l glutamine  (Thermo Fisher)


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    Name:
    Penicillin Streptomycin 10 000 U mL
    Description:
    The antibiotics penicillin and streptomycin are used to prevent bacterial contamination of cell cultures due to their effective combined action against gram positive and gram negative bacteria Penicillin was originally purified from the fungi Penicillium and acts by interfering directly with the turnover of the bacterial cell wall and indirectly by triggering the release of enzymes that further alter the cell wall Streptomycin was originally purified from Streptomyces griseus It acts by binding to the 30S subunit of the bacterial ribosome leading to inhibition of protein synthesis and death in susceptible bacteria This solution contains 10 000 units mL of penicillin and 10 000 µg mL of streptomycin We offer a wide range of antibiotics and antimycotics in both powder and liquid formats See the complete list or find products for • Contamination control• Eukaryotic and bacterial selectionSee recommended working concentrations for selection antibiotics Learn more about the use of antibiotics and antimycotics in cell culture and review guidelines for decontaminating cultures
    Catalog Number:
    15140122
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Cell Culture|Mammalian Cell Culture
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    Structured Review

    Thermo Fisher l glutamine
    The antibiotics penicillin and streptomycin are used to prevent bacterial contamination of cell cultures due to their effective combined action against gram positive and gram negative bacteria Penicillin was originally purified from the fungi Penicillium and acts by interfering directly with the turnover of the bacterial cell wall and indirectly by triggering the release of enzymes that further alter the cell wall Streptomycin was originally purified from Streptomyces griseus It acts by binding to the 30S subunit of the bacterial ribosome leading to inhibition of protein synthesis and death in susceptible bacteria This solution contains 10 000 units mL of penicillin and 10 000 µg mL of streptomycin We offer a wide range of antibiotics and antimycotics in both powder and liquid formats See the complete list or find products for • Contamination control• Eukaryotic and bacterial selectionSee recommended working concentrations for selection antibiotics Learn more about the use of antibiotics and antimycotics in cell culture and review guidelines for decontaminating cultures
    https://www.bioz.com/result/l glutamine/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    l glutamine - by Bioz Stars, 2021-03
    86/100 stars

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    Related Articles

    Cell Culture:

    Article Title: Autophagy-mediated metabolic effects of aspirin
    Article Snippet: .. J. Yuan, Harvard University) and HeLa cells were cultured in DMEM medium (#41966-02, Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS; #F7524, Sigma Aldrich), 100 mg/L sodium pyruvate (#11360070, Thermo Fisher Scientific), 10 mM HEPES buffer (#15630080, Thermo Fisher Scientific), 100 UI/ml penicillin G sodium salt, 100 μg/mL streptomycin sulfate (#15140122, Thermo Fisher Scientific) and, for the (GFP)-LC3-expressing derivatives cell, 500 µg/mL geneticin (G418 sulfate) (#10131-27, Thermo Fisher Scientific). .. MEFs (mouse embryonic fibroblast) cells were cultured in the same DMEM with additional supplementation of 1 mM nonessential amino acids (#11140-035, Thermo Fisher Scientific).

    Article Title: Induction of 3-hydroxy-3-methylglutaryl-CoA reductase mediates statin resistance in breast cancer cells
    Article Snippet: The human breast cancer cell lines MDA-MB-231, MDA-MB-468, MCF-7, and T47D were obtained from ATCC (Manassas, VA, USA). .. MDA-MB-231, MCF-7, and T47D cells were cultured in DMEM/Ham´s F12 (Gibco Life Technologies, Darmstadt, Germany) and MDA-MB-468 cells in DMEM (Gibco Life Technologies, Darmstadt, Germany), supplemented with 10% fetal bovine serum (FBS; Biochrome, Berlin, Germany) and 1% penicillin/streptomycin (Gibco Life Technologies). ..

    Article Title: Gain-of-function assay for SARS-CoV-2 Mpro inhibition in living cells
    Article Snippet: .. Cell culture and flow cytometry 293T cells were maintained at 37°C/5%CO2 in RPMI-1640 (Gibco #11875093) supplemented with 10% fetal bovine serum (Gibco #10091148) and penicillin/streptomycin (Gibco #15140122) 293T cells were seeded in a 24-well plate at 1.5×105 cells/well and transfected 24h later with 200 ng of the wildtype or mutant chimeric reporter construct (TransIT-LT1, Mirus #MIR2304). .. 48h post-transfection cells were washed twice with PBS and resuspended in 500 μL PBS.

    Article Title: Jet‐Printing Microfluidic Devices on Demand, Jet‐Printing Microfluidic Devices on Demand
    Article Snippet: Adherent human embryonic kidney cells (HEKs) were grown in DMEM (Gibco, Gaithersburg, MD) + 10% FBS; these HEKs were genetically modified reporter cells (NF‐kB/293/GFP‐Luc Transcriptional Reporter Cell Line; System Biosciences, catalogue number TR860A‐I), but reporter activity is not relevant here. .. Mouse embryonic stem (ES) cells (EK.CCE line, derived from a single XY blastocyst‐stage embryo of strain 129/Sv/Ec)[ ] were routinely cultured in DMEM supplemented with 15% FBS, 1% penicillin/streptomycin (Gibco, #15140122), 0.1 × 10−3 m 2‐mercaptoethanol, 1% glutamine (Invitrogen, Loughborough, UK), 1% minimum essential media nonessential amino acids (Gibco), 1 × 10−3 m sodium pyruvate, and 1000 U mL−1 leukemia inhibitory factor (LIF, ESGRO; Merck, Darmstadt, Germany; #L5158) on gelatin‐coated plates (Merck, #ES‐006‐B). .. Mouse mammary tumor cells (NM18, a derivative of the NMuMG line)[ ] were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.1% insulin (Sigma Aldrich, #10516).

    Article Title: Jet-printing microfluidic devices on demand
    Article Snippet: Adherent human embryonic kidney cells (HEKs) were grown in DMEM (Gibco, Gaithersburg, MD) + 10% FBS; these HEKs were genetically-modified reporter cells (NF-κB/293/GFP-Luc™ Transcriptional Reporter Cell Line; System Biosciences, catalogue number TR860A-I), but reporter activity is not relevant here. .. Mouse embryonic stem (ES) cells (EK.CCE line, derived from a single XY blastocyst-stage embryo of strain 129/Sv/Ec; ) were routinely cultured in DMEM supplemented with 15% FBS, 1% penicillin/streptomycin (Gibco, #15140122), 0.1 mM 2-mercaptoethanol, 1% glutamine (Invitrogen, Loughborough, UK), 1% minimum essential media nonessential amino acids (Gibco), 1 mM sodium pyruvate, and 1000 U/mL leukemia inhibitory factor (LIF, ESGRO; Merck, Darmstadt, Germany; #L5158) on gelatin-coated plates (Merck, #ES-006-B). .. Mouse mammary tumor cells (NM18, a derivative of the NMuMG line; ) were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.1% insulin (Sigma Aldrich, #10516).

    Genetically Modified:

    Article Title: Jet-printing microfluidic devices on demand
    Article Snippet: Adherent human embryonic kidney cells (HEKs) were grown in DMEM (Gibco, Gaithersburg, MD) + 10% FBS; these HEKs were genetically-modified reporter cells (NF-κB/293/GFP-Luc™ Transcriptional Reporter Cell Line; System Biosciences, catalogue number TR860A-I), but reporter activity is not relevant here. .. Adherent human embryonic kidney cells (HEKs) were grown in DMEM (Gibco, Gaithersburg, MD) + 10% FBS; these HEKs were genetically-modified reporter cells (NF-κB/293/GFP-Luc™ Transcriptional Reporter Cell Line; System Biosciences, catalogue number TR860A-I), but reporter activity is not relevant here. .. Mouse embryonic stem (ES) cells (EK.CCE line, derived from a single XY blastocyst-stage embryo of strain 129/Sv/Ec; ) were routinely cultured in DMEM supplemented with 15% FBS, 1% penicillin/streptomycin (Gibco, #15140122), 0.1 mM 2-mercaptoethanol, 1% glutamine (Invitrogen, Loughborough, UK), 1% minimum essential media nonessential amino acids (Gibco), 1 mM sodium pyruvate, and 1000 U/mL leukemia inhibitory factor (LIF, ESGRO; Merck, Darmstadt, Germany; #L5158) on gelatin-coated plates (Merck, #ES-006-B).

    Activity Assay:

    Article Title: Jet-printing microfluidic devices on demand
    Article Snippet: Adherent human embryonic kidney cells (HEKs) were grown in DMEM (Gibco, Gaithersburg, MD) + 10% FBS; these HEKs were genetically-modified reporter cells (NF-κB/293/GFP-Luc™ Transcriptional Reporter Cell Line; System Biosciences, catalogue number TR860A-I), but reporter activity is not relevant here. .. Adherent human embryonic kidney cells (HEKs) were grown in DMEM (Gibco, Gaithersburg, MD) + 10% FBS; these HEKs were genetically-modified reporter cells (NF-κB/293/GFP-Luc™ Transcriptional Reporter Cell Line; System Biosciences, catalogue number TR860A-I), but reporter activity is not relevant here. .. Mouse embryonic stem (ES) cells (EK.CCE line, derived from a single XY blastocyst-stage embryo of strain 129/Sv/Ec; ) were routinely cultured in DMEM supplemented with 15% FBS, 1% penicillin/streptomycin (Gibco, #15140122), 0.1 mM 2-mercaptoethanol, 1% glutamine (Invitrogen, Loughborough, UK), 1% minimum essential media nonessential amino acids (Gibco), 1 mM sodium pyruvate, and 1000 U/mL leukemia inhibitory factor (LIF, ESGRO; Merck, Darmstadt, Germany; #L5158) on gelatin-coated plates (Merck, #ES-006-B).

    Multiple Displacement Amplification:

    Article Title: Induction of 3-hydroxy-3-methylglutaryl-CoA reductase mediates statin resistance in breast cancer cells
    Article Snippet: The human breast cancer cell lines MDA-MB-231, MDA-MB-468, MCF-7, and T47D were obtained from ATCC (Manassas, VA, USA). .. MDA-MB-231, MCF-7, and T47D cells were cultured in DMEM/Ham´s F12 (Gibco Life Technologies, Darmstadt, Germany) and MDA-MB-468 cells in DMEM (Gibco Life Technologies, Darmstadt, Germany), supplemented with 10% fetal bovine serum (FBS; Biochrome, Berlin, Germany) and 1% penicillin/streptomycin (Gibco Life Technologies). ..

    Flow Cytometry:

    Article Title: Gain-of-function assay for SARS-CoV-2 Mpro inhibition in living cells
    Article Snippet: .. Cell culture and flow cytometry 293T cells were maintained at 37°C/5%CO2 in RPMI-1640 (Gibco #11875093) supplemented with 10% fetal bovine serum (Gibco #10091148) and penicillin/streptomycin (Gibco #15140122) 293T cells were seeded in a 24-well plate at 1.5×105 cells/well and transfected 24h later with 200 ng of the wildtype or mutant chimeric reporter construct (TransIT-LT1, Mirus #MIR2304). .. 48h post-transfection cells were washed twice with PBS and resuspended in 500 μL PBS.

    Transfection:

    Article Title: Gain-of-function assay for SARS-CoV-2 Mpro inhibition in living cells
    Article Snippet: .. Cell culture and flow cytometry 293T cells were maintained at 37°C/5%CO2 in RPMI-1640 (Gibco #11875093) supplemented with 10% fetal bovine serum (Gibco #10091148) and penicillin/streptomycin (Gibco #15140122) 293T cells were seeded in a 24-well plate at 1.5×105 cells/well and transfected 24h later with 200 ng of the wildtype or mutant chimeric reporter construct (TransIT-LT1, Mirus #MIR2304). .. 48h post-transfection cells were washed twice with PBS and resuspended in 500 μL PBS.

    Mutagenesis:

    Article Title: Gain-of-function assay for SARS-CoV-2 Mpro inhibition in living cells
    Article Snippet: .. Cell culture and flow cytometry 293T cells were maintained at 37°C/5%CO2 in RPMI-1640 (Gibco #11875093) supplemented with 10% fetal bovine serum (Gibco #10091148) and penicillin/streptomycin (Gibco #15140122) 293T cells were seeded in a 24-well plate at 1.5×105 cells/well and transfected 24h later with 200 ng of the wildtype or mutant chimeric reporter construct (TransIT-LT1, Mirus #MIR2304). .. 48h post-transfection cells were washed twice with PBS and resuspended in 500 μL PBS.

    Construct:

    Article Title: Gain-of-function assay for SARS-CoV-2 Mpro inhibition in living cells
    Article Snippet: .. Cell culture and flow cytometry 293T cells were maintained at 37°C/5%CO2 in RPMI-1640 (Gibco #11875093) supplemented with 10% fetal bovine serum (Gibco #10091148) and penicillin/streptomycin (Gibco #15140122) 293T cells were seeded in a 24-well plate at 1.5×105 cells/well and transfected 24h later with 200 ng of the wildtype or mutant chimeric reporter construct (TransIT-LT1, Mirus #MIR2304). .. 48h post-transfection cells were washed twice with PBS and resuspended in 500 μL PBS.

    Derivative Assay:

    Article Title: Jet‐Printing Microfluidic Devices on Demand, Jet‐Printing Microfluidic Devices on Demand
    Article Snippet: Adherent human embryonic kidney cells (HEKs) were grown in DMEM (Gibco, Gaithersburg, MD) + 10% FBS; these HEKs were genetically modified reporter cells (NF‐kB/293/GFP‐Luc Transcriptional Reporter Cell Line; System Biosciences, catalogue number TR860A‐I), but reporter activity is not relevant here. .. Mouse embryonic stem (ES) cells (EK.CCE line, derived from a single XY blastocyst‐stage embryo of strain 129/Sv/Ec)[ ] were routinely cultured in DMEM supplemented with 15% FBS, 1% penicillin/streptomycin (Gibco, #15140122), 0.1 × 10−3 m 2‐mercaptoethanol, 1% glutamine (Invitrogen, Loughborough, UK), 1% minimum essential media nonessential amino acids (Gibco), 1 × 10−3 m sodium pyruvate, and 1000 U mL−1 leukemia inhibitory factor (LIF, ESGRO; Merck, Darmstadt, Germany; #L5158) on gelatin‐coated plates (Merck, #ES‐006‐B). .. Mouse mammary tumor cells (NM18, a derivative of the NMuMG line)[ ] were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.1% insulin (Sigma Aldrich, #10516).

    Article Title: Jet-printing microfluidic devices on demand
    Article Snippet: Adherent human embryonic kidney cells (HEKs) were grown in DMEM (Gibco, Gaithersburg, MD) + 10% FBS; these HEKs were genetically-modified reporter cells (NF-κB/293/GFP-Luc™ Transcriptional Reporter Cell Line; System Biosciences, catalogue number TR860A-I), but reporter activity is not relevant here. .. Mouse embryonic stem (ES) cells (EK.CCE line, derived from a single XY blastocyst-stage embryo of strain 129/Sv/Ec; ) were routinely cultured in DMEM supplemented with 15% FBS, 1% penicillin/streptomycin (Gibco, #15140122), 0.1 mM 2-mercaptoethanol, 1% glutamine (Invitrogen, Loughborough, UK), 1% minimum essential media nonessential amino acids (Gibco), 1 mM sodium pyruvate, and 1000 U/mL leukemia inhibitory factor (LIF, ESGRO; Merck, Darmstadt, Germany; #L5158) on gelatin-coated plates (Merck, #ES-006-B). .. Mouse mammary tumor cells (NM18, a derivative of the NMuMG line; ) were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.1% insulin (Sigma Aldrich, #10516).

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  • 99
    Thermo Fisher glucose l glutamine dulbecco s modified eagle s medium
    siRNA knockdown of α-AP-2 reduces CFTR endocytosis. A , HEK 293 cells expressing CFTR were incubated overnight in <t>Dulbecco's</t> modified <t>Eagle's</t> medium with 10% fetal bovine serum and allowed to grow to 70% confluency. The cells were transfected with α-AP-2 or scrambled siRNA and analyzed by Western blot at 72 h to detect α-AP-2 and β-actin ( A ). B , cell surface proteins were labeled using NHS SS-biotin and allowed to internalize for 1 and 5 min at 37 °C. Following MESNA treatment, the cells were lysed, surface biotinylated proteins were bound to streptavidin-agarose beads, and CFTR was detected by Western blot. C , the percentage of cell surface CFTR internalized over time is shown. The data are expressed as the means ± S.E. ( n > 3).
    Glucose L Glutamine Dulbecco S Modified Eagle S Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glucose l glutamine dulbecco s modified eagle s medium/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
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    93
    Thermo Fisher keratinocyte cells
    NME4 is a direct regulatory target of miR-196. (A) Differential expression of miR-196a/b and NME4 mRNA in two immortalized normal <t>keratinocyte</t> and four oral cancer cell lines, as determined by RT-qPCR. Relative levels of miR-196a/b were determined after normalization to the U6 RNA level (internal control) for each sample. The NME4 RNA level was normalized to actin for each sample. (B) Differential expression of NME4 mRNA and protein in two immortalized normal keratinocyte and four oral cancer cell lines, as determined by RT-PCR and western blotting. Actin expression was also determined as an internal control. (C) Inverse correlation between NME4 and miR-196a/b expression which determined by RT-qPCR. (D) Effect of miR-196 modulation on NME4 mRNA expression. OECM1 and SAS cells transfected with miR-196a/b–specific antagomirs or overexpression plasmids were harvested. Total protein was extracted and subjected to western blot analysis of NME4 expression. The relative expression was determined after normalization to actin as an internal control. (E) The mature sequences of miR-196a and miR-196b, as well as the 3′-UTR sequence of the human NME4 gene, are shown. The mutant sequence of the 3′-UTR region of NEM4 was designed. Luciferase reporter assay was performed to determine whether NME4 is a direct miR-196a/b target gene. Cells transfected with miR-196–specific antagomirs or overexpression plasmids were co-transfected with pMIR, p-UTR-WT, or p-UTR-mut. Renilla luciferase was also transfected as a reference control for each condition. Firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay. (* p
    Keratinocyte Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/keratinocyte cells/product/Thermo Fisher
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    siRNA knockdown of α-AP-2 reduces CFTR endocytosis. A , HEK 293 cells expressing CFTR were incubated overnight in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and allowed to grow to 70% confluency. The cells were transfected with α-AP-2 or scrambled siRNA and analyzed by Western blot at 72 h to detect α-AP-2 and β-actin ( A ). B , cell surface proteins were labeled using NHS SS-biotin and allowed to internalize for 1 and 5 min at 37 °C. Following MESNA treatment, the cells were lysed, surface biotinylated proteins were bound to streptavidin-agarose beads, and CFTR was detected by Western blot. C , the percentage of cell surface CFTR internalized over time is shown. The data are expressed as the means ± S.E. ( n > 3).

    Journal: The Journal of Biological Chemistry

    Article Title: ?-AP-2 Directs Myosin VI-dependent Endocytosis of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels in the Intestine *

    doi: 10.1074/jbc.M110.127613

    Figure Lengend Snippet: siRNA knockdown of α-AP-2 reduces CFTR endocytosis. A , HEK 293 cells expressing CFTR were incubated overnight in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and allowed to grow to 70% confluency. The cells were transfected with α-AP-2 or scrambled siRNA and analyzed by Western blot at 72 h to detect α-AP-2 and β-actin ( A ). B , cell surface proteins were labeled using NHS SS-biotin and allowed to internalize for 1 and 5 min at 37 °C. Following MESNA treatment, the cells were lysed, surface biotinylated proteins were bound to streptavidin-agarose beads, and CFTR was detected by Western blot. C , the percentage of cell surface CFTR internalized over time is shown. The data are expressed as the means ± S.E. ( n > 3).

    Article Snippet: The cells were grown in high glucose l -glutamine Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Invitrogen) and hygromycin B (150 μg/ml; Invitrogen) at 37 °C in 5% CO2 , 90% air atmosphere.

    Techniques: Expressing, Incubation, Modification, Transfection, Western Blot, Labeling

    NNAT interaction with the SPC and modulation of preproinsulin handling. ( A ) Overview of affinity purification/mass spectrometry (AP/MS) screen for novel interaction partners of NNAT. Endogenous NNAT was immunoprecipitated (IP) from MIN6 cell lysates and interacting partners in co-IPs analyzed by liquid chromatography/mass spectrometry (LC/MS). ( B ) Heatmap from AP/MS analysis of top protein hits in IPs using antibodies against NNAT (NNAT Ab) and control IPs with rabbit immunoglobulins (IgG). Relatively high abundance is shown in yellow and relatively low abundance in blue. ( C ) Lysates from HEK293T cells expressing c-Myc–tagged SEC11A and FLAG-tagged NNAT were immunoprecipitated using anti–c-Myc antibodies. Proteins in input and IP samples were detected by Western blotting using anti–c-Myc and anti-FLAG antibodies. Panel shows a representative blot of 3 independent experiments. ( D ) INS1E cells transiently transfected with siRNA targeting Nnat or Sec11a were assayed in vitro for GSIS at low (3 mM) and high (25 mM) glucose. A scramble siRNA served as a control with data expressed as mean insulin secretion per unit cellular protein. Graph on the right shows total insulin content in cell lysates. ( n = 9 independent cultures per group, 3 independent experiments, 2-way ANOVA [left graph] and 1-way ANOVA [right graph].) ( E ) INS1E cells transfected with c-Myc–tagged preproinsulin and siRNAs targeting Nnat or Sec11a were pulse-labeled with 35 S-Cys/Met. Lysates immunoprecipitated with anti–c-Myc agarose were analyzed by autoradiography. Associated bar chart shows preproinsulin and proinsulin band intensities in multiple experiments quantified by densitometry and expressed as percentage processing of preproinsulin ( n = 3 cultures per group from 3 independent experiments, 1-way ANOVA). ( F ) Similar experiments performed as in E , from 3-hour chase cell lysates (C) and media (M), quantified as in E ( n = 4 cultures per group from 3 independent experiments, 1-way ANOVA for both C and M). (* P

    Journal: The Journal of Clinical Investigation

    Article Title: Neuronatin regulates pancreatic β cell insulin content and secretion

    doi: 10.1172/JCI120115

    Figure Lengend Snippet: NNAT interaction with the SPC and modulation of preproinsulin handling. ( A ) Overview of affinity purification/mass spectrometry (AP/MS) screen for novel interaction partners of NNAT. Endogenous NNAT was immunoprecipitated (IP) from MIN6 cell lysates and interacting partners in co-IPs analyzed by liquid chromatography/mass spectrometry (LC/MS). ( B ) Heatmap from AP/MS analysis of top protein hits in IPs using antibodies against NNAT (NNAT Ab) and control IPs with rabbit immunoglobulins (IgG). Relatively high abundance is shown in yellow and relatively low abundance in blue. ( C ) Lysates from HEK293T cells expressing c-Myc–tagged SEC11A and FLAG-tagged NNAT were immunoprecipitated using anti–c-Myc antibodies. Proteins in input and IP samples were detected by Western blotting using anti–c-Myc and anti-FLAG antibodies. Panel shows a representative blot of 3 independent experiments. ( D ) INS1E cells transiently transfected with siRNA targeting Nnat or Sec11a were assayed in vitro for GSIS at low (3 mM) and high (25 mM) glucose. A scramble siRNA served as a control with data expressed as mean insulin secretion per unit cellular protein. Graph on the right shows total insulin content in cell lysates. ( n = 9 independent cultures per group, 3 independent experiments, 2-way ANOVA [left graph] and 1-way ANOVA [right graph].) ( E ) INS1E cells transfected with c-Myc–tagged preproinsulin and siRNAs targeting Nnat or Sec11a were pulse-labeled with 35 S-Cys/Met. Lysates immunoprecipitated with anti–c-Myc agarose were analyzed by autoradiography. Associated bar chart shows preproinsulin and proinsulin band intensities in multiple experiments quantified by densitometry and expressed as percentage processing of preproinsulin ( n = 3 cultures per group from 3 independent experiments, 1-way ANOVA). ( F ) Similar experiments performed as in E , from 3-hour chase cell lysates (C) and media (M), quantified as in E ( n = 4 cultures per group from 3 independent experiments, 1-way ANOVA for both C and M). (* P

    Article Snippet: For siRNA silencing, INS1E cells ( ) (cultured in RPMI, 10% FBS, 2 mM l -glutamine, 50 μM β-mercaptoethanol, 37°C, 5% CO2 ) were transfected with 50 nM of Silencer Select siRNAs ( Nnat , s137247; Sec11a , s80747; both Ambion) using Dharmafect 1 reagent (Dharmacon).

    Techniques: Affinity Purification, Mass Spectrometry, Immunoprecipitation, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, Transfection, In Vitro, Labeling, Autoradiography

    Role of the nuclear transcription factor RARB in altering the expression of genes involved in steroidogenesis. ( A ) H295R cells were transfected with the human −1.05 kb HSD3B2, −1.3 kb StAR and −3.7 kb CYP17A1, -2327 bp CYP11A1 promoter constructs along with an expression vector for the RARB transcription factor. Transfection medium was changed after 6 h and cells were cultivated in serum free medium for 24 h. Thereafter, cells were grown in the presence or absence of ATRA in serum-free medium for another 24 h. Promoter activation was assessed by the dual luciferase assay (Promega) using pRL-TK as internal control. Data are expressed in Relative Light Units (RLU). Error bars show the mean ± SD of three independent experiments. * p

    Journal: Scientific Reports

    Article Title: Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

    doi: 10.1038/srep10132

    Figure Lengend Snippet: Role of the nuclear transcription factor RARB in altering the expression of genes involved in steroidogenesis. ( A ) H295R cells were transfected with the human −1.05 kb HSD3B2, −1.3 kb StAR and −3.7 kb CYP17A1, -2327 bp CYP11A1 promoter constructs along with an expression vector for the RARB transcription factor. Transfection medium was changed after 6 h and cells were cultivated in serum free medium for 24 h. Thereafter, cells were grown in the presence or absence of ATRA in serum-free medium for another 24 h. Promoter activation was assessed by the dual luciferase assay (Promega) using pRL-TK as internal control. Data are expressed in Relative Light Units (RLU). Error bars show the mean ± SD of three independent experiments. * p

    Article Snippet: Transfection medium [DMEM containing L-glutamine and 15 mm HEPES (GIBCO) supplemented with 10% NU-I serum (BD biosciences) and 0.1% selenium/insulin/transferrin (GIBCO)] was changed after 6 h and cells were cultivated in serum free medium for 24 h. Thereafter, cells were grown in the presence or absence of 1 μM ΑΤRA in serum-free medium for another 24 h. Finally, 48 hours after transfection, cells were washed with phosphate buffered saline (PBS) and then assayed for luciferase activity using the Dual Luciferase Reporter Assay system and the protocol by Promega.

    Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Activation Assay, Luciferase

    RARB and Nur77 co-operation regulate HSD3B2 in H295R cells. H295R cells were transfected with the human −1.05 kb HSD3B2 promoter constructs along with an expression vector RARB, Nur77 and in combination of RARB/Nur77. Transfection medium was changed after 6 h and cells were cultivated in either serum free or in normal growth medium for 24 h. Thereafter, cells were grown in the presence or absence of ATRA for another 24 h. ( A ) RARB and Nur77 co-operation study under serum starvation condition. ( B ) RARB and Nur77 co-operation study under normal growth condition. Promoter activation was assessed by the dual luciferase assay (Promega) using pRL-TK as internal control. Data are expressed in Relative Light Units (RLU). Error bars show the mean ± SD of four independent experiments. * p

    Journal: Scientific Reports

    Article Title: Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

    doi: 10.1038/srep10132

    Figure Lengend Snippet: RARB and Nur77 co-operation regulate HSD3B2 in H295R cells. H295R cells were transfected with the human −1.05 kb HSD3B2 promoter constructs along with an expression vector RARB, Nur77 and in combination of RARB/Nur77. Transfection medium was changed after 6 h and cells were cultivated in either serum free or in normal growth medium for 24 h. Thereafter, cells were grown in the presence or absence of ATRA for another 24 h. ( A ) RARB and Nur77 co-operation study under serum starvation condition. ( B ) RARB and Nur77 co-operation study under normal growth condition. Promoter activation was assessed by the dual luciferase assay (Promega) using pRL-TK as internal control. Data are expressed in Relative Light Units (RLU). Error bars show the mean ± SD of four independent experiments. * p

    Article Snippet: Transfection medium [DMEM containing L-glutamine and 15 mm HEPES (GIBCO) supplemented with 10% NU-I serum (BD biosciences) and 0.1% selenium/insulin/transferrin (GIBCO)] was changed after 6 h and cells were cultivated in serum free medium for 24 h. Thereafter, cells were grown in the presence or absence of 1 μM ΑΤRA in serum-free medium for another 24 h. Finally, 48 hours after transfection, cells were washed with phosphate buffered saline (PBS) and then assayed for luciferase activity using the Dual Luciferase Reporter Assay system and the protocol by Promega.

    Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Activation Assay, Luciferase

    NME4 is a direct regulatory target of miR-196. (A) Differential expression of miR-196a/b and NME4 mRNA in two immortalized normal keratinocyte and four oral cancer cell lines, as determined by RT-qPCR. Relative levels of miR-196a/b were determined after normalization to the U6 RNA level (internal control) for each sample. The NME4 RNA level was normalized to actin for each sample. (B) Differential expression of NME4 mRNA and protein in two immortalized normal keratinocyte and four oral cancer cell lines, as determined by RT-PCR and western blotting. Actin expression was also determined as an internal control. (C) Inverse correlation between NME4 and miR-196a/b expression which determined by RT-qPCR. (D) Effect of miR-196 modulation on NME4 mRNA expression. OECM1 and SAS cells transfected with miR-196a/b–specific antagomirs or overexpression plasmids were harvested. Total protein was extracted and subjected to western blot analysis of NME4 expression. The relative expression was determined after normalization to actin as an internal control. (E) The mature sequences of miR-196a and miR-196b, as well as the 3′-UTR sequence of the human NME4 gene, are shown. The mutant sequence of the 3′-UTR region of NEM4 was designed. Luciferase reporter assay was performed to determine whether NME4 is a direct miR-196a/b target gene. Cells transfected with miR-196–specific antagomirs or overexpression plasmids were co-transfected with pMIR, p-UTR-WT, or p-UTR-mut. Renilla luciferase was also transfected as a reference control for each condition. Firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay. (* p

    Journal: Molecular Cancer

    Article Title: OncomiR-196 promotes an invasive phenotype in oral cancer through the NME4-JNK-TIMP1-MMP signaling pathway

    doi: 10.1186/1476-4598-13-218

    Figure Lengend Snippet: NME4 is a direct regulatory target of miR-196. (A) Differential expression of miR-196a/b and NME4 mRNA in two immortalized normal keratinocyte and four oral cancer cell lines, as determined by RT-qPCR. Relative levels of miR-196a/b were determined after normalization to the U6 RNA level (internal control) for each sample. The NME4 RNA level was normalized to actin for each sample. (B) Differential expression of NME4 mRNA and protein in two immortalized normal keratinocyte and four oral cancer cell lines, as determined by RT-PCR and western blotting. Actin expression was also determined as an internal control. (C) Inverse correlation between NME4 and miR-196a/b expression which determined by RT-qPCR. (D) Effect of miR-196 modulation on NME4 mRNA expression. OECM1 and SAS cells transfected with miR-196a/b–specific antagomirs or overexpression plasmids were harvested. Total protein was extracted and subjected to western blot analysis of NME4 expression. The relative expression was determined after normalization to actin as an internal control. (E) The mature sequences of miR-196a and miR-196b, as well as the 3′-UTR sequence of the human NME4 gene, are shown. The mutant sequence of the 3′-UTR region of NEM4 was designed. Luciferase reporter assay was performed to determine whether NME4 is a direct miR-196a/b target gene. Cells transfected with miR-196–specific antagomirs or overexpression plasmids were co-transfected with pMIR, p-UTR-WT, or p-UTR-mut. Renilla luciferase was also transfected as a reference control for each condition. Firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay. (* p

    Article Snippet: The immortalized normal keratinocyte cells were maintained in KSFM medium (Life Technologies, Inc., Gibco BRL, Rockville, MD, USA).

    Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Over Expression, Sequencing, Mutagenesis, Luciferase, Reporter Assay