fatty acid-free bovine serum albumin bsa Search Results


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  • 99
    Thermo Fisher fatty acid free bovine serum albumin
    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% <t>BSA</t> or 10% <t>FCS.</t> 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
    Fatty Acid Free Bovine Serum Albumin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore fatty acid free bsa
    Modulation of S1P homeostasis of K562 cells by sodium butyrate-induced differentiation into erythroblast-like cells. K562 cells were treated with 2 mM sodium butyrate (NaB); 72 hours later, we investigated the modulation of S1P homeostasis. (A) The mRNA levels of GYPA and ALAS were determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (B, C) The expression of CD235a was investigated with a flow cytometer (n = 3/group). (D) C 17 S1P formation assay. NaB-treated or vehicle-treated K562 cells were treated with 10 μM of C 17 sphingosine for 20 minutes. Then, we replaced the supernatant with <t>PBS</t> containing 0.5% <t>BSA</t> and incubated the cells at 37°C for another 20 minutes. Then, the supernatants and cells were collected and used for the C 17 S1P measurements (n = 6/group). (E) The expression of key enzymes in S1P metabolism was determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (F) SK activity assay. The SK activity assay was performed using NaB-treated or vehicle-treated K562 cells (n = 6/group). (G) Reverse transcription PCR was performed using cDNAs prepared from NaB-treated K562 cells (K), HepG2 cells (He), and HUVECs (Hu). (H) The expression of possible S1P transporters was determined using real-time PCR. GAPDH was utilized as an internal control (n = 4/group). (I) Real-time PCR of Band3. GAPDH was utilized as an internal control (n = 8/group). (J) Western blot of Band3 with membranous protein. The whole cell lysate of RBCs (2 μg) was placed as a positive control. Pan-cadherin was utilized as an internal control (n = 3/group).
    Fatty Acid Free Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8267 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bovine serum albumin
    Modulation of S1P homeostasis of K562 cells by sodium butyrate-induced differentiation into erythroblast-like cells. K562 cells were treated with 2 mM sodium butyrate (NaB); 72 hours later, we investigated the modulation of S1P homeostasis. (A) The mRNA levels of GYPA and ALAS were determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (B, C) The expression of CD235a was investigated with a flow cytometer (n = 3/group). (D) C 17 S1P formation assay. NaB-treated or vehicle-treated K562 cells were treated with 10 μM of C 17 sphingosine for 20 minutes. Then, we replaced the supernatant with <t>PBS</t> containing 0.5% <t>BSA</t> and incubated the cells at 37°C for another 20 minutes. Then, the supernatants and cells were collected and used for the C 17 S1P measurements (n = 6/group). (E) The expression of key enzymes in S1P metabolism was determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (F) SK activity assay. The SK activity assay was performed using NaB-treated or vehicle-treated K562 cells (n = 6/group). (G) Reverse transcription PCR was performed using cDNAs prepared from NaB-treated K562 cells (K), HepG2 cells (He), and HUVECs (Hu). (H) The expression of possible S1P transporters was determined using real-time PCR. GAPDH was utilized as an internal control (n = 4/group). (I) Real-time PCR of Band3. GAPDH was utilized as an internal control (n = 8/group). (J) Western blot of Band3 with membranous protein. The whole cell lysate of RBCs (2 μg) was placed as a positive control. Pan-cadherin was utilized as an internal control (n = 3/group).
    Bovine Serum Albumin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 96597 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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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    Millipore bsa fatty acid free
    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 Fatty Acid Free, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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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    92
    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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    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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    Thermo Fisher fatty acid free bovine serum albumin 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 Bovine Serum Albumin Bsa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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
    Faf Bsa, supplied by Proliant, used in various techniques. Bioz Stars score: 89/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    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:

    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

    Modulation of S1P homeostasis of K562 cells by sodium butyrate-induced differentiation into erythroblast-like cells. K562 cells were treated with 2 mM sodium butyrate (NaB); 72 hours later, we investigated the modulation of S1P homeostasis. (A) The mRNA levels of GYPA and ALAS were determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (B, C) The expression of CD235a was investigated with a flow cytometer (n = 3/group). (D) C 17 S1P formation assay. NaB-treated or vehicle-treated K562 cells were treated with 10 μM of C 17 sphingosine for 20 minutes. Then, we replaced the supernatant with PBS containing 0.5% BSA and incubated the cells at 37°C for another 20 minutes. Then, the supernatants and cells were collected and used for the C 17 S1P measurements (n = 6/group). (E) The expression of key enzymes in S1P metabolism was determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (F) SK activity assay. The SK activity assay was performed using NaB-treated or vehicle-treated K562 cells (n = 6/group). (G) Reverse transcription PCR was performed using cDNAs prepared from NaB-treated K562 cells (K), HepG2 cells (He), and HUVECs (Hu). (H) The expression of possible S1P transporters was determined using real-time PCR. GAPDH was utilized as an internal control (n = 4/group). (I) Real-time PCR of Band3. GAPDH was utilized as an internal control (n = 8/group). (J) Western blot of Band3 with membranous protein. The whole cell lysate of RBCs (2 μg) was placed as a positive control. Pan-cadherin was utilized as an internal control (n = 3/group).

    Journal: PLoS ONE

    Article Title: Involvement of Band3 in the efflux of sphingosine 1-phosphate from erythrocytes

    doi: 10.1371/journal.pone.0177543

    Figure Lengend Snippet: Modulation of S1P homeostasis of K562 cells by sodium butyrate-induced differentiation into erythroblast-like cells. K562 cells were treated with 2 mM sodium butyrate (NaB); 72 hours later, we investigated the modulation of S1P homeostasis. (A) The mRNA levels of GYPA and ALAS were determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (B, C) The expression of CD235a was investigated with a flow cytometer (n = 3/group). (D) C 17 S1P formation assay. NaB-treated or vehicle-treated K562 cells were treated with 10 μM of C 17 sphingosine for 20 minutes. Then, we replaced the supernatant with PBS containing 0.5% BSA and incubated the cells at 37°C for another 20 minutes. Then, the supernatants and cells were collected and used for the C 17 S1P measurements (n = 6/group). (E) The expression of key enzymes in S1P metabolism was determined using real-time PCR. GAPDH was utilized as an internal control (n = 8/group). (F) SK activity assay. The SK activity assay was performed using NaB-treated or vehicle-treated K562 cells (n = 6/group). (G) Reverse transcription PCR was performed using cDNAs prepared from NaB-treated K562 cells (K), HepG2 cells (He), and HUVECs (Hu). (H) The expression of possible S1P transporters was determined using real-time PCR. GAPDH was utilized as an internal control (n = 4/group). (I) Real-time PCR of Band3. GAPDH was utilized as an internal control (n = 8/group). (J) Western blot of Band3 with membranous protein. The whole cell lysate of RBCs (2 μg) was placed as a positive control. Pan-cadherin was utilized as an internal control (n = 3/group).

    Article Snippet: Then, we replaced the supernatant with PBS containing 0.5% fatty acid-free BSA (A8806; Sigma-Aldrich Co.) and incubated the cells at 37°C for another 20 minutes.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Tube Formation Assay, Incubation, Activity Assay, Polymerase Chain Reaction, Western Blot, Positive Control

    Valsartan or aliskiren prevented palmitic acid (PA; 0.8 mM)-induced endoplasmic reticulum (ER) stress in cultured human proximal tubule epithelial cells (HK2) cells after a 24-h treatment. A and B : protein abundance of binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP) was unchanged in HK2 cells treated with BSA (2 and 10 mg/ml) and with or without valsartan (10 −6 M) or aliskiren (10 −7 M). C : MTT assays of HK2 cells. HK2 cells were incubated with PA at different concentrations for 24 h. After the cells were incubated with tetrazolium salt solution for 2 h, the quantity of formazan product was determined from the absorbance at 560 nm. * P

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Intrarenal renin-angiotensin system mediates fatty acid-induced ER stress in the kidney

    doi: 10.1152/ajprenal.00223.2015

    Figure Lengend Snippet: Valsartan or aliskiren prevented palmitic acid (PA; 0.8 mM)-induced endoplasmic reticulum (ER) stress in cultured human proximal tubule epithelial cells (HK2) cells after a 24-h treatment. A and B : protein abundance of binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP) was unchanged in HK2 cells treated with BSA (2 and 10 mg/ml) and with or without valsartan (10 −6 M) or aliskiren (10 −7 M). C : MTT assays of HK2 cells. HK2 cells were incubated with PA at different concentrations for 24 h. After the cells were incubated with tetrazolium salt solution for 2 h, the quantity of formazan product was determined from the absorbance at 560 nm. * P

    Article Snippet: PA, fatty acid-free bovine serum albumin (BSA), tunicamycin, and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-dipthenyltetrazolium bromide) were purchased from Sigma-Aldrich; anti-BiP (3177), anti-CHOP (2895), anti-peIF2α (3597)/eIF2α (9722), anti-IRE1α (3294), and anti-activated caspase-3 (9664) antibodies from Cell Signaling; anti-GRP78 (BiP) (SC-13968 for immunohistochemistry) from Santa-Cruz Biotechnology; anti-ATF4 from Abcam (ab50546); and anti-β-actin from Sigma.

    Techniques: Cell Culture, Binding Assay, MTT Assay, Incubation

    Stern-Volmer plots of HSA and BSA fluorescence quenching by C 60 nanoparticles. (F 0 : The fluorescence intensity of BSA/HSA in the absence of C 60 ; F: The fluorescence intensity of BSA/HSA in the presence of C 60 ).

    Journal: Nanoscale Research Letters

    Article Title: Spectroscopic study on the interaction of pristine C60 and serum albumins in solution

    doi: 10.1186/1556-276X-7-433

    Figure Lengend Snippet: Stern-Volmer plots of HSA and BSA fluorescence quenching by C 60 nanoparticles. (F 0 : The fluorescence intensity of BSA/HSA in the absence of C 60 ; F: The fluorescence intensity of BSA/HSA in the presence of C 60 ).

    Article Snippet: HSA and BSA (free fatty acid fraction V) were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Fluorescence

    The CD spectra of HSA (a) and BSA (b). Conditions: HSA/BSA: 1.0 × 10 -6 mol/L; C 60 : (1 to 4): 0, 2.78, 5.56, 11.12 × 10 -6 mol/L; pH = 7.4.

    Journal: Nanoscale Research Letters

    Article Title: Spectroscopic study on the interaction of pristine C60 and serum albumins in solution

    doi: 10.1186/1556-276X-7-433

    Figure Lengend Snippet: The CD spectra of HSA (a) and BSA (b). Conditions: HSA/BSA: 1.0 × 10 -6 mol/L; C 60 : (1 to 4): 0, 2.78, 5.56, 11.12 × 10 -6 mol/L; pH = 7.4.

    Article Snippet: HSA and BSA (free fatty acid fraction V) were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques:

    Stern-Volmer plots of HSA (a) and BSA (b) synchronous fluorescence quenching by C 60 . (1): Δλ=60 nm; (2): Δλ=20 nm.

    Journal: Nanoscale Research Letters

    Article Title: Spectroscopic study on the interaction of pristine C60 and serum albumins in solution

    doi: 10.1186/1556-276X-7-433

    Figure Lengend Snippet: Stern-Volmer plots of HSA (a) and BSA (b) synchronous fluorescence quenching by C 60 . (1): Δλ=60 nm; (2): Δλ=20 nm.

    Article Snippet: HSA and BSA (free fatty acid fraction V) were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Fluorescence

    The fluorescence spectra of HSA (a) and BSA (b) in the absence and presence of C 60 nanoparticles. Conditions: HSA/BSA: 1.0 × 10 -5 mol/L; C 60 (from up to down): 0, 1.39, 2.78, 5.56, 8.34, 11.12 × 10 -6 mol/L; pH = 7.4; ex = 292 nm.

    Journal: Nanoscale Research Letters

    Article Title: Spectroscopic study on the interaction of pristine C60 and serum albumins in solution

    doi: 10.1186/1556-276X-7-433

    Figure Lengend Snippet: The fluorescence spectra of HSA (a) and BSA (b) in the absence and presence of C 60 nanoparticles. Conditions: HSA/BSA: 1.0 × 10 -5 mol/L; C 60 (from up to down): 0, 1.39, 2.78, 5.56, 8.34, 11.12 × 10 -6 mol/L; pH = 7.4; ex = 292 nm.

    Article Snippet: HSA and BSA (free fatty acid fraction V) were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Fluorescence

    The synchronous fluorescence spectra of HSA (a, b)and BSA (c, d) in the absence and presence of C 60 nanoparticles. ( a. HSA, Δλ = 20 nm; b. HSA, Δλ = 60 nm; c. BSA, Δλ = 20 nm; d. BSA, Δλ = 60 nm.) Conditions: HSA/BSA: 1.0 × 10 -5 mol/L; C 60 (from up to down): 0, 1.39, 2.78, 5.56, 8.34, 11.12 × 10 -6 mol/L; pH = 7.4; ex = 292 nm.

    Journal: Nanoscale Research Letters

    Article Title: Spectroscopic study on the interaction of pristine C60 and serum albumins in solution

    doi: 10.1186/1556-276X-7-433

    Figure Lengend Snippet: The synchronous fluorescence spectra of HSA (a, b)and BSA (c, d) in the absence and presence of C 60 nanoparticles. ( a. HSA, Δλ = 20 nm; b. HSA, Δλ = 60 nm; c. BSA, Δλ = 20 nm; d. BSA, Δλ = 60 nm.) Conditions: HSA/BSA: 1.0 × 10 -5 mol/L; C 60 (from up to down): 0, 1.39, 2.78, 5.56, 8.34, 11.12 × 10 -6 mol/L; pH = 7.4; ex = 292 nm.

    Article Snippet: HSA and BSA (free fatty acid fraction V) were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Fluorescence

    Adipose tissue deposition and lipolysis in EPRS A/A and EPRS D/D mice a , Length of mice was measured from head to beginning of tail using a digital caliper (Fisherbrand Traceable). Data shown are mean ± SEM, n = 15 for 20-week male mice. b , Ventral view of wild-type and EPRS A/A mice abdominal cavity. c , Weights of adipose and non-adipose tissues from 20-week male EPRS D/D and control mice (mean ± SEM, n = 14/group, P value from unpaired t-test). d , Scanning electron micrographs of EWAT in 20-week male EPRS S/S , EPRS A/A , and EPRS D/D mice. e , Total adipocyte cell number in EWAT of EPRS A/A knock-in and wild-type mice. Data represent mean ± SEM, n = 5/group. f , Elevated lipolysis in adipocytes from EPRS A/A , but not EPRS D/D , mice (mean ± SEM; n = 6/group). g , Elevated β-oxidation in WAT explants from EPRS A/A mice as determined by release of 14 CO 2 from 14 C-oleic acid (mean ± SEM; n = 5/group). h , Serum levels of insulin, glucose, triglycerides (TG) and free fatty acids (FFA) in 12-h fasted and 1-h post-prandial (fed) 16-week old male mice (mean ± SEM, n = 10/group, * P

    Journal: Nature

    Article Title: EPRS is a critical mTORC1-S6K1 effector that influences adiposity in mice

    doi: 10.1038/nature21380

    Figure Lengend Snippet: Adipose tissue deposition and lipolysis in EPRS A/A and EPRS D/D mice a , Length of mice was measured from head to beginning of tail using a digital caliper (Fisherbrand Traceable). Data shown are mean ± SEM, n = 15 for 20-week male mice. b , Ventral view of wild-type and EPRS A/A mice abdominal cavity. c , Weights of adipose and non-adipose tissues from 20-week male EPRS D/D and control mice (mean ± SEM, n = 14/group, P value from unpaired t-test). d , Scanning electron micrographs of EWAT in 20-week male EPRS S/S , EPRS A/A , and EPRS D/D mice. e , Total adipocyte cell number in EWAT of EPRS A/A knock-in and wild-type mice. Data represent mean ± SEM, n = 5/group. f , Elevated lipolysis in adipocytes from EPRS A/A , but not EPRS D/D , mice (mean ± SEM; n = 6/group). g , Elevated β-oxidation in WAT explants from EPRS A/A mice as determined by release of 14 CO 2 from 14 C-oleic acid (mean ± SEM; n = 5/group). h , Serum levels of insulin, glucose, triglycerides (TG) and free fatty acids (FFA) in 12-h fasted and 1-h post-prandial (fed) 16-week old male mice (mean ± SEM, n = 10/group, * P

    Article Snippet: Briefly, after mouse sacrifice, fat pads were removed and minced in Krebs-Ringer-bicarbonate-HEPES (KRBH) buffer pH 7.4 containing 10 mM sodium bicarbonate, 30 mM HEPES, 200 nM adenosine, and 1% fatty acid-free bovine serum albumin (BSA, Sigma).

    Techniques: Mouse Assay, Knock-In

    Insulin, metformin, BLX-1002, pioglitazone and candesartan protect HCAECs against palmitate-induced caspase-3 activation . Palmitate-induced caspase-3 activity, a measure of apoptosis, was evaluated using the EnzChek ® Caspase-3 Assay Kit. HCAECs were incubated in medium containing 5 mM glucose, supplemented with 0.5% FBS, with or without insulin lispro (1 nM), BLX-1002 (1 μM), metformin (500 μM), pioglitazone (2.5 μM), candesartan (100 nM) in the presence of 0.125 mM palmitate or vehicle (ethanol and BSA) for 24 h. * denotes P

    Journal: Cardiovascular Diabetology

    Article Title: Effects of some anti-diabetic and cardioprotective agents on proliferation and apoptosis of human coronary artery endothelial cells

    doi: 10.1186/1475-2840-11-27

    Figure Lengend Snippet: Insulin, metformin, BLX-1002, pioglitazone and candesartan protect HCAECs against palmitate-induced caspase-3 activation . Palmitate-induced caspase-3 activity, a measure of apoptosis, was evaluated using the EnzChek ® Caspase-3 Assay Kit. HCAECs were incubated in medium containing 5 mM glucose, supplemented with 0.5% FBS, with or without insulin lispro (1 nM), BLX-1002 (1 μM), metformin (500 μM), pioglitazone (2.5 μM), candesartan (100 nM) in the presence of 0.125 mM palmitate or vehicle (ethanol and BSA) for 24 h. * denotes P

    Article Snippet: Metformin, glimepiride, sodium palmitate, and bovine serum albumin (BSA) (fatty acid free) were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques: Activation Assay, Activity Assay, Caspase-3 Assay, Incubation

    HPN improves glucose uptake ability of HepG2 cells. ( A ) Insulin-stimulated pAkt (S473) was up-regulated during HPN treatment in PA-induced HepG2 cells. HepG2 cells were serum-starved with 0.5% FFA-free BSA medium, and treated with 0.25 mM PA for 16 h after HPN incubation. Subsequently, cells were stimulated with 100 nM insulin for 30 min. Western blot assay was used to determine the changes of pAkt (S473) and total Akt. β-Actin was used as loading control. ### p

    Journal: Marine Drugs

    Article Title: Marine Bromophenol Derivative 3,4-Dibromo-5-(2-bromo-3,4-dihydroxy-6-isopropoxymethyl benzyl)benzene-1,2-diol Protects Hepatocytes from Lipid-Induced Cell Damage and Insulin Resistance via PTP1B Inhibition

    doi: 10.3390/md13074452

    Figure Lengend Snippet: HPN improves glucose uptake ability of HepG2 cells. ( A ) Insulin-stimulated pAkt (S473) was up-regulated during HPN treatment in PA-induced HepG2 cells. HepG2 cells were serum-starved with 0.5% FFA-free BSA medium, and treated with 0.25 mM PA for 16 h after HPN incubation. Subsequently, cells were stimulated with 100 nM insulin for 30 min. Western blot assay was used to determine the changes of pAkt (S473) and total Akt. β-Actin was used as loading control. ### p

    Article Snippet: Palmitate, insulin and FFA-free BSA were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Incubation, Western Blot

    Effects of OF and CM on the mRNA expressions of lipogenesis-related genes. Real-time RT-PCR analysis of sterol regulatory element-binding protein-1 ( SREBP-1c ) (a) and glycerol-3-phosphate acyltransferase ( GPAT ) (b) mRNA levels in 1 mM FFA/BSA-treated HepG2 cells. All data were normalized to GAPDH mRNA, and the fold changes in expression were calculated relative to control cells (treated with 1% BSA). Each experiment was independently performed three times. Data were presented as the mean ± SD. Values not sharing common superscripts are significantly different ( p

    Journal: Mediators of Inflammation

    Article Title: Antilipotoxicity Activity of Osmanthus fragrans and Chrysanthemum morifolium Flower Extracts in Hepatocytes and Renal Glomerular Mesangial Cells

    doi: 10.1155/2017/4856095

    Figure Lengend Snippet: Effects of OF and CM on the mRNA expressions of lipogenesis-related genes. Real-time RT-PCR analysis of sterol regulatory element-binding protein-1 ( SREBP-1c ) (a) and glycerol-3-phosphate acyltransferase ( GPAT ) (b) mRNA levels in 1 mM FFA/BSA-treated HepG2 cells. All data were normalized to GAPDH mRNA, and the fold changes in expression were calculated relative to control cells (treated with 1% BSA). Each experiment was independently performed three times. Data were presented as the mean ± SD. Values not sharing common superscripts are significantly different ( p

    Article Snippet: The FFA/BSA or oleic acid (OA)/BSA complex solution was sterile-filtered through 0.22 μ m sterile filters (Millipore S.A.S., Molsheim, France) and then stored at −20°C until use.

    Techniques: Quantitative RT-PCR, Binding Assay, Expressing

    Effect of OF and CM on FFA-induced ROS production. HepG2 cells were incubated with 1 mM FFAs/BSA for 24 h in the presence of OF or CM. Vehicle cells were incubated with 0.1% DMSO in the presence of FFAs/BSA. Control cells were incubated with 1% BSA. Intracellular ROS production was quantified using the fluorescent probe DCFDA. Data were presented as mean ± SD of three independent experiments. Values not sharing common superscripts are significantly different ( p

    Journal: Mediators of Inflammation

    Article Title: Antilipotoxicity Activity of Osmanthus fragrans and Chrysanthemum morifolium Flower Extracts in Hepatocytes and Renal Glomerular Mesangial Cells

    doi: 10.1155/2017/4856095

    Figure Lengend Snippet: Effect of OF and CM on FFA-induced ROS production. HepG2 cells were incubated with 1 mM FFAs/BSA for 24 h in the presence of OF or CM. Vehicle cells were incubated with 0.1% DMSO in the presence of FFAs/BSA. Control cells were incubated with 1% BSA. Intracellular ROS production was quantified using the fluorescent probe DCFDA. Data were presented as mean ± SD of three independent experiments. Values not sharing common superscripts are significantly different ( p

    Article Snippet: The FFA/BSA or oleic acid (OA)/BSA complex solution was sterile-filtered through 0.22 μ m sterile filters (Millipore S.A.S., Molsheim, France) and then stored at −20°C until use.

    Techniques: Incubation

    Effects of OF and CM on lipid accumulation in free fatty acid-overloaded HepG2 cells. HepG2 cells were incubated with 1 mM FFAs/BSA and cotreated with various concentrations of OF and CM for 24 h. Vehicle cells were incubated with 0.1% DMSO in the presence of FFAs/BSA. Control cells were incubated with 1% BSA. Cell viability was measured by the MTT assay (a). Quantitative analysis of lipid deposition in cells by the OD 500 nm values using Oil Red O staining (b). Intracellular triglyceride (c) and cholesterol (d) contents were determined in cell lysates by an enzymatic colorimetric method using a commercially available kit. Total cholesterol and TG levels of the control cells were 25.3 ± 7.1 and 28.7 ± 2.7 μ g/mg of cellular protein, respectively. Data were presented as mean ± SD of three independent experiments. Values not sharing common superscripts are significantly different ( p

    Journal: Mediators of Inflammation

    Article Title: Antilipotoxicity Activity of Osmanthus fragrans and Chrysanthemum morifolium Flower Extracts in Hepatocytes and Renal Glomerular Mesangial Cells

    doi: 10.1155/2017/4856095

    Figure Lengend Snippet: Effects of OF and CM on lipid accumulation in free fatty acid-overloaded HepG2 cells. HepG2 cells were incubated with 1 mM FFAs/BSA and cotreated with various concentrations of OF and CM for 24 h. Vehicle cells were incubated with 0.1% DMSO in the presence of FFAs/BSA. Control cells were incubated with 1% BSA. Cell viability was measured by the MTT assay (a). Quantitative analysis of lipid deposition in cells by the OD 500 nm values using Oil Red O staining (b). Intracellular triglyceride (c) and cholesterol (d) contents were determined in cell lysates by an enzymatic colorimetric method using a commercially available kit. Total cholesterol and TG levels of the control cells were 25.3 ± 7.1 and 28.7 ± 2.7 μ g/mg of cellular protein, respectively. Data were presented as mean ± SD of three independent experiments. Values not sharing common superscripts are significantly different ( p

    Article Snippet: The FFA/BSA or oleic acid (OA)/BSA complex solution was sterile-filtered through 0.22 μ m sterile filters (Millipore S.A.S., Molsheim, France) and then stored at −20°C until use.

    Techniques: Incubation, MTT Assay, Staining

    4BA inhibits dark adaptation in 11-cis-retinal–regenerated WT-HEK293S cells. Giant cells were regenerated with 50 μM 11-cis-retinal and cell surface area–normalized R 2 charge motion obtained for the first bleach cycle (Control, c). 4BA complexed to FAF-BSA was then perfused through the chamber and the amount of R 2 charge motion obtained in successive bleach cycles determined (1Æ5). Recovery time between bleach cycles was 10 min in all cases and flash stimulation was 500 nm. After the fifth bleach cycle, 4BA was washed out of the chamber and the recovery of R 2 charge determined (washout, w). Control and washout conditions were not statistically different. The decay of R 2 charge motion in 4BA (0.5 mM) was reliably fit with a single exponential model with a decay constant of 0.45 ± 0.01 cycles ( P

    Journal: The Journal of General Physiology

    Article Title: HEK293S Cells Have Functional Retinoid Processing Machinery

    doi: 10.1085/jgp.20018495

    Figure Lengend Snippet: 4BA inhibits dark adaptation in 11-cis-retinal–regenerated WT-HEK293S cells. Giant cells were regenerated with 50 μM 11-cis-retinal and cell surface area–normalized R 2 charge motion obtained for the first bleach cycle (Control, c). 4BA complexed to FAF-BSA was then perfused through the chamber and the amount of R 2 charge motion obtained in successive bleach cycles determined (1Æ5). Recovery time between bleach cycles was 10 min in all cases and flash stimulation was 500 nm. After the fifth bleach cycle, 4BA was washed out of the chamber and the recovery of R 2 charge determined (washout, w). Control and washout conditions were not statistically different. The decay of R 2 charge motion in 4BA (0.5 mM) was reliably fit with a single exponential model with a decay constant of 0.45 ± 0.01 cycles ( P

    Article Snippet: Cellular Chromophore Loading and Rhodopsin Regeneration Coverslips with attached giant cells were placed in a polystyrene dish in a light-tight container at room temperature (21–23°C) in regeneration buffer (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2 , 1.0 MgCl2 , 10 glucose, 10 HEPES-NaOH, pH 7.2, and 2% (wt/vol) FAF-BSA (290 μM) (Sigma-Aldrich).

    Techniques:

    ERC evidence for retinoid conversions in giant WT-HEK293S cells. Cells were loaded with 50 μM chromophore complexed to FAF-BSA in all cases. ERCs were obtained upon the first 500-nm flash from cells regenerated for 40 min at room temperature in darkness (A and C) or overnight at 4°C in darkness (B and D). Each panel is from a single cell. Cells were regenerated with all-trans-retinal (A and B) or Vitamin A (C and D). Cells are representative of larger populations of cells examined (all-trans-retinal, 40 min: n = 43, overnight at 4°C: n = 7; Vitamin A, 40 min: n = 2 cells, overnight at 4°C: n = 23 cells).

    Journal: The Journal of General Physiology

    Article Title: HEK293S Cells Have Functional Retinoid Processing Machinery

    doi: 10.1085/jgp.20018495

    Figure Lengend Snippet: ERC evidence for retinoid conversions in giant WT-HEK293S cells. Cells were loaded with 50 μM chromophore complexed to FAF-BSA in all cases. ERCs were obtained upon the first 500-nm flash from cells regenerated for 40 min at room temperature in darkness (A and C) or overnight at 4°C in darkness (B and D). Each panel is from a single cell. Cells were regenerated with all-trans-retinal (A and B) or Vitamin A (C and D). Cells are representative of larger populations of cells examined (all-trans-retinal, 40 min: n = 43, overnight at 4°C: n = 7; Vitamin A, 40 min: n = 2 cells, overnight at 4°C: n = 23 cells).

    Article Snippet: Cellular Chromophore Loading and Rhodopsin Regeneration Coverslips with attached giant cells were placed in a polystyrene dish in a light-tight container at room temperature (21–23°C) in regeneration buffer (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2 , 1.0 MgCl2 , 10 glucose, 10 HEPES-NaOH, pH 7.2, and 2% (wt/vol) FAF-BSA (290 μM) (Sigma-Aldrich).

    Techniques:

    Lack of bulk BSA uptake into single or giant WT-HEK293S cells. Single (A and B) or giant PEG-fused (C and D) WT-HEK293S cells were exposed to regeneration buffer containing 1.9% (wt/vol) FAF-BSA plus 0.1% FITC-BSA. Hoffman contrast images of representative fields of single (A) or PEG-fused (C) WT-HEK293S cells are shown beside fluorescence images from the same respective fields (B and D). Arrows in A and B indicate healthy single cells and arrowheads indicate unhealthy single cells and single cells or debris taking up FITC-BSA. Scale marker is 20 μm in all fields and approximates the size of a single unfused HEK293S cell.

    Journal: The Journal of General Physiology

    Article Title: HEK293S Cells Have Functional Retinoid Processing Machinery

    doi: 10.1085/jgp.20018495

    Figure Lengend Snippet: Lack of bulk BSA uptake into single or giant WT-HEK293S cells. Single (A and B) or giant PEG-fused (C and D) WT-HEK293S cells were exposed to regeneration buffer containing 1.9% (wt/vol) FAF-BSA plus 0.1% FITC-BSA. Hoffman contrast images of representative fields of single (A) or PEG-fused (C) WT-HEK293S cells are shown beside fluorescence images from the same respective fields (B and D). Arrows in A and B indicate healthy single cells and arrowheads indicate unhealthy single cells and single cells or debris taking up FITC-BSA. Scale marker is 20 μm in all fields and approximates the size of a single unfused HEK293S cell.

    Article Snippet: Cellular Chromophore Loading and Rhodopsin Regeneration Coverslips with attached giant cells were placed in a polystyrene dish in a light-tight container at room temperature (21–23°C) in regeneration buffer (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2 , 1.0 MgCl2 , 10 glucose, 10 HEPES-NaOH, pH 7.2, and 2% (wt/vol) FAF-BSA (290 μM) (Sigma-Aldrich).

    Techniques: Fluorescence, Marker

    ERC signals from WT-HEK293S cells regenerated with different cis-retinaldehydes. Fused WT-HEK293S giant cells were loaded with 25 μM 11-cis-retinal or 9-cis-retinal or 50 μM 13-cis-retinal complexed to FAF-BSA. ERC signals on the first 500-nm flash during the primary bleaching extinction (A, C, and E) and secondary bleaching extinctions (B, D, and F) are shown for 11-cis-retinal– (A and B), 9-cis-retinal– (C and D), and 13-cis-retinal– (E and F) loaded representative cells. Membrane capacitances of the cells are indicated. The arrow indicates the timing of the flash stimulus. Responses from each single cell are representative of larger populations of cells regenerated with 11-cis-retinal ( n = 54), 9-cis-retinal ( n = 5), and 13-cis-retinal ( n = 4).

    Journal: The Journal of General Physiology

    Article Title: HEK293S Cells Have Functional Retinoid Processing Machinery

    doi: 10.1085/jgp.20018495

    Figure Lengend Snippet: ERC signals from WT-HEK293S cells regenerated with different cis-retinaldehydes. Fused WT-HEK293S giant cells were loaded with 25 μM 11-cis-retinal or 9-cis-retinal or 50 μM 13-cis-retinal complexed to FAF-BSA. ERC signals on the first 500-nm flash during the primary bleaching extinction (A, C, and E) and secondary bleaching extinctions (B, D, and F) are shown for 11-cis-retinal– (A and B), 9-cis-retinal– (C and D), and 13-cis-retinal– (E and F) loaded representative cells. Membrane capacitances of the cells are indicated. The arrow indicates the timing of the flash stimulus. Responses from each single cell are representative of larger populations of cells regenerated with 11-cis-retinal ( n = 54), 9-cis-retinal ( n = 5), and 13-cis-retinal ( n = 4).

    Article Snippet: Cellular Chromophore Loading and Rhodopsin Regeneration Coverslips with attached giant cells were placed in a polystyrene dish in a light-tight container at room temperature (21–23°C) in regeneration buffer (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2 , 1.0 MgCl2 , 10 glucose, 10 HEPES-NaOH, pH 7.2, and 2% (wt/vol) FAF-BSA (290 μM) (Sigma-Aldrich).

    Techniques:

    Palmitate induced oxidative stress in INS-1 cells. INS-1 cells were treated with control medium, FFA-free BSA (0.5 mol/ml), or indicated concentration of palmitate 24 h or 0.5 mM of palmitate for indicated times. The results showed that palmitate increased the level of ROS in INS-1 cells, which was measured by flow cytometry (B, D) and fluorescence microscopy (A, C). Data are expressed as means ± SEM of 3 independent experiments; * P

    Journal: PLoS ONE

    Article Title: Interleukin-22 Alleviated Palmitate-Induced Endoplasmic Reticulum Stress in INS-1 Cells through Activation of Autophagy

    doi: 10.1371/journal.pone.0146818

    Figure Lengend Snippet: Palmitate induced oxidative stress in INS-1 cells. INS-1 cells were treated with control medium, FFA-free BSA (0.5 mol/ml), or indicated concentration of palmitate 24 h or 0.5 mM of palmitate for indicated times. The results showed that palmitate increased the level of ROS in INS-1 cells, which was measured by flow cytometry (B, D) and fluorescence microscopy (A, C). Data are expressed as means ± SEM of 3 independent experiments; * P

    Article Snippet: A 5% (w/v) solution of FFA-free bovine serum albumin (BSA) (Sigma-Aldrich, Milano, Italy) was prepared in serum-free RPMI medium.

    Techniques: Concentration Assay, Flow Cytometry, Cytometry, Fluorescence, Microscopy

    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