fatty acid free bovine serum albumin bsa Search Results


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  • 99
    Thermo Fisher bsa
    Total amount of lipids in bovine oocytes matured in vitro for 24 h. A) Lipid content in oocytes with different protein sources (0.4% <t>BSA</t> or 10% <t>FCS)</t> with or without phosphodiesterase 5 inhibitor (10 −5 M SDF) associated or not with PKG inhibitor (10 -5 M KT5823). B) Representative image (20x magnification) of Nile Red stained FCS and FCS + SDF treated oocytes. The control group consists of COCs matured with 0.4% BSA without addition of FCS or SDF. Data are expressed as the mean ± SEM of six replicates. Values with different superscript letters differ significantly (p
    Bsa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25730 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore fatty acid free bsa
    Comparison of Δh ads values for the adsorption of <t>BSA</t> and <t>β‐lactoglobulin</t> to Butyl Sepharose 4 FF at 1.2 mol/kg (NH 4 ) 2 SO 4 . Adsorption of BSA (A) and β‐lactoglobulin (B) at 308 K. Adsorption of BSA (C) and β‐lactoglobulin (D) at 303 K. Adsorption of BSA (E) and β‐lactoglobulin (F) at 298 K
    Fatty Acid Free Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20755 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Equitech-Bio fatty acid free bsa
    Effect of diabetes on cholesterol efflux to <t>apoA1</t> or HDL and regulation of cholesterol transporters by Ager in primary murine BMDMs. Primary BMDMs were retrieved from nondiabetic and diabetic WT (2 months of hyperglycemia) and Ager −/− mice and were cultured in concentrations of glucose consistent with the glycemic state of the mice from which they were retrieved. BMDMs were labeled with 3 H-cholesterol and treated with acetylated LDL and fatty acid–free <t>BSA</t> (1%) for 24 h. A and B : To mediate cholesterol efflux, cells were treated with apoA1 (5 μg/mL) ( A ) or HDL (100 μg/mL) ( B ) for 6 h, and supernatant was collected. Percent cholesterol efflux is the radioactivity in the supernatant divided by the sum of the radioactivity measured in the supernatant and the cell lysates from n ≥ 5 mice/group. C and D : BMDMs were retrieved from WT or Ager −/− mice and subjected to quantitative real-time PCR for detection of Abca1 mRNA transcript ( C ) and ABCA1 protein ( D ) analysis ( n = 3 mice/group). E and F : BMDMs were prepared as in C and D and assessed for levels of Abcg1 mRNA transcripts ( E ) and ABCG1 protein ( F ) ( n = 3 mice/group). Error bars represent mean ± SEM. hr, hour; NS, not statistically significant.
    Fatty Acid Free Bsa, supplied by Equitech-Bio, used in various techniques. Bioz Stars score: 93/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim fatty acid free bsa
    Effect of diabetes on cholesterol efflux to <t>apoA1</t> or HDL and regulation of cholesterol transporters by Ager in primary murine BMDMs. Primary BMDMs were retrieved from nondiabetic and diabetic WT (2 months of hyperglycemia) and Ager −/− mice and were cultured in concentrations of glucose consistent with the glycemic state of the mice from which they were retrieved. BMDMs were labeled with 3 H-cholesterol and treated with acetylated LDL and fatty acid–free <t>BSA</t> (1%) for 24 h. A and B : To mediate cholesterol efflux, cells were treated with apoA1 (5 μg/mL) ( A ) or HDL (100 μg/mL) ( B ) for 6 h, and supernatant was collected. Percent cholesterol efflux is the radioactivity in the supernatant divided by the sum of the radioactivity measured in the supernatant and the cell lysates from n ≥ 5 mice/group. C and D : BMDMs were retrieved from WT or Ager −/− mice and subjected to quantitative real-time PCR for detection of Abca1 mRNA transcript ( C ) and ABCA1 protein ( D ) analysis ( n = 3 mice/group). E and F : BMDMs were prepared as in C and D and assessed for levels of Abcg1 mRNA transcripts ( E ) and ABCG1 protein ( F ) ( n = 3 mice/group). Error bars represent mean ± SEM. hr, hour; NS, not statistically significant.
    Fatty Acid Free Bsa, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant fatty acid free bsa
    Analyses of flippase activity in different human cancer cell types A. Flippase activity assay: Indicated cell types were incubated with <t>NBD-PS</t> for indicated time periods and subjected to <t>BSA</t> extraction and sodium dithionite treatment. % nonextractable NBD-PS (after BSA extraction and sodium dithionite treatment) represents internalized NBD-PS, indicative of flippase activity (Schwann ◊, U87ΔEGFR-Luc □, H1299 Δ, MDA-MB-231 ○, MDA-MB-231-Luc-D3H2LN ◆, Gli36 ▲, U373 ●) B. NBD PS incorporation rate. C. NBD PS incorporation at 15 minutes time point.
    Fatty Acid Free Bsa, supplied by Valiant, used in various techniques. Bioz Stars score: 93/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FUJIFILM fatty acid free bsa
    C-terminus of ATP11C is required for inhibition of flippase activities by PKC activation. a – c Ba/F3 cells stably expressing HA-tagged ATP11A, ATP11C, and ATP11AAC chimeric proteins were treated with vehicle alone ( a ), 400 nM PMA ( b ), or 1 μM A23187 in the presence of CaCl 2 ( c ) for 15 min. The cells were fixed and immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 5 μm. d – f Ba/F3 cells were treated with vehicle alone (white bars), 400 nM PMA (black bars), or 400 nM PMA and 2 μM of BIM-1 (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated at 15 °C for 5 min with <t>NBD-PS</t> ( d ), or 15 min with NBD-PE ( e ) or NBD-PC ( f ). After extraction with fatty acid-free <t>BSA,</t> the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-lipids uptake is shown relative to that in Mock-treated parental Ba/F3 cells (−). Graphs display averages from three independent experiments ± SD. ** p
    Fatty Acid Free Bsa, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fatty acid free bsa
    C-terminus of ATP11C is required for inhibition of flippase activities by PKC activation. a – c Ba/F3 cells stably expressing HA-tagged ATP11A, ATP11C, and ATP11AAC chimeric proteins were treated with vehicle alone ( a ), 400 nM PMA ( b ), or 1 μM A23187 in the presence of CaCl 2 ( c ) for 15 min. The cells were fixed and immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 5 μm. d – f Ba/F3 cells were treated with vehicle alone (white bars), 400 nM PMA (black bars), or 400 nM PMA and 2 μM of BIM-1 (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated at 15 °C for 5 min with <t>NBD-PS</t> ( d ), or 15 min with NBD-PE ( e ) or NBD-PC ( f ). After extraction with fatty acid-free <t>BSA,</t> the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-lipids uptake is shown relative to that in Mock-treated parental Ba/F3 cells (−). Graphs display averages from three independent experiments ± SD. ** p
    Fatty Acid Free Bsa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore fatty acid free bovine serum albumin faf bsa
    C-terminus of ATP11C is required for inhibition of flippase activities by PKC activation. a – c Ba/F3 cells stably expressing HA-tagged ATP11A, ATP11C, and ATP11AAC chimeric proteins were treated with vehicle alone ( a ), 400 nM PMA ( b ), or 1 μM A23187 in the presence of CaCl 2 ( c ) for 15 min. The cells were fixed and immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 5 μm. d – f Ba/F3 cells were treated with vehicle alone (white bars), 400 nM PMA (black bars), or 400 nM PMA and 2 μM of BIM-1 (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated at 15 °C for 5 min with <t>NBD-PS</t> ( d ), or 15 min with NBD-PE ( e ) or NBD-PC ( f ). After extraction with fatty acid-free <t>BSA,</t> the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-lipids uptake is shown relative to that in Mock-treated parental Ba/F3 cells (−). Graphs display averages from three independent experiments ± SD. ** p
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    Valiant fatty acid free bovine serum albumin
    C-terminus of ATP11C is required for inhibition of flippase activities by PKC activation. a – c Ba/F3 cells stably expressing HA-tagged ATP11A, ATP11C, and ATP11AAC chimeric proteins were treated with vehicle alone ( a ), 400 nM PMA ( b ), or 1 μM A23187 in the presence of CaCl 2 ( c ) for 15 min. The cells were fixed and immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 5 μm. d – f Ba/F3 cells were treated with vehicle alone (white bars), 400 nM PMA (black bars), or 400 nM PMA and 2 μM of BIM-1 (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated at 15 °C for 5 min with <t>NBD-PS</t> ( d ), or 15 min with NBD-PE ( e ) or NBD-PC ( f ). After extraction with fatty acid-free <t>BSA,</t> the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-lipids uptake is shown relative to that in Mock-treated parental Ba/F3 cells (−). Graphs display averages from three independent experiments ± SD. ** p
    Fatty Acid Free Bovine Serum Albumin, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bsa fraction v igg free fatty acid poor custom product
    C-terminus of ATP11C is required for inhibition of flippase activities by PKC activation. a – c Ba/F3 cells stably expressing HA-tagged ATP11A, ATP11C, and ATP11AAC chimeric proteins were treated with vehicle alone ( a ), 400 nM PMA ( b ), or 1 μM A23187 in the presence of CaCl 2 ( c ) for 15 min. The cells were fixed and immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 5 μm. d – f Ba/F3 cells were treated with vehicle alone (white bars), 400 nM PMA (black bars), or 400 nM PMA and 2 μM of BIM-1 (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated at 15 °C for 5 min with <t>NBD-PS</t> ( d ), or 15 min with NBD-PE ( e ) or NBD-PC ( f ). After extraction with fatty acid-free <t>BSA,</t> the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-lipids uptake is shown relative to that in Mock-treated parental Ba/F3 cells (−). Graphs display averages from three independent experiments ± SD. ** p
    Bsa Fraction V Igg Free Fatty Acid Poor Custom Product, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare fatty acid free bsa
    C-terminus of ATP11C is required for inhibition of flippase activities by PKC activation. a – c Ba/F3 cells stably expressing HA-tagged ATP11A, ATP11C, and ATP11AAC chimeric proteins were treated with vehicle alone ( a ), 400 nM PMA ( b ), or 1 μM A23187 in the presence of CaCl 2 ( c ) for 15 min. The cells were fixed and immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 5 μm. d – f Ba/F3 cells were treated with vehicle alone (white bars), 400 nM PMA (black bars), or 400 nM PMA and 2 μM of BIM-1 (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated at 15 °C for 5 min with <t>NBD-PS</t> ( d ), or 15 min with NBD-PE ( e ) or NBD-PC ( f ). After extraction with fatty acid-free <t>BSA,</t> the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-lipids uptake is shown relative to that in Mock-treated parental Ba/F3 cells (−). Graphs display averages from three independent experiments ± SD. ** p
    Fatty Acid Free Bsa, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PAA Laboratories fatty acid free bsa
    C-terminus of ATP11C is required for inhibition of flippase activities by PKC activation. a – c Ba/F3 cells stably expressing HA-tagged ATP11A, ATP11C, and ATP11AAC chimeric proteins were treated with vehicle alone ( a ), 400 nM PMA ( b ), or 1 μM A23187 in the presence of CaCl 2 ( c ) for 15 min. The cells were fixed and immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 5 μm. d – f Ba/F3 cells were treated with vehicle alone (white bars), 400 nM PMA (black bars), or 400 nM PMA and 2 μM of BIM-1 (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated at 15 °C for 5 min with <t>NBD-PS</t> ( d ), or 15 min with NBD-PE ( e ) or NBD-PC ( f ). After extraction with fatty acid-free <t>BSA,</t> the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-lipids uptake is shown relative to that in Mock-treated parental Ba/F3 cells (−). Graphs display averages from three independent experiments ± SD. ** p
    Fatty Acid Free Bsa, supplied by PAA Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Seahorse Biosciences fatty acid free bsa
    C-terminus of ATP11C is required for inhibition of flippase activities by PKC activation. a – c Ba/F3 cells stably expressing HA-tagged ATP11A, ATP11C, and ATP11AAC chimeric proteins were treated with vehicle alone ( a ), 400 nM PMA ( b ), or 1 μM A23187 in the presence of CaCl 2 ( c ) for 15 min. The cells were fixed and immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 5 μm. d – f Ba/F3 cells were treated with vehicle alone (white bars), 400 nM PMA (black bars), or 400 nM PMA and 2 μM of BIM-1 (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated at 15 °C for 5 min with <t>NBD-PS</t> ( d ), or 15 min with NBD-PE ( e ) or NBD-PC ( f ). After extraction with fatty acid-free <t>BSA,</t> the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-lipids uptake is shown relative to that in Mock-treated parental Ba/F3 cells (−). Graphs display averages from three independent experiments ± SD. ** p
    Fatty Acid Free Bsa, supplied by Seahorse Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proliant faf bsa
    C-terminus of ATP11C is required for inhibition of flippase activities by PKC activation. a – c Ba/F3 cells stably expressing HA-tagged ATP11A, ATP11C, and ATP11AAC chimeric proteins were treated with vehicle alone ( a ), 400 nM PMA ( b ), or 1 μM A23187 in the presence of CaCl 2 ( c ) for 15 min. The cells were fixed and immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 5 μm. d – f Ba/F3 cells were treated with vehicle alone (white bars), 400 nM PMA (black bars), or 400 nM PMA and 2 μM of BIM-1 (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated at 15 °C for 5 min with <t>NBD-PS</t> ( d ), or 15 min with NBD-PE ( e ) or NBD-PC ( f ). After extraction with fatty acid-free <t>BSA,</t> the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-lipids uptake is shown relative to that in Mock-treated parental Ba/F3 cells (−). Graphs display averages from three independent experiments ± SD. ** p
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    Image Search Results


    Total amount of lipids in bovine oocytes matured in vitro for 24 h. A) Lipid content in oocytes with different protein sources (0.4% BSA or 10% FCS) with or without phosphodiesterase 5 inhibitor (10 −5 M SDF) associated or not with PKG inhibitor (10 -5 M KT5823). B) Representative image (20x magnification) of Nile Red stained FCS and FCS + SDF treated oocytes. The control group consists of COCs matured with 0.4% BSA without addition of FCS or SDF. Data are expressed as the mean ± SEM of six replicates. Values with different superscript letters differ significantly (p

    Journal: PLoS ONE

    Article Title: The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation

    doi: 10.1371/journal.pone.0191023

    Figure Lengend Snippet: Total amount of lipids in bovine oocytes matured in vitro for 24 h. A) Lipid content in oocytes with different protein sources (0.4% BSA or 10% FCS) with or without phosphodiesterase 5 inhibitor (10 −5 M SDF) associated or not with PKG inhibitor (10 -5 M KT5823). B) Representative image (20x magnification) of Nile Red stained FCS and FCS + SDF treated oocytes. The control group consists of COCs matured with 0.4% BSA without addition of FCS or SDF. Data are expressed as the mean ± SEM of six replicates. Values with different superscript letters differ significantly (p

    Article Snippet: The culture medium was synthetic oviduct fluid with amino acids (SOFaa) [ ] supplemented with 2.7 mM/mL myo-inositol, 0.2 mM/mL pyruvate, 2.0% fetal calf serum (FCS; v/v), 5 mg/mL BSA (fatty acid-free), 100 μg/mL streptomycin sulfate and 100 IU/mL penicillin (Gibco).

    Techniques: In Vitro, Staining

    Relative abundance of transcripts for genes related to lipid metabolism in cumulus cells after 24 h IVM with BSA (control) or 10% FCS with or without phosphodiesterase 5 inhibitor (10 −5 M SDF) associated or not with PKG inhibitor (10 −5 M KT). Data are expressed as the mean ± SEM of five replicates. (A) ATGL, adipose triglyceride lipase; (B) PLIN2, perilipin 2.

    Journal: PLoS ONE

    Article Title: The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation

    doi: 10.1371/journal.pone.0191023

    Figure Lengend Snippet: Relative abundance of transcripts for genes related to lipid metabolism in cumulus cells after 24 h IVM with BSA (control) or 10% FCS with or without phosphodiesterase 5 inhibitor (10 −5 M SDF) associated or not with PKG inhibitor (10 −5 M KT). Data are expressed as the mean ± SEM of five replicates. (A) ATGL, adipose triglyceride lipase; (B) PLIN2, perilipin 2.

    Article Snippet: The culture medium was synthetic oviduct fluid with amino acids (SOFaa) [ ] supplemented with 2.7 mM/mL myo-inositol, 0.2 mM/mL pyruvate, 2.0% fetal calf serum (FCS; v/v), 5 mg/mL BSA (fatty acid-free), 100 μg/mL streptomycin sulfate and 100 IU/mL penicillin (Gibco).

    Techniques:

    cGMP levels in bovine COCs matured in vitro for 24 h in in the presence or not of the PDE5 inhibitor (10 −5 M SDF) in TCM199 supplemented with 0.4% BSA or 10% FCS. The control group consists of COCs matured with 0.4% BSA without addition of FCS or SDF. Data are the mean ± SEM of four replicates. Different letters indicate significant differences (p

    Journal: PLoS ONE

    Article Title: The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation

    doi: 10.1371/journal.pone.0191023

    Figure Lengend Snippet: cGMP levels in bovine COCs matured in vitro for 24 h in in the presence or not of the PDE5 inhibitor (10 −5 M SDF) in TCM199 supplemented with 0.4% BSA or 10% FCS. The control group consists of COCs matured with 0.4% BSA without addition of FCS or SDF. Data are the mean ± SEM of four replicates. Different letters indicate significant differences (p

    Article Snippet: The culture medium was synthetic oviduct fluid with amino acids (SOFaa) [ ] supplemented with 2.7 mM/mL myo-inositol, 0.2 mM/mL pyruvate, 2.0% fetal calf serum (FCS; v/v), 5 mg/mL BSA (fatty acid-free), 100 μg/mL streptomycin sulfate and 100 IU/mL penicillin (Gibco).

    Techniques: In Vitro

    Comparison of Δh ads values for the adsorption of BSA and β‐lactoglobulin to Butyl Sepharose 4 FF at 1.2 mol/kg (NH 4 ) 2 SO 4 . Adsorption of BSA (A) and β‐lactoglobulin (B) at 308 K. Adsorption of BSA (C) and β‐lactoglobulin (D) at 303 K. Adsorption of BSA (E) and β‐lactoglobulin (F) at 298 K

    Journal: Journal of Separation Science

    Article Title: Hydrophobic interaction chromatography of proteins: Studies of unfolding upon adsorption by isothermal titration calorimetry. Hydrophobic interaction chromatography of proteins: Studies of unfolding upon adsorption by isothermal titration calorimetry

    doi: 10.1002/jssc.201800016

    Figure Lengend Snippet: Comparison of Δh ads values for the adsorption of BSA and β‐lactoglobulin to Butyl Sepharose 4 FF at 1.2 mol/kg (NH 4 ) 2 SO 4 . Adsorption of BSA (A) and β‐lactoglobulin (B) at 308 K. Adsorption of BSA (C) and β‐lactoglobulin (D) at 303 K. Adsorption of BSA (E) and β‐lactoglobulin (F) at 298 K

    Article Snippet: BSA (A6003; essentially fatty acid free) and β‐lactoglobulin (L3908) were purchased from Sigma–Aldrich (Vienna, Austria).

    Techniques: Adsorption

    Values of Δh ads for the adsorption of BSA and β‐lactoglobulin to Toyopearl Butyl‐650 M at an (NH 4 ) 2 SO 4 concentration of 1.2 mol/kg (A and C) and at 0.7 mol/kg (NH 4 ) 2 SO 4 (B and D)

    Journal: Journal of Separation Science

    Article Title: Hydrophobic interaction chromatography of proteins: Studies of unfolding upon adsorption by isothermal titration calorimetry. Hydrophobic interaction chromatography of proteins: Studies of unfolding upon adsorption by isothermal titration calorimetry

    doi: 10.1002/jssc.201800016

    Figure Lengend Snippet: Values of Δh ads for the adsorption of BSA and β‐lactoglobulin to Toyopearl Butyl‐650 M at an (NH 4 ) 2 SO 4 concentration of 1.2 mol/kg (A and C) and at 0.7 mol/kg (NH 4 ) 2 SO 4 (B and D)

    Article Snippet: BSA (A6003; essentially fatty acid free) and β‐lactoglobulin (L3908) were purchased from Sigma–Aldrich (Vienna, Austria).

    Techniques: Adsorption, Concentration Assay

    Competition affinity experiments with FAF-BSA. Global curve fitting of the three affinity experiments used to determine the equilibrium dissociation constants for the individual LPA species (A–E) or S1P (F) binding the antibody or FAF-BSA in solution.

    Journal: Journal of Lipid Research

    Article Title: A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay [S]

    doi: 10.1194/jlr.D068866

    Figure Lengend Snippet: Competition affinity experiments with FAF-BSA. Global curve fitting of the three affinity experiments used to determine the equilibrium dissociation constants for the individual LPA species (A–E) or S1P (F) binding the antibody or FAF-BSA in solution.

    Article Snippet: The concentrations of the protein stock solutions were determined by measuring the absorbance at 280 nm and using an extinction coefficient of 1.4 ml/mg for LT1009 and LT3015 and 0.66 ml/mg for FAF-BSA and FAF-HSA (Sigma-Aldrich) ( – ).

    Techniques: Binding Assay

    Error curves for FAF-BSA experiments. Competition n-curve error curves for the experiments in . The K d values for LPA (A–E) and S1P (F) binding either the antibody ( K d 1 , red dotted curve) or FAF-BSA ( K d 2 , black dotted curve) correspond to

    Journal: Journal of Lipid Research

    Article Title: A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay [S]

    doi: 10.1194/jlr.D068866

    Figure Lengend Snippet: Error curves for FAF-BSA experiments. Competition n-curve error curves for the experiments in . The K d values for LPA (A–E) and S1P (F) binding either the antibody ( K d 1 , red dotted curve) or FAF-BSA ( K d 2 , black dotted curve) correspond to

    Article Snippet: The concentrations of the protein stock solutions were determined by measuring the absorbance at 280 nm and using an extinction coefficient of 1.4 ml/mg for LT1009 and LT3015 and 0.66 ml/mg for FAF-BSA and FAF-HSA (Sigma-Aldrich) ( – ).

    Techniques: Binding Assay

    Effect of diabetes on cholesterol efflux to apoA1 or HDL and regulation of cholesterol transporters by Ager in primary murine BMDMs. Primary BMDMs were retrieved from nondiabetic and diabetic WT (2 months of hyperglycemia) and Ager −/− mice and were cultured in concentrations of glucose consistent with the glycemic state of the mice from which they were retrieved. BMDMs were labeled with 3 H-cholesterol and treated with acetylated LDL and fatty acid–free BSA (1%) for 24 h. A and B : To mediate cholesterol efflux, cells were treated with apoA1 (5 μg/mL) ( A ) or HDL (100 μg/mL) ( B ) for 6 h, and supernatant was collected. Percent cholesterol efflux is the radioactivity in the supernatant divided by the sum of the radioactivity measured in the supernatant and the cell lysates from n ≥ 5 mice/group. C and D : BMDMs were retrieved from WT or Ager −/− mice and subjected to quantitative real-time PCR for detection of Abca1 mRNA transcript ( C ) and ABCA1 protein ( D ) analysis ( n = 3 mice/group). E and F : BMDMs were prepared as in C and D and assessed for levels of Abcg1 mRNA transcripts ( E ) and ABCG1 protein ( F ) ( n = 3 mice/group). Error bars represent mean ± SEM. hr, hour; NS, not statistically significant.

    Journal: Diabetes

    Article Title: RAGE Suppresses ABCG1-Mediated Macrophage Cholesterol Efflux in Diabetes

    doi: 10.2337/db15-0575

    Figure Lengend Snippet: Effect of diabetes on cholesterol efflux to apoA1 or HDL and regulation of cholesterol transporters by Ager in primary murine BMDMs. Primary BMDMs were retrieved from nondiabetic and diabetic WT (2 months of hyperglycemia) and Ager −/− mice and were cultured in concentrations of glucose consistent with the glycemic state of the mice from which they were retrieved. BMDMs were labeled with 3 H-cholesterol and treated with acetylated LDL and fatty acid–free BSA (1%) for 24 h. A and B : To mediate cholesterol efflux, cells were treated with apoA1 (5 μg/mL) ( A ) or HDL (100 μg/mL) ( B ) for 6 h, and supernatant was collected. Percent cholesterol efflux is the radioactivity in the supernatant divided by the sum of the radioactivity measured in the supernatant and the cell lysates from n ≥ 5 mice/group. C and D : BMDMs were retrieved from WT or Ager −/− mice and subjected to quantitative real-time PCR for detection of Abca1 mRNA transcript ( C ) and ABCA1 protein ( D ) analysis ( n = 3 mice/group). E and F : BMDMs were prepared as in C and D and assessed for levels of Abcg1 mRNA transcripts ( E ) and ABCG1 protein ( F ) ( n = 3 mice/group). Error bars represent mean ± SEM. hr, hour; NS, not statistically significant.

    Article Snippet: Reagents The following materials were purchased: apolipoprotein A1 (apoA1), HDL, and acetylated LDL (Biomedical Technologies, Inc.); fatty acid–free BSA (Equitech-Bio); T 0901317 (Tocris Bioscience); human plasma LDL (Sigma-Aldrich); assays for measurements of HDL, total cholesterol, and triglycerides (Wako Diagnostics); rosiglitazone (Sigma-Aldrich); and U0126 and PD98509 (Cell Signaling).

    Techniques: Mouse Assay, Cell Culture, Labeling, Radioactivity, Real-time Polymerase Chain Reaction

    Analyses of flippase activity in different human cancer cell types A. Flippase activity assay: Indicated cell types were incubated with NBD-PS for indicated time periods and subjected to BSA extraction and sodium dithionite treatment. % nonextractable NBD-PS (after BSA extraction and sodium dithionite treatment) represents internalized NBD-PS, indicative of flippase activity (Schwann ◊, U87ΔEGFR-Luc □, H1299 Δ, MDA-MB-231 ○, MDA-MB-231-Luc-D3H2LN ◆, Gli36 ▲, U373 ●) B. NBD PS incorporation rate. C. NBD PS incorporation at 15 minutes time point.

    Journal: Oncotarget

    Article Title: Variation in human cancer cell external phosphatidylserine is regulated by flippase activity and intracellular calcium

    doi:

    Figure Lengend Snippet: Analyses of flippase activity in different human cancer cell types A. Flippase activity assay: Indicated cell types were incubated with NBD-PS for indicated time periods and subjected to BSA extraction and sodium dithionite treatment. % nonextractable NBD-PS (after BSA extraction and sodium dithionite treatment) represents internalized NBD-PS, indicative of flippase activity (Schwann ◊, U87ΔEGFR-Luc □, H1299 Δ, MDA-MB-231 ○, MDA-MB-231-Luc-D3H2LN ◆, Gli36 ▲, U373 ●) B. NBD PS incorporation rate. C. NBD PS incorporation at 15 minutes time point.

    Article Snippet: The remaining half was spun down to remove non inserted NBD-PS and subjected to BSA extraction of NBD-PS from the outer leaflet by adding 3% fatty acid free BSA (MP biomedicals) in flippase assay buffer.

    Techniques: Activity Assay, Incubation, Multiple Displacement Amplification

    Inhibition of flippase activity by NEM reveals involvement of flippase activity in the regulation of surface PS A . Low surface PS cells were either treated with NEM or left untreated, incubated with NBD-PS for indicated time periods and subjected to BSA extraction and sodium dithionite treatment. % nonextractable NBD-PS (after BSA extraction and sodium dithionite treatment) represents internalized NBD-PS, indicative of flippase activity B . Flippase activity was inhibited by use of NEM in low surface PS cell lines and surface PS levels were measured by annexin V FITC binding assay, by flow cytometry. The graph shows annexin V FITC fold change compared to mock treated cells. C . High surface PS cells were either treated with NEM or left untreated and incubated with NBD-PS for indicated time periods and subjected to BSA extraction and sodium dithionite treatment. % nonextractable NBD-PS (after BSA extraction and sodium dithionite treatment) represents internalized NBD-PS, indicative of flippase activity D . Flippase activity was inhibited by use of NEM in the high surface PS cell lines and surface PS levels were measured by annexin V FITC binding assay by flow cytometry. The graph shows annexin V FITC fold change compared to mock treated cells.

    Journal: Oncotarget

    Article Title: Variation in human cancer cell external phosphatidylserine is regulated by flippase activity and intracellular calcium

    doi:

    Figure Lengend Snippet: Inhibition of flippase activity by NEM reveals involvement of flippase activity in the regulation of surface PS A . Low surface PS cells were either treated with NEM or left untreated, incubated with NBD-PS for indicated time periods and subjected to BSA extraction and sodium dithionite treatment. % nonextractable NBD-PS (after BSA extraction and sodium dithionite treatment) represents internalized NBD-PS, indicative of flippase activity B . Flippase activity was inhibited by use of NEM in low surface PS cell lines and surface PS levels were measured by annexin V FITC binding assay, by flow cytometry. The graph shows annexin V FITC fold change compared to mock treated cells. C . High surface PS cells were either treated with NEM or left untreated and incubated with NBD-PS for indicated time periods and subjected to BSA extraction and sodium dithionite treatment. % nonextractable NBD-PS (after BSA extraction and sodium dithionite treatment) represents internalized NBD-PS, indicative of flippase activity D . Flippase activity was inhibited by use of NEM in the high surface PS cell lines and surface PS levels were measured by annexin V FITC binding assay by flow cytometry. The graph shows annexin V FITC fold change compared to mock treated cells.

    Article Snippet: The remaining half was spun down to remove non inserted NBD-PS and subjected to BSA extraction of NBD-PS from the outer leaflet by adding 3% fatty acid free BSA (MP biomedicals) in flippase assay buffer.

    Techniques: Inhibition, Activity Assay, Incubation, Binding Assay, Flow Cytometry, Cytometry

    C-terminus of ATP11C is required for inhibition of flippase activities by PKC activation. a – c Ba/F3 cells stably expressing HA-tagged ATP11A, ATP11C, and ATP11AAC chimeric proteins were treated with vehicle alone ( a ), 400 nM PMA ( b ), or 1 μM A23187 in the presence of CaCl 2 ( c ) for 15 min. The cells were fixed and immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 5 μm. d – f Ba/F3 cells were treated with vehicle alone (white bars), 400 nM PMA (black bars), or 400 nM PMA and 2 μM of BIM-1 (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated at 15 °C for 5 min with NBD-PS ( d ), or 15 min with NBD-PE ( e ) or NBD-PC ( f ). After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-lipids uptake is shown relative to that in Mock-treated parental Ba/F3 cells (−). Graphs display averages from three independent experiments ± SD. ** p

    Journal: Nature Communications

    Article Title: Phospholipid flippase ATP11C is endocytosed and downregulated following Ca2+-mediated protein kinase C activation

    doi: 10.1038/s41467-017-01338-1

    Figure Lengend Snippet: C-terminus of ATP11C is required for inhibition of flippase activities by PKC activation. a – c Ba/F3 cells stably expressing HA-tagged ATP11A, ATP11C, and ATP11AAC chimeric proteins were treated with vehicle alone ( a ), 400 nM PMA ( b ), or 1 μM A23187 in the presence of CaCl 2 ( c ) for 15 min. The cells were fixed and immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 5 μm. d – f Ba/F3 cells were treated with vehicle alone (white bars), 400 nM PMA (black bars), or 400 nM PMA and 2 μM of BIM-1 (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated at 15 °C for 5 min with NBD-PS ( d ), or 15 min with NBD-PE ( e ) or NBD-PC ( f ). After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-lipids uptake is shown relative to that in Mock-treated parental Ba/F3 cells (−). Graphs display averages from three independent experiments ± SD. ** p

    Article Snippet: At each time point, 200 μl of the cell suspension was mixed with 200 μl of ice-cold HBSS-glucose containing 5% fatty acid-free BSA (Wako) to extract NBD-lipids incorporated into the exoplasmic leaflet of the plasma membrane, as well as unincorporated lipids.

    Techniques: Inhibition, Activation Assay, Stable Transfection, Expressing, Incubation, Fluorescence, Flow Cytometry, Cytometry

    ATP11C is endocytosed by treatment with phorbol 12-myristate 13-acetate (PMA) and increasing cytosolic Ca 2+ . a HeLa cells stably expressing C-terminally HA-tagged ATP11A, and ATP11C were treated for 15 min at 37 °C with vehicle alone (Mock); with either 400 nM of PMA (PMA) or PMA and 2 μM of BIM-1 (PMA + BIM); or with 1 μM A23187 in the presence of either 1.8 mM of CaCl 2 (A23187) or 1.8 mM CaCl 2 and 2 μM BIM-1 (A23187 + BIM). The cells were fixed and immunostained with anti-HA antibody, followed by Cy3-conjugated anti-rat secondary antibody. See Supplementary Fig. 1 and Supplementary Movie 1 . b Cell-surface expression levels of ATP11A and ATP11C following treatment with PMA or A23187 and CaCl 2 were analyzed after surface biotinylation. Proteins precipitated with streptavidin-agarose beads were subjected to immunoblot analysis (left panels, biotinylated). 15% of the input of the biotinylation reaction was loaded in each lane (right panels, total lysate). Expression of ATP11A and ATP11C proteins was analyzed by immunoblotting with anti-HA and anti-ATP1A1 (as an internal control) antibodies. c HeLa cells stably expressing HA-tagged ATP11A and ATP11C, and parental cells (−) were treated with vehicle alone (white bars), 400 nM of PMA (black bars), or 400 nM of PMA and 2 μM of BIM-1, simultaneously (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated with NBD-PS at 15 °C for 5 min. After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-PS uptake is shown relative to that in Mock-treated parental HeLa cells (−). Graph displays averages from four independent experiments ± SD. *** p

    Journal: Nature Communications

    Article Title: Phospholipid flippase ATP11C is endocytosed and downregulated following Ca2+-mediated protein kinase C activation

    doi: 10.1038/s41467-017-01338-1

    Figure Lengend Snippet: ATP11C is endocytosed by treatment with phorbol 12-myristate 13-acetate (PMA) and increasing cytosolic Ca 2+ . a HeLa cells stably expressing C-terminally HA-tagged ATP11A, and ATP11C were treated for 15 min at 37 °C with vehicle alone (Mock); with either 400 nM of PMA (PMA) or PMA and 2 μM of BIM-1 (PMA + BIM); or with 1 μM A23187 in the presence of either 1.8 mM of CaCl 2 (A23187) or 1.8 mM CaCl 2 and 2 μM BIM-1 (A23187 + BIM). The cells were fixed and immunostained with anti-HA antibody, followed by Cy3-conjugated anti-rat secondary antibody. See Supplementary Fig. 1 and Supplementary Movie 1 . b Cell-surface expression levels of ATP11A and ATP11C following treatment with PMA or A23187 and CaCl 2 were analyzed after surface biotinylation. Proteins precipitated with streptavidin-agarose beads were subjected to immunoblot analysis (left panels, biotinylated). 15% of the input of the biotinylation reaction was loaded in each lane (right panels, total lysate). Expression of ATP11A and ATP11C proteins was analyzed by immunoblotting with anti-HA and anti-ATP1A1 (as an internal control) antibodies. c HeLa cells stably expressing HA-tagged ATP11A and ATP11C, and parental cells (−) were treated with vehicle alone (white bars), 400 nM of PMA (black bars), or 400 nM of PMA and 2 μM of BIM-1, simultaneously (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated with NBD-PS at 15 °C for 5 min. After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-PS uptake is shown relative to that in Mock-treated parental HeLa cells (−). Graph displays averages from four independent experiments ± SD. *** p

    Article Snippet: At each time point, 200 μl of the cell suspension was mixed with 200 μl of ice-cold HBSS-glucose containing 5% fatty acid-free BSA (Wako) to extract NBD-lipids incorporated into the exoplasmic leaflet of the plasma membrane, as well as unincorporated lipids.

    Techniques: Stable Transfection, Expressing, Incubation, Fluorescence, Flow Cytometry, Cytometry