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    ATCC hek293 cells hek293 cells
    a Changes in the SPTLC2 phosphorylation pattern after phosphatase treatment. Total protein extract of CHO cells were extracted in the presence of phosphatase inhibitors (−P’ase) or alkaline phosphatase (+P’ase). The extract was subjected to isoelectric focusing in the first dimension and separated on a 12 % SDS-PAGE in the second dimension. SPTLC2 was detected with a polyclonal anti-SPTLC2 antibody. Phosphatase treatment resulted a single spot with a uniform isoelectric point (pI). b Extracts from <t>HEK293</t> cells expressing SPTLC2wt, S384A, S384F or S384D were analyzed by 2D-PAGE. SPTLC2wt and mutants were detected using an anti V5-tag antibody. Isoelectric focusing showed a reduced heterogeneity in the pI for the mutants in comparison with wild type. In vitro SPT activity was analyzed in cell extract of SPTLC2wt, S384F, S384D or S384A expressing HEK293 cells either in the presence of l -serine ( c ) or l -alanine ( d ). Cells were grown in the presence of isotope-labeled d3- l -serine (1 mM) and (C 13 )-labeled l -alanine (5 mM) for 48 h. De novo formed d2-sphinganine (d2-SA) and d2-sphingosine (d2-SO) ( e ) and de novo formed C13 -deoxy-sphinganine (C13-deoxySA) and C13 -deoxy-sphingosine (C13-deoxySO) ( f ) ( Note during the SPT reaction, one of the deuteriums of the d3- l -serine is replaced by an unlabeled hydrogen which results in the formation of d2 labeled sphinganine). Data are shown as mean with standard deviations ( N = 3, ** p
    Hek293 Cells Hek293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 361 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Novus Biologicals hek 293 cell lysate
    Caspase cleavage and LC3B-I/II turnover. A ) CCRF-CEM cells treated with drug/fatty acid vehicle (C), sphinganine (1 µM, S), or sphinganine (1 µM) + GT-11 (0.5 µM) (SG), were supplemented with the indicated fatty acids (5 µM). After 12 hours, total proteins were extracted and procaspase-3 (35 kb), activated caspase-3 (17/19 kb), and LCB3-I/II (14/16 kb), were detected by immunoblotting. β-Actin served as loading control. Treatment with pan-Bcl-2 inhibitor, ABT-737 (1 µM, A), and LC3B-transfected, <t>HEK-293</t> cell lysate, were used as positive controls. C22:1-fatty acid was used as a negative control for C22:0-fatty acid. Lanes rearranged to ease interpretation. Data representative of three separate experiments are shown. B ) Assessment of LC3B-II flux. CCRF-CEM cells were pretreated with or without protease inhibitors (Pepstatin-A and E64d) and treated as described. After 12 hours, total proteins were extracted and LC3B-I/II analyzed by immunoblotting. Data representative of three separate experiments are shown, except for C22:1-fatty acid.
    Hek 293 Cell Lysate, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ATCC hek293 cells
    Endogenously-expressed human TLR5 in <t>HEK293</t> cells mediate IL-8 responses to Flagellin and T. gondii- derived profilin
    Hek293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 14474 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    a Changes in the SPTLC2 phosphorylation pattern after phosphatase treatment. Total protein extract of CHO cells were extracted in the presence of phosphatase inhibitors (−P’ase) or alkaline phosphatase (+P’ase). The extract was subjected to isoelectric focusing in the first dimension and separated on a 12 % SDS-PAGE in the second dimension. SPTLC2 was detected with a polyclonal anti-SPTLC2 antibody. Phosphatase treatment resulted a single spot with a uniform isoelectric point (pI). b Extracts from HEK293 cells expressing SPTLC2wt, S384A, S384F or S384D were analyzed by 2D-PAGE. SPTLC2wt and mutants were detected using an anti V5-tag antibody. Isoelectric focusing showed a reduced heterogeneity in the pI for the mutants in comparison with wild type. In vitro SPT activity was analyzed in cell extract of SPTLC2wt, S384F, S384D or S384A expressing HEK293 cells either in the presence of l -serine ( c ) or l -alanine ( d ). Cells were grown in the presence of isotope-labeled d3- l -serine (1 mM) and (C 13 )-labeled l -alanine (5 mM) for 48 h. De novo formed d2-sphinganine (d2-SA) and d2-sphingosine (d2-SO) ( e ) and de novo formed C13 -deoxy-sphinganine (C13-deoxySA) and C13 -deoxy-sphingosine (C13-deoxySO) ( f ) ( Note during the SPT reaction, one of the deuteriums of the d3- l -serine is replaced by an unlabeled hydrogen which results in the formation of d2 labeled sphinganine). Data are shown as mean with standard deviations ( N = 3, ** p

    Journal: Neuromolecular Medicine

    Article Title: Novel HSAN1 Mutation in Serine Palmitoyltransferase Resides at a Putative Phosphorylation Site That Is Involved in Regulating Substrate Specificity

    doi: 10.1007/s12017-014-8339-1

    Figure Lengend Snippet: a Changes in the SPTLC2 phosphorylation pattern after phosphatase treatment. Total protein extract of CHO cells were extracted in the presence of phosphatase inhibitors (−P’ase) or alkaline phosphatase (+P’ase). The extract was subjected to isoelectric focusing in the first dimension and separated on a 12 % SDS-PAGE in the second dimension. SPTLC2 was detected with a polyclonal anti-SPTLC2 antibody. Phosphatase treatment resulted a single spot with a uniform isoelectric point (pI). b Extracts from HEK293 cells expressing SPTLC2wt, S384A, S384F or S384D were analyzed by 2D-PAGE. SPTLC2wt and mutants were detected using an anti V5-tag antibody. Isoelectric focusing showed a reduced heterogeneity in the pI for the mutants in comparison with wild type. In vitro SPT activity was analyzed in cell extract of SPTLC2wt, S384F, S384D or S384A expressing HEK293 cells either in the presence of l -serine ( c ) or l -alanine ( d ). Cells were grown in the presence of isotope-labeled d3- l -serine (1 mM) and (C 13 )-labeled l -alanine (5 mM) for 48 h. De novo formed d2-sphinganine (d2-SA) and d2-sphingosine (d2-SO) ( e ) and de novo formed C13 -deoxy-sphinganine (C13-deoxySA) and C13 -deoxy-sphingosine (C13-deoxySO) ( f ) ( Note during the SPT reaction, one of the deuteriums of the d3- l -serine is replaced by an unlabeled hydrogen which results in the formation of d2 labeled sphinganine). Data are shown as mean with standard deviations ( N = 3, ** p

    Article Snippet: Stable Expression of Mutant and Wild-type SPTLC2 in HEK293 Cells HEK293 cells (ATCC) were cultured in DMEM (Sigma-Aldrich) with 10 % fetal calf serum (FisherScientific FSA15-043) and penicillin/streptomycin (100 U/ml and 0.1 mg/ml, respectively, Sigma-Aldrich).

    Techniques: SDS Page, Expressing, Polyacrylamide Gel Electrophoresis, In Vitro, Single-particle Tracking, Activity Assay, Labeling

    (A) Representative western blot images depicting the presence or absence of TRPA1, p25 or Cdk5 in HEK293 cells that were either non-transfected or transfected with TRPA1 only or Cdk5, p25 and TRPA1. GAPDH was probed as a loading control. (B) Representative trace depicting [Ca 2+ ] i transients induced by propofol and AITC (TRPA1 agonists) in the absence and presence of roscovitine (50 µM) in TRPA1/TRPV1-transfected HEK293 cells (which lack Cdk5 activity). Cells were exposed to propofol (Pro, 10 µM) and AITC (100 µM) for 60 seconds at the time points marked by arrows. (C) Representative trace depicting [Ca 2+ ] i transients induced by propofol and AITC in the absence and presence of roscovitine (50 µM) in DRG neurons. Cells were exposed to potassium chloride (KCl, 50 mM) for 30 seconds to demonstrate cell viability after exposure to roscovitine. (D) Representative control trace depicting [Ca 2+ ] i transients induced by propofol and AITC in the absence and presence of DMSO vehicle (0.1%) in DRG neurons. E) Summarized data for experiments represented in panels B-D expressed as % of control ± SEM. NS = Not significant. n = 8 separate experiments, p

    Journal: Channels

    Article Title: Modulation of TRPA1 channel activity by Cdk5 in sensory neurons

    doi: 10.1080/19336950.2018.1424282

    Figure Lengend Snippet: (A) Representative western blot images depicting the presence or absence of TRPA1, p25 or Cdk5 in HEK293 cells that were either non-transfected or transfected with TRPA1 only or Cdk5, p25 and TRPA1. GAPDH was probed as a loading control. (B) Representative trace depicting [Ca 2+ ] i transients induced by propofol and AITC (TRPA1 agonists) in the absence and presence of roscovitine (50 µM) in TRPA1/TRPV1-transfected HEK293 cells (which lack Cdk5 activity). Cells were exposed to propofol (Pro, 10 µM) and AITC (100 µM) for 60 seconds at the time points marked by arrows. (C) Representative trace depicting [Ca 2+ ] i transients induced by propofol and AITC in the absence and presence of roscovitine (50 µM) in DRG neurons. Cells were exposed to potassium chloride (KCl, 50 mM) for 30 seconds to demonstrate cell viability after exposure to roscovitine. (D) Representative control trace depicting [Ca 2+ ] i transients induced by propofol and AITC in the absence and presence of DMSO vehicle (0.1%) in DRG neurons. E) Summarized data for experiments represented in panels B-D expressed as % of control ± SEM. NS = Not significant. n = 8 separate experiments, p

    Article Snippet: HEK293 cell culture HEK293 cells were obtained from the American Type Culture Collection and grown in 10 cm dishes in Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and antibiotics (penicillin (100 U/ml), streptomycin (100 mg/ml)) in a humidified atmosphere at 37° Celsius and 5% CO2 .

    Techniques: Western Blot, Transfection, Activity Assay

    (A) Representative western blot depicting phosphoserine immunoreactivity in HEK293 lysates from myc-tagged TRPA1 (myc-TRPA1) expressing cells co-transfected with either control RFP vector, Cdk5/p25 (in the presence or absence of roscovitine) or TRPA1 S449A mutant cDNAs. Lower blot shows myc-TRPA1 immunoreactivity in the same lanes after stripping and reprobing. (B) Summarized data for panel A, illustrating normalized TRPA1 phosphoserine immunoreactivities ± SEM. n = 6, p

    Journal: Channels

    Article Title: Modulation of TRPA1 channel activity by Cdk5 in sensory neurons

    doi: 10.1080/19336950.2018.1424282

    Figure Lengend Snippet: (A) Representative western blot depicting phosphoserine immunoreactivity in HEK293 lysates from myc-tagged TRPA1 (myc-TRPA1) expressing cells co-transfected with either control RFP vector, Cdk5/p25 (in the presence or absence of roscovitine) or TRPA1 S449A mutant cDNAs. Lower blot shows myc-TRPA1 immunoreactivity in the same lanes after stripping and reprobing. (B) Summarized data for panel A, illustrating normalized TRPA1 phosphoserine immunoreactivities ± SEM. n = 6, p

    Article Snippet: HEK293 cell culture HEK293 cells were obtained from the American Type Culture Collection and grown in 10 cm dishes in Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and antibiotics (penicillin (100 U/ml), streptomycin (100 mg/ml)) in a humidified atmosphere at 37° Celsius and 5% CO2 .

    Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Stripping Membranes

    NK1R activation in HEK293 cells does not cause apoptosis. HEK293-NK1R cells were grown in normal medium for 48 h. Cells were then treated either with vehicle, Sar9 (100 n m ), or staurosporine (0.5 μ m ). Cells were then grown for an additional

    Journal: The Journal of Biological Chemistry

    Article Title: Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism

    doi: 10.1074/jbc.M808825200

    Figure Lengend Snippet: NK1R activation in HEK293 cells does not cause apoptosis. HEK293-NK1R cells were grown in normal medium for 48 h. Cells were then treated either with vehicle, Sar9 (100 n m ), or staurosporine (0.5 μ m ). Cells were then grown for an additional

    Article Snippet: Cells —HEK293 cells (human embryonic kidney cells; American Type Culture Collection, Manassas, VA) were grown at 37 °C and 5% CO2 in 100-mm cell culture dishes (Fisher) in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 584 mg/liter l -glutamine, and 110 mg/liter sodium pyruvate (Mediatech, Manassas, VA), supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 10% fetal bovine serum (Hyclone, Logan, UT).

    Techniques: Activation Assay

    Impedance changes induced by NK1R agonists in HEK293 cells. A , representative real time cell electronic sensor ( RT-CES ) recordings in HEK293-NK1R cells treated with varying doses of SP. SP was added where indicated by an arrow. B , dose-response curve

    Journal: The Journal of Biological Chemistry

    Article Title: Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism

    doi: 10.1074/jbc.M808825200

    Figure Lengend Snippet: Impedance changes induced by NK1R agonists in HEK293 cells. A , representative real time cell electronic sensor ( RT-CES ) recordings in HEK293-NK1R cells treated with varying doses of SP. SP was added where indicated by an arrow. B , dose-response curve

    Article Snippet: Cells —HEK293 cells (human embryonic kidney cells; American Type Culture Collection, Manassas, VA) were grown at 37 °C and 5% CO2 in 100-mm cell culture dishes (Fisher) in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 584 mg/liter l -glutamine, and 110 mg/liter sodium pyruvate (Mediatech, Manassas, VA), supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 10% fetal bovine serum (Hyclone, Logan, UT).

    Techniques:

    NK1R antagonists block intracellular calcium increase and shape changes. HEK293-NK1R cells were preincubated with solvent or aprepitant (1 μ m ), L-73060 (3 μ m ), or L-73061 (3 μ m ) for 10 min. A , representative intracellular of

    Journal: The Journal of Biological Chemistry

    Article Title: Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism

    doi: 10.1074/jbc.M808825200

    Figure Lengend Snippet: NK1R antagonists block intracellular calcium increase and shape changes. HEK293-NK1R cells were preincubated with solvent or aprepitant (1 μ m ), L-73060 (3 μ m ), or L-73061 (3 μ m ) for 10 min. A , representative intracellular of

    Article Snippet: Cells —HEK293 cells (human embryonic kidney cells; American Type Culture Collection, Manassas, VA) were grown at 37 °C and 5% CO2 in 100-mm cell culture dishes (Fisher) in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 584 mg/liter l -glutamine, and 110 mg/liter sodium pyruvate (Mediatech, Manassas, VA), supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 10% fetal bovine serum (Hyclone, Logan, UT).

    Techniques: Blocking Assay

    Cellular blebbing induced by NK1R activation is not blocked by the PKC inhibitor GF109203X. Representative cell impedance measurements of HEK293-NK1R cells pretreated with either vehicle ( A and C ) or 5 μ m GF109203X ( B and D ) for 30 min and

    Journal: The Journal of Biological Chemistry

    Article Title: Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism

    doi: 10.1074/jbc.M808825200

    Figure Lengend Snippet: Cellular blebbing induced by NK1R activation is not blocked by the PKC inhibitor GF109203X. Representative cell impedance measurements of HEK293-NK1R cells pretreated with either vehicle ( A and C ) or 5 μ m GF109203X ( B and D ) for 30 min and

    Article Snippet: Cells —HEK293 cells (human embryonic kidney cells; American Type Culture Collection, Manassas, VA) were grown at 37 °C and 5% CO2 in 100-mm cell culture dishes (Fisher) in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 584 mg/liter l -glutamine, and 110 mg/liter sodium pyruvate (Mediatech, Manassas, VA), supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 10% fetal bovine serum (Hyclone, Logan, UT).

    Techniques: Activation Assay

    . HEK293-NK1R cells were pretreated with either vehicle or 10 μ

    Journal: The Journal of Biological Chemistry

    Article Title: Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism

    doi: 10.1074/jbc.M808825200

    Figure Lengend Snippet: . HEK293-NK1R cells were pretreated with either vehicle or 10 μ

    Article Snippet: Cells —HEK293 cells (human embryonic kidney cells; American Type Culture Collection, Manassas, VA) were grown at 37 °C and 5% CO2 in 100-mm cell culture dishes (Fisher) in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 584 mg/liter l -glutamine, and 110 mg/liter sodium pyruvate (Mediatech, Manassas, VA), supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 10% fetal bovine serum (Hyclone, Logan, UT).

    Techniques:

    ATP, an agonist at the P2Y 1 and P2Y 2 receptors, induces impedance changes that are distinct from those mediated by NK1R. HEK293-NK1R cells were treated with 1 μ m ATP. Shown are representative intracellular calcium ( A ) and cell impedance (

    Journal: The Journal of Biological Chemistry

    Article Title: Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism

    doi: 10.1074/jbc.M808825200

    Figure Lengend Snippet: ATP, an agonist at the P2Y 1 and P2Y 2 receptors, induces impedance changes that are distinct from those mediated by NK1R. HEK293-NK1R cells were treated with 1 μ m ATP. Shown are representative intracellular calcium ( A ) and cell impedance (

    Article Snippet: Cells —HEK293 cells (human embryonic kidney cells; American Type Culture Collection, Manassas, VA) were grown at 37 °C and 5% CO2 in 100-mm cell culture dishes (Fisher) in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 584 mg/liter l -glutamine, and 110 mg/liter sodium pyruvate (Mediatech, Manassas, VA), supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 10% fetal bovine serum (Hyclone, Logan, UT).

    Techniques:

    NK1R activation leads to cell shape changes in HEK293 cells. Scanning electron micrographs ( A ) and phase-contrast micrographs ( B ) of HEK293-NK1R cells treated with 100 n m SP for either 2 or 5 min. Untreated cells (0 min) are shown for comparison.

    Journal: The Journal of Biological Chemistry

    Article Title: Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism

    doi: 10.1074/jbc.M808825200

    Figure Lengend Snippet: NK1R activation leads to cell shape changes in HEK293 cells. Scanning electron micrographs ( A ) and phase-contrast micrographs ( B ) of HEK293-NK1R cells treated with 100 n m SP for either 2 or 5 min. Untreated cells (0 min) are shown for comparison.

    Article Snippet: Cells —HEK293 cells (human embryonic kidney cells; American Type Culture Collection, Manassas, VA) were grown at 37 °C and 5% CO2 in 100-mm cell culture dishes (Fisher) in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 584 mg/liter l -glutamine, and 110 mg/liter sodium pyruvate (Mediatech, Manassas, VA), supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 10% fetal bovine serum (Hyclone, Logan, UT).

    Techniques: Activation Assay

    Cellular blebbing induced by NK1R activation is mediated by the ROCK/Rho/MLCK pathway. A and B , HEK293-NK1R cells were pretreated with solvent ( left ), 10 μ m Y27632 for 1 h ( middle ), or 2.0 μg/ml C3 transferase for 24 h ( right ) and

    Journal: The Journal of Biological Chemistry

    Article Title: Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism

    doi: 10.1074/jbc.M808825200

    Figure Lengend Snippet: Cellular blebbing induced by NK1R activation is mediated by the ROCK/Rho/MLCK pathway. A and B , HEK293-NK1R cells were pretreated with solvent ( left ), 10 μ m Y27632 for 1 h ( middle ), or 2.0 μg/ml C3 transferase for 24 h ( right ) and

    Article Snippet: Cells —HEK293 cells (human embryonic kidney cells; American Type Culture Collection, Manassas, VA) were grown at 37 °C and 5% CO2 in 100-mm cell culture dishes (Fisher) in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 584 mg/liter l -glutamine, and 110 mg/liter sodium pyruvate (Mediatech, Manassas, VA), supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 10% fetal bovine serum (Hyclone, Logan, UT).

    Techniques: Activation Assay

    Stoichiometric FRET analysis of PKC ε -RhoA interaction in live cells. HEK293 cells were cotransfected with mCherry-PKC ε and eGFP-RhoA. Fluorescence images were captured prior to and every 15 seconds after PMA (100 nM) stimulation for 12.5 minutes and then subjected to quantitative FRET analysis. The images presented at each time point represent mCherry-PKC ε (acceptor image, IA), eGFP-RhoA (donor image, ID), and the FRET interaction between mCherry-PKC ε and eGFP-RhoA (ED). The color bars at the end of the panels indicate the scaling of the ED images with warmer colors representing higher values. Time course of normalized ED for the cell membrane and cytoplasm is presented. ED was normalized to the ED of the cytoplasm at the 0 minute time point. Cell membrane is defined as ±1 μ m from the cell membrane border. Cytoplasm is defined as all intracellular space, including the nucleus, 1 μ m from the cell membrane border.

    Journal: ISRN Oncology

    Article Title: PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

    doi: 10.1155/2013/329063

    Figure Lengend Snippet: Stoichiometric FRET analysis of PKC ε -RhoA interaction in live cells. HEK293 cells were cotransfected with mCherry-PKC ε and eGFP-RhoA. Fluorescence images were captured prior to and every 15 seconds after PMA (100 nM) stimulation for 12.5 minutes and then subjected to quantitative FRET analysis. The images presented at each time point represent mCherry-PKC ε (acceptor image, IA), eGFP-RhoA (donor image, ID), and the FRET interaction between mCherry-PKC ε and eGFP-RhoA (ED). The color bars at the end of the panels indicate the scaling of the ED images with warmer colors representing higher values. Time course of normalized ED for the cell membrane and cytoplasm is presented. ED was normalized to the ED of the cytoplasm at the 0 minute time point. Cell membrane is defined as ±1 μ m from the cell membrane border. Cytoplasm is defined as all intracellular space, including the nucleus, 1 μ m from the cell membrane border.

    Article Snippet: Cell Line HEK293 cells were purchased from ATCC (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium supplemented with penicillin (100 units/mL), streptomycin (100 μ g/mL), and 10% fetal bovine serum.

    Techniques: Fluorescence, IA

    Kinase-inactive PKC ε colocalizes with RhoA at the cell membrane. (a) Colocalization of kinase-inactive PKC ε and RhoA. HEK293 cells were cotransfected with mCherry-PKC ε /K437R and eGFP-RhoA. Fluorescence images were captured prior to and 15 minutes after PMA (100 nM) stimulation. (b) Stoichiometric FRET analysis of kinase-inactive PKC ε and RhoA interaction. HEK293 cells were cotransfected with mCherry-PKC ε /K437R and eGFP-RhoA. Fluorescence images were captured prior to and every 15 seconds after PMA (100 nM) stimulation for 12.5 minutes and then subjected to quantitative FRET analysis. The images presented at each time point represent mCherry-PKC ε /K437R (acceptor image, IA), eGFP-RhoA (donor image, ID), and the FRET interaction between mCherry-PKC ε /K437R and eGFP-RhoA (ED). The color bars at the end of the panels indicate the scaling of the ED images with warmer colors representing higher values. Time course of normalized ED for the cell membrane and cytoplasm is presented. ED was normalized to the ED of the cytoplasm at the 0 minute time point. Cell membrane is defined as ±1 μ m from the cell membrane border. Cytoplasm is defined as all intracellular space, including the nucleus, 1 μ m from the cell membrane border.

    Journal: ISRN Oncology

    Article Title: PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

    doi: 10.1155/2013/329063

    Figure Lengend Snippet: Kinase-inactive PKC ε colocalizes with RhoA at the cell membrane. (a) Colocalization of kinase-inactive PKC ε and RhoA. HEK293 cells were cotransfected with mCherry-PKC ε /K437R and eGFP-RhoA. Fluorescence images were captured prior to and 15 minutes after PMA (100 nM) stimulation. (b) Stoichiometric FRET analysis of kinase-inactive PKC ε and RhoA interaction. HEK293 cells were cotransfected with mCherry-PKC ε /K437R and eGFP-RhoA. Fluorescence images were captured prior to and every 15 seconds after PMA (100 nM) stimulation for 12.5 minutes and then subjected to quantitative FRET analysis. The images presented at each time point represent mCherry-PKC ε /K437R (acceptor image, IA), eGFP-RhoA (donor image, ID), and the FRET interaction between mCherry-PKC ε /K437R and eGFP-RhoA (ED). The color bars at the end of the panels indicate the scaling of the ED images with warmer colors representing higher values. Time course of normalized ED for the cell membrane and cytoplasm is presented. ED was normalized to the ED of the cytoplasm at the 0 minute time point. Cell membrane is defined as ±1 μ m from the cell membrane border. Cytoplasm is defined as all intracellular space, including the nucleus, 1 μ m from the cell membrane border.

    Article Snippet: Cell Line HEK293 cells were purchased from ATCC (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium supplemented with penicillin (100 units/mL), streptomycin (100 μ g/mL), and 10% fetal bovine serum.

    Techniques: Fluorescence, IA

    PKC ε colocalizes with RhoA at the cell membrane. (a) PKC ε translocates to the cell membrane, and RhoA remains localized at the cytoplasm in response to PMA. HEK293 cells were transfected with mCherry-PKC ε (left panel) or eGFP-RhoA (right panel). Fluorescence images were captured prior to and 15 minutes after PMA (100 nM) stimulation. (b) PKC ε colocalizes with RhoA at the cell membrane. HEK293 cells were cotransfected with mCherry-PKC ε and eGFP-RhoA. Fluorescence images were captured prior to and 15 minutes after PMA (100 nM) stimulation.

    Journal: ISRN Oncology

    Article Title: PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

    doi: 10.1155/2013/329063

    Figure Lengend Snippet: PKC ε colocalizes with RhoA at the cell membrane. (a) PKC ε translocates to the cell membrane, and RhoA remains localized at the cytoplasm in response to PMA. HEK293 cells were transfected with mCherry-PKC ε (left panel) or eGFP-RhoA (right panel). Fluorescence images were captured prior to and 15 minutes after PMA (100 nM) stimulation. (b) PKC ε colocalizes with RhoA at the cell membrane. HEK293 cells were cotransfected with mCherry-PKC ε and eGFP-RhoA. Fluorescence images were captured prior to and 15 minutes after PMA (100 nM) stimulation.

    Article Snippet: Cell Line HEK293 cells were purchased from ATCC (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium supplemented with penicillin (100 units/mL), streptomycin (100 μ g/mL), and 10% fetal bovine serum.

    Techniques: Transfection, Fluorescence

    The Arc binding site is located in the PICK1 BAR domain. (a) A schematic representation of full length PICK1 and PICK1B highlights the structural domains of the protein (PDZ in green; BAR domain in red; and an acidic region in purple). PICK1B consists of the first 278 amino acids and the last 86 amino acids at the C-terminal end resulting in a truncated protein that is 52 amino acids shorter than the original sequence. ( b ) HEK293 cells were transfected with PICK1 (lane 1–3, positive control) or PICK1B (lanes 4–6). The lysates were loaded (lane 1 and 4, full lysate as a second positive control) or were subjected to a pull-down with purified GST-Arc (lane 3 and 6) or purified GST alone (lane 2 and 4, negative control), followed by Westernblot (WB) blotted for PICK1. ( c – e ) Representative intensity images of co-transfected SH-SY5Y cells containing Arc-mCherry and EGFP-PICK1-BAR or (g–i) EGFP-PICK1-PDZ under depolarizing conditions indicate that Arc-mCherry can associate with the PICK1-BAR domain but not the PICK1-PDZ. ( f , j ) Cross-correlation of the Arc-mCherry and EGFP-PICK1 fluctuation signals during RICS analysis produces (f) a characteristic autocorrelation function (ACF) indicating protein-protein interaction for cells transfected with the PICK1-BAR construct ( j ) while the PICK1-PDZ domain showed little to no correlation with Arc-mCherry. n = 15 cells for each condition. (The red to light blue distribution indicates the intensity of pixels that have correlated diffusion in both channels. Broader histograms (more red) indicate less correlation of the signals).

    Journal: Scientific Reports

    Article Title: Arc/Arg3.1 has an activity-regulated interaction with PICK1 that results in altered spatial dynamics

    doi: 10.1038/s41598-018-32821-4

    Figure Lengend Snippet: The Arc binding site is located in the PICK1 BAR domain. (a) A schematic representation of full length PICK1 and PICK1B highlights the structural domains of the protein (PDZ in green; BAR domain in red; and an acidic region in purple). PICK1B consists of the first 278 amino acids and the last 86 amino acids at the C-terminal end resulting in a truncated protein that is 52 amino acids shorter than the original sequence. ( b ) HEK293 cells were transfected with PICK1 (lane 1–3, positive control) or PICK1B (lanes 4–6). The lysates were loaded (lane 1 and 4, full lysate as a second positive control) or were subjected to a pull-down with purified GST-Arc (lane 3 and 6) or purified GST alone (lane 2 and 4, negative control), followed by Westernblot (WB) blotted for PICK1. ( c – e ) Representative intensity images of co-transfected SH-SY5Y cells containing Arc-mCherry and EGFP-PICK1-BAR or (g–i) EGFP-PICK1-PDZ under depolarizing conditions indicate that Arc-mCherry can associate with the PICK1-BAR domain but not the PICK1-PDZ. ( f , j ) Cross-correlation of the Arc-mCherry and EGFP-PICK1 fluctuation signals during RICS analysis produces (f) a characteristic autocorrelation function (ACF) indicating protein-protein interaction for cells transfected with the PICK1-BAR construct ( j ) while the PICK1-PDZ domain showed little to no correlation with Arc-mCherry. n = 15 cells for each condition. (The red to light blue distribution indicates the intensity of pixels that have correlated diffusion in both channels. Broader histograms (more red) indicate less correlation of the signals).

    Article Snippet: Cell culture HEK293 cells and SH-SY5Y cells (ATCC) were cultured in DMEM F12 containing 15 mM HEPES, 1.5 mM sodium pyruvate, 1x Antibiotic-Antimycotic and 10% FBS in tissue culture-treated T75 flasks at 37 °C and 5% CO2 .

    Techniques: Binding Assay, Sequencing, Transfection, Positive Control, Purification, Negative Control, Western Blot, Construct, Diffusion-based Assay

    The dimer selectivity for cosmosiin and angolensin was demonstrated in dose-response BRET assays (A and B) and reporter assays (C and D) in HEK293 cells. ER dimer-specific BRET assays were performed over a range of compound concentrations of cosmosiin (A) and angolensin (B). HEK293T ERE-luciferase transcriptional assays reveal each compound's ability to transcriptionally activate various dimer pairs (C and D). ERα alone, ERβ alone, or ERα+ERβ was transfected along with an ERE-luciferase element in order to test the ability of cosmosiin (C) and angolensin (D) to transcriptionally activate these various ER dimer pairs. RLU, relative luciferase units. Error bars represent standard deviations from the mean of triplicate samples. In BRET (A), p values indicate all pairs with statistical significance by the Student's T-Test.

    Journal: PLoS ONE

    Article Title: Identification of Estrogen Receptor Dimer Selective Ligands Reveals Growth-Inhibitory Effects on Cells That Co-Express ER? and ER?

    doi: 10.1371/journal.pone.0030993

    Figure Lengend Snippet: The dimer selectivity for cosmosiin and angolensin was demonstrated in dose-response BRET assays (A and B) and reporter assays (C and D) in HEK293 cells. ER dimer-specific BRET assays were performed over a range of compound concentrations of cosmosiin (A) and angolensin (B). HEK293T ERE-luciferase transcriptional assays reveal each compound's ability to transcriptionally activate various dimer pairs (C and D). ERα alone, ERβ alone, or ERα+ERβ was transfected along with an ERE-luciferase element in order to test the ability of cosmosiin (C) and angolensin (D) to transcriptionally activate these various ER dimer pairs. RLU, relative luciferase units. Error bars represent standard deviations from the mean of triplicate samples. In BRET (A), p values indicate all pairs with statistical significance by the Student's T-Test.

    Article Snippet: In vivo BRET assays to monitor ER dimer formation in living cells HEK293 cells (ATCC, CRL-1573) were either transfected with a single BRET fusion plasmid (pCMX-ERα-RLuc or pCMX-RLuc-ERβ) or co-transfected with RLuc and YFP BRET fusions (pCMX-ERα-RLuc+pCMX-YFP-ERβ for ERα/ERβ heterodimers; pCMX-ERα-RLuc+pCMX-ERα-YFP for ERα homodimers; or pCMX-RLuc-ERβ+pCMX-YFP-ERβ for ERβ homodimers) .

    Techniques: Bioluminescence Resonance Energy Transfer, Luciferase, Transfection

    IGF1 stimulation enhances endosomal PtdIns(3)P and recruitment of GFP-SGK3 to the endosomes. HEK293 cells were serum-starved overnight then treated as indicated with or without VPS34-IN1 (1 µM) and/or GDC-0941 (0.5 µM) for 60 min prior to stimulation with 50 ng/ml IGF1 for 15 min. Cells were subsequently fixed with 4% (v/v) paraformaldehyde and stained with recombinant wild-type (WT) PtdIns(3)P-binding probe FYVE domain of HRS[147–223] conjugated to Alexa Fluor 488 conjugate (green channel) ( A , C , E and G ) or with the mutant non-PtdIns(3)P-binding FYVE domain of HRS[147–223, H176A/H177A] (red channel, Alexa 594). ( B , D , F and H ). ( I ) The histogram displays a representative quantitation of the sum fluorescence intensity of PX domain staining on endosomal structures ± SEM (increase in PtdIns(3)P levels upon IGF1 stimulation ranged from 30 to 50% compared with serum-starved cells). ( J–Q ) GFP-SGK3 knock-in HEK293 cells were serum-starved overnight and treated as above prior to IGF1 stimulation. Cells were fixed with 4% (v/v) paraformaldehyde and GFP distribution was visualised using chicken anti-GFP primary and anti-chicken Alexa Fluor 488 to enhance the GFP signal (upper panel). SGK3 co-localisation with the early endosomal marker EEA1 was visualised using rabbit anti-EEA1 primary and anti-rabbit Alexa Fluor 594 secondary antibody (lower panel). ( R ) The histogram displays a representative quantitation of the sum of intensity of fluorescent signal from GFP-SGK3, co-localising with the EEA1 marker at the endosomes ± SEM following the various treatments (increase in GFP-SGK3 levels at the endosomes ranged from 30 to 60% upon the addition of IGF1 compared with serum-starved conditions).

    Journal: Biochemical Journal

    Article Title: Mechanism of activation of SGK3 by growth factors via the Class 1 and Class 3 PI3Ks

    doi: 10.1042/BCJ20170650

    Figure Lengend Snippet: IGF1 stimulation enhances endosomal PtdIns(3)P and recruitment of GFP-SGK3 to the endosomes. HEK293 cells were serum-starved overnight then treated as indicated with or without VPS34-IN1 (1 µM) and/or GDC-0941 (0.5 µM) for 60 min prior to stimulation with 50 ng/ml IGF1 for 15 min. Cells were subsequently fixed with 4% (v/v) paraformaldehyde and stained with recombinant wild-type (WT) PtdIns(3)P-binding probe FYVE domain of HRS[147–223] conjugated to Alexa Fluor 488 conjugate (green channel) ( A , C , E and G ) or with the mutant non-PtdIns(3)P-binding FYVE domain of HRS[147–223, H176A/H177A] (red channel, Alexa 594). ( B , D , F and H ). ( I ) The histogram displays a representative quantitation of the sum fluorescence intensity of PX domain staining on endosomal structures ± SEM (increase in PtdIns(3)P levels upon IGF1 stimulation ranged from 30 to 50% compared with serum-starved cells). ( J–Q ) GFP-SGK3 knock-in HEK293 cells were serum-starved overnight and treated as above prior to IGF1 stimulation. Cells were fixed with 4% (v/v) paraformaldehyde and GFP distribution was visualised using chicken anti-GFP primary and anti-chicken Alexa Fluor 488 to enhance the GFP signal (upper panel). SGK3 co-localisation with the early endosomal marker EEA1 was visualised using rabbit anti-EEA1 primary and anti-rabbit Alexa Fluor 594 secondary antibody (lower panel). ( R ) The histogram displays a representative quantitation of the sum of intensity of fluorescent signal from GFP-SGK3, co-localising with the EEA1 marker at the endosomes ± SEM following the various treatments (increase in GFP-SGK3 levels at the endosomes ranged from 30 to 60% upon the addition of IGF1 compared with serum-starved conditions).

    Article Snippet: Cell culture, transfection and cell lysis HEK293 cells were purchased from the American Tissue Culture Collection and cultured in DMEM supplemented with 10% (v/v) foetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.

    Techniques: Staining, Recombinant, Binding Assay, Mutagenesis, Quantitation Assay, Fluorescence, Knock-In, Marker

    AdPROM-mediated knock-down of hVPS34 and UV-RAG reduces IGF1-induced SGK3 activation. GFP-Vps34 ( A ) or GFP-UV-RAG ( B ) knock-in HEK293 cells that stably express either the anti-GFP nanobody (Control-aGFP16) or the VHL-E3-ligase–anti-GFP nanobody (VHL-aGFP16) were generated as described in the Materials and Methods. Cells were serum-starved overnight and treated as indicated with or without VPS34-IN1 (1 µM) and/or GDC-0941 (0.5 µM) for 60 min prior to stimulation with 50 ng/ml IGF1 for 15 min. Endogenous SGK3 was immunoprecipitated from cell lysates and kinase activity was assessed by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32 PγATP in a 30 min reaction. Both immunoprecipitates and lysates were subjected to western blotting with the indicated antibodies. Kinase reactions are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments for all data shown.

    Journal: Biochemical Journal

    Article Title: Mechanism of activation of SGK3 by growth factors via the Class 1 and Class 3 PI3Ks

    doi: 10.1042/BCJ20170650

    Figure Lengend Snippet: AdPROM-mediated knock-down of hVPS34 and UV-RAG reduces IGF1-induced SGK3 activation. GFP-Vps34 ( A ) or GFP-UV-RAG ( B ) knock-in HEK293 cells that stably express either the anti-GFP nanobody (Control-aGFP16) or the VHL-E3-ligase–anti-GFP nanobody (VHL-aGFP16) were generated as described in the Materials and Methods. Cells were serum-starved overnight and treated as indicated with or without VPS34-IN1 (1 µM) and/or GDC-0941 (0.5 µM) for 60 min prior to stimulation with 50 ng/ml IGF1 for 15 min. Endogenous SGK3 was immunoprecipitated from cell lysates and kinase activity was assessed by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32 PγATP in a 30 min reaction. Both immunoprecipitates and lysates were subjected to western blotting with the indicated antibodies. Kinase reactions are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments for all data shown.

    Article Snippet: Cell culture, transfection and cell lysis HEK293 cells were purchased from the American Tissue Culture Collection and cultured in DMEM supplemented with 10% (v/v) foetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.

    Techniques: Activation Assay, Knock-In, Stable Transfection, Generated, Immunoprecipitation, Activity Assay, Western Blot

    SGK3 is activated by IGF1. ( A ) Wild-type (WT) and SGK3 knock-out (KO) HEK293 cells were serum-starved for 16 h prior to stimulation with or without 50 ng/ml IGF1 for 10 min. Subsequently, cells were lysed and endogenous SGK3 was immunoprecipitated with anti-SGK3 antibody. Immunoprecipitates were assayed for SGK3 kinase activity by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32 PγATP in a 30 min reaction. The lysates and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies following the kinase assay. Kinase reactions are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments. ( B ) As in ( A ), except cells were treated with or without 3 µM 14H and/or 1 µM MK2206 for 1 h before stimulation with or without 50 ng/ml IGF1 for 10 min. Cell lysates were subjected to immunoblot analysis with the indicated antibodies.

    Journal: Biochemical Journal

    Article Title: Mechanism of activation of SGK3 by growth factors via the Class 1 and Class 3 PI3Ks

    doi: 10.1042/BCJ20170650

    Figure Lengend Snippet: SGK3 is activated by IGF1. ( A ) Wild-type (WT) and SGK3 knock-out (KO) HEK293 cells were serum-starved for 16 h prior to stimulation with or without 50 ng/ml IGF1 for 10 min. Subsequently, cells were lysed and endogenous SGK3 was immunoprecipitated with anti-SGK3 antibody. Immunoprecipitates were assayed for SGK3 kinase activity by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32 PγATP in a 30 min reaction. The lysates and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies following the kinase assay. Kinase reactions are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments. ( B ) As in ( A ), except cells were treated with or without 3 µM 14H and/or 1 µM MK2206 for 1 h before stimulation with or without 50 ng/ml IGF1 for 10 min. Cell lysates were subjected to immunoblot analysis with the indicated antibodies.

    Article Snippet: Cell culture, transfection and cell lysis HEK293 cells were purchased from the American Tissue Culture Collection and cultured in DMEM supplemented with 10% (v/v) foetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.

    Techniques: Knock-Out, Immunoprecipitation, Activity Assay, Kinase Assay

    SGK3 activity is induced by growth factors, insulin and oncogenic Ras. ( A ) HEK293 cells were serum-starved for 16 h prior to stimulation with or without the indicated concentrations of IGF1, EGF, insulin, FGF, TPA, H 2 O 2 and serum for 15 min. Endogenous SGK3 was immunoprecipitated from the lysates and SGK3 kinase activity was assessed by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32 PγATP in a 30 min reaction. Both immunoprecipitates and lysates were subjected to western blotting with the indicated antibodies. ( B ) As in ( A ), except that HEK293 cells were transiently transfected when 60% confluent, with KRAS[G12C], KRAS[G12D], NRAS[G12D], ERBB2[V842I] and EGFR[L833R] (all HA epitope tagged). Forty eight hours later, WT and transfected cells were serum-starved for 16 h before cells lysed without further stimulation. ( C ) As in ( B ), except that HEK293 cells were transiently transfected with KRAS[G12C] and KRAS[G12D] and treated as indicated with VPS34-IN1 (1 µM) and/or GDC-0941 (0.5 µM) prior to stimulation with 50 ng/ml IGF1 for 10 min. Kinase reactions are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments for all data shown.

    Journal: Biochemical Journal

    Article Title: Mechanism of activation of SGK3 by growth factors via the Class 1 and Class 3 PI3Ks

    doi: 10.1042/BCJ20170650

    Figure Lengend Snippet: SGK3 activity is induced by growth factors, insulin and oncogenic Ras. ( A ) HEK293 cells were serum-starved for 16 h prior to stimulation with or without the indicated concentrations of IGF1, EGF, insulin, FGF, TPA, H 2 O 2 and serum for 15 min. Endogenous SGK3 was immunoprecipitated from the lysates and SGK3 kinase activity was assessed by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32 PγATP in a 30 min reaction. Both immunoprecipitates and lysates were subjected to western blotting with the indicated antibodies. ( B ) As in ( A ), except that HEK293 cells were transiently transfected when 60% confluent, with KRAS[G12C], KRAS[G12D], NRAS[G12D], ERBB2[V842I] and EGFR[L833R] (all HA epitope tagged). Forty eight hours later, WT and transfected cells were serum-starved for 16 h before cells lysed without further stimulation. ( C ) As in ( B ), except that HEK293 cells were transiently transfected with KRAS[G12C] and KRAS[G12D] and treated as indicated with VPS34-IN1 (1 µM) and/or GDC-0941 (0.5 µM) prior to stimulation with 50 ng/ml IGF1 for 10 min. Kinase reactions are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments for all data shown.

    Article Snippet: Cell culture, transfection and cell lysis HEK293 cells were purchased from the American Tissue Culture Collection and cultured in DMEM supplemented with 10% (v/v) foetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.

    Techniques: Activity Assay, Immunoprecipitation, Western Blot, Transfection

    Class 1 and Class 3 PI3Ks mediate IGF1-induced SGK3 activity. HEK293 cells were serum-starved for 16 h. One hour prior to stimulation, cells were left untreated ( A and B ) or treated with 1 µM VPS34-IN1 ( C and D ) or 0.5 µM GDC-0941 ( E and F ) or 1 µM VPS34-IN1 and 0.5 µM GDC-0941 ( G and H ). Cells were then stimulated with 50 ng/ml IGF1 for the indicated times in the presence of inhibitors. Cells were lysed and endogenous SGK3 was immunoprecipitated with anti-SGK3 antibody. The lysates and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies, after being assayed for SGK3 kinase activity by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32 PγATP in a 30 min reaction. Kinase reactions are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments for all data shown.

    Journal: Biochemical Journal

    Article Title: Mechanism of activation of SGK3 by growth factors via the Class 1 and Class 3 PI3Ks

    doi: 10.1042/BCJ20170650

    Figure Lengend Snippet: Class 1 and Class 3 PI3Ks mediate IGF1-induced SGK3 activity. HEK293 cells were serum-starved for 16 h. One hour prior to stimulation, cells were left untreated ( A and B ) or treated with 1 µM VPS34-IN1 ( C and D ) or 0.5 µM GDC-0941 ( E and F ) or 1 µM VPS34-IN1 and 0.5 µM GDC-0941 ( G and H ). Cells were then stimulated with 50 ng/ml IGF1 for the indicated times in the presence of inhibitors. Cells were lysed and endogenous SGK3 was immunoprecipitated with anti-SGK3 antibody. The lysates and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies, after being assayed for SGK3 kinase activity by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32 PγATP in a 30 min reaction. Kinase reactions are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments for all data shown.

    Article Snippet: Cell culture, transfection and cell lysis HEK293 cells were purchased from the American Tissue Culture Collection and cultured in DMEM supplemented with 10% (v/v) foetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.

    Techniques: Activity Assay, Immunoprecipitation

    IGF1 does not stimulate hVPS34 activity. Wild-type (WT), GFP-Vps34 or GFP-UV-RAG knock-in HEK293 cells were cultured in serum-lysed and subjected to immunoblot analysis with the indicated antibodies. ( B ) WT, GFP-Vps34 or GFP-UV-RAG knock-in HEK293 cells were serum-starved overnight prior to treatment with or without 50 ng/ml IGF1 for 15 min. Cells were subsequently lysed using a 1% (v/v) NP-40 lysis buffer and immunoprecipitations undertaken using GFP-TRAP antibodies. The immunoprecipitates were subjected to a kinase assay in the presence of PtdIns (5 µg) and 0.1 mM 32 PγATP for a 30 min reaction measure in the absence or presence of VPS43-IN1 (1 µM) as indicated. Reactions were chromatographed on a Silica 60 TLC plate and 32 P-radioactivity associated with the spot, corresponding to PtdIns(3)P, was visualised by autoradiography and quantified by Cerenkov scintillation. Immunoprecipitates were also subjected to immunoblot analysis with the indicated antibodies. In the top panel, we show the quantitation of the VPS34 PI 3-kinase activity. The data are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments for all data shown.

    Journal: Biochemical Journal

    Article Title: Mechanism of activation of SGK3 by growth factors via the Class 1 and Class 3 PI3Ks

    doi: 10.1042/BCJ20170650

    Figure Lengend Snippet: IGF1 does not stimulate hVPS34 activity. Wild-type (WT), GFP-Vps34 or GFP-UV-RAG knock-in HEK293 cells were cultured in serum-lysed and subjected to immunoblot analysis with the indicated antibodies. ( B ) WT, GFP-Vps34 or GFP-UV-RAG knock-in HEK293 cells were serum-starved overnight prior to treatment with or without 50 ng/ml IGF1 for 15 min. Cells were subsequently lysed using a 1% (v/v) NP-40 lysis buffer and immunoprecipitations undertaken using GFP-TRAP antibodies. The immunoprecipitates were subjected to a kinase assay in the presence of PtdIns (5 µg) and 0.1 mM 32 PγATP for a 30 min reaction measure in the absence or presence of VPS43-IN1 (1 µM) as indicated. Reactions were chromatographed on a Silica 60 TLC plate and 32 P-radioactivity associated with the spot, corresponding to PtdIns(3)P, was visualised by autoradiography and quantified by Cerenkov scintillation. Immunoprecipitates were also subjected to immunoblot analysis with the indicated antibodies. In the top panel, we show the quantitation of the VPS34 PI 3-kinase activity. The data are presented as means ± SEM for triplicate reactions. Similar results were obtained in at least two independent experiments for all data shown.

    Article Snippet: Cell culture, transfection and cell lysis HEK293 cells were purchased from the American Tissue Culture Collection and cultured in DMEM supplemented with 10% (v/v) foetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.

    Techniques: Activity Assay, Knock-In, Cell Culture, Lysis, Kinase Assay, Thin Layer Chromatography, Radioactivity, Autoradiography, Quantitation Assay

    Loss of mTORC2 activity ablates IGF1-induced SGK3 activity. Wild-type and SIN1 knock-out HEK293 cells were serum-starved overnight prior to 50 ng/ml IGF1 stimulation for 15 min. Endogenous SGK3 was immunoprecipitated from the lysates and SGK3 kinase activity was assessed by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32 PγATP in a 30 min reaction. Both immunoprecipitates and lysates were subjected to western blotting with the indicated antibodies.

    Journal: Biochemical Journal

    Article Title: Mechanism of activation of SGK3 by growth factors via the Class 1 and Class 3 PI3Ks

    doi: 10.1042/BCJ20170650

    Figure Lengend Snippet: Loss of mTORC2 activity ablates IGF1-induced SGK3 activity. Wild-type and SIN1 knock-out HEK293 cells were serum-starved overnight prior to 50 ng/ml IGF1 stimulation for 15 min. Endogenous SGK3 was immunoprecipitated from the lysates and SGK3 kinase activity was assessed by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM 32 PγATP in a 30 min reaction. Both immunoprecipitates and lysates were subjected to western blotting with the indicated antibodies.

    Article Snippet: Cell culture, transfection and cell lysis HEK293 cells were purchased from the American Tissue Culture Collection and cultured in DMEM supplemented with 10% (v/v) foetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.

    Techniques: Activity Assay, Knock-Out, Immunoprecipitation, Western Blot

    IGF1 does not affect localisation of hVPS34 and UV-RAG. GFP-Vps34 ( A – I ) or GFP-UV-RAG ( J – R ) knock-in HEK293 cells were serum-starved overnight and treated as indicated with or without VPS34-IN1 (1 µM) and/or GDC-0941 (0.5 µM) for 60 min prior to stimulation with IGF1 for 15 min. Cells were subsequently fixed with 4% (v/v) paraformaldehyde and GFP distribution was visualised using chicken anti-GFP primary and anti-chicken Alexa Fluor 488 to enhance the GFP signal. Co-localisation of hVPS34 or UV-RAG with an early endosomal EEA1 marker was visualised using rabbit anti-EEA1 primary and anti-rabbit Alexa Fluor 594 secondary antibody. The histograms display quantitation of the sum of intensity of fluorescent signal from GFP-Vps34 ( I ) or GFP-UV-RAG ( R ), co-localising with the EEA1 marker ± SEM following the various treatments.

    Journal: Biochemical Journal

    Article Title: Mechanism of activation of SGK3 by growth factors via the Class 1 and Class 3 PI3Ks

    doi: 10.1042/BCJ20170650

    Figure Lengend Snippet: IGF1 does not affect localisation of hVPS34 and UV-RAG. GFP-Vps34 ( A – I ) or GFP-UV-RAG ( J – R ) knock-in HEK293 cells were serum-starved overnight and treated as indicated with or without VPS34-IN1 (1 µM) and/or GDC-0941 (0.5 µM) for 60 min prior to stimulation with IGF1 for 15 min. Cells were subsequently fixed with 4% (v/v) paraformaldehyde and GFP distribution was visualised using chicken anti-GFP primary and anti-chicken Alexa Fluor 488 to enhance the GFP signal. Co-localisation of hVPS34 or UV-RAG with an early endosomal EEA1 marker was visualised using rabbit anti-EEA1 primary and anti-rabbit Alexa Fluor 594 secondary antibody. The histograms display quantitation of the sum of intensity of fluorescent signal from GFP-Vps34 ( I ) or GFP-UV-RAG ( R ), co-localising with the EEA1 marker ± SEM following the various treatments.

    Article Snippet: Cell culture, transfection and cell lysis HEK293 cells were purchased from the American Tissue Culture Collection and cultured in DMEM supplemented with 10% (v/v) foetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.

    Techniques: Knock-In, Marker, Quantitation Assay

    PV-encoded miRNAs are active in RISC. RISC reporter assays for the PV-encoded miRNAs where HEK293 cells were co-transfected with a firefly luciferase transfection control and Renilla luciferase reporter with either perfectly complementary sequence matches for each indicated miRNA, or its respective negative control seed complement mutant. Either of these were co-transfected with control empty miRNA expression vector (blue), the relevant PV miRNA-expression vector (orange), or negative control irrelevant miRNA expression vector (SV40) (gray). Average Renilla luciferase activity relative to firefly luciferase normalized to empty miRNA expression vector control is shown for fcpv1-miR-F1 (N = 3), fcpv1-miR-F2 (N = 5), hpv41-miR-H1 (N = 5), hpv17-miR-H1 (N = 3), and hpv37-miR-H1 (N = 4). Statistical test performed was a Two-Sample t Test. The average Renilla luciferase activity normalized to firefly luciferase activity is shown, error bars indicate Standard Error, and asterisks indicate statistical significance, (*) p≤0.05; (**) p≤0.01.

    Journal: PLoS Pathogens

    Article Title: Identification of virus-encoded microRNAs in divergent Papillomaviruses

    doi: 10.1371/journal.ppat.1007156

    Figure Lengend Snippet: PV-encoded miRNAs are active in RISC. RISC reporter assays for the PV-encoded miRNAs where HEK293 cells were co-transfected with a firefly luciferase transfection control and Renilla luciferase reporter with either perfectly complementary sequence matches for each indicated miRNA, or its respective negative control seed complement mutant. Either of these were co-transfected with control empty miRNA expression vector (blue), the relevant PV miRNA-expression vector (orange), or negative control irrelevant miRNA expression vector (SV40) (gray). Average Renilla luciferase activity relative to firefly luciferase normalized to empty miRNA expression vector control is shown for fcpv1-miR-F1 (N = 3), fcpv1-miR-F2 (N = 5), hpv41-miR-H1 (N = 5), hpv17-miR-H1 (N = 3), and hpv37-miR-H1 (N = 4). Statistical test performed was a Two-Sample t Test. The average Renilla luciferase activity normalized to firefly luciferase activity is shown, error bars indicate Standard Error, and asterisks indicate statistical significance, (*) p≤0.05; (**) p≤0.01.

    Article Snippet: Plasmids and cells HEK293 or HEK293T cells were originally obtained from ATCC and maintained in DMEM supplemented with 10% (vol/vol) FBS and Pen/Strep (Cellgro).

    Techniques: Transfection, Luciferase, Sequencing, Negative Control, Mutagenesis, Expressing, Plasmid Preparation, Activity Assay

    Heterologous expression of the extracellular domains of TLR1 and TLR2 together with heterologous expression of the TIR domains of TLR1 and TLR2 is sufficient for NF κ B signal activation. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1 TIR] DNA encoding chimeric proteins or wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam 3 CSK 4 , or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of cotransfection of TLR [1–2] and TLR [2–1 TIR] chimeric proteins. Co-transfection of TLR [1–2] chimeric protein containing the entire intracellular domain of TLR2 (including the TIR domain) with the TLR [2–1 TIR] chimeric protein containing the TIR domain of TLR1, confers responsiveness. The transfected cells express the extracellular domains of TLR1 and TLR2 (ligand recognition) as well as both the TIR domains of TLR1 and TLR2 (signal transduction). With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.

    Journal: The Journal of Cell Biology

    Article Title: Importance of extra- and intracellular domains of TLR1 and TLR2 in NF?B signaling

    doi: 10.1083/jcb.200304093

    Figure Lengend Snippet: Heterologous expression of the extracellular domains of TLR1 and TLR2 together with heterologous expression of the TIR domains of TLR1 and TLR2 is sufficient for NF κ B signal activation. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1 TIR] DNA encoding chimeric proteins or wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam 3 CSK 4 , or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of cotransfection of TLR [1–2] and TLR [2–1 TIR] chimeric proteins. Co-transfection of TLR [1–2] chimeric protein containing the entire intracellular domain of TLR2 (including the TIR domain) with the TLR [2–1 TIR] chimeric protein containing the TIR domain of TLR1, confers responsiveness. The transfected cells express the extracellular domains of TLR1 and TLR2 (ligand recognition) as well as both the TIR domains of TLR1 and TLR2 (signal transduction). With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.

    Article Snippet: Transfection and reporter gene assays in HEK 293-CD14 cells HEK293 cells (CRL-1573; American Type Culture Collection) stably expressing human CD14 (HEK293-CD14) were cloned as described previously ( ).

    Techniques: Expressing, Activation Assay, Luciferase, Activity Assay, Positive Control, Cotransfection, Transfection, Transduction

    Neither the intracellular nor the extracellular domain of TLR2 is sufficient to confer NF κ B signal activation. (A) HEK293-CD14 cells were transfected with either TLR [1–2] or TLR [2–1] DNA encoding chimeric protein and wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam 3 CSK 4 , or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of separate TLR1 and TLR2 chimeric protein transfection. With TLR1 endogenously expressed, transfection only of the intracellular portion of TLR2 (TLR [1–2]) does not confer responsiveness as a result of missing TLR2 extracellular domain causing ligand recognition failure. Transfection with only the extracellular portion of TLR2 (TLR [2–1]) is not sufficient to confer responsiveness because the TLR1 intracellular domain lacks the TLR2 intracellular domain for effective initiation of signaling pathways. With TLR1 endogenously present, transfection of TLR2-WT protein confers responsiveness by providing both extra- and intracellular domains needed for ligand recognition and signal activation respectively.

    Journal: The Journal of Cell Biology

    Article Title: Importance of extra- and intracellular domains of TLR1 and TLR2 in NF?B signaling

    doi: 10.1083/jcb.200304093

    Figure Lengend Snippet: Neither the intracellular nor the extracellular domain of TLR2 is sufficient to confer NF κ B signal activation. (A) HEK293-CD14 cells were transfected with either TLR [1–2] or TLR [2–1] DNA encoding chimeric protein and wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam 3 CSK 4 , or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of separate TLR1 and TLR2 chimeric protein transfection. With TLR1 endogenously expressed, transfection only of the intracellular portion of TLR2 (TLR [1–2]) does not confer responsiveness as a result of missing TLR2 extracellular domain causing ligand recognition failure. Transfection with only the extracellular portion of TLR2 (TLR [2–1]) is not sufficient to confer responsiveness because the TLR1 intracellular domain lacks the TLR2 intracellular domain for effective initiation of signaling pathways. With TLR1 endogenously present, transfection of TLR2-WT protein confers responsiveness by providing both extra- and intracellular domains needed for ligand recognition and signal activation respectively.

    Article Snippet: Transfection and reporter gene assays in HEK 293-CD14 cells HEK293 cells (CRL-1573; American Type Culture Collection) stably expressing human CD14 (HEK293-CD14) were cloned as described previously ( ).

    Techniques: Activation Assay, Transfection, Luciferase, Activity Assay, Positive Control

    TLR2 and TLR1 mutants inhibit NF κ B activation in HEK 293 cells in stimulation with araLAM and zymosan. (A) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by a single point mutated TLR2 construct, TLR2-P681H. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-P681H (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. Luciferase activity is expressed in normalized RLU as the ratio of NFκB-dependent firefly luciferase activity to NFκB-independent renilla luciferase activity. Data shown are the mean ± SD of triplicate wells. (B) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a TLR2 mutant missing the TIR domain TLR2-ΔTIR. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-ΔTIR (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (C) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a cytoplasmic deletion mutant of TLR1, TLR1-Δcyt. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR1-Δcyt (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control.

    Journal: The Journal of Cell Biology

    Article Title: Importance of extra- and intracellular domains of TLR1 and TLR2 in NF?B signaling

    doi: 10.1083/jcb.200304093

    Figure Lengend Snippet: TLR2 and TLR1 mutants inhibit NF κ B activation in HEK 293 cells in stimulation with araLAM and zymosan. (A) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by a single point mutated TLR2 construct, TLR2-P681H. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-P681H (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. Luciferase activity is expressed in normalized RLU as the ratio of NFκB-dependent firefly luciferase activity to NFκB-independent renilla luciferase activity. Data shown are the mean ± SD of triplicate wells. (B) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a TLR2 mutant missing the TIR domain TLR2-ΔTIR. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-ΔTIR (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (C) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a cytoplasmic deletion mutant of TLR1, TLR1-Δcyt. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR1-Δcyt (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control.

    Article Snippet: Transfection and reporter gene assays in HEK 293-CD14 cells HEK293 cells (CRL-1573; American Type Culture Collection) stably expressing human CD14 (HEK293-CD14) were cloned as described previously ( ).

    Techniques: Activation Assay, Construct, Luciferase, Activity Assay, Positive Control, Expressing, Mutagenesis

    NF κ B signal activation requires both extracellular and intracellular domains of TLR1 and TLR2. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1] DNA encoding chimeric proteins or wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam 3 CSK 4 , or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of cotransfection of TLR [1–2] and TLR [2–1] chimeric proteins. Co-transfection of both chimeric proteins confers responsiveness as a result of concomitant expression of both intracellular and extracellular domains of TLR1 and TLR2. With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.

    Journal: The Journal of Cell Biology

    Article Title: Importance of extra- and intracellular domains of TLR1 and TLR2 in NF?B signaling

    doi: 10.1083/jcb.200304093

    Figure Lengend Snippet: NF κ B signal activation requires both extracellular and intracellular domains of TLR1 and TLR2. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1] DNA encoding chimeric proteins or wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam 3 CSK 4 , or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of cotransfection of TLR [1–2] and TLR [2–1] chimeric proteins. Co-transfection of both chimeric proteins confers responsiveness as a result of concomitant expression of both intracellular and extracellular domains of TLR1 and TLR2. With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.

    Article Snippet: Transfection and reporter gene assays in HEK 293-CD14 cells HEK293 cells (CRL-1573; American Type Culture Collection) stably expressing human CD14 (HEK293-CD14) were cloned as described previously ( ).

    Techniques: Activation Assay, Luciferase, Activity Assay, Positive Control, Cotransfection, Expressing, Transfection

    PAL in HEK293 cells and mouse pancreas. A , representative PAL gel in HEK293 WCL. Band numbering was kept consistent with similar sized bands observed in SKBR3 cells. B , ratio of densitometry of the indicated bands ( bands 1–12 ) from multiple independent

    Journal: The Journal of Biological Chemistry

    Article Title: Photoaffinity Labeling of Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Targets in Mammalian Cells *Photoaffinity Labeling of Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Targets in Mammalian Cells * ♦

    doi: 10.1074/jbc.M111.305813

    Figure Lengend Snippet: PAL in HEK293 cells and mouse pancreas. A , representative PAL gel in HEK293 WCL. Band numbering was kept consistent with similar sized bands observed in SKBR3 cells. B , ratio of densitometry of the indicated bands ( bands 1–12 ) from multiple independent

    Article Snippet: SKBR3 (human breast carcinoma) and HEK293 (human embryonic kidney) cells were from ATCC.

    Techniques:

    AZ7914 ( A ), AZ4237 ( B ), AZ1395 ( C ) and Zn 2+ ( D ) concentration response curves in back-scattering Interferometry binding assays. Compounds were incubated with membranes from HEK293s-hGPR39 and HEK293s cells. AZ7914, AZ4237 and AZ1395 were incubated in the presence of 5 μM ZnCl 2 . Signals from HEK293s membranes (reference curves) were subtracted from HEK293s-hGPR39 membrane signals at each concentration point to derive GPR39 specific binding (difference curves). Values shown are means ± SD of three (AZ7914, AZ4237 and AZ1395) or four (Zn 2+ ) independent experiments. One-site binding models were used to calculate K D values for AZ7914, AZ4237 and AZ1395 summarized in Table 1 . For Zn 2+ , data was fitted to a two-site binding model.

    Journal: PLoS ONE

    Article Title: Novel Zn2+ Modulated GPR39 Receptor Agonists Do Not Drive Acute Insulin Secretion in Rodents

    doi: 10.1371/journal.pone.0145849

    Figure Lengend Snippet: AZ7914 ( A ), AZ4237 ( B ), AZ1395 ( C ) and Zn 2+ ( D ) concentration response curves in back-scattering Interferometry binding assays. Compounds were incubated with membranes from HEK293s-hGPR39 and HEK293s cells. AZ7914, AZ4237 and AZ1395 were incubated in the presence of 5 μM ZnCl 2 . Signals from HEK293s membranes (reference curves) were subtracted from HEK293s-hGPR39 membrane signals at each concentration point to derive GPR39 specific binding (difference curves). Values shown are means ± SD of three (AZ7914, AZ4237 and AZ1395) or four (Zn 2+ ) independent experiments. One-site binding models were used to calculate K D values for AZ7914, AZ4237 and AZ1395 summarized in Table 1 . For Zn 2+ , data was fitted to a two-site binding model.

    Article Snippet: The resulting expression vectors hGPR39-, rGPR39- and mGPR39-pIRESneo3 were transfected into HEK293s cells (ATCC CRL-1573) using Lipofectamine 2000 (Life Technologies).

    Techniques: Concentration Assay, Binding Assay, Incubation

    AZ7914 ( A ), AZ4237 ( B ) and AZ1395 ( C ) ten point concentration response curves for in vitro screens. Concentration responses of compounds were measured with DMR, IP 1 and cAMP assays in the absence (-) or presence of 5 μM Zn 2+ (+) as indicated in the figure. Lines represent fits to Eq 1 . Cells employed were HEK293s-hGPR39 (hGPR39), untransfected HEK293s (HEK), and NIT-1 cells endogenously expressing GPR39. Responses were normalized to AZ1395 in each assay and presented as % effect of control. For HEK293s and HEK293s-hGPR39 cells, individual DMR assays were run in singlicates and IP 1 and cAMP assays were run in triplicates. Values shown are means ± SEM of typically three (two to five) independent experiments. For NIT-1 cells, IP 1 assays were typically run in duplicates (one to four experiments), and values are shown as means with error bars representing range. EC 50 values calculated from the data in Fig 2 are summarized in Table 1 .

    Journal: PLoS ONE

    Article Title: Novel Zn2+ Modulated GPR39 Receptor Agonists Do Not Drive Acute Insulin Secretion in Rodents

    doi: 10.1371/journal.pone.0145849

    Figure Lengend Snippet: AZ7914 ( A ), AZ4237 ( B ) and AZ1395 ( C ) ten point concentration response curves for in vitro screens. Concentration responses of compounds were measured with DMR, IP 1 and cAMP assays in the absence (-) or presence of 5 μM Zn 2+ (+) as indicated in the figure. Lines represent fits to Eq 1 . Cells employed were HEK293s-hGPR39 (hGPR39), untransfected HEK293s (HEK), and NIT-1 cells endogenously expressing GPR39. Responses were normalized to AZ1395 in each assay and presented as % effect of control. For HEK293s and HEK293s-hGPR39 cells, individual DMR assays were run in singlicates and IP 1 and cAMP assays were run in triplicates. Values shown are means ± SEM of typically three (two to five) independent experiments. For NIT-1 cells, IP 1 assays were typically run in duplicates (one to four experiments), and values are shown as means with error bars representing range. EC 50 values calculated from the data in Fig 2 are summarized in Table 1 .

    Article Snippet: The resulting expression vectors hGPR39-, rGPR39- and mGPR39-pIRESneo3 were transfected into HEK293s cells (ATCC CRL-1573) using Lipofectamine 2000 (Life Technologies).

    Techniques: Concentration Assay, In Vitro, Expressing

    TRPM1 colocalizes with the ER in heterologous cells. A , B , HEK293 ( A ) or CHO ( B ) cells were transiently transfected with 1D4 epitope-tagged ( A ) or untagged ( B ) TRPM1 and Emerald-Sec61 (green). Cells were labeled with 1D4 ( A ) or TRPM1 antibody ( B ; magenta). C , CHO cells were transiently transfected with HA-tagged TRPM1, and labeled with HA (magenta) and BiP (green) antibodies.

    Journal: eNeuro

    Article Title: A Large Endoplasmic Reticulum-Resident Pool of TRPM1 in Retinal ON-Bipolar Cells

    doi: 10.1523/ENEURO.0143-18.2018

    Figure Lengend Snippet: TRPM1 colocalizes with the ER in heterologous cells. A , B , HEK293 ( A ) or CHO ( B ) cells were transiently transfected with 1D4 epitope-tagged ( A ) or untagged ( B ) TRPM1 and Emerald-Sec61 (green). Cells were labeled with 1D4 ( A ) or TRPM1 antibody ( B ; magenta). C , CHO cells were transiently transfected with HA-tagged TRPM1, and labeled with HA (magenta) and BiP (green) antibodies.

    Article Snippet: Cells Human embryonic kidney (HEK293) cells were obtained from the ATCC (#CRL-1573, RRID: CVCL_0045 ) and maintained in DMEM (Corning) supplemented with 10% FBS (HyClone or Sigma).

    Techniques: Transfection, Labeling

    RNF114 increases the stability of both I κ B α and A20. ( a ) HEK293 cells were transfected with different amounts of AU5-RNF114. Western blot analysis with anti-I κ B α , -A20, -AU5 and -GAPDH (charge control) antibodies are presented. Graphs representing the relative amounts of I κ B α or A20 versus the charge control (GAPDH) of the western blots are shown. Representative result of different experiments is presented. ( b ) HEK293 cells were transfected with different amounts of RNF114 and treated for the indicated times with CHX (10 μ g/ml). A representative graph of three independent experiments is shown. ( c ) RNF114-specific gene silencing by lentivirus-mediated shRNA in Jurkat T cells. (Left panel) The percentage of infected cells was quantified by flow cytometry. (Right panel) Levels of protein expression were analyzed by western blot with the indicated antibodies. ( d ) Analysis of RNF114, A20 and I κ B α protein expressions in Jurkat T cells transduced with shControl or shRNF1 and 2. Western blot analysis with RNF114, A20 and I κ B α antibodies are presented as well as a corresponding quantification. Representative result of different experiments is presented

    Journal: Cell Death & Disease

    Article Title: The RING ubiquitin E3 RNF114 interacts with A20 and modulates NF-κB activity and T-cell activation

    doi: 10.1038/cddis.2014.366

    Figure Lengend Snippet: RNF114 increases the stability of both I κ B α and A20. ( a ) HEK293 cells were transfected with different amounts of AU5-RNF114. Western blot analysis with anti-I κ B α , -A20, -AU5 and -GAPDH (charge control) antibodies are presented. Graphs representing the relative amounts of I κ B α or A20 versus the charge control (GAPDH) of the western blots are shown. Representative result of different experiments is presented. ( b ) HEK293 cells were transfected with different amounts of RNF114 and treated for the indicated times with CHX (10 μ g/ml). A representative graph of three independent experiments is shown. ( c ) RNF114-specific gene silencing by lentivirus-mediated shRNA in Jurkat T cells. (Left panel) The percentage of infected cells was quantified by flow cytometry. (Right panel) Levels of protein expression were analyzed by western blot with the indicated antibodies. ( d ) Analysis of RNF114, A20 and I κ B α protein expressions in Jurkat T cells transduced with shControl or shRNF1 and 2. Western blot analysis with RNF114, A20 and I κ B α antibodies are presented as well as a corresponding quantification. Representative result of different experiments is presented

    Article Snippet: Cell culture and cell-based assays HEK293 cells (ATCC, Manassas, VA, USA) were grown in DMEM (Gibco, Life Technologies, Carlsbad, CA, USA) and Jurkat cells (ATCC) in RPMI (Gibco), all supplemented with 10% FBS and antibiotics.

    Techniques: Transfection, Western Blot, shRNA, Infection, Flow Cytometry, Cytometry, Expressing, Transduction

    RNF114 is involved in the regulation of NF- κ B activity. ( a ) Luciferase assay using the 3- κ B enhancer ConA-luciferase plasmid (3-EnhConA) in the presence of the indicated vectors in HEK293 cells stimulated or not with TNF α (5 h). The graph represents the mean of three independent experiments done in triplicate. Levels of protein expression were analyzed by western blot with the indicated antibodies. ( b ) Luciferase assay using the 3-EnhConA reporter plasmids in the presence of the indicated vectors in Jurkat T cells stimulated for 5 h or not with TNF α or with the anti-CD3/CD28 antibodies. The graph represents the mean of three independent experiments done in triplicate. Levels of protein expression were analyzed by western blot with the indicated antibodies. ( c ) Luciferase assay using the 3-EnhConA reporter plasmids in Jurkat T cells stably transduced with shcontrol, shRNF1 or shRNF2 and stimulated or not with anti-CD3/CD28 antibodies (left panel) or with the TNF α (right panel). Each graph represents the mean of three independent experiments done in triplicate

    Journal: Cell Death & Disease

    Article Title: The RING ubiquitin E3 RNF114 interacts with A20 and modulates NF-κB activity and T-cell activation

    doi: 10.1038/cddis.2014.366

    Figure Lengend Snippet: RNF114 is involved in the regulation of NF- κ B activity. ( a ) Luciferase assay using the 3- κ B enhancer ConA-luciferase plasmid (3-EnhConA) in the presence of the indicated vectors in HEK293 cells stimulated or not with TNF α (5 h). The graph represents the mean of three independent experiments done in triplicate. Levels of protein expression were analyzed by western blot with the indicated antibodies. ( b ) Luciferase assay using the 3-EnhConA reporter plasmids in the presence of the indicated vectors in Jurkat T cells stimulated for 5 h or not with TNF α or with the anti-CD3/CD28 antibodies. The graph represents the mean of three independent experiments done in triplicate. Levels of protein expression were analyzed by western blot with the indicated antibodies. ( c ) Luciferase assay using the 3-EnhConA reporter plasmids in Jurkat T cells stably transduced with shcontrol, shRNF1 or shRNF2 and stimulated or not with anti-CD3/CD28 antibodies (left panel) or with the TNF α (right panel). Each graph represents the mean of three independent experiments done in triplicate

    Article Snippet: Cell culture and cell-based assays HEK293 cells (ATCC, Manassas, VA, USA) were grown in DMEM (Gibco, Life Technologies, Carlsbad, CA, USA) and Jurkat cells (ATCC) in RPMI (Gibco), all supplemented with 10% FBS and antibiotics.

    Techniques: Activity Assay, Luciferase, Plasmid Preparation, Expressing, Western Blot, Stable Transfection, Transduction

    Effect of RNF114 on A20 ubiquitylation. ( a ) HEK293 cells were transfected with FLAG-A20WT in the presence or not of His6-ubiquitin and AU5-RNF114 as indicated. His 6 -ubiquitylated proteins were purified using denaturing conditions and Ni 2+ chromatography. EV, empty vector; UB, His 6 -ubiquitin. ( b ) HEK293 cells were transfected with FLAG-A20WT in the presence or not of His6-ubiquitin and different amounts of AU5-RNF114 as indicated. Cells were stimulated for 30 min or not with PMA and ionomycin. His 6 -ubiquitylated proteins were purified using denaturing conditions and Ni 2+ chromatography

    Journal: Cell Death & Disease

    Article Title: The RING ubiquitin E3 RNF114 interacts with A20 and modulates NF-κB activity and T-cell activation

    doi: 10.1038/cddis.2014.366

    Figure Lengend Snippet: Effect of RNF114 on A20 ubiquitylation. ( a ) HEK293 cells were transfected with FLAG-A20WT in the presence or not of His6-ubiquitin and AU5-RNF114 as indicated. His 6 -ubiquitylated proteins were purified using denaturing conditions and Ni 2+ chromatography. EV, empty vector; UB, His 6 -ubiquitin. ( b ) HEK293 cells were transfected with FLAG-A20WT in the presence or not of His6-ubiquitin and different amounts of AU5-RNF114 as indicated. Cells were stimulated for 30 min or not with PMA and ionomycin. His 6 -ubiquitylated proteins were purified using denaturing conditions and Ni 2+ chromatography

    Article Snippet: Cell culture and cell-based assays HEK293 cells (ATCC, Manassas, VA, USA) were grown in DMEM (Gibco, Life Technologies, Carlsbad, CA, USA) and Jurkat cells (ATCC) in RPMI (Gibco), all supplemented with 10% FBS and antibiotics.

    Techniques: Transfection, Purification, Chromatography, Plasmid Preparation

    RNF114/ZNF313 interacts with A20. ( a ) Pull-down experiment using GST-A20 or GST fusion proteins and lysates of HEK293 cells transfected with FLAG-A20WT or FLAG-RNF114 is shown (* indicates unspecific band). ( b ) HEK293 cells were transfected with FLAG-A20WT and when indicated with AU5-RNF114. AU5-RNF114 immunoprecipitation was used to confirm the interaction with FLAG-A20. ( c ) HEK293 cells were transfected with AU5-RNF114 and FLAG-A20WT as indicated. Cells were treated with TNF α for 20 min and lysates were submitted to anti-AU5 immunoprecipitation ( d ) HEK293 cells were transfected with different forms of FLAG-A20 (WT, N-terminal: 1–390, C-terminal: 390–790) and AU5-RNF114 to determine which domains were involved in the interaction between A20 and RNF114. ( e ) Different constructs of A20 were prepared to define its interaction domain with RNF114. Results of immunoprecipitation experiments are shown. T he symbol ‘−' indicates no interaction and ‘+' indicates interaction

    Journal: Cell Death & Disease

    Article Title: The RING ubiquitin E3 RNF114 interacts with A20 and modulates NF-κB activity and T-cell activation

    doi: 10.1038/cddis.2014.366

    Figure Lengend Snippet: RNF114/ZNF313 interacts with A20. ( a ) Pull-down experiment using GST-A20 or GST fusion proteins and lysates of HEK293 cells transfected with FLAG-A20WT or FLAG-RNF114 is shown (* indicates unspecific band). ( b ) HEK293 cells were transfected with FLAG-A20WT and when indicated with AU5-RNF114. AU5-RNF114 immunoprecipitation was used to confirm the interaction with FLAG-A20. ( c ) HEK293 cells were transfected with AU5-RNF114 and FLAG-A20WT as indicated. Cells were treated with TNF α for 20 min and lysates were submitted to anti-AU5 immunoprecipitation ( d ) HEK293 cells were transfected with different forms of FLAG-A20 (WT, N-terminal: 1–390, C-terminal: 390–790) and AU5-RNF114 to determine which domains were involved in the interaction between A20 and RNF114. ( e ) Different constructs of A20 were prepared to define its interaction domain with RNF114. Results of immunoprecipitation experiments are shown. T he symbol ‘−' indicates no interaction and ‘+' indicates interaction

    Article Snippet: Cell culture and cell-based assays HEK293 cells (ATCC, Manassas, VA, USA) were grown in DMEM (Gibco, Life Technologies, Carlsbad, CA, USA) and Jurkat cells (ATCC) in RPMI (Gibco), all supplemented with 10% FBS and antibiotics.

    Techniques: Transfection, Immunoprecipitation, Construct

    Identification of RhoB and MLC2 as direct targets of miR-223. Luciferase reporter gene assay on the interaction between miR-223 and 3′-UTR of RhoB ( A ) and MLC2 ( B ) in HEK293 cells. Upper panel: putative miR-223 binding sites in the 3′-UTR of RhoB or MLC2 along with the mutation sites. TS1,target site 1 (a pooly conserved binding site); TS2, target site 2 (a conserved binding site); Bottom panel: 3′-UTR luciferase reporter assay with targets and their mutant along with miR-223/miR-Con overexpressing vectors. Bar charts of luciferase reporter analysis represent means ± SE (n = 3), firefly luciferase activity was normalized to renilla luciferase activity. ( C ) Active RhoB proteins in miR-223 or miRNA control overexpressing PASMC were pulled down by rhotekin-RBD beads and detected by western blotting analysis using anti-RhoB antibody (n = 3). ( D ) Expression of RhoB/Rho-kinase signaling pathway related components in PASMC, with overexpression or inhibition of miR-223, was measured by western blotting (n = 3). ( E ) Expression of RhoB, MLC2 and PCNA in lungs of hypoxia-exposed rats administered agomirs miR-223/miR-Con was determined by western blotting (n = 3). β-actin was used as a loading control for the above western blot analysis. Two representative blots are shown. * p

    Journal: Scientific Reports

    Article Title: MicroRNA-223 Attenuates Hypoxia-induced Vascular Remodeling by Targeting RhoB/MLC2 in Pulmonary Arterial Smooth Muscle Cells

    doi: 10.1038/srep24900

    Figure Lengend Snippet: Identification of RhoB and MLC2 as direct targets of miR-223. Luciferase reporter gene assay on the interaction between miR-223 and 3′-UTR of RhoB ( A ) and MLC2 ( B ) in HEK293 cells. Upper panel: putative miR-223 binding sites in the 3′-UTR of RhoB or MLC2 along with the mutation sites. TS1,target site 1 (a pooly conserved binding site); TS2, target site 2 (a conserved binding site); Bottom panel: 3′-UTR luciferase reporter assay with targets and their mutant along with miR-223/miR-Con overexpressing vectors. Bar charts of luciferase reporter analysis represent means ± SE (n = 3), firefly luciferase activity was normalized to renilla luciferase activity. ( C ) Active RhoB proteins in miR-223 or miRNA control overexpressing PASMC were pulled down by rhotekin-RBD beads and detected by western blotting analysis using anti-RhoB antibody (n = 3). ( D ) Expression of RhoB/Rho-kinase signaling pathway related components in PASMC, with overexpression or inhibition of miR-223, was measured by western blotting (n = 3). ( E ) Expression of RhoB, MLC2 and PCNA in lungs of hypoxia-exposed rats administered agomirs miR-223/miR-Con was determined by western blotting (n = 3). β-actin was used as a loading control for the above western blot analysis. Two representative blots are shown. * p

    Article Snippet: Cell culture Human HEK293 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

    Techniques: Luciferase, Reporter Gene Assay, Binding Assay, Mutagenesis, Reporter Assay, Activity Assay, Western Blot, Expressing, Over Expression, Inhibition

    Caspase cleavage and LC3B-I/II turnover. A ) CCRF-CEM cells treated with drug/fatty acid vehicle (C), sphinganine (1 µM, S), or sphinganine (1 µM) + GT-11 (0.5 µM) (SG), were supplemented with the indicated fatty acids (5 µM). After 12 hours, total proteins were extracted and procaspase-3 (35 kb), activated caspase-3 (17/19 kb), and LCB3-I/II (14/16 kb), were detected by immunoblotting. β-Actin served as loading control. Treatment with pan-Bcl-2 inhibitor, ABT-737 (1 µM, A), and LC3B-transfected, HEK-293 cell lysate, were used as positive controls. C22:1-fatty acid was used as a negative control for C22:0-fatty acid. Lanes rearranged to ease interpretation. Data representative of three separate experiments are shown. B ) Assessment of LC3B-II flux. CCRF-CEM cells were pretreated with or without protease inhibitors (Pepstatin-A and E64d) and treated as described. After 12 hours, total proteins were extracted and LC3B-I/II analyzed by immunoblotting. Data representative of three separate experiments are shown, except for C22:1-fatty acid.

    Journal: PLoS ONE

    Article Title: C22:0- and C24:0-dihydroceramides Confer Mixed Cytotoxicity in T-Cell Acute Lymphoblastic Leukemia Cell Lines

    doi: 10.1371/journal.pone.0074768

    Figure Lengend Snippet: Caspase cleavage and LC3B-I/II turnover. A ) CCRF-CEM cells treated with drug/fatty acid vehicle (C), sphinganine (1 µM, S), or sphinganine (1 µM) + GT-11 (0.5 µM) (SG), were supplemented with the indicated fatty acids (5 µM). After 12 hours, total proteins were extracted and procaspase-3 (35 kb), activated caspase-3 (17/19 kb), and LCB3-I/II (14/16 kb), were detected by immunoblotting. β-Actin served as loading control. Treatment with pan-Bcl-2 inhibitor, ABT-737 (1 µM, A), and LC3B-transfected, HEK-293 cell lysate, were used as positive controls. C22:1-fatty acid was used as a negative control for C22:0-fatty acid. Lanes rearranged to ease interpretation. Data representative of three separate experiments are shown. B ) Assessment of LC3B-II flux. CCRF-CEM cells were pretreated with or without protease inhibitors (Pepstatin-A and E64d) and treated as described. After 12 hours, total proteins were extracted and LC3B-I/II analyzed by immunoblotting. Data representative of three separate experiments are shown, except for C22:1-fatty acid.

    Article Snippet: LC3B-transfected, HEK-293 cell lysate was included as a positive control (Novus Biologicals, Littleton, CO).

    Techniques: Transfection, Negative Control

    Identification, recombinant expression, purification and nick sealing activity of PfLigI. Endogenous expression of PfLigI ( A ) 25 μg of uninfected erythrocyte lysate (E) and stage specific parasite lysates (R, T and S for ring, trophozoite and schizont stages, respectively) were analyzed by Western blot using anti-PfLigI affinity purified antibody at 1:2500. 0.1 μg of PfLigI was used as a control (+). PfLigI-FLAG ® was expressed in HEK-293 cells and purified using an anti-FLAG ® affinity column. ( B ) upper panel; Coomassie stained, 10% SDS-PAGE gel of FLAG ® column fractions. Lane 1, molecular weight standards; lanes 2–4, pre-column, flow-through and wash fractions, respectively. Lane 5, 3μg of FLAG ® column protein eluate. Western blot of FLAG ® column fractions (lower panel). Fractions are identical to the Coomassie gel with the exception that 0.5 μg of protein was loaded in lane 5. Anti-FLAG ® -M2 monoclonal was used at a 1:5000 dilution and the Western blot was visualized using 3,3′-diaminobenzadine. ( C ) Western blot of FLAG ® column fractions using anti-human ligase I antibody at 1:1000 dilution. Gel was loaded as described in B , upper panel. ( D ) Schematic representation of the nicked DNA substrate utilized for ligation activity assays. Star denotes the location of the 5′- 32 P radiolabel; OH represents the 3′-OH end of the nick and P represents the 5′-phosphate end of the nick. ( E ) Nick sealing activity of FLAG ® purified PfLigI. Ligation reactions (20 μl) containing 67 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 5 mM DTT, 1mM ATP and 1 pmol of nicked substrate were incubated for 30 min at 37°C. Lane 1, no enzyme; Lanes 2 and 3, PfLigI (0.1 and 1 pmol, respectively) and Lane 4, 1 pmol T4 DNA ligase. Upper bands represent ligated 44-mer and lower bands represent unligated radiolabeled 26-mer. Samples were separated on a 20% denaturing polyacrylamide gel.

    Journal: Molecular and biochemical parasitology

    Article Title: Expression and Biochemical Characterization of Plasmodium falciparum DNA Ligase I

    doi: 10.1016/j.molbiopara.2007.06.011

    Figure Lengend Snippet: Identification, recombinant expression, purification and nick sealing activity of PfLigI. Endogenous expression of PfLigI ( A ) 25 μg of uninfected erythrocyte lysate (E) and stage specific parasite lysates (R, T and S for ring, trophozoite and schizont stages, respectively) were analyzed by Western blot using anti-PfLigI affinity purified antibody at 1:2500. 0.1 μg of PfLigI was used as a control (+). PfLigI-FLAG ® was expressed in HEK-293 cells and purified using an anti-FLAG ® affinity column. ( B ) upper panel; Coomassie stained, 10% SDS-PAGE gel of FLAG ® column fractions. Lane 1, molecular weight standards; lanes 2–4, pre-column, flow-through and wash fractions, respectively. Lane 5, 3μg of FLAG ® column protein eluate. Western blot of FLAG ® column fractions (lower panel). Fractions are identical to the Coomassie gel with the exception that 0.5 μg of protein was loaded in lane 5. Anti-FLAG ® -M2 monoclonal was used at a 1:5000 dilution and the Western blot was visualized using 3,3′-diaminobenzadine. ( C ) Western blot of FLAG ® column fractions using anti-human ligase I antibody at 1:1000 dilution. Gel was loaded as described in B , upper panel. ( D ) Schematic representation of the nicked DNA substrate utilized for ligation activity assays. Star denotes the location of the 5′- 32 P radiolabel; OH represents the 3′-OH end of the nick and P represents the 5′-phosphate end of the nick. ( E ) Nick sealing activity of FLAG ® purified PfLigI. Ligation reactions (20 μl) containing 67 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 5 mM DTT, 1mM ATP and 1 pmol of nicked substrate were incubated for 30 min at 37°C. Lane 1, no enzyme; Lanes 2 and 3, PfLigI (0.1 and 1 pmol, respectively) and Lane 4, 1 pmol T4 DNA ligase. Upper bands represent ligated 44-mer and lower bands represent unligated radiolabeled 26-mer. Samples were separated on a 20% denaturing polyacrylamide gel.

    Article Snippet: Since HEK 293 cells contain endogenous ligase I, column fractions were also probed with a human DNA ligase I antibody (IMGENEX, Ca.), generated towards the N-terminal, non-homologous region of the protein.

    Techniques: Recombinant, Expressing, Purification, Activity Assay, Western Blot, Affinity Purification, Affinity Column, Staining, SDS Page, Molecular Weight, Flow Cytometry, Ligation, Incubation

    Endogenously-expressed human TLR5 in HEK293 cells mediate IL-8 responses to Flagellin and T. gondii- derived profilin

    Journal: Journal of innate immunity

    Article Title: Toxoplasma gondii-derived profilin triggers Human TLR5-dependent cytokine production

    doi: 10.1159/000362367

    Figure Lengend Snippet: Endogenously-expressed human TLR5 in HEK293 cells mediate IL-8 responses to Flagellin and T. gondii- derived profilin

    Article Snippet: HEK293 cells were purchased from ATCC (CRL-1573.3) and grown in 10% FCS RPMI medium.

    Techniques: Derivative Assay