ryanodine  (Abcam)

 
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    Name:
    Ryanodine
    Description:

    Catalog Number:
    ab120083
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    Structured Review

    Abcam ryanodine
    Comparison of increases in [Ca 2+ ] cyt due to inward Ca 2+ transportation through Na + /Ca 2+ exchanger (NCX) in CASMC and PASMC. A : representative records showing different patterns of [Ca 2+ ] cyt changes ( patterns 1 and 3 ) in CASMC and PASMC by removal of extracellular Na + (0Na). B : summarized data (means ± SE) showing the amplitude ( left ) and AUC ( middle ) of increases in [Ca 2+ ] cyt and frequency ( right ) of Na + -free medium-induced [Ca 2+ ] cyt oscillations in CASMC and PASMC. C : summarized data showing the percentage of cells exhibiting different patterns of [Ca 2+ ] cyt changes induced by removal of extracellular Na + in CASMC and PASMC. D : summarized data (means ± SE) showing the percentage of responsive cells in CASMC ( n = 212 cells from 8 coverslips) and PASMC ( n = 179 cells from 8 coverslips). E : representative records showing different patterns of [Ca 2+ ] cyt changes induced by removal of extracellular Na + in the presence of 50 μM <t>ryanodine</t> (an RyR inhibitor) in CASMC and PASMC. F : summarized data (means ± SE) showing the amplitude ( left ) and AUC ( middle ) of increases in [Ca 2+ ] cyt and the frequency ( right ) of Na + -free medium-induced [Ca 2+ ] cyt oscillations in CASMC and PASMC. G : summarized data showing the percentage of cells exhibiting a different pattern of [Ca 2+ ] cyt changes induced by removal of extracellular Na + in the presence of 50 μM ryanodine in CASMC and PASMC. H : summarized data (means ± SE) showing the percentage of responsive cells in CASMC ( n = 385 cells from 6 coverslips) and PASMC ( n = 379 cells from 6 coverslips). *** P

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    ryanodine - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Hypoxia selectively upregulates cation channels and increases cytosolic [Ca2+] in pulmonary, but not coronary, arterial smooth muscle cells"

    Article Title: Hypoxia selectively upregulates cation channels and increases cytosolic [Ca2+] in pulmonary, but not coronary, arterial smooth muscle cells

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00272.2017

    Comparison of increases in [Ca 2+ ] cyt due to inward Ca 2+ transportation through Na + /Ca 2+ exchanger (NCX) in CASMC and PASMC. A : representative records showing different patterns of [Ca 2+ ] cyt changes ( patterns 1 and 3 ) in CASMC and PASMC by removal of extracellular Na + (0Na). B : summarized data (means ± SE) showing the amplitude ( left ) and AUC ( middle ) of increases in [Ca 2+ ] cyt and frequency ( right ) of Na + -free medium-induced [Ca 2+ ] cyt oscillations in CASMC and PASMC. C : summarized data showing the percentage of cells exhibiting different patterns of [Ca 2+ ] cyt changes induced by removal of extracellular Na + in CASMC and PASMC. D : summarized data (means ± SE) showing the percentage of responsive cells in CASMC ( n = 212 cells from 8 coverslips) and PASMC ( n = 179 cells from 8 coverslips). E : representative records showing different patterns of [Ca 2+ ] cyt changes induced by removal of extracellular Na + in the presence of 50 μM ryanodine (an RyR inhibitor) in CASMC and PASMC. F : summarized data (means ± SE) showing the amplitude ( left ) and AUC ( middle ) of increases in [Ca 2+ ] cyt and the frequency ( right ) of Na + -free medium-induced [Ca 2+ ] cyt oscillations in CASMC and PASMC. G : summarized data showing the percentage of cells exhibiting a different pattern of [Ca 2+ ] cyt changes induced by removal of extracellular Na + in the presence of 50 μM ryanodine in CASMC and PASMC. H : summarized data (means ± SE) showing the percentage of responsive cells in CASMC ( n = 385 cells from 6 coverslips) and PASMC ( n = 379 cells from 6 coverslips). *** P
    Figure Legend Snippet: Comparison of increases in [Ca 2+ ] cyt due to inward Ca 2+ transportation through Na + /Ca 2+ exchanger (NCX) in CASMC and PASMC. A : representative records showing different patterns of [Ca 2+ ] cyt changes ( patterns 1 and 3 ) in CASMC and PASMC by removal of extracellular Na + (0Na). B : summarized data (means ± SE) showing the amplitude ( left ) and AUC ( middle ) of increases in [Ca 2+ ] cyt and frequency ( right ) of Na + -free medium-induced [Ca 2+ ] cyt oscillations in CASMC and PASMC. C : summarized data showing the percentage of cells exhibiting different patterns of [Ca 2+ ] cyt changes induced by removal of extracellular Na + in CASMC and PASMC. D : summarized data (means ± SE) showing the percentage of responsive cells in CASMC ( n = 212 cells from 8 coverslips) and PASMC ( n = 179 cells from 8 coverslips). E : representative records showing different patterns of [Ca 2+ ] cyt changes induced by removal of extracellular Na + in the presence of 50 μM ryanodine (an RyR inhibitor) in CASMC and PASMC. F : summarized data (means ± SE) showing the amplitude ( left ) and AUC ( middle ) of increases in [Ca 2+ ] cyt and the frequency ( right ) of Na + -free medium-induced [Ca 2+ ] cyt oscillations in CASMC and PASMC. G : summarized data showing the percentage of cells exhibiting a different pattern of [Ca 2+ ] cyt changes induced by removal of extracellular Na + in the presence of 50 μM ryanodine in CASMC and PASMC. H : summarized data (means ± SE) showing the percentage of responsive cells in CASMC ( n = 385 cells from 6 coverslips) and PASMC ( n = 379 cells from 6 coverslips). *** P

    Techniques Used:

    2) Product Images from "The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes"

    Article Title: The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes

    Journal: eLife

    doi: 10.7554/eLife.15887

    The ER Ca 2+ store regulates lysosome Ca 2+ stores. ( A ) In un-transfected HEK293T cells, ATP induced Ca 2+ release through IP3-receptors on the ER, and GPN induced lysosomal Ca 2+ release. ( B ) A 2-min application of TPEN, a membrane-permeable chelator of luminal ER Ca 2+ , attenuated Ca 2+ release from IP3-receptors stimulated by ATP in HEK293T cells. ( C ) A 2-min TPEN application did not significantly reduce GPN-induced lysosomal Ca 2+ release in HEK293T cells. ( D ) Long-term TPEN treatment (20 min) abolished ER Ca 2+ release upon ATP stimulation and GPN-induced lysosomal Ca 2+ release in HEK293T cells loaded with Fura-2. ( E ) In HEK293T cells transfected with the IP3R-ligand binding domain with ER targeting sequence (IP3R-LBD-ER), the responses to ATP and GPN were reduced compared to un-transfected cells on the same coverslip. ( F ) Caffeine stimulates Ca 2+ release from ryanodine receptors and ATP stimulates Ca 2+ release from IP3Rs in HEK-GCaMP3-ML1 cells loaded with Fura-2. Panels A – C and E show the average response of 30–40 cells from one representative experiment. DOI: http://dx.doi.org/10.7554/eLife.15887.011
    Figure Legend Snippet: The ER Ca 2+ store regulates lysosome Ca 2+ stores. ( A ) In un-transfected HEK293T cells, ATP induced Ca 2+ release through IP3-receptors on the ER, and GPN induced lysosomal Ca 2+ release. ( B ) A 2-min application of TPEN, a membrane-permeable chelator of luminal ER Ca 2+ , attenuated Ca 2+ release from IP3-receptors stimulated by ATP in HEK293T cells. ( C ) A 2-min TPEN application did not significantly reduce GPN-induced lysosomal Ca 2+ release in HEK293T cells. ( D ) Long-term TPEN treatment (20 min) abolished ER Ca 2+ release upon ATP stimulation and GPN-induced lysosomal Ca 2+ release in HEK293T cells loaded with Fura-2. ( E ) In HEK293T cells transfected with the IP3R-ligand binding domain with ER targeting sequence (IP3R-LBD-ER), the responses to ATP and GPN were reduced compared to un-transfected cells on the same coverslip. ( F ) Caffeine stimulates Ca 2+ release from ryanodine receptors and ATP stimulates Ca 2+ release from IP3Rs in HEK-GCaMP3-ML1 cells loaded with Fura-2. Panels A – C and E show the average response of 30–40 cells from one representative experiment. DOI: http://dx.doi.org/10.7554/eLife.15887.011

    Techniques Used: Transfection, Ligand Binding Assay, Sequencing

    Lysosomal Ca2+ refilling is compromised in IP3R TKO DT40 cells. ( A ) Ryanodine receptor blocker DHBP (50 μM) did not block Ca 2+ refilling of lysosomes. ( B ) Quantification of the 1st, 2nd and 3rd ML-SA1 responses in GCaMP3-ML1-transfected WT and IP3R-TKO DT40 cells ( Figure 3 — source data 1 ). ( C ) Time-dependence of lysosomal Ca 2+ store refilling in WT and IP3R TKO DT40 cells. ( D ) GCaMP3-ML1-transfected IP3R-TKO DT40 cells still showed refilling after 5 min of DHBP application to block RYRs. ( E ) RYR inhibitors Diltiazem (50 µM) and Dantrolene (50 µM) did not block lysosomal Ca 2+ refilling in GCaMP3-ML1-transfected IP3R-TKO DT40 cells. Panels A , D and E show the average response of 30–40 cells from one representative experiment. DOI: http://dx.doi.org/10.7554/eLife.15887.015
    Figure Legend Snippet: Lysosomal Ca2+ refilling is compromised in IP3R TKO DT40 cells. ( A ) Ryanodine receptor blocker DHBP (50 μM) did not block Ca 2+ refilling of lysosomes. ( B ) Quantification of the 1st, 2nd and 3rd ML-SA1 responses in GCaMP3-ML1-transfected WT and IP3R-TKO DT40 cells ( Figure 3 — source data 1 ). ( C ) Time-dependence of lysosomal Ca 2+ store refilling in WT and IP3R TKO DT40 cells. ( D ) GCaMP3-ML1-transfected IP3R-TKO DT40 cells still showed refilling after 5 min of DHBP application to block RYRs. ( E ) RYR inhibitors Diltiazem (50 µM) and Dantrolene (50 µM) did not block lysosomal Ca 2+ refilling in GCaMP3-ML1-transfected IP3R-TKO DT40 cells. Panels A , D and E show the average response of 30–40 cells from one representative experiment. DOI: http://dx.doi.org/10.7554/eLife.15887.015

    Techniques Used: Blocking Assay, Transfection

    Blocking ER IP3-receptors Ca 2+ channels refill lysosome Ca 2+ stores to prevent lysosomal dysfunction. ( A ) Upper panels: Western blotting analyses of Lamp1 in HEK293T cells treated with 2-APB (50 μM), TPEN (0.1 μM), Xesto (10 μM), and DHBP (5 μM) compared to DMSO for 24 hr (n=4 separate experiments for each condition). Lower panel: treating HEK293T cells with 2-APB (p=0.05) and Xesto (p=0.013), as well as TPEN (p=0.02), significantly increased Lamp1 expression. DHBP did not significantly change Lamp1 expression (p=0.23) ( Figure 4 — source data 1 ). ( B ) The effects of Xesto (10 μM, 18 hr; p=0.0001) and DHBP (50 μM, 18 hr; p=0.063) treatment compared to DMSO on the lysosomal compartments detected by LysoTracker staining in HEK293T cells (average of 20–30 cells in each of 3 experiments; Figure 4 — source data 1 ). Scale bar = 15 μm. ( C ) The effect of Xesto (10 μM, 18 h) treatment on accumulation of the autofluorescent lipofuscin materials in non-transfected HEK293T cells. Autofluorescence was observed in a wide spectrum but shown at two excitation wavelengths (488 and 561 nm). ML1 KO MEFs are shown for comparison. Scale bar = 15 μm. ( D ) A proposed model of Ca 2+ transfer from the ER to lysosomes. The ER is a Ca 2+ store with [Ca 2+ ] ER ~ 0.3–0.7 mM; lysosomes are acidic (pH Ly ~ 4.6) Ca 2+ stores ([Ca 2+ ] Ly ~ 0.5 mM). IP3Rs on the ER release Ca 2+ to produce a local high Ca 2+ concentration, from which an unknown low-affinity Ca 2+ transport mechanism refills Ca 2+ to a lysosome. Unidentified tether proteins may link the ER membrane proteins directly with the lysosomal membrane proteins to maintain contact sites of 20–30 nm for purposes of Ca 2+ exchange. Ca 2+ released from lysosomes or a reduction/depletion in [Ca 2+ ] Ly may, through unidentified mechanisms, 'promote' or 'stabilize' ER-lysosome interaction ( Phillips and Voeltz, 2016 ; Eden, 2016 ). At the functional ER-lysosome contact sites, Ca 2+ can be transferred from the ER to lysosomes through a passive Ca 2+ transporter or channel based on the large chemical gradient of Ca 2+ that is created when lysosome stores are depleted. Baf-A and Con-A are specific V-ATPase inhibitors; Xesto and 2APB are IP3R blockers; U73122 is a PLC inhibitor that blocks the constitutive production of IP3; DHBP and Ryanodine ( > 10 μM) are specific RyR blockers; TG and CPA are SERCA pump inhibitors; and TPEN is a luminal Ca 2+ chelator. DOI: http://dx.doi.org/10.7554/eLife.15887.017 10.7554/eLife.15887.018 Source data of Figure 4A,B : Quantifications of Lamp-1 protein levels ( A ) or LysoTracker staining ( B ) under different experimental conditions and manipulations. DOI: http://dx.doi.org/10.7554/eLife.15887.018
    Figure Legend Snippet: Blocking ER IP3-receptors Ca 2+ channels refill lysosome Ca 2+ stores to prevent lysosomal dysfunction. ( A ) Upper panels: Western blotting analyses of Lamp1 in HEK293T cells treated with 2-APB (50 μM), TPEN (0.1 μM), Xesto (10 μM), and DHBP (5 μM) compared to DMSO for 24 hr (n=4 separate experiments for each condition). Lower panel: treating HEK293T cells with 2-APB (p=0.05) and Xesto (p=0.013), as well as TPEN (p=0.02), significantly increased Lamp1 expression. DHBP did not significantly change Lamp1 expression (p=0.23) ( Figure 4 — source data 1 ). ( B ) The effects of Xesto (10 μM, 18 hr; p=0.0001) and DHBP (50 μM, 18 hr; p=0.063) treatment compared to DMSO on the lysosomal compartments detected by LysoTracker staining in HEK293T cells (average of 20–30 cells in each of 3 experiments; Figure 4 — source data 1 ). Scale bar = 15 μm. ( C ) The effect of Xesto (10 μM, 18 h) treatment on accumulation of the autofluorescent lipofuscin materials in non-transfected HEK293T cells. Autofluorescence was observed in a wide spectrum but shown at two excitation wavelengths (488 and 561 nm). ML1 KO MEFs are shown for comparison. Scale bar = 15 μm. ( D ) A proposed model of Ca 2+ transfer from the ER to lysosomes. The ER is a Ca 2+ store with [Ca 2+ ] ER ~ 0.3–0.7 mM; lysosomes are acidic (pH Ly ~ 4.6) Ca 2+ stores ([Ca 2+ ] Ly ~ 0.5 mM). IP3Rs on the ER release Ca 2+ to produce a local high Ca 2+ concentration, from which an unknown low-affinity Ca 2+ transport mechanism refills Ca 2+ to a lysosome. Unidentified tether proteins may link the ER membrane proteins directly with the lysosomal membrane proteins to maintain contact sites of 20–30 nm for purposes of Ca 2+ exchange. Ca 2+ released from lysosomes or a reduction/depletion in [Ca 2+ ] Ly may, through unidentified mechanisms, 'promote' or 'stabilize' ER-lysosome interaction ( Phillips and Voeltz, 2016 ; Eden, 2016 ). At the functional ER-lysosome contact sites, Ca 2+ can be transferred from the ER to lysosomes through a passive Ca 2+ transporter or channel based on the large chemical gradient of Ca 2+ that is created when lysosome stores are depleted. Baf-A and Con-A are specific V-ATPase inhibitors; Xesto and 2APB are IP3R blockers; U73122 is a PLC inhibitor that blocks the constitutive production of IP3; DHBP and Ryanodine ( > 10 μM) are specific RyR blockers; TG and CPA are SERCA pump inhibitors; and TPEN is a luminal Ca 2+ chelator. DOI: http://dx.doi.org/10.7554/eLife.15887.017 10.7554/eLife.15887.018 Source data of Figure 4A,B : Quantifications of Lamp-1 protein levels ( A ) or LysoTracker staining ( B ) under different experimental conditions and manipulations. DOI: http://dx.doi.org/10.7554/eLife.15887.018

    Techniques Used: Blocking Assay, Western Blot, Expressing, Staining, Transfection, Concentration Assay, Functional Assay, Planar Chromatography

    3) Product Images from "Insights into the Gating Mechanism of the Ryanodine-Modified Human Cardiac Ca2+-Release Channel (Ryanodine Receptor 2)"

    Article Title: Insights into the Gating Mechanism of the Ryanodine-Modified Human Cardiac Ca2+-Release Channel (Ryanodine Receptor 2)

    Journal: Molecular pharmacology

    doi: 10.1124/mol.114.093757

    Cytosolic Ba 2+ inhibition of ryanodine-modified channel Po also abolishes voltage dependence.
    Figure Legend Snippet: Cytosolic Ba 2+ inhibition of ryanodine-modified channel Po also abolishes voltage dependence.

    Techniques Used: Inhibition, Modification

    Voltage dependence of ryanodine-modified RyR2 at zero Ca 2+ .
    Figure Legend Snippet: Voltage dependence of ryanodine-modified RyR2 at zero Ca 2+ .

    Techniques Used: Modification

    Kinetic parameters of ryanodine-modified RyR2 gating (A–D) are represented by black bars (1 μ M Ca 2+ ) and gray bars (nominally zero Ca 2+ ).
    Figure Legend Snippet: Kinetic parameters of ryanodine-modified RyR2 gating (A–D) are represented by black bars (1 μ M Ca 2+ ) and gray bars (nominally zero Ca 2+ ).

    Techniques Used: Modification

    Detailed examination of ryanodine-modified RyR2 gating in the absence of activating Ca 2+ at various holding potentials reveals steep voltage dependence.
    Figure Legend Snippet: Detailed examination of ryanodine-modified RyR2 gating in the absence of activating Ca 2+ at various holding potentials reveals steep voltage dependence.

    Techniques Used: Modification

    4) Product Images from "Role of transient receptor potential vanilloid 1 in the modulation of airway smooth muscle tone and calcium handling"

    Article Title: Role of transient receptor potential vanilloid 1 in the modulation of airway smooth muscle tone and calcium handling

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00064.2017

    Absence of capsazepine effect on store-operated calcium entry (SOCE) in ASM cells in mouse PCLS. Mouse peripheral lung slices loaded with an intracellular calcium indicator dye were exposed to caffeine (20 mM) and ryanodine (25 μM) simultaneously to “lock” the ryanodine receptor of sarcoplasmic reticulum (SR) in the open state, to irreversibly deplete the SR of calcium, and to induce SOCE in the presence of zero-calcium external buffer. The slices were then reexposed to 1.3 mM Ca 2+ extracellular buffer, and the resultant increase in ASM calcium-mediated fluorescence was considered a measure of SOCE. GSK 7575A, 100 µM, a SOCE inhibitor, significantly inhibited SOCE in this assay; however, 100 µM capsazepine did not. This suggests SOCE inhibition is not the mechanism by which capsazepine inhibits calcium oscillations in ASM. A : combined Ca 2+ -mediated fluorescent tracings. B : quantification of SOCE-mediate calcium influx. Values are means ± SE; n = 3. *** P
    Figure Legend Snippet: Absence of capsazepine effect on store-operated calcium entry (SOCE) in ASM cells in mouse PCLS. Mouse peripheral lung slices loaded with an intracellular calcium indicator dye were exposed to caffeine (20 mM) and ryanodine (25 μM) simultaneously to “lock” the ryanodine receptor of sarcoplasmic reticulum (SR) in the open state, to irreversibly deplete the SR of calcium, and to induce SOCE in the presence of zero-calcium external buffer. The slices were then reexposed to 1.3 mM Ca 2+ extracellular buffer, and the resultant increase in ASM calcium-mediated fluorescence was considered a measure of SOCE. GSK 7575A, 100 µM, a SOCE inhibitor, significantly inhibited SOCE in this assay; however, 100 µM capsazepine did not. This suggests SOCE inhibition is not the mechanism by which capsazepine inhibits calcium oscillations in ASM. A : combined Ca 2+ -mediated fluorescent tracings. B : quantification of SOCE-mediate calcium influx. Values are means ± SE; n = 3. *** P

    Techniques Used: Fluorescence, Inhibition

    5) Product Images from "Ca2+ oscillations, Ca2+ sensitization, and contraction activated by protein kinase C in small airway smooth muscle"

    Article Title: Ca2+ oscillations, Ca2+ sensitization, and contraction activated by protein kinase C in small airway smooth muscle

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201210876

    Phosphorylation of CPI-17 and rMLC induced by PMA and inhibited by GF-109203X. Separation of (A) phosphorylated (p-CPI-17) and unphosphorylated (u-CPI-17) CPI-17 and (B and C) mono-phosphorylated (1p-rMLC), bi-phosphorylated (2p-rMLC), and unphosphorylated (u-rMLC) rMLC by SDS-PAGE with polyacrylamide-bound phosphate-binding tag (Phos-tag SDS-PAGE) and detected by specific antibodies using Western blot. The Western blots in B and C were obtained after stripping previous antibodies and reprobing the membrane in A with antibodies directed against total rMLC and subsequently against phosphorylated rMLC at serine 19 (p-ser19-rMLC), respectively. Samples were prepared from selected lung slices that contained airways but not arteries and were incubated with 20 mM caffeine plus 25 µM ryanodine for 5 min, washed with sHBSS, and then incubated for 40 min with sHBSS (CTRL), 10 µM PMA, or PMA followed by 10-min exposure to 1 µM GF-109203X (upper labels). The antibody against CPI-17 detected two CPI-17 splice variants (arrow pairs) along with a nonspecific (nsp) band, as indicated by the manufacturer. Parallel samples were separated in an SDS-PAGE without Phos-tag to identify the phosphorylated/unphosphorylated CPI-17 and rMLC by verifying that each of these forms migrated in the same position in the gel without Phos-tag. SMC α actin was detected in all samples. (D) Ratio values (mean ± SEM) of p-CPI-17 to total CPI-17 and 1p+2p-rMLC to total rMLC from three experiments similar to that presented in A and B from three mice are shown.
    Figure Legend Snippet: Phosphorylation of CPI-17 and rMLC induced by PMA and inhibited by GF-109203X. Separation of (A) phosphorylated (p-CPI-17) and unphosphorylated (u-CPI-17) CPI-17 and (B and C) mono-phosphorylated (1p-rMLC), bi-phosphorylated (2p-rMLC), and unphosphorylated (u-rMLC) rMLC by SDS-PAGE with polyacrylamide-bound phosphate-binding tag (Phos-tag SDS-PAGE) and detected by specific antibodies using Western blot. The Western blots in B and C were obtained after stripping previous antibodies and reprobing the membrane in A with antibodies directed against total rMLC and subsequently against phosphorylated rMLC at serine 19 (p-ser19-rMLC), respectively. Samples were prepared from selected lung slices that contained airways but not arteries and were incubated with 20 mM caffeine plus 25 µM ryanodine for 5 min, washed with sHBSS, and then incubated for 40 min with sHBSS (CTRL), 10 µM PMA, or PMA followed by 10-min exposure to 1 µM GF-109203X (upper labels). The antibody against CPI-17 detected two CPI-17 splice variants (arrow pairs) along with a nonspecific (nsp) band, as indicated by the manufacturer. Parallel samples were separated in an SDS-PAGE without Phos-tag to identify the phosphorylated/unphosphorylated CPI-17 and rMLC by verifying that each of these forms migrated in the same position in the gel without Phos-tag. SMC α actin was detected in all samples. (D) Ratio values (mean ± SEM) of p-CPI-17 to total CPI-17 and 1p+2p-rMLC to total rMLC from three experiments similar to that presented in A and B from three mice are shown.

    Techniques Used: SDS Page, Binding Assay, Western Blot, Stripping Membranes, Incubation, Mouse Assay

    Effect of PKC inhibitor GF-109203X on Ca 2+ sensitization induced by PMA or contractile agonists. Lung slices were first exposed to 20 mM caffeine plus 25 µM ryanodine (upper bars) to induce Ca 2+ permeabilization. (A) Representative experiment showing the effect of airway exposure to 1 µM GF-109203X before and during stimulation with 10 µM PMA. Contractile response of the airways after Ca 2+ permeabilization was accessed with 0.5 µM 5-HT before their exposure to GF-109203X and PMA. This experiment is representative of six lung slices from three mice. (B and C) Airway contraction induced by PMA or 0.1 U/ml thrombin and the subsequent relaxation induced by GF-109203X. (D) Summary of the effect of GF-109203X on airway contraction in Ca 2+ -permeabilized airways precontracted with 10 µM PMA, 0.25 µM ACh, 0.5 µM 5-HT, and 0.1 U/ml thrombin. Airway relaxation (mean ± SEM; n = 5 lung slices from 3 mice) was obtained from experiments similar to that shown in B and C using the appropriated contractile agonist or PMA.
    Figure Legend Snippet: Effect of PKC inhibitor GF-109203X on Ca 2+ sensitization induced by PMA or contractile agonists. Lung slices were first exposed to 20 mM caffeine plus 25 µM ryanodine (upper bars) to induce Ca 2+ permeabilization. (A) Representative experiment showing the effect of airway exposure to 1 µM GF-109203X before and during stimulation with 10 µM PMA. Contractile response of the airways after Ca 2+ permeabilization was accessed with 0.5 µM 5-HT before their exposure to GF-109203X and PMA. This experiment is representative of six lung slices from three mice. (B and C) Airway contraction induced by PMA or 0.1 U/ml thrombin and the subsequent relaxation induced by GF-109203X. (D) Summary of the effect of GF-109203X on airway contraction in Ca 2+ -permeabilized airways precontracted with 10 µM PMA, 0.25 µM ACh, 0.5 µM 5-HT, and 0.1 U/ml thrombin. Airway relaxation (mean ± SEM; n = 5 lung slices from 3 mice) was obtained from experiments similar to that shown in B and C using the appropriated contractile agonist or PMA.

    Techniques Used: Mouse Assay

    Effect of extracellular Ca 2+ removal, nifedipine, CPA, and ryanodine on PMA-induced airway SMC twitching. Line scans from airway wall regions obtained from phase-contrast images (similar to that presented in Fig. 1 ) showing airway SMC twitching induced by 10 µM PMA (added 30 min before the recordings started) and their sensitivity to: (A) Ca 2+ -free sHBSS, (B) 10 µM nifedipine, (C) 10 µM CPA, or (D) 25 µM ryanodine. PMA-induced SMC twitching was inhibited by removal of extracellular Ca 2+ , nifedipine, CPA, and ryanodine. Each line scan is representative of three experiments in lung slices from two mice.
    Figure Legend Snippet: Effect of extracellular Ca 2+ removal, nifedipine, CPA, and ryanodine on PMA-induced airway SMC twitching. Line scans from airway wall regions obtained from phase-contrast images (similar to that presented in Fig. 1 ) showing airway SMC twitching induced by 10 µM PMA (added 30 min before the recordings started) and their sensitivity to: (A) Ca 2+ -free sHBSS, (B) 10 µM nifedipine, (C) 10 µM CPA, or (D) 25 µM ryanodine. PMA-induced SMC twitching was inhibited by removal of extracellular Ca 2+ , nifedipine, CPA, and ryanodine. Each line scan is representative of three experiments in lung slices from two mice.

    Techniques Used: Mouse Assay

    Effect of extracellular Ca 2+ removal, nifedipine, CPA, and ryanodine on PMA-induced Ca 2+ oscillations. Ca 2+ oscillations in single airway SMCs induced by 10 µM PMA (added 30 min before the recording started) and its inhibition by: (A) superfusion with Ca 2+ -free sHBSS, (B) 10 µM nifedipine, (C) 10 µM CPA, or (D) 25 µM ryanodine. Each trace is representative of four experiments in lung slices from three mice.
    Figure Legend Snippet: Effect of extracellular Ca 2+ removal, nifedipine, CPA, and ryanodine on PMA-induced Ca 2+ oscillations. Ca 2+ oscillations in single airway SMCs induced by 10 µM PMA (added 30 min before the recording started) and its inhibition by: (A) superfusion with Ca 2+ -free sHBSS, (B) 10 µM nifedipine, (C) 10 µM CPA, or (D) 25 µM ryanodine. Each trace is representative of four experiments in lung slices from three mice.

    Techniques Used: Inhibition, Mouse Assay

    Ca 2+ sensitization induced by agonists and phorbol esters. Ca 2+ sensitization of airway SMCs studied in Ca 2+ -permeabilized lung slices with 20 mM caffeine plus 25 µM ryanodine. (A) Representative phase-contrast images of a Ca 2+ -permeabilized lung slice showing an airway before (1) and after stimulation with 5-HT (2) or PMA (3) at the times indicated by the corresponding numbers in B. (B) Traces from two similar experiments showing the changes in airway lumen area during perfusion of caffeine plus ryanodine, 0.5 µM 5-HT, and 10 µM PMA (black trace) or 1 µM PDBu (gray trace) at the times indicated by the upper lines. (C) Summary of airway contractions induced by 5-HT, 50 mM of isosmotic KCl, PMA, or PDBu (mean ± SEM; eight airways from slices from four mice) in Ca 2+ -permeabilized lung slices obtained from experiments similar to that shown in B. *, different from baseline airway lumen area (P
    Figure Legend Snippet: Ca 2+ sensitization induced by agonists and phorbol esters. Ca 2+ sensitization of airway SMCs studied in Ca 2+ -permeabilized lung slices with 20 mM caffeine plus 25 µM ryanodine. (A) Representative phase-contrast images of a Ca 2+ -permeabilized lung slice showing an airway before (1) and after stimulation with 5-HT (2) or PMA (3) at the times indicated by the corresponding numbers in B. (B) Traces from two similar experiments showing the changes in airway lumen area during perfusion of caffeine plus ryanodine, 0.5 µM 5-HT, and 10 µM PMA (black trace) or 1 µM PDBu (gray trace) at the times indicated by the upper lines. (C) Summary of airway contractions induced by 5-HT, 50 mM of isosmotic KCl, PMA, or PDBu (mean ± SEM; eight airways from slices from four mice) in Ca 2+ -permeabilized lung slices obtained from experiments similar to that shown in B. *, different from baseline airway lumen area (P

    Techniques Used: Mouse Assay

    6) Product Images from "Rapid sensing of l-leucine by human and murine hypothalamic neurons: Neurochemical and mechanistic insights"

    Article Title: Rapid sensing of l-leucine by human and murine hypothalamic neurons: Neurochemical and mechanistic insights

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2018.01.021

    Role of calcium channels in hypothalamic leucine sensing. Traces of a typical response of neurons in culture to thapsigargin exposure (A). Trace of a leucine-activated neuron in the presence of thapsigargin (B). Percentage of leucine-activated and leucine-inhibited neurons in the presence of thapsigargin (C). Percentage of leucineactivated and inhibited neurons in the presence of Ryanodine (D) and traces of a leucine-activated (E) and a leucine-inhibited (F) neuron in the presence of ryanodine. Percentage of leucine-activated and inhibited neurons in the presence of 2-APB (G). Trace of a leucine-activated neuron in the presence of 2-APB (H). Trace of a leucine-inhibited neurons in the presence of 2-APB (I) showing the effect of thapsigargin on calcium concentration following a 5 min treatment in calcium depleted media (J). Trace showing the response to the calcium depleted media in neurons with a calcium leak (K). Percentage of leucine-activated neurons in calcium depleted conditions (L) and trace of a leucine-activated neurons in calcium depleted conditions (M). Data are means SEM. ∗: p
    Figure Legend Snippet: Role of calcium channels in hypothalamic leucine sensing. Traces of a typical response of neurons in culture to thapsigargin exposure (A). Trace of a leucine-activated neuron in the presence of thapsigargin (B). Percentage of leucine-activated and leucine-inhibited neurons in the presence of thapsigargin (C). Percentage of leucineactivated and inhibited neurons in the presence of Ryanodine (D) and traces of a leucine-activated (E) and a leucine-inhibited (F) neuron in the presence of ryanodine. Percentage of leucine-activated and inhibited neurons in the presence of 2-APB (G). Trace of a leucine-activated neuron in the presence of 2-APB (H). Trace of a leucine-inhibited neurons in the presence of 2-APB (I) showing the effect of thapsigargin on calcium concentration following a 5 min treatment in calcium depleted media (J). Trace showing the response to the calcium depleted media in neurons with a calcium leak (K). Percentage of leucine-activated neurons in calcium depleted conditions (L) and trace of a leucine-activated neurons in calcium depleted conditions (M). Data are means SEM. ∗: p

    Techniques Used: Concentration Assay

    7) Product Images from "Regulation of Spontaneous Contractions in Intact Rat Bladder Strips and the Effects of Hydrogen Peroxide"

    Article Title: Regulation of Spontaneous Contractions in Intact Rat Bladder Strips and the Effects of Hydrogen Peroxide

    Journal: BioMed Research International

    doi: 10.1155/2018/2925985

    Representative tracings of spontaneous contractions of intact rat bladder strips before (Phase 1) and during (Phase 2) treatment with vehicle (0.1% DMSO) or test agent [nisoldipine (100 nM), SKF96365 (10 μ M), ryanodine (10 μ M), iberiotoxin (100 nM), NS1619 (30 μ M), apamin (100 nM), NS309 (10 μ M), Y27632 (10 μ M), BIS-1 (2 μ M), and Y27632 + NS309 (each 10 μ M)] and then incubated with 0.003 g% H 2 O 2 (Phase 3).
    Figure Legend Snippet: Representative tracings of spontaneous contractions of intact rat bladder strips before (Phase 1) and during (Phase 2) treatment with vehicle (0.1% DMSO) or test agent [nisoldipine (100 nM), SKF96365 (10 μ M), ryanodine (10 μ M), iberiotoxin (100 nM), NS1619 (30 μ M), apamin (100 nM), NS309 (10 μ M), Y27632 (10 μ M), BIS-1 (2 μ M), and Y27632 + NS309 (each 10 μ M)] and then incubated with 0.003 g% H 2 O 2 (Phase 3).

    Techniques Used: Incubation

    8) Product Images from "3,5-Diiodo-l-Thyronine Increases Glucose Consumption in Cardiomyoblasts Without Affecting the Contractile Performance in Rat Heart"

    Article Title: 3,5-Diiodo-l-Thyronine Increases Glucose Consumption in Cardiomyoblasts Without Affecting the Contractile Performance in Rat Heart

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2018.00282

    Ryanodine binding. Effect of T2 on the density of ryanodine binding sites [Bmax, fmol/mg, (A) ] and affinity for ryanodine [Kd, nM, (B) ] in crude cardiac homogenates. Ryanodine binding was determined in ventricle homogenate after 50 min of perfusion. Histograms represent mean ± SEM derived from three hearts per group. Unpaired t -test yielded P = ns for the difference between groups.
    Figure Legend Snippet: Ryanodine binding. Effect of T2 on the density of ryanodine binding sites [Bmax, fmol/mg, (A) ] and affinity for ryanodine [Kd, nM, (B) ] in crude cardiac homogenates. Ryanodine binding was determined in ventricle homogenate after 50 min of perfusion. Histograms represent mean ± SEM derived from three hearts per group. Unpaired t -test yielded P = ns for the difference between groups.

    Techniques Used: Binding Assay, Derivative Assay

    9) Product Images from "Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells"

    Article Title: Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.12239

    Histamine H 1 receptors stimulate an increase in [Ca 2+ ] i via IP 3 . (A) Histamine (100 μM, bar) stimulates an increase in [Ca 2+ ] i in populations of ASMC incubated in HBS or Ca 2+ -free HBS. Results show means ± SEM from three wells on a single 96-well plate and are typical of results from four independent plates. (B) Concentration-dependent effects of histamine on the peak [Ca 2+ ] i in the presence or absence of extracellular Ca 2+ , and on the sustained Ca 2+ entry (measured ∼230 s after histamine addition). Results are means ± SEM from four independent plates, with one to three wells on each plate. (C) Effect of mepyramine (0.5 μM) and cimetidine (50 μM), each added 5 min before histamine, on histamine-evoked Ca 2+ signals. Results are means ± SEM from nine independent plates, with one to three wells on each plate. (D) Effects of the indicated concentrations of U73122 and U73343 (added 5 min before histamine) on the peak increase in [Ca 2+ ] i evoked by histamine (100 μM). Results are means ± SEM from three independent plates, with two wells on each plate. (E) Effects of trans -Ned-19 (1 μM) and ryanodine (100 μM), each added 5 min before histamine, on the peak increase in [Ca 2+ ] i evoked by histamine. Results (percentage of the maximal response) are means ± SEM from three independent plates with one to three wells on each plate. (F) Effect of indomethacin (10 μM, added 5 min before histamine) on the peak Ca 2+ signals evoked by histamine. Results are means ± SEM from four independent plates, each with three wells. (B–F) Ct denotes control.
    Figure Legend Snippet: Histamine H 1 receptors stimulate an increase in [Ca 2+ ] i via IP 3 . (A) Histamine (100 μM, bar) stimulates an increase in [Ca 2+ ] i in populations of ASMC incubated in HBS or Ca 2+ -free HBS. Results show means ± SEM from three wells on a single 96-well plate and are typical of results from four independent plates. (B) Concentration-dependent effects of histamine on the peak [Ca 2+ ] i in the presence or absence of extracellular Ca 2+ , and on the sustained Ca 2+ entry (measured ∼230 s after histamine addition). Results are means ± SEM from four independent plates, with one to three wells on each plate. (C) Effect of mepyramine (0.5 μM) and cimetidine (50 μM), each added 5 min before histamine, on histamine-evoked Ca 2+ signals. Results are means ± SEM from nine independent plates, with one to three wells on each plate. (D) Effects of the indicated concentrations of U73122 and U73343 (added 5 min before histamine) on the peak increase in [Ca 2+ ] i evoked by histamine (100 μM). Results are means ± SEM from three independent plates, with two wells on each plate. (E) Effects of trans -Ned-19 (1 μM) and ryanodine (100 μM), each added 5 min before histamine, on the peak increase in [Ca 2+ ] i evoked by histamine. Results (percentage of the maximal response) are means ± SEM from three independent plates with one to three wells on each plate. (F) Effect of indomethacin (10 μM, added 5 min before histamine) on the peak Ca 2+ signals evoked by histamine. Results are means ± SEM from four independent plates, each with three wells. (B–F) Ct denotes control.

    Techniques Used: Incubation, Concentration Assay

    10) Product Images from "Distance-dependent gradient in NMDAR-driven spine calcium signals along tapering dendrites"

    Article Title: Distance-dependent gradient in NMDAR-driven spine calcium signals along tapering dendrites

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

    doi: 10.1073/pnas.1607462114

    mCaT distribution is unaffected by blocking AMPA receptors, ryanodine receptors, VGCCs, or GluN2B receptor subtypes. ( A–Ei ) Cumulative frequency distributions for mCaT amplitude for each spine where each plot is color matched to a different bin
    Figure Legend Snippet: mCaT distribution is unaffected by blocking AMPA receptors, ryanodine receptors, VGCCs, or GluN2B receptor subtypes. ( A–Ei ) Cumulative frequency distributions for mCaT amplitude for each spine where each plot is color matched to a different bin

    Techniques Used: Blocking Assay

    mCaT amplitude and frequency following pharmacological blockade. ( A–C ) Summary of mCaT amplitude and frequency following treatment with 10 μM NBQX ( A ) (green data), 30 μM ryanodine ( B ) (pink data), and 500 nM SNX-482 ( C ) (orange
    Figure Legend Snippet: mCaT amplitude and frequency following pharmacological blockade. ( A–C ) Summary of mCaT amplitude and frequency following treatment with 10 μM NBQX ( A ) (green data), 30 μM ryanodine ( B ) (pink data), and 500 nM SNX-482 ( C ) (orange

    Techniques Used:

    11) Product Images from "Rapid sensing of l-leucine by human and murine hypothalamic neurons: Neurochemical and mechanistic insights"

    Article Title: Rapid sensing of l-leucine by human and murine hypothalamic neurons: Neurochemical and mechanistic insights

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2018.01.021

    Role of calcium channels in hypothalamic leucine sensing. Traces of a typical response of neurons in culture to thapsigargin exposure (A). Trace of a leucine-activated neuron in the presence of thapsigargin (B). Percentage of leucine-activated and leucine-inhibited neurons in the presence of thapsigargin (C). Percentage of leucineactivated and inhibited neurons in the presence of Ryanodine (D) and traces of a leucine-activated (E) and a leucine-inhibited (F) neuron in the presence of ryanodine. Percentage of leucine-activated and inhibited neurons in the presence of 2-APB (G). Trace of a leucine-activated neuron in the presence of 2-APB (H). Trace of a leucine-inhibited neurons in the presence of 2-APB (I) showing the effect of thapsigargin on calcium concentration following a 5 min treatment in calcium depleted media (J). Trace showing the response to the calcium depleted media in neurons with a calcium leak (K). Percentage of leucine-activated neurons in calcium depleted conditions (L) and trace of a leucine-activated neurons in calcium depleted conditions (M). Data are means SEM. ∗: p
    Figure Legend Snippet: Role of calcium channels in hypothalamic leucine sensing. Traces of a typical response of neurons in culture to thapsigargin exposure (A). Trace of a leucine-activated neuron in the presence of thapsigargin (B). Percentage of leucine-activated and leucine-inhibited neurons in the presence of thapsigargin (C). Percentage of leucineactivated and inhibited neurons in the presence of Ryanodine (D) and traces of a leucine-activated (E) and a leucine-inhibited (F) neuron in the presence of ryanodine. Percentage of leucine-activated and inhibited neurons in the presence of 2-APB (G). Trace of a leucine-activated neuron in the presence of 2-APB (H). Trace of a leucine-inhibited neurons in the presence of 2-APB (I) showing the effect of thapsigargin on calcium concentration following a 5 min treatment in calcium depleted media (J). Trace showing the response to the calcium depleted media in neurons with a calcium leak (K). Percentage of leucine-activated neurons in calcium depleted conditions (L) and trace of a leucine-activated neurons in calcium depleted conditions (M). Data are means SEM. ∗: p

    Techniques Used: Concentration Assay

    12) Product Images from "Ryanodine receptors are uncoupled from contraction in rat vena cava"

    Article Title: Ryanodine receptors are uncoupled from contraction in rat vena cava

    Journal: Cell calcium

    doi: 10.1016/j.ceca.2012.10.006

    Measurement of 20 mM caffeine-induced contraction of rat aorta, in the presence of the ryanodine receptor antagonists ryanodine (10 μM) or tetracaine (100 μM). Vehicle or antagonists were incubated with tissue for 1 h prior to caffeine exposure. White bars represent vehicle-exposed aorta. Black bars represent ryanodine-exposed aorta. Gray bars represent tetracaine-exposed aorta. Bars represent mean ± SEM for the number of animals indicated in parentheses. * p
    Figure Legend Snippet: Measurement of 20 mM caffeine-induced contraction of rat aorta, in the presence of the ryanodine receptor antagonists ryanodine (10 μM) or tetracaine (100 μM). Vehicle or antagonists were incubated with tissue for 1 h prior to caffeine exposure. White bars represent vehicle-exposed aorta. Black bars represent ryanodine-exposed aorta. Gray bars represent tetracaine-exposed aorta. Bars represent mean ± SEM for the number of animals indicated in parentheses. * p

    Techniques Used: Incubation

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    Immunocytochemistry:

    Article Title: PKC? Signalling Activates ERK1/2, and Regulates Aggrecan, ADAMTS5, and miR377 Gene Expression in Human Nucleus Pulposus Cells
    Article Snippet: .. We used the following antibodies (and dilutions): polyclonal antibodies to tropomyocin (1:400) from Sigma, to phosphorylated species of PKCε (1:1,000) and myristoylated alanine-rich C-kinase substrate (MARCKS) (1:1,000), to PKCε (1:1,000), ERK1/2 (1:2,000), and to activator protein 1 (AP-1) (1:1,000) from Santa Cruz, to cAMP responsive element binding protein 1(CREB1) (1:1,000) from Millipore; mouse monoclonal antibodies to MARCKS (1:1,000) and phosho-ERK1/2 (1:2,000) from Santa Cruz, to p120GAP (1:2,000) from Upstate Biotechnology, to neo-epitope ARG on aggrecan, clone BC-3 (1:1,000 for Western blotting and 1:200 for immunocytochemistry) from ABCAM, and to β-tubulin (1:500) from Sigma; and secondary antibodies conjugated to horseradish peroxidase or fluorochromes from Santa Cruz. .. The PKCε-selective activator peptide, ψεRACK [PKCε (85-92), HDAPIGYD] [ ] was synthesized and conjugated to a peptide derived from the trans-activating transcriptional activator (TAT) (amino acids 45-57, YGRKKRRQRRR) by Peptide 2.0 Inc. PD98059, U0126, and SB203580 were from Calbiochem.

    Blocking Assay:

    Article Title: Macrophage-Derived Extracellular Succinate Licenses Neural Stem Cells to Suppress Chronic Neuroinflammation
    Article Snippet: .. The following primary antibodies diluted in blocking buffer were used: anti-nestin (1:200, Abcam), anti-SOX2 (1:100, Abcam), anti-glial fibrillary acidic protein (GFAP) (1:500, Abcam), anti-β-tubulin-III (1:500, Covance), anti-O4 (1:400, R & D). .. Cells were then washed in PBS with 0.1% Triton X-100 and incubated with the appropriate fluorescent secondary antibodies (1:1,000 Alexa Fluor 405, 488, 555, 647, Invitrogen) 1 hr at RT.

    Incubation:

    Article Title: Mesenchymal stem cell-derived exosomes improve motor function and attenuate neuropathology in a mouse model of Machado-Joseph disease
    Article Snippet: .. The sections were then incubated overnight at 4 °C with the following primary antibodies: mouse anti-calbindin D28K (1:500, catalog number C9848, Sigma-Aldrich, USA), mouse anti-ATXN3 (1H9) (1:200, catalog number MAB5360, Millipore, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (1:200, catalog number ab48050, Abcam, USA), and rat anti-myelin basic protein (MBP) (1:200, catalog number ab7349, Abcam, USA), followed by incubation with the following secondary antibodies: goat-anti mouse IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 488 conjugated) antibody (1:300, catalog number 4408S, Cell Signaling, USA), goat-anti mouse IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 555 conjugated) antibody (1:300, catalog number 4409S, Cell Signaling), goat-anti rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 488 conjugated) antibody (1:300, catalog number 4412S, Cell Signaling), and goat-anti rat IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 488 conjugated) antibody (1:300, catalog number 4416S, Cell Signaling) for 1 h at room temperature. ..

    Article Title: Targeted Gene Transfer to the Brain via the Delivery of Brain-Penetrating DNA Nanoparticles with Focused Ultrasound
    Article Snippet: .. Sections were next incubated overnight with mouse anti-mCherry (1:200; Abcam). .. After washing 3× for 10 min in PBS, sections were incubated for 1 hr at room temp with Alexa Fluor 647 conjugated goat anti-mouse IgG (Invitrogen).

    other:

    Article Title: Hypoxia selectively upregulates cation channels and increases cytosolic [Ca2+] in pulmonary, but not coronary, arterial smooth muscle cells
    Article Snippet: Ryanodine was purchased from Abcam and soluble in ethanol.

    Marker:

    Article Title: Dental enamel cells express functional SOCE channels
    Article Snippet: .. The following antibodies and dilutions were used: anti-STIM1 (1:200, Sigma-Aldrich), anti-calnexin (a marker for ER) (1:1000, Abcam), anti-ORAI1 (1:50, described in ) as well as anti-IP3 R1 (1:50, Novusbio), anti-IP3R2 (1:100, Sigma-Aldrich), anti-IP3R3 (1:1000, Abcam), anti-SERCA2 (1:200, Abcam), anti-RyR1 (1: 500, LSBio), anti-RyR2 (1:200, Sigma-Aldrich) and anti-RyR3 (1:300, Abcam). .. Negative controls were provided by incubating sections with Dako Diluent lacking primary antibody.

    Western Blot:

    Article Title: PKC? Signalling Activates ERK1/2, and Regulates Aggrecan, ADAMTS5, and miR377 Gene Expression in Human Nucleus Pulposus Cells
    Article Snippet: .. We used the following antibodies (and dilutions): polyclonal antibodies to tropomyocin (1:400) from Sigma, to phosphorylated species of PKCε (1:1,000) and myristoylated alanine-rich C-kinase substrate (MARCKS) (1:1,000), to PKCε (1:1,000), ERK1/2 (1:2,000), and to activator protein 1 (AP-1) (1:1,000) from Santa Cruz, to cAMP responsive element binding protein 1(CREB1) (1:1,000) from Millipore; mouse monoclonal antibodies to MARCKS (1:1,000) and phosho-ERK1/2 (1:2,000) from Santa Cruz, to p120GAP (1:2,000) from Upstate Biotechnology, to neo-epitope ARG on aggrecan, clone BC-3 (1:1,000 for Western blotting and 1:200 for immunocytochemistry) from ABCAM, and to β-tubulin (1:500) from Sigma; and secondary antibodies conjugated to horseradish peroxidase or fluorochromes from Santa Cruz. .. The PKCε-selective activator peptide, ψεRACK [PKCε (85-92), HDAPIGYD] [ ] was synthesized and conjugated to a peptide derived from the trans-activating transcriptional activator (TAT) (amino acids 45-57, YGRKKRRQRRR) by Peptide 2.0 Inc. PD98059, U0126, and SB203580 were from Calbiochem.

    Article Title: Muscle Creatine Kinase Deficiency Triggers Both Actin Depolymerization and Desmin Disorganization by Advanced Glycation End Products in Dilated Cardiomyopathy
    Article Snippet: .. The following primary antibodies were used for Western blot: mouse monoclonal anti-sarcomeric α-actin (1:400, Sigma, recognize both cardiac and skeletal α-actin), mouse monoclonal anti-α-tropomyosin, rabbit polyclonal anti-GAPDH (1:400, Santa Cruz), rabbit polyclonal anti-Histone-3 (1:400, Sigma), mouse monoclonal anti-αB-crystallin (1:200, Abcam), and anti-phosphorylated serines 19, 45, and 59 on αB-crystallin (1:500, StressGen). ..

    Binding Assay:

    Article Title: PKC? Signalling Activates ERK1/2, and Regulates Aggrecan, ADAMTS5, and miR377 Gene Expression in Human Nucleus Pulposus Cells
    Article Snippet: .. We used the following antibodies (and dilutions): polyclonal antibodies to tropomyocin (1:400) from Sigma, to phosphorylated species of PKCε (1:1,000) and myristoylated alanine-rich C-kinase substrate (MARCKS) (1:1,000), to PKCε (1:1,000), ERK1/2 (1:2,000), and to activator protein 1 (AP-1) (1:1,000) from Santa Cruz, to cAMP responsive element binding protein 1(CREB1) (1:1,000) from Millipore; mouse monoclonal antibodies to MARCKS (1:1,000) and phosho-ERK1/2 (1:2,000) from Santa Cruz, to p120GAP (1:2,000) from Upstate Biotechnology, to neo-epitope ARG on aggrecan, clone BC-3 (1:1,000 for Western blotting and 1:200 for immunocytochemistry) from ABCAM, and to β-tubulin (1:500) from Sigma; and secondary antibodies conjugated to horseradish peroxidase or fluorochromes from Santa Cruz. .. The PKCε-selective activator peptide, ψεRACK [PKCε (85-92), HDAPIGYD] [ ] was synthesized and conjugated to a peptide derived from the trans-activating transcriptional activator (TAT) (amino acids 45-57, YGRKKRRQRRR) by Peptide 2.0 Inc. PD98059, U0126, and SB203580 were from Calbiochem.

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    Abcam rabbit anti glial fibrillary acidic protein gfap
    Injection of MSC-derived exosomes significantly reduced the activation of astrocytes. a Immunofluorescence images of astrocytes in pons among YACMJD84.2 mice with exosomal treatment (Exosome-Tg, n = 4), YACMJD84.2 mice without exosomal treatment (Control-Tg, n = 4), and wild-type littermates (Control-Wt, n = 4). The scale bar was 50 μm. b Quantitative analysis of <t>GFAP</t> signal intensity ( n = 4). ( c ) Quantitative analysis of the number of nuclear inclusions stained by 1H9 ( n = 4). d Western blot analysis of mutant (Mut) and wild-type (WT) <t>ATXN3</t> expression among YACMJD84.2 mice with exosomal treatment (Exosome-Tg, n = 4), YACMJD84.2 mice without exosomal treatment (Control-Tg, n = 4), and wild-type littermates (Control-Wt, n = 4) in the cerebellum. e Quantitative analysis of mutant ATXN3 expression. One-way ANOVA analysis; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001
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    Injection of MSC-derived exosomes significantly reduced the activation of astrocytes. a Immunofluorescence images of astrocytes in pons among YACMJD84.2 mice with exosomal treatment (Exosome-Tg, n = 4), YACMJD84.2 mice without exosomal treatment (Control-Tg, n = 4), and wild-type littermates (Control-Wt, n = 4). The scale bar was 50 μm. b Quantitative analysis of GFAP signal intensity ( n = 4). ( c ) Quantitative analysis of the number of nuclear inclusions stained by 1H9 ( n = 4). d Western blot analysis of mutant (Mut) and wild-type (WT) ATXN3 expression among YACMJD84.2 mice with exosomal treatment (Exosome-Tg, n = 4), YACMJD84.2 mice without exosomal treatment (Control-Tg, n = 4), and wild-type littermates (Control-Wt, n = 4) in the cerebellum. e Quantitative analysis of mutant ATXN3 expression. One-way ANOVA analysis; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

    Journal: Stem Cell Research & Therapy

    Article Title: Mesenchymal stem cell-derived exosomes improve motor function and attenuate neuropathology in a mouse model of Machado-Joseph disease

    doi: 10.1186/s13287-020-01727-2

    Figure Lengend Snippet: Injection of MSC-derived exosomes significantly reduced the activation of astrocytes. a Immunofluorescence images of astrocytes in pons among YACMJD84.2 mice with exosomal treatment (Exosome-Tg, n = 4), YACMJD84.2 mice without exosomal treatment (Control-Tg, n = 4), and wild-type littermates (Control-Wt, n = 4). The scale bar was 50 μm. b Quantitative analysis of GFAP signal intensity ( n = 4). ( c ) Quantitative analysis of the number of nuclear inclusions stained by 1H9 ( n = 4). d Western blot analysis of mutant (Mut) and wild-type (WT) ATXN3 expression among YACMJD84.2 mice with exosomal treatment (Exosome-Tg, n = 4), YACMJD84.2 mice without exosomal treatment (Control-Tg, n = 4), and wild-type littermates (Control-Wt, n = 4) in the cerebellum. e Quantitative analysis of mutant ATXN3 expression. One-way ANOVA analysis; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

    Article Snippet: The sections were then incubated overnight at 4 °C with the following primary antibodies: mouse anti-calbindin D28K (1:500, catalog number C9848, Sigma-Aldrich, USA), mouse anti-ATXN3 (1H9) (1:200, catalog number MAB5360, Millipore, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (1:200, catalog number ab48050, Abcam, USA), and rat anti-myelin basic protein (MBP) (1:200, catalog number ab7349, Abcam, USA), followed by incubation with the following secondary antibodies: goat-anti mouse IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 488 conjugated) antibody (1:300, catalog number 4408S, Cell Signaling, USA), goat-anti mouse IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 555 conjugated) antibody (1:300, catalog number 4409S, Cell Signaling), goat-anti rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 488 conjugated) antibody (1:300, catalog number 4412S, Cell Signaling), and goat-anti rat IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 488 conjugated) antibody (1:300, catalog number 4416S, Cell Signaling) for 1 h at room temperature.

    Techniques: Injection, Derivative Assay, Activation Assay, Immunofluorescence, Mouse Assay, Staining, Western Blot, Mutagenesis, Expressing

    NSCs Transplantation Ameliorates Chronic Neuroinflammation and Reduces Succinate Levels in the CSF of EAE Mice (A–D) Representative images of fGFP + iNSCs at 30 dpt expressing the proliferation marker Ki67 (A, arrowheads) and the neural marker Nestin (A), the mature neuronal marker NeuN (B, arrowhead), the astroglial lineage marker GFAP (C), or the oligodendroglial lineage marker OLIG2 (D, arrowhead). (E) Confocal microscopy image of a perivascular area with several fGFP + iNSCs in juxtaposition to fGFP − /F4/80 + MPs. Nuclei in (A)–(E) are stained with DAPI (blue). (F) Behavioral outcome of iNSCs/NSCs-transplanted EAE mice. Data are mean EAE score (±SEM) from n ≥ 7 mice/group over n = 2 independent experiments. EAE mice injected icv with mouse fibroblasts (MFs) or PBS were used as controls. (G and H) Flow-cytometry-based ex vivo quantification of the expression levels of type 1 inflammatory (CD80) and anti-inflammatory (MRC1) markers in CX3CR1 + microglial cells (G) and CCR2 + monocyte-derived infiltrating macrophages (H) from the CNS of iNSC- and NSC-transplanted EAE mice at 30 dpt. Quantitative data are shown on the left, whereas representative density plots are shown on the right. Data are min to max % of marker-positive cells from n ≥ 4 pools of mice/group. (I) Representative confocal microscopy image and comparative histograms of a perivascular area with several fGFP + iNSCs in juxtaposition to F4/80 + MPs. Low iNOS and prevalent MRC1 expression is detected in F4/80 + MPs close to fGFP + iNSCs (inset on the left), whereas high iNOS expression is observed in the remaining MP infiltrate (inset on the right). Nuclei are stained with DAPI. (J) Expression levels (qRT-PCR) of pro- and anti-inflammatory genes in the brain and spinal cord of EAE mice. Data are mean fold change over HC from n ≥ 3 mice/group. (K and L) Quantification and representative 3D reconstructions of spinal cord damage in iNSC- and NSC-transplanted EAE mice. Data are mean % of Bielschowsky negative-stained axonal loss (K) or Luxol fast blue (LFB) negative-stained demyelinated (L) areas/spinal cord section (±SEM) from n ≥ 5 mice/group over n = 2 independent experiments. (M) Levels of CSF metabolites significantly changed during EAE (versus HC). Corresponding levels in matched plasma samples are also shown. Data are mean a.u. (±SEM) from n ≥ 3 mice/group. The scale bars represent 25 μm (A–E), 50 μm (I), and 2 mm (K and L). ∗ p ≤ 0.05 and ∗∗ p ≤ 0.01 versus PBS; # .

    Journal: Cell Stem Cell

    Article Title: Macrophage-Derived Extracellular Succinate Licenses Neural Stem Cells to Suppress Chronic Neuroinflammation

    doi: 10.1016/j.stem.2018.01.020

    Figure Lengend Snippet: NSCs Transplantation Ameliorates Chronic Neuroinflammation and Reduces Succinate Levels in the CSF of EAE Mice (A–D) Representative images of fGFP + iNSCs at 30 dpt expressing the proliferation marker Ki67 (A, arrowheads) and the neural marker Nestin (A), the mature neuronal marker NeuN (B, arrowhead), the astroglial lineage marker GFAP (C), or the oligodendroglial lineage marker OLIG2 (D, arrowhead). (E) Confocal microscopy image of a perivascular area with several fGFP + iNSCs in juxtaposition to fGFP − /F4/80 + MPs. Nuclei in (A)–(E) are stained with DAPI (blue). (F) Behavioral outcome of iNSCs/NSCs-transplanted EAE mice. Data are mean EAE score (±SEM) from n ≥ 7 mice/group over n = 2 independent experiments. EAE mice injected icv with mouse fibroblasts (MFs) or PBS were used as controls. (G and H) Flow-cytometry-based ex vivo quantification of the expression levels of type 1 inflammatory (CD80) and anti-inflammatory (MRC1) markers in CX3CR1 + microglial cells (G) and CCR2 + monocyte-derived infiltrating macrophages (H) from the CNS of iNSC- and NSC-transplanted EAE mice at 30 dpt. Quantitative data are shown on the left, whereas representative density plots are shown on the right. Data are min to max % of marker-positive cells from n ≥ 4 pools of mice/group. (I) Representative confocal microscopy image and comparative histograms of a perivascular area with several fGFP + iNSCs in juxtaposition to F4/80 + MPs. Low iNOS and prevalent MRC1 expression is detected in F4/80 + MPs close to fGFP + iNSCs (inset on the left), whereas high iNOS expression is observed in the remaining MP infiltrate (inset on the right). Nuclei are stained with DAPI. (J) Expression levels (qRT-PCR) of pro- and anti-inflammatory genes in the brain and spinal cord of EAE mice. Data are mean fold change over HC from n ≥ 3 mice/group. (K and L) Quantification and representative 3D reconstructions of spinal cord damage in iNSC- and NSC-transplanted EAE mice. Data are mean % of Bielschowsky negative-stained axonal loss (K) or Luxol fast blue (LFB) negative-stained demyelinated (L) areas/spinal cord section (±SEM) from n ≥ 5 mice/group over n = 2 independent experiments. (M) Levels of CSF metabolites significantly changed during EAE (versus HC). Corresponding levels in matched plasma samples are also shown. Data are mean a.u. (±SEM) from n ≥ 3 mice/group. The scale bars represent 25 μm (A–E), 50 μm (I), and 2 mm (K and L). ∗ p ≤ 0.05 and ∗∗ p ≤ 0.01 versus PBS; # .

    Article Snippet: The following primary antibodies diluted in blocking buffer were used: anti-nestin (1:200, Abcam), anti-SOX2 (1:100, Abcam), anti-glial fibrillary acidic protein (GFAP) (1:500, Abcam), anti-β-tubulin-III (1:500, Covance), anti-O4 (1:400, R & D).

    Techniques: Transplantation Assay, Mouse Assay, Expressing, Marker, Confocal Microscopy, Staining, Injection, Flow Cytometry, Cytometry, Ex Vivo, Derivative Assay, Quantitative RT-PCR

    Changes in αB-crystallin and desmin localization in SRF HKO cardiomyocytes. Immunofluorescent co-staining for total αB-crystallin ( CryAB ) ( A–C ) or phospho-Ser 59 CryAB ( D–I ) in green and F-actin using phalloidin-TRITC ( A–F

    Journal: The Journal of Biological Chemistry

    Article Title: Muscle Creatine Kinase Deficiency Triggers Both Actin Depolymerization and Desmin Disorganization by Advanced Glycation End Products in Dilated Cardiomyopathy

    doi: 10.1074/jbc.M111.252395

    Figure Lengend Snippet: Changes in αB-crystallin and desmin localization in SRF HKO cardiomyocytes. Immunofluorescent co-staining for total αB-crystallin ( CryAB ) ( A–C ) or phospho-Ser 59 CryAB ( D–I ) in green and F-actin using phalloidin-TRITC ( A–F

    Article Snippet: The following primary antibodies were used for Western blot: mouse monoclonal anti-sarcomeric α-actin (1:400, Sigma, recognize both cardiac and skeletal α-actin), mouse monoclonal anti-α-tropomyosin, rabbit polyclonal anti-GAPDH (1:400, Santa Cruz), rabbit polyclonal anti-Histone-3 (1:400, Sigma), mouse monoclonal anti-αB-crystallin (1:200, Abcam), and anti-phosphorylated serines 19, 45, and 59 on αB-crystallin (1:500, StressGen).

    Techniques: Staining

    Induction of phosphorylation on αB-crystallin in SRF HKO hearts. Upper panel , Western blot analysis of control and mutant hearts at D25 and D45 with anti-αB-crystallin ( CryAB ), -phospho-Ser 45 -CryAB, -phospho-Ser 59 -CryAB, and -GAPDH antibodies.

    Journal: The Journal of Biological Chemistry

    Article Title: Muscle Creatine Kinase Deficiency Triggers Both Actin Depolymerization and Desmin Disorganization by Advanced Glycation End Products in Dilated Cardiomyopathy

    doi: 10.1074/jbc.M111.252395

    Figure Lengend Snippet: Induction of phosphorylation on αB-crystallin in SRF HKO hearts. Upper panel , Western blot analysis of control and mutant hearts at D25 and D45 with anti-αB-crystallin ( CryAB ), -phospho-Ser 45 -CryAB, -phospho-Ser 59 -CryAB, and -GAPDH antibodies.

    Article Snippet: The following primary antibodies were used for Western blot: mouse monoclonal anti-sarcomeric α-actin (1:400, Sigma, recognize both cardiac and skeletal α-actin), mouse monoclonal anti-α-tropomyosin, rabbit polyclonal anti-GAPDH (1:400, Santa Cruz), rabbit polyclonal anti-Histone-3 (1:400, Sigma), mouse monoclonal anti-αB-crystallin (1:200, Abcam), and anti-phosphorylated serines 19, 45, and 59 on αB-crystallin (1:500, StressGen).

    Techniques: Western Blot, Mutagenesis

    Preactivation of αB-crystallin phosphorylation protects desmin network from short-term treatment with glyoxal or cytochalasin D. A , immunofluorescent co-staining for phospho-Ser 59 αB-crystallin ( orange ), desmin ( green ), and F-actin (phalloidin-TRITC,

    Journal: The Journal of Biological Chemistry

    Article Title: Muscle Creatine Kinase Deficiency Triggers Both Actin Depolymerization and Desmin Disorganization by Advanced Glycation End Products in Dilated Cardiomyopathy

    doi: 10.1074/jbc.M111.252395

    Figure Lengend Snippet: Preactivation of αB-crystallin phosphorylation protects desmin network from short-term treatment with glyoxal or cytochalasin D. A , immunofluorescent co-staining for phospho-Ser 59 αB-crystallin ( orange ), desmin ( green ), and F-actin (phalloidin-TRITC,

    Article Snippet: The following primary antibodies were used for Western blot: mouse monoclonal anti-sarcomeric α-actin (1:400, Sigma, recognize both cardiac and skeletal α-actin), mouse monoclonal anti-α-tropomyosin, rabbit polyclonal anti-GAPDH (1:400, Santa Cruz), rabbit polyclonal anti-Histone-3 (1:400, Sigma), mouse monoclonal anti-αB-crystallin (1:200, Abcam), and anti-phosphorylated serines 19, 45, and 59 on αB-crystallin (1:500, StressGen).

    Techniques: Staining

    Schematic model representing calcium entry in enamel cells. Working model for Ca 2+  uptake by enamel cells showing maturation stage ameloblasts forming a cell barrier joined by tight junctions at the apical pole. In the endoplasmic reticulum (ER) we find that enamel cells express the sarco/endoplasmic reticulum SERCA2 as the main Ca 2+  refilling pump. Inositol 1,4,5-trisphosphate receptors (IP 3 R) and ryanodine receptors (RyR) are also identified as release channels with the former likely being the active release system. STIM1 has a wide distribution throughout the ER and ORAI1 is found in the plasma membrane of enamel cells. As Ca 2+  pools are depleted in the ER, STIM1 clusters enable Ca 2+  entry via the ORAI1 channel.

    Journal: Scientific Reports

    Article Title: Dental enamel cells express functional SOCE channels

    doi: 10.1038/srep15803

    Figure Lengend Snippet: Schematic model representing calcium entry in enamel cells. Working model for Ca 2+ uptake by enamel cells showing maturation stage ameloblasts forming a cell barrier joined by tight junctions at the apical pole. In the endoplasmic reticulum (ER) we find that enamel cells express the sarco/endoplasmic reticulum SERCA2 as the main Ca 2+ refilling pump. Inositol 1,4,5-trisphosphate receptors (IP 3 R) and ryanodine receptors (RyR) are also identified as release channels with the former likely being the active release system. STIM1 has a wide distribution throughout the ER and ORAI1 is found in the plasma membrane of enamel cells. As Ca 2+ pools are depleted in the ER, STIM1 clusters enable Ca 2+ entry via the ORAI1 channel.

    Article Snippet: The following antibodies and dilutions were used: anti-STIM1 (1:200, Sigma-Aldrich), anti-calnexin (a marker for ER) (1:1000, Abcam), anti-ORAI1 (1:50, described in ) as well as anti-IP3 R1 (1:50, Novusbio), anti-IP3R2 (1:100, Sigma-Aldrich), anti-IP3R3 (1:1000, Abcam), anti-SERCA2 (1:200, Abcam), anti-RyR1 (1: 500, LSBio), anti-RyR2 (1:200, Sigma-Aldrich) and anti-RyR3 (1:300, Abcam).

    Techniques: