructs. Dysgenic myotubes (Ca V 1.1 -/- ) were reconstituted with GFP-Ca V 1.2, GFP-Ca V 1.2-D4N, GFP-Ca V 1.2-ΔE33, or GFP-Ca V 1.2-ΔE33-D4N and double immunofluorescence labeled with anti-GFP and anti-RyR. All channel constructs are equally expressed and colocalized in clusters with the RyR1, indicative of their correct targeting into t-tubule/SR or plasma membrane/SR junctions. Scale bar, 10 μm.

    Journal: Channels

    Article Title: Role of putative voltage-sensor countercharge D4 in regulating gating properties of CaV1.2 and CaV1.3 calcium channels

    doi: 10.1080/19336950.2018.1482183

    Figure Lengend Snippet: Expression and triad targeting of Ca V 1.2 constructs. Dysgenic myotubes (Ca V 1.1 -/- ) were reconstituted with GFP-Ca V 1.2, GFP-Ca V 1.2-D4N, GFP-Ca V 1.2-ΔE33, or GFP-Ca V 1.2-ΔE33-D4N and double immunofluorescence labeled with anti-GFP and anti-RyR. All channel constructs are equally expressed and colocalized in clusters with the RyR1, indicative of their correct targeting into t-tubule/SR or plasma membrane/SR junctions. Scale bar, 10 μm.

    Article Snippet: Immunofluorescence and antibodies Paraformaldehyde-fixed cultures were immunolabeled as previously described [ ] with rabbit polyclonal anti-GFP (1:10,000; Molecular Probes, Eugene, OR) and mouse monoclonal anti-RyR (34-C; 1:1000; Alexis Biochemicals, Lausanne, Switzerland) and fluorescently labeled with goat anti-rabbit Alexa-488 and secondary goat anti-mouse Alexa-594 (1:4000; Molecular Probes), respectively.

    Techniques: Expressing, Construct, Immunofluorescence, Labeling

    Immunogold labeling shows i) preferential I band localization of STIM1 and ii) increased presence of Orai1 at the I band following exercise. Left) Representative immunogold EM images showing the localization of RyR1 (top), STIM1 (center), and Orai1 (bottom) under control and exercised conditions. Right) Histograms of distances of immunogold particles from the Z line. The cyan line marks the position of the TT at the triad. Brackets with numeric values indicate the percentage of gold particles that fall within the triad-area vs. those that fall within the I-band area. Grey arrow points to the increased presence of Orai1 at the I-band following exercise. Sample size: control, 2 mice/4–8 fibers analyzed; exercised, 3 mice/3–8 fibers analyzed . *p

    Journal: Scientific Reports

    Article Title: Exercise-dependent formation of new junctions that promote STIM1-Orai1 assembly in skeletal muscle

    doi: 10.1038/s41598-017-14134-0

    Figure Lengend Snippet: Immunogold labeling shows i) preferential I band localization of STIM1 and ii) increased presence of Orai1 at the I band following exercise. Left) Representative immunogold EM images showing the localization of RyR1 (top), STIM1 (center), and Orai1 (bottom) under control and exercised conditions. Right) Histograms of distances of immunogold particles from the Z line. The cyan line marks the position of the TT at the triad. Brackets with numeric values indicate the percentage of gold particles that fall within the triad-area vs. those that fall within the I-band area. Grey arrow points to the increased presence of Orai1 at the I-band following exercise. Sample size: control, 2 mice/4–8 fibers analyzed; exercised, 3 mice/3–8 fibers analyzed . *p

    Article Snippet: Primary antibodies used: a) mouse monoclonal anti-RyR1/RyR3 (34 C antibody, 1:30, Developmental Studies Hybridoma Bank); b) rabbit polyclonal anti-STIM1 (1:100, Sigma Aldrich); c) mouse monoclonal anti-STIM1 (1:50, BD Biosciences); or d) rabbit polyclonal anti-Orai1, (1:20, Thermo Scientific).

    Techniques: Labeling, Mouse Assay

    Co-localization between STIM1 and Orai1, low in control samples, increases significantly following exercise. Representative immunofluorescence images obtained from EDL fibers double-labeled for RyR1-STIM1 ( A and D ), RyR1-Orai1 ( B and E ), and STIM1-Orai1 ( C and F ). Each panel also contains a fluorescence intensity profile along 4 sarcomeres (see dashed line in A ) and the Pearson’s correlation coefficient value, a measure of covariance of pixel intensities, given as the mean ± SEM; *p

    Journal: Scientific Reports

    Article Title: Exercise-dependent formation of new junctions that promote STIM1-Orai1 assembly in skeletal muscle

    doi: 10.1038/s41598-017-14134-0

    Figure Lengend Snippet: Co-localization between STIM1 and Orai1, low in control samples, increases significantly following exercise. Representative immunofluorescence images obtained from EDL fibers double-labeled for RyR1-STIM1 ( A and D ), RyR1-Orai1 ( B and E ), and STIM1-Orai1 ( C and F ). Each panel also contains a fluorescence intensity profile along 4 sarcomeres (see dashed line in A ) and the Pearson’s correlation coefficient value, a measure of covariance of pixel intensities, given as the mean ± SEM; *p

    Article Snippet: Primary antibodies used: a) mouse monoclonal anti-RyR1/RyR3 (34 C antibody, 1:30, Developmental Studies Hybridoma Bank); b) rabbit polyclonal anti-STIM1 (1:100, Sigma Aldrich); c) mouse monoclonal anti-STIM1 (1:50, BD Biosciences); or d) rabbit polyclonal anti-Orai1, (1:20, Thermo Scientific).

    Techniques: Immunofluorescence, Labeling, Fluorescence

    Shown for each panel is the myosin from the Stain Free gel, indicative of total protein (top) and the representative Western blot protein (bottom) for MHC1 (A, black line indicates non-contiguous lanes from the same gel), actin (B), DHPR (C) and RyR1 (D) in 28 d and 70 d WT, mdx and mdx tau mice.

    Journal: PLoS Currents

    Article Title: Benefits of Prenatal Taurine Supplementation in Preventing the Onset of Acute Damage in the Mdx Mouse

    doi: 10.1371/currents.md.9a3e357a0154d01050b591601cbd4fdb

    Figure Lengend Snippet: Shown for each panel is the myosin from the Stain Free gel, indicative of total protein (top) and the representative Western blot protein (bottom) for MHC1 (A, black line indicates non-contiguous lanes from the same gel), actin (B), DHPR (C) and RyR1 (D) in 28 d and 70 d WT, mdx and mdx tau mice.

    Article Snippet: Antibody details and dilutions are as follows: rabbit polyclonal monoclonal anti-Actin (Batch A 2066, SIGMA, 1:300); mouse monoclonal anti-MHC1 (A4.840, Developmental Studies Hybridoma Bank (DSHB), IgM, 1:200), mouse monoclonal anti-RyR1 (34C, DSHB, 1:300), mouse monoclonal anti-SERCA1 (CaF2-5D2, DSHB, 1:1,000), mouse monoclonal anti-DHPR (IIID5E1, DSHB, 1:400), mouse monoclonal anti-CSQ1 (ab2824, Abcam, 1:2000), rabbit polyclonal anti-CSQ2, (ab3516, Abcam, 1:1,000), rabbit polyclonal anti-Taurine Transporter (TauT, Professor David Pow, RMIT University, 1:10,000), mouse monoclonal anti-Utrophin (Mancho3 clone 8A4, DSHB, 1:200), mouse monoclonal anti-Dystrophin (MANDYS1 clone 3B7, DSHB, 1:500) and mouse monoclonal anti-Myogenin (F5D, DSHB, 1:250).

    Techniques: Staining, Western Blot, Mouse Assay

    Immunohistochemical analysis and subcellular localization of RYR1 and of the α 1 subunit of the DHPR in single muscle fibers from WT and ryr3 −/− mice. (A) Left and central panels show the staining obtained using rabbit anti-RYR1 mAb D4E1 (green in merged image) and mouse anti-Ca v 1.1 (red in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). (B) Left and central panels show the staining obtained using mouse anti-Ca v 1.1 (red in merged image) and rabbit anti-Ca v 1.2 (green in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). The bottom panels show staining of mouse EDL fibers, which are negative for Ca v 1.2 and were used as staining control. All images in B were acquired using the same settings for the laser intensities and acquisition parameters. Images were acquired using a Nikon A1 plus confocal microscope equipped with a Plan Apo 60× oil objective (numerical aperture, 1.4) and stained as described in Materials and methods. Orange pixels show areas of colocalization. Scale bars, 30 µm.

    Journal: The Journal of General Physiology

    Article Title: Extraocular muscle function is impaired inryr3−/− mice

    doi: 10.1085/jgp.201912333

    Figure Lengend Snippet: Immunohistochemical analysis and subcellular localization of RYR1 and of the α 1 subunit of the DHPR in single muscle fibers from WT and ryr3 −/− mice. (A) Left and central panels show the staining obtained using rabbit anti-RYR1 mAb D4E1 (green in merged image) and mouse anti-Ca v 1.1 (red in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). (B) Left and central panels show the staining obtained using mouse anti-Ca v 1.1 (red in merged image) and rabbit anti-Ca v 1.2 (green in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). The bottom panels show staining of mouse EDL fibers, which are negative for Ca v 1.2 and were used as staining control. All images in B were acquired using the same settings for the laser intensities and acquisition parameters. Images were acquired using a Nikon A1 plus confocal microscope equipped with a Plan Apo 60× oil objective (numerical aperture, 1.4) and stained as described in Materials and methods. Orange pixels show areas of colocalization. Scale bars, 30 µm.

    Article Snippet: The following primary antibodies were used for Western blotting: rabbit anti-RYR1 (8153S; Cell Signaling), goat anti-Cav 1.1 (sc-8160; Santa Cruz), rabbit anti-Cav 1.2 (sc-25686; Santa Cruz) rabbit anti-calsequestrin-1 (C-0743; Sigma) and calsequestrin-2 (ab-3516; Abcam), goat anti-SERCA1 (sc-8093; Santa Cruz), goat anti-SERCA2 (sc-8095; Santa Cruz), mouse anti-MyHC (05–716; Millipore), mouse anti-MyHC13 (4A6; DSHB Iowa), rabbit anti-Desmin (sc-14026; Santa Cruz) and rabbit anti-parvalbumin (PV25; Swant), and rabbit anti-JP-45 ( ).

    Techniques: Immunohistochemistry, Mouse Assay, Staining, Microscopy

    Hypoxic increase in [Ca 2+ ] i and contraction are diminished in RyR1 −/− and RyR1 +/− PASMCs. a Original recordings of hypoxic increase in [Ca 2+ ] i in an RyR1 +/+ and RyR1 −/− cell. Bar graph summarizes the effect of hypoxia

    Journal: Pflugers Archiv : European journal of physiology

    Article Title: Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

    doi: 10.1007/s00424-008-0556-8

    Figure Lengend Snippet: Hypoxic increase in [Ca 2+ ] i and contraction are diminished in RyR1 −/− and RyR1 +/− PASMCs. a Original recordings of hypoxic increase in [Ca 2+ ] i in an RyR1 +/+ and RyR1 −/− cell. Bar graph summarizes the effect of hypoxia

    Article Snippet: After that, cells were incubated with monoclonal anti-RyR1 antibody (1:100 dilution; Upstate Biotechnology, Lake Placid, NY, USA) overnight at 4°C, followed by Alexa 488-conjugated antibody (1:500; Molecular Probes, Eugene, OR, USA) for 2 h. Immunofluorescence staining was examined using a Zeiss LSM510 laser-scanning confocal microscope.

    Techniques:

    Neurotransmitter-induced Ca 2+ release is inhibited in RyR1 −/− mouse PASMCs. a Recordings display an increase in [Ca 2+ ] i induced by ATP in an RyR1 +/+ and RyR1 −/− PASMC. Graph shows summary of ATP-induced increase in RyR1

    Journal: Pflugers Archiv : European journal of physiology

    Article Title: Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

    doi: 10.1007/s00424-008-0556-8

    Figure Lengend Snippet: Neurotransmitter-induced Ca 2+ release is inhibited in RyR1 −/− mouse PASMCs. a Recordings display an increase in [Ca 2+ ] i induced by ATP in an RyR1 +/+ and RyR1 −/− PASMC. Graph shows summary of ATP-induced increase in RyR1

    Article Snippet: After that, cells were incubated with monoclonal anti-RyR1 antibody (1:100 dilution; Upstate Biotechnology, Lake Placid, NY, USA) overnight at 4°C, followed by Alexa 488-conjugated antibody (1:500; Molecular Probes, Eugene, OR, USA) for 2 h. Immunofluorescence staining was examined using a Zeiss LSM510 laser-scanning confocal microscope.

    Techniques:

    Neurotransmitter-induced Ca 2+ release and contraction are depressed in RyR1 +/− mouse PASMCs. a ATP-induced increase in [Ca 2+ ] i in RyR1 +/+ and RyR1 +/− PASMCs in normal Ca 2+ PSS. * P

    Journal: Pflugers Archiv : European journal of physiology

    Article Title: Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

    doi: 10.1007/s00424-008-0556-8

    Figure Lengend Snippet: Neurotransmitter-induced Ca 2+ release and contraction are depressed in RyR1 +/− mouse PASMCs. a ATP-induced increase in [Ca 2+ ] i in RyR1 +/+ and RyR1 +/− PASMCs in normal Ca 2+ PSS. * P

    Article Snippet: After that, cells were incubated with monoclonal anti-RyR1 antibody (1:100 dilution; Upstate Biotechnology, Lake Placid, NY, USA) overnight at 4°C, followed by Alexa 488-conjugated antibody (1:500; Molecular Probes, Eugene, OR, USA) for 2 h. Immunofluorescence staining was examined using a Zeiss LSM510 laser-scanning confocal microscope.

    Techniques:

    Voltage-dependent Ca 2+ currents are unaltered in RyR1 +/− mouse PASMCs. Representative current traces show I Ca recorded in an RyR1 +/+ and RyR1 +/− cell before (control, Ctl ) and after treatment with nifedipine ( Nif , 1 μM) for 5 min.

    Journal: Pflugers Archiv : European journal of physiology

    Article Title: Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

    doi: 10.1007/s00424-008-0556-8

    Figure Lengend Snippet: Voltage-dependent Ca 2+ currents are unaltered in RyR1 +/− mouse PASMCs. Representative current traces show I Ca recorded in an RyR1 +/+ and RyR1 +/− cell before (control, Ctl ) and after treatment with nifedipine ( Nif , 1 μM) for 5 min.

    Article Snippet: After that, cells were incubated with monoclonal anti-RyR1 antibody (1:100 dilution; Upstate Biotechnology, Lake Placid, NY, USA) overnight at 4°C, followed by Alexa 488-conjugated antibody (1:500; Molecular Probes, Eugene, OR, USA) for 2 h. Immunofluorescence staining was examined using a Zeiss LSM510 laser-scanning confocal microscope.

    Techniques: CTL Assay

    Spontaneous local Ca 2+ release is inhibited in RyR1 −/− mouse PASMCs. a Original line-scan confocal images show Ca 2+ sparks in an RyR1 +/+ and RyR1 −/− PASMC in normal Ca 2+ (1.8 mM) extracellular solution. The images were

    Journal: Pflugers Archiv : European journal of physiology

    Article Title: Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

    doi: 10.1007/s00424-008-0556-8

    Figure Lengend Snippet: Spontaneous local Ca 2+ release is inhibited in RyR1 −/− mouse PASMCs. a Original line-scan confocal images show Ca 2+ sparks in an RyR1 +/+ and RyR1 −/− PASMC in normal Ca 2+ (1.8 mM) extracellular solution. The images were

    Article Snippet: After that, cells were incubated with monoclonal anti-RyR1 antibody (1:100 dilution; Upstate Biotechnology, Lake Placid, NY, USA) overnight at 4°C, followed by Alexa 488-conjugated antibody (1:500; Molecular Probes, Eugene, OR, USA) for 2 h. Immunofluorescence staining was examined using a Zeiss LSM510 laser-scanning confocal microscope.

    Techniques:

    Caffeine-induced global Ca 2+ release is reduced in RyR1 −/− and RyR1 +/− mouse PASMCs. a Original recordings show caffeine ( Caf )-induced increase in fluo-4 fluorescence ([Ca 2+ ] i ), determined using a Leica TCS SP2 laser-scanning confocal

    Journal: Pflugers Archiv : European journal of physiology

    Article Title: Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

    doi: 10.1007/s00424-008-0556-8

    Figure Lengend Snippet: Caffeine-induced global Ca 2+ release is reduced in RyR1 −/− and RyR1 +/− mouse PASMCs. a Original recordings show caffeine ( Caf )-induced increase in fluo-4 fluorescence ([Ca 2+ ] i ), determined using a Leica TCS SP2 laser-scanning confocal

    Article Snippet: After that, cells were incubated with monoclonal anti-RyR1 antibody (1:100 dilution; Upstate Biotechnology, Lake Placid, NY, USA) overnight at 4°C, followed by Alexa 488-conjugated antibody (1:500; Molecular Probes, Eugene, OR, USA) for 2 h. Immunofluorescence staining was examined using a Zeiss LSM510 laser-scanning confocal microscope.

    Techniques: Fluorescence

    RyR1 protein expression is absent in RyR1 −/− mouse PASMCs and reduced in RyR1 +/− mouse PASMCs. Original images show an embryonic RyR1 +/+ and RyR1 −/− , as well as adult RyR1 +/+ and RyR1 +/− cell stained with

    Journal: Pflugers Archiv : European journal of physiology

    Article Title: Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

    doi: 10.1007/s00424-008-0556-8

    Figure Lengend Snippet: RyR1 protein expression is absent in RyR1 −/− mouse PASMCs and reduced in RyR1 +/− mouse PASMCs. Original images show an embryonic RyR1 +/+ and RyR1 −/− , as well as adult RyR1 +/+ and RyR1 +/− cell stained with

    Article Snippet: After that, cells were incubated with monoclonal anti-RyR1 antibody (1:100 dilution; Upstate Biotechnology, Lake Placid, NY, USA) overnight at 4°C, followed by Alexa 488-conjugated antibody (1:500; Molecular Probes, Eugene, OR, USA) for 2 h. Immunofluorescence staining was examined using a Zeiss LSM510 laser-scanning confocal microscope.

    Techniques: Expressing, Staining

    Membrane depolarization-induced increase in [Ca 2+ ] i is blocked in RyR1 −/− mouse PASMCs. a Recording traces illustrate an increase in [Ca 2+ ] i induced by membrane depolarization with high K + in an RyR1 +/+ and RyR1 −/− PASMC

    Journal: Pflugers Archiv : European journal of physiology

    Article Title: Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

    doi: 10.1007/s00424-008-0556-8

    Figure Lengend Snippet: Membrane depolarization-induced increase in [Ca 2+ ] i is blocked in RyR1 −/− mouse PASMCs. a Recording traces illustrate an increase in [Ca 2+ ] i induced by membrane depolarization with high K + in an RyR1 +/+ and RyR1 −/− PASMC

    Article Snippet: After that, cells were incubated with monoclonal anti-RyR1 antibody (1:100 dilution; Upstate Biotechnology, Lake Placid, NY, USA) overnight at 4°C, followed by Alexa 488-conjugated antibody (1:500; Molecular Probes, Eugene, OR, USA) for 2 h. Immunofluorescence staining was examined using a Zeiss LSM510 laser-scanning confocal microscope.

    Techniques:

    Membrane depolarization-caused muscle contraction is reduced in RyR1 +/− mouse pulmonary arteries. a Original recordings show muscle contraction caused by membrane depolarization with high K + in an RyR1 +/+ and RyR1 +/− pulmonary artery in

    Journal: Pflugers Archiv : European journal of physiology

    Article Title: Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

    doi: 10.1007/s00424-008-0556-8

    Figure Lengend Snippet: Membrane depolarization-caused muscle contraction is reduced in RyR1 +/− mouse pulmonary arteries. a Original recordings show muscle contraction caused by membrane depolarization with high K + in an RyR1 +/+ and RyR1 +/− pulmonary artery in

    Article Snippet: After that, cells were incubated with monoclonal anti-RyR1 antibody (1:100 dilution; Upstate Biotechnology, Lake Placid, NY, USA) overnight at 4°C, followed by Alexa 488-conjugated antibody (1:500; Molecular Probes, Eugene, OR, USA) for 2 h. Immunofluorescence staining was examined using a Zeiss LSM510 laser-scanning confocal microscope.

    Techniques:

    Membrane depolarization-induced increase in [Ca 2+ ] i is attenuated as well in RyR1 +/− mouse PASMCs. a Representative recordings exhibit high K + -induced increase in [Ca 2+ ] i in an RyR1 +/+ and RyR1 +/− PASMC in normal Ca 2+ extracellular solution.

    Journal: Pflugers Archiv : European journal of physiology

    Article Title: Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

    doi: 10.1007/s00424-008-0556-8

    Figure Lengend Snippet: Membrane depolarization-induced increase in [Ca 2+ ] i is attenuated as well in RyR1 +/− mouse PASMCs. a Representative recordings exhibit high K + -induced increase in [Ca 2+ ] i in an RyR1 +/+ and RyR1 +/− PASMC in normal Ca 2+ extracellular solution.

    Article Snippet: After that, cells were incubated with monoclonal anti-RyR1 antibody (1:100 dilution; Upstate Biotechnology, Lake Placid, NY, USA) overnight at 4°C, followed by Alexa 488-conjugated antibody (1:500; Molecular Probes, Eugene, OR, USA) for 2 h. Immunofluorescence staining was examined using a Zeiss LSM510 laser-scanning confocal microscope.

    Techniques:

    Quantitative analysis in histologic sections of EDL fibers presenting structural damage following the ES protocol. A – C ) Histologic examination of EDL muscles after ES protocol allowed classification of fibers in 3 main classes: fibers with no apparent sign of damage ( A ); fibers losing striation (dashed oval; B ); and fibers with contractures (asterisks; C ). D ) Quantitative analysis showing the percentage of EDL fibers presenting the different features classified in A – C . Azu, azumolene; Y522S, RYR1 Y522S/WT . Scale bar, 10 µm. * P

    Journal: The FASEB Journal

    Article Title: Strenuous exercise triggers a life-threatening response in mice susceptible to malignant hyperthermia

    doi: 10.1096/fj.201601292R

    Figure Lengend Snippet: Quantitative analysis in histologic sections of EDL fibers presenting structural damage following the ES protocol. A – C ) Histologic examination of EDL muscles after ES protocol allowed classification of fibers in 3 main classes: fibers with no apparent sign of damage ( A ); fibers losing striation (dashed oval; B ); and fibers with contractures (asterisks; C ). D ) Quantitative analysis showing the percentage of EDL fibers presenting the different features classified in A – C . Azu, azumolene; Y522S, RYR1 Y522S/WT . Scale bar, 10 µm. * P

    Article Snippet: Primary Abs used were as follows: mouse monoclonal anti-RYR1/RYR3 34C (1:20; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA); and rabbit polyclonal anti-mitochondrial preprotein translocases of the outer membrane homolog 20, (TOM20) (1:50; Santa Cruz Biotechnology).

    Techniques:

    ryr3 −/− EOM-derived myotubes show fewer calcium waves than WT EOM-derived myotubes. (A) WT (left) and ryr3 −/− (right) confocal image of myotubes loaded with Fluo-4. Spontaneous calcium waves can be seen as lines during image acquisition. Scale bars, 30 µm. (B) Representative Fluo-4 line scan traces of WT (top, black line) and ryr3 −/− (bottom, gray line) myotubes. a.u., arbitrary units. (C) Analysis of the frequency of the spontaneous Ca 2+ transient. Each point represents the number of transients per minute recorded in a single myotube. Each myotube was recorded for 30 s, and the frequency output is given as frequency per minute. Experiments were performed at room temperature. The horizontal black line represents the mean value. Empty circles, WT ( n = 95); gray circles, ryr3 −/− ( n = 22). (D) ΔF/F of the Fluo-4 transients. The number of transients analyzed was n = 9,319 and n = 1,368 for WT and ryr3 −/− , respectively. **, P

    Journal: The Journal of General Physiology

    Article Title: Extraocular muscle function is impaired inryr3−/− mice

    doi: 10.1085/jgp.201912333

    Figure Lengend Snippet: ryr3 −/− EOM-derived myotubes show fewer calcium waves than WT EOM-derived myotubes. (A) WT (left) and ryr3 −/− (right) confocal image of myotubes loaded with Fluo-4. Spontaneous calcium waves can be seen as lines during image acquisition. Scale bars, 30 µm. (B) Representative Fluo-4 line scan traces of WT (top, black line) and ryr3 −/− (bottom, gray line) myotubes. a.u., arbitrary units. (C) Analysis of the frequency of the spontaneous Ca 2+ transient. Each point represents the number of transients per minute recorded in a single myotube. Each myotube was recorded for 30 s, and the frequency output is given as frequency per minute. Experiments were performed at room temperature. The horizontal black line represents the mean value. Empty circles, WT ( n = 95); gray circles, ryr3 −/− ( n = 22). (D) ΔF/F of the Fluo-4 transients. The number of transients analyzed was n = 9,319 and n = 1,368 for WT and ryr3 −/− , respectively. **, P

    Article Snippet: The following primary and secondary antibodies were used: rabbit monoclonal anti-RYR1 (D4E1, #8153; Cell Signaling), mouse monoclonal anti-Cav 1.1 (IIC12D4 and IIID5E1; Iowa Hybridoma bank), rabbit anti-Cav 1.2 (sc-25686; Santa Cruz), Alexa Fluor 568 donkey anti-mouse IgG (A10037; Invitrogen), and Alexa Fluor 488 chicken anti-rabbit IgG (A21441; Invitrogen).

    Techniques: Derivative Assay

    ryr3 −/− mice exhibit visual impairment . (A) Cued water maze. Top: Representative heatmaps of the swimming path of WT (left) and ryr3 −/− (right) mice. Bottom left: Total swimming distance (mean ± SEM) required to reach the platform. Bottom right: Time required to reach the platform. Experiments were performed on 10 mice per strain, and data from four different pool entry points were combined. (B) OKR. Top: OptoMotry setup. From Prusky et al. (2004) , Fig. 2 B is reprinted with permission from Investigative Ophthalmology Visual Science . Bottom: Visual acuity of two independent measurements performed under scotopic conditions on each mouse. Each symbol represents the visual acuity of a single mouse; empty circles, WT mice ( n = 20); gray circles, ryr3 −/− mice ( n = 20). The unit of visual acuity is cycles/degree (c/d). (C and D) Scotopic ERG results of n = 9 WT (empty boxes) and n = 10 ryr3 −/− (gray boxes). Amplitude (in millivolts) of A-waves (C) and B-waves (D); the x axes represent the intensity of the illuminating flash. *, P

    Journal: The Journal of General Physiology

    Article Title: Extraocular muscle function is impaired inryr3−/− mice

    doi: 10.1085/jgp.201912333

    Figure Lengend Snippet: ryr3 −/− mice exhibit visual impairment . (A) Cued water maze. Top: Representative heatmaps of the swimming path of WT (left) and ryr3 −/− (right) mice. Bottom left: Total swimming distance (mean ± SEM) required to reach the platform. Bottom right: Time required to reach the platform. Experiments were performed on 10 mice per strain, and data from four different pool entry points were combined. (B) OKR. Top: OptoMotry setup. From Prusky et al. (2004) , Fig. 2 B is reprinted with permission from Investigative Ophthalmology Visual Science . Bottom: Visual acuity of two independent measurements performed under scotopic conditions on each mouse. Each symbol represents the visual acuity of a single mouse; empty circles, WT mice ( n = 20); gray circles, ryr3 −/− mice ( n = 20). The unit of visual acuity is cycles/degree (c/d). (C and D) Scotopic ERG results of n = 9 WT (empty boxes) and n = 10 ryr3 −/− (gray boxes). Amplitude (in millivolts) of A-waves (C) and B-waves (D); the x axes represent the intensity of the illuminating flash. *, P

    Article Snippet: The following primary and secondary antibodies were used: rabbit monoclonal anti-RYR1 (D4E1, #8153; Cell Signaling), mouse monoclonal anti-Cav 1.1 (IIC12D4 and IIID5E1; Iowa Hybridoma bank), rabbit anti-Cav 1.2 (sc-25686; Santa Cruz), Alexa Fluor 568 donkey anti-mouse IgG (A10037; Invitrogen), and Alexa Fluor 488 chicken anti-rabbit IgG (A21441; Invitrogen).

    Techniques: Mouse Assay

    Calcium homeostasis in isolated EOM fibers. (A) Confocal images of WT (a and b) and ryr3 −/− (c and d) fibers; a and c show the transmitted light channel, and b and d show Mag-Fluo4 fluorescence, excited at 488 nm and recorded at an emission between 500 and 550 nm. Scale bar, 30 µm. (B) Representative line scan traces of MagFluo4 calcium transients in EOM fibers from WT (black) and ryr3 −/− (gray), recorded at 7,921 lines per second. (C) Measurements of resting Ca 2+ expressed as ratio (340/380 nm) using the fluorescent indicator fura-2. Each symbol represents the ratio obtained from a single fiber. The horizontal black line shows the mean value. Fibers were isolated from a total of three mice per group, and a total of 49 fibers from WT and 40 fibers from ryr3 −/− were analyzed. (D) Peak Ca 2+ (ΔF/F) of the MagFluo4 fluorescence obtained by stimulating EOM fibers by electrical field stimulation with a 0.5-ms bipolar pulse. All experiments were performed at room temperature. Each symbol represents the value from a single fiber. The horizontal black line shows the mean value. A total of 14 fibers from four WT mice and 21 fibers from nine ryr3 −/− mice were analyzed. (E) Analysis of the kinetics of the Ca 2+ transients; TTHP, TTP, and HRT of the calcium transients are plotted. White dots, WT; gray dots, ryr3 −/− . A total of 12 fibers from four WT mice and 19 fibers from nine ryr3 −/− mice were analyzed. *, P

    Journal: The Journal of General Physiology

    Article Title: Extraocular muscle function is impaired inryr3−/− mice

    doi: 10.1085/jgp.201912333

    Figure Lengend Snippet: Calcium homeostasis in isolated EOM fibers. (A) Confocal images of WT (a and b) and ryr3 −/− (c and d) fibers; a and c show the transmitted light channel, and b and d show Mag-Fluo4 fluorescence, excited at 488 nm and recorded at an emission between 500 and 550 nm. Scale bar, 30 µm. (B) Representative line scan traces of MagFluo4 calcium transients in EOM fibers from WT (black) and ryr3 −/− (gray), recorded at 7,921 lines per second. (C) Measurements of resting Ca 2+ expressed as ratio (340/380 nm) using the fluorescent indicator fura-2. Each symbol represents the ratio obtained from a single fiber. The horizontal black line shows the mean value. Fibers were isolated from a total of three mice per group, and a total of 49 fibers from WT and 40 fibers from ryr3 −/− were analyzed. (D) Peak Ca 2+ (ΔF/F) of the MagFluo4 fluorescence obtained by stimulating EOM fibers by electrical field stimulation with a 0.5-ms bipolar pulse. All experiments were performed at room temperature. Each symbol represents the value from a single fiber. The horizontal black line shows the mean value. A total of 14 fibers from four WT mice and 21 fibers from nine ryr3 −/− mice were analyzed. (E) Analysis of the kinetics of the Ca 2+ transients; TTHP, TTP, and HRT of the calcium transients are plotted. White dots, WT; gray dots, ryr3 −/− . A total of 12 fibers from four WT mice and 19 fibers from nine ryr3 −/− mice were analyzed. *, P

    Article Snippet: The following primary and secondary antibodies were used: rabbit monoclonal anti-RYR1 (D4E1, #8153; Cell Signaling), mouse monoclonal anti-Cav 1.1 (IIC12D4 and IIID5E1; Iowa Hybridoma bank), rabbit anti-Cav 1.2 (sc-25686; Santa Cruz), Alexa Fluor 568 donkey anti-mouse IgG (A10037; Invitrogen), and Alexa Fluor 488 chicken anti-rabbit IgG (A21441; Invitrogen).

    Techniques: Isolation, Fluorescence, Mouse Assay, Mass Spectrometry

    Transcript expression and protein content of key players involved in skeletal muscle calcium homeostasis. (A) Expression levels of transcripts encoding ECC proteins measured by qPCR. Values are plotted as mean (±SEM) fold change in ryr3 −/− vs. WT levels (which were set as 1). ACTN2 was used as housekeeping gene. The mean expression level of duplicate determinations obtained from pooled EOM from four to six mice is shown. Transcript levels of RYR1 and CASQ1 were reduced by ∼50%. *, P

    Journal: The Journal of General Physiology

    Article Title: Extraocular muscle function is impaired inryr3−/− mice

    doi: 10.1085/jgp.201912333

    Figure Lengend Snippet: Transcript expression and protein content of key players involved in skeletal muscle calcium homeostasis. (A) Expression levels of transcripts encoding ECC proteins measured by qPCR. Values are plotted as mean (±SEM) fold change in ryr3 −/− vs. WT levels (which were set as 1). ACTN2 was used as housekeeping gene. The mean expression level of duplicate determinations obtained from pooled EOM from four to six mice is shown. Transcript levels of RYR1 and CASQ1 were reduced by ∼50%. *, P

    Article Snippet: The following primary and secondary antibodies were used: rabbit monoclonal anti-RYR1 (D4E1, #8153; Cell Signaling), mouse monoclonal anti-Cav 1.1 (IIC12D4 and IIID5E1; Iowa Hybridoma bank), rabbit anti-Cav 1.2 (sc-25686; Santa Cruz), Alexa Fluor 568 donkey anti-mouse IgG (A10037; Invitrogen), and Alexa Fluor 488 chicken anti-rabbit IgG (A21441; Invitrogen).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

    Ex vivo isometric force measurements in mouse EOMs . (A) Representative trace of the absolute twitch force obtained from isolated single extraocular rectus muscles from WT (black line; n = 8) and ryr3 −/− (gray line, n = 9) muscles. Muscles were stimulated by electric field stimulation with a pulse of 24.6 V having a duration of 0.5 ms. (B) Objective classification of force measurements into four groups. The force was normalized to the maximum of each transient to illustrate the differences in the kinetics as well as the presence or absence of the fast and slow component of the twitch. (C) Kinetic twitch parameters (TTHP, TTP, and HRT). Each symbol represents the kinetic parameters of a single rectus EOM from a single mouse; empty circles, WT mice; gray circles, ryr3 −/− mice. *, P

    Journal: The Journal of General Physiology

    Article Title: Extraocular muscle function is impaired inryr3−/− mice

    doi: 10.1085/jgp.201912333

    Figure Lengend Snippet: Ex vivo isometric force measurements in mouse EOMs . (A) Representative trace of the absolute twitch force obtained from isolated single extraocular rectus muscles from WT (black line; n = 8) and ryr3 −/− (gray line, n = 9) muscles. Muscles were stimulated by electric field stimulation with a pulse of 24.6 V having a duration of 0.5 ms. (B) Objective classification of force measurements into four groups. The force was normalized to the maximum of each transient to illustrate the differences in the kinetics as well as the presence or absence of the fast and slow component of the twitch. (C) Kinetic twitch parameters (TTHP, TTP, and HRT). Each symbol represents the kinetic parameters of a single rectus EOM from a single mouse; empty circles, WT mice; gray circles, ryr3 −/− mice. *, P

    Article Snippet: The following primary and secondary antibodies were used: rabbit monoclonal anti-RYR1 (D4E1, #8153; Cell Signaling), mouse monoclonal anti-Cav 1.1 (IIC12D4 and IIID5E1; Iowa Hybridoma bank), rabbit anti-Cav 1.2 (sc-25686; Santa Cruz), Alexa Fluor 568 donkey anti-mouse IgG (A10037; Invitrogen), and Alexa Fluor 488 chicken anti-rabbit IgG (A21441; Invitrogen).

    Techniques: Ex Vivo, Isolation, Mass Spectrometry, Mouse Assay

    Fiber type distribution and MyHC composition is similar in EOM muscles from WT and ryr3 −/− mice. (A) EOM from WT and ryr3 −/− mice were sectioned, stained with anti-MyHC recognizing all isoforms and DAPI, and observed by fluorescent microscopy. Scale bars represent 500 µm (5× images) and 200 µm (10× images). (B) The fiber size distribution of EOMs was determined using the minimal Feret’s diameter using MyHC immunohistochemistry ( Delbono et al., 2007 ; black bars, WT; red bars ryr3 −/− ). (C) High-resolution SDS-PAGE separation of MyHC isoforms in WT and ryr3 −/− EOM muscles ( Talmadge and Roy, 1993 ). The bottom bar graphs show the percent specific MyHC isoform vs. total MyHC content in ryr3 −/− relative to WT (mean ± SEM, n = 5 WT and n = 5 ryr3 −/− ), which was set to 100%.

    Journal: The Journal of General Physiology

    Article Title: Extraocular muscle function is impaired inryr3−/− mice

    doi: 10.1085/jgp.201912333

    Figure Lengend Snippet: Fiber type distribution and MyHC composition is similar in EOM muscles from WT and ryr3 −/− mice. (A) EOM from WT and ryr3 −/− mice were sectioned, stained with anti-MyHC recognizing all isoforms and DAPI, and observed by fluorescent microscopy. Scale bars represent 500 µm (5× images) and 200 µm (10× images). (B) The fiber size distribution of EOMs was determined using the minimal Feret’s diameter using MyHC immunohistochemistry ( Delbono et al., 2007 ; black bars, WT; red bars ryr3 −/− ). (C) High-resolution SDS-PAGE separation of MyHC isoforms in WT and ryr3 −/− EOM muscles ( Talmadge and Roy, 1993 ). The bottom bar graphs show the percent specific MyHC isoform vs. total MyHC content in ryr3 −/− relative to WT (mean ± SEM, n = 5 WT and n = 5 ryr3 −/− ), which was set to 100%.

    Article Snippet: The following primary and secondary antibodies were used: rabbit monoclonal anti-RYR1 (D4E1, #8153; Cell Signaling), mouse monoclonal anti-Cav 1.1 (IIC12D4 and IIID5E1; Iowa Hybridoma bank), rabbit anti-Cav 1.2 (sc-25686; Santa Cruz), Alexa Fluor 568 donkey anti-mouse IgG (A10037; Invitrogen), and Alexa Fluor 488 chicken anti-rabbit IgG (A21441; Invitrogen).

    Techniques: Mouse Assay, Staining, Microscopy, Immunohistochemistry, SDS Page

    RYR3 is expressed in murine EOM. (A) Expression of ECC transcripts in mouse EOM muscles. Transcript levels were quantified by qPCR in muscles from three WT mice (performed twice in triplicate). Values are plotted as mean (SEM) change in EOM vs. hindlimb muscles, which was set as 1. **, P

    Journal: The Journal of General Physiology

    Article Title: Extraocular muscle function is impaired inryr3−/− mice

    doi: 10.1085/jgp.201912333

    Figure Lengend Snippet: RYR3 is expressed in murine EOM. (A) Expression of ECC transcripts in mouse EOM muscles. Transcript levels were quantified by qPCR in muscles from three WT mice (performed twice in triplicate). Values are plotted as mean (SEM) change in EOM vs. hindlimb muscles, which was set as 1. **, P

    Article Snippet: The following primary and secondary antibodies were used: rabbit monoclonal anti-RYR1 (D4E1, #8153; Cell Signaling), mouse monoclonal anti-Cav 1.1 (IIC12D4 and IIID5E1; Iowa Hybridoma bank), rabbit anti-Cav 1.2 (sc-25686; Santa Cruz), Alexa Fluor 568 donkey anti-mouse IgG (A10037; Invitrogen), and Alexa Fluor 488 chicken anti-rabbit IgG (A21441; Invitrogen).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

    Immunohistochemical analysis and subcellular localization of RYR1 and of the α 1 subunit of the DHPR in single muscle fibers from WT and ryr3 −/− mice. (A) Left and central panels show the staining obtained using rabbit anti-RYR1 mAb D4E1 (green in merged image) and mouse anti-Ca v 1.1 (red in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). (B) Left and central panels show the staining obtained using mouse anti-Ca v 1.1 (red in merged image) and rabbit anti-Ca v 1.2 (green in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). The bottom panels show staining of mouse EDL fibers, which are negative for Ca v 1.2 and were used as staining control. All images in B were acquired using the same settings for the laser intensities and acquisition parameters. Images were acquired using a Nikon A1 plus confocal microscope equipped with a Plan Apo 60× oil objective (numerical aperture, 1.4) and stained as described in Materials and methods. Orange pixels show areas of colocalization. Scale bars, 30 µm.

    Journal: The Journal of General Physiology

    Article Title: Extraocular muscle function is impaired inryr3−/− mice

    doi: 10.1085/jgp.201912333

    Figure Lengend Snippet: Immunohistochemical analysis and subcellular localization of RYR1 and of the α 1 subunit of the DHPR in single muscle fibers from WT and ryr3 −/− mice. (A) Left and central panels show the staining obtained using rabbit anti-RYR1 mAb D4E1 (green in merged image) and mouse anti-Ca v 1.1 (red in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). (B) Left and central panels show the staining obtained using mouse anti-Ca v 1.1 (red in merged image) and rabbit anti-Ca v 1.2 (green in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). The bottom panels show staining of mouse EDL fibers, which are negative for Ca v 1.2 and were used as staining control. All images in B were acquired using the same settings for the laser intensities and acquisition parameters. Images were acquired using a Nikon A1 plus confocal microscope equipped with a Plan Apo 60× oil objective (numerical aperture, 1.4) and stained as described in Materials and methods. Orange pixels show areas of colocalization. Scale bars, 30 µm.

    Article Snippet: The following primary and secondary antibodies were used: rabbit monoclonal anti-RYR1 (D4E1, #8153; Cell Signaling), mouse monoclonal anti-Cav 1.1 (IIC12D4 and IIID5E1; Iowa Hybridoma bank), rabbit anti-Cav 1.2 (sc-25686; Santa Cruz), Alexa Fluor 568 donkey anti-mouse IgG (A10037; Invitrogen), and Alexa Fluor 488 chicken anti-rabbit IgG (A21441; Invitrogen).

    Techniques: Immunohistochemistry, Mouse Assay, Staining, Microscopy

    Distribution of RyR2 signals in cytoplasm of SMB cells. Representative images double-immunofluorescently stained with the antibodies for RyR2 (green) and ER (red, A) or membrane (red, B) on the slices of SMB-S15 and SMB-PS cells.

    Journal: Prion

    Article Title: Decrease of RyR2 in the prion infected cell line and in the brains of the scrapie infected mice models and the patients of human prion diseases

    doi: 10.1080/19336896.2018.1465162

    Figure Lengend Snippet: Distribution of RyR2 signals in cytoplasm of SMB cells. Representative images double-immunofluorescently stained with the antibodies for RyR2 (green) and ER (red, A) or membrane (red, B) on the slices of SMB-S15 and SMB-PS cells.

    Article Snippet: Paraffin embedded brain tissue slices were permeabilizated by 0.3% Triton X-100 in PBS for 30 min and blocked with normal goat serum for 1 h. After blocked, samples were probed with 1:100 diluted mAb anti-RyR2 (Abcam, UK), 1:100 diluted polyclonal antibody (pAb) for ER (Thermo Fisher, UK), 1:100 diluted pAb anti-membrane (Thermo Fisher, UK), 1:200 diluted pAb anti-NeuN (Millipore, USA) or 1:200-diluted pAb anti-GFAP (Cell Signaling Technology, USA) in dilution solution (PBS with 2% BSA and 0.3% Triton X-100) at 4°C overnight.

    Techniques: Staining

    Colocalization of RyR2 signals with NeuN signals in the brain slices of mice. Representative images of double stained IFA by the antibodies for RyR2 (green) and NeuN (red, A) or GFAP (red, B).

    Journal: Prion

    Article Title: Decrease of RyR2 in the prion infected cell line and in the brains of the scrapie infected mice models and the patients of human prion diseases

    doi: 10.1080/19336896.2018.1465162

    Figure Lengend Snippet: Colocalization of RyR2 signals with NeuN signals in the brain slices of mice. Representative images of double stained IFA by the antibodies for RyR2 (green) and NeuN (red, A) or GFAP (red, B).

    Article Snippet: Paraffin embedded brain tissue slices were permeabilizated by 0.3% Triton X-100 in PBS for 30 min and blocked with normal goat serum for 1 h. After blocked, samples were probed with 1:100 diluted mAb anti-RyR2 (Abcam, UK), 1:100 diluted polyclonal antibody (pAb) for ER (Thermo Fisher, UK), 1:100 diluted pAb anti-membrane (Thermo Fisher, UK), 1:200 diluted pAb anti-NeuN (Millipore, USA) or 1:200-diluted pAb anti-GFAP (Cell Signaling Technology, USA) in dilution solution (PBS with 2% BSA and 0.3% Triton X-100) at 4°C overnight.

    Techniques: Mouse Assay, Staining, Immunofluorescence

    Dynamic decreases of RyR2 in the brains of the mice infected with scrapie agents 139A and ME7. The brain samples collected at 0, 80, 120, 150 and180 dpi were pooled from three individual mice and tested by RyR2 specific Western blot. The densities of signals are determined by densitometry and shown relative to RyR2/β-actin.

    Journal: Prion

    Article Title: Decrease of RyR2 in the prion infected cell line and in the brains of the scrapie infected mice models and the patients of human prion diseases

    doi: 10.1080/19336896.2018.1465162

    Figure Lengend Snippet: Dynamic decreases of RyR2 in the brains of the mice infected with scrapie agents 139A and ME7. The brain samples collected at 0, 80, 120, 150 and180 dpi were pooled from three individual mice and tested by RyR2 specific Western blot. The densities of signals are determined by densitometry and shown relative to RyR2/β-actin.

    Article Snippet: Paraffin embedded brain tissue slices were permeabilizated by 0.3% Triton X-100 in PBS for 30 min and blocked with normal goat serum for 1 h. After blocked, samples were probed with 1:100 diluted mAb anti-RyR2 (Abcam, UK), 1:100 diluted polyclonal antibody (pAb) for ER (Thermo Fisher, UK), 1:100 diluted pAb anti-membrane (Thermo Fisher, UK), 1:200 diluted pAb anti-NeuN (Millipore, USA) or 1:200-diluted pAb anti-GFAP (Cell Signaling Technology, USA) in dilution solution (PBS with 2% BSA and 0.3% Triton X-100) at 4°C overnight.

    Techniques: Mouse Assay, Infection, Western Blot

    Decrease of RyR2 in the postmortem cortical tissues of the patients of sCJD, D178N-FFI and G114V-gCJD. The RyR2 levels in the brain samples were tested by RyR2 specific Western blot. The brain samples donated by healthy persons (n = 2) were used as control. The densities of signals are determined by densitometry and shown relative to RyR2/β-actin. Graphical data denote mean+SD. Statistical differences compared with controls are illustrated on the top.

    Journal: Prion

    Article Title: Decrease of RyR2 in the prion infected cell line and in the brains of the scrapie infected mice models and the patients of human prion diseases

    doi: 10.1080/19336896.2018.1465162

    Figure Lengend Snippet: Decrease of RyR2 in the postmortem cortical tissues of the patients of sCJD, D178N-FFI and G114V-gCJD. The RyR2 levels in the brain samples were tested by RyR2 specific Western blot. The brain samples donated by healthy persons (n = 2) were used as control. The densities of signals are determined by densitometry and shown relative to RyR2/β-actin. Graphical data denote mean+SD. Statistical differences compared with controls are illustrated on the top.

    Article Snippet: Paraffin embedded brain tissue slices were permeabilizated by 0.3% Triton X-100 in PBS for 30 min and blocked with normal goat serum for 1 h. After blocked, samples were probed with 1:100 diluted mAb anti-RyR2 (Abcam, UK), 1:100 diluted polyclonal antibody (pAb) for ER (Thermo Fisher, UK), 1:100 diluted pAb anti-membrane (Thermo Fisher, UK), 1:200 diluted pAb anti-NeuN (Millipore, USA) or 1:200-diluted pAb anti-GFAP (Cell Signaling Technology, USA) in dilution solution (PBS with 2% BSA and 0.3% Triton X-100) at 4°C overnight.

    Techniques: Western Blot

    Decrease of RyR2 in the prion infected cell line SMB-S15 cells. The same amounts of the lysates from SMB-15 and SMB-PS cells were loaded onto 8% SDS-PAGE and the levels of RyR2 were evaluated by RyR2-specific Western blot analysis. β-actin was used as an internal control. The densities of signals are determined by densitometry and shown relative to RyR2/β-actin. Graphical data denote mean+SD (n = 3, three different batches). Statistical differences compared with controls are illustrated on the top.

    Journal: Prion

    Article Title: Decrease of RyR2 in the prion infected cell line and in the brains of the scrapie infected mice models and the patients of human prion diseases

    doi: 10.1080/19336896.2018.1465162

    Figure Lengend Snippet: Decrease of RyR2 in the prion infected cell line SMB-S15 cells. The same amounts of the lysates from SMB-15 and SMB-PS cells were loaded onto 8% SDS-PAGE and the levels of RyR2 were evaluated by RyR2-specific Western blot analysis. β-actin was used as an internal control. The densities of signals are determined by densitometry and shown relative to RyR2/β-actin. Graphical data denote mean+SD (n = 3, three different batches). Statistical differences compared with controls are illustrated on the top.

    Article Snippet: Paraffin embedded brain tissue slices were permeabilizated by 0.3% Triton X-100 in PBS for 30 min and blocked with normal goat serum for 1 h. After blocked, samples were probed with 1:100 diluted mAb anti-RyR2 (Abcam, UK), 1:100 diluted polyclonal antibody (pAb) for ER (Thermo Fisher, UK), 1:100 diluted pAb anti-membrane (Thermo Fisher, UK), 1:200 diluted pAb anti-NeuN (Millipore, USA) or 1:200-diluted pAb anti-GFAP (Cell Signaling Technology, USA) in dilution solution (PBS with 2% BSA and 0.3% Triton X-100) at 4°C overnight.

    Techniques: Infection, SDS Page, Western Blot

    Decrease of RyR2 in the brains of the mice infected with the lysates of SMB-S15 cells. The levels of RyR2 in the brain homogenates of the SMB-S15 infected mice (n = 3) at terminal stage and SMB-PS inoculated mice (n = 3) were evaluated with Western blot. β-actin was used as an internal control. The densities of signals are determined by densitometry and shown relative to RyR2/β-actin. Graphical data denote mean+SD. Statistical differences compared with controls are illustrated on the top.

    Journal: Prion

    Article Title: Decrease of RyR2 in the prion infected cell line and in the brains of the scrapie infected mice models and the patients of human prion diseases

    doi: 10.1080/19336896.2018.1465162

    Figure Lengend Snippet: Decrease of RyR2 in the brains of the mice infected with the lysates of SMB-S15 cells. The levels of RyR2 in the brain homogenates of the SMB-S15 infected mice (n = 3) at terminal stage and SMB-PS inoculated mice (n = 3) were evaluated with Western blot. β-actin was used as an internal control. The densities of signals are determined by densitometry and shown relative to RyR2/β-actin. Graphical data denote mean+SD. Statistical differences compared with controls are illustrated on the top.

    Article Snippet: Paraffin embedded brain tissue slices were permeabilizated by 0.3% Triton X-100 in PBS for 30 min and blocked with normal goat serum for 1 h. After blocked, samples were probed with 1:100 diluted mAb anti-RyR2 (Abcam, UK), 1:100 diluted polyclonal antibody (pAb) for ER (Thermo Fisher, UK), 1:100 diluted pAb anti-membrane (Thermo Fisher, UK), 1:200 diluted pAb anti-NeuN (Millipore, USA) or 1:200-diluted pAb anti-GFAP (Cell Signaling Technology, USA) in dilution solution (PBS with 2% BSA and 0.3% Triton X-100) at 4°C overnight.

    Techniques: Mouse Assay, Infection, Western Blot

    Slight recovery of RyR2 in the cell line of SMB-S15-Res after removal of prion infection. Western blot of RyR2 in the cell lines of SMB-PS, SMB-S15 and SMB-S15-Res. The same amounts of the lysates from three cell lines were loaded into 8% SDS-PAGE. The densities of signals are determined by densitometry and shown relative to RyR2/β-actin. Graphical data denote mean+SD. Statistical differences compared with controls are illustrated on the top.

    Journal: Prion

    Article Title: Decrease of RyR2 in the prion infected cell line and in the brains of the scrapie infected mice models and the patients of human prion diseases

    doi: 10.1080/19336896.2018.1465162

    Figure Lengend Snippet: Slight recovery of RyR2 in the cell line of SMB-S15-Res after removal of prion infection. Western blot of RyR2 in the cell lines of SMB-PS, SMB-S15 and SMB-S15-Res. The same amounts of the lysates from three cell lines were loaded into 8% SDS-PAGE. The densities of signals are determined by densitometry and shown relative to RyR2/β-actin. Graphical data denote mean+SD. Statistical differences compared with controls are illustrated on the top.

    Article Snippet: Paraffin embedded brain tissue slices were permeabilizated by 0.3% Triton X-100 in PBS for 30 min and blocked with normal goat serum for 1 h. After blocked, samples were probed with 1:100 diluted mAb anti-RyR2 (Abcam, UK), 1:100 diluted polyclonal antibody (pAb) for ER (Thermo Fisher, UK), 1:100 diluted pAb anti-membrane (Thermo Fisher, UK), 1:200 diluted pAb anti-NeuN (Millipore, USA) or 1:200-diluted pAb anti-GFAP (Cell Signaling Technology, USA) in dilution solution (PBS with 2% BSA and 0.3% Triton X-100) at 4°C overnight.

    Techniques: Infection, Western Blot, SDS Page

    Comparative analysis of the mRNA transcriptions of RyR2 by qRT-PCR. The brain tissues of 139- and ME7-infected mice (n = 3) were collected at end stage, while those of the age-matched normal mice (n- = 3) were used as the normal control. The transcription level of RyR2 mRNA each reaction was determined relative to that of the individual GAPDH. The relative intensity (2 −△△Ct values) of brain RyR2 transcript of 139A- or ME7-infected mice was relative to that of normal mice that was set to 1. Data are representative of three independent experiments. Statistical differences between normal and infected animals are indicated above.

    Journal: Prion

    Article Title: Decrease of RyR2 in the prion infected cell line and in the brains of the scrapie infected mice models and the patients of human prion diseases

    doi: 10.1080/19336896.2018.1465162

    Figure Lengend Snippet: Comparative analysis of the mRNA transcriptions of RyR2 by qRT-PCR. The brain tissues of 139- and ME7-infected mice (n = 3) were collected at end stage, while those of the age-matched normal mice (n- = 3) were used as the normal control. The transcription level of RyR2 mRNA each reaction was determined relative to that of the individual GAPDH. The relative intensity (2 −△△Ct values) of brain RyR2 transcript of 139A- or ME7-infected mice was relative to that of normal mice that was set to 1. Data are representative of three independent experiments. Statistical differences between normal and infected animals are indicated above.

    Article Snippet: Paraffin embedded brain tissue slices were permeabilizated by 0.3% Triton X-100 in PBS for 30 min and blocked with normal goat serum for 1 h. After blocked, samples were probed with 1:100 diluted mAb anti-RyR2 (Abcam, UK), 1:100 diluted polyclonal antibody (pAb) for ER (Thermo Fisher, UK), 1:100 diluted pAb anti-membrane (Thermo Fisher, UK), 1:200 diluted pAb anti-NeuN (Millipore, USA) or 1:200-diluted pAb anti-GFAP (Cell Signaling Technology, USA) in dilution solution (PBS with 2% BSA and 0.3% Triton X-100) at 4°C overnight.

    Techniques: Quantitative RT-PCR, Infection, Mouse Assay

    Immunoblot analysis of Ca 2+ -cycling proteins from WT and KO mice Cardiac protein samples were separated by SDS-PAGE and immunoblotting was performed with antibodies against various Ca 2+ -cycling proteins. a: Representative western blot results of SR Ca 2+ -cycling protein levels in WT and HRC-KO hearts. b: Quantification of SR protein levels in WT and HRC-KO hearts. p-RyR2 indicates phosphorylated RyR2 at serine 2809 and 2814; PLN, phospholamban; CSQ, calsequestrin. Data are means ± SEM; n=6 hearts for WT or HRC KO mice. *P

    Journal: Basic research in cardiology

    Article Title: Targeted ablation of the histidine-rich Ca2+-binding protein (HRC) gene is associated with abnormal SR Ca2+-cycling and severe pathology under pressure-overload stress

    doi: 10.1007/s00395-013-0344-2

    Figure Lengend Snippet: Immunoblot analysis of Ca 2+ -cycling proteins from WT and KO mice Cardiac protein samples were separated by SDS-PAGE and immunoblotting was performed with antibodies against various Ca 2+ -cycling proteins. a: Representative western blot results of SR Ca 2+ -cycling protein levels in WT and HRC-KO hearts. b: Quantification of SR protein levels in WT and HRC-KO hearts. p-RyR2 indicates phosphorylated RyR2 at serine 2809 and 2814; PLN, phospholamban; CSQ, calsequestrin. Data are means ± SEM; n=6 hearts for WT or HRC KO mice. *P

    Article Snippet: The monoclonal anti-RyR2 antibody was from Sigma (Sigma-Aldrich Co., RBI, Natick, MA).

    Techniques: Mouse Assay, SDS Page, Western Blot

    Visualization of RyR2 in the rat heart mitochondrial fractions using immuno-organelle chemistry. pRHM, cRHM, and RHSR “glued” to CellTak-coated coverslip were fixed with paraformaldehyde and labeled with mouse monoclonal or rabbit polyclonal

    Journal:

    Article Title: Physical Coupling Supports the Local Ca2+ Transfer between Sarcoplasmic Reticulum Subdomains and the Mitochondria in Heart Muscle *

    doi: 10.1074/jbc.M803385200

    Figure Lengend Snippet: Visualization of RyR2 in the rat heart mitochondrial fractions using immuno-organelle chemistry. pRHM, cRHM, and RHSR “glued” to CellTak-coated coverslip were fixed with paraformaldehyde and labeled with mouse monoclonal or rabbit polyclonal

    Article Snippet: Primary antibodies were obtained as follows: mouse monoclonal: anti-RyR2 from ABR (MA3–916), anti-phospholamban from Abcam (ab2865), and anti-cytochrome oxidase subunit 1 from Molecular Probes (A6403); rabbit polyclonal: anti-RyR2 from Chemicon (AB9080), anti-SERCA2 from ABR (MA3–919), anti-VDAC from ABR (PA1–954A), and anti-calsequestrin from Upstate (catalog number 06-382).

    Techniques: Labeling