kinase detection reagent Search Results


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  • 99
    Thermo Fisher t4 kinase
    T4 Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 627 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore luciferin luciferase detection reagent
    Luciferin Luciferase Detection Reagent, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega kinase detection reagent
    Kinase Detection Reagent, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega kinase glo detection reagent
    Kinase Glo Detection Reagent, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega kinase glo kit
    Kinase Glo Kit, supplied by Promega, used in various techniques. Bioz Stars score: 98/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Lonza adenylate kinase detection reagent
    ZIKV replication and cytopathicity. ( a ) Kinetics of MR766 replication (left) and INMI-1 (right) in T-HESC (blue) and dT-HESC (red) were measured by retrotitration of culture supernatants on Vero cells by PFA. Mean ± SEM of 3 independent experiments is reported. ( b ) Kinetics of cell death were measured by the activity of cell-associated <t>adenylate</t> kinase (AK) released in cell culture supernatants from T-HESC (left) and dT-HESC (right). Two-way ANOVA with Bonferroni post-tests was used. *Represents statistical comparison between the viral strains and uninfected cultures (****p
    Adenylate Kinase Detection Reagent, supplied by Lonza, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti map kinase
    ZIKV replication and cytopathicity. ( a ) Kinetics of MR766 replication (left) and INMI-1 (right) in T-HESC (blue) and dT-HESC (red) were measured by retrotitration of culture supernatants on Vero cells by PFA. Mean ± SEM of 3 independent experiments is reported. ( b ) Kinetics of cell death were measured by the activity of cell-associated <t>adenylate</t> kinase (AK) released in cell culture supernatants from T-HESC (left) and dT-HESC (right). Two-way ANOVA with Bonferroni post-tests was used. *Represents statistical comparison between the viral strains and uninfected cultures (****p
    Anti Map Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher antibodies against erk2 kinase
    DDR2/-KD is localized on the plasma membrane. a) Confocal images of HEK293 cells transiently transfected with DDR2/-KD and immunolabeled with an antibody against the DDR2 ECD; nontransfected cells show a low level of endogenous DDR2 while transfected cells show an increased level of DDR2 ECD on the membrane. b) Cell surface biotinylation of MC3T3 cells expressing DDR2/-KD construct, demonstrating surface localization of the truncated receptor. Cell samples were biotinylated (as indicated) and surface proteins were precipitated from the lysates with streptavidin-agarose beads ( s lanes); the supernatant from the pull-down was recovered and probed for intracellular proteins ( i lanes). Following SDS-PAGE, samples were analyzed by Western blotting with antibodies against V5 tag (DDR2/-KD) or <t>ERK2</t> as indicated. DDR2/-KD is mainly present on the cell surface, while a lower molecular weight band (probably indicating the immature, non glycosylated receptor) is only found in the intracellular lane. The intracellular protein ERK2 is only present in the intracellular lane, demonstrating that biotinylation is specific to cell surface proteins. As an additional control, non-biotinylated samples, show that the streptavidin pull down is specific to biotinylated proteins.
    Antibodies Against Erk2 Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher neomycin phosphotransferase ii nptii
    DDR2/-KD is localized on the plasma membrane. a) Confocal images of HEK293 cells transiently transfected with DDR2/-KD and immunolabeled with an antibody against the DDR2 ECD; nontransfected cells show a low level of endogenous DDR2 while transfected cells show an increased level of DDR2 ECD on the membrane. b) Cell surface biotinylation of MC3T3 cells expressing DDR2/-KD construct, demonstrating surface localization of the truncated receptor. Cell samples were biotinylated (as indicated) and surface proteins were precipitated from the lysates with streptavidin-agarose beads ( s lanes); the supernatant from the pull-down was recovered and probed for intracellular proteins ( i lanes). Following SDS-PAGE, samples were analyzed by Western blotting with antibodies against V5 tag (DDR2/-KD) or <t>ERK2</t> as indicated. DDR2/-KD is mainly present on the cell surface, while a lower molecular weight band (probably indicating the immature, non glycosylated receptor) is only found in the intracellular lane. The intracellular protein ERK2 is only present in the intracellular lane, demonstrating that biotinylation is specific to cell surface proteins. As an additional control, non-biotinylated samples, show that the streptavidin pull down is specific to biotinylated proteins.
    Neomycin Phosphotransferase Ii Nptii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phosphorylated p70s6 kinase p70s6k antibody
    Pamidronate and clodronate inhibited PI-3K/Akt/mTOR signaling pathways activated by IGF-1 in MCF-7 cells. ( a ) Serum-starved MCF-7 cells were treated with IGF-1 (40 ng/mL) for different time intervals, and phosphorylated ERK1/2, AKT, mTOR, <t>p70S6K</t> and 4E-BP1
    Phosphorylated P70s6 Kinase P70s6k Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phospho p70s6 kinase thr389 antibody
    Pamidronate and clodronate inhibited PI-3K/Akt/mTOR signaling pathways activated by IGF-1 in MCF-7 cells. ( a ) Serum-starved MCF-7 cells were treated with IGF-1 (40 ng/mL) for different time intervals, and phosphorylated ERK1/2, AKT, mTOR, <t>p70S6K</t> and 4E-BP1
    Phospho P70s6 Kinase Thr389 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phospho ser thr kinase substrate antibody immobilized protein
    Pamidronate and clodronate inhibited PI-3K/Akt/mTOR signaling pathways activated by IGF-1 in MCF-7 cells. ( a ) Serum-starved MCF-7 cells were treated with IGF-1 (40 ng/mL) for different time intervals, and phosphorylated ERK1/2, AKT, mTOR, <t>p70S6K</t> and 4E-BP1
    Phospho Ser Thr Kinase Substrate Antibody Immobilized Protein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phosphoglycerate kinase
    Sec35p is a peripheral membrane protein. ( a ) Sec35p partitions into soluble and membrane-associated pools. A wild-type strain was grown to logarithmic phase, lysed with glass beads, and then centrifuged at 1,000 g for 3 min to generate the S1 supernatant fraction. The S1 supernatant was centrifuged at 175,000 g to obtain the S175 supernatant and P175 pellet fractions. ( b ) The membrane-associated portion of Sec35p fractionates similarly to the Golgi protein, <t>Sed5p.</t> The S1 supernatant was centrifuged at 10,000 g to generate the S10 supernatant and P10 pellet fractions, and the S10 was subsequently centrifuged at 175,000 g to obtain the S175 supernatant and P175 pellet fractions. ( c ) Sec35p behaves as a peripheral membrane protein. The S1 supernatant was incubated with buffer 88, 1% Triton X-100, 1 M NaCl, or 100 mM Na 2 CO 3 , pH 11, centrifuged at 175,000 g , and separated into supernatant and pellet fractions. ( a–c ) Aliquots of each fraction were separated by SDS-12%PAGE and immunoblotted with affinity-purified anti-Sec35p ( top panel ), anti-Sed5p ( center panel ), or <t>anti-PGK</t> ( bottom panel ) antibodies. The samples loaded in each lane were derived from equivalent amounts of starting material.
    Phosphoglycerate Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 168 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher thioltracker violet
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Thioltracker Violet, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp detection reagent
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Atp Detection Reagent, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher creatine kinase mb
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Creatine Kinase Mb, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mixed lineage kinase domain like protein mlkl polyclonal antibody
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Mixed Lineage Kinase Domain Like Protein Mlkl Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher monoclonal anti cyclin dependent kinase cdk 2
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Monoclonal Anti Cyclin Dependent Kinase Cdk 2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phospho p70 s6 kinase thr389 antibody
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Phospho P70 S6 Kinase Thr389 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phospho p70 s6 kinase pa5 17884
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Phospho P70 S6 Kinase Pa5 17884, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher horseradish peroxidase hrp conjugated phospho kinase antibody
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Horseradish Peroxidase Hrp Conjugated Phospho Kinase Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher primary antibodies anti phospho pi3 kinase p85 ptyr458 p55 ptyr199
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Primary Antibodies Anti Phospho Pi3 Kinase P85 Ptyr458 P55 Ptyr199, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phospho p70 s6k thr421 ser424
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Phospho P70 S6k Thr421 Ser424, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti phospho janus kinase 2 jak2 pab
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Anti Phospho Janus Kinase 2 Jak2 Pab, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti phosphorylated phosphatidylinositol 3 kinase p pi3k
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Anti Phosphorylated Phosphatidylinositol 3 Kinase P Pi3k, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher monoclonal anti rhodopsin kinase 1a antibody
    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe <t>ThiolTracker</t> Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p
    Monoclonal Anti Rhodopsin Kinase 1a Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti map kinase erk1 2 mabs
    Expression of GIPC(LG > AE) inhibits NGF-induced neurite outgrowth and <t>MAP</t> kinase activation. (A) Bar graph showing the percentages of cells that have differentiated cells expressing different GIPC constructs. In nontransfected cells that were not stimulated with NGF (−), only 4% of the cells differentiated and grew out at least one neurite. After NGF treatment (+), 50% of the nontransfected cells, 41% of the mock-transfected cells (empty vector), and 37% of the GIPC-transfected cells differentiated, whereas in the cells transfected with the GIPC(LG > AE) mutant, only 27% of the cells differentiated. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG > AE), incubated with NGF (50 ng/ml) for 16 h, fixed, permeabilized, and incubated with anti-FLAG to label transfected cells. Cells were scored for differentiation (neurite outgrowth) as described in Materials and Methods. Average differentiation rates (±standard errors) are for six experiments (≥50 cells/experiment). **, P ≤ 0.01. (B) Overexpression of the FLAG-tagged GIPC(LG > AE) mutant strongly inhibits NGF-induced <t>Erk1/2</t> activation. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG > AE), serum starved, treated with NGF (100 ng/ml) for 5 min, and lysed for immunoblotting with anti-pErk1/2 antibody. Per lane, 20 μg protein was loaded; the levels of total Erk1/2 (bottom) are the same in all samples. (C) Quantification of the pErk1/2 levels from panel B, shown as normalized percentages of the mock control.
    Anti Map Kinase Erk1 2 Mabs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher antihuman α extracellular signal regulated kinases
    Expression of GIPC(LG > AE) inhibits NGF-induced neurite outgrowth and <t>MAP</t> kinase activation. (A) Bar graph showing the percentages of cells that have differentiated cells expressing different GIPC constructs. In nontransfected cells that were not stimulated with NGF (−), only 4% of the cells differentiated and grew out at least one neurite. After NGF treatment (+), 50% of the nontransfected cells, 41% of the mock-transfected cells (empty vector), and 37% of the GIPC-transfected cells differentiated, whereas in the cells transfected with the GIPC(LG > AE) mutant, only 27% of the cells differentiated. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG > AE), incubated with NGF (50 ng/ml) for 16 h, fixed, permeabilized, and incubated with anti-FLAG to label transfected cells. Cells were scored for differentiation (neurite outgrowth) as described in Materials and Methods. Average differentiation rates (±standard errors) are for six experiments (≥50 cells/experiment). **, P ≤ 0.01. (B) Overexpression of the FLAG-tagged GIPC(LG > AE) mutant strongly inhibits NGF-induced <t>Erk1/2</t> activation. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG > AE), serum starved, treated with NGF (100 ng/ml) for 5 min, and lysed for immunoblotting with anti-pErk1/2 antibody. Per lane, 20 μg protein was loaded; the levels of total Erk1/2 (bottom) are the same in all samples. (C) Quantification of the pErk1/2 levels from panel B, shown as normalized percentages of the mock control.
    Antihuman α Extracellular Signal Regulated Kinases, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rabbit polyclonal primary antibody against cam kinase ii
    Expression of GIPC(LG > AE) inhibits NGF-induced neurite outgrowth and <t>MAP</t> kinase activation. (A) Bar graph showing the percentages of cells that have differentiated cells expressing different GIPC constructs. In nontransfected cells that were not stimulated with NGF (−), only 4% of the cells differentiated and grew out at least one neurite. After NGF treatment (+), 50% of the nontransfected cells, 41% of the mock-transfected cells (empty vector), and 37% of the GIPC-transfected cells differentiated, whereas in the cells transfected with the GIPC(LG > AE) mutant, only 27% of the cells differentiated. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG > AE), incubated with NGF (50 ng/ml) for 16 h, fixed, permeabilized, and incubated with anti-FLAG to label transfected cells. Cells were scored for differentiation (neurite outgrowth) as described in Materials and Methods. Average differentiation rates (±standard errors) are for six experiments (≥50 cells/experiment). **, P ≤ 0.01. (B) Overexpression of the FLAG-tagged GIPC(LG > AE) mutant strongly inhibits NGF-induced <t>Erk1/2</t> activation. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG > AE), serum starved, treated with NGF (100 ng/ml) for 5 min, and lysed for immunoblotting with anti-pErk1/2 antibody. Per lane, 20 μg protein was loaded; the levels of total Erk1/2 (bottom) are the same in all samples. (C) Quantification of the pErk1/2 levels from panel B, shown as normalized percentages of the mock control.
    Rabbit Polyclonal Primary Antibody Against Cam Kinase Ii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher antibodies against focal adhesion kinase fak anti fak anti fak py397
    Expression of GIPC(LG > AE) inhibits NGF-induced neurite outgrowth and <t>MAP</t> kinase activation. (A) Bar graph showing the percentages of cells that have differentiated cells expressing different GIPC constructs. In nontransfected cells that were not stimulated with NGF (−), only 4% of the cells differentiated and grew out at least one neurite. After NGF treatment (+), 50% of the nontransfected cells, 41% of the mock-transfected cells (empty vector), and 37% of the GIPC-transfected cells differentiated, whereas in the cells transfected with the GIPC(LG > AE) mutant, only 27% of the cells differentiated. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG > AE), incubated with NGF (50 ng/ml) for 16 h, fixed, permeabilized, and incubated with anti-FLAG to label transfected cells. Cells were scored for differentiation (neurite outgrowth) as described in Materials and Methods. Average differentiation rates (±standard errors) are for six experiments (≥50 cells/experiment). **, P ≤ 0.01. (B) Overexpression of the FLAG-tagged GIPC(LG > AE) mutant strongly inhibits NGF-induced <t>Erk1/2</t> activation. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG > AE), serum starved, treated with NGF (100 ng/ml) for 5 min, and lysed for immunoblotting with anti-pErk1/2 antibody. Per lane, 20 μg protein was loaded; the levels of total Erk1/2 (bottom) are the same in all samples. (C) Quantification of the pErk1/2 levels from panel B, shown as normalized percentages of the mock control.
    Antibodies Against Focal Adhesion Kinase Fak Anti Fak Anti Fak Py397, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ZIKV replication and cytopathicity. ( a ) Kinetics of MR766 replication (left) and INMI-1 (right) in T-HESC (blue) and dT-HESC (red) were measured by retrotitration of culture supernatants on Vero cells by PFA. Mean ± SEM of 3 independent experiments is reported. ( b ) Kinetics of cell death were measured by the activity of cell-associated adenylate kinase (AK) released in cell culture supernatants from T-HESC (left) and dT-HESC (right). Two-way ANOVA with Bonferroni post-tests was used. *Represents statistical comparison between the viral strains and uninfected cultures (****p

    Journal: Scientific Reports

    Article Title: Human Endometrial Stromal Cells Are Highly Permissive To Productive Infection by Zika Virus

    doi: 10.1038/srep44286

    Figure Lengend Snippet: ZIKV replication and cytopathicity. ( a ) Kinetics of MR766 replication (left) and INMI-1 (right) in T-HESC (blue) and dT-HESC (red) were measured by retrotitration of culture supernatants on Vero cells by PFA. Mean ± SEM of 3 independent experiments is reported. ( b ) Kinetics of cell death were measured by the activity of cell-associated adenylate kinase (AK) released in cell culture supernatants from T-HESC (left) and dT-HESC (right). Two-way ANOVA with Bonferroni post-tests was used. *Represents statistical comparison between the viral strains and uninfected cultures (****p

    Article Snippet: To each well, 50 μl of the adenylate kinase detection reagent (ToxiLight® BioAssay, Lonza) was added and the plate was incubated for 10 min at room temperature.

    Techniques: Activity Assay, Cell Culture

    DDR2/-KD is localized on the plasma membrane. a) Confocal images of HEK293 cells transiently transfected with DDR2/-KD and immunolabeled with an antibody against the DDR2 ECD; nontransfected cells show a low level of endogenous DDR2 while transfected cells show an increased level of DDR2 ECD on the membrane. b) Cell surface biotinylation of MC3T3 cells expressing DDR2/-KD construct, demonstrating surface localization of the truncated receptor. Cell samples were biotinylated (as indicated) and surface proteins were precipitated from the lysates with streptavidin-agarose beads ( s lanes); the supernatant from the pull-down was recovered and probed for intracellular proteins ( i lanes). Following SDS-PAGE, samples were analyzed by Western blotting with antibodies against V5 tag (DDR2/-KD) or ERK2 as indicated. DDR2/-KD is mainly present on the cell surface, while a lower molecular weight band (probably indicating the immature, non glycosylated receptor) is only found in the intracellular lane. The intracellular protein ERK2 is only present in the intracellular lane, demonstrating that biotinylation is specific to cell surface proteins. As an additional control, non-biotinylated samples, show that the streptavidin pull down is specific to biotinylated proteins.

    Journal: Journal of molecular biology

    Article Title: Regulation Of Collagen Fibrillogenesis By Cell Surface Expression Of Kinase Dead DDR2

    doi: 10.1016/j.jmb.2008.10.060

    Figure Lengend Snippet: DDR2/-KD is localized on the plasma membrane. a) Confocal images of HEK293 cells transiently transfected with DDR2/-KD and immunolabeled with an antibody against the DDR2 ECD; nontransfected cells show a low level of endogenous DDR2 while transfected cells show an increased level of DDR2 ECD on the membrane. b) Cell surface biotinylation of MC3T3 cells expressing DDR2/-KD construct, demonstrating surface localization of the truncated receptor. Cell samples were biotinylated (as indicated) and surface proteins were precipitated from the lysates with streptavidin-agarose beads ( s lanes); the supernatant from the pull-down was recovered and probed for intracellular proteins ( i lanes). Following SDS-PAGE, samples were analyzed by Western blotting with antibodies against V5 tag (DDR2/-KD) or ERK2 as indicated. DDR2/-KD is mainly present on the cell surface, while a lower molecular weight band (probably indicating the immature, non glycosylated receptor) is only found in the intracellular lane. The intracellular protein ERK2 is only present in the intracellular lane, demonstrating that biotinylation is specific to cell surface proteins. As an additional control, non-biotinylated samples, show that the streptavidin pull down is specific to biotinylated proteins.

    Article Snippet: To confirm that the biotinylation protocol did not resulted in nonspecific labeling of intracellular proteins, aliquots of the biotinylated surface protein pool and intracellular protein pool were analyzed by Western blotting and probed with antibodies against ERK2 kinase (Invitrogen, Carlsbad, CA), an intracellular protein.

    Techniques: Transfection, Immunolabeling, Expressing, Construct, SDS Page, Western Blot, Molecular Weight

    Pamidronate and clodronate inhibited PI-3K/Akt/mTOR signaling pathways activated by IGF-1 in MCF-7 cells. ( a ) Serum-starved MCF-7 cells were treated with IGF-1 (40 ng/mL) for different time intervals, and phosphorylated ERK1/2, AKT, mTOR, p70S6K and 4E-BP1

    Journal: International journal of cancer. Journal international du cancer

    Article Title: Bisphosphonates suppress insulin-like growth factor 1-induced angiogenesis via the HIF-1?/VEGF signaling pathways in human breast cancer cells

    doi: 10.1002/ijc.24710

    Figure Lengend Snippet: Pamidronate and clodronate inhibited PI-3K/Akt/mTOR signaling pathways activated by IGF-1 in MCF-7 cells. ( a ) Serum-starved MCF-7 cells were treated with IGF-1 (40 ng/mL) for different time intervals, and phosphorylated ERK1/2, AKT, mTOR, p70S6K and 4E-BP1

    Article Snippet: After blocking with TBS/5% nonfat dry milk for 2 h, the membrane was incubated overnight at 4°C with antibodies against HIF-1α, total or phosphorylated ERK1/2 (Thr202 /Tyr204 ) or Akt (Ser473 ), total and phosphorylated mTOR, p70S6K, or 4E-BP1, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000) (Pierce) for 45 minutes at room temperature, and the signals were visualized by enhanced chemiluminescence detection (ECL).

    Techniques:

    Sec35p is a peripheral membrane protein. ( a ) Sec35p partitions into soluble and membrane-associated pools. A wild-type strain was grown to logarithmic phase, lysed with glass beads, and then centrifuged at 1,000 g for 3 min to generate the S1 supernatant fraction. The S1 supernatant was centrifuged at 175,000 g to obtain the S175 supernatant and P175 pellet fractions. ( b ) The membrane-associated portion of Sec35p fractionates similarly to the Golgi protein, Sed5p. The S1 supernatant was centrifuged at 10,000 g to generate the S10 supernatant and P10 pellet fractions, and the S10 was subsequently centrifuged at 175,000 g to obtain the S175 supernatant and P175 pellet fractions. ( c ) Sec35p behaves as a peripheral membrane protein. The S1 supernatant was incubated with buffer 88, 1% Triton X-100, 1 M NaCl, or 100 mM Na 2 CO 3 , pH 11, centrifuged at 175,000 g , and separated into supernatant and pellet fractions. ( a–c ) Aliquots of each fraction were separated by SDS-12%PAGE and immunoblotted with affinity-purified anti-Sec35p ( top panel ), anti-Sed5p ( center panel ), or anti-PGK ( bottom panel ) antibodies. The samples loaded in each lane were derived from equivalent amounts of starting material.

    Journal: The Journal of Cell Biology

    Article Title: Sec35p, a Novel Peripheral Membrane Protein, Is Required for ER to Golgi Vesicle Docking

    doi:

    Figure Lengend Snippet: Sec35p is a peripheral membrane protein. ( a ) Sec35p partitions into soluble and membrane-associated pools. A wild-type strain was grown to logarithmic phase, lysed with glass beads, and then centrifuged at 1,000 g for 3 min to generate the S1 supernatant fraction. The S1 supernatant was centrifuged at 175,000 g to obtain the S175 supernatant and P175 pellet fractions. ( b ) The membrane-associated portion of Sec35p fractionates similarly to the Golgi protein, Sed5p. The S1 supernatant was centrifuged at 10,000 g to generate the S10 supernatant and P10 pellet fractions, and the S10 was subsequently centrifuged at 175,000 g to obtain the S175 supernatant and P175 pellet fractions. ( c ) Sec35p behaves as a peripheral membrane protein. The S1 supernatant was incubated with buffer 88, 1% Triton X-100, 1 M NaCl, or 100 mM Na 2 CO 3 , pH 11, centrifuged at 175,000 g , and separated into supernatant and pellet fractions. ( a–c ) Aliquots of each fraction were separated by SDS-12%PAGE and immunoblotted with affinity-purified anti-Sec35p ( top panel ), anti-Sed5p ( center panel ), or anti-PGK ( bottom panel ) antibodies. The samples loaded in each lane were derived from equivalent amounts of starting material.

    Article Snippet: Affinity-purified antibodies against Sec35p were made as described above, antibodies against Sed5p were generated and affinity purified as described previously , and monoclonal antibodies against phosphoglycerate kinase (PGK) were from Molecular Probes, Inc. (Eugene, OR).

    Techniques: Incubation, Polyacrylamide Gel Electrophoresis, Affinity Purification, Derivative Assay

    Intracellular PGK activity in the metabolism of 4′-Ed4T in Tet-On PGK RKO cells. (A) Expression of PGK in Tet-On PGK RKO cells, as detected by Western blotting. APE-1 was used as the internal control. (B) Doxycycline-induced PGK RKO cells (5 ×

    Journal:

    Article Title: Intracellular Metabolism and Persistence of the Anti-Human Immunodeficiency Virus Activity of 2?,3?-Didehydro-3?-Deoxy-4?-Ethynylthymidine, a Novel Thymidine Analog ▿

    doi: 10.1128/AAC.00692-07

    Figure Lengend Snippet: Intracellular PGK activity in the metabolism of 4′-Ed4T in Tet-On PGK RKO cells. (A) Expression of PGK in Tet-On PGK RKO cells, as detected by Western blotting. APE-1 was used as the internal control. (B) Doxycycline-induced PGK RKO cells (5 ×

    Article Snippet: For PGK overexpression in Tet-On RKO cells , the coding sequence of phosphoglycerate kinase (PGK) was cloned into the pcDNA5/TO vector (Invitrogen Corporation, Carlsbad, CA) with a Tet-responsive element between the 5′ XhoI and 3′ BamHI sites (Invitrogen Corporation, Carlsbad, CA).

    Techniques: Activity Assay, Expressing, Western Blot

    Function of the Hsp31 family in stationary growth phase of cells. (A) Pulse-chase analysis was done with exponentially-growing yeast cells expressing ΔssCL*myc. Cells were lysed at the indicated time points. Proteins were immunoprecipitated, separated by SDS-PAGE and analysed using a Phosphorimager (Storm 860; Molecular Dynamics) and the ImageQuant software (Amersham Biosciences). Plotted data represent the mean values of three independent experiments. Error bars represent the standard error of the mean. (B) Steady state analysis of the amount of the ΔssCL*myc substrate in stationary growth phase. Equal amounts of cells were harvested and cell lysates were subjected to SDS-PAGE followed by immunodetection using c-Myc antibody. As reference protein, 3-phosphoglycerate kinase (PGK) was used. (C) Solubility assays of the misfolded protein ΔssCL*myc expressed in the temperature-sensitive Hsp70 (Ssa) mutant strains Δssa2Δssa3Δssa4ssa1-45 ts and Δssa2Δssa3Δssa4ssa1-45 ts Δubr1 . Cells were grown at 25°C before splitting into two halves. One half of the yeast culture was shifted to 37°C for 1 h prior to harvesting, lysis and fractionation into supernatant (S) and pellet (P) fractions. The total (T) fraction represents the precleared cell lysate. Exponentially growing cells were harvested at an OD 600 value of 1.0 whereas stationary cells were grown 3 days prior to temperature shift, cell lysis and fractionation. The different fractions were subjected to TCA precipitation prior to SDS-PAGE and immunoblotting using c-Myc antibody for substrate detection. PGK served as loading control and reference for a soluble protein. (D) Solubility assays were performed as described for Fig 4C. Strains defective in either Ubr1 or/and the Hsp31 chaperones were used in this assay. The cells were grown at 30°C and harvested either in exponential phase or stationary phase (72h growth), lysed and subjected to fractionation into supernatant (S) and pellet (P) fractions. The samples were subjected to TCA precipitation, SDS-PAGE and immunoblotting using c-Myc antibody. PGK served as loading control and reference for a soluble protein. (E) Growth tests were performed as described earlier. The used strains express the substrate ΔssCL*myc from a HIS3 -marker-containing plasmid (pIA1). In addition, the strains were transformed either with the empty plasmid pRS426 containing a URA3 marker or a pRS426-based plasmid expressing functional histidine-tagged Ssa1 under control of the GPD promoter (pAM25).

    Journal: PLoS ONE

    Article Title: Absence of the Yeast Hsp31 Chaperones of the DJ-1 Superfamily Perturbs Cytoplasmic Protein Quality Control in Late Growth Phase

    doi: 10.1371/journal.pone.0140363

    Figure Lengend Snippet: Function of the Hsp31 family in stationary growth phase of cells. (A) Pulse-chase analysis was done with exponentially-growing yeast cells expressing ΔssCL*myc. Cells were lysed at the indicated time points. Proteins were immunoprecipitated, separated by SDS-PAGE and analysed using a Phosphorimager (Storm 860; Molecular Dynamics) and the ImageQuant software (Amersham Biosciences). Plotted data represent the mean values of three independent experiments. Error bars represent the standard error of the mean. (B) Steady state analysis of the amount of the ΔssCL*myc substrate in stationary growth phase. Equal amounts of cells were harvested and cell lysates were subjected to SDS-PAGE followed by immunodetection using c-Myc antibody. As reference protein, 3-phosphoglycerate kinase (PGK) was used. (C) Solubility assays of the misfolded protein ΔssCL*myc expressed in the temperature-sensitive Hsp70 (Ssa) mutant strains Δssa2Δssa3Δssa4ssa1-45 ts and Δssa2Δssa3Δssa4ssa1-45 ts Δubr1 . Cells were grown at 25°C before splitting into two halves. One half of the yeast culture was shifted to 37°C for 1 h prior to harvesting, lysis and fractionation into supernatant (S) and pellet (P) fractions. The total (T) fraction represents the precleared cell lysate. Exponentially growing cells were harvested at an OD 600 value of 1.0 whereas stationary cells were grown 3 days prior to temperature shift, cell lysis and fractionation. The different fractions were subjected to TCA precipitation prior to SDS-PAGE and immunoblotting using c-Myc antibody for substrate detection. PGK served as loading control and reference for a soluble protein. (D) Solubility assays were performed as described for Fig 4C. Strains defective in either Ubr1 or/and the Hsp31 chaperones were used in this assay. The cells were grown at 30°C and harvested either in exponential phase or stationary phase (72h growth), lysed and subjected to fractionation into supernatant (S) and pellet (P) fractions. The samples were subjected to TCA precipitation, SDS-PAGE and immunoblotting using c-Myc antibody. PGK served as loading control and reference for a soluble protein. (E) Growth tests were performed as described earlier. The used strains express the substrate ΔssCL*myc from a HIS3 -marker-containing plasmid (pIA1). In addition, the strains were transformed either with the empty plasmid pRS426 containing a URA3 marker or a pRS426-based plasmid expressing functional histidine-tagged Ssa1 under control of the GPD promoter (pAM25).

    Article Snippet: For detection of 3-phosphoglycerate kinase (PGK) monoclonal PGK antibody was used in a 1:10,000 dilution (Molecular Probes; clone 22C5).

    Techniques: Pulse Chase, Expressing, Immunoprecipitation, SDS Page, Software, Immunodetection, Solubility, Mutagenesis, Lysis, Fractionation, TCA Precipitation, Marker, Plasmid Preparation, Transformation Assay, Functional Assay

    The α7 C-terminal tail is important for Ecm29 proteasomal association in vivo . ( a , b ) Indicated strains were serially diluted (four-fold) and (a) spotted onto YPD medium and grown at the indicated temperatures for 3 days or (b) spotted on SD plates lacking arginine with or without canvanine and grown at 30 °C for 5 to 6 days. The α 7 -Δ 40 truncation resemble a deletion of ECM29 in the different backgrounds. (c) Equal protein amount of total cell lysates from the indicated strains were analyzed on 3.5% nondenaturing gel and stained using the LLVY-AMC activity assay (upper panel). Protein extracts were also resolved on SDS-PAGE, followed by immuno blotting to determine the levels Ecm29 and Rpn8. Immunoblot against Pgk1 was used as a loading control.

    Journal: Scientific Reports

    Article Title: Phosphorylation of the C-terminal tail of proteasome subunit α7 is required for binding of the proteasome quality control factor Ecm29

    doi: 10.1038/srep27873

    Figure Lengend Snippet: The α7 C-terminal tail is important for Ecm29 proteasomal association in vivo . ( a , b ) Indicated strains were serially diluted (four-fold) and (a) spotted onto YPD medium and grown at the indicated temperatures for 3 days or (b) spotted on SD plates lacking arginine with or without canvanine and grown at 30 °C for 5 to 6 days. The α 7 -Δ 40 truncation resemble a deletion of ECM29 in the different backgrounds. (c) Equal protein amount of total cell lysates from the indicated strains were analyzed on 3.5% nondenaturing gel and stained using the LLVY-AMC activity assay (upper panel). Protein extracts were also resolved on SDS-PAGE, followed by immuno blotting to determine the levels Ecm29 and Rpn8. Immunoblot against Pgk1 was used as a loading control.

    Article Snippet: Anti-α7 monoclonal antibody and anti-Blm10 poly clonal antibody were purchased from Enzo Life Sciences and anti-Pgk1 monoclonal antibody was purchased from Invitrogen.

    Techniques: In Vivo, Staining, Activity Assay, SDS Page

    Binding of Ecm29 to proteasomes depends on the phosphorylation of the C-terminal tail of α7. (a) Schematic overview of α7 truncations and mutations used. A shorter truncation of the α7 C-terminal tail, α 7 -Δ 19 (1–269), was created. This truncation retains the acidic patch as well as three serine phosphorylation sites (S258, S263 and S264). Mutation of these phosphorylation sites to alanine in this background resulted in the α 7 -Δ 19 (S/A) strain. (b) Ecm29 levels in indicated strains was analyzed by immunoblotting of whole cell lysates using the anti-Ecm29 antibody and an anti-Pgk1 antibody as loading control. ( c,d ) Proteasome complexes were purified from the indicated strains and equal amount of purified proteasomes were analyzed on the SDS-PAGE and Coomassie Blue stained or used for immunoblotting to determine level of Ecm29. Immublots against α7 were used to confirm truncations and immunoblots agains Rpn8 served as loading control. To determine phosphorylation state, samples resolved by SDS-PAGE were stained with Pro-Q Diamond Phosphoprotein Gel Staining assay (right panels).

    Journal: Scientific Reports

    Article Title: Phosphorylation of the C-terminal tail of proteasome subunit α7 is required for binding of the proteasome quality control factor Ecm29

    doi: 10.1038/srep27873

    Figure Lengend Snippet: Binding of Ecm29 to proteasomes depends on the phosphorylation of the C-terminal tail of α7. (a) Schematic overview of α7 truncations and mutations used. A shorter truncation of the α7 C-terminal tail, α 7 -Δ 19 (1–269), was created. This truncation retains the acidic patch as well as three serine phosphorylation sites (S258, S263 and S264). Mutation of these phosphorylation sites to alanine in this background resulted in the α 7 -Δ 19 (S/A) strain. (b) Ecm29 levels in indicated strains was analyzed by immunoblotting of whole cell lysates using the anti-Ecm29 antibody and an anti-Pgk1 antibody as loading control. ( c,d ) Proteasome complexes were purified from the indicated strains and equal amount of purified proteasomes were analyzed on the SDS-PAGE and Coomassie Blue stained or used for immunoblotting to determine level of Ecm29. Immublots against α7 were used to confirm truncations and immunoblots agains Rpn8 served as loading control. To determine phosphorylation state, samples resolved by SDS-PAGE were stained with Pro-Q Diamond Phosphoprotein Gel Staining assay (right panels).

    Article Snippet: Anti-α7 monoclonal antibody and anti-Blm10 poly clonal antibody were purchased from Enzo Life Sciences and anti-Pgk1 monoclonal antibody was purchased from Invitrogen.

    Techniques: Binding Assay, Mutagenesis, Purification, SDS Page, Staining, Western Blot

    ( A ) The erb1 [R470E] mutation confers slow growth and temperature sensitivity. A null erb1 strain containing YCplac111 plasmid-borne wild-type ERB1 or the erb1 [R470E] allele were grown in liquid YPD, diluted to an OD 600 of 0.05 and spotted in 10-fold serial dilutions onto YPD plates. Plates were incubated at 30°C and 37°C for 3 days. ( B ) The amounts of Erb1[R470E], Nop7 and Ytm1 proteins are reduced in the erb1 [R470E] mutant. Whole cell extracts were prepared from the indicated strains, growing in exponential phase at 30°C and 37°C in YPD. Equivalent amounts of extracts were analyzed by western blotting using antibodies against Erb1, Nop7, Ytm1, L1 and Pgk1. ( C ) The erb1 [R470E] mutation results in a deficit in 60S r-subunits. The indicated strains were grown in YPD at 37°C to exponential phase. Cell extracts were prepared and 10 A 260 of each extract was resolved on 7–50% sucrose gradients. The A 254 was continuously measured. Sedimentation is from left to right. The peaks of free 40S, 60S r-subunits, 80S vacant ribosomes/monosomes and polysomes are indicated. Half-mers are labeled by arrows.

    Journal: Nucleic Acids Research

    Article Title: The structure of Erb1-Ytm1 complex reveals the functional importance of a high-affinity binding between two β-propellers during the assembly of large ribosomal subunits in eukaryotes

    doi: 10.1093/nar/gkv1043

    Figure Lengend Snippet: ( A ) The erb1 [R470E] mutation confers slow growth and temperature sensitivity. A null erb1 strain containing YCplac111 plasmid-borne wild-type ERB1 or the erb1 [R470E] allele were grown in liquid YPD, diluted to an OD 600 of 0.05 and spotted in 10-fold serial dilutions onto YPD plates. Plates were incubated at 30°C and 37°C for 3 days. ( B ) The amounts of Erb1[R470E], Nop7 and Ytm1 proteins are reduced in the erb1 [R470E] mutant. Whole cell extracts were prepared from the indicated strains, growing in exponential phase at 30°C and 37°C in YPD. Equivalent amounts of extracts were analyzed by western blotting using antibodies against Erb1, Nop7, Ytm1, L1 and Pgk1. ( C ) The erb1 [R470E] mutation results in a deficit in 60S r-subunits. The indicated strains were grown in YPD at 37°C to exponential phase. Cell extracts were prepared and 10 A 260 of each extract was resolved on 7–50% sucrose gradients. The A 254 was continuously measured. Sedimentation is from left to right. The peaks of free 40S, 60S r-subunits, 80S vacant ribosomes/monosomes and polysomes are indicated. Half-mers are labeled by arrows.

    Article Snippet: As a loading control for whole cell extracts, monoclonal anti-Pgk1 antibodies (Invitrogen) were used.

    Techniques: Mutagenesis, Plasmid Preparation, Incubation, Western Blot, Sedimentation, Labeling

    Sucrose density gradient fractionation of cells (YTS3) expressing Ha-tagged Gid2p from the chromosome. After 16 h of growth on ethanol medium, the cells were shifted to glucose medium. The cells were analyzed before (A) and 30 min after shift to glucose medium (B). From the sucrose gradient, fractions were collected from the top of the gradient (fraction 1) and analyzed by immunoblotting with antibodies against Ha (Ha-tagged Gid2p), cytosolic PGK, Fas (cytosolic fatty acid synthase), CPY (vacuolar carboxypeptidase yscY); vacuolar API, and ER lumenal Kar2p. The fractions were further tested for their Guanosine diphosphatase (Golgi) activity. Further details are given in the text.

    Journal: Molecular Biology of the Cell

    Article Title: Catabolite Degradation of Fructose-1,6-bisphosphatase in the Yeast Saccharomyces cerevisiae: A Genome-wide Screen Identifies Eight Novel GID Genes and Indicates the Existence of Two Degradation Pathways

    doi: 10.1091/mbc.E02-08-0456

    Figure Lengend Snippet: Sucrose density gradient fractionation of cells (YTS3) expressing Ha-tagged Gid2p from the chromosome. After 16 h of growth on ethanol medium, the cells were shifted to glucose medium. The cells were analyzed before (A) and 30 min after shift to glucose medium (B). From the sucrose gradient, fractions were collected from the top of the gradient (fraction 1) and analyzed by immunoblotting with antibodies against Ha (Ha-tagged Gid2p), cytosolic PGK, Fas (cytosolic fatty acid synthase), CPY (vacuolar carboxypeptidase yscY); vacuolar API, and ER lumenal Kar2p. The fractions were further tested for their Guanosine diphosphatase (Golgi) activity. Further details are given in the text.

    Article Snippet: After immunoblotting, samples were analyzed with antibodies against phosphoglycerate kinase (PGK), carboxypeptidase yscY (CPY) (Molecular Probes, Leiden, The Netherlands), Kar2p (R. Scheckman, Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California, Berkeley, CA), and aminopeptidase I (API) ( ).

    Techniques: Fractionation, Expressing, Activity Assay

    BTN2 expression under mild ethanol stress. Cells carrying a FLAG-tagged chromosomal copy of BTN2 gene in the exponential growth phase were treated with the indicated concentrations of ethanol for 60 min. (A) BTN2 mRNA level was determined by performing qRT-PCR and was normalized to that of ACT1 . (B) Btn2 protein level was determined by performing Western blotting with anti-FLAG antibody. Pgk1 was used as the loading control. Btn2 protein level was normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are expressed as mean ± SD ( n = 3).

    Journal: Frontiers in Microbiology

    Article Title: Prioritized Expression of BTN2 of Saccharomyces cerevisiae under Pronounced Translation Repression Induced by Severe Ethanol Stress

    doi: 10.3389/fmicb.2016.01319

    Figure Lengend Snippet: BTN2 expression under mild ethanol stress. Cells carrying a FLAG-tagged chromosomal copy of BTN2 gene in the exponential growth phase were treated with the indicated concentrations of ethanol for 60 min. (A) BTN2 mRNA level was determined by performing qRT-PCR and was normalized to that of ACT1 . (B) Btn2 protein level was determined by performing Western blotting with anti-FLAG antibody. Pgk1 was used as the loading control. Btn2 protein level was normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are expressed as mean ± SD ( n = 3).

    Article Snippet: Pgk1 was used as a loading control, and its level was monitored using a monoclonal anti-Pgk1 primary antibody (22C5D8; dilution, 1:4,000; Life Technologies, Frederick, MD, USA).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    BTN2 was preferentially expressed under severe ethanol stress. Yeast cells carrying a FLAG-tagged chromosomal copy of BTN2 in the exponential growth phase were treated with 9 or 10% (v/v) ethanol (severe ethanol stress) for the indicated time (A–D) or treated with 9 or 10% (v/v) ethanol and 0.1 mg/ml cycloheximide (CHX) for 60 min (E) . (A,C) BTN2 mRNA level was determined by performing qRT-PCR and was normalized to that of ACT1 . (B,D,E) Btn2 protein level was determined by performing Western blotting with anti-FLAG antibody. Pgk1 was used as the loading control. Btn2 protein level was normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are expressed as mean ± SD ( n = 3). ND, not detected.

    Journal: Frontiers in Microbiology

    Article Title: Prioritized Expression of BTN2 of Saccharomyces cerevisiae under Pronounced Translation Repression Induced by Severe Ethanol Stress

    doi: 10.3389/fmicb.2016.01319

    Figure Lengend Snippet: BTN2 was preferentially expressed under severe ethanol stress. Yeast cells carrying a FLAG-tagged chromosomal copy of BTN2 in the exponential growth phase were treated with 9 or 10% (v/v) ethanol (severe ethanol stress) for the indicated time (A–D) or treated with 9 or 10% (v/v) ethanol and 0.1 mg/ml cycloheximide (CHX) for 60 min (E) . (A,C) BTN2 mRNA level was determined by performing qRT-PCR and was normalized to that of ACT1 . (B,D,E) Btn2 protein level was determined by performing Western blotting with anti-FLAG antibody. Pgk1 was used as the loading control. Btn2 protein level was normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are expressed as mean ± SD ( n = 3). ND, not detected.

    Article Snippet: Pgk1 was used as a loading control, and its level was monitored using a monoclonal anti-Pgk1 primary antibody (22C5D8; dilution, 1:4,000; Life Technologies, Frederick, MD, USA).

    Techniques: Quantitative RT-PCR, Western Blot

    Btn2 protein level decreased during recovery from severe ethanol stress. Cells carrying a FLAG-tagged chromosomal copy of BTN2 gene in the exponential growth phase were treated with severe ethanol stress (10% v/v) for 60 min. After the treatment with ethanol stress, cells were transferred to a fresh SD medium lacking ethanol (A,B) or containing 5% ethanol (C,D) and were incubated further at 28°C for the indicated time. (A,C) BTN2 mRNA level was determined by performing qRT-PCR and was normalized to that of ACT1 . (B,D) Btn2 protein level was determined by performing Western blotting with anti-FLAG antibody. Pgk1 was used as the loading control. Btn2 protein level was normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are expressed as mean ± SD ( n = 3).

    Journal: Frontiers in Microbiology

    Article Title: Prioritized Expression of BTN2 of Saccharomyces cerevisiae under Pronounced Translation Repression Induced by Severe Ethanol Stress

    doi: 10.3389/fmicb.2016.01319

    Figure Lengend Snippet: Btn2 protein level decreased during recovery from severe ethanol stress. Cells carrying a FLAG-tagged chromosomal copy of BTN2 gene in the exponential growth phase were treated with severe ethanol stress (10% v/v) for 60 min. After the treatment with ethanol stress, cells were transferred to a fresh SD medium lacking ethanol (A,B) or containing 5% ethanol (C,D) and were incubated further at 28°C for the indicated time. (A,C) BTN2 mRNA level was determined by performing qRT-PCR and was normalized to that of ACT1 . (B,D) Btn2 protein level was determined by performing Western blotting with anti-FLAG antibody. Pgk1 was used as the loading control. Btn2 protein level was normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are expressed as mean ± SD ( n = 3).

    Article Snippet: Pgk1 was used as a loading control, and its level was monitored using a monoclonal anti-Pgk1 primary antibody (22C5D8; dilution, 1:4,000; Life Technologies, Frederick, MD, USA).

    Techniques: Incubation, Quantitative RT-PCR, Western Blot

    The BTN2 promoter region induced the protein synthesis of non-native genes under severe ethanol stress. (A,B) Cells harboring the pYY plasmid series ( BTN2 promoter-driven expression system) in the exponential growth phase were treated with 9 or 10% ethanol for the indicated time. (A) The mRNA levels of each FLAG-tagged gene were analyzed by performing qRT-PCR and were normalized to that of ACT1 . (B) Protein levels of Cur1-FLAG, Gic2-FLAG, and Yur1-FLAG were determined by performing Western blotting with anti-FLAG antibody. (C) The BTN2 terminator region had a negligible effect on preferential translation upon severe ethanol stress. Cells carrying YIp- BTN2-FLAG-ADH6 Ter or YIp- BTN2-FLAG-TEF1 Ter in the exponential phase of growth were treated with 9 or 10% ethanol for 1 h. Protein levels of Btn2-FLAG were determined by Western blot analysis using an anti-FLAG antibody. Pgk1 was used as the loading control. Their protein levels were normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are shown as mean ± SD ( n = 3). Ctrl, control cells not treated with ethanol stress.

    Journal: Frontiers in Microbiology

    Article Title: Prioritized Expression of BTN2 of Saccharomyces cerevisiae under Pronounced Translation Repression Induced by Severe Ethanol Stress

    doi: 10.3389/fmicb.2016.01319

    Figure Lengend Snippet: The BTN2 promoter region induced the protein synthesis of non-native genes under severe ethanol stress. (A,B) Cells harboring the pYY plasmid series ( BTN2 promoter-driven expression system) in the exponential growth phase were treated with 9 or 10% ethanol for the indicated time. (A) The mRNA levels of each FLAG-tagged gene were analyzed by performing qRT-PCR and were normalized to that of ACT1 . (B) Protein levels of Cur1-FLAG, Gic2-FLAG, and Yur1-FLAG were determined by performing Western blotting with anti-FLAG antibody. (C) The BTN2 terminator region had a negligible effect on preferential translation upon severe ethanol stress. Cells carrying YIp- BTN2-FLAG-ADH6 Ter or YIp- BTN2-FLAG-TEF1 Ter in the exponential phase of growth were treated with 9 or 10% ethanol for 1 h. Protein levels of Btn2-FLAG were determined by Western blot analysis using an anti-FLAG antibody. Pgk1 was used as the loading control. Their protein levels were normalized to that of Pgk1, and the intensity of the Pgk1 band in each lane was set at 100%. Data are shown as mean ± SD ( n = 3). Ctrl, control cells not treated with ethanol stress.

    Article Snippet: Pgk1 was used as a loading control, and its level was monitored using a monoclonal anti-Pgk1 primary antibody (22C5D8; dilution, 1:4,000; Life Technologies, Frederick, MD, USA).

    Techniques: Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot

    PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe ThiolTracker Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p

    Journal: Neurobiology of Disease

    Article Title: Mitochondrial iron and energetic dysfunction distinguish fibroblasts and induced neurons from pantothenate kinase-associated neurodegeneration patients

    doi: 10.1016/j.nbd.2015.02.030

    Figure Lengend Snippet: PKAN fibroblasts show altered oxidative status. (A) The determination of the PANK2 protein content in fibroblast soluble extracts. The arrows point to PANK2 mature peptide (47 kDa) and to SDH70 used as loading control. The other lower band, present in patients and controls, is a non-specific reaction of the antibody. (B) The fibroblasts were treated or not with 100 μM deferoxamine (DFO) for 18 h, and the levels of carbonylated proteins in soluble cell extracts were then analysed by Oxyblot. One representative of three independent experiments is shown. (C) Measurements of total and reduced glutathione content in fibroblasts evaluated using the specific fluorescent probe ThiolTracker Violet. The images were acquired by fluorescence microscopy, and the quantified signal, relative to controls, was plotted as the mean and standard deviation of three independent experiments in triplicate. **p

    Article Snippet: Glutathione measurement Fibroblasts, treated or not with 1 mM TCEP (tris[2-carboxyethyl]phosphine) for 30 min at 37 °C, were incubated with 20 μM ThiolTracker Violet (Invitrogen) for 30 min at 37 °C, washed with PBS and fixed in 4% paraformaldehyde in PBS for 20 min at room temperature.

    Techniques: Fluorescence, Microscopy, Standard Deviation

    Expression of GIPC(LG > AE) inhibits NGF-induced neurite outgrowth and MAP kinase activation. (A) Bar graph showing the percentages of cells that have differentiated cells expressing different GIPC constructs. In nontransfected cells that were not stimulated with NGF (−), only 4% of the cells differentiated and grew out at least one neurite. After NGF treatment (+), 50% of the nontransfected cells, 41% of the mock-transfected cells (empty vector), and 37% of the GIPC-transfected cells differentiated, whereas in the cells transfected with the GIPC(LG > AE) mutant, only 27% of the cells differentiated. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG > AE), incubated with NGF (50 ng/ml) for 16 h, fixed, permeabilized, and incubated with anti-FLAG to label transfected cells. Cells were scored for differentiation (neurite outgrowth) as described in Materials and Methods. Average differentiation rates (±standard errors) are for six experiments (≥50 cells/experiment). **, P ≤ 0.01. (B) Overexpression of the FLAG-tagged GIPC(LG > AE) mutant strongly inhibits NGF-induced Erk1/2 activation. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG > AE), serum starved, treated with NGF (100 ng/ml) for 5 min, and lysed for immunoblotting with anti-pErk1/2 antibody. Per lane, 20 μg protein was loaded; the levels of total Erk1/2 (bottom) are the same in all samples. (C) Quantification of the pErk1/2 levels from panel B, shown as normalized percentages of the mock control.

    Journal: Molecular and Cellular Biology

    Article Title: GIPC Is Recruited by APPL to Peripheral TrkA Endosomes and Regulates TrkA Trafficking and Signaling ▿

    doi: 10.1128/MCB.00305-06

    Figure Lengend Snippet: Expression of GIPC(LG > AE) inhibits NGF-induced neurite outgrowth and MAP kinase activation. (A) Bar graph showing the percentages of cells that have differentiated cells expressing different GIPC constructs. In nontransfected cells that were not stimulated with NGF (−), only 4% of the cells differentiated and grew out at least one neurite. After NGF treatment (+), 50% of the nontransfected cells, 41% of the mock-transfected cells (empty vector), and 37% of the GIPC-transfected cells differentiated, whereas in the cells transfected with the GIPC(LG > AE) mutant, only 27% of the cells differentiated. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG > AE), incubated with NGF (50 ng/ml) for 16 h, fixed, permeabilized, and incubated with anti-FLAG to label transfected cells. Cells were scored for differentiation (neurite outgrowth) as described in Materials and Methods. Average differentiation rates (±standard errors) are for six experiments (≥50 cells/experiment). **, P ≤ 0.01. (B) Overexpression of the FLAG-tagged GIPC(LG > AE) mutant strongly inhibits NGF-induced Erk1/2 activation. PC12(615) cells were transfected with empty vector, FLAG-tagged GIPC, or GIPC(LG > AE), serum starved, treated with NGF (100 ng/ml) for 5 min, and lysed for immunoblotting with anti-pErk1/2 antibody. Per lane, 20 μg protein was loaded; the levels of total Erk1/2 (bottom) are the same in all samples. (C) Quantification of the pErk1/2 levels from panel B, shown as normalized percentages of the mock control.

    Article Snippet: Anti-MAP kinase (Erk1/2) MAbs were purchased from Zymed Laboratories (San Francisco, CA).

    Techniques: Expressing, Activation Assay, Construct, Transfection, Plasmid Preparation, Mutagenesis, Incubation, Over Expression