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pdk1 overexpression construct  (OriGene)


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    Structured Review

    OriGene pdk1 overexpression construct
    Pdk1 Overexpression Construct, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+pdk1/PDK1+(NM_002610)+Human+Tagged+ORF+Clone/pm40086584-70-3-0
    Average 90 stars, based on 4 article reviews
    pdk1 overexpression construct - by Bioz Stars, 2026-09
    90/100 stars

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    Related Articles

    Transfection:

    Article Title: Overexpression of Pyruvate Dehydrogenase Kinase 1 and Lactate Dehydrogenase A in Nerve Cells Confers Resistance to Amyloid ? and Other Toxins by Decreasing Mitochondrial Respiration and Reactive Oxygen Species Production
    Article Snippet: For stable expression of PDK1 and LDHA, B12 cells were transfected with Lipofectamine (Invitrogen) according to the manufacturer's directions. .. Cells were plated at a density to achieve 70–80% confluency and transfected with vectors containing human PDK1 (pCMV6-AC) or LDHA (pCMV6-XL4) cDNA (Origene, Rockville, MD). ..

    Article Title: Overexpression of Pyruvate Dehydrogenase Kinase 1 and Lactate Dehydrogenase A in Nerve Cells Confers Resistance to Amyloid β and Other Toxins by Decreasing Mitochondrial Respiration and Reactive Oxygen Species Production
    Article Snippet: Derivation of PDK1- and LDHA-overexpressing Cell Lines— For stable expression of PDK1 and LDHA, B12 cells were transfected with Lipofectamine (Invitrogen) according to the manufacturer’s directions. .. Cells were plated at a density to achieve 70–80% confluency and transfected with vectors containing human PDK1 (pCMV6-AC) or LDHA (pCMV6-XL4) cDNA (Origene, Rockville, MD). ..

    Expressing:

    Article Title: Discovery of a novel class of AKT pleckstrin homology domain inhibitors
    Article Snippet: .. Expression and Purification of Recombinant PH Domains Recombinant mouse AKT1 PH domain amino acids 1 to 111 (UBI/Millipore), human AKT1 PH domain amino acids 1 to 111 (Origene, NM005163.2), human IRS1 PH domain amino acids 12 to 133 (Invitrogen), and human PDK1 PH domain amino acids 407 to 549 (Origene, NM002613.3) were cloned by PCR into EcoRI/XhoI sites in pGEX-4T1 inducible bacterial expression plasmid (GeneStorm, InVitrogen) transformed into BL21(DE3) Escherichia coli . .. Surface Plasmon Resonance Spectroscopy Binding Assays All interaction analyses were done with a Biacore 2000 Control Software v3.2, and BIAevaluation v4.1 analysis software (Biacore).

    Purification:

    Article Title: Discovery of a novel class of AKT pleckstrin homology domain inhibitors
    Article Snippet: .. Expression and Purification of Recombinant PH Domains Recombinant mouse AKT1 PH domain amino acids 1 to 111 (UBI/Millipore), human AKT1 PH domain amino acids 1 to 111 (Origene, NM005163.2), human IRS1 PH domain amino acids 12 to 133 (Invitrogen), and human PDK1 PH domain amino acids 407 to 549 (Origene, NM002613.3) were cloned by PCR into EcoRI/XhoI sites in pGEX-4T1 inducible bacterial expression plasmid (GeneStorm, InVitrogen) transformed into BL21(DE3) Escherichia coli . .. Surface Plasmon Resonance Spectroscopy Binding Assays All interaction analyses were done with a Biacore 2000 Control Software v3.2, and BIAevaluation v4.1 analysis software (Biacore).

    Recombinant:

    Article Title: Discovery of a novel class of AKT pleckstrin homology domain inhibitors
    Article Snippet: .. Expression and Purification of Recombinant PH Domains Recombinant mouse AKT1 PH domain amino acids 1 to 111 (UBI/Millipore), human AKT1 PH domain amino acids 1 to 111 (Origene, NM005163.2), human IRS1 PH domain amino acids 12 to 133 (Invitrogen), and human PDK1 PH domain amino acids 407 to 549 (Origene, NM002613.3) were cloned by PCR into EcoRI/XhoI sites in pGEX-4T1 inducible bacterial expression plasmid (GeneStorm, InVitrogen) transformed into BL21(DE3) Escherichia coli . .. Surface Plasmon Resonance Spectroscopy Binding Assays All interaction analyses were done with a Biacore 2000 Control Software v3.2, and BIAevaluation v4.1 analysis software (Biacore).

    Clone Assay:

    Article Title: Discovery of a novel class of AKT pleckstrin homology domain inhibitors
    Article Snippet: .. Expression and Purification of Recombinant PH Domains Recombinant mouse AKT1 PH domain amino acids 1 to 111 (UBI/Millipore), human AKT1 PH domain amino acids 1 to 111 (Origene, NM005163.2), human IRS1 PH domain amino acids 12 to 133 (Invitrogen), and human PDK1 PH domain amino acids 407 to 549 (Origene, NM002613.3) were cloned by PCR into EcoRI/XhoI sites in pGEX-4T1 inducible bacterial expression plasmid (GeneStorm, InVitrogen) transformed into BL21(DE3) Escherichia coli . .. Surface Plasmon Resonance Spectroscopy Binding Assays All interaction analyses were done with a Biacore 2000 Control Software v3.2, and BIAevaluation v4.1 analysis software (Biacore).

    Polymerase Chain Reaction:

    Article Title: Discovery of a novel class of AKT pleckstrin homology domain inhibitors
    Article Snippet: .. Expression and Purification of Recombinant PH Domains Recombinant mouse AKT1 PH domain amino acids 1 to 111 (UBI/Millipore), human AKT1 PH domain amino acids 1 to 111 (Origene, NM005163.2), human IRS1 PH domain amino acids 12 to 133 (Invitrogen), and human PDK1 PH domain amino acids 407 to 549 (Origene, NM002613.3) were cloned by PCR into EcoRI/XhoI sites in pGEX-4T1 inducible bacterial expression plasmid (GeneStorm, InVitrogen) transformed into BL21(DE3) Escherichia coli . .. Surface Plasmon Resonance Spectroscopy Binding Assays All interaction analyses were done with a Biacore 2000 Control Software v3.2, and BIAevaluation v4.1 analysis software (Biacore).

    Plasmid Preparation:

    Article Title: Discovery of a novel class of AKT pleckstrin homology domain inhibitors
    Article Snippet: .. Expression and Purification of Recombinant PH Domains Recombinant mouse AKT1 PH domain amino acids 1 to 111 (UBI/Millipore), human AKT1 PH domain amino acids 1 to 111 (Origene, NM005163.2), human IRS1 PH domain amino acids 12 to 133 (Invitrogen), and human PDK1 PH domain amino acids 407 to 549 (Origene, NM002613.3) were cloned by PCR into EcoRI/XhoI sites in pGEX-4T1 inducible bacterial expression plasmid (GeneStorm, InVitrogen) transformed into BL21(DE3) Escherichia coli . .. Surface Plasmon Resonance Spectroscopy Binding Assays All interaction analyses were done with a Biacore 2000 Control Software v3.2, and BIAevaluation v4.1 analysis software (Biacore).

    Transformation Assay:

    Article Title: Discovery of a novel class of AKT pleckstrin homology domain inhibitors
    Article Snippet: .. Expression and Purification of Recombinant PH Domains Recombinant mouse AKT1 PH domain amino acids 1 to 111 (UBI/Millipore), human AKT1 PH domain amino acids 1 to 111 (Origene, NM005163.2), human IRS1 PH domain amino acids 12 to 133 (Invitrogen), and human PDK1 PH domain amino acids 407 to 549 (Origene, NM002613.3) were cloned by PCR into EcoRI/XhoI sites in pGEX-4T1 inducible bacterial expression plasmid (GeneStorm, InVitrogen) transformed into BL21(DE3) Escherichia coli . .. Surface Plasmon Resonance Spectroscopy Binding Assays All interaction analyses were done with a Biacore 2000 Control Software v3.2, and BIAevaluation v4.1 analysis software (Biacore).



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    Image Search Results


    The serum levels of PDK1, LDH and PDK1/LDH in different patients with ICU ( A – C ). The serum levels of PDK1, LDH and PDK1/LDH were measured in ICU control groups, sepsis group, and septic shock group. P<0.05 were considered statistically significant. T -test (the serum level of LDH) or Mann–Whitney U -test (the serum level of PDK1, PDK1/LDH) between the two groups. *Denotes P< 0.05.

    Journal: Journal of Inflammation Research

    Article Title: The Values of PDK1 and LDH Levels in Patients with Sepsis and Septic Shock: A Prospective Observational Study

    doi: 10.2147/JIR.S477495

    Figure Lengend Snippet: The serum levels of PDK1, LDH and PDK1/LDH in different patients with ICU ( A – C ). The serum levels of PDK1, LDH and PDK1/LDH were measured in ICU control groups, sepsis group, and septic shock group. P<0.05 were considered statistically significant. T -test (the serum level of LDH) or Mann–Whitney U -test (the serum level of PDK1, PDK1/LDH) between the two groups. *Denotes P< 0.05.

    Article Snippet: After defrosting the frozen serum, we used the human PDK1 ELISA kit (LifeSpan Biosciences, Inc., Seattle, America, Catalog # LS-F12294) to detect the concentration of PDK1, and human LDH ELISA kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China, Catalog # BC0685) to detect the level of LDH in the serum.

    Techniques: Control, MANN-WHITNEY

    Correlation between PDK1/LDH and clinical indicators in septic patients ( A ) Correlation between serum PDK1 and both ScvO 2 and lactate in septic shock group. ( B) Correlation between serum LDH and both ScvO 2 and lactate in septic shock group. ( C) Correlation between PDK1/LDH and both ScvO 2 and lactate in sepsis group. ( D) Correlation between PDK1/LDH and both ScvO 2 and lactate in septic shock group. P<0.05 were considered statistically significant. All data analyzed by Spearman rank correlation coefficient.

    Journal: Journal of Inflammation Research

    Article Title: The Values of PDK1 and LDH Levels in Patients with Sepsis and Septic Shock: A Prospective Observational Study

    doi: 10.2147/JIR.S477495

    Figure Lengend Snippet: Correlation between PDK1/LDH and clinical indicators in septic patients ( A ) Correlation between serum PDK1 and both ScvO 2 and lactate in septic shock group. ( B) Correlation between serum LDH and both ScvO 2 and lactate in septic shock group. ( C) Correlation between PDK1/LDH and both ScvO 2 and lactate in sepsis group. ( D) Correlation between PDK1/LDH and both ScvO 2 and lactate in septic shock group. P<0.05 were considered statistically significant. All data analyzed by Spearman rank correlation coefficient.

    Article Snippet: After defrosting the frozen serum, we used the human PDK1 ELISA kit (LifeSpan Biosciences, Inc., Seattle, America, Catalog # LS-F12294) to detect the concentration of PDK1, and human LDH ELISA kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China, Catalog # BC0685) to detect the level of LDH in the serum.

    Techniques:

    The serum levels of PDK1, LDH and PDK1/LDH in survival groups and no-survival group ( A–C ) The serum levels of PDK1, LDH and PDK1/LDH were measured in survival groups and no-survival group. P<0.05 was considered statistically significant. T -test (the serum level of LDH) or Mann–Whitney U -test (the serum level of PDK1, PDK1/LDH) between the two groups. *Denotes P< 0.05, **Denotes P<0.01.

    Journal: Journal of Inflammation Research

    Article Title: The Values of PDK1 and LDH Levels in Patients with Sepsis and Septic Shock: A Prospective Observational Study

    doi: 10.2147/JIR.S477495

    Figure Lengend Snippet: The serum levels of PDK1, LDH and PDK1/LDH in survival groups and no-survival group ( A–C ) The serum levels of PDK1, LDH and PDK1/LDH were measured in survival groups and no-survival group. P<0.05 was considered statistically significant. T -test (the serum level of LDH) or Mann–Whitney U -test (the serum level of PDK1, PDK1/LDH) between the two groups. *Denotes P< 0.05, **Denotes P<0.01.

    Article Snippet: After defrosting the frozen serum, we used the human PDK1 ELISA kit (LifeSpan Biosciences, Inc., Seattle, America, Catalog # LS-F12294) to detect the concentration of PDK1, and human LDH ELISA kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China, Catalog # BC0685) to detect the level of LDH in the serum.

    Techniques: MANN-WHITNEY

    AUC and Optimal Cutoff Points with Their Corresponding Validity Indexes for SOFA and Biomarkers in Relation to 28-Day Mortality of Patients with Sepsis

    Journal: Journal of Inflammation Research

    Article Title: The Values of PDK1 and LDH Levels in Patients with Sepsis and Septic Shock: A Prospective Observational Study

    doi: 10.2147/JIR.S477495

    Figure Lengend Snippet: AUC and Optimal Cutoff Points with Their Corresponding Validity Indexes for SOFA and Biomarkers in Relation to 28-Day Mortality of Patients with Sepsis

    Article Snippet: After defrosting the frozen serum, we used the human PDK1 ELISA kit (LifeSpan Biosciences, Inc., Seattle, America, Catalog # LS-F12294) to detect the concentration of PDK1, and human LDH ELISA kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China, Catalog # BC0685) to detect the level of LDH in the serum.

    Techniques:

    The ROC curves of indicators in relation to 28-day mortality of patients with sepsis. AUC was 0.713 (95% CI, 0.589–0.836, p= 0.002) for serum PDK1/LDH levels at admission in the patients with sepsis. AUC: area under the curve, ROC curve: Receiver operating characteristic curve. P<0.05 were considered statistically significant. See legend for AUC and the corresponding validity indexes of other indicators.

    Journal: Journal of Inflammation Research

    Article Title: The Values of PDK1 and LDH Levels in Patients with Sepsis and Septic Shock: A Prospective Observational Study

    doi: 10.2147/JIR.S477495

    Figure Lengend Snippet: The ROC curves of indicators in relation to 28-day mortality of patients with sepsis. AUC was 0.713 (95% CI, 0.589–0.836, p= 0.002) for serum PDK1/LDH levels at admission in the patients with sepsis. AUC: area under the curve, ROC curve: Receiver operating characteristic curve. P<0.05 were considered statistically significant. See legend for AUC and the corresponding validity indexes of other indicators.

    Article Snippet: After defrosting the frozen serum, we used the human PDK1 ELISA kit (LifeSpan Biosciences, Inc., Seattle, America, Catalog # LS-F12294) to detect the concentration of PDK1, and human LDH ELISA kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China, Catalog # BC0685) to detect the level of LDH in the serum.

    Techniques:

    Kaplan-Meier survival curves of patients with sepsis based on the PDK1/LDH cutoff value (0.1407). Significant difference was measured between the two curves (P <0.001, Log rank test).

    Journal: Journal of Inflammation Research

    Article Title: The Values of PDK1 and LDH Levels in Patients with Sepsis and Septic Shock: A Prospective Observational Study

    doi: 10.2147/JIR.S477495

    Figure Lengend Snippet: Kaplan-Meier survival curves of patients with sepsis based on the PDK1/LDH cutoff value (0.1407). Significant difference was measured between the two curves (P <0.001, Log rank test).

    Article Snippet: After defrosting the frozen serum, we used the human PDK1 ELISA kit (LifeSpan Biosciences, Inc., Seattle, America, Catalog # LS-F12294) to detect the concentration of PDK1, and human LDH ELISA kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China, Catalog # BC0685) to detect the level of LDH in the serum.

    Techniques:

    Kaplan–Meier estimator analysis of PDK/LDH for 28 day survival based on the median for PDK1/LDH (0.1808). Significant difference was measured between the two curves (P =0.004, Log rank test).

    Journal: Journal of Inflammation Research

    Article Title: The Values of PDK1 and LDH Levels in Patients with Sepsis and Septic Shock: A Prospective Observational Study

    doi: 10.2147/JIR.S477495

    Figure Lengend Snippet: Kaplan–Meier estimator analysis of PDK/LDH for 28 day survival based on the median for PDK1/LDH (0.1808). Significant difference was measured between the two curves (P =0.004, Log rank test).

    Article Snippet: After defrosting the frozen serum, we used the human PDK1 ELISA kit (LifeSpan Biosciences, Inc., Seattle, America, Catalog # LS-F12294) to detect the concentration of PDK1, and human LDH ELISA kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China, Catalog # BC0685) to detect the level of LDH in the serum.

    Techniques:

    A ) Schematic shows upstream and downstream kinases in the AKT signaling network. B ) BMDMs were primed, or not, with LPS and then stimulated with oxPAPC (100, 50 or 25 μg ml −1 ). Phosphorylation of upstream AKT regulators (TBK1, IKKε, PDK1, PTEN and PI3K) was analyzed by immunoblot. Images are representative of three independent experiments. C ) BMDMs were primed, or not, with LPS (1 μg ml −1 ) and then stimulated with oxPAPC (100, 50 or 25 μg ml −1 ). The phosphorylation of the indicated mTORC1/2-associated proteins was analyzed after 1h by immunoblotting. Data are representative of three independent experiments. D ) WT and Nfe2l2 −/− BMDMs were primed, or not, with LPS (1 μg ml −1 ) and treated or not with oxPAPC (100 μg ml −1 ) for 1h. The indicated transcripts were analyzed by qPCR. n = 3, data are representative of three independent experiments and show means ± SEM. Statistical significance was calculated using two-way ANOVA and Sidak’s multiple comparisons test. E-F ) BMDMs were primed, or not, with LPS (1 μg ml −1 ) and treated or not with 15d-PGJ 2 (20, 10 or 1 μM in E and 10 μM in F ) for 1h ( E ) or 24h ( F ). AKT phosphorylation and NRF2 accumulation were assessed by immunoblot ( E ). Data are representative of three independent experiments. IL-10 and TNF release was quantified by ELISA ( F ). n = 6, graphs are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Sidak’s test. G ) Expression of Akt isoforms (RNA-seq, Immgen.org database) in the indicated murine macrophage populations. H ) Normalized expression of Akt1 , Akt2 and Akt3 in BMDMs, analyzed by qPCR. n = 3, data are representative of three independent experiments and show means ± SEM. I, J ) Microscale thermophoresis (MST) analysis of oxPAPC interactions with AKT2 ( I ) and AKT3 ( J ). Traces of fluorescently labeled human recombinant AKT incubated with oxPAPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) or DPPC (500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM). oxPAPC-AKT binding curve was derived from the quantification of normalized fluorescence changes. n = 3, graph shows means ± SD. Images are representative of three independent experiments. K, L ) Active human recombinant AKT2 ( K ) or AKT3 ( L ) were incubated with oxPAPC and a kinase-specific FRET-peptide substrate (Z-’LYTE). Kinase inhibition was measured as the ability of lipid to block substrate phosphorylation. n = 4, graph shows means ± SD. Data are representative of three independent experiments.

    Journal: bioRxiv

    Article Title: Host-derived oxidized phospholipids initiate effector-triggered immunity fostering lethality upon microbial encounter

    doi: 10.1101/2023.11.21.568047

    Figure Lengend Snippet: A ) Schematic shows upstream and downstream kinases in the AKT signaling network. B ) BMDMs were primed, or not, with LPS and then stimulated with oxPAPC (100, 50 or 25 μg ml −1 ). Phosphorylation of upstream AKT regulators (TBK1, IKKε, PDK1, PTEN and PI3K) was analyzed by immunoblot. Images are representative of three independent experiments. C ) BMDMs were primed, or not, with LPS (1 μg ml −1 ) and then stimulated with oxPAPC (100, 50 or 25 μg ml −1 ). The phosphorylation of the indicated mTORC1/2-associated proteins was analyzed after 1h by immunoblotting. Data are representative of three independent experiments. D ) WT and Nfe2l2 −/− BMDMs were primed, or not, with LPS (1 μg ml −1 ) and treated or not with oxPAPC (100 μg ml −1 ) for 1h. The indicated transcripts were analyzed by qPCR. n = 3, data are representative of three independent experiments and show means ± SEM. Statistical significance was calculated using two-way ANOVA and Sidak’s multiple comparisons test. E-F ) BMDMs were primed, or not, with LPS (1 μg ml −1 ) and treated or not with 15d-PGJ 2 (20, 10 or 1 μM in E and 10 μM in F ) for 1h ( E ) or 24h ( F ). AKT phosphorylation and NRF2 accumulation were assessed by immunoblot ( E ). Data are representative of three independent experiments. IL-10 and TNF release was quantified by ELISA ( F ). n = 6, graphs are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Sidak’s test. G ) Expression of Akt isoforms (RNA-seq, Immgen.org database) in the indicated murine macrophage populations. H ) Normalized expression of Akt1 , Akt2 and Akt3 in BMDMs, analyzed by qPCR. n = 3, data are representative of three independent experiments and show means ± SEM. I, J ) Microscale thermophoresis (MST) analysis of oxPAPC interactions with AKT2 ( I ) and AKT3 ( J ). Traces of fluorescently labeled human recombinant AKT incubated with oxPAPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) or DPPC (500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM). oxPAPC-AKT binding curve was derived from the quantification of normalized fluorescence changes. n = 3, graph shows means ± SD. Images are representative of three independent experiments. K, L ) Active human recombinant AKT2 ( K ) or AKT3 ( L ) were incubated with oxPAPC and a kinase-specific FRET-peptide substrate (Z-’LYTE). Kinase inhibition was measured as the ability of lipid to block substrate phosphorylation. n = 4, graph shows means ± SD. Data are representative of three independent experiments.

    Article Snippet: Briefly, recombinant human AKT1 (BPS Bioscience, Cat# 40003), AKT2 (BPS Bioscience, Cat# 40011) and AKT3 (BPS Bioscience, Cat# 40012) were used at 7.5 ng per reaction and recombinant human PDK1 (BPS Bioscience, Cat# 40080) was used at 50 ng per reaction.

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, RNA Sequencing Assay, Microscale Thermophoresis, Labeling, Recombinant, Incubation, Binding Assay, Derivative Assay, Fluorescence, Inhibition, Blocking Assay

    A-E ) WT and Tlr4 −/− ( A ), Tlr2 −/− ( B ), Cd14 −/− ( C ), Cd36 −/− ( D ) or Nfe2l2 −/− ( E ) BMDMs were primed, or not, with R848 (1 μg ml −1 ) ( A, C ) or LPS (1 μg ml −1 ) ( B-E ) and treated or not with oxPAPC (100 μg ml −1 ) for 1h ( left, center ) or 24h ( right ). AKT phosphorylation ( A-E ) or NRF2 accumulation ( E ) were assessed by immunoblot ( left ). Data are representative of three independent experiments. ECAR ( center ) was measured using a Seahorse analyzer. n = 6 ( A-C, E ), n = 5 ( D ), data are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Tukey’s test. IL-10 and TNF release ( right ) was quantified by ELISA. n = 3, graphs are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Tukey’s test. F ) oxPAPC binding capacity of AKT was determined by pull down assay. Cellular lysate of 293T cells expressing HA-tagged human AKT1 was incubated with oxPAPE-N-Biotin and the indicated doses of oxPAPC. AKT associated with biotinylated lipids was captured by streptavidin beads and revealed by immunoblotting using anti-HA antibody. β-actin was used as a negative control. Data are representative of three independent experiments. G ) Microscale thermophoresis (MST) analysis of oxPAPC and AKT interactions. Traces of fluorescently labeled human recombinant AKT1 incubated with oxPAPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) or DPPC (500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) ( left and central ). oxPAPC-AKT binding curve was derived from the quantification of normalized fluorescence changes ( right ). n = 3, graph shows means ± SD. Images are representative of three independent experiments. H ) Cellular lysate of 293T cells expressing the indicated HA-tagged AKT1 truncated forms were incubated with oxPAPE-N-Biotin. HA-proteins associated with biotinylated lipid were captured by streptavidin beads and revealed by immunoblotting. Data shown are representative of three independent experiments. I ) Active human recombinant AKT1 or PDK1 were incubated with oxPAPC or DPPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, and 1.9 μM) and kinase-specific FRET-peptide substrates (Z-’LYTE). Kinase inhibition was measured as the ability of lipid to block substrate phosphorylation. n = 4, graph shows means ± SD. Data are representative of three independent experiments.

    Journal: bioRxiv

    Article Title: Host-derived oxidized phospholipids initiate effector-triggered immunity fostering lethality upon microbial encounter

    doi: 10.1101/2023.11.21.568047

    Figure Lengend Snippet: A-E ) WT and Tlr4 −/− ( A ), Tlr2 −/− ( B ), Cd14 −/− ( C ), Cd36 −/− ( D ) or Nfe2l2 −/− ( E ) BMDMs were primed, or not, with R848 (1 μg ml −1 ) ( A, C ) or LPS (1 μg ml −1 ) ( B-E ) and treated or not with oxPAPC (100 μg ml −1 ) for 1h ( left, center ) or 24h ( right ). AKT phosphorylation ( A-E ) or NRF2 accumulation ( E ) were assessed by immunoblot ( left ). Data are representative of three independent experiments. ECAR ( center ) was measured using a Seahorse analyzer. n = 6 ( A-C, E ), n = 5 ( D ), data are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Tukey’s test. IL-10 and TNF release ( right ) was quantified by ELISA. n = 3, graphs are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Tukey’s test. F ) oxPAPC binding capacity of AKT was determined by pull down assay. Cellular lysate of 293T cells expressing HA-tagged human AKT1 was incubated with oxPAPE-N-Biotin and the indicated doses of oxPAPC. AKT associated with biotinylated lipids was captured by streptavidin beads and revealed by immunoblotting using anti-HA antibody. β-actin was used as a negative control. Data are representative of three independent experiments. G ) Microscale thermophoresis (MST) analysis of oxPAPC and AKT interactions. Traces of fluorescently labeled human recombinant AKT1 incubated with oxPAPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) or DPPC (500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) ( left and central ). oxPAPC-AKT binding curve was derived from the quantification of normalized fluorescence changes ( right ). n = 3, graph shows means ± SD. Images are representative of three independent experiments. H ) Cellular lysate of 293T cells expressing the indicated HA-tagged AKT1 truncated forms were incubated with oxPAPE-N-Biotin. HA-proteins associated with biotinylated lipid were captured by streptavidin beads and revealed by immunoblotting. Data shown are representative of three independent experiments. I ) Active human recombinant AKT1 or PDK1 were incubated with oxPAPC or DPPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, and 1.9 μM) and kinase-specific FRET-peptide substrates (Z-’LYTE). Kinase inhibition was measured as the ability of lipid to block substrate phosphorylation. n = 4, graph shows means ± SD. Data are representative of three independent experiments.

    Article Snippet: Briefly, recombinant human AKT1 (BPS Bioscience, Cat# 40003), AKT2 (BPS Bioscience, Cat# 40011) and AKT3 (BPS Bioscience, Cat# 40012) were used at 7.5 ng per reaction and recombinant human PDK1 (BPS Bioscience, Cat# 40080) was used at 50 ng per reaction.

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Pull Down Assay, Expressing, Incubation, Negative Control, Microscale Thermophoresis, Labeling, Recombinant, Derivative Assay, Fluorescence, Inhibition, Blocking Assay