Structured Review

Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1 ryr3
Mouse Monoclonal Anti Ryr1 Ryr3, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse monoclonal anti ryr1 ryr3/product/Developmental Studies Hybridoma Bank
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse monoclonal anti ryr1 ryr3 - by Bioz Stars, 2023-01
86/100 stars

Images


Structured Review

Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1 ryr3 34c
Structural cores and blood markers of muscle damage (CK and LDH) at 4 months of age. (a–c) Representative histological images of normal fibers (a), fibers with unstructured cores (b), and fibers with contracture cores (c). (d) Percentage of EDL fibers presenting the features classified in (a–c) (white: normal fibers ; grey: fibers with unstructured cores ; and dark grey: fibers with contracture cores ). See also Table S1. (e and f) Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH). In (e) and (f), data are given as mean ± SEM; ∗ p < 0.05, WT versus <t>RYR1</t> Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (d), n = number of fibers analyzed; in (e-f), n = number of mice. Scale bar: 10 μm .
Mouse Monoclonal Anti Ryr1 Ryr3 34c, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse monoclonal anti ryr1 ryr3 34c/product/Developmental Studies Hybridoma Bank
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse monoclonal anti ryr1 ryr3 34c - by Bioz Stars, 2023-01
94/100 stars

Images

1) Product Images from "Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice"

Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2017/6792694

Structural cores and blood markers of muscle damage (CK and LDH) at 4 months of age. (a–c) Representative histological images of normal fibers (a), fibers with unstructured cores (b), and fibers with contracture cores (c). (d) Percentage of EDL fibers presenting the features classified in (a–c) (white: normal fibers ; grey: fibers with unstructured cores ; and dark grey: fibers with contracture cores ). See also Table S1. (e and f) Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH). In (e) and (f), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (d), n = number of fibers analyzed; in (e-f), n = number of mice. Scale bar: 10 μm .
Figure Legend Snippet: Structural cores and blood markers of muscle damage (CK and LDH) at 4 months of age. (a–c) Representative histological images of normal fibers (a), fibers with unstructured cores (b), and fibers with contracture cores (c). (d) Percentage of EDL fibers presenting the features classified in (a–c) (white: normal fibers ; grey: fibers with unstructured cores ; and dark grey: fibers with contracture cores ). See also Table S1. (e and f) Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH). In (e) and (f), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (d), n = number of fibers analyzed; in (e-f), n = number of mice. Scale bar: 10 μm .

Techniques Used:

Fiber ultrastructure, calpain activity, and carbonyl protein content at 4 months of age. (a–f) Representative immunofluorescence and EM images showing fluorescent cross striation (a, c, and e) and myofibrillar/sarcomeric organization (b, d, and f) in EDL fibers. In (b) and (f), small arrows point at Z -lines, while in (c and d) large arrows point to areas in which cross striation and sarcomeric structure are compromised. (g) Calpain activity expressed in total hind limb muscle homogenates. (h) Carbonyl protein content in EDL muscle homogenates. In (g and h), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (g and h), n = number of mice. Scale bars in (a, c, and e) 10 μ m (insets 5 μ m); in (b, d, and f) 1 μ m.
Figure Legend Snippet: Fiber ultrastructure, calpain activity, and carbonyl protein content at 4 months of age. (a–f) Representative immunofluorescence and EM images showing fluorescent cross striation (a, c, and e) and myofibrillar/sarcomeric organization (b, d, and f) in EDL fibers. In (b) and (f), small arrows point at Z -lines, while in (c and d) large arrows point to areas in which cross striation and sarcomeric structure are compromised. (g) Calpain activity expressed in total hind limb muscle homogenates. (h) Carbonyl protein content in EDL muscle homogenates. In (g and h), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (g and h), n = number of mice. Scale bars in (a, c, and e) 10 μ m (insets 5 μ m); in (b, d, and f) 1 μ m.

Techniques Used: Activity Assay, Immunofluorescence

In vivo grip strength and ex vivo specific force at 4 months of age. (a) Time-course of grip strength from 2 (beginning of short-term NAC treatment) to 4 months (end of short-term NAC treatment) of age expressed as force on body weight (N/g). (b) Change in grip strength from 2 to 4 months of age (shown as a percentage). (c and d) Force-frequency (1–140 Hz) relationship curves of specific force (c) and specific force during a single 2 s, 120 Hz stimulation train (d) recorded for the same EDL muscles. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (b), n = number of mice; in (d), n = number of EDL muscles.
Figure Legend Snippet: In vivo grip strength and ex vivo specific force at 4 months of age. (a) Time-course of grip strength from 2 (beginning of short-term NAC treatment) to 4 months (end of short-term NAC treatment) of age expressed as force on body weight (N/g). (b) Change in grip strength from 2 to 4 months of age (shown as a percentage). (c and d) Force-frequency (1–140 Hz) relationship curves of specific force (c) and specific force during a single 2 s, 120 Hz stimulation train (d) recorded for the same EDL muscles. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (b), n = number of mice; in (d), n = number of EDL muscles.

Techniques Used: In Vivo, Ex Vivo

Mitochondrial damage, size, and volume at 4 months of age. (a–c) Representative EM images displaying apparently normal (a) and damaged mitochondria (b and c). (d) Percentage of damaged mitochondria. (e) Average number of damaged mitochondria/area of EM section. (f) Average size of apparently normal mitochondria (i.e., mitochondria not included in the quantitative analysis of (d and e)). (g) Percentage of fiber volume occupied by mitochondria. See also Table S2. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (d and f), n = number of measurements; in (e and g), n = number of fibers analyzed. Scale bar: 0.1 μ m.
Figure Legend Snippet: Mitochondrial damage, size, and volume at 4 months of age. (a–c) Representative EM images displaying apparently normal (a) and damaged mitochondria (b and c). (d) Percentage of damaged mitochondria. (e) Average number of damaged mitochondria/area of EM section. (f) Average size of apparently normal mitochondria (i.e., mitochondria not included in the quantitative analysis of (d and e)). (g) Percentage of fiber volume occupied by mitochondria. See also Table S2. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (d and f), n = number of measurements; in (e and g), n = number of fibers analyzed. Scale bar: 0.1 μ m.

Techniques Used:

Effects of long-term NAC treatment at 10 months of age. (a) Quantitative analysis of EDL fibers presenting the features classified in Figures , , and shown as percentage of fibers analyzed (white: normal fibers ; grey: fibers with unstructured cores ; and dark grey: fibers with contracture cores ). See also Table S4. (b and c) Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH). (d) Time course of grip strength from 4 (beginning of long-term NAC treatment) to 10 months (end of long-term NAC treatment) of age expressed as force on body weight (N/g). (e) Change in grip strength from 4 to 10 months of age (shown as a percentage). (f and g) Force-frequency (1–140 Hz) relationship curves of specific force (f) and specific force during a single 2 s, 120 Hz stimulation train (g) recorded for the same EDL muscles. In s (b–g), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (a), n = number of EDL fibers analyzed; in (b–e), n = number of mice; in (f and g), n = number of EDL muscles.
Figure Legend Snippet: Effects of long-term NAC treatment at 10 months of age. (a) Quantitative analysis of EDL fibers presenting the features classified in Figures , , and shown as percentage of fibers analyzed (white: normal fibers ; grey: fibers with unstructured cores ; and dark grey: fibers with contracture cores ). See also Table S4. (b and c) Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH). (d) Time course of grip strength from 4 (beginning of long-term NAC treatment) to 10 months (end of long-term NAC treatment) of age expressed as force on body weight (N/g). (e) Change in grip strength from 4 to 10 months of age (shown as a percentage). (f and g) Force-frequency (1–140 Hz) relationship curves of specific force (f) and specific force during a single 2 s, 120 Hz stimulation train (g) recorded for the same EDL muscles. In s (b–g), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (a), n = number of EDL fibers analyzed; in (b–e), n = number of mice; in (f and g), n = number of EDL muscles.

Techniques Used:

Oxidative stress markers at 4 and 10 months of age following either short- or long-term treatment. (a and e) Representative immunoblots showing expression of 3-Nitrotyrosine (3-NT), SOD1, and SOD2 in total hind limb muscle homogenates. (b–d) and (f–h) Relative band densities normalized to GAPDH levels. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. n = number of mice.
Figure Legend Snippet: Oxidative stress markers at 4 and 10 months of age following either short- or long-term treatment. (a and e) Representative immunoblots showing expression of 3-Nitrotyrosine (3-NT), SOD1, and SOD2 in total hind limb muscle homogenates. (b–d) and (f–h) Relative band densities normalized to GAPDH levels. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. n = number of mice.

Techniques Used: Western Blot, Expressing


Structured Review

Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1
Schematic diagrams of excitation–contraction coupling (ECC) at the triad, and SPEG domains and pathogenic variants. (A) The triads are made of transverse tubules (T-tubules) flanked by terminal cisternae of the sarcoplasmic reticulum (SR). Junctional membranes (JM) are connected by the interaction of the <t>ryanodine</t> <t>receptor</t> <t>(RyR1)</t> at the SR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with <t>RyR1,</t> leading to its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the Ca 2+ transporter sarco/endoplasmic reticulum Ca 2+ -ATPase [SERCA; also known as ATP2A; regulated by phospholamban (PLN)] returns Ca 2+ into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J) and triadin (T). Striated muscle enriched protein kinase (SPEG) is localized at the triad, but its role remains elusive. (B) SPEG contains immunoglobulin domains (Ig; light purple shaded), fibronectin domains (Fn; light green shaded) and two kinase domains (yellow shaded). SPEG directly binds to myotubularin 1 (MTM1; dark green; SPEG 2530-2674 a.a.) and desmin (DES; magenta; SPEG 2200-2960 a.a.) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Dfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue font), a cardiomyopathy only (e.g. dilated cardiomyopathy, red font) or both (black font), with no clear genotype–phenotype correlation. Note that patient Q2233* died before a cardiac evaluation. Illustrations were made using Illustrator for Biological Sciences (IBS) .
Mouse Monoclonal Anti Ryr1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse monoclonal anti ryr1/product/Developmental Studies Hybridoma Bank
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse monoclonal anti ryr1 - by Bioz Stars, 2023-01
94/100 stars

Images

1) Product Images from "Characterization of a novel zebrafish model of SPEG -related centronuclear myopathy"

Article Title: Characterization of a novel zebrafish model of SPEG -related centronuclear myopathy

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.049437

Schematic diagrams of excitation–contraction coupling (ECC) at the triad, and SPEG domains and pathogenic variants. (A) The triads are made of transverse tubules (T-tubules) flanked by terminal cisternae of the sarcoplasmic reticulum (SR). Junctional membranes (JM) are connected by the interaction of the ryanodine receptor (RyR1) at the SR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with RyR1, leading to its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the Ca 2+ transporter sarco/endoplasmic reticulum Ca 2+ -ATPase [SERCA; also known as ATP2A; regulated by phospholamban (PLN)] returns Ca 2+ into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J) and triadin (T). Striated muscle enriched protein kinase (SPEG) is localized at the triad, but its role remains elusive. (B) SPEG contains immunoglobulin domains (Ig; light purple shaded), fibronectin domains (Fn; light green shaded) and two kinase domains (yellow shaded). SPEG directly binds to myotubularin 1 (MTM1; dark green; SPEG 2530-2674 a.a.) and desmin (DES; magenta; SPEG 2200-2960 a.a.) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Dfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue font), a cardiomyopathy only (e.g. dilated cardiomyopathy, red font) or both (black font), with no clear genotype–phenotype correlation. Note that patient Q2233* died before a cardiac evaluation. Illustrations were made using Illustrator for Biological Sciences (IBS) .
Figure Legend Snippet: Schematic diagrams of excitation–contraction coupling (ECC) at the triad, and SPEG domains and pathogenic variants. (A) The triads are made of transverse tubules (T-tubules) flanked by terminal cisternae of the sarcoplasmic reticulum (SR). Junctional membranes (JM) are connected by the interaction of the ryanodine receptor (RyR1) at the SR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with RyR1, leading to its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the Ca 2+ transporter sarco/endoplasmic reticulum Ca 2+ -ATPase [SERCA; also known as ATP2A; regulated by phospholamban (PLN)] returns Ca 2+ into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J) and triadin (T). Striated muscle enriched protein kinase (SPEG) is localized at the triad, but its role remains elusive. (B) SPEG contains immunoglobulin domains (Ig; light purple shaded), fibronectin domains (Fn; light green shaded) and two kinase domains (yellow shaded). SPEG directly binds to myotubularin 1 (MTM1; dark green; SPEG 2530-2674 a.a.) and desmin (DES; magenta; SPEG 2200-2960 a.a.) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Dfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue font), a cardiomyopathy only (e.g. dilated cardiomyopathy, red font) or both (black font), with no clear genotype–phenotype correlation. Note that patient Q2233* died before a cardiac evaluation. Illustrations were made using Illustrator for Biological Sciences (IBS) .

Techniques Used: Activation Assay, Binding Assay

Speg expression during zebrafish development. There are two Speg genes in zebrafish, spega and spegb . Each encodes a single transcript that is highly conserved with human SPEG . (A,B) RT-qPCR shows similar temporal expression patterns of spega (A) versus spegb (B) from 2 days post-fertilization (dpf) to 7 dpf. Both Speg mRNA transcripts are significantly upregulated in the heads (light blue bars; 20-fold for spega , 7-fold for spegb ), but stay at relatively similar levels in the skeletal-muscle-predominant tails (white bars). Each data point represents the average of technical triplicates, and three independent experiments are included. Columns and error bars represent mean±s.e.m. Unpaired two-tailed Student's t -test was performed: ** P <0.01; ns, not significant. (C) Whole-mount in situ hybridization using DIG-conjugated RNA probes in 1 dpf embryos shows distinct spatial expression patterns of spega versus spegb . spega is predominantly expressed in the developing brain (yellow arrowhead) and along the neural tube (red arrowheads), but absent from the notochord (cyan arrowheads). spegb staining, however, is predominantly detected at the chevron-shaped developing somites (black arrowheads). Scale bars: 200 µm (top and middle rows) or 100 µm (bottom row). (D,E) Confocal images showing 2 dpf (D) or 5 dpf (E) isolated wild-type (WT) skeletal myofibers double-stained with anti-RyR1 (34C, DSHB) and anti-SPEG (PA553875, Invitrogen). (D,D′) At 2 dpf, SPEG is predominantly localized at the sarcolemma (yellow arrows) and perinuclear regions (cyan arrows; nucleus, yellow asterisks), with weak expression in transverse striations (red arrow), whereas RyR1 is localized in transverse striations labeling the terminal SR. Little colocalization (white in Merge) of SPEG (green) and RyR1 (magenta) can be observed at this stage. (E,E′) At 5 dpf, SPEG expression becomes restricted in the transverse striations (red arrows) that overlap with RyR1. Scale bars: 10 µm (D,E); 5 µm (D′,E′).
Figure Legend Snippet: Speg expression during zebrafish development. There are two Speg genes in zebrafish, spega and spegb . Each encodes a single transcript that is highly conserved with human SPEG . (A,B) RT-qPCR shows similar temporal expression patterns of spega (A) versus spegb (B) from 2 days post-fertilization (dpf) to 7 dpf. Both Speg mRNA transcripts are significantly upregulated in the heads (light blue bars; 20-fold for spega , 7-fold for spegb ), but stay at relatively similar levels in the skeletal-muscle-predominant tails (white bars). Each data point represents the average of technical triplicates, and three independent experiments are included. Columns and error bars represent mean±s.e.m. Unpaired two-tailed Student's t -test was performed: ** P <0.01; ns, not significant. (C) Whole-mount in situ hybridization using DIG-conjugated RNA probes in 1 dpf embryos shows distinct spatial expression patterns of spega versus spegb . spega is predominantly expressed in the developing brain (yellow arrowhead) and along the neural tube (red arrowheads), but absent from the notochord (cyan arrowheads). spegb staining, however, is predominantly detected at the chevron-shaped developing somites (black arrowheads). Scale bars: 200 µm (top and middle rows) or 100 µm (bottom row). (D,E) Confocal images showing 2 dpf (D) or 5 dpf (E) isolated wild-type (WT) skeletal myofibers double-stained with anti-RyR1 (34C, DSHB) and anti-SPEG (PA553875, Invitrogen). (D,D′) At 2 dpf, SPEG is predominantly localized at the sarcolemma (yellow arrows) and perinuclear regions (cyan arrows; nucleus, yellow asterisks), with weak expression in transverse striations (red arrow), whereas RyR1 is localized in transverse striations labeling the terminal SR. Little colocalization (white in Merge) of SPEG (green) and RyR1 (magenta) can be observed at this stage. (E,E′) At 5 dpf, SPEG expression becomes restricted in the transverse striations (red arrows) that overlap with RyR1. Scale bars: 10 µm (D,E); 5 µm (D′,E′).

Techniques Used: Expressing, Quantitative RT-PCR, Two Tailed Test, In Situ Hybridization, Staining, Isolation, Labeling

spega/b deficiency in zebrafish disrupts triad protein organization and triad ultrastructure, leading to reduced triad numbers. (A-D′) IF staining was performed on 2 dpf (A,B,C,D) and 5 dpf (A′,B′,C′,D′) isolated myofibers. Confocal images show disrupted transverse pattern of RyR1 (A,A′), DHPR (B,B′) and SERCA1a (D,D′) in speg -DKO starting from 2 dpf, while α-Actinin (D,D′) is not affected. Scale bars: 50 µm. (E-F′) Electron micrographs of 7 dpf WT and speg -DKO muscles. (E) In WT zebrafish skeletal muscle, normal triads are physically above the sarcomeric Z-disks and composed of centrally-located T-tubules flanked by terminal sarcoplasmic reticulum (yellow arrowhead). (E′) Triads in speg -DKO appear structurally disrupted, losing the obvious terminal cisternae of the sarcoplasmic membrane (tSR)/T-tubule/tSR pattern (red arrowhead). (F,F′) More importantly, the majority of sarcomeric Z-disks in speg -DKO do not have adjacent triads (yellow arrows). Scale bars: 0.5 µm (E,E′); 1 µm (F,F′). (G) The total number of triads per 60 µm 2 (under an electron microscope) was significantly reduced in speg -DKO. Each dot represents the average of technical triplicates, and three biological replicates are included. Data are mean±s.e.m. Unpaired two-tailed Student's t -test: * P <0.05.
Figure Legend Snippet: spega/b deficiency in zebrafish disrupts triad protein organization and triad ultrastructure, leading to reduced triad numbers. (A-D′) IF staining was performed on 2 dpf (A,B,C,D) and 5 dpf (A′,B′,C′,D′) isolated myofibers. Confocal images show disrupted transverse pattern of RyR1 (A,A′), DHPR (B,B′) and SERCA1a (D,D′) in speg -DKO starting from 2 dpf, while α-Actinin (D,D′) is not affected. Scale bars: 50 µm. (E-F′) Electron micrographs of 7 dpf WT and speg -DKO muscles. (E) In WT zebrafish skeletal muscle, normal triads are physically above the sarcomeric Z-disks and composed of centrally-located T-tubules flanked by terminal sarcoplasmic reticulum (yellow arrowhead). (E′) Triads in speg -DKO appear structurally disrupted, losing the obvious terminal cisternae of the sarcoplasmic membrane (tSR)/T-tubule/tSR pattern (red arrowhead). (F,F′) More importantly, the majority of sarcomeric Z-disks in speg -DKO do not have adjacent triads (yellow arrows). Scale bars: 0.5 µm (E,E′); 1 µm (F,F′). (G) The total number of triads per 60 µm 2 (under an electron microscope) was significantly reduced in speg -DKO. Each dot represents the average of technical triplicates, and three biological replicates are included. Data are mean±s.e.m. Unpaired two-tailed Student's t -test: * P <0.05.

Techniques Used: Staining, Isolation, Microscopy, Two Tailed Test


Structured Review

Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1
( A ) The triads are made of transverse tubules (T-tubules) flanked by terminal sarcoplasmic reticulum (tSR). Junctional membranes (JM) are connected by the interaction of the <t>ryanodine</t> <t>receptor</t> <t>(RyR1)</t> at the tSR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with <t>RyR1,</t> causing its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the calcium transporter SERCA (sarco/endoplasmic reticulum Ca2+-ATPase, regulated by PLN or phospholamban) returns calcium into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J); and triadin (T). SPEG (striated muscle enriched protein kinase) is localized at the triad, but its role remains elusive. ( B ) SPEG contains immunoglobulin domains (Ig, light purple shaded ), fibronectin domains (Fn, light green shaded ), and two kinase domains ( yellow shaded ). SPEG directly binds to Myotubularin 1 (MTM1, dark green ; SPEG 2530∼2674 a.a.) (Agrawal et al., 2014), and Desmin (DES, magenta ; SPEG 2200∼2960 a.a.) (Luo et al., 2021) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Nfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue fonts ), a cardiomyopathy only (e.g. dilated cardiomyopathy, red fonts ), or both ( black fonts ), with no clear genotype-phenotype correlation. Illustrations were made using Illustrator for Biological Sciences (IBS) .
Mouse Monoclonal Anti Ryr1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse monoclonal anti ryr1/product/Developmental Studies Hybridoma Bank
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse monoclonal anti ryr1 - by Bioz Stars, 2023-01
94/100 stars

Images

1) Product Images from "A novel zebrafish model of SPEG -related centronuclear myopathy (CNM): characterization and comparison with other CNM model zebrafish"

Article Title: A novel zebrafish model of SPEG -related centronuclear myopathy (CNM): characterization and comparison with other CNM model zebrafish

Journal: bioRxiv

doi: 10.1101/2021.12.22.473918

( A ) The triads are made of transverse tubules (T-tubules) flanked by terminal sarcoplasmic reticulum (tSR). Junctional membranes (JM) are connected by the interaction of the ryanodine receptor (RyR1) at the tSR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with RyR1, causing its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the calcium transporter SERCA (sarco/endoplasmic reticulum Ca2+-ATPase, regulated by PLN or phospholamban) returns calcium into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J); and triadin (T). SPEG (striated muscle enriched protein kinase) is localized at the triad, but its role remains elusive. ( B ) SPEG contains immunoglobulin domains (Ig, light purple shaded ), fibronectin domains (Fn, light green shaded ), and two kinase domains ( yellow shaded ). SPEG directly binds to Myotubularin 1 (MTM1, dark green ; SPEG 2530∼2674 a.a.) (Agrawal et al., 2014), and Desmin (DES, magenta ; SPEG 2200∼2960 a.a.) (Luo et al., 2021) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Nfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue fonts ), a cardiomyopathy only (e.g. dilated cardiomyopathy, red fonts ), or both ( black fonts ), with no clear genotype-phenotype correlation. Illustrations were made using Illustrator for Biological Sciences (IBS) .
Figure Legend Snippet: ( A ) The triads are made of transverse tubules (T-tubules) flanked by terminal sarcoplasmic reticulum (tSR). Junctional membranes (JM) are connected by the interaction of the ryanodine receptor (RyR1) at the tSR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with RyR1, causing its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the calcium transporter SERCA (sarco/endoplasmic reticulum Ca2+-ATPase, regulated by PLN or phospholamban) returns calcium into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J); and triadin (T). SPEG (striated muscle enriched protein kinase) is localized at the triad, but its role remains elusive. ( B ) SPEG contains immunoglobulin domains (Ig, light purple shaded ), fibronectin domains (Fn, light green shaded ), and two kinase domains ( yellow shaded ). SPEG directly binds to Myotubularin 1 (MTM1, dark green ; SPEG 2530∼2674 a.a.) (Agrawal et al., 2014), and Desmin (DES, magenta ; SPEG 2200∼2960 a.a.) (Luo et al., 2021) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Nfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue fonts ), a cardiomyopathy only (e.g. dilated cardiomyopathy, red fonts ), or both ( black fonts ), with no clear genotype-phenotype correlation. Illustrations were made using Illustrator for Biological Sciences (IBS) .

Techniques Used: Activation Assay, Binding Assay

There are two SPEG genes in zebrafish, spega and spegb . Each encodes a single transcript that is highly conserved with human SPEG. ( A-B ) RT-qPCR shows similar temporal expression patterns of spega ( A ) vs spegb ( B ) from 2 dpf (day-post-fertilization) to 7 dpf. Both SPEG mRNA transcripts are significantly upregulated in the heads ( gray bars ; 20-fold for spega , and 7-fold for spegb ), but stay at relatively similar levels in the skeletal-muscle-predominant tails ( white bars ). Each data point represents the average of technical triplicates, and three independent experiments are included. Columns and error bars represent Mean ± SEM. Two-tailed Student’s t -test was performed: **, P <0.01; ns, not significant. ( C ) Wholemount in situ hybridization using DIG-conjugated RNA probes in 1 dpf embryos show distinct spatial expression patterns of spega vs spegb . spega is predominantly expressed in the developing brain ( yellow arrowhead ) and along the neural tube ( red arrowheads ), but absent from the notochord ( cyan arrowheads ). spegb staining, however, is predominantly detected at the chevron-shaped developing somites ( black arrowheads ). In situ images - scale bars : 200 μm (upper, middle) or 100 μm (bottom). ( D-E ) Average Z-projections of confocal images showing 2 dpf ( D ) or 5 dpf ( E ) isolated wildtype skeletal myofibres double-stained with anti-RyR1 (34C, DSHB) and anti-SPEG (PA553875, Invitrogen). ( D and D’ ) At 2 dpf, SPEG is predominantly localized at the sarcolemma ( yellow arrows ) and perinuclear regions ( cyan arrows ; nucleus: yellow asterisks ) with weak expression in transverse striations ( orange arrows ), whereas RyR1 is localized in transverse striations labeling the terminal sarcoplasmic reticulum (SR). Little co-localization ( white in Merge) of SPEG ( green ) and RyR1 ( magenta ) can be observed at this stage. ( E and E’ ) At 5 dpf, SPEG expression becomes restricted in the transverse striations ( orange arrows ) that overlap with RyR1. IF images - scale bars : 10 μm for D and E, 5 μm for D’ and E’.
Figure Legend Snippet: There are two SPEG genes in zebrafish, spega and spegb . Each encodes a single transcript that is highly conserved with human SPEG. ( A-B ) RT-qPCR shows similar temporal expression patterns of spega ( A ) vs spegb ( B ) from 2 dpf (day-post-fertilization) to 7 dpf. Both SPEG mRNA transcripts are significantly upregulated in the heads ( gray bars ; 20-fold for spega , and 7-fold for spegb ), but stay at relatively similar levels in the skeletal-muscle-predominant tails ( white bars ). Each data point represents the average of technical triplicates, and three independent experiments are included. Columns and error bars represent Mean ± SEM. Two-tailed Student’s t -test was performed: **, P <0.01; ns, not significant. ( C ) Wholemount in situ hybridization using DIG-conjugated RNA probes in 1 dpf embryos show distinct spatial expression patterns of spega vs spegb . spega is predominantly expressed in the developing brain ( yellow arrowhead ) and along the neural tube ( red arrowheads ), but absent from the notochord ( cyan arrowheads ). spegb staining, however, is predominantly detected at the chevron-shaped developing somites ( black arrowheads ). In situ images - scale bars : 200 μm (upper, middle) or 100 μm (bottom). ( D-E ) Average Z-projections of confocal images showing 2 dpf ( D ) or 5 dpf ( E ) isolated wildtype skeletal myofibres double-stained with anti-RyR1 (34C, DSHB) and anti-SPEG (PA553875, Invitrogen). ( D and D’ ) At 2 dpf, SPEG is predominantly localized at the sarcolemma ( yellow arrows ) and perinuclear regions ( cyan arrows ; nucleus: yellow asterisks ) with weak expression in transverse striations ( orange arrows ), whereas RyR1 is localized in transverse striations labeling the terminal sarcoplasmic reticulum (SR). Little co-localization ( white in Merge) of SPEG ( green ) and RyR1 ( magenta ) can be observed at this stage. ( E and E’ ) At 5 dpf, SPEG expression becomes restricted in the transverse striations ( orange arrows ) that overlap with RyR1. IF images - scale bars : 10 μm for D and E, 5 μm for D’ and E’.

Techniques Used: Quantitative RT-PCR, Expressing, Two Tailed Test, In Situ Hybridization, Staining, In Situ, Isolation, Labeling

Immunofluorescence staining was performed on 2 dpf ( A-D ) and 5 dpf ( A’-D’ ) isolated myofibres. Average projections of confocal Z-stacks show disrupted transverse pattern of RyR1 ( A and A’ ), DHPR ( B and B’ ), and SERCA1a ( D ad D’ ) in speg -DKO starting from 2 dpf, while α-Actinin ( D and D’ ) is not affected. IF images - scale bars : 50 μm. ( E-F ) Electron micrographs of 7 dpf WT and speg -DKO muscles. ( E ) In WT zebrafish skeletal muscle, normal triads are physically above the sarcomeric Z-disks, and composed of centrally-located T-tubules flanked by terminal sarcoplasmic reticulum ( yellow arrows ). ( E’ ) Triads in speg -DKO appear structurally disrupted, losing the obvious tSR/T-tubule/tSR pattern ( red arrows ). More importantly, ( F ) the majority of sarcomeric Z-disks in speg -DKO do not have adjacent triads ( yellow arrows ). EM images – scale bars: (E) – 0.5 μm; (F) – 1 μm. ( G ) The total number of triads per 60 μm 2 (under electron microscopy) was significantly reduced in speg -DKO. Each dot represents the average of technical triplicates, and three biological replicates are included. Error bars are shown as Mean ± SEM. Two-tailed Student’s t -test: *, P <0.05.
Figure Legend Snippet: Immunofluorescence staining was performed on 2 dpf ( A-D ) and 5 dpf ( A’-D’ ) isolated myofibres. Average projections of confocal Z-stacks show disrupted transverse pattern of RyR1 ( A and A’ ), DHPR ( B and B’ ), and SERCA1a ( D ad D’ ) in speg -DKO starting from 2 dpf, while α-Actinin ( D and D’ ) is not affected. IF images - scale bars : 50 μm. ( E-F ) Electron micrographs of 7 dpf WT and speg -DKO muscles. ( E ) In WT zebrafish skeletal muscle, normal triads are physically above the sarcomeric Z-disks, and composed of centrally-located T-tubules flanked by terminal sarcoplasmic reticulum ( yellow arrows ). ( E’ ) Triads in speg -DKO appear structurally disrupted, losing the obvious tSR/T-tubule/tSR pattern ( red arrows ). More importantly, ( F ) the majority of sarcomeric Z-disks in speg -DKO do not have adjacent triads ( yellow arrows ). EM images – scale bars: (E) – 0.5 μm; (F) – 1 μm. ( G ) The total number of triads per 60 μm 2 (under electron microscopy) was significantly reduced in speg -DKO. Each dot represents the average of technical triplicates, and three biological replicates are included. Error bars are shown as Mean ± SEM. Two-tailed Student’s t -test: *, P <0.05.

Techniques Used: Immunofluorescence, Staining, Isolation, Electron Microscopy, Two Tailed Test


Structured Review

Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1
Immunofluorescence and EM analysis of EDL fibers from aged-control mice. (A,B) Representative immunofluorescence images obtained from aged mice, double-labeled for <t>RYR1</t> (red) and STIM1 (green) in panel (A) and RYR1 (red) and ORAI1 in panel (B) . Raw images for the individual fluorescence channel used to construct these overlays are shown in . (C,D) Representative EM images of longitudinal (C) and transversal (D) sections with TAs false-labeled in green (arrows point to TTs stained with ferrocyanide) and TTs stained with ferrocyanide (dark precipitate). Black arrows in panel (C,D) point to TTs within the interior of the TA. Inset in panel (D) small bridges, pointed by small arrows, are visible between membranes of adjacent cross-sectioned tubes. Scale bars: (A,B) , 5 μm (insets 2 μm); (C,D) , 1 μm (inset 0.1 μm).
Mouse Monoclonal Anti Ryr1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Long-Term Exercise Reduces Formation of Tubular Aggregates and Promotes Maintenance of Ca 2+ Entry Units in Aged Muscle"

Article Title: Long-Term Exercise Reduces Formation of Tubular Aggregates and Promotes Maintenance of Ca 2+ Entry Units in Aged Muscle

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2020.601057

Immunofluorescence and EM analysis of EDL fibers from aged-control mice. (A,B) Representative immunofluorescence images obtained from aged mice, double-labeled for RYR1 (red) and STIM1 (green) in panel (A) and RYR1 (red) and ORAI1 in panel (B) . Raw images for the individual fluorescence channel used to construct these overlays are shown in . (C,D) Representative EM images of longitudinal (C) and transversal (D) sections with TAs false-labeled in green (arrows point to TTs stained with ferrocyanide) and TTs stained with ferrocyanide (dark precipitate). Black arrows in panel (C,D) point to TTs within the interior of the TA. Inset in panel (D) small bridges, pointed by small arrows, are visible between membranes of adjacent cross-sectioned tubes. Scale bars: (A,B) , 5 μm (insets 2 μm); (C,D) , 1 μm (inset 0.1 μm).
Figure Legend Snippet: Immunofluorescence and EM analysis of EDL fibers from aged-control mice. (A,B) Representative immunofluorescence images obtained from aged mice, double-labeled for RYR1 (red) and STIM1 (green) in panel (A) and RYR1 (red) and ORAI1 in panel (B) . Raw images for the individual fluorescence channel used to construct these overlays are shown in . (C,D) Representative EM images of longitudinal (C) and transversal (D) sections with TAs false-labeled in green (arrows point to TTs stained with ferrocyanide) and TTs stained with ferrocyanide (dark precipitate). Black arrows in panel (C,D) point to TTs within the interior of the TA. Inset in panel (D) small bridges, pointed by small arrows, are visible between membranes of adjacent cross-sectioned tubes. Scale bars: (A,B) , 5 μm (insets 2 μm); (C,D) , 1 μm (inset 0.1 μm).

Techniques Used: Immunofluorescence, Labeling, Fluorescence, Construct, Staining


Structured Review

Developmental Studies Hybridoma Bank chicken pectoral muscle ryanodine receptor mouse igg1 mab
Antibody information
Chicken Pectoral Muscle Ryanodine Receptor Mouse Igg1 Mab, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/chicken pectoral muscle ryanodine receptor mouse igg1 mab/product/Developmental Studies Hybridoma Bank
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
chicken pectoral muscle ryanodine receptor mouse igg1 mab - by Bioz Stars, 2023-01
86/100 stars

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1) Product Images from "Neuronal ER–plasma membrane junctions organized by Kv2–VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis"

Article Title: Neuronal ER–plasma membrane junctions organized by Kv2–VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA119.007635

Antibody information
Figure Legend Snippet: Antibody information

Techniques Used: Concentration Assay, Affinity Purification, Purification, Western Blot, Cell Culture, Recombinant


Structured Review

Developmental Studies Hybridoma Bank chicken pectoral muscle ryanodine receptor mouse igg1 mab
Antibody information
Chicken Pectoral Muscle Ryanodine Receptor Mouse Igg1 Mab, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/chicken pectoral muscle ryanodine receptor mouse igg1 mab/product/Developmental Studies Hybridoma Bank
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
chicken pectoral muscle ryanodine receptor mouse igg1 mab - by Bioz Stars, 2023-01
94/100 stars

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1) Product Images from "Neuronal ER–plasma membrane junctions organized by Kv2–VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis"

Article Title: Neuronal ER–plasma membrane junctions organized by Kv2–VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA119.007635

Antibody information
Figure Legend Snippet: Antibody information

Techniques Used: Concentration Assay, Affinity Purification, Purification, Western Blot, Cell Culture, Recombinant


Structured Review

Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1
IF and EM analysis of EDL fibers from aged and aged-trained mice. A and B) Representative IF images showing <t>RyR1</t> and TOM20 double-staining (marking the position of CRUs and mitochondria, respectively). C-F) Representative EM images showing the position of mitochondria (pointed by small arrows in C and D) and the organization of TTs (pointed by arrowheads in E and F). In C, the empty arrow point to an area with abnormal mitochondria distribution. G-L) Quantitative analysis of mitochondrial n./area, volume, disposition, and association with CRUs. Data are shown as mean ± SEM; n = number of EDL fibers analyzed; *p < 0.05 as evaluated by two-tailed unpaired Student’s t-test. Scale bars: A and B, 5 μm (insets 2 μm); C–F, 2 μm.
Mouse Monoclonal Anti Ryr1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse monoclonal anti ryr1/product/Developmental Studies Hybridoma Bank
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse monoclonal anti ryr1 - by Bioz Stars, 2023-01
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Images

1) Product Images from "Muscle activity prevents the uncoupling of mitochondria from Ca 2+ Release Units induced by ageing and disuse"

Article Title: Muscle activity prevents the uncoupling of mitochondria from Ca 2+ Release Units induced by ageing and disuse

Journal: Archives of biochemistry and biophysics

doi: 10.1016/j.abb.2018.12.017

IF and EM analysis of EDL fibers from aged and aged-trained mice. A and B) Representative IF images showing RyR1 and TOM20 double-staining (marking the position of CRUs and mitochondria, respectively). C-F) Representative EM images showing the position of mitochondria (pointed by small arrows in C and D) and the organization of TTs (pointed by arrowheads in E and F). In C, the empty arrow point to an area with abnormal mitochondria distribution. G-L) Quantitative analysis of mitochondrial n./area, volume, disposition, and association with CRUs. Data are shown as mean ± SEM; n = number of EDL fibers analyzed; *p < 0.05 as evaluated by two-tailed unpaired Student’s t-test. Scale bars: A and B, 5 μm (insets 2 μm); C–F, 2 μm.
Figure Legend Snippet: IF and EM analysis of EDL fibers from aged and aged-trained mice. A and B) Representative IF images showing RyR1 and TOM20 double-staining (marking the position of CRUs and mitochondria, respectively). C-F) Representative EM images showing the position of mitochondria (pointed by small arrows in C and D) and the organization of TTs (pointed by arrowheads in E and F). In C, the empty arrow point to an area with abnormal mitochondria distribution. G-L) Quantitative analysis of mitochondrial n./area, volume, disposition, and association with CRUs. Data are shown as mean ± SEM; n = number of EDL fibers analyzed; *p < 0.05 as evaluated by two-tailed unpaired Student’s t-test. Scale bars: A and B, 5 μm (insets 2 μm); C–F, 2 μm.

Techniques Used: Double Staining, Two Tailed Test

IF and EM analysis of EDL fibers from control, denervated, and reinnervated rats. A, C and E) Representative IF images showing fibers double immuno-labeled with RyR1 and TOM20 antibodies marking the position of CRUs and mitochondria, respectively. B, D and F) Representative EM images showing the disposition of mitochondria in EDL fibers from control, denervated, and reinnervated rats. In B and F black arrows point to mitochondria properly positioned at the I band, while in D empty arrows point to mitochondria forming longitudinal columns between myofibrils. G-L) Quantitative analysis of mitochondrial n./area, volume, disposition, and association with CRUs. Data are shown as mean ± SEM; n = number of fibers analyzed; *p < 0.05 as evaluated by one-way ANOVA followed by post-hoc Tuckey test. Scale bars: A, C and E, 5 μm (insets 2 μm); B, D and F, 1 μm.
Figure Legend Snippet: IF and EM analysis of EDL fibers from control, denervated, and reinnervated rats. A, C and E) Representative IF images showing fibers double immuno-labeled with RyR1 and TOM20 antibodies marking the position of CRUs and mitochondria, respectively. B, D and F) Representative EM images showing the disposition of mitochondria in EDL fibers from control, denervated, and reinnervated rats. In B and F black arrows point to mitochondria properly positioned at the I band, while in D empty arrows point to mitochondria forming longitudinal columns between myofibrils. G-L) Quantitative analysis of mitochondrial n./area, volume, disposition, and association with CRUs. Data are shown as mean ± SEM; n = number of fibers analyzed; *p < 0.05 as evaluated by one-way ANOVA followed by post-hoc Tuckey test. Scale bars: A, C and E, 5 μm (insets 2 μm); B, D and F, 1 μm.

Techniques Used: Labeling


Structured Review

Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1
IF and EM analysis of EDL fibers from aged and aged-trained mice. A and B) Representative IF images showing <t>RyR1</t> and TOM20 double-staining (marking the position of CRUs and mitochondria, respectively). C-F) Representative EM images showing the position of mitochondria (pointed by small arrows in C and D) and the organization of TTs (pointed by arrowheads in E and F). In C, the empty arrow point to an area with abnormal mitochondria distribution. G-L) Quantitative analysis of mitochondrial n./area, volume, disposition, and association with CRUs. Data are shown as mean ± SEM; n = number of EDL fibers analyzed; *p < 0.05 as evaluated by two-tailed unpaired Student’s t-test. Scale bars: A and B, 5 μm (insets 2 μm); C–F, 2 μm.
Mouse Monoclonal Anti Ryr1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse monoclonal anti ryr1/product/Developmental Studies Hybridoma Bank
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse monoclonal anti ryr1 - by Bioz Stars, 2023-01
86/100 stars

Images

1) Product Images from "Muscle activity prevents the uncoupling of mitochondria from Ca 2+ Release Units induced by ageing and disuse"

Article Title: Muscle activity prevents the uncoupling of mitochondria from Ca 2+ Release Units induced by ageing and disuse

Journal: Archives of biochemistry and biophysics

doi: 10.1016/j.abb.2018.12.017

IF and EM analysis of EDL fibers from aged and aged-trained mice. A and B) Representative IF images showing RyR1 and TOM20 double-staining (marking the position of CRUs and mitochondria, respectively). C-F) Representative EM images showing the position of mitochondria (pointed by small arrows in C and D) and the organization of TTs (pointed by arrowheads in E and F). In C, the empty arrow point to an area with abnormal mitochondria distribution. G-L) Quantitative analysis of mitochondrial n./area, volume, disposition, and association with CRUs. Data are shown as mean ± SEM; n = number of EDL fibers analyzed; *p < 0.05 as evaluated by two-tailed unpaired Student’s t-test. Scale bars: A and B, 5 μm (insets 2 μm); C–F, 2 μm.
Figure Legend Snippet: IF and EM analysis of EDL fibers from aged and aged-trained mice. A and B) Representative IF images showing RyR1 and TOM20 double-staining (marking the position of CRUs and mitochondria, respectively). C-F) Representative EM images showing the position of mitochondria (pointed by small arrows in C and D) and the organization of TTs (pointed by arrowheads in E and F). In C, the empty arrow point to an area with abnormal mitochondria distribution. G-L) Quantitative analysis of mitochondrial n./area, volume, disposition, and association with CRUs. Data are shown as mean ± SEM; n = number of EDL fibers analyzed; *p < 0.05 as evaluated by two-tailed unpaired Student’s t-test. Scale bars: A and B, 5 μm (insets 2 μm); C–F, 2 μm.

Techniques Used: Double Staining, Two Tailed Test

IF and EM analysis of EDL fibers from control, denervated, and reinnervated rats. A, C and E) Representative IF images showing fibers double immuno-labeled with RyR1 and TOM20 antibodies marking the position of CRUs and mitochondria, respectively. B, D and F) Representative EM images showing the disposition of mitochondria in EDL fibers from control, denervated, and reinnervated rats. In B and F black arrows point to mitochondria properly positioned at the I band, while in D empty arrows point to mitochondria forming longitudinal columns between myofibrils. G-L) Quantitative analysis of mitochondrial n./area, volume, disposition, and association with CRUs. Data are shown as mean ± SEM; n = number of fibers analyzed; *p < 0.05 as evaluated by one-way ANOVA followed by post-hoc Tuckey test. Scale bars: A, C and E, 5 μm (insets 2 μm); B, D and F, 1 μm.
Figure Legend Snippet: IF and EM analysis of EDL fibers from control, denervated, and reinnervated rats. A, C and E) Representative IF images showing fibers double immuno-labeled with RyR1 and TOM20 antibodies marking the position of CRUs and mitochondria, respectively. B, D and F) Representative EM images showing the disposition of mitochondria in EDL fibers from control, denervated, and reinnervated rats. In B and F black arrows point to mitochondria properly positioned at the I band, while in D empty arrows point to mitochondria forming longitudinal columns between myofibrils. G-L) Quantitative analysis of mitochondrial n./area, volume, disposition, and association with CRUs. Data are shown as mean ± SEM; n = number of fibers analyzed; *p < 0.05 as evaluated by one-way ANOVA followed by post-hoc Tuckey test. Scale bars: A, C and E, 5 μm (insets 2 μm); B, D and F, 1 μm.

Techniques Used: Labeling

34c partially purified chicken pectoral muscle ryanodine receptor mouse igg1 mab  (Developmental Studies Hybridoma Bank)

 
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    Structured Review

    Developmental Studies Hybridoma Bank 34c partially purified chicken pectoral muscle ryanodine receptor mouse igg1 mab
    Antibody information
    34c Partially Purified Chicken Pectoral Muscle Ryanodine Receptor Mouse Igg1 Mab, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/34c partially purified chicken pectoral muscle ryanodine receptor mouse igg1 mab/product/Developmental Studies Hybridoma Bank
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    34c partially purified chicken pectoral muscle ryanodine receptor mouse igg1 mab - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Identification of VAPA and VAPB as Kv2 Channel-Interacting Proteins Defining Endoplasmic Reticulum–Plasma Membrane Junctions in Mammalian Brain Neurons"

    Article Title: Identification of VAPA and VAPB as Kv2 Channel-Interacting Proteins Defining Endoplasmic Reticulum–Plasma Membrane Junctions in Mammalian Brain Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0893-18.2018

    Antibody information
    Figure Legend Snippet: Antibody information

    Techniques Used: Concentration Assay, Affinity Purification, Purification, Recombinant

    CRISPR-mediated KO of VAPA expression affects Kv2.1 localization in RAW264.7 cells A. Representative TIRF images of SEP-Kv2.1 expression in a live WT RAW264.7 cell expressing SEP-Kv2.1 (green is cell surface pHluorin fluorescence, magenta is total cellular mCherry fluorescence). Scale bar is 5 μm and holds for all panels in figure. B, Representative TIRF images of GFP-Kv2.1 (green) and BFP-SEC61β (magenta) in live WT (left) or VAPA KO (right) RAW264.7 cells coexpressing GFP-Kv2.1 and BFP-SEC61β. Note the reduction in clustered Kv2.1 expression in the VAPA KO cell. C, Line scan analysis of selection indicated in merged image of WT RAW264.7 cell coexpressing GFP-Kv2.1 and BFP-SEC61β. D, Line scan analysis of selection indicated in merged image of VAPA KO RAW264.7 cell coexpressing GFP-Kv2.1 and BFP-SEC61β. E, Representative TIRF image of live WT (left) or VAPA KO (right) RAW264.7 cells expressing GFP-Kv1.4. F, Summary graph of CV measurements of GFP-Kv2.1 in WT and VAPA KO RAW264.7 cells (****p = 6.648 × 10−9, n = 29 cells, two-tailed unpaired t test). G, Summary graph of CV measurement of GFP-Kv1.4 in WT and VAPA KO RAW264.7 cells (ns, 0.5603, n = 10 cells, two-tailed unpaired t test). H-K. Images of WT (left) or VAPA KO (right) RAW264.7 cells used for high-content analysis of immunolabeling (magenta) and with Hoechst labeling of nuclei (green). H, No primary (anti-IgG2b secondary antibody). I, Positive control anti-mortalin mAb N52A/42 (IgG1). J, Anti-VAPA mAb N479/22 (IgG2a). K, Anti-VAPA/B mAb N479/107 (IgG2b). Scale bar in H is 65 μm and holds for H–K. L, Mean immunolabeling intensity of all samples across 1300–2800 individual cells in each sample. See Materials and Methods for further details of these antibody validation results.
    Figure Legend Snippet: CRISPR-mediated KO of VAPA expression affects Kv2.1 localization in RAW264.7 cells A. Representative TIRF images of SEP-Kv2.1 expression in a live WT RAW264.7 cell expressing SEP-Kv2.1 (green is cell surface pHluorin fluorescence, magenta is total cellular mCherry fluorescence). Scale bar is 5 μm and holds for all panels in figure. B, Representative TIRF images of GFP-Kv2.1 (green) and BFP-SEC61β (magenta) in live WT (left) or VAPA KO (right) RAW264.7 cells coexpressing GFP-Kv2.1 and BFP-SEC61β. Note the reduction in clustered Kv2.1 expression in the VAPA KO cell. C, Line scan analysis of selection indicated in merged image of WT RAW264.7 cell coexpressing GFP-Kv2.1 and BFP-SEC61β. D, Line scan analysis of selection indicated in merged image of VAPA KO RAW264.7 cell coexpressing GFP-Kv2.1 and BFP-SEC61β. E, Representative TIRF image of live WT (left) or VAPA KO (right) RAW264.7 cells expressing GFP-Kv1.4. F, Summary graph of CV measurements of GFP-Kv2.1 in WT and VAPA KO RAW264.7 cells (****p = 6.648 × 10−9, n = 29 cells, two-tailed unpaired t test). G, Summary graph of CV measurement of GFP-Kv1.4 in WT and VAPA KO RAW264.7 cells (ns, 0.5603, n = 10 cells, two-tailed unpaired t test). H-K. Images of WT (left) or VAPA KO (right) RAW264.7 cells used for high-content analysis of immunolabeling (magenta) and with Hoechst labeling of nuclei (green). H, No primary (anti-IgG2b secondary antibody). I, Positive control anti-mortalin mAb N52A/42 (IgG1). J, Anti-VAPA mAb N479/22 (IgG2a). K, Anti-VAPA/B mAb N479/107 (IgG2b). Scale bar in H is 65 μm and holds for H–K. L, Mean immunolabeling intensity of all samples across 1300–2800 individual cells in each sample. See Materials and Methods for further details of these antibody validation results.

    Techniques Used: CRISPR, Expressing, Fluorescence, Selection, Two Tailed Test, High Content Screening, Immunolabeling, Labeling, Positive Control

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    Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1 ryr3
    Mouse Monoclonal Anti Ryr1 Ryr3, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1 ryr3 34c
    Structural cores and blood markers of muscle damage (CK and LDH) at 4 months of age. (a–c) Representative histological images of normal fibers (a), fibers with unstructured cores (b), and fibers with contracture cores (c). (d) Percentage of EDL fibers presenting the features classified in (a–c) (white: normal fibers ; grey: fibers with unstructured cores ; and dark grey: fibers with contracture cores ). See also Table S1. (e and f) Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH). In (e) and (f), data are given as mean ± SEM; ∗ p < 0.05, WT versus <t>RYR1</t> Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (d), n = number of fibers analyzed; in (e-f), n = number of mice. Scale bar: 10 μm .
    Mouse Monoclonal Anti Ryr1 Ryr3 34c, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank mouse monoclonal anti ryr1
    Schematic diagrams of excitation–contraction coupling (ECC) at the triad, and SPEG domains and pathogenic variants. (A) The triads are made of transverse tubules (T-tubules) flanked by terminal cisternae of the sarcoplasmic reticulum (SR). Junctional membranes (JM) are connected by the interaction of the <t>ryanodine</t> <t>receptor</t> <t>(RyR1)</t> at the SR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with <t>RyR1,</t> leading to its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the Ca 2+ transporter sarco/endoplasmic reticulum Ca 2+ -ATPase [SERCA; also known as ATP2A; regulated by phospholamban (PLN)] returns Ca 2+ into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J) and triadin (T). Striated muscle enriched protein kinase (SPEG) is localized at the triad, but its role remains elusive. (B) SPEG contains immunoglobulin domains (Ig; light purple shaded), fibronectin domains (Fn; light green shaded) and two kinase domains (yellow shaded). SPEG directly binds to myotubularin 1 (MTM1; dark green; SPEG 2530-2674 a.a.) and desmin (DES; magenta; SPEG 2200-2960 a.a.) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Dfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue font), a cardiomyopathy only (e.g. dilated cardiomyopathy, red font) or both (black font), with no clear genotype–phenotype correlation. Note that patient Q2233* died before a cardiac evaluation. Illustrations were made using Illustrator for Biological Sciences (IBS) .
    Mouse Monoclonal Anti Ryr1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank chicken pectoral muscle ryanodine receptor mouse igg1 mab
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    Chicken Pectoral Muscle Ryanodine Receptor Mouse Igg1 Mab, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank 34c partially purified chicken pectoral muscle ryanodine receptor mouse igg1 mab
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    Image Search Results


    Structural cores and blood markers of muscle damage (CK and LDH) at 4 months of age. (a–c) Representative histological images of normal fibers (a), fibers with unstructured cores (b), and fibers with contracture cores (c). (d) Percentage of EDL fibers presenting the features classified in (a–c) (white: normal fibers ; grey: fibers with unstructured cores ; and dark grey: fibers with contracture cores ). See also Table S1. (e and f) Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH). In (e) and (f), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (d), n = number of fibers analyzed; in (e-f), n = number of mice. Scale bar: 10 μm .

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

    doi: 10.1155/2017/6792694

    Figure Lengend Snippet: Structural cores and blood markers of muscle damage (CK and LDH) at 4 months of age. (a–c) Representative histological images of normal fibers (a), fibers with unstructured cores (b), and fibers with contracture cores (c). (d) Percentage of EDL fibers presenting the features classified in (a–c) (white: normal fibers ; grey: fibers with unstructured cores ; and dark grey: fibers with contracture cores ). See also Table S1. (e and f) Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH). In (e) and (f), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (d), n = number of fibers analyzed; in (e-f), n = number of mice. Scale bar: 10 μm .

    Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques:

    Fiber ultrastructure, calpain activity, and carbonyl protein content at 4 months of age. (a–f) Representative immunofluorescence and EM images showing fluorescent cross striation (a, c, and e) and myofibrillar/sarcomeric organization (b, d, and f) in EDL fibers. In (b) and (f), small arrows point at Z -lines, while in (c and d) large arrows point to areas in which cross striation and sarcomeric structure are compromised. (g) Calpain activity expressed in total hind limb muscle homogenates. (h) Carbonyl protein content in EDL muscle homogenates. In (g and h), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (g and h), n = number of mice. Scale bars in (a, c, and e) 10 μ m (insets 5 μ m); in (b, d, and f) 1 μ m.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

    doi: 10.1155/2017/6792694

    Figure Lengend Snippet: Fiber ultrastructure, calpain activity, and carbonyl protein content at 4 months of age. (a–f) Representative immunofluorescence and EM images showing fluorescent cross striation (a, c, and e) and myofibrillar/sarcomeric organization (b, d, and f) in EDL fibers. In (b) and (f), small arrows point at Z -lines, while in (c and d) large arrows point to areas in which cross striation and sarcomeric structure are compromised. (g) Calpain activity expressed in total hind limb muscle homogenates. (h) Carbonyl protein content in EDL muscle homogenates. In (g and h), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (g and h), n = number of mice. Scale bars in (a, c, and e) 10 μ m (insets 5 μ m); in (b, d, and f) 1 μ m.

    Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Activity Assay, Immunofluorescence

    In vivo grip strength and ex vivo specific force at 4 months of age. (a) Time-course of grip strength from 2 (beginning of short-term NAC treatment) to 4 months (end of short-term NAC treatment) of age expressed as force on body weight (N/g). (b) Change in grip strength from 2 to 4 months of age (shown as a percentage). (c and d) Force-frequency (1–140 Hz) relationship curves of specific force (c) and specific force during a single 2 s, 120 Hz stimulation train (d) recorded for the same EDL muscles. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (b), n = number of mice; in (d), n = number of EDL muscles.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

    doi: 10.1155/2017/6792694

    Figure Lengend Snippet: In vivo grip strength and ex vivo specific force at 4 months of age. (a) Time-course of grip strength from 2 (beginning of short-term NAC treatment) to 4 months (end of short-term NAC treatment) of age expressed as force on body weight (N/g). (b) Change in grip strength from 2 to 4 months of age (shown as a percentage). (c and d) Force-frequency (1–140 Hz) relationship curves of specific force (c) and specific force during a single 2 s, 120 Hz stimulation train (d) recorded for the same EDL muscles. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (b), n = number of mice; in (d), n = number of EDL muscles.

    Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: In Vivo, Ex Vivo

    Mitochondrial damage, size, and volume at 4 months of age. (a–c) Representative EM images displaying apparently normal (a) and damaged mitochondria (b and c). (d) Percentage of damaged mitochondria. (e) Average number of damaged mitochondria/area of EM section. (f) Average size of apparently normal mitochondria (i.e., mitochondria not included in the quantitative analysis of (d and e)). (g) Percentage of fiber volume occupied by mitochondria. See also Table S2. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (d and f), n = number of measurements; in (e and g), n = number of fibers analyzed. Scale bar: 0.1 μ m.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

    doi: 10.1155/2017/6792694

    Figure Lengend Snippet: Mitochondrial damage, size, and volume at 4 months of age. (a–c) Representative EM images displaying apparently normal (a) and damaged mitochondria (b and c). (d) Percentage of damaged mitochondria. (e) Average number of damaged mitochondria/area of EM section. (f) Average size of apparently normal mitochondria (i.e., mitochondria not included in the quantitative analysis of (d and e)). (g) Percentage of fiber volume occupied by mitochondria. See also Table S2. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (d and f), n = number of measurements; in (e and g), n = number of fibers analyzed. Scale bar: 0.1 μ m.

    Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques:

    Effects of long-term NAC treatment at 10 months of age. (a) Quantitative analysis of EDL fibers presenting the features classified in Figures , , and shown as percentage of fibers analyzed (white: normal fibers ; grey: fibers with unstructured cores ; and dark grey: fibers with contracture cores ). See also Table S4. (b and c) Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH). (d) Time course of grip strength from 4 (beginning of long-term NAC treatment) to 10 months (end of long-term NAC treatment) of age expressed as force on body weight (N/g). (e) Change in grip strength from 4 to 10 months of age (shown as a percentage). (f and g) Force-frequency (1–140 Hz) relationship curves of specific force (f) and specific force during a single 2 s, 120 Hz stimulation train (g) recorded for the same EDL muscles. In s (b–g), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (a), n = number of EDL fibers analyzed; in (b–e), n = number of mice; in (f and g), n = number of EDL muscles.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

    doi: 10.1155/2017/6792694

    Figure Lengend Snippet: Effects of long-term NAC treatment at 10 months of age. (a) Quantitative analysis of EDL fibers presenting the features classified in Figures , , and shown as percentage of fibers analyzed (white: normal fibers ; grey: fibers with unstructured cores ; and dark grey: fibers with contracture cores ). See also Table S4. (b and c) Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH). (d) Time course of grip strength from 4 (beginning of long-term NAC treatment) to 10 months (end of long-term NAC treatment) of age expressed as force on body weight (N/g). (e) Change in grip strength from 4 to 10 months of age (shown as a percentage). (f and g) Force-frequency (1–140 Hz) relationship curves of specific force (f) and specific force during a single 2 s, 120 Hz stimulation train (g) recorded for the same EDL muscles. In s (b–g), data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. In (a), n = number of EDL fibers analyzed; in (b–e), n = number of mice; in (f and g), n = number of EDL muscles.

    Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques:

    Oxidative stress markers at 4 and 10 months of age following either short- or long-term treatment. (a and e) Representative immunoblots showing expression of 3-Nitrotyrosine (3-NT), SOD1, and SOD2 in total hind limb muscle homogenates. (b–d) and (f–h) Relative band densities normalized to GAPDH levels. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. n = number of mice.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1 Y522S/WT Mice

    doi: 10.1155/2017/6792694

    Figure Lengend Snippet: Oxidative stress markers at 4 and 10 months of age following either short- or long-term treatment. (a and e) Representative immunoblots showing expression of 3-Nitrotyrosine (3-NT), SOD1, and SOD2 in total hind limb muscle homogenates. (b–d) and (f–h) Relative band densities normalized to GAPDH levels. Data are given as mean ± SEM; ∗ p < 0.05, WT versus RYR1 Y522S/WT mice; # p < 0.05, untreated RYR1 Y522S/WT versus NAC-treated RYR1 Y522S/WT mice. n = number of mice.

    Article Snippet: Primary antibodies used (a) mouse monoclonal anti-RyR1/RyR3 34C (1 : 20) (Developmental Studies Hybridoma Bank, University of Iowa, Iowa, USA) and (b) rabbit polyclonal antimitochondrial preprotein translocases of the outer membrane, TOM20 (1 : 50) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

    Techniques: Western Blot, Expressing

    Schematic diagrams of excitation–contraction coupling (ECC) at the triad, and SPEG domains and pathogenic variants. (A) The triads are made of transverse tubules (T-tubules) flanked by terminal cisternae of the sarcoplasmic reticulum (SR). Junctional membranes (JM) are connected by the interaction of the ryanodine receptor (RyR1) at the SR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with RyR1, leading to its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the Ca 2+ transporter sarco/endoplasmic reticulum Ca 2+ -ATPase [SERCA; also known as ATP2A; regulated by phospholamban (PLN)] returns Ca 2+ into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J) and triadin (T). Striated muscle enriched protein kinase (SPEG) is localized at the triad, but its role remains elusive. (B) SPEG contains immunoglobulin domains (Ig; light purple shaded), fibronectin domains (Fn; light green shaded) and two kinase domains (yellow shaded). SPEG directly binds to myotubularin 1 (MTM1; dark green; SPEG 2530-2674 a.a.) and desmin (DES; magenta; SPEG 2200-2960 a.a.) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Dfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue font), a cardiomyopathy only (e.g. dilated cardiomyopathy, red font) or both (black font), with no clear genotype–phenotype correlation. Note that patient Q2233* died before a cardiac evaluation. Illustrations were made using Illustrator for Biological Sciences (IBS) .

    Journal: Disease Models & Mechanisms

    Article Title: Characterization of a novel zebrafish model of SPEG -related centronuclear myopathy

    doi: 10.1242/dmm.049437

    Figure Lengend Snippet: Schematic diagrams of excitation–contraction coupling (ECC) at the triad, and SPEG domains and pathogenic variants. (A) The triads are made of transverse tubules (T-tubules) flanked by terminal cisternae of the sarcoplasmic reticulum (SR). Junctional membranes (JM) are connected by the interaction of the ryanodine receptor (RyR1) at the SR, and dihydropyridine receptor (DHPR) at the T-tubules, forming the core components of the ECC machinery. ECC starts when a neuronal action potential arrives via T-tubules and causes a conformational change in DHPR, allowing it to interact with RyR1, leading to its activation. As a result, calcium (Ca 2+ ) leaves the SR via RyR1 channel opening and moves into the cytosol, promoting sarcomeric contraction. Finally, RyR1 is closed and the Ca 2+ transporter sarco/endoplasmic reticulum Ca 2+ -ATPase [SERCA; also known as ATP2A; regulated by phospholamban (PLN)] returns Ca 2+ into the SR, where it is largely bound to calsequestrin (C). The terminal SR also contains RyR1 modulators, such as junctophilin-1 (JPH1), FK506-binding protein 1A (F), junctin (J) and triadin (T). Striated muscle enriched protein kinase (SPEG) is localized at the triad, but its role remains elusive. (B) SPEG contains immunoglobulin domains (Ig; light purple shaded), fibronectin domains (Fn; light green shaded) and two kinase domains (yellow shaded). SPEG directly binds to myotubularin 1 (MTM1; dark green; SPEG 2530-2674 a.a.) and desmin (DES; magenta; SPEG 2200-2960 a.a.) at the inter-kinase domain. Pathogenic variants in SPEG span different regions of the gene and are typically nonsense mutations that result in decreased SPEG levels. The variants exist either in compound heterozygosity (e.g. K359Vfs*35; R1467*), or homozygosity (e.g. T544Dfs*48). SPEG mutations can cause a skeletal muscle disorder only (i.e. centronuclear myopathy or CNM, blue font), a cardiomyopathy only (e.g. dilated cardiomyopathy, red font) or both (black font), with no clear genotype–phenotype correlation. Note that patient Q2233* died before a cardiac evaluation. Illustrations were made using Illustrator for Biological Sciences (IBS) .

    Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-Speg (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB) , rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819, Abcam) , rabbit polyclonal anti-Desmin (1:100; D8281, Sigma-Aldrich) , rabbit polyclonal anti-Dnm2 (1:100; GTX127330, GeneTex) ( ) and mouse monoclonal anti-α-Actinin (1:100; A7811, Sigma-Aldrich) ( ).

    Techniques: Activation Assay, Binding Assay

    Speg expression during zebrafish development. There are two Speg genes in zebrafish, spega and spegb . Each encodes a single transcript that is highly conserved with human SPEG . (A,B) RT-qPCR shows similar temporal expression patterns of spega (A) versus spegb (B) from 2 days post-fertilization (dpf) to 7 dpf. Both Speg mRNA transcripts are significantly upregulated in the heads (light blue bars; 20-fold for spega , 7-fold for spegb ), but stay at relatively similar levels in the skeletal-muscle-predominant tails (white bars). Each data point represents the average of technical triplicates, and three independent experiments are included. Columns and error bars represent mean±s.e.m. Unpaired two-tailed Student's t -test was performed: ** P <0.01; ns, not significant. (C) Whole-mount in situ hybridization using DIG-conjugated RNA probes in 1 dpf embryos shows distinct spatial expression patterns of spega versus spegb . spega is predominantly expressed in the developing brain (yellow arrowhead) and along the neural tube (red arrowheads), but absent from the notochord (cyan arrowheads). spegb staining, however, is predominantly detected at the chevron-shaped developing somites (black arrowheads). Scale bars: 200 µm (top and middle rows) or 100 µm (bottom row). (D,E) Confocal images showing 2 dpf (D) or 5 dpf (E) isolated wild-type (WT) skeletal myofibers double-stained with anti-RyR1 (34C, DSHB) and anti-SPEG (PA553875, Invitrogen). (D,D′) At 2 dpf, SPEG is predominantly localized at the sarcolemma (yellow arrows) and perinuclear regions (cyan arrows; nucleus, yellow asterisks), with weak expression in transverse striations (red arrow), whereas RyR1 is localized in transverse striations labeling the terminal SR. Little colocalization (white in Merge) of SPEG (green) and RyR1 (magenta) can be observed at this stage. (E,E′) At 5 dpf, SPEG expression becomes restricted in the transverse striations (red arrows) that overlap with RyR1. Scale bars: 10 µm (D,E); 5 µm (D′,E′).

    Journal: Disease Models & Mechanisms

    Article Title: Characterization of a novel zebrafish model of SPEG -related centronuclear myopathy

    doi: 10.1242/dmm.049437

    Figure Lengend Snippet: Speg expression during zebrafish development. There are two Speg genes in zebrafish, spega and spegb . Each encodes a single transcript that is highly conserved with human SPEG . (A,B) RT-qPCR shows similar temporal expression patterns of spega (A) versus spegb (B) from 2 days post-fertilization (dpf) to 7 dpf. Both Speg mRNA transcripts are significantly upregulated in the heads (light blue bars; 20-fold for spega , 7-fold for spegb ), but stay at relatively similar levels in the skeletal-muscle-predominant tails (white bars). Each data point represents the average of technical triplicates, and three independent experiments are included. Columns and error bars represent mean±s.e.m. Unpaired two-tailed Student's t -test was performed: ** P <0.01; ns, not significant. (C) Whole-mount in situ hybridization using DIG-conjugated RNA probes in 1 dpf embryos shows distinct spatial expression patterns of spega versus spegb . spega is predominantly expressed in the developing brain (yellow arrowhead) and along the neural tube (red arrowheads), but absent from the notochord (cyan arrowheads). spegb staining, however, is predominantly detected at the chevron-shaped developing somites (black arrowheads). Scale bars: 200 µm (top and middle rows) or 100 µm (bottom row). (D,E) Confocal images showing 2 dpf (D) or 5 dpf (E) isolated wild-type (WT) skeletal myofibers double-stained with anti-RyR1 (34C, DSHB) and anti-SPEG (PA553875, Invitrogen). (D,D′) At 2 dpf, SPEG is predominantly localized at the sarcolemma (yellow arrows) and perinuclear regions (cyan arrows; nucleus, yellow asterisks), with weak expression in transverse striations (red arrow), whereas RyR1 is localized in transverse striations labeling the terminal SR. Little colocalization (white in Merge) of SPEG (green) and RyR1 (magenta) can be observed at this stage. (E,E′) At 5 dpf, SPEG expression becomes restricted in the transverse striations (red arrows) that overlap with RyR1. Scale bars: 10 µm (D,E); 5 µm (D′,E′).

    Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-Speg (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB) , rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819, Abcam) , rabbit polyclonal anti-Desmin (1:100; D8281, Sigma-Aldrich) , rabbit polyclonal anti-Dnm2 (1:100; GTX127330, GeneTex) ( ) and mouse monoclonal anti-α-Actinin (1:100; A7811, Sigma-Aldrich) ( ).

    Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, In Situ Hybridization, Staining, Isolation, Labeling

    spega/b deficiency in zebrafish disrupts triad protein organization and triad ultrastructure, leading to reduced triad numbers. (A-D′) IF staining was performed on 2 dpf (A,B,C,D) and 5 dpf (A′,B′,C′,D′) isolated myofibers. Confocal images show disrupted transverse pattern of RyR1 (A,A′), DHPR (B,B′) and SERCA1a (D,D′) in speg -DKO starting from 2 dpf, while α-Actinin (D,D′) is not affected. Scale bars: 50 µm. (E-F′) Electron micrographs of 7 dpf WT and speg -DKO muscles. (E) In WT zebrafish skeletal muscle, normal triads are physically above the sarcomeric Z-disks and composed of centrally-located T-tubules flanked by terminal sarcoplasmic reticulum (yellow arrowhead). (E′) Triads in speg -DKO appear structurally disrupted, losing the obvious terminal cisternae of the sarcoplasmic membrane (tSR)/T-tubule/tSR pattern (red arrowhead). (F,F′) More importantly, the majority of sarcomeric Z-disks in speg -DKO do not have adjacent triads (yellow arrows). Scale bars: 0.5 µm (E,E′); 1 µm (F,F′). (G) The total number of triads per 60 µm 2 (under an electron microscope) was significantly reduced in speg -DKO. Each dot represents the average of technical triplicates, and three biological replicates are included. Data are mean±s.e.m. Unpaired two-tailed Student's t -test: * P <0.05.

    Journal: Disease Models & Mechanisms

    Article Title: Characterization of a novel zebrafish model of SPEG -related centronuclear myopathy

    doi: 10.1242/dmm.049437

    Figure Lengend Snippet: spega/b deficiency in zebrafish disrupts triad protein organization and triad ultrastructure, leading to reduced triad numbers. (A-D′) IF staining was performed on 2 dpf (A,B,C,D) and 5 dpf (A′,B′,C′,D′) isolated myofibers. Confocal images show disrupted transverse pattern of RyR1 (A,A′), DHPR (B,B′) and SERCA1a (D,D′) in speg -DKO starting from 2 dpf, while α-Actinin (D,D′) is not affected. Scale bars: 50 µm. (E-F′) Electron micrographs of 7 dpf WT and speg -DKO muscles. (E) In WT zebrafish skeletal muscle, normal triads are physically above the sarcomeric Z-disks and composed of centrally-located T-tubules flanked by terminal sarcoplasmic reticulum (yellow arrowhead). (E′) Triads in speg -DKO appear structurally disrupted, losing the obvious terminal cisternae of the sarcoplasmic membrane (tSR)/T-tubule/tSR pattern (red arrowhead). (F,F′) More importantly, the majority of sarcomeric Z-disks in speg -DKO do not have adjacent triads (yellow arrows). Scale bars: 0.5 µm (E,E′); 1 µm (F,F′). (G) The total number of triads per 60 µm 2 (under an electron microscope) was significantly reduced in speg -DKO. Each dot represents the average of technical triplicates, and three biological replicates are included. Data are mean±s.e.m. Unpaired two-tailed Student's t -test: * P <0.05.

    Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-Speg (1:100; PA553875, Invitrogen), mouse monoclonal anti-RyR1 (1:100; 34C, DSHB) , rabbit polyclonal anti-CACNA1S (1:100; ab203662, Abcam; which labels the DHPR protein), mouse monoclonal anti-SERCA1a (1:200; ab2819, Abcam) , rabbit polyclonal anti-Desmin (1:100; D8281, Sigma-Aldrich) , rabbit polyclonal anti-Dnm2 (1:100; GTX127330, GeneTex) ( ) and mouse monoclonal anti-α-Actinin (1:100; A7811, Sigma-Aldrich) ( ).

    Techniques: Staining, Isolation, Microscopy, Two Tailed Test

    Antibody information

    Journal: The Journal of Biological Chemistry

    Article Title: Neuronal ER–plasma membrane junctions organized by Kv2–VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis

    doi: 10.1074/jbc.RA119.007635

    Figure Lengend Snippet: Antibody information

    Article Snippet: RRID:AB_2722711 Tissue cultured supernatant (1:5) 6 Nir2 Recombinant fragment of human Nir2 aa 589–651 Rabbit pAb, Abcam catalog no. ab221381, RRID:AB_2801563 10 μg/ml ( A ), 1 μg/ml ( E ) S1 Nir2 Recombinant peptide of human Nir2 aa 365–444 Rabbit pAb, Novus Biologicals catalog no. NBP2–34132, RRID: AB_2801564 10 μg/ml ( A ) 6, S1 RyRs (34C) Partially purified chicken pectoral muscle ryanodine receptor Mouse IgG1 mAb, Developmental Studies Hybridoma Bank, RRID: AB_528457 1 μg/ml 6 Open in a separate window Antibody information Immunopurification and proteomic analyses Preparation of cross-linked mouse brain samples for immunopurification was as described previously ( 43 ).

    Techniques: Concentration Assay, Affinity Purification, Purification, Western Blot, Cell Culture, Recombinant

    Antibody information

    Journal: The Journal of Neuroscience

    Article Title: Identification of VAPA and VAPB as Kv2 Channel-Interacting Proteins Defining Endoplasmic Reticulum–Plasma Membrane Junctions in Mammalian Brain Neurons

    doi: 10.1523/JNEUROSCI.0893-18.2018

    Figure Lengend Snippet: Antibody information

    Article Snippet: For the three VAPA-specific mAbs, N479/12, N479/22, and N479/24, we saw a reduction of the immunolabeling signal in the VAPA KO cells to levels approaching those of subclass-matched irrelevant primary and no primary control levels and, for the VAPA/B mAb N479/107, a smaller but still substantial reduction of signal in the VAPA KO cells. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Immunogen Manufacturer information Concentration used Figure(s) N479/12 Fusion protein aa 1-219 of rat VAPA Mouse IgG1 mAb, NeuroMab, RRID: AB_2722707 Tissue culture supernatant, 1:5 A–F , N479/22 Fusion protein aa 1-219 of rat VAPA Mouse IgG2a mAb, NeuroMab, RRID: AB_2722708 Tissue culture supernatant, neat , 1:5 , 1:10 ( ) G–J , N479/24 Fusion protein aa 1-219 of rat VAPA Mouse IgG2a mAb, RRID: AB_2722709 Tissue culture supernatant, 1:5 N479/107 Fusion protein aa 1-219 of rat VAPA Mouse IgG2b mAb, NeuroMab, RRID: AB_2722711 Tissue culture supernatant, 1:5 , KC Synthetic peptide aa 837-853 of rat Kv2.1 Rabbit pAb, in-house (Trimmer Laboratory), RRID: AB_2315767 Affinity purified, 1:150 , , K89/34 Synthetic peptide aa 837-853 of rat Kv2.1 Mouse IgG1 mAb, NeuroMab catalog #73-014, RRID: AB_10672253 Tissue culture supernatant, 1:10 34C Partially purified chicken pectoral muscle ryanodine receptor Mouse IgG1 mAb, Thermo Fisher catalog #MA3-925 RRID: AB_2254138 Purified, 1 μg/ml 34C Partially purified chicken pectoral muscle ryanodine receptor Mouse IgG1 mAb, Developmental Studies Hybridoma Bank, RRID: AB_528457 Concentrated tissue culture supernatant, 1 μg/ml N106/36 Full-length recombinant human ankG Mouse IgG2b mAb, NeuroMab catalog #75-147, RRID: AB_10675130 Tissue culture supernatant, 1:5 N372B/1 Fusion protein aa 717-907 of rat Kv2.2 long isoform Mouse IgG1 mAb, NeuroMab catalog #73-369, RRID: AB_2315869 Tissue culture supernatant, 1:2 , N372B/60 Fusion protein aa 717-907 of rat Kv2.2 long isoform Mouse IgG2a mAb, NeuroMab catalog #73-360, RRID: AB_2315867 Purified, 10 μg/ml , , Kv1.5E Synthetic peptide aa 271-284 of rat Kv1.5 Rabbit pAb, in-house (Trimmer Laboratory) RRID: AB_2722698 Affinity purified, 10 μg/ml 2–2.20.14, DyLight 488 HA peptide YPYDVPDYA Mouse IgG1 mAb, Thermo Fisher Scientific catalog #26183-D488; RRID: AB_2533051 Purified, 0.3 μg/ml Open in a separate window Antibody information fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 5. caption a7 CRISPR-mediated KO of VAPA expression affects Kv2.1 localization in RAW264.7 cells A .

    Techniques: Concentration Assay, Affinity Purification, Purification, Recombinant

    CRISPR-mediated KO of VAPA expression affects Kv2.1 localization in RAW264.7 cells A. Representative TIRF images of SEP-Kv2.1 expression in a live WT RAW264.7 cell expressing SEP-Kv2.1 (green is cell surface pHluorin fluorescence, magenta is total cellular mCherry fluorescence). Scale bar is 5 μm and holds for all panels in figure. B, Representative TIRF images of GFP-Kv2.1 (green) and BFP-SEC61β (magenta) in live WT (left) or VAPA KO (right) RAW264.7 cells coexpressing GFP-Kv2.1 and BFP-SEC61β. Note the reduction in clustered Kv2.1 expression in the VAPA KO cell. C, Line scan analysis of selection indicated in merged image of WT RAW264.7 cell coexpressing GFP-Kv2.1 and BFP-SEC61β. D, Line scan analysis of selection indicated in merged image of VAPA KO RAW264.7 cell coexpressing GFP-Kv2.1 and BFP-SEC61β. E, Representative TIRF image of live WT (left) or VAPA KO (right) RAW264.7 cells expressing GFP-Kv1.4. F, Summary graph of CV measurements of GFP-Kv2.1 in WT and VAPA KO RAW264.7 cells (****p = 6.648 × 10−9, n = 29 cells, two-tailed unpaired t test). G, Summary graph of CV measurement of GFP-Kv1.4 in WT and VAPA KO RAW264.7 cells (ns, 0.5603, n = 10 cells, two-tailed unpaired t test). H-K. Images of WT (left) or VAPA KO (right) RAW264.7 cells used for high-content analysis of immunolabeling (magenta) and with Hoechst labeling of nuclei (green). H, No primary (anti-IgG2b secondary antibody). I, Positive control anti-mortalin mAb N52A/42 (IgG1). J, Anti-VAPA mAb N479/22 (IgG2a). K, Anti-VAPA/B mAb N479/107 (IgG2b). Scale bar in H is 65 μm and holds for H–K. L, Mean immunolabeling intensity of all samples across 1300–2800 individual cells in each sample. See Materials and Methods for further details of these antibody validation results.

    Journal: The Journal of Neuroscience

    Article Title: Identification of VAPA and VAPB as Kv2 Channel-Interacting Proteins Defining Endoplasmic Reticulum–Plasma Membrane Junctions in Mammalian Brain Neurons

    doi: 10.1523/JNEUROSCI.0893-18.2018

    Figure Lengend Snippet: CRISPR-mediated KO of VAPA expression affects Kv2.1 localization in RAW264.7 cells A. Representative TIRF images of SEP-Kv2.1 expression in a live WT RAW264.7 cell expressing SEP-Kv2.1 (green is cell surface pHluorin fluorescence, magenta is total cellular mCherry fluorescence). Scale bar is 5 μm and holds for all panels in figure. B, Representative TIRF images of GFP-Kv2.1 (green) and BFP-SEC61β (magenta) in live WT (left) or VAPA KO (right) RAW264.7 cells coexpressing GFP-Kv2.1 and BFP-SEC61β. Note the reduction in clustered Kv2.1 expression in the VAPA KO cell. C, Line scan analysis of selection indicated in merged image of WT RAW264.7 cell coexpressing GFP-Kv2.1 and BFP-SEC61β. D, Line scan analysis of selection indicated in merged image of VAPA KO RAW264.7 cell coexpressing GFP-Kv2.1 and BFP-SEC61β. E, Representative TIRF image of live WT (left) or VAPA KO (right) RAW264.7 cells expressing GFP-Kv1.4. F, Summary graph of CV measurements of GFP-Kv2.1 in WT and VAPA KO RAW264.7 cells (****p = 6.648 × 10−9, n = 29 cells, two-tailed unpaired t test). G, Summary graph of CV measurement of GFP-Kv1.4 in WT and VAPA KO RAW264.7 cells (ns, 0.5603, n = 10 cells, two-tailed unpaired t test). H-K. Images of WT (left) or VAPA KO (right) RAW264.7 cells used for high-content analysis of immunolabeling (magenta) and with Hoechst labeling of nuclei (green). H, No primary (anti-IgG2b secondary antibody). I, Positive control anti-mortalin mAb N52A/42 (IgG1). J, Anti-VAPA mAb N479/22 (IgG2a). K, Anti-VAPA/B mAb N479/107 (IgG2b). Scale bar in H is 65 μm and holds for H–K. L, Mean immunolabeling intensity of all samples across 1300–2800 individual cells in each sample. See Materials and Methods for further details of these antibody validation results.

    Article Snippet: For the three VAPA-specific mAbs, N479/12, N479/22, and N479/24, we saw a reduction of the immunolabeling signal in the VAPA KO cells to levels approaching those of subclass-matched irrelevant primary and no primary control levels and, for the VAPA/B mAb N479/107, a smaller but still substantial reduction of signal in the VAPA KO cells. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Immunogen Manufacturer information Concentration used Figure(s) N479/12 Fusion protein aa 1-219 of rat VAPA Mouse IgG1 mAb, NeuroMab, RRID: AB_2722707 Tissue culture supernatant, 1:5 A–F , N479/22 Fusion protein aa 1-219 of rat VAPA Mouse IgG2a mAb, NeuroMab, RRID: AB_2722708 Tissue culture supernatant, neat , 1:5 , 1:10 ( ) G–J , N479/24 Fusion protein aa 1-219 of rat VAPA Mouse IgG2a mAb, RRID: AB_2722709 Tissue culture supernatant, 1:5 N479/107 Fusion protein aa 1-219 of rat VAPA Mouse IgG2b mAb, NeuroMab, RRID: AB_2722711 Tissue culture supernatant, 1:5 , KC Synthetic peptide aa 837-853 of rat Kv2.1 Rabbit pAb, in-house (Trimmer Laboratory), RRID: AB_2315767 Affinity purified, 1:150 , , K89/34 Synthetic peptide aa 837-853 of rat Kv2.1 Mouse IgG1 mAb, NeuroMab catalog #73-014, RRID: AB_10672253 Tissue culture supernatant, 1:10 34C Partially purified chicken pectoral muscle ryanodine receptor Mouse IgG1 mAb, Thermo Fisher catalog #MA3-925 RRID: AB_2254138 Purified, 1 μg/ml 34C Partially purified chicken pectoral muscle ryanodine receptor Mouse IgG1 mAb, Developmental Studies Hybridoma Bank, RRID: AB_528457 Concentrated tissue culture supernatant, 1 μg/ml N106/36 Full-length recombinant human ankG Mouse IgG2b mAb, NeuroMab catalog #75-147, RRID: AB_10675130 Tissue culture supernatant, 1:5 N372B/1 Fusion protein aa 717-907 of rat Kv2.2 long isoform Mouse IgG1 mAb, NeuroMab catalog #73-369, RRID: AB_2315869 Tissue culture supernatant, 1:2 , N372B/60 Fusion protein aa 717-907 of rat Kv2.2 long isoform Mouse IgG2a mAb, NeuroMab catalog #73-360, RRID: AB_2315867 Purified, 10 μg/ml , , Kv1.5E Synthetic peptide aa 271-284 of rat Kv1.5 Rabbit pAb, in-house (Trimmer Laboratory) RRID: AB_2722698 Affinity purified, 10 μg/ml 2–2.20.14, DyLight 488 HA peptide YPYDVPDYA Mouse IgG1 mAb, Thermo Fisher Scientific catalog #26183-D488; RRID: AB_2533051 Purified, 0.3 μg/ml Open in a separate window Antibody information fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 5. caption a7 CRISPR-mediated KO of VAPA expression affects Kv2.1 localization in RAW264.7 cells A .

    Techniques: CRISPR, Expressing, Fluorescence, Selection, Two Tailed Test, High Content Screening, Immunolabeling, Labeling, Positive Control