ryr1  (Alomone Labs)


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    Name:
    Anti Ryanodine Receptor 1 Antibody
    Description:
    Anti Ryanodine Receptor 1 Antibody ARR 001 is a highly specific antibody directed against an epitope of human RyR1 The antibody can be used in western blot immunocytochemistry and immunohistochemistry applications It has been designed to recognize RyR1 from human rat and mouse samples
    Catalog Number:
    ARR-001
    Price:
    495.0
    Category:
    Primary Antibody
    Applications:
    Immunocytochemistry, Immunofluorescence, Immunohistochemistry, Western Blot
    Purity:
    Affinity purified on immobilized antigen.
    Immunogen:
    Synthetic peptide
    Size:
    50 mcl
    Antibody Type:
    Polyclonal Primary Antibodies
    Format:
    Lyophilized Powder
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Alomone Labs ryr1
    Anti Ryanodine Receptor 1 Antibody
    Anti Ryanodine Receptor 1 Antibody ARR 001 is a highly specific antibody directed against an epitope of human RyR1 The antibody can be used in western blot immunocytochemistry and immunohistochemistry applications It has been designed to recognize RyR1 from human rat and mouse samples
    https://www.bioz.com/result/ryr1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ryr1 - by Bioz Stars, 2021-09
    94/100 stars

    Images

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

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

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

    doi: 10.1152/ajpregu.00032.2011

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

    Techniques Used: Labeling

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

    Techniques Used: Staining

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

    Techniques Used: Expressing, Western Blot

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

    Techniques Used: Labeling

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

    Techniques Used: Labeling

    Related Articles

    Immunoprecipitation:

    Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons
    Article Snippet: .. Cells were either lysed 40 h post-transfection and processed for immunoprecipitation as described above or fixed in 4% paraformaldehyde for 15 min, permeabilized in 0.1% Triton-X100 in PBS for 20 min and immunostained using mouse monoclonal antibodies against RyR2 (1:1000; Clone c3-33, Pierce), or RyR1 (1:500; Alomone Labs, Israel), followed by subsequent detection using secondary antibodies conjugated to Alexa488 (1:1000; Invitrogen). ..

    Western Blot:

    Article Title: Upregulation of the CaV 1.1-ryanodine receptor complex in a rat model of critical illness myopathy
    Article Snippet: .. Primary antibodies used against RYR1 (1:100 Alomone Labs), RYR2 (1:100 Alomone Labs), fast myosin II (Abcam), slow myosin 1 (Sigma), calpain II (Calbiochem), and Cav 1.1 (1:200 Abcam) were the same antibodies used for Western blot analysis. ..

    Incubation:

    Article Title: Implication of the ryanodine receptor in TRPV4-induced calcium response in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats.
    Article Snippet: .. There is a growing body of evidence indicating that transient receptor potential (TRP) channels are implicated in calcium signaling and various cellular functions in the pulmonary vasculature.. The aim of this study was to investigate the expression, functional role, and coupling to reticulum calcium channels of the type 4 vanilloid TRP subfamily (TRPV4) in the pulmonary artery from both normoxic (Nx) and chronically hypoxic (CH) rats. ..

    Article Title: Ginkgolide B Maintains Calcium Homeostasis in Hypoxic Hippocampal Neurons by Inhibiting Calcium Influx and Intracellular Calcium Release
    Article Snippet: .. The membranes were blocked for 1 h in non-fat milk and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-Cav1.2 (1:200, cat. #ACC-005, Alomone, Jerusalem, Israel), rabbit anti-stromal interaction molecule-1 (STIM1; 1:500, cat. #ACC-063, Alomone), rabbit anti-ryanodine receptor type-1 (RyR2; 1:200, cat. #ARR-001, Alomone) and rabbit anti-β-actin (1:2,000, cat. #4970, Cell Signaling Technology, Danvers, MA, USA). ..

    Article Title: Caveolae are involved in mechanotransduction during pulmonary hypertension.
    Article Snippet: .. Cells were then incubated overnight in PTB 4% with the ryanodine receptors (RyR) 1 (1:200; ARR-001, Alomone Labs) or RyR3 antibodies (1:200; ARR-003, Alomone Labs), plus the Cav-1 [7C8] antibody (1:500; ab37141, abcam), at 4°C. ..

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    Alomone Labs rabbit anti nr1
    Exposure to high glucose (HG) increases expression of NMDA receptor subunits in primary cultures of rat mesangial cells. A : Representative results of RT-PCR showing significantly increased abundance of transcripts encoding <t>NR1,</t> NR2B, and NR2C subunits but not of NR2A or NR2D in cells cultured for 24 h in HG medium compared with cells cultured in normal glucose (control [Con]). B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in primary cultures of rat mesangial cells cultured in HG. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P
    Rabbit Anti Nr1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti nr1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti nr1 - by Bioz Stars, 2021-09
    86/100 stars
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    93
    Alomone Labs p2x1 primary antibody
    Localization of 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modification of the human <t>P2X1</t> receptor. (a) P2X1 homology model (based on zP2X4 crystal structure model – Kawate et al . 2009 ) depicted as a trimeric cartoon. Lysine residues modified DTSSP and identified by mass spectrometry are indicated in red. Lysine residue K70 that was not observed in any mass spectrometry runs is shown in dark grey. Residues observed and not modified by DTSSP in some runs are shown in grey. Residue K68 that was not observed to be modified by DTSSP (11/11) is shown in black. The serine residues 130, 286 (blue spheres) and tyrosine 274 (green spheres) were also modified by DTSSP. (b) Human P2X1 protein sequence with DTSSP modification of the P2X1 receptor marked as observed from mass spectrometry data. Transmembrane regions 1 and 2 are highlighted with a bold line. Residues are coloured as indicated in panel (a). Arrows indicate residues modified by DTSSP, an arrow with a cross show a residue that was not modified by DTSSP, and arrows with a question mark where it remains unknown either because of non-coverage of the protein sequence or modification not detected. Note that all the DTSSP modifications occur in the extracellular region.
    P2x1 Primary Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2x1 primary antibody/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p2x1 primary antibody - by Bioz Stars, 2021-09
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    93
    Alomone Labs polyclonal anti endothelin receptor a
    The response to ET-1 is enhanced in BALB-STD mice. A: Vasoconstriction in small pulmonary artery profiles in BALB-STD mice is significantly greater over the range of 10 −10 to 10 −9 ET-1. B: This coincides with a significant increase in the ET A R in BALB-STD mice total lung tissue as indicated by quantification of Western blot analysis. Data are expressed as means ± SEM. n = 4 to 5 ( B ) or n = 16 to 35 profiles per group across 4 to 5 mice ( A ) in two independent experiments. ∗ P ≤ 0.05 versus IFN-γ-STD and controls. CON, control; ET A R, <t>endothelin</t> receptor A; ET-1, endothelin-1; IFN, interferon; STD, short-term depletion.
    Polyclonal Anti Endothelin Receptor A, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal anti endothelin receptor a/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti endothelin receptor a - by Bioz Stars, 2021-09
    93/100 stars
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    Image Search Results


    Exposure to high glucose (HG) increases expression of NMDA receptor subunits in primary cultures of rat mesangial cells. A : Representative results of RT-PCR showing significantly increased abundance of transcripts encoding NR1, NR2B, and NR2C subunits but not of NR2A or NR2D in cells cultured for 24 h in HG medium compared with cells cultured in normal glucose (control [Con]). B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in primary cultures of rat mesangial cells cultured in HG. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Exposure to high glucose (HG) increases expression of NMDA receptor subunits in primary cultures of rat mesangial cells. A : Representative results of RT-PCR showing significantly increased abundance of transcripts encoding NR1, NR2B, and NR2C subunits but not of NR2A or NR2D in cells cultured for 24 h in HG medium compared with cells cultured in normal glucose (control [Con]). B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in primary cultures of rat mesangial cells cultured in HG. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    Increased expression of NMDA receptor subunits in renal cortex of Akita mice. A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, and NR2C subunits but not in NR2B or NR2D in renal cortex in 12-week-old Akita mice compared with 12-week-old DBA/2J control mice. B : Densitometric analysis from four mice per group. C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in Akita mice compared with DBA/2J control mice. D : Densitometric analysis from four mice per group. Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Increased expression of NMDA receptor subunits in renal cortex of Akita mice. A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, and NR2C subunits but not in NR2B or NR2D in renal cortex in 12-week-old Akita mice compared with 12-week-old DBA/2J control mice. B : Densitometric analysis from four mice per group. C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in Akita mice compared with DBA/2J control mice. D : Densitometric analysis from four mice per group. Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    Immunohistochemistry (IHC) suggests increased abundance of NMDA receptor subunits throughout the kidney of 12-week-old Akita mice. IHC was carried out in paraffin sections. Negative control sections shown at the top were not exposed to a primary antibody. A : Especially large increases in NR1, NR2A, and NR2C in renal tubules. B : Signal in glomeruli for NR1, NR2A, and NR2C. Primary processes were visible in some of the cells within glomeruli. C : Staining intensity per square micron in the whole kidney (top) and within glomeruli (bottom). Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Immunohistochemistry (IHC) suggests increased abundance of NMDA receptor subunits throughout the kidney of 12-week-old Akita mice. IHC was carried out in paraffin sections. Negative control sections shown at the top were not exposed to a primary antibody. A : Especially large increases in NR1, NR2A, and NR2C in renal tubules. B : Signal in glomeruli for NR1, NR2A, and NR2C. Primary processes were visible in some of the cells within glomeruli. C : Staining intensity per square micron in the whole kidney (top) and within glomeruli (bottom). Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Immunohistochemistry, Mouse Assay, Negative Control, Staining

    Exposure to high glucose (HG) increases expression of NMDA receptor subunits in cultured mouse podocytes (MPC-5 cells). A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, NR2B, and NR2C subunits in cells cultured for 24 h in a medium containing 25 mmol/L glucose (HG). There was no change in NR2D. Control cells (Con) were cultured in medium containing 9 mmol/L glucose, with 16 mmol/L mannitol as an osmotic control. B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in podocytes cultured in HG compared with Con. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Journal: Diabetes

    Article Title: NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801

    doi: 10.2337/db16-0209

    Figure Lengend Snippet: Exposure to high glucose (HG) increases expression of NMDA receptor subunits in cultured mouse podocytes (MPC-5 cells). A : Representative results of RT-PCR showing increased abundance of transcripts encoding NR1, NR2A, NR2B, and NR2C subunits in cells cultured for 24 h in a medium containing 25 mmol/L glucose (HG). There was no change in NR2D. Control cells (Con) were cultured in medium containing 9 mmol/L glucose, with 16 mmol/L mannitol as an osmotic control. B : Densitometric analysis of three repetitions of the experiments shown in A . C : Immunoblot analysis showing increased abundance of NMDA receptor subunits in podocytes cultured in HG compared with Con. D : Densitometric analysis of three repetitions of the experiments shown in C . Data are mean ± SD. * P

    Article Snippet: Primary antibodies were rabbit anti-NR1 (AGC-001 1:100; Alomone Labs) or rabbit anti-NR2A (AGC-002 1:100; Alomone Labs) for 24 h at 4°C.

    Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    Localization of 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modification of the human P2X1 receptor. (a) P2X1 homology model (based on zP2X4 crystal structure model – Kawate et al . 2009 ) depicted as a trimeric cartoon. Lysine residues modified DTSSP and identified by mass spectrometry are indicated in red. Lysine residue K70 that was not observed in any mass spectrometry runs is shown in dark grey. Residues observed and not modified by DTSSP in some runs are shown in grey. Residue K68 that was not observed to be modified by DTSSP (11/11) is shown in black. The serine residues 130, 286 (blue spheres) and tyrosine 274 (green spheres) were also modified by DTSSP. (b) Human P2X1 protein sequence with DTSSP modification of the P2X1 receptor marked as observed from mass spectrometry data. Transmembrane regions 1 and 2 are highlighted with a bold line. Residues are coloured as indicated in panel (a). Arrows indicate residues modified by DTSSP, an arrow with a cross show a residue that was not modified by DTSSP, and arrows with a question mark where it remains unknown either because of non-coverage of the protein sequence or modification not detected. Note that all the DTSSP modifications occur in the extracellular region.

    Journal: Journal of Neurochemistry

    Article Title: Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

    doi: 10.1111/jnc.12012

    Figure Lengend Snippet: Localization of 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modification of the human P2X1 receptor. (a) P2X1 homology model (based on zP2X4 crystal structure model – Kawate et al . 2009 ) depicted as a trimeric cartoon. Lysine residues modified DTSSP and identified by mass spectrometry are indicated in red. Lysine residue K70 that was not observed in any mass spectrometry runs is shown in dark grey. Residues observed and not modified by DTSSP in some runs are shown in grey. Residue K68 that was not observed to be modified by DTSSP (11/11) is shown in black. The serine residues 130, 286 (blue spheres) and tyrosine 274 (green spheres) were also modified by DTSSP. (b) Human P2X1 protein sequence with DTSSP modification of the P2X1 receptor marked as observed from mass spectrometry data. Transmembrane regions 1 and 2 are highlighted with a bold line. Residues are coloured as indicated in panel (a). Arrows indicate residues modified by DTSSP, an arrow with a cross show a residue that was not modified by DTSSP, and arrows with a question mark where it remains unknown either because of non-coverage of the protein sequence or modification not detected. Note that all the DTSSP modifications occur in the extracellular region.

    Article Snippet: Proteins were transferred to nitrocellulose, probed with P2X1 primary antibody (1 : 1000; Alomone Labs) followed by secondary goat anti-rabbit antibody (A6154, 1 : 3500 dilution; Sigma).

    Techniques: Modification, Mass Spectrometry, Sequencing

    Effect of 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modification on Human P2X1 receptor function. (a) Application of 100 μM ATP to Xenopus oocytes expressing P2X1 wildtype receptors evoked a large inward current recorded by two electrode voltage clamp. Pre-incubation with 100 μM DTSSP for 30 min almost abolished responses. Inset traces are representative of normalized responses to 100 μM ATP in the presence or absence of 100 μM DTSSP indicating no significant change in the current time course. (b) Pooled electrophysiology data depicting the decrease in channel function post-DTSSP treatment. (c) P2X1 receptor ATP-binding site analysis utilizing uv cross-linked 32 P 2Azido ATP (2AzATP) shows a marked reduction in radioactivity of the P2X1 receptor protein band following pre-treatment with 100 μM DTSSP. (d) Pooled densitometry data collected from autoradiography of the DTSSP treated and non-treated 2AzATP radioactive P2X1 receptor bands ( n = 4) *** p

    Journal: Journal of Neurochemistry

    Article Title: Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

    doi: 10.1111/jnc.12012

    Figure Lengend Snippet: Effect of 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modification on Human P2X1 receptor function. (a) Application of 100 μM ATP to Xenopus oocytes expressing P2X1 wildtype receptors evoked a large inward current recorded by two electrode voltage clamp. Pre-incubation with 100 μM DTSSP for 30 min almost abolished responses. Inset traces are representative of normalized responses to 100 μM ATP in the presence or absence of 100 μM DTSSP indicating no significant change in the current time course. (b) Pooled electrophysiology data depicting the decrease in channel function post-DTSSP treatment. (c) P2X1 receptor ATP-binding site analysis utilizing uv cross-linked 32 P 2Azido ATP (2AzATP) shows a marked reduction in radioactivity of the P2X1 receptor protein band following pre-treatment with 100 μM DTSSP. (d) Pooled densitometry data collected from autoradiography of the DTSSP treated and non-treated 2AzATP radioactive P2X1 receptor bands ( n = 4) *** p

    Article Snippet: Proteins were transferred to nitrocellulose, probed with P2X1 primary antibody (1 : 1000; Alomone Labs) followed by secondary goat anti-rabbit antibody (A6154, 1 : 3500 dilution; Sigma).

    Techniques: Modification, Expressing, Incubation, Binding Assay, Radioactivity, Autoradiography

    Purification and mass spectrometry analysis of the Human P2X1 receptor. (a) Anti-P2X1 receptor antibody western blot analysis of 3X FLAG peptide eluted fractions from anti-FLAG agarose beads. FT, flow through; W1–2, washes; E1–E10, FLAG peptide eluted fractions; E11–13, 0.1 M Glycine pH 3.5 eluted fractions. P2X1 protein of the correct mass is observed in fractions E2–E8. (b) InstantBlue (Expedeon) stained gel of eluted fractions from anti-FLAG agarose beads (lanes labelled as Fig. 1a ). (c) Combined and concentrated fractions were run on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with InstantBlue (Expedeon). Clean purified FLAG tagged P2X1 protein can be observed along with PNGase F deglycosylated P2X1 receptor protein and the PNGase F protein (star indicates confirmed by mass spectrometry) (d) Identification of the P2X1 receptor with 26.8 ± 6% percentage coverage on single mass spectrometry runs was achieved with trypsin digest though other enzymes were tested (e.g. chymotrypsin, 11.2 ± 5% coverage, data not shown). Coverage of the P2X1 receptor protein was increased (26.8 ± 6% vs. 37.8 ± 3%) by deglycosylating the receptors with PNGase F. Use of Orbitrap versus Qtrap mass spectrometer increased mass accuracy and peptide identification increasing coverage even further on average to 59.2 ± 2%. (e) Human P2X1 receptor protein sequence showing the total coverage of all observed peptides (26 runs). Transmembrane regions 1 and 2 are highlighted with a bold line. Amino acid residues shown in bold lowercase were not observed on mass spectrometry of the P2X1 receptor protein most probably because their masses were below the limit of detection (∼500 Da) (Table S1). Other areas of predicted low mass were only observed as a result of partial digestion and therefore identified as part of a larger peptide mass. Vertical lines indicate sites for trypsin digestion at arginine and lysine residues.

    Journal: Journal of Neurochemistry

    Article Title: Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

    doi: 10.1111/jnc.12012

    Figure Lengend Snippet: Purification and mass spectrometry analysis of the Human P2X1 receptor. (a) Anti-P2X1 receptor antibody western blot analysis of 3X FLAG peptide eluted fractions from anti-FLAG agarose beads. FT, flow through; W1–2, washes; E1–E10, FLAG peptide eluted fractions; E11–13, 0.1 M Glycine pH 3.5 eluted fractions. P2X1 protein of the correct mass is observed in fractions E2–E8. (b) InstantBlue (Expedeon) stained gel of eluted fractions from anti-FLAG agarose beads (lanes labelled as Fig. 1a ). (c) Combined and concentrated fractions were run on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with InstantBlue (Expedeon). Clean purified FLAG tagged P2X1 protein can be observed along with PNGase F deglycosylated P2X1 receptor protein and the PNGase F protein (star indicates confirmed by mass spectrometry) (d) Identification of the P2X1 receptor with 26.8 ± 6% percentage coverage on single mass spectrometry runs was achieved with trypsin digest though other enzymes were tested (e.g. chymotrypsin, 11.2 ± 5% coverage, data not shown). Coverage of the P2X1 receptor protein was increased (26.8 ± 6% vs. 37.8 ± 3%) by deglycosylating the receptors with PNGase F. Use of Orbitrap versus Qtrap mass spectrometer increased mass accuracy and peptide identification increasing coverage even further on average to 59.2 ± 2%. (e) Human P2X1 receptor protein sequence showing the total coverage of all observed peptides (26 runs). Transmembrane regions 1 and 2 are highlighted with a bold line. Amino acid residues shown in bold lowercase were not observed on mass spectrometry of the P2X1 receptor protein most probably because their masses were below the limit of detection (∼500 Da) (Table S1). Other areas of predicted low mass were only observed as a result of partial digestion and therefore identified as part of a larger peptide mass. Vertical lines indicate sites for trypsin digestion at arginine and lysine residues.

    Article Snippet: Proteins were transferred to nitrocellulose, probed with P2X1 primary antibody (1 : 1000; Alomone Labs) followed by secondary goat anti-rabbit antibody (A6154, 1 : 3500 dilution; Sigma).

    Techniques: Purification, Mass Spectrometry, Western Blot, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Sequencing

    Site directed mutagenesis of human P2X1 receptor to discover the molecular basis for 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) inhibition. (a) Cartoon representation of P2X1 receptor structure highlighting the residues K199 and K221 (blue spheres) positioned on different subunits approximately 12 angstroms apart. The diameter of the circle shown is approximately 24 angstroms. Residues in yellow correspond to positions 196 and 320 in adjacent subunits where introduced cysteine residues form a disulphide bond and inhibit channel activation. Red spheres correspond to lysine residues that when mutated had no effect on DTSSP inhibition of ATP evoked responses. (b) Combining mass spectrometry and crystal structure to map possible cross-linked pairs, multiple mutations were designed to discover the residues responsible for DTSSP inhibition and cross-linking. Residues 70 : 140; 70 : 309; 70 : 286; 140 : 215; 190 : 283; 190 : 286; 199 : 221 and 322 : 322 are all within 12 angstroms and on separate P2X1 subunits and therefore possible candidates for causing functional inhibition and dimer/trimer formation with DTSSP. Only double mutant K199R:K221R showed a significant reduction of DTSSP inhibition also reflected in the single mutants K199R and K221R ( n = 3–25). No mutations were observed to disrupt the DTSSP formation of dimers/trimers on western blot (data not shown). Inset example trace data for human P2X1 wildtype and P2X1 double mutant K199R: K221R in the presence and absence of 100 μM DTSSP ** p

    Journal: Journal of Neurochemistry

    Article Title: Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

    doi: 10.1111/jnc.12012

    Figure Lengend Snippet: Site directed mutagenesis of human P2X1 receptor to discover the molecular basis for 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) inhibition. (a) Cartoon representation of P2X1 receptor structure highlighting the residues K199 and K221 (blue spheres) positioned on different subunits approximately 12 angstroms apart. The diameter of the circle shown is approximately 24 angstroms. Residues in yellow correspond to positions 196 and 320 in adjacent subunits where introduced cysteine residues form a disulphide bond and inhibit channel activation. Red spheres correspond to lysine residues that when mutated had no effect on DTSSP inhibition of ATP evoked responses. (b) Combining mass spectrometry and crystal structure to map possible cross-linked pairs, multiple mutations were designed to discover the residues responsible for DTSSP inhibition and cross-linking. Residues 70 : 140; 70 : 309; 70 : 286; 140 : 215; 190 : 283; 190 : 286; 199 : 221 and 322 : 322 are all within 12 angstroms and on separate P2X1 subunits and therefore possible candidates for causing functional inhibition and dimer/trimer formation with DTSSP. Only double mutant K199R:K221R showed a significant reduction of DTSSP inhibition also reflected in the single mutants K199R and K221R ( n = 3–25). No mutations were observed to disrupt the DTSSP formation of dimers/trimers on western blot (data not shown). Inset example trace data for human P2X1 wildtype and P2X1 double mutant K199R: K221R in the presence and absence of 100 μM DTSSP ** p

    Article Snippet: Proteins were transferred to nitrocellulose, probed with P2X1 primary antibody (1 : 1000; Alomone Labs) followed by secondary goat anti-rabbit antibody (A6154, 1 : 3500 dilution; Sigma).

    Techniques: Mutagenesis, Inhibition, Activation Assay, Mass Spectrometry, Functional Assay, Western Blot

    Phosphorylation status of the Human P2X1 receptor. (a) P2X1 receptor protein sequence of the N- and C-termini. Intracellular modifications made by membrane permeable Dithio bis (sulfosuccinimidylpropionate) (DSP) were identified by mass spectrometry and are indicated on the protein sequence. Membrane impermeable 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modifications were only observed on extracellular P2X1 protein residues. DSP modification highlights the lipid/transmembrane boundaries with K27 and K28 modified showing accessibility. In silico analysis of the N-terminal protein sequence reveals residues Y16 and T18 as potential phosphorylation sites (bold type). Mass spectrometry analysis did not show modification at these residues even after enriching for phosphorylated peptides. In silico analysis of the C-terminal protein sequence reveals tyrosine residues Y362, 363 and 370 and serine and threonine residues 386–389 and 398–399 as potential phosphorylation sites (bold type). Phosphorylation was initially observed at residues S387 and S388 and residues S388, T389 and S399 were identified on mass spectrometry runs enriched for phosphorylated peptides (P *). These residues were the only phosphorylated residues observed on mass spectrometry of the P2X1 receptor protein. (b) ATP evoked currents (period indicated by bar) from P2X1 wildtype and mutant receptors (S387A and S388A – SS-AA, S387A, S388A and T389A – SST-AAA). Traces show reproducible response evoked at a 5-minute interval and level of recovery from desensitization at 60 s between ATP applications. (c, d) Time to peak (10–90%) and decay time (100–50%) of currents evoked by 100 μM ATP for wildtype and mutant P2X1 receptors. (e) Recovery from desensitization at 60 s for wildtype and mutant P2X1 receptors. * p

    Journal: Journal of Neurochemistry

    Article Title: Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

    doi: 10.1111/jnc.12012

    Figure Lengend Snippet: Phosphorylation status of the Human P2X1 receptor. (a) P2X1 receptor protein sequence of the N- and C-termini. Intracellular modifications made by membrane permeable Dithio bis (sulfosuccinimidylpropionate) (DSP) were identified by mass spectrometry and are indicated on the protein sequence. Membrane impermeable 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modifications were only observed on extracellular P2X1 protein residues. DSP modification highlights the lipid/transmembrane boundaries with K27 and K28 modified showing accessibility. In silico analysis of the N-terminal protein sequence reveals residues Y16 and T18 as potential phosphorylation sites (bold type). Mass spectrometry analysis did not show modification at these residues even after enriching for phosphorylated peptides. In silico analysis of the C-terminal protein sequence reveals tyrosine residues Y362, 363 and 370 and serine and threonine residues 386–389 and 398–399 as potential phosphorylation sites (bold type). Phosphorylation was initially observed at residues S387 and S388 and residues S388, T389 and S399 were identified on mass spectrometry runs enriched for phosphorylated peptides (P *). These residues were the only phosphorylated residues observed on mass spectrometry of the P2X1 receptor protein. (b) ATP evoked currents (period indicated by bar) from P2X1 wildtype and mutant receptors (S387A and S388A – SS-AA, S387A, S388A and T389A – SST-AAA). Traces show reproducible response evoked at a 5-minute interval and level of recovery from desensitization at 60 s between ATP applications. (c, d) Time to peak (10–90%) and decay time (100–50%) of currents evoked by 100 μM ATP for wildtype and mutant P2X1 receptors. (e) Recovery from desensitization at 60 s for wildtype and mutant P2X1 receptors. * p

    Article Snippet: Proteins were transferred to nitrocellulose, probed with P2X1 primary antibody (1 : 1000; Alomone Labs) followed by secondary goat anti-rabbit antibody (A6154, 1 : 3500 dilution; Sigma).

    Techniques: Sequencing, Mass Spectrometry, Modification, In Silico, Mutagenesis

    The response to ET-1 is enhanced in BALB-STD mice. A: Vasoconstriction in small pulmonary artery profiles in BALB-STD mice is significantly greater over the range of 10 −10 to 10 −9 ET-1. B: This coincides with a significant increase in the ET A R in BALB-STD mice total lung tissue as indicated by quantification of Western blot analysis. Data are expressed as means ± SEM. n = 4 to 5 ( B ) or n = 16 to 35 profiles per group across 4 to 5 mice ( A ) in two independent experiments. ∗ P ≤ 0.05 versus IFN-γ-STD and controls. CON, control; ET A R, endothelin receptor A; ET-1, endothelin-1; IFN, interferon; STD, short-term depletion.

    Journal: The American Journal of Pathology

    Article Title: Vascular Dysfunction in Pneumocystis-Associated Pulmonary Hypertension Is Related to Endothelin Response and Adrenomedullin Concentration

    doi: 10.1016/j.ajpath.2015.10.008

    Figure Lengend Snippet: The response to ET-1 is enhanced in BALB-STD mice. A: Vasoconstriction in small pulmonary artery profiles in BALB-STD mice is significantly greater over the range of 10 −10 to 10 −9 ET-1. B: This coincides with a significant increase in the ET A R in BALB-STD mice total lung tissue as indicated by quantification of Western blot analysis. Data are expressed as means ± SEM. n = 4 to 5 ( B ) or n = 16 to 35 profiles per group across 4 to 5 mice ( A ) in two independent experiments. ∗ P ≤ 0.05 versus IFN-γ-STD and controls. CON, control; ET A R, endothelin receptor A; ET-1, endothelin-1; IFN, interferon; STD, short-term depletion.

    Article Snippet: Polyclonal anti-endothelin receptor A (ETA R) and the control peptide to verify specificity were from Alomone Labs (no. AER-001; Jerusalem, Israel).

    Techniques: Mouse Assay, Western Blot

    Small pulmonary artery profiles show increasing vasoconstriction when treated with a cumulative dose response of ET-1. Shown is a representative profile from a BALB-STD mouse precision cut lung slices sample. ET-1, endothelin-1; STD, short-term depletion.

    Journal: The American Journal of Pathology

    Article Title: Vascular Dysfunction in Pneumocystis-Associated Pulmonary Hypertension Is Related to Endothelin Response and Adrenomedullin Concentration

    doi: 10.1016/j.ajpath.2015.10.008

    Figure Lengend Snippet: Small pulmonary artery profiles show increasing vasoconstriction when treated with a cumulative dose response of ET-1. Shown is a representative profile from a BALB-STD mouse precision cut lung slices sample. ET-1, endothelin-1; STD, short-term depletion.

    Article Snippet: Polyclonal anti-endothelin receptor A (ETA R) and the control peptide to verify specificity were from Alomone Labs (no. AER-001; Jerusalem, Israel).

    Techniques:

    ET-1 concentrations are not significantly different in BALB-STD or IFN-γ-STD mice compared with controls. Samples are peptide extracts from total lung homogenates. Data are expressed as means ± SEM. n = 5 mice in two independent experiments. CON, control; ET-1, endothelin-1; IFN, interferon; STD, short-term depletion.

    Journal: The American Journal of Pathology

    Article Title: Vascular Dysfunction in Pneumocystis-Associated Pulmonary Hypertension Is Related to Endothelin Response and Adrenomedullin Concentration

    doi: 10.1016/j.ajpath.2015.10.008

    Figure Lengend Snippet: ET-1 concentrations are not significantly different in BALB-STD or IFN-γ-STD mice compared with controls. Samples are peptide extracts from total lung homogenates. Data are expressed as means ± SEM. n = 5 mice in two independent experiments. CON, control; ET-1, endothelin-1; IFN, interferon; STD, short-term depletion.

    Article Snippet: Polyclonal anti-endothelin receptor A (ETA R) and the control peptide to verify specificity were from Alomone Labs (no. AER-001; Jerusalem, Israel).

    Techniques: Mouse Assay