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  • 99
    Thermo Fisher whole cell extraction buffer
    Whole Cell Extraction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    whole cell extraction buffer - by Bioz Stars, 2020-05
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    99
    Millipore yeast whole cell extracts
    Yeast Whole Cell Extracts, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/yeast whole cell extracts/product/Millipore
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    yeast whole cell extracts - by Bioz Stars, 2020-05
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    85
    Active Motif hela whole cell extracts
    Hela Whole Cell Extracts, supplied by Active Motif, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam whole cell extract kit
    Whole Cell Extract Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore hela whole cell extracts
    Hela Whole Cell Extracts, supplied by Millipore, 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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    94
    Millipore imr 90 whole cell extracts
    Proliferative capacity of RECQ1-depleted glioblastoma cells . A) Western blot analysis of T98G, U-87 and <t>IMR-90</t> cell lines transiently transfected with an siRNA against RECQ1. L indicates the Luciferase siRNA control, R the siRNA against RECQ1. B) Clonogenic assays performed in RECQ1-depleted T98G, U-87, and IMR-90 cell lines. Pictures show colonies formed after seeding 800 cells. C) Graphs showing the plating efficiencies expressed as colony forming capacity. Values represent the average ratio of the number of formed colonies to the number of cells seeded, expressed as percentage.
    Imr 90 Whole Cell Extracts, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore antibodies whole cell extracts
    Total FcγRI, RIIB, and RIII protein in MɸN and MɸP <t>cell</t> <t>extracts.</t> ( A ) Cells from three different passages of MɸN and cells from passages 72, 83 and 87 of MɸP were lysed and FcγRI, FcγRIIB, FcγRIII and GAPDH proteins in cell extracts were determined by <t>Western</t> <t>blot</t> with specific antibodies; ( B ) The relative amount of each FcγR isoform was determined as the ratio of the densitometric intensity of the FcγR band to the intensity of the GAPDH band in the correspondent cell extract. Results are expressed as mean ± 1 SD (* indicates p
    Antibodies Whole Cell Extracts, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Beckman Coulter final whole cell extracts
    Total FcγRI, RIIB, and RIII protein in MɸN and MɸP <t>cell</t> <t>extracts.</t> ( A ) Cells from three different passages of MɸN and cells from passages 72, 83 and 87 of MɸP were lysed and FcγRI, FcγRIIB, FcγRIII and GAPDH proteins in cell extracts were determined by <t>Western</t> <t>blot</t> with specific antibodies; ( B ) The relative amount of each FcγR isoform was determined as the ratio of the densitometric intensity of the FcγR band to the intensity of the GAPDH band in the correspondent cell extract. Results are expressed as mean ± 1 SD (* indicates p
    Final Whole Cell Extracts, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Active Motif whole cell extracts wce
    Total FcγRI, RIIB, and RIII protein in MɸN and MɸP <t>cell</t> <t>extracts.</t> ( A ) Cells from three different passages of MɸN and cells from passages 72, 83 and 87 of MɸP were lysed and FcγRI, FcγRIIB, FcγRIII and GAPDH proteins in cell extracts were determined by <t>Western</t> <t>blot</t> with specific antibodies; ( B ) The relative amount of each FcγR isoform was determined as the ratio of the densitometric intensity of the FcγR band to the intensity of the GAPDH band in the correspondent cell extract. Results are expressed as mean ± 1 SD (* indicates p
    Whole Cell Extracts Wce, supplied by Active Motif, used in various techniques. Bioz Stars score: 89/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore immunochemical methods whole cell extracts
    Total FcγRI, RIIB, and RIII protein in MɸN and MɸP <t>cell</t> <t>extracts.</t> ( A ) Cells from three different passages of MɸN and cells from passages 72, 83 and 87 of MɸP were lysed and FcγRI, FcγRIIB, FcγRIII and GAPDH proteins in cell extracts were determined by <t>Western</t> <t>blot</t> with specific antibodies; ( B ) The relative amount of each FcγR isoform was determined as the ratio of the densitometric intensity of the FcγR band to the intensity of the GAPDH band in the correspondent cell extract. Results are expressed as mean ± 1 SD (* indicates p
    Immunochemical Methods Whole Cell Extracts, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    GE Healthcare whole protein cell extracts
    Total FcγRI, RIIB, and RIII protein in MɸN and MɸP <t>cell</t> <t>extracts.</t> ( A ) Cells from three different passages of MɸN and cells from passages 72, 83 and 87 of MɸP were lysed and FcγRI, FcγRIIB, FcγRIII and GAPDH proteins in cell extracts were determined by <t>Western</t> <t>blot</t> with specific antibodies; ( B ) The relative amount of each FcγR isoform was determined as the ratio of the densitometric intensity of the FcγR band to the intensity of the GAPDH band in the correspondent cell extract. Results are expressed as mean ± 1 SD (* indicates p
    Whole Protein Cell Extracts, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher western blotting whole cell extracts
    Effects of 8-Cl-Ado on DNA relaxation and on p53, p21 and p53R2 expression. ( A ) A549 and H1299 <t>cells</t> were exposed to 2 μM 8-Cl-Ado for 48 h, and nuclear <t>extracts</t> (NE) were prepared. Relaxation activities in NE were tested by incubating with supercoiled pUC19 DNA in the reaction conditions as indicated on the top. After ethanol precipitated, extracted DNA samples were subjected to 1% agarose gel electrophoresis. The pUC19 DNA is used as markers for supercoiled and relaxed DNA; ( B , C ) <t>Western</t> <t>blotting</t> for p53, p21 and p53R2 expression. β-Actin as a loading control. The numbers below the blots and histograms in lower panels show the relative levels of p53, p21 and p53R2 in Western blotting. The ratio of target protein/Actin from control cells was designated as “1”. * p
    Western Blotting Whole Cell Extracts, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology 293t whole cell extracts
    Treatment of immune cells with filtered and unfiltered Ebola-transfected cell supernatants. <t>293T</t> cells were transfected with either VP40, GP, or NP plasmids with Attractene Transfection Reagent (Qiagen) as per the manufacturer’s instructions and incubated for 2 days. Transfection complexes were removed and cells were treated with antibiotics for plasmid selection for 14 days. Transfection supernatants were either left unfiltered or passed through a 0.22 micron filter. Control 293T cells were not transfected nor treated with antibiotics (Hygromycin B, Zeocin, or Geneticin). Seventy-five microliters (0.45 mU AChE) of both filtered and unfiltered supernatants were used to treat CEM (A) , Jurkat (B) and U937 (C) cells, which were subsequently assayed for cell viability via CellTiter-Glo after 4 days of incubation. Statistical significance was determined using 2-tailed student’s t -test, with filtered or unfiltered experimental values compared to the Control values of like type. (D) U937 cells were treated with PMA (50 nM) for 5 days to stimulate differentiation into macrophages. On day 5 media was replaced and differentiated U937 macrophages were treated with either E. coli -purified free VP40 protein, or supernatants from VP40-transfected 293T cells (filtered and unfiltered). Untreated macrophages received no external supernatant treatment. Cells were incubated for an additional 5 days and cell viability was assayed with CellTiter-Glo. (E) Primary monocytes from 3 healthy donors were treated with PMA (10 nM) for 2 days to stimulate differentiation into macrophages. Primary macrophages were then treated with supernatants from either VP40-transfected 293T cells (filtered and unfiltered), or from VP40-resistant clone EVTR2C cells (filtered). Control primary macrophages were treated with filtered supernatant from normal 293T cells. Cells were incubated for 2 days before being assayed for viability with CellTiter-Glo. Statistical significance was determined using 2-tailed student’s t -test, with filtered or unfiltered experimental groups compared to the Control groups of like type. MØ, Macrophage; RLU, Relative Luminescent Units. ∗ p
    293t Whole Cell Extracts, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore hek293 whole cell extracts
    Activation of the Gαq signaling pathway promotes calpain-mediated β-catenin proteolysis in a Ca 2+ -dependent manner. ( a ) The inhibitory effect of the Gq signaling pathway on TCF/LEF-1-mediated transcription is not mediated by PKC. SW480 cells were transfected with empty plasmid as control, Q209L-Gαq, or M3R expression plasmids. PKC inhibitor bisindolylmaleimide I (1 μM) was added on transfection and carbachol (1 mM) was added 24 h posttransfection. Relative TCF/LEF-1 activity was assayed 48 h after transfection. ( b ) Thapsigargin mimics the effect of Q209L–Gαq. SW480 cells were transfected with indicated expression plasmids. Thapsigargin (50 nM) was added on transfection. Relative TCF/LEF-1 activity was assayed 48 h after transfection. ( c ) Q209L–Gαq induces reduction in cytosolic β-catenin levels. SW480 cells were transfected with empty plasmid as control or Q209L–Gαq expression plasmid. Cycloheximide (25 μg/ml) was added 24 h after transfection. Cells were harvested at the indicated times after cycloheximide addition. Cytosolic fraction was prepared, resolved by SDS/PAGE, and blotted with antibody specific against the C terminus of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. ( d ) Q209L–Gαq induces cleavage of cytosolic β-catenin. Cytosolic fractions of SW480 cells were prepared at the indicated time points after cycloheximide treatment, electrophoretically resolved, and blotted with β-catenin antibody specific for the C terminus. Proteolytic products of β-catenin are indicated by arrowheads. ( e ) Thapsigargin induces calpain-mediated proteolysis of β-catenin in a calcium-dependent manner. SW480 cells were pretreated with DMSO or inhibitors for 10 min, and then treated with thapsigargin (50 nM) for 30 min. The untreated cells were used as control. The concentrations of the various inhibitors were BAPTA/AM (40 μM), NH 4 Cl (1 mM), calpeptin (50 μM), calpastatin (10 μM), lactacystin (10 μM), and MG-132 (10 μM). Whole-cell extract was prepared, resolved by SDS/PAGE, and blotted with the C-terminal β-catenin antibody. Proteolytic products of β-catenin are indicated by arrowheads. ( f ) Activation of Gq-coupled M3R induces calpain-mediated proteolysis of β-catenin. SW480 cells stably expressing M3R were treated with carbachol (1 mM), lactacystin (10 μM), or calpeptin (10 μM) as indicated. Whole-cell lysates were prepared at the indicated time points, and β-catenin fragments were detected by antibody specific for the C terminus of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. ( g and h ) Calpain-mediated cleavage occurs at the N-terminal region of β-catenin. As described in Materials and Methods , <t>HEK293</t> cell extracts were incubated at 37°C for 30 min alone as control or with CaCl 2 (0.1 mM) and μ-calpain in the presence of the indicated reagents: EGTA (1 mM), ALLN (10 μM), ALLM (10 μM), E-64 (25 μM), calpastatin (10 μM), lactacystin (10 μM), and MG-132 (10 μM). Cleavage of β-catenin was assessed with antibodies specific for the N-terminal or C-terminal regions of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. WB, Western blot; NT, N terminus; CT, C terminus.
    Hek293 Whole Cell Extracts, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore skeletal muscle whole cell extracts
    Activation of the Gαq signaling pathway promotes calpain-mediated β-catenin proteolysis in a Ca 2+ -dependent manner. ( a ) The inhibitory effect of the Gq signaling pathway on TCF/LEF-1-mediated transcription is not mediated by PKC. SW480 cells were transfected with empty plasmid as control, Q209L-Gαq, or M3R expression plasmids. PKC inhibitor bisindolylmaleimide I (1 μM) was added on transfection and carbachol (1 mM) was added 24 h posttransfection. Relative TCF/LEF-1 activity was assayed 48 h after transfection. ( b ) Thapsigargin mimics the effect of Q209L–Gαq. SW480 cells were transfected with indicated expression plasmids. Thapsigargin (50 nM) was added on transfection. Relative TCF/LEF-1 activity was assayed 48 h after transfection. ( c ) Q209L–Gαq induces reduction in cytosolic β-catenin levels. SW480 cells were transfected with empty plasmid as control or Q209L–Gαq expression plasmid. Cycloheximide (25 μg/ml) was added 24 h after transfection. Cells were harvested at the indicated times after cycloheximide addition. Cytosolic fraction was prepared, resolved by SDS/PAGE, and blotted with antibody specific against the C terminus of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. ( d ) Q209L–Gαq induces cleavage of cytosolic β-catenin. Cytosolic fractions of SW480 cells were prepared at the indicated time points after cycloheximide treatment, electrophoretically resolved, and blotted with β-catenin antibody specific for the C terminus. Proteolytic products of β-catenin are indicated by arrowheads. ( e ) Thapsigargin induces calpain-mediated proteolysis of β-catenin in a calcium-dependent manner. SW480 cells were pretreated with DMSO or inhibitors for 10 min, and then treated with thapsigargin (50 nM) for 30 min. The untreated cells were used as control. The concentrations of the various inhibitors were BAPTA/AM (40 μM), NH 4 Cl (1 mM), calpeptin (50 μM), calpastatin (10 μM), lactacystin (10 μM), and MG-132 (10 μM). Whole-cell extract was prepared, resolved by SDS/PAGE, and blotted with the C-terminal β-catenin antibody. Proteolytic products of β-catenin are indicated by arrowheads. ( f ) Activation of Gq-coupled M3R induces calpain-mediated proteolysis of β-catenin. SW480 cells stably expressing M3R were treated with carbachol (1 mM), lactacystin (10 μM), or calpeptin (10 μM) as indicated. Whole-cell lysates were prepared at the indicated time points, and β-catenin fragments were detected by antibody specific for the C terminus of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. ( g and h ) Calpain-mediated cleavage occurs at the N-terminal region of β-catenin. As described in Materials and Methods , <t>HEK293</t> cell extracts were incubated at 37°C for 30 min alone as control or with CaCl 2 (0.1 mM) and μ-calpain in the presence of the indicated reagents: EGTA (1 mM), ALLN (10 μM), ALLM (10 μM), E-64 (25 μM), calpastatin (10 μM), lactacystin (10 μM), and MG-132 (10 μM). Cleavage of β-catenin was assessed with antibodies specific for the N-terminal or C-terminal regions of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. WB, Western blot; NT, N terminus; CT, C terminus.
    Skeletal Muscle Whole Cell Extracts, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore detection yeast whole cell extracts
    PRX Tsa1 Undergoes Cycles of Oxidation across the YRO (A) Three PRXs in the <t>yeast</t> S. cerevisiae have the nine amino acid sequence recognized by the PRX-SO 2/3 antisera when over-oxidized. (B) Western blot showing that over-oxidized Tsa1 is recognized by the PRX-SO 2/3 antisera in a redox-dependent manner. Samples were harvested immediately before (−) or 15 min after (+) the addition of hydrogen peroxide (1 mM final concentration). The asterisk marks a cross-reacting band. (C) YROs were obtained using 0.1 or 0.12 dilutions/hr (sampled every 20 or 12 min, respectively) and monitored using the dissolved oxygen trace (top). Bottom: <t>whole-cell</t> <t>extracts</t> obtained from samples taken across the oscillation were analyzed for PRX over-oxidation by western blotting. (D) Grouped data showing the normalized PRX-SO 2/3 intensity over the YRO, peaking around late OX phase (mean ± SEM, n = 3, p = 0.024 for time effect by two-way ANOVA).
    Detection Yeast Whole Cell Extracts, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    CEM Corporation cem whole cell extracts
    PRX Tsa1 Undergoes Cycles of Oxidation across the YRO (A) Three PRXs in the <t>yeast</t> S. cerevisiae have the nine amino acid sequence recognized by the PRX-SO 2/3 antisera when over-oxidized. (B) Western blot showing that over-oxidized Tsa1 is recognized by the PRX-SO 2/3 antisera in a redox-dependent manner. Samples were harvested immediately before (−) or 15 min after (+) the addition of hydrogen peroxide (1 mM final concentration). The asterisk marks a cross-reacting band. (C) YROs were obtained using 0.1 or 0.12 dilutions/hr (sampled every 20 or 12 min, respectively) and monitored using the dissolved oxygen trace (top). Bottom: <t>whole-cell</t> <t>extracts</t> obtained from samples taken across the oscillation were analyzed for PRX over-oxidation by western blotting. (D) Grouped data showing the normalized PRX-SO 2/3 intensity over the YRO, peaking around late OX phase (mean ± SEM, n = 3, p = 0.024 for time effect by two-way ANOVA).
    Cem Whole Cell Extracts, supplied by CEM Corporation, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Active Motif whole cell extract kit
    PRX Tsa1 Undergoes Cycles of Oxidation across the YRO (A) Three PRXs in the <t>yeast</t> S. cerevisiae have the nine amino acid sequence recognized by the PRX-SO 2/3 antisera when over-oxidized. (B) Western blot showing that over-oxidized Tsa1 is recognized by the PRX-SO 2/3 antisera in a redox-dependent manner. Samples were harvested immediately before (−) or 15 min after (+) the addition of hydrogen peroxide (1 mM final concentration). The asterisk marks a cross-reacting band. (C) YROs were obtained using 0.1 or 0.12 dilutions/hr (sampled every 20 or 12 min, respectively) and monitored using the dissolved oxygen trace (top). Bottom: <t>whole-cell</t> <t>extracts</t> obtained from samples taken across the oscillation were analyzed for PRX over-oxidation by western blotting. (D) Grouped data showing the normalized PRX-SO 2/3 intensity over the YRO, peaking around late OX phase (mean ± SEM, n = 3, p = 0.024 for time effect by two-way ANOVA).
    Whole Cell Extract Kit, supplied by Active Motif, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam hela cell extracts
    PRX Tsa1 Undergoes Cycles of Oxidation across the YRO (A) Three PRXs in the <t>yeast</t> S. cerevisiae have the nine amino acid sequence recognized by the PRX-SO 2/3 antisera when over-oxidized. (B) Western blot showing that over-oxidized Tsa1 is recognized by the PRX-SO 2/3 antisera in a redox-dependent manner. Samples were harvested immediately before (−) or 15 min after (+) the addition of hydrogen peroxide (1 mM final concentration). The asterisk marks a cross-reacting band. (C) YROs were obtained using 0.1 or 0.12 dilutions/hr (sampled every 20 or 12 min, respectively) and monitored using the dissolved oxygen trace (top). Bottom: <t>whole-cell</t> <t>extracts</t> obtained from samples taken across the oscillation were analyzed for PRX over-oxidation by western blotting. (D) Grouped data showing the normalized PRX-SO 2/3 intensity over the YRO, peaking around late OX phase (mean ± SEM, n = 3, p = 0.024 for time effect by two-way ANOVA).
    Hela Cell Extracts, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Beyotime whole cell extraction kit
    PRX Tsa1 Undergoes Cycles of Oxidation across the YRO (A) Three PRXs in the <t>yeast</t> S. cerevisiae have the nine amino acid sequence recognized by the PRX-SO 2/3 antisera when over-oxidized. (B) Western blot showing that over-oxidized Tsa1 is recognized by the PRX-SO 2/3 antisera in a redox-dependent manner. Samples were harvested immediately before (−) or 15 min after (+) the addition of hydrogen peroxide (1 mM final concentration). The asterisk marks a cross-reacting band. (C) YROs were obtained using 0.1 or 0.12 dilutions/hr (sampled every 20 or 12 min, respectively) and monitored using the dissolved oxygen trace (top). Bottom: <t>whole-cell</t> <t>extracts</t> obtained from samples taken across the oscillation were analyzed for PRX over-oxidation by western blotting. (D) Grouped data showing the normalized PRX-SO 2/3 intensity over the YRO, peaking around late OX phase (mean ± SEM, n = 3, p = 0.024 for time effect by two-way ANOVA).
    Whole Cell Extraction Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 88/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore whole cell extract buffer
    PRX Tsa1 Undergoes Cycles of Oxidation across the YRO (A) Three PRXs in the <t>yeast</t> S. cerevisiae have the nine amino acid sequence recognized by the PRX-SO 2/3 antisera when over-oxidized. (B) Western blot showing that over-oxidized Tsa1 is recognized by the PRX-SO 2/3 antisera in a redox-dependent manner. Samples were harvested immediately before (−) or 15 min after (+) the addition of hydrogen peroxide (1 mM final concentration). The asterisk marks a cross-reacting band. (C) YROs were obtained using 0.1 or 0.12 dilutions/hr (sampled every 20 or 12 min, respectively) and monitored using the dissolved oxygen trace (top). Bottom: <t>whole-cell</t> <t>extracts</t> obtained from samples taken across the oscillation were analyzed for PRX over-oxidation by western blotting. (D) Grouped data showing the normalized PRX-SO 2/3 intensity over the YRO, peaking around late OX phase (mean ± SEM, n = 3, p = 0.024 for time effect by two-way ANOVA).
    Whole Cell Extract Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology u937 whole cell extract
    Western blots are shown in which all 9 tissues are present on each blot. On the PPAR α blot, all tissues shown by dark bars were from a single 91-day-old fetus, and adrenal and kidney (white bars) were from different 91-day-old fetuses, and thymus was from a 101-day-old fetus. The blots for PPAR β and PPAR γ used samples from a 91-day-old fetus (dark bars, the same set of samples for both PPAR β and γ ) with kidney and thymus samples (white bars) from different fetuses (91 and 108 days, resp.). Blot images are labeled to show the location of the PPAR band, the GAPDH band, and lane containing the positive control (Hep G2 whole cell extract, Jurkat cell nuclear extract, and <t>U937</t> whole cell extract, for expression of PPAR α , β , or γ , resp.). The densitometry data (PPAR expression normalized to GAPDH) for each gel is shown above the blot image. Lanes 1–9 contain the samples listed on the x -axis of the bar graphs.
    U937 Whole Cell Extract, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u937 whole cell extract/product/Santa Cruz Biotechnology
    Average 85 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    u937 whole cell extract - by Bioz Stars, 2020-05
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    85
    Millipore hek293t 17 whole cell extracts
    Western blots are shown in which all 9 tissues are present on each blot. On the PPAR α blot, all tissues shown by dark bars were from a single 91-day-old fetus, and adrenal and kidney (white bars) were from different 91-day-old fetuses, and thymus was from a 101-day-old fetus. The blots for PPAR β and PPAR γ used samples from a 91-day-old fetus (dark bars, the same set of samples for both PPAR β and γ ) with kidney and thymus samples (white bars) from different fetuses (91 and 108 days, resp.). Blot images are labeled to show the location of the PPAR band, the GAPDH band, and lane containing the positive control (Hep G2 whole cell extract, Jurkat cell nuclear extract, and <t>U937</t> whole cell extract, for expression of PPAR α , β , or γ , resp.). The densitometry data (PPAR expression normalized to GAPDH) for each gel is shown above the blot image. Lanes 1–9 contain the samples listed on the x -axis of the bar graphs.
    Hek293t 17 Whole Cell Extracts, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hek293t 17 whole cell extracts/product/Millipore
    Average 85 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    hek293t 17 whole cell extracts - by Bioz Stars, 2020-05
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    Image Search Results


    Proliferative capacity of RECQ1-depleted glioblastoma cells . A) Western blot analysis of T98G, U-87 and IMR-90 cell lines transiently transfected with an siRNA against RECQ1. L indicates the Luciferase siRNA control, R the siRNA against RECQ1. B) Clonogenic assays performed in RECQ1-depleted T98G, U-87, and IMR-90 cell lines. Pictures show colonies formed after seeding 800 cells. C) Graphs showing the plating efficiencies expressed as colony forming capacity. Values represent the average ratio of the number of formed colonies to the number of cells seeded, expressed as percentage.

    Journal: Molecular Cancer

    Article Title: The human RECQ1 helicase is highly expressed in glioblastoma and plays an important role in tumor cell proliferation

    doi: 10.1186/1476-4598-10-83

    Figure Lengend Snippet: Proliferative capacity of RECQ1-depleted glioblastoma cells . A) Western blot analysis of T98G, U-87 and IMR-90 cell lines transiently transfected with an siRNA against RECQ1. L indicates the Luciferase siRNA control, R the siRNA against RECQ1. B) Clonogenic assays performed in RECQ1-depleted T98G, U-87, and IMR-90 cell lines. Pictures show colonies formed after seeding 800 cells. C) Graphs showing the plating efficiencies expressed as colony forming capacity. Values represent the average ratio of the number of formed colonies to the number of cells seeded, expressed as percentage.

    Article Snippet: T98G, U-87, and IMR-90 whole cell extracts were prepared in HNNG buffer (15 mM Hepes pH 7.5, 250 mM NaCl, 1% (v/v) NP-40, 10% (v/v) glycerol, 1 mM PMSF) supplemented with 0.2 mM sodium orthovandate (Sigma), 10 mM sodium glycerol-2-phosphate (Sigma), 25 mM NaF (Sigma) and protease inhibitors cocktail tablet (Roche).

    Techniques: Western Blot, Transfection, Luciferase

    Total FcγRI, RIIB, and RIII protein in MɸN and MɸP cell extracts. ( A ) Cells from three different passages of MɸN and cells from passages 72, 83 and 87 of MɸP were lysed and FcγRI, FcγRIIB, FcγRIII and GAPDH proteins in cell extracts were determined by Western blot with specific antibodies; ( B ) The relative amount of each FcγR isoform was determined as the ratio of the densitometric intensity of the FcγR band to the intensity of the GAPDH band in the correspondent cell extract. Results are expressed as mean ± 1 SD (* indicates p

    Journal: Viruses

    Article Title: Respiratory Syncytial Virus Persistence in Macrophages Upregulates Fcgamma Receptors Expression

    doi: 10.3390/v6020624

    Figure Lengend Snippet: Total FcγRI, RIIB, and RIII protein in MɸN and MɸP cell extracts. ( A ) Cells from three different passages of MɸN and cells from passages 72, 83 and 87 of MɸP were lysed and FcγRI, FcγRIIB, FcγRIII and GAPDH proteins in cell extracts were determined by Western blot with specific antibodies; ( B ) The relative amount of each FcγR isoform was determined as the ratio of the densitometric intensity of the FcγR band to the intensity of the GAPDH band in the correspondent cell extract. Results are expressed as mean ± 1 SD (* indicates p

    Article Snippet: Western Blot Whole-cell extracts were prepared from MɸN or MɸP (3 × 106 cells) with ice-cold lysing buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) containing 1× protease inhibitors cocktail (Sigma Aldrich Corp., St. Louis, MO, USA).

    Techniques: Western Blot

    Effects of 8-Cl-Ado on DNA relaxation and on p53, p21 and p53R2 expression. ( A ) A549 and H1299 cells were exposed to 2 μM 8-Cl-Ado for 48 h, and nuclear extracts (NE) were prepared. Relaxation activities in NE were tested by incubating with supercoiled pUC19 DNA in the reaction conditions as indicated on the top. After ethanol precipitated, extracted DNA samples were subjected to 1% agarose gel electrophoresis. The pUC19 DNA is used as markers for supercoiled and relaxed DNA; ( B , C ) Western blotting for p53, p21 and p53R2 expression. β-Actin as a loading control. The numbers below the blots and histograms in lower panels show the relative levels of p53, p21 and p53R2 in Western blotting. The ratio of target protein/Actin from control cells was designated as “1”. * p

    Journal: International Journal of Molecular Sciences

    Article Title: DNA Damage-Response Pathway Heterogeneity of Human Lung Cancer A549 and H1299 Cells Determines Sensitivity to 8-Chloro-Adenosine

    doi: 10.3390/ijms19061587

    Figure Lengend Snippet: Effects of 8-Cl-Ado on DNA relaxation and on p53, p21 and p53R2 expression. ( A ) A549 and H1299 cells were exposed to 2 μM 8-Cl-Ado for 48 h, and nuclear extracts (NE) were prepared. Relaxation activities in NE were tested by incubating with supercoiled pUC19 DNA in the reaction conditions as indicated on the top. After ethanol precipitated, extracted DNA samples were subjected to 1% agarose gel electrophoresis. The pUC19 DNA is used as markers for supercoiled and relaxed DNA; ( B , C ) Western blotting for p53, p21 and p53R2 expression. β-Actin as a loading control. The numbers below the blots and histograms in lower panels show the relative levels of p53, p21 and p53R2 in Western blotting. The ratio of target protein/Actin from control cells was designated as “1”. * p

    Article Snippet: Western Blotting Whole-cell extracts were prepared in lysis buffer and protein concentration was determined using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA).

    Techniques: Expressing, Agarose Gel Electrophoresis, Western Blot

    Treatment of immune cells with filtered and unfiltered Ebola-transfected cell supernatants. 293T cells were transfected with either VP40, GP, or NP plasmids with Attractene Transfection Reagent (Qiagen) as per the manufacturer’s instructions and incubated for 2 days. Transfection complexes were removed and cells were treated with antibiotics for plasmid selection for 14 days. Transfection supernatants were either left unfiltered or passed through a 0.22 micron filter. Control 293T cells were not transfected nor treated with antibiotics (Hygromycin B, Zeocin, or Geneticin). Seventy-five microliters (0.45 mU AChE) of both filtered and unfiltered supernatants were used to treat CEM (A) , Jurkat (B) and U937 (C) cells, which were subsequently assayed for cell viability via CellTiter-Glo after 4 days of incubation. Statistical significance was determined using 2-tailed student’s t -test, with filtered or unfiltered experimental values compared to the Control values of like type. (D) U937 cells were treated with PMA (50 nM) for 5 days to stimulate differentiation into macrophages. On day 5 media was replaced and differentiated U937 macrophages were treated with either E. coli -purified free VP40 protein, or supernatants from VP40-transfected 293T cells (filtered and unfiltered). Untreated macrophages received no external supernatant treatment. Cells were incubated for an additional 5 days and cell viability was assayed with CellTiter-Glo. (E) Primary monocytes from 3 healthy donors were treated with PMA (10 nM) for 2 days to stimulate differentiation into macrophages. Primary macrophages were then treated with supernatants from either VP40-transfected 293T cells (filtered and unfiltered), or from VP40-resistant clone EVTR2C cells (filtered). Control primary macrophages were treated with filtered supernatant from normal 293T cells. Cells were incubated for 2 days before being assayed for viability with CellTiter-Glo. Statistical significance was determined using 2-tailed student’s t -test, with filtered or unfiltered experimental groups compared to the Control groups of like type. MØ, Macrophage; RLU, Relative Luminescent Units. ∗ p

    Journal: Frontiers in Microbiology

    Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

    doi: 10.3389/fmicb.2016.01765

    Figure Lengend Snippet: Treatment of immune cells with filtered and unfiltered Ebola-transfected cell supernatants. 293T cells were transfected with either VP40, GP, or NP plasmids with Attractene Transfection Reagent (Qiagen) as per the manufacturer’s instructions and incubated for 2 days. Transfection complexes were removed and cells were treated with antibiotics for plasmid selection for 14 days. Transfection supernatants were either left unfiltered or passed through a 0.22 micron filter. Control 293T cells were not transfected nor treated with antibiotics (Hygromycin B, Zeocin, or Geneticin). Seventy-five microliters (0.45 mU AChE) of both filtered and unfiltered supernatants were used to treat CEM (A) , Jurkat (B) and U937 (C) cells, which were subsequently assayed for cell viability via CellTiter-Glo after 4 days of incubation. Statistical significance was determined using 2-tailed student’s t -test, with filtered or unfiltered experimental values compared to the Control values of like type. (D) U937 cells were treated with PMA (50 nM) for 5 days to stimulate differentiation into macrophages. On day 5 media was replaced and differentiated U937 macrophages were treated with either E. coli -purified free VP40 protein, or supernatants from VP40-transfected 293T cells (filtered and unfiltered). Untreated macrophages received no external supernatant treatment. Cells were incubated for an additional 5 days and cell viability was assayed with CellTiter-Glo. (E) Primary monocytes from 3 healthy donors were treated with PMA (10 nM) for 2 days to stimulate differentiation into macrophages. Primary macrophages were then treated with supernatants from either VP40-transfected 293T cells (filtered and unfiltered), or from VP40-resistant clone EVTR2C cells (filtered). Control primary macrophages were treated with filtered supernatant from normal 293T cells. Cells were incubated for 2 days before being assayed for viability with CellTiter-Glo. Statistical significance was determined using 2-tailed student’s t -test, with filtered or unfiltered experimental groups compared to the Control groups of like type. MØ, Macrophage; RLU, Relative Luminescent Units. ∗ p

    Article Snippet: Immunoprecipication, in vivo Labeling, and Kinase Assays Immunoprecipitation (IP) was performed by incubation of 500 μg of CEM or transfected and treated 293T whole cell extracts with 10 μg of appropriate primary antibody (α-Cdk2, α-CycE, α-CycA, α-normal rabbit IgG; Santa Cruz Biotechnology) and 100 μL TNE50 + 0.1% NP-40 for 48 h at 4°C.

    Techniques: Transfection, Incubation, Plasmid Preparation, Selection, Purification

    Inhibition of miRNA machinery in transfected donor and recipient cells. (A) Mid-log phase 293T cells were transfected with VP40-producing plasmids via electroporation and incubated for 24 h, followed by treatment with Hygromycin B for plasmid selection until cells were confluent (9 days; lane 2). Mock transfected cells were electroporated without plasmid (lane 1). Cells were lysed with lysis buffer, whole cell extract was resuspended in Laemmli buffer, run on a 4–20% Tris-glycine SDS gel, and subsequently analyzed by Western blot for levels of miRNA machinery components Dicer, Drosha, Ago 1, Exportin 5, DGCR8, and TRBP. Presence of VP40 protein was analyzed as a control, along with Actin. NS, non-Specific binding (B) VP40-transfected 293T cells were grown under antibiotic selection for 14 days, followed by removal and filtering (0.22 micron) of the supernatant. CEM cells were treated with 100, 250, or 500 μL of filtered transfection supernatant (0.3, 0.75, and 1.5 mU AChE, respectively) and incubated for 5 days at 37°C. Cell pellets were isolated, lysed with lysis buffer, and analyzed by Western blot for miRNA machinery components Dicer, Drosha, Ago 1, Exportin 5, DCGR8, and TRBP. Actin levels were also analyzed.

    Journal: Frontiers in Microbiology

    Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

    doi: 10.3389/fmicb.2016.01765

    Figure Lengend Snippet: Inhibition of miRNA machinery in transfected donor and recipient cells. (A) Mid-log phase 293T cells were transfected with VP40-producing plasmids via electroporation and incubated for 24 h, followed by treatment with Hygromycin B for plasmid selection until cells were confluent (9 days; lane 2). Mock transfected cells were electroporated without plasmid (lane 1). Cells were lysed with lysis buffer, whole cell extract was resuspended in Laemmli buffer, run on a 4–20% Tris-glycine SDS gel, and subsequently analyzed by Western blot for levels of miRNA machinery components Dicer, Drosha, Ago 1, Exportin 5, DGCR8, and TRBP. Presence of VP40 protein was analyzed as a control, along with Actin. NS, non-Specific binding (B) VP40-transfected 293T cells were grown under antibiotic selection for 14 days, followed by removal and filtering (0.22 micron) of the supernatant. CEM cells were treated with 100, 250, or 500 μL of filtered transfection supernatant (0.3, 0.75, and 1.5 mU AChE, respectively) and incubated for 5 days at 37°C. Cell pellets were isolated, lysed with lysis buffer, and analyzed by Western blot for miRNA machinery components Dicer, Drosha, Ago 1, Exportin 5, DCGR8, and TRBP. Actin levels were also analyzed.

    Article Snippet: Immunoprecipication, in vivo Labeling, and Kinase Assays Immunoprecipitation (IP) was performed by incubation of 500 μg of CEM or transfected and treated 293T whole cell extracts with 10 μg of appropriate primary antibody (α-Cdk2, α-CycE, α-CycA, α-normal rabbit IgG; Santa Cruz Biotechnology) and 100 μL TNE50 + 0.1% NP-40 for 48 h at 4°C.

    Techniques: Inhibition, Transfection, Electroporation, Incubation, Plasmid Preparation, Selection, Lysis, SDS-Gel, Western Blot, Binding Assay, Isolation

    Potential activity and sites of Cdk2/Cyclin phosphorylation on Ebola VP40. (A) The amino acid sequence of Ebola VP40 with potential phosphorylation sites ( ∗ ) and hypothesized Cdk2 phosphorylation site (red). (B) Crystal structure of EBOV VP40 monomer. Potential phosphorylation sites are marked on two serine residues (red) and two threonine residues (blue). Previously demonstrated c-Abl1 phosphorylation site tyrosine-13 ( García et al., 2012 ) is indicated (green). The Ebola VP40 crystal structure modified was from Dessen et al. (2000) . (C) CEM extracts were used for immunoprecipitation using α-Cdk2, α-Cyclin E, α-Cyclin A or IgG as control. Five hundred micrograms of CEM whole cell extract was used with 10 μg of each antibody and 100 μL TNE50 + 0.1% NP-40 for 48 h at 4°C, followed by next day addition of Protein A/G for 2 h at 4°C. The complexes were washed with twice with TNE50 + 0.1% NP-40 and twice with kinase buffer, and successively used for in vitro kinase assay usi ng purified VP40 protein (lanes 3–9) and [γ- 32 P] ATP. Cdk inhibitors Alsterpaullone, Indirubin-3′-monoxime, Purvalanol A and r-Roscovitine (lanes 6–9) were used at final 1 μM concentration for 1 h at 37°C. Densitometry analysis of kinase activity as a percentage is shown in the bottom panel with α-Cdk2+VP40 set to 100%. (D) 293T cells were transfected with 20 μg of VP40 DNA using electroporation and were placed under antibiotic selection (Hygromycin B). After 4 days, cells (30–40% confluent) were either untreated or blocked at G1/S phase of cell cycle with Hydroxyurea (1 mM) for 1 day. Cells were subsequently radio-labeled with [γ- 32 P] ATP and a few samples were treated with r-Roscovitine (10 μM) for 4 h in complete media. Radioactive material was subsequently removed, washed and chased with complete media for 2 h. Next, cells were removed (cell scraper), lysed with lysis buffer, and immunoprecipitation was performed overnight with α-VP40 antibody in TNE150 + 0.1% NP-40 buffer. Protein A/G was added for 2 h and complexes were washed twice with TNE150 + 0.1% NP-40 and once with kinase buffer. Radioactive immunoprecipitated complexes were resuspended in Laemmli buffer, run on a 4–20% Tris-glycine SDS gel, dried, and exposed to phosphoroImager cassette. Densitometry analysis of kinase activity as determined by ImageJ software is shown as a percentage in the bottom panel, with untreated VP40-transfected 293T cells set to 100%. (E) Five hundred micrograms of CEM whole cell extract was immunoprecipitated with 10 μg of α-Cyclin E or normal rabbit IgG antibody in TNE50 + 0.1% NP-40, and incubated overnight at 4°C. Fifty microliters of a 30% slurry of Protein A/G was added next day, incubated for 2 h, washed twice with PBS and once with kinase buffer, and then resuspended in kinase buffer. Fifty microgram of 10–12 mer peptides matching potential phosphorylation sites on EBOV VP40 were added to 15 μL of sample IP and 2 μL of a [γ- 32 P] ATP and kinase buffer solution (1:3). Samples were incubated for 1 h before being dotted onto Whatman glass microfibre filters and dried for 30 min before being submerged in 1x TE buffer with gentle agitation for 2 days. DPM2 counts were then taken with a scintillation counter (QuantaSmart TM ). The peptide sequences used are illustrated in the boxes. Underlined letters indicate the potentially phosphorylated residue. Residues that were altered from the wild type sequence are indicated (red).

    Journal: Frontiers in Microbiology

    Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

    doi: 10.3389/fmicb.2016.01765

    Figure Lengend Snippet: Potential activity and sites of Cdk2/Cyclin phosphorylation on Ebola VP40. (A) The amino acid sequence of Ebola VP40 with potential phosphorylation sites ( ∗ ) and hypothesized Cdk2 phosphorylation site (red). (B) Crystal structure of EBOV VP40 monomer. Potential phosphorylation sites are marked on two serine residues (red) and two threonine residues (blue). Previously demonstrated c-Abl1 phosphorylation site tyrosine-13 ( García et al., 2012 ) is indicated (green). The Ebola VP40 crystal structure modified was from Dessen et al. (2000) . (C) CEM extracts were used for immunoprecipitation using α-Cdk2, α-Cyclin E, α-Cyclin A or IgG as control. Five hundred micrograms of CEM whole cell extract was used with 10 μg of each antibody and 100 μL TNE50 + 0.1% NP-40 for 48 h at 4°C, followed by next day addition of Protein A/G for 2 h at 4°C. The complexes were washed with twice with TNE50 + 0.1% NP-40 and twice with kinase buffer, and successively used for in vitro kinase assay usi ng purified VP40 protein (lanes 3–9) and [γ- 32 P] ATP. Cdk inhibitors Alsterpaullone, Indirubin-3′-monoxime, Purvalanol A and r-Roscovitine (lanes 6–9) were used at final 1 μM concentration for 1 h at 37°C. Densitometry analysis of kinase activity as a percentage is shown in the bottom panel with α-Cdk2+VP40 set to 100%. (D) 293T cells were transfected with 20 μg of VP40 DNA using electroporation and were placed under antibiotic selection (Hygromycin B). After 4 days, cells (30–40% confluent) were either untreated or blocked at G1/S phase of cell cycle with Hydroxyurea (1 mM) for 1 day. Cells were subsequently radio-labeled with [γ- 32 P] ATP and a few samples were treated with r-Roscovitine (10 μM) for 4 h in complete media. Radioactive material was subsequently removed, washed and chased with complete media for 2 h. Next, cells were removed (cell scraper), lysed with lysis buffer, and immunoprecipitation was performed overnight with α-VP40 antibody in TNE150 + 0.1% NP-40 buffer. Protein A/G was added for 2 h and complexes were washed twice with TNE150 + 0.1% NP-40 and once with kinase buffer. Radioactive immunoprecipitated complexes were resuspended in Laemmli buffer, run on a 4–20% Tris-glycine SDS gel, dried, and exposed to phosphoroImager cassette. Densitometry analysis of kinase activity as determined by ImageJ software is shown as a percentage in the bottom panel, with untreated VP40-transfected 293T cells set to 100%. (E) Five hundred micrograms of CEM whole cell extract was immunoprecipitated with 10 μg of α-Cyclin E or normal rabbit IgG antibody in TNE50 + 0.1% NP-40, and incubated overnight at 4°C. Fifty microliters of a 30% slurry of Protein A/G was added next day, incubated for 2 h, washed twice with PBS and once with kinase buffer, and then resuspended in kinase buffer. Fifty microgram of 10–12 mer peptides matching potential phosphorylation sites on EBOV VP40 were added to 15 μL of sample IP and 2 μL of a [γ- 32 P] ATP and kinase buffer solution (1:3). Samples were incubated for 1 h before being dotted onto Whatman glass microfibre filters and dried for 30 min before being submerged in 1x TE buffer with gentle agitation for 2 days. DPM2 counts were then taken with a scintillation counter (QuantaSmart TM ). The peptide sequences used are illustrated in the boxes. Underlined letters indicate the potentially phosphorylated residue. Residues that were altered from the wild type sequence are indicated (red).

    Article Snippet: Immunoprecipication, in vivo Labeling, and Kinase Assays Immunoprecipitation (IP) was performed by incubation of 500 μg of CEM or transfected and treated 293T whole cell extracts with 10 μg of appropriate primary antibody (α-Cdk2, α-CycE, α-CycA, α-normal rabbit IgG; Santa Cruz Biotechnology) and 100 μL TNE50 + 0.1% NP-40 for 48 h at 4°C.

    Techniques: Activity Assay, Sequencing, Modification, Immunoprecipitation, In Vitro, Kinase Assay, Purification, Concentration Assay, Transfection, Electroporation, Selection, Labeling, Lysis, SDS-Gel, Software, Incubation

    Effect of FDA approved drugs on EBOV transfected donor and recipient immune cells. (A) Varying concentrations of Cambinol and Esomepraole (1 nM, 1 μM, 10 μM), Oxytetracycline (1 nM, 10 nM, 10 μM), and DMSO (0.00001, 0.0001, 0.001%) treatments were administered to VP40-transfected 293T cells, followed by incubation for 3 days. Supernatant was centrifuged to remove cellular material and subsequently incubated for 72 h at 4°C with 20 μL of 30% slurry of NT80/82 beads. The NT pellet was washed twice with PBS and resuspended in SDS Laemmli buffer, followed by Western blot analysis for levels of exosomal markers CD63 and Alix. Actin levels were also analyzed. Mid-log phase EVTR2C cells (containing VP40) were treated with either low (0.1 μM), medium (1 μM), or high (10 μM) concentrations of Oxytetracycline and incubated for 5 days along with untreated 293T cells. Cell-free supernatants were harvested, filtered, and used to treat CEM (B) , Jurkat (C) , and U937 (D) cells. Cells were incubated for 5 days and assayed with CellTiter-Glo for viability. Statistical significance was determined using two-tailed student’s t -test with all groups compared to the untreated 293T cell control groups. RLU, Relative Luminescent Units. ∗ p

    Journal: Frontiers in Microbiology

    Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

    doi: 10.3389/fmicb.2016.01765

    Figure Lengend Snippet: Effect of FDA approved drugs on EBOV transfected donor and recipient immune cells. (A) Varying concentrations of Cambinol and Esomepraole (1 nM, 1 μM, 10 μM), Oxytetracycline (1 nM, 10 nM, 10 μM), and DMSO (0.00001, 0.0001, 0.001%) treatments were administered to VP40-transfected 293T cells, followed by incubation for 3 days. Supernatant was centrifuged to remove cellular material and subsequently incubated for 72 h at 4°C with 20 μL of 30% slurry of NT80/82 beads. The NT pellet was washed twice with PBS and resuspended in SDS Laemmli buffer, followed by Western blot analysis for levels of exosomal markers CD63 and Alix. Actin levels were also analyzed. Mid-log phase EVTR2C cells (containing VP40) were treated with either low (0.1 μM), medium (1 μM), or high (10 μM) concentrations of Oxytetracycline and incubated for 5 days along with untreated 293T cells. Cell-free supernatants were harvested, filtered, and used to treat CEM (B) , Jurkat (C) , and U937 (D) cells. Cells were incubated for 5 days and assayed with CellTiter-Glo for viability. Statistical significance was determined using two-tailed student’s t -test with all groups compared to the untreated 293T cell control groups. RLU, Relative Luminescent Units. ∗ p

    Article Snippet: Immunoprecipication, in vivo Labeling, and Kinase Assays Immunoprecipitation (IP) was performed by incubation of 500 μg of CEM or transfected and treated 293T whole cell extracts with 10 μg of appropriate primary antibody (α-Cdk2, α-CycE, α-CycA, α-normal rabbit IgG; Santa Cruz Biotechnology) and 100 μL TNE50 + 0.1% NP-40 for 48 h at 4°C.

    Techniques: Transfection, Incubation, Western Blot, Two Tailed Test

    Effect of VP40 presence on exosomal markers and ESCRT machinery components. (A) 293T cells and VP40-resistant clones (EVTR2C cells) were grown for 5 days, followed by harvesting of both cells and supernatants. Cells were lysed and analyzed by Western blot for levels of CD63, Alix, and Actin. Supernatants were centrifuged and passed through a 0.22 micron filter, followed by incubation at 4°C with 30% slurry of NT80/82 beads for 72 h. Beads were spun down next day and resuspended in Laemmli buffer, followed by SDS/PAGE and Western blot analysis for levels of CD63, Alix, and Actin. (B) Whole cell extracts from 293T, EVTR2C, and EVTR2C cells treated for 5 days with 10 uM Oxytetracycline were run on a 4–20% Tris-glycine SDS gel and analyzed by Western blot for levels of ESCRT pathway components TSG101 (ESCRT-I), EAP20 (ESCRT-II), EAP45 (ESCRT-II), and CHMP6 (ESCRT-III), as well as VP40 and Actin.

    Journal: Frontiers in Microbiology

    Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

    doi: 10.3389/fmicb.2016.01765

    Figure Lengend Snippet: Effect of VP40 presence on exosomal markers and ESCRT machinery components. (A) 293T cells and VP40-resistant clones (EVTR2C cells) were grown for 5 days, followed by harvesting of both cells and supernatants. Cells were lysed and analyzed by Western blot for levels of CD63, Alix, and Actin. Supernatants were centrifuged and passed through a 0.22 micron filter, followed by incubation at 4°C with 30% slurry of NT80/82 beads for 72 h. Beads were spun down next day and resuspended in Laemmli buffer, followed by SDS/PAGE and Western blot analysis for levels of CD63, Alix, and Actin. (B) Whole cell extracts from 293T, EVTR2C, and EVTR2C cells treated for 5 days with 10 uM Oxytetracycline were run on a 4–20% Tris-glycine SDS gel and analyzed by Western blot for levels of ESCRT pathway components TSG101 (ESCRT-I), EAP20 (ESCRT-II), EAP45 (ESCRT-II), and CHMP6 (ESCRT-III), as well as VP40 and Actin.

    Article Snippet: Immunoprecipication, in vivo Labeling, and Kinase Assays Immunoprecipitation (IP) was performed by incubation of 500 μg of CEM or transfected and treated 293T whole cell extracts with 10 μg of appropriate primary antibody (α-Cdk2, α-CycE, α-CycA, α-normal rabbit IgG; Santa Cruz Biotechnology) and 100 μL TNE50 + 0.1% NP-40 for 48 h at 4°C.

    Techniques: Clone Assay, Western Blot, Incubation, SDS Page, SDS-Gel

    Activation of the Gαq signaling pathway promotes calpain-mediated β-catenin proteolysis in a Ca 2+ -dependent manner. ( a ) The inhibitory effect of the Gq signaling pathway on TCF/LEF-1-mediated transcription is not mediated by PKC. SW480 cells were transfected with empty plasmid as control, Q209L-Gαq, or M3R expression plasmids. PKC inhibitor bisindolylmaleimide I (1 μM) was added on transfection and carbachol (1 mM) was added 24 h posttransfection. Relative TCF/LEF-1 activity was assayed 48 h after transfection. ( b ) Thapsigargin mimics the effect of Q209L–Gαq. SW480 cells were transfected with indicated expression plasmids. Thapsigargin (50 nM) was added on transfection. Relative TCF/LEF-1 activity was assayed 48 h after transfection. ( c ) Q209L–Gαq induces reduction in cytosolic β-catenin levels. SW480 cells were transfected with empty plasmid as control or Q209L–Gαq expression plasmid. Cycloheximide (25 μg/ml) was added 24 h after transfection. Cells were harvested at the indicated times after cycloheximide addition. Cytosolic fraction was prepared, resolved by SDS/PAGE, and blotted with antibody specific against the C terminus of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. ( d ) Q209L–Gαq induces cleavage of cytosolic β-catenin. Cytosolic fractions of SW480 cells were prepared at the indicated time points after cycloheximide treatment, electrophoretically resolved, and blotted with β-catenin antibody specific for the C terminus. Proteolytic products of β-catenin are indicated by arrowheads. ( e ) Thapsigargin induces calpain-mediated proteolysis of β-catenin in a calcium-dependent manner. SW480 cells were pretreated with DMSO or inhibitors for 10 min, and then treated with thapsigargin (50 nM) for 30 min. The untreated cells were used as control. The concentrations of the various inhibitors were BAPTA/AM (40 μM), NH 4 Cl (1 mM), calpeptin (50 μM), calpastatin (10 μM), lactacystin (10 μM), and MG-132 (10 μM). Whole-cell extract was prepared, resolved by SDS/PAGE, and blotted with the C-terminal β-catenin antibody. Proteolytic products of β-catenin are indicated by arrowheads. ( f ) Activation of Gq-coupled M3R induces calpain-mediated proteolysis of β-catenin. SW480 cells stably expressing M3R were treated with carbachol (1 mM), lactacystin (10 μM), or calpeptin (10 μM) as indicated. Whole-cell lysates were prepared at the indicated time points, and β-catenin fragments were detected by antibody specific for the C terminus of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. ( g and h ) Calpain-mediated cleavage occurs at the N-terminal region of β-catenin. As described in Materials and Methods , HEK293 cell extracts were incubated at 37°C for 30 min alone as control or with CaCl 2 (0.1 mM) and μ-calpain in the presence of the indicated reagents: EGTA (1 mM), ALLN (10 μM), ALLM (10 μM), E-64 (25 μM), calpastatin (10 μM), lactacystin (10 μM), and MG-132 (10 μM). Cleavage of β-catenin was assessed with antibodies specific for the N-terminal or C-terminal regions of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. WB, Western blot; NT, N terminus; CT, C terminus.

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

    Article Title: Calpain as an effector of the Gq signaling pathway for inhibition of Wnt/?-catenin-regulated cell proliferation

    doi: 10.1073/pnas.202355799

    Figure Lengend Snippet: Activation of the Gαq signaling pathway promotes calpain-mediated β-catenin proteolysis in a Ca 2+ -dependent manner. ( a ) The inhibitory effect of the Gq signaling pathway on TCF/LEF-1-mediated transcription is not mediated by PKC. SW480 cells were transfected with empty plasmid as control, Q209L-Gαq, or M3R expression plasmids. PKC inhibitor bisindolylmaleimide I (1 μM) was added on transfection and carbachol (1 mM) was added 24 h posttransfection. Relative TCF/LEF-1 activity was assayed 48 h after transfection. ( b ) Thapsigargin mimics the effect of Q209L–Gαq. SW480 cells were transfected with indicated expression plasmids. Thapsigargin (50 nM) was added on transfection. Relative TCF/LEF-1 activity was assayed 48 h after transfection. ( c ) Q209L–Gαq induces reduction in cytosolic β-catenin levels. SW480 cells were transfected with empty plasmid as control or Q209L–Gαq expression plasmid. Cycloheximide (25 μg/ml) was added 24 h after transfection. Cells were harvested at the indicated times after cycloheximide addition. Cytosolic fraction was prepared, resolved by SDS/PAGE, and blotted with antibody specific against the C terminus of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. ( d ) Q209L–Gαq induces cleavage of cytosolic β-catenin. Cytosolic fractions of SW480 cells were prepared at the indicated time points after cycloheximide treatment, electrophoretically resolved, and blotted with β-catenin antibody specific for the C terminus. Proteolytic products of β-catenin are indicated by arrowheads. ( e ) Thapsigargin induces calpain-mediated proteolysis of β-catenin in a calcium-dependent manner. SW480 cells were pretreated with DMSO or inhibitors for 10 min, and then treated with thapsigargin (50 nM) for 30 min. The untreated cells were used as control. The concentrations of the various inhibitors were BAPTA/AM (40 μM), NH 4 Cl (1 mM), calpeptin (50 μM), calpastatin (10 μM), lactacystin (10 μM), and MG-132 (10 μM). Whole-cell extract was prepared, resolved by SDS/PAGE, and blotted with the C-terminal β-catenin antibody. Proteolytic products of β-catenin are indicated by arrowheads. ( f ) Activation of Gq-coupled M3R induces calpain-mediated proteolysis of β-catenin. SW480 cells stably expressing M3R were treated with carbachol (1 mM), lactacystin (10 μM), or calpeptin (10 μM) as indicated. Whole-cell lysates were prepared at the indicated time points, and β-catenin fragments were detected by antibody specific for the C terminus of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. ( g and h ) Calpain-mediated cleavage occurs at the N-terminal region of β-catenin. As described in Materials and Methods , HEK293 cell extracts were incubated at 37°C for 30 min alone as control or with CaCl 2 (0.1 mM) and μ-calpain in the presence of the indicated reagents: EGTA (1 mM), ALLN (10 μM), ALLM (10 μM), E-64 (25 μM), calpastatin (10 μM), lactacystin (10 μM), and MG-132 (10 μM). Cleavage of β-catenin was assessed with antibodies specific for the N-terminal or C-terminal regions of β-catenin. Proteolytic products of β-catenin are indicated by arrowheads. WB, Western blot; NT, N terminus; CT, C terminus.

    Article Snippet: HEK293 whole-cell extracts (1 mg/ml) were prepared and incubated with μ-calpain (10 μg/ml, Calbiochem) and an appropriate amount of CaCl2 in the buffer (30 mM Tris⋅HCl, pH 7.5/1.5 mM DTT) at 37°C for 30 min.

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Activity Assay, SDS Page, Stable Transfection, Incubation, Western Blot

    PRX Tsa1 Undergoes Cycles of Oxidation across the YRO (A) Three PRXs in the yeast S. cerevisiae have the nine amino acid sequence recognized by the PRX-SO 2/3 antisera when over-oxidized. (B) Western blot showing that over-oxidized Tsa1 is recognized by the PRX-SO 2/3 antisera in a redox-dependent manner. Samples were harvested immediately before (−) or 15 min after (+) the addition of hydrogen peroxide (1 mM final concentration). The asterisk marks a cross-reacting band. (C) YROs were obtained using 0.1 or 0.12 dilutions/hr (sampled every 20 or 12 min, respectively) and monitored using the dissolved oxygen trace (top). Bottom: whole-cell extracts obtained from samples taken across the oscillation were analyzed for PRX over-oxidation by western blotting. (D) Grouped data showing the normalized PRX-SO 2/3 intensity over the YRO, peaking around late OX phase (mean ± SEM, n = 3, p = 0.024 for time effect by two-way ANOVA).

    Journal: Current Biology

    Article Title: Metabolic Cycles in Yeast Share Features Conserved among Circadian Rhythms

    doi: 10.1016/j.cub.2015.02.035

    Figure Lengend Snippet: PRX Tsa1 Undergoes Cycles of Oxidation across the YRO (A) Three PRXs in the yeast S. cerevisiae have the nine amino acid sequence recognized by the PRX-SO 2/3 antisera when over-oxidized. (B) Western blot showing that over-oxidized Tsa1 is recognized by the PRX-SO 2/3 antisera in a redox-dependent manner. Samples were harvested immediately before (−) or 15 min after (+) the addition of hydrogen peroxide (1 mM final concentration). The asterisk marks a cross-reacting band. (C) YROs were obtained using 0.1 or 0.12 dilutions/hr (sampled every 20 or 12 min, respectively) and monitored using the dissolved oxygen trace (top). Bottom: whole-cell extracts obtained from samples taken across the oscillation were analyzed for PRX over-oxidation by western blotting. (D) Grouped data showing the normalized PRX-SO 2/3 intensity over the YRO, peaking around late OX phase (mean ± SEM, n = 3, p = 0.024 for time effect by two-way ANOVA).

    Article Snippet: Protein Preparation and Detection Yeast whole-cell extracts were prepared by trichloroacetic acid (TCA) precipitation as described [ ], with the addition of the following protease inhibitors (Sigma): aminobenzamide dihydrochloride (200 μg/ml), antipain (1 μg/ml), aprotinin (1 μg/ml), leupeptin (1 μg/ml), chymostatin (1 μg/ml), PMSF (200 μg/ml), TPCK (50 μg/ml), and pepstatin (1 μg/ml).

    Techniques: Sequencing, Western Blot, Concentration Assay

    Western blots are shown in which all 9 tissues are present on each blot. On the PPAR α blot, all tissues shown by dark bars were from a single 91-day-old fetus, and adrenal and kidney (white bars) were from different 91-day-old fetuses, and thymus was from a 101-day-old fetus. The blots for PPAR β and PPAR γ used samples from a 91-day-old fetus (dark bars, the same set of samples for both PPAR β and γ ) with kidney and thymus samples (white bars) from different fetuses (91 and 108 days, resp.). Blot images are labeled to show the location of the PPAR band, the GAPDH band, and lane containing the positive control (Hep G2 whole cell extract, Jurkat cell nuclear extract, and U937 whole cell extract, for expression of PPAR α , β , or γ , resp.). The densitometry data (PPAR expression normalized to GAPDH) for each gel is shown above the blot image. Lanes 1–9 contain the samples listed on the x -axis of the bar graphs.

    Journal: PPAR Research

    Article Title: Peroxisome Proliferator-Activated Receptors Alpha, Beta, and Gamma mRNA and Protein Expression in Human Fetal Tissues

    doi: 10.1155/2010/690907

    Figure Lengend Snippet: Western blots are shown in which all 9 tissues are present on each blot. On the PPAR α blot, all tissues shown by dark bars were from a single 91-day-old fetus, and adrenal and kidney (white bars) were from different 91-day-old fetuses, and thymus was from a 101-day-old fetus. The blots for PPAR β and PPAR γ used samples from a 91-day-old fetus (dark bars, the same set of samples for both PPAR β and γ ) with kidney and thymus samples (white bars) from different fetuses (91 and 108 days, resp.). Blot images are labeled to show the location of the PPAR band, the GAPDH band, and lane containing the positive control (Hep G2 whole cell extract, Jurkat cell nuclear extract, and U937 whole cell extract, for expression of PPAR α , β , or γ , resp.). The densitometry data (PPAR expression normalized to GAPDH) for each gel is shown above the blot image. Lanes 1–9 contain the samples listed on the x -axis of the bar graphs.

    Article Snippet: Positive controls were Hep G2 whole cell extract (Santa Cruz, SC-2227), Jurkat cell nuclear extract (Santa Cruz, SC-2132), and U937 whole cell extract (Santa Cruz, SC-2239), for expression of PPARα , β , or γ , respectively.

    Techniques: Western Blot, Labeling, Positive Control, Expressing