total akt Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 96
    Millipore total akt
    Activation of PI3K and MAPK pathways by ZnR/GPR39 in estrogen independent breast cancer cells. ( A ) Immuno-blots of phospho and total <t>AKT</t> in TAMR cells transfected with siGPR39 or siControl and treated with Zn 2+ (200 μM) or IGF-1 (100 nM) or a combination of Zn 2+ and IGF-1 or EDTA (100 μM) as control (top panel). Bottom panel shows densitometry analysis. ( B ) Immunoblot and denistometry analysis of pAKT and tAKT, as in A, of TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min) and following Zn 2+ (200 μM) application. ( C ) Immunoblot and denistometry analysis of phospho-mTOR levels in TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min) and following Zn 2+ (200 μM) application. ( D ) Immunoblot and densitometry analysis of pERK1/2, relative to total <t>ERK1/2</t> in TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min) and following Zn 2+ (200 μM) application. ( E ) Immunoblot and denistometry analysis of pERK1/2, relative to total ERK1/2 in TAMR cells transfected with siGPR39 or siControl and treated with or without Zn 2+ (200 μM). ( F ) Immunoblot and denistometry analysis of pERK1/2, relative to total ERK1/2 in BT20 cells transfected with several siGPR39 constructs or siControl and treated with or without Zn 2+ (200 μM) (n = 2 independent experiments). Densitometry quantification in the bar graphs are averages of at least n = 3 independent experiments performed in triplicates for each condition (p
    Total Akt, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/total akt/product/Millipore
    Average 96 stars, based on 217 article reviews
    Price from $9.99 to $1999.99
    total akt - by Bioz Stars, 2020-10
    96/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc total akt
    Activation of PI3K and MAPK pathways by ZnR/GPR39 in estrogen independent breast cancer cells. ( A ) Immuno-blots of phospho and total <t>AKT</t> in TAMR cells transfected with siGPR39 or siControl and treated with Zn 2+ (200 μM) or IGF-1 (100 nM) or a combination of Zn 2+ and IGF-1 or EDTA (100 μM) as control (top panel). Bottom panel shows densitometry analysis. ( B ) Immunoblot and denistometry analysis of pAKT and tAKT, as in A, of TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min) and following Zn 2+ (200 μM) application. ( C ) Immunoblot and denistometry analysis of phospho-mTOR levels in TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min) and following Zn 2+ (200 μM) application. ( D ) Immunoblot and densitometry analysis of pERK1/2, relative to total <t>ERK1/2</t> in TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min) and following Zn 2+ (200 μM) application. ( E ) Immunoblot and denistometry analysis of pERK1/2, relative to total ERK1/2 in TAMR cells transfected with siGPR39 or siControl and treated with or without Zn 2+ (200 μM). ( F ) Immunoblot and denistometry analysis of pERK1/2, relative to total ERK1/2 in BT20 cells transfected with several siGPR39 constructs or siControl and treated with or without Zn 2+ (200 μM) (n = 2 independent experiments). Densitometry quantification in the bar graphs are averages of at least n = 3 independent experiments performed in triplicates for each condition (p
    Total Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 9314 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/total akt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 9314 article reviews
    Price from $9.99 to $1999.99
    total akt - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc anti total akt
    Oligomeric Aβ 1–42 specifically binds to TREM2 and activates TREM2 signaling pathway . a Schematic representation of human TREM2 extracellular domain (sTREM2, amino acid residues 1–171) tagged with human IgG1 Fc. SP: signal peptide. b The cDNA encoding sTREM2-Fc, sTREM1-Fc or Fc alone was transfected into HEK 293 T cells. Each protein was purified from the conditioned medium and analyzed by silver stained SDS-PAGE. c The prepared oAβ 1–42 peptides were analyzed by Western blotting using 4–12% Bis-Tris NuPAGE gel. d Solid phase binding assay showing the saturation binding curve and equilibrium dissociation constant (K D ) of oAβ 1–42 binding to sTREM2-Fc. Fc and sTREM1-Fc served as negative controls ( n = 3). e The Fc, sTREM2-Fc or sTREM1-Fc control was pre-bound to protein A agarose beads and used as baits for immunoprecipitation of oAβ 1–42 . The precipitated products were separated on 4–12% Bis-Tris NuPAGE gel and further subjected to Western blotting. ( f and g ) The binding profiles of oAβ 1–42 to different concentrations of sTREM2-Fc f or Fc g were generated by SPR assay. h The prepared monomeric Aβ 1–42 (mAβ 1–42 ) peptides were analyzed by Western blotting using 4–12% Bis-Tris NuPAGE gel. i The binding profiles of mAβ 1–42 to different concentrations of sTREM2-Fc were generated by SPR assay. j The binding profiles of scrambled Aβ 42 (scAβ 42 ) to different concentrations of sTREM2-Fc were generated by SPR assay. k Wild-type (WT) or Trem2 -knockout (KO) microglia were stimulated with 1.0 μM oAβ 1–42 for 1 h. Cell lysates were analyzed by Western blotting using antibodies specific for either total <t>(T-Syk)</t> or phosphorylated form (p-Syk) of Syk. l Quantification of Western blots as ratios of p-Syk/T-Syk. β-Actin was used as an internal control ( n = 3, two-way ANOVA). m WT or Trem2 -KO microglia were stimulated with 1.0 μM oAβ 1–42 for the indicated time. Cell lysates at each time point were analyzed by Western blotting using antibodies specific for either total <t>(T-Akt)</t> or phosphorylated form (p-Akt) of Akt. n Quantification of Western blots as ratios of p-Akt/T-Akt. β-Actin was used as an internal control (n = 3, two-way ANOVA). Data information: Data represent mean ± SD. **, p
    Anti Total Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1509 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti total akt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1509 article reviews
    Price from $9.99 to $1999.99
    anti total akt - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    94
    Santa Cruz Biotechnology total akt
    Role of <t>Notch1</t> and IKKα in TNBC. (A) Working hypothesis: IKKα as a central mediator of mitochondrial and nuclear non-canonical Notch signaling in TNBC. Diagram representing our working hypothesis whereby Jagged1 activates Notch1, which in turn acts through IKKα to trigger two parallel and potentially interacting non-canonical pathways: a mitochondrial pathway culminating in mTORC2-dependent <t>AKT</t> activation and increased oxidative phosphorylation and a nuclear pathway whereby IKKα binds to NF-κB-responsive elements and triggers transcriptional activation of survival genes, such as c-IAP2. Dashed arrows indicate possible secondary effects of AKT on NF-κB activation and survival gene expression, which have been documented in the literature in other systems but not explored in this study. (B) Expression of NOTCH1 and IKKα (Gene symbol (CHUK) significantly correlates with poor relapse-free survival in TNBC but not ER + breast cancers. Using the Kaplan-Meier Plotter ( http://kmplot.com/analysis/ ; 73 ) Breast Cancer 2017 dataset, the correlation between Relapse Free Survival (RFS) and expression of these two transcripts was determined. TNBC ( n = 801) and ER + tumors ( n = 2565) were analyzed separately.
    Total Akt, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 961 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/total akt/product/Santa Cruz Biotechnology
    Average 94 stars, based on 961 article reviews
    Price from $9.99 to $1999.99
    total akt - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc t akt antibodies
    Decreased Pten leads to brain region-specific increases in <t>phosphorylated-Akt</t> and <t>-Erk1/2</t> in Pten m3m4 mice. ( A–C ) Western blot showing Pten, phosphorylated (Ser473) and total Akt, phosphorylated (Thr202/Tyr204) and total Erk1/2 in (A) cortex, (B) hippocampus, and (C) cerebellum. Quantification of band intensity for each target protein is shown to the right of the western images. *indicates P
    T Akt Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 13750 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t akt antibodies/product/Cell Signaling Technology Inc
    Average 99 stars, based on 13750 article reviews
    Price from $9.99 to $1999.99
    t akt antibodies - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    t akt  (Abcam)
    96
    Abcam t akt
    Decreased Pten leads to brain region-specific increases in <t>phosphorylated-Akt</t> and <t>-Erk1/2</t> in Pten m3m4 mice. ( A–C ) Western blot showing Pten, phosphorylated (Ser473) and total Akt, phosphorylated (Thr202/Tyr204) and total Erk1/2 in (A) cortex, (B) hippocampus, and (C) cerebellum. Quantification of band intensity for each target protein is shown to the right of the western images. *indicates P
    T Akt, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t akt/product/Abcam
    Average 96 stars, based on 128 article reviews
    Price from $9.99 to $1999.99
    t akt - by Bioz Stars, 2020-10
    96/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology anti total akt
    Activation of <t>PLC-PKC-ERK1-2-AKT-dependent</t> transduction pathway in presence of SOD1 wt and SOD1 G93A in NSC-34 cells. Western Blotting analysis of the levels of <t>P-ERK</t> 1-2 (A) and P-AKT (B) in NSC-34 cells incubated with 400 ng/ml of SOD1 wt and with 400 ng/ml of SOD1 G93A for times of 10 and 30 min. The data represent the means ± SEM of three independent experiments relative to control obtained by densitometric analysis of P-ERK1-2 and P-AKT protein bands normalized to ERK1-2 and AKT, respectively. ∗ p
    Anti Total Akt, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti total akt/product/Santa Cruz Biotechnology
    Average 93 stars, based on 258 article reviews
    Price from $9.99 to $1999.99
    anti total akt - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc antibodies against total phospho akt
    Activation of <t>PLC-PKC-ERK1-2-AKT-dependent</t> transduction pathway in presence of SOD1 wt and SOD1 G93A in NSC-34 cells. Western Blotting analysis of the levels of <t>P-ERK</t> 1-2 (A) and P-AKT (B) in NSC-34 cells incubated with 400 ng/ml of SOD1 wt and with 400 ng/ml of SOD1 G93A for times of 10 and 30 min. The data represent the means ± SEM of three independent experiments relative to control obtained by densitometric analysis of P-ERK1-2 and P-AKT protein bands normalized to ERK1-2 and AKT, respectively. ∗ p
    Antibodies Against Total Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against total phospho akt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    antibodies against total phospho akt - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc phospho total akt
    DPPIV inhibits in vitro migration of Neuro-2a cells in association with decreased <t>ERK1/2</t> and <t>AKT</t> signaling. A . Effect of exogenous DPPIV on SDF1 mediated Neuro-2a cell migration. Cells were cultured on transwell inserts coated with ECM. Four experimental groups included 1) Untreated control, Neuro-2a cells were cultured in media alone, 2) SDF1 (100 ng/ml), 3) SDF1 and DPPIV (500 ng/ml), and 4) 5 mM Diprotin A +SDF1+DPPIV. Cells were grown for 24 hours. Optical density values at 540 nm correlating with cell migration were plotted. Results are presented as mean ± SD of triplicates. ** p
    Phospho Total Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho total akt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    phospho total akt - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    92
    Meso Scale Diagnostics LLC total akt
    <t>XMetA</t> improves hyperglycemia and other metabolic markers of disease in diabetic mice. A : CHO-mINSR cells were incubated with increasing concentrations of XMetA (■), isotype control antibody (○), or insulin (▲), and <t>Akt</t> phosphorylation was measured by ELISA ( n = 3). B : Fasting blood glucose measurements were obtained weekly for 6 weeks from control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (■) or isotype control antibody (●). C : After 3 weeks of treatment, fasting blood glucose was measured in control mice treated with 10 mg/kg isotype control antibody (white bar), diabetic mice treated with 10 mg/kg isotype control antibody (gray bar), and diabetic mice treated with the indicated doses of XMetA (black bars). D : Nonfasted blood glucose measurements were obtained weekly for 6 weeks from control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (■) or isotype control antibody (●). After 6 weeks of treatment, blood hemoglobin A 1c ( E ) and nonfasted plasma β-hydroxybutyrate ( F ) were measured in control mice treated with 10 mg/kg isotype control antibody (white bar) and diabetic mice treated with either 10 mg/kg isotype control antibody (gray bar) or XMetA (black bar). Values shown are mean ± SEM. * P
    Total Akt, supplied by Meso Scale Diagnostics LLC, used in various techniques. Bioz Stars score: 92/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/total akt/product/Meso Scale Diagnostics LLC
    Average 92 stars, based on 84 article reviews
    Price from $9.99 to $1999.99
    total akt - by Bioz Stars, 2020-10
    92/100 stars
      Buy from Supplier

    99
    Millipore anti akt antibody
    Baicalein inhibits cell proliferation, promotes apoptosis and increases cisplatin sensitivity in A549 and H460 cells via up‐regulation of PTEN and suppression of the <t>PI3K/Akt</t> pathway. (A) A549 and H460 cells were treated with 0 or 40 μmol/L baicalein for 0‐72 h, and CCK‐8 was performed to measure cell proliferation. (B) Clone formation assay was used to detect number of colonies 24 h after baicalein treatment. (C) Annexin V‐FITC/PI double staining and flow cytometry were used to detect apoptosis in A549 and H460 cells treated with 0, 40, 80 μmol/L baicalein for 24 h. (D) Caspase‐3/7 activity assay kit was used to detect caspase‐3/7 activity in A549 and H460 cells. (E) Western blotting was performed to detect expression levels of survivin, Bcl‐xL and proteins involved in the PTEN/PI3K/Akt pathway 48 h after baicalein treatment. (F) Cells treated with 0 or 40 μmol/L baicalein were cotreated with different concentrations of cisplatin for 24 h, CCK‐8 was used to detect cell viability, and the IC50 was calculated. IC50 indicates the concentration of cisplatin at which cell viability is inhibited by 50%. (G,H) Xenograft mice were divided into four groups: vehicle control, baicalein, cisplatin and baicalein combined with cisplatin. Average radiance of each mouse was observed weekly, and tumour weights were recorded at week 4. * P
    Anti Akt Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti akt antibody/product/Millipore
    Average 99 stars, based on 128 article reviews
    Price from $9.99 to $1999.99
    anti akt antibody - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Abcam anti pan akt antibody
    Dihydromyricetin (DHM) promoted autophagy by inhibiting <t>phosphatidylinositol</t> 3’-kinase/protein kinase B/mammalian target of rapamycin <t>(PI3K/AKT/mTOR)</t> signaling pathway. The pathway-related proteins were detected by western blotting in NRK-52E cells. ** p
    Anti Pan Akt Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti pan akt antibody/product/Abcam
    Average 99 stars, based on 173 article reviews
    Price from $9.99 to $1999.99
    anti pan akt antibody - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    Image Search Results


    Activation of PI3K and MAPK pathways by ZnR/GPR39 in estrogen independent breast cancer cells. ( A ) Immuno-blots of phospho and total AKT in TAMR cells transfected with siGPR39 or siControl and treated with Zn 2+ (200 μM) or IGF-1 (100 nM) or a combination of Zn 2+ and IGF-1 or EDTA (100 μM) as control (top panel). Bottom panel shows densitometry analysis. ( B ) Immunoblot and denistometry analysis of pAKT and tAKT, as in A, of TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min) and following Zn 2+ (200 μM) application. ( C ) Immunoblot and denistometry analysis of phospho-mTOR levels in TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min) and following Zn 2+ (200 μM) application. ( D ) Immunoblot and densitometry analysis of pERK1/2, relative to total ERK1/2 in TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min) and following Zn 2+ (200 μM) application. ( E ) Immunoblot and denistometry analysis of pERK1/2, relative to total ERK1/2 in TAMR cells transfected with siGPR39 or siControl and treated with or without Zn 2+ (200 μM). ( F ) Immunoblot and denistometry analysis of pERK1/2, relative to total ERK1/2 in BT20 cells transfected with several siGPR39 constructs or siControl and treated with or without Zn 2+ (200 μM) (n = 2 independent experiments). Densitometry quantification in the bar graphs are averages of at least n = 3 independent experiments performed in triplicates for each condition (p

    Journal: Scientific Reports

    Article Title: Enhanced ZnR/GPR39 Activity in Breast Cancer, an Alternative Trigger of Signaling Leading to Cell Growth

    doi: 10.1038/s41598-018-26459-5

    Figure Lengend Snippet: Activation of PI3K and MAPK pathways by ZnR/GPR39 in estrogen independent breast cancer cells. ( A ) Immuno-blots of phospho and total AKT in TAMR cells transfected with siGPR39 or siControl and treated with Zn 2+ (200 μM) or IGF-1 (100 nM) or a combination of Zn 2+ and IGF-1 or EDTA (100 μM) as control (top panel). Bottom panel shows densitometry analysis. ( B ) Immunoblot and denistometry analysis of pAKT and tAKT, as in A, of TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min) and following Zn 2+ (200 μM) application. ( C ) Immunoblot and denistometry analysis of phospho-mTOR levels in TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min) and following Zn 2+ (200 μM) application. ( D ) Immunoblot and densitometry analysis of pERK1/2, relative to total ERK1/2 in TAMR cells treated with or without the Gαq inhibitor, YM-254890 (1 μM, 30 min) and following Zn 2+ (200 μM) application. ( E ) Immunoblot and denistometry analysis of pERK1/2, relative to total ERK1/2 in TAMR cells transfected with siGPR39 or siControl and treated with or without Zn 2+ (200 μM). ( F ) Immunoblot and denistometry analysis of pERK1/2, relative to total ERK1/2 in BT20 cells transfected with several siGPR39 constructs or siControl and treated with or without Zn 2+ (200 μM) (n = 2 independent experiments). Densitometry quantification in the bar graphs are averages of at least n = 3 independent experiments performed in triplicates for each condition (p

    Article Snippet: Kinase phosphorylation was assayed using cell lysates (25 μg), separated on a SDS-PAGE (10% for ERK1/2, AKT and occludin or 7.5% for mTOR) and analyzed using antibodies raised against the doubly phosphorylated ERK1/2, total ERK1/2 and total AKT (Sigma-Aldrich, Israel) or phosphorylated AKT (Santa-Cruz Biotechnology, TX, USA) phosphorylated mTOR (1:500, Cell Signaling, MA, USA), actin (1:50,000,MP-Biomedicals, UK).

    Techniques: Activation Assay, Western Blot, Transfection, Construct

    Oligomeric Aβ 1–42 specifically binds to TREM2 and activates TREM2 signaling pathway . a Schematic representation of human TREM2 extracellular domain (sTREM2, amino acid residues 1–171) tagged with human IgG1 Fc. SP: signal peptide. b The cDNA encoding sTREM2-Fc, sTREM1-Fc or Fc alone was transfected into HEK 293 T cells. Each protein was purified from the conditioned medium and analyzed by silver stained SDS-PAGE. c The prepared oAβ 1–42 peptides were analyzed by Western blotting using 4–12% Bis-Tris NuPAGE gel. d Solid phase binding assay showing the saturation binding curve and equilibrium dissociation constant (K D ) of oAβ 1–42 binding to sTREM2-Fc. Fc and sTREM1-Fc served as negative controls ( n = 3). e The Fc, sTREM2-Fc or sTREM1-Fc control was pre-bound to protein A agarose beads and used as baits for immunoprecipitation of oAβ 1–42 . The precipitated products were separated on 4–12% Bis-Tris NuPAGE gel and further subjected to Western blotting. ( f and g ) The binding profiles of oAβ 1–42 to different concentrations of sTREM2-Fc f or Fc g were generated by SPR assay. h The prepared monomeric Aβ 1–42 (mAβ 1–42 ) peptides were analyzed by Western blotting using 4–12% Bis-Tris NuPAGE gel. i The binding profiles of mAβ 1–42 to different concentrations of sTREM2-Fc were generated by SPR assay. j The binding profiles of scrambled Aβ 42 (scAβ 42 ) to different concentrations of sTREM2-Fc were generated by SPR assay. k Wild-type (WT) or Trem2 -knockout (KO) microglia were stimulated with 1.0 μM oAβ 1–42 for 1 h. Cell lysates were analyzed by Western blotting using antibodies specific for either total (T-Syk) or phosphorylated form (p-Syk) of Syk. l Quantification of Western blots as ratios of p-Syk/T-Syk. β-Actin was used as an internal control ( n = 3, two-way ANOVA). m WT or Trem2 -KO microglia were stimulated with 1.0 μM oAβ 1–42 for the indicated time. Cell lysates at each time point were analyzed by Western blotting using antibodies specific for either total (T-Akt) or phosphorylated form (p-Akt) of Akt. n Quantification of Western blots as ratios of p-Akt/T-Akt. β-Actin was used as an internal control (n = 3, two-way ANOVA). Data information: Data represent mean ± SD. **, p

    Journal: Molecular Neurodegeneration

    Article Title: Amyloid-beta modulates microglial responses by binding to the triggering receptor expressed on myeloid cells 2 (TREM2)

    doi: 10.1186/s13024-018-0247-7

    Figure Lengend Snippet: Oligomeric Aβ 1–42 specifically binds to TREM2 and activates TREM2 signaling pathway . a Schematic representation of human TREM2 extracellular domain (sTREM2, amino acid residues 1–171) tagged with human IgG1 Fc. SP: signal peptide. b The cDNA encoding sTREM2-Fc, sTREM1-Fc or Fc alone was transfected into HEK 293 T cells. Each protein was purified from the conditioned medium and analyzed by silver stained SDS-PAGE. c The prepared oAβ 1–42 peptides were analyzed by Western blotting using 4–12% Bis-Tris NuPAGE gel. d Solid phase binding assay showing the saturation binding curve and equilibrium dissociation constant (K D ) of oAβ 1–42 binding to sTREM2-Fc. Fc and sTREM1-Fc served as negative controls ( n = 3). e The Fc, sTREM2-Fc or sTREM1-Fc control was pre-bound to protein A agarose beads and used as baits for immunoprecipitation of oAβ 1–42 . The precipitated products were separated on 4–12% Bis-Tris NuPAGE gel and further subjected to Western blotting. ( f and g ) The binding profiles of oAβ 1–42 to different concentrations of sTREM2-Fc f or Fc g were generated by SPR assay. h The prepared monomeric Aβ 1–42 (mAβ 1–42 ) peptides were analyzed by Western blotting using 4–12% Bis-Tris NuPAGE gel. i The binding profiles of mAβ 1–42 to different concentrations of sTREM2-Fc were generated by SPR assay. j The binding profiles of scrambled Aβ 42 (scAβ 42 ) to different concentrations of sTREM2-Fc were generated by SPR assay. k Wild-type (WT) or Trem2 -knockout (KO) microglia were stimulated with 1.0 μM oAβ 1–42 for 1 h. Cell lysates were analyzed by Western blotting using antibodies specific for either total (T-Syk) or phosphorylated form (p-Syk) of Syk. l Quantification of Western blots as ratios of p-Syk/T-Syk. β-Actin was used as an internal control ( n = 3, two-way ANOVA). m WT or Trem2 -KO microglia were stimulated with 1.0 μM oAβ 1–42 for the indicated time. Cell lysates at each time point were analyzed by Western blotting using antibodies specific for either total (T-Akt) or phosphorylated form (p-Akt) of Akt. n Quantification of Western blots as ratios of p-Akt/T-Akt. β-Actin was used as an internal control (n = 3, two-way ANOVA). Data information: Data represent mean ± SD. **, p

    Article Snippet: Anti-Phospho-Syk (Tyr525/526) (2711 s), anti-total-Syk (13,198 s), anti-Phospho-Akt (Ser473) (4060 s), anti-total-Akt (4685 s) and anti-β-actin antibody (4970 s) were purchased from Cell Signaling Technology.

    Techniques: Transfection, Purification, Staining, SDS Page, Western Blot, Binding Assay, Immunoprecipitation, Generated, SPR Assay, Knock-Out

    Increased Glut-1 translocation to the membrane is mediated via axitinib-induced activation of Akt. ( a ) Mouse PDAC cells were treated with 1 μ M axitinib for the indicated times and pAkt and total Akt were analyzed by immunoblotting. ( b ) Graphical representation of ( a ) ratio of pAKT protein levels, normalized to total AKT levels and relative to untreated. Data represent mean and S.D. and experiments were repeated at least three times. ( c ) Glut-1 surface expression was quantified by flow cytometry using Glut-1 conjugated to PE. Cells were treated with either 1 μ M LY294002 or axitinib at the indicated concentrations and in combination for 24 h. ( d ) pAKT and total AKT were assessed by western blotting in cells treated with either 1 μ M LY294002 or axitinib at the indicated concentrations and in combination for 24 h

    Journal: Cell Death & Disease

    Article Title: Resistance to the tyrosine kinase inhibitor axitinib is associated with increased glucose metabolism in pancreatic adenocarcinoma

    doi: 10.1038/cddis.2014.125

    Figure Lengend Snippet: Increased Glut-1 translocation to the membrane is mediated via axitinib-induced activation of Akt. ( a ) Mouse PDAC cells were treated with 1 μ M axitinib for the indicated times and pAkt and total Akt were analyzed by immunoblotting. ( b ) Graphical representation of ( a ) ratio of pAKT protein levels, normalized to total AKT levels and relative to untreated. Data represent mean and S.D. and experiments were repeated at least three times. ( c ) Glut-1 surface expression was quantified by flow cytometry using Glut-1 conjugated to PE. Cells were treated with either 1 μ M LY294002 or axitinib at the indicated concentrations and in combination for 24 h. ( d ) pAKT and total AKT were assessed by western blotting in cells treated with either 1 μ M LY294002 or axitinib at the indicated concentrations and in combination for 24 h

    Article Snippet: The antibodies used were Akt (Total) and pAkt (ser473) from Cell Signaling (Danvers, MA, USA), Glut-1 (Millipore, Billerica, MA, USA), MCT-4 and β -tubulin (Santa Cruz, Santa Cruz, CA, USA).

    Techniques: Translocation Assay, Activation Assay, Expressing, Flow Cytometry, Cytometry, Western Blot

    Solid stress signal transduction is mediated by Akt/CREB1 to regulate GDF15 expression. ( a . ( b ) Prediction of CREB1 (Matrix) trascription factor-binding sites on the nucleotide sequence of GDF15 (Seq. name) as predicted by MatInspector tool. ( c ) qPCR was used to quantify the mRNA levels of GDF15 in compressed MIA PaCa-2 cells treated with 10 μΜ BKM120 compared to compressed cells treated with DMSO. ΔΔCt method was used to quantify the gene expression in each sample using as a reference the expression in compressed and treated with DMSO cells (control). Bar graphs represent the mean fold change ± SE of two independent experiments (n = 6) and statistical changes are indicated with an asterisk (*) (p

    Journal: Scientific Reports

    Article Title: Solid stress-induced migration is mediated by GDF15 through Akt pathway activation in pancreatic cancer cells

    doi: 10.1038/s41598-018-37425-6

    Figure Lengend Snippet: Solid stress signal transduction is mediated by Akt/CREB1 to regulate GDF15 expression. ( a . ( b ) Prediction of CREB1 (Matrix) trascription factor-binding sites on the nucleotide sequence of GDF15 (Seq. name) as predicted by MatInspector tool. ( c ) qPCR was used to quantify the mRNA levels of GDF15 in compressed MIA PaCa-2 cells treated with 10 μΜ BKM120 compared to compressed cells treated with DMSO. ΔΔCt method was used to quantify the gene expression in each sample using as a reference the expression in compressed and treated with DMSO cells (control). Bar graphs represent the mean fold change ± SE of two independent experiments (n = 6) and statistical changes are indicated with an asterisk (*) (p

    Article Snippet: Proteins were transferred to a PVDF membrane which was then blocked in 5% non-fat milk or BSA in TBS-T buffer and incubated with anti-Growth Differentiation Factor 15 (GDF15) (Cell Signalling), anti-phospho-Akt (Ser 473) (Cell signalling), anti-Akt total (Cell Signalling), anti-phospho-CREB1 (Ser 133) (Abcam) or anti-CREB1 total (Abcam) antibodies overnight.

    Techniques: Transduction, Expressing, Binding Assay, Sequencing, Real-time Polymerase Chain Reaction

    Proposed mechanism of how solid stress signal transduction via Akt pathway regulates GDF15 expression to induce pancreatic cancer cell migration. The development of solid stress during the growth of several solid tumors, such as pancreatic cancer, activates Akt pathway which in turn phosphorylates CREB1. Subsequently, the activated CREB1 acts as transcription factor by a direct binding onto the promoter region of GDF15. GDF15 is then secreted and acts in an autocrine manner to promote pancreatic cancer cell migration.

    Journal: Scientific Reports

    Article Title: Solid stress-induced migration is mediated by GDF15 through Akt pathway activation in pancreatic cancer cells

    doi: 10.1038/s41598-018-37425-6

    Figure Lengend Snippet: Proposed mechanism of how solid stress signal transduction via Akt pathway regulates GDF15 expression to induce pancreatic cancer cell migration. The development of solid stress during the growth of several solid tumors, such as pancreatic cancer, activates Akt pathway which in turn phosphorylates CREB1. Subsequently, the activated CREB1 acts as transcription factor by a direct binding onto the promoter region of GDF15. GDF15 is then secreted and acts in an autocrine manner to promote pancreatic cancer cell migration.

    Article Snippet: Proteins were transferred to a PVDF membrane which was then blocked in 5% non-fat milk or BSA in TBS-T buffer and incubated with anti-Growth Differentiation Factor 15 (GDF15) (Cell Signalling), anti-phospho-Akt (Ser 473) (Cell signalling), anti-Akt total (Cell Signalling), anti-phospho-CREB1 (Ser 133) (Abcam) or anti-CREB1 total (Abcam) antibodies overnight.

    Techniques: Transduction, Expressing, Migration, Binding Assay

    Role of Notch1 and IKKα in TNBC. (A) Working hypothesis: IKKα as a central mediator of mitochondrial and nuclear non-canonical Notch signaling in TNBC. Diagram representing our working hypothesis whereby Jagged1 activates Notch1, which in turn acts through IKKα to trigger two parallel and potentially interacting non-canonical pathways: a mitochondrial pathway culminating in mTORC2-dependent AKT activation and increased oxidative phosphorylation and a nuclear pathway whereby IKKα binds to NF-κB-responsive elements and triggers transcriptional activation of survival genes, such as c-IAP2. Dashed arrows indicate possible secondary effects of AKT on NF-κB activation and survival gene expression, which have been documented in the literature in other systems but not explored in this study. (B) Expression of NOTCH1 and IKKα (Gene symbol (CHUK) significantly correlates with poor relapse-free survival in TNBC but not ER + breast cancers. Using the Kaplan-Meier Plotter ( http://kmplot.com/analysis/ ; 73 ) Breast Cancer 2017 dataset, the correlation between Relapse Free Survival (RFS) and expression of these two transcripts was determined. TNBC ( n = 801) and ER + tumors ( n = 2565) were analyzed separately.

    Journal: Frontiers in Oncology

    Article Title: Notch Signaling Regulates Mitochondrial Metabolism and NF-κB Activity in Triple-Negative Breast Cancer Cells via IKKα-Dependent Non-canonical Pathways

    doi: 10.3389/fonc.2018.00575

    Figure Lengend Snippet: Role of Notch1 and IKKα in TNBC. (A) Working hypothesis: IKKα as a central mediator of mitochondrial and nuclear non-canonical Notch signaling in TNBC. Diagram representing our working hypothesis whereby Jagged1 activates Notch1, which in turn acts through IKKα to trigger two parallel and potentially interacting non-canonical pathways: a mitochondrial pathway culminating in mTORC2-dependent AKT activation and increased oxidative phosphorylation and a nuclear pathway whereby IKKα binds to NF-κB-responsive elements and triggers transcriptional activation of survival genes, such as c-IAP2. Dashed arrows indicate possible secondary effects of AKT on NF-κB activation and survival gene expression, which have been documented in the literature in other systems but not explored in this study. (B) Expression of NOTCH1 and IKKα (Gene symbol (CHUK) significantly correlates with poor relapse-free survival in TNBC but not ER + breast cancers. Using the Kaplan-Meier Plotter ( http://kmplot.com/analysis/ ; 73 ) Breast Cancer 2017 dataset, the correlation between Relapse Free Survival (RFS) and expression of these two transcripts was determined. TNBC ( n = 801) and ER + tumors ( n = 2565) were analyzed separately.

    Article Snippet: Odyssey blocking buffer (LI-COR) was used to block non-specific binding sites and then membranes were incubated with primary antibodies to evaluate the expression of Notch1 (C-20-R), Notch4 (H-225), pIKKα (Thr 23), Total AKT (H-136), RBP-J (CSL), and β-actin (AC-15) (Santa Cruz Biotechnology); IKKα and pAKT (Ser473, D9E) (Cell Signaling Technology).

    Techniques: Activation Assay, Expressing

    Jagged1 regulates Cellular Bioenergetics in Notch and IKKα dependent fashion. (A) MDA-MB-231 cells were plated on control or Jagged1 coated XF24 Cell Culture plate in the presence or absence of AKT inhibitor (MK-2206, 5 μM). Cell energy phenotype profile, mitochondrial respiration as Oxygen Consumption Rate (OCR) and fermentation as indicated by Extracellular Acidification Rate (ECAR) was measured using a Seahorse Analyzer as described in the Methods section. (B,C) MDA-MB-231 cells were transfected with control siRNA or Notch1 siRNA or IKKα siRNA. Forty eight hours following transfection, equal number of live cells were plated on Jagged1 coated XF24 cell culture plate and analyzed for OCR and ECAR by Seahorse Analyzer. Basal OCR was the difference between the OCR before Oligomycin and after Rotenone Antimycin A (R/A). (D) Control and Notch1 or IKKa siRNA transfected cells were stained with 50 nM MitoTracker Green FM (Invitrogen) to detect Mitochondrial Mass by Flow Cytometer according to the manufacturer protocol. Data were analyzed using Beckman Coulter Kaluza Analysis Software.

    Journal: Frontiers in Oncology

    Article Title: Notch Signaling Regulates Mitochondrial Metabolism and NF-κB Activity in Triple-Negative Breast Cancer Cells via IKKα-Dependent Non-canonical Pathways

    doi: 10.3389/fonc.2018.00575

    Figure Lengend Snippet: Jagged1 regulates Cellular Bioenergetics in Notch and IKKα dependent fashion. (A) MDA-MB-231 cells were plated on control or Jagged1 coated XF24 Cell Culture plate in the presence or absence of AKT inhibitor (MK-2206, 5 μM). Cell energy phenotype profile, mitochondrial respiration as Oxygen Consumption Rate (OCR) and fermentation as indicated by Extracellular Acidification Rate (ECAR) was measured using a Seahorse Analyzer as described in the Methods section. (B,C) MDA-MB-231 cells were transfected with control siRNA or Notch1 siRNA or IKKα siRNA. Forty eight hours following transfection, equal number of live cells were plated on Jagged1 coated XF24 cell culture plate and analyzed for OCR and ECAR by Seahorse Analyzer. Basal OCR was the difference between the OCR before Oligomycin and after Rotenone Antimycin A (R/A). (D) Control and Notch1 or IKKa siRNA transfected cells were stained with 50 nM MitoTracker Green FM (Invitrogen) to detect Mitochondrial Mass by Flow Cytometer according to the manufacturer protocol. Data were analyzed using Beckman Coulter Kaluza Analysis Software.

    Article Snippet: Odyssey blocking buffer (LI-COR) was used to block non-specific binding sites and then membranes were incubated with primary antibodies to evaluate the expression of Notch1 (C-20-R), Notch4 (H-225), pIKKα (Thr 23), Total AKT (H-136), RBP-J (CSL), and β-actin (AC-15) (Santa Cruz Biotechnology); IKKα and pAKT (Ser473, D9E) (Cell Signaling Technology).

    Techniques: Multiple Displacement Amplification, Cell Culture, Transfection, Staining, Flow Cytometry, Cytometry, Software

    MDA-MB-231 Mammospheres have increased oxidative metabolism, which is sensitive to GSI, AKT inhibition or IKK inhibition. Mammospheres were enriched from MDA-MB-231 cells using human Mammocult media as per the manufacturer's protocol (STEMCELL Technologies). (A) Expression of Notch1 and AKT phosphorylation was determined in mammospheres and monolayer cell lysates by Western blotting. (B) OCR including Reserve Capacity and ECAR were compared between mammospheres and monolayer cells using a Seahorse Analyzer. Mammospheres were treated with (C) AKT inhibitor (MK-2206, 5 μM); (D) IKK inhibitor (Bay11-7082, 1 μM) or (E) GSI PF-03084014 (PF, 5 μM) and then OCR and ECAR were measured using a Seahorse Analyzer.

    Journal: Frontiers in Oncology

    Article Title: Notch Signaling Regulates Mitochondrial Metabolism and NF-κB Activity in Triple-Negative Breast Cancer Cells via IKKα-Dependent Non-canonical Pathways

    doi: 10.3389/fonc.2018.00575

    Figure Lengend Snippet: MDA-MB-231 Mammospheres have increased oxidative metabolism, which is sensitive to GSI, AKT inhibition or IKK inhibition. Mammospheres were enriched from MDA-MB-231 cells using human Mammocult media as per the manufacturer's protocol (STEMCELL Technologies). (A) Expression of Notch1 and AKT phosphorylation was determined in mammospheres and monolayer cell lysates by Western blotting. (B) OCR including Reserve Capacity and ECAR were compared between mammospheres and monolayer cells using a Seahorse Analyzer. Mammospheres were treated with (C) AKT inhibitor (MK-2206, 5 μM); (D) IKK inhibitor (Bay11-7082, 1 μM) or (E) GSI PF-03084014 (PF, 5 μM) and then OCR and ECAR were measured using a Seahorse Analyzer.

    Article Snippet: Odyssey blocking buffer (LI-COR) was used to block non-specific binding sites and then membranes were incubated with primary antibodies to evaluate the expression of Notch1 (C-20-R), Notch4 (H-225), pIKKα (Thr 23), Total AKT (H-136), RBP-J (CSL), and β-actin (AC-15) (Santa Cruz Biotechnology); IKKα and pAKT (Ser473, D9E) (Cell Signaling Technology).

    Techniques: Multiple Displacement Amplification, Inhibition, Expressing, Western Blot

    Notch activation induces AKT phosphorylation independently of RBP-Jκ but dependent on IKKα in PTEN-wt TNBC cells. (A,B) MDA-MB-231 (MSL, PTEN-wt) and HCC1143 (BL1, PTEN-wt) cells were plated on gelatin (0.2%) (Control) or human recombinant Jagged1 (1 μg/ml) in gelatin (Jagged) coated plates in the presence of indicated drugs (AKT inhibitor MK-2206, 5 μM), GSI PF-03084014 (5 μM), BAY11-7082 (IKK inhibitor, 5 μM), Everolimus (mTORC1 selective inhibitor, 5 μM), and KU-0063794 (dual mTORC1/mTORC2 inhibitor, 5 μM) for an hour. Western blotting was carried out using whole cell lysates. (C) Similarly, MDA-MB-468 (BL1, PTEN null) cells were plated on control or Jagged1 coated plates for an hour in the presence of indicated drugs and western blot was carried out using whole cell lysates. (D,E) MDA-MB-231 cells were transfected with control siRNA or Notch1siRNA or IKKα siRNA. Forty-eight hours following transfection, equal number of cells were plated on Jagged coated plates for an hour and AKT activation was measured by Western Blot. (F,G) RBP-Jκ was silenced in MDA-MB-231 cells using siRNA. RBP-Jκ silenced and or control cells were plated on Jagged1 coated plates for an hour and phosphorylation of AKT was determined using Western Blot. (H) MDA-MB-231 cells were transfected with a dominant negative form of MAML1 (DN-MAMAL1) or control vector. Cells were then plated on Jagged1 coated plates for an hour and phosphorylation of AKT was determined using Western Blot.

    Journal: Frontiers in Oncology

    Article Title: Notch Signaling Regulates Mitochondrial Metabolism and NF-κB Activity in Triple-Negative Breast Cancer Cells via IKKα-Dependent Non-canonical Pathways

    doi: 10.3389/fonc.2018.00575

    Figure Lengend Snippet: Notch activation induces AKT phosphorylation independently of RBP-Jκ but dependent on IKKα in PTEN-wt TNBC cells. (A,B) MDA-MB-231 (MSL, PTEN-wt) and HCC1143 (BL1, PTEN-wt) cells were plated on gelatin (0.2%) (Control) or human recombinant Jagged1 (1 μg/ml) in gelatin (Jagged) coated plates in the presence of indicated drugs (AKT inhibitor MK-2206, 5 μM), GSI PF-03084014 (5 μM), BAY11-7082 (IKK inhibitor, 5 μM), Everolimus (mTORC1 selective inhibitor, 5 μM), and KU-0063794 (dual mTORC1/mTORC2 inhibitor, 5 μM) for an hour. Western blotting was carried out using whole cell lysates. (C) Similarly, MDA-MB-468 (BL1, PTEN null) cells were plated on control or Jagged1 coated plates for an hour in the presence of indicated drugs and western blot was carried out using whole cell lysates. (D,E) MDA-MB-231 cells were transfected with control siRNA or Notch1siRNA or IKKα siRNA. Forty-eight hours following transfection, equal number of cells were plated on Jagged coated plates for an hour and AKT activation was measured by Western Blot. (F,G) RBP-Jκ was silenced in MDA-MB-231 cells using siRNA. RBP-Jκ silenced and or control cells were plated on Jagged1 coated plates for an hour and phosphorylation of AKT was determined using Western Blot. (H) MDA-MB-231 cells were transfected with a dominant negative form of MAML1 (DN-MAMAL1) or control vector. Cells were then plated on Jagged1 coated plates for an hour and phosphorylation of AKT was determined using Western Blot.

    Article Snippet: Odyssey blocking buffer (LI-COR) was used to block non-specific binding sites and then membranes were incubated with primary antibodies to evaluate the expression of Notch1 (C-20-R), Notch4 (H-225), pIKKα (Thr 23), Total AKT (H-136), RBP-J (CSL), and β-actin (AC-15) (Santa Cruz Biotechnology); IKKα and pAKT (Ser473, D9E) (Cell Signaling Technology).

    Techniques: Activation Assay, Multiple Displacement Amplification, Recombinant, Western Blot, Transfection, Dominant Negative Mutation, Plasmid Preparation

    Decreased Pten leads to brain region-specific increases in phosphorylated-Akt and -Erk1/2 in Pten m3m4 mice. ( A–C ) Western blot showing Pten, phosphorylated (Ser473) and total Akt, phosphorylated (Thr202/Tyr204) and total Erk1/2 in (A) cortex, (B) hippocampus, and (C) cerebellum. Quantification of band intensity for each target protein is shown to the right of the western images. *indicates P

    Journal: Human Molecular Genetics

    Article Title: Germline disruption of Pten localization causes enhanced sex-dependent social motivation and increased glial production

    doi: 10.1093/hmg/ddu031

    Figure Lengend Snippet: Decreased Pten leads to brain region-specific increases in phosphorylated-Akt and -Erk1/2 in Pten m3m4 mice. ( A–C ) Western blot showing Pten, phosphorylated (Ser473) and total Akt, phosphorylated (Thr202/Tyr204) and total Erk1/2 in (A) cortex, (B) hippocampus, and (C) cerebellum. Quantification of band intensity for each target protein is shown to the right of the western images. *indicates P

    Article Snippet: The primary antibodies used in this study were mouse anti-human PTEN (Cascade Biosciences, Winchester, MA, USA, 1 : 5000), rabbit anti-HSP 90 (Cell Signaling, 1:5000), rabbit anti-phosphorylated-ERK1/2 (Cell Signaling, 1:2000), rabbit anti-total ERK1/2 (Cell Signaling, 1:2000), rabbit anti-total AKT (Cell Signaling, 1:2000), rabbit anti-phosphorylated AKT (Cell Signaling, 1:250), mouse anti-Gfap (Santa Cruz, Santa Cruz, CA, USA, 1:10 000) and rabbit anti-GAPDH (Cell Signaling, 1:20 000).

    Techniques: Mouse Assay, Western Blot

    Activation of PLC-PKC-ERK1-2-AKT-dependent transduction pathway in presence of SOD1 wt and SOD1 G93A in NSC-34 cells. Western Blotting analysis of the levels of P-ERK 1-2 (A) and P-AKT (B) in NSC-34 cells incubated with 400 ng/ml of SOD1 wt and with 400 ng/ml of SOD1 G93A for times of 10 and 30 min. The data represent the means ± SEM of three independent experiments relative to control obtained by densitometric analysis of P-ERK1-2 and P-AKT protein bands normalized to ERK1-2 and AKT, respectively. ∗ p

    Journal: Frontiers in Physiology

    Article Title: Effect of Mutated Cu, Zn Superoxide Dismutase (SOD1G93A) on Modulation of Transductional Pathway Mediated by M1 Muscarinic Receptor in SK-N-BE and NSC-34 Cells

    doi: 10.3389/fphys.2018.00611

    Figure Lengend Snippet: Activation of PLC-PKC-ERK1-2-AKT-dependent transduction pathway in presence of SOD1 wt and SOD1 G93A in NSC-34 cells. Western Blotting analysis of the levels of P-ERK 1-2 (A) and P-AKT (B) in NSC-34 cells incubated with 400 ng/ml of SOD1 wt and with 400 ng/ml of SOD1 G93A for times of 10 and 30 min. The data represent the means ± SEM of three independent experiments relative to control obtained by densitometric analysis of P-ERK1-2 and P-AKT protein bands normalized to ERK1-2 and AKT, respectively. ∗ p

    Article Snippet: The quantitative analysis was carried out by densitometry, after incubating the membranes with rabbit polyclonal antibodies anti total ERK and anti-total AKT respectively diluted 1:1000 in TBST 0.1% and incubated for 1 h at room temperature, and then incubated with a peroxidase-conjugated secondary antibody diluted 1:2000 in TBST 0.1% for 1 h at room temperature (Santa Cruz Biotechnology Inc.).

    Techniques: Activation Assay, Planar Chromatography, Transduction, Western Blot, Incubation

    Activation of ERK1-2 and AKT trasductional pathways in presence of SOD1 wt and SOD1 G93A in SK-N-BE cells. Western blotting analysis of P-ERK (A) and P-AKT (B) in cells incubated with 400 ng/ml of SOD1 wt and SOD1 G93A for 10 and 30 min. The histograms show the mean values (+SE) evaluated by densitometric analysis of three independent experiments. The results were normalized to ERK1-2 and AKT respectively. ∗ p

    Journal: Frontiers in Physiology

    Article Title: Effect of Mutated Cu, Zn Superoxide Dismutase (SOD1G93A) on Modulation of Transductional Pathway Mediated by M1 Muscarinic Receptor in SK-N-BE and NSC-34 Cells

    doi: 10.3389/fphys.2018.00611

    Figure Lengend Snippet: Activation of ERK1-2 and AKT trasductional pathways in presence of SOD1 wt and SOD1 G93A in SK-N-BE cells. Western blotting analysis of P-ERK (A) and P-AKT (B) in cells incubated with 400 ng/ml of SOD1 wt and SOD1 G93A for 10 and 30 min. The histograms show the mean values (+SE) evaluated by densitometric analysis of three independent experiments. The results were normalized to ERK1-2 and AKT respectively. ∗ p

    Article Snippet: The quantitative analysis was carried out by densitometry, after incubating the membranes with rabbit polyclonal antibodies anti total ERK and anti-total AKT respectively diluted 1:1000 in TBST 0.1% and incubated for 1 h at room temperature, and then incubated with a peroxidase-conjugated secondary antibody diluted 1:2000 in TBST 0.1% for 1 h at room temperature (Santa Cruz Biotechnology Inc.).

    Techniques: Activation Assay, Western Blot, Incubation

    Activation of PLC-PKC-ERK 1-2-AKT-dependent transduction pathway in presence of SOD1 wt and SOD1 G93A . Western Blotting analysis of the levels of P-ERK 1-2 and P-AKT in SK-N-BE (A,B) and NSC-34 (C,D) cells incubated for 10 min with 400 ng/ml of SOD1 wt and with 400 ng/ml of SOD1 G93A in absence and in presence of Pirenzepine 10 μM for 5 min. The data represent the means ± SEM of three independent experiments relative to control obtained by densitometric analysis of P-ERK1/2 and P-AKT protein bands normalized to ERK1-2 and AKT respectively. ∗ p

    Journal: Frontiers in Physiology

    Article Title: Effect of Mutated Cu, Zn Superoxide Dismutase (SOD1G93A) on Modulation of Transductional Pathway Mediated by M1 Muscarinic Receptor in SK-N-BE and NSC-34 Cells

    doi: 10.3389/fphys.2018.00611

    Figure Lengend Snippet: Activation of PLC-PKC-ERK 1-2-AKT-dependent transduction pathway in presence of SOD1 wt and SOD1 G93A . Western Blotting analysis of the levels of P-ERK 1-2 and P-AKT in SK-N-BE (A,B) and NSC-34 (C,D) cells incubated for 10 min with 400 ng/ml of SOD1 wt and with 400 ng/ml of SOD1 G93A in absence and in presence of Pirenzepine 10 μM for 5 min. The data represent the means ± SEM of three independent experiments relative to control obtained by densitometric analysis of P-ERK1/2 and P-AKT protein bands normalized to ERK1-2 and AKT respectively. ∗ p

    Article Snippet: The quantitative analysis was carried out by densitometry, after incubating the membranes with rabbit polyclonal antibodies anti total ERK and anti-total AKT respectively diluted 1:1000 in TBST 0.1% and incubated for 1 h at room temperature, and then incubated with a peroxidase-conjugated secondary antibody diluted 1:2000 in TBST 0.1% for 1 h at room temperature (Santa Cruz Biotechnology Inc.).

    Techniques: Activation Assay, Planar Chromatography, Transduction, Western Blot, Incubation

    DPPIV inhibits in vitro migration of Neuro-2a cells in association with decreased ERK1/2 and AKT signaling. A . Effect of exogenous DPPIV on SDF1 mediated Neuro-2a cell migration. Cells were cultured on transwell inserts coated with ECM. Four experimental groups included 1) Untreated control, Neuro-2a cells were cultured in media alone, 2) SDF1 (100 ng/ml), 3) SDF1 and DPPIV (500 ng/ml), and 4) 5 mM Diprotin A +SDF1+DPPIV. Cells were grown for 24 hours. Optical density values at 540 nm correlating with cell migration were plotted. Results are presented as mean ± SD of triplicates. ** p

    Journal: Molecular neurobiology

    Article Title: Regulation of dipeptidyl peptidase IV in the post-stroke rat brain and In vitro ischemia: Implications for chemokine mediated neural progenitor cell migration and angiogenesis

    doi: 10.1007/s12035-016-0039-4

    Figure Lengend Snippet: DPPIV inhibits in vitro migration of Neuro-2a cells in association with decreased ERK1/2 and AKT signaling. A . Effect of exogenous DPPIV on SDF1 mediated Neuro-2a cell migration. Cells were cultured on transwell inserts coated with ECM. Four experimental groups included 1) Untreated control, Neuro-2a cells were cultured in media alone, 2) SDF1 (100 ng/ml), 3) SDF1 and DPPIV (500 ng/ml), and 4) 5 mM Diprotin A +SDF1+DPPIV. Cells were grown for 24 hours. Optical density values at 540 nm correlating with cell migration were plotted. Results are presented as mean ± SD of triplicates. ** p

    Article Snippet: Briefly, 30 μg of total protein was separated by SDS-PAGE and probed with respective antibodies, 1:1000 for phospho/total AKT, phospho/total ERK1/2 (Cell Signaling, Danvers, MA) followed by incubation with secondary antibody conjugated to horseradish peroxidase for 1 hour.

    Techniques: In Vitro, Migration, Cell Culture

    XMetA improves hyperglycemia and other metabolic markers of disease in diabetic mice. A : CHO-mINSR cells were incubated with increasing concentrations of XMetA (■), isotype control antibody (○), or insulin (▲), and Akt phosphorylation was measured by ELISA ( n = 3). B : Fasting blood glucose measurements were obtained weekly for 6 weeks from control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (■) or isotype control antibody (●). C : After 3 weeks of treatment, fasting blood glucose was measured in control mice treated with 10 mg/kg isotype control antibody (white bar), diabetic mice treated with 10 mg/kg isotype control antibody (gray bar), and diabetic mice treated with the indicated doses of XMetA (black bars). D : Nonfasted blood glucose measurements were obtained weekly for 6 weeks from control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (■) or isotype control antibody (●). After 6 weeks of treatment, blood hemoglobin A 1c ( E ) and nonfasted plasma β-hydroxybutyrate ( F ) were measured in control mice treated with 10 mg/kg isotype control antibody (white bar) and diabetic mice treated with either 10 mg/kg isotype control antibody (gray bar) or XMetA (black bar). Values shown are mean ± SEM. * P

    Journal: Diabetes

    Article Title: A Fully Human, Allosteric Monoclonal Antibody That Activates the Insulin Receptor and Improves Glycemic Control

    doi: 10.2337/db11-1578

    Figure Lengend Snippet: XMetA improves hyperglycemia and other metabolic markers of disease in diabetic mice. A : CHO-mINSR cells were incubated with increasing concentrations of XMetA (■), isotype control antibody (○), or insulin (▲), and Akt phosphorylation was measured by ELISA ( n = 3). B : Fasting blood glucose measurements were obtained weekly for 6 weeks from control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (■) or isotype control antibody (●). C : After 3 weeks of treatment, fasting blood glucose was measured in control mice treated with 10 mg/kg isotype control antibody (white bar), diabetic mice treated with 10 mg/kg isotype control antibody (gray bar), and diabetic mice treated with the indicated doses of XMetA (black bars). D : Nonfasted blood glucose measurements were obtained weekly for 6 weeks from control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (■) or isotype control antibody (●). After 6 weeks of treatment, blood hemoglobin A 1c ( E ) and nonfasted plasma β-hydroxybutyrate ( F ) were measured in control mice treated with 10 mg/kg isotype control antibody (white bar) and diabetic mice treated with either 10 mg/kg isotype control antibody (gray bar) or XMetA (black bar). Values shown are mean ± SEM. * P

    Article Snippet: CHO cells at 37°C expressing mouse or human INSR or IGF-IR were incubated in serum-free culture medium with increasing concentrations of insulin, control antibody, or XMetA for 10 min. Total Akt and Akt phosphorylated at Ser473 and total Erk1/2 and Erk1/2 phosphorylated at Thr202/Tyr204; Thr185/Tyr187 were measured by enzyme-linked immunosorbent assay (ELISA; Meso Scale Discovery, Gaithersburg, MD).

    Techniques: Mouse Assay, Incubation, Enzyme-linked Immunosorbent Assay

    XMetA is a partial agonist of the INSR that selectively activates the PI3K/Akt pathway. A : CHO-hINSR cells were incubated with increasing concentrations of either XMetA (■), isotype control antibody (○), or insulin (▲), and INSR autophosphorylation was measured by ELISA ( n = 3). B : CHO-hINSR cells were incubated with either 33 nmol/L XMetA (■) or isotype control antibody (○) with increasing concentrations of insulin. INSR autophosphorylation was then measured ( n = 3). C : CHO-hINSR cells were incubated with increasing concentrations of XMetA (■), isotype control antibody (○), or insulin (▲), and Akt phosphorylation was measured by ELISA ( n = 3). D : CHO-hINSR cells were incubated with either 33 nmol/L XMetA (■) or isotype control antibody (○) with increasing concentrations of insulin. Akt phosphorylation was then measured ( n = 3). E : CHO-hINSR cells were incubated with increasing concentrations of XMetA (■), isotype control antibody (○), or insulin (▲), and extracellular signal–related kinase (Erk)1/2 phosphorylation was measured by ELISA ( n = 3). mAb, monoclonal antibody.

    Journal: Diabetes

    Article Title: A Fully Human, Allosteric Monoclonal Antibody That Activates the Insulin Receptor and Improves Glycemic Control

    doi: 10.2337/db11-1578

    Figure Lengend Snippet: XMetA is a partial agonist of the INSR that selectively activates the PI3K/Akt pathway. A : CHO-hINSR cells were incubated with increasing concentrations of either XMetA (■), isotype control antibody (○), or insulin (▲), and INSR autophosphorylation was measured by ELISA ( n = 3). B : CHO-hINSR cells were incubated with either 33 nmol/L XMetA (■) or isotype control antibody (○) with increasing concentrations of insulin. INSR autophosphorylation was then measured ( n = 3). C : CHO-hINSR cells were incubated with increasing concentrations of XMetA (■), isotype control antibody (○), or insulin (▲), and Akt phosphorylation was measured by ELISA ( n = 3). D : CHO-hINSR cells were incubated with either 33 nmol/L XMetA (■) or isotype control antibody (○) with increasing concentrations of insulin. Akt phosphorylation was then measured ( n = 3). E : CHO-hINSR cells were incubated with increasing concentrations of XMetA (■), isotype control antibody (○), or insulin (▲), and extracellular signal–related kinase (Erk)1/2 phosphorylation was measured by ELISA ( n = 3). mAb, monoclonal antibody.

    Article Snippet: CHO cells at 37°C expressing mouse or human INSR or IGF-IR were incubated in serum-free culture medium with increasing concentrations of insulin, control antibody, or XMetA for 10 min. Total Akt and Akt phosphorylated at Ser473 and total Erk1/2 and Erk1/2 phosphorylated at Thr202/Tyr204; Thr185/Tyr187 were measured by enzyme-linked immunosorbent assay (ELISA; Meso Scale Discovery, Gaithersburg, MD).

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay

    Baicalein inhibits cell proliferation, promotes apoptosis and increases cisplatin sensitivity in A549 and H460 cells via up‐regulation of PTEN and suppression of the PI3K/Akt pathway. (A) A549 and H460 cells were treated with 0 or 40 μmol/L baicalein for 0‐72 h, and CCK‐8 was performed to measure cell proliferation. (B) Clone formation assay was used to detect number of colonies 24 h after baicalein treatment. (C) Annexin V‐FITC/PI double staining and flow cytometry were used to detect apoptosis in A549 and H460 cells treated with 0, 40, 80 μmol/L baicalein for 24 h. (D) Caspase‐3/7 activity assay kit was used to detect caspase‐3/7 activity in A549 and H460 cells. (E) Western blotting was performed to detect expression levels of survivin, Bcl‐xL and proteins involved in the PTEN/PI3K/Akt pathway 48 h after baicalein treatment. (F) Cells treated with 0 or 40 μmol/L baicalein were cotreated with different concentrations of cisplatin for 24 h, CCK‐8 was used to detect cell viability, and the IC50 was calculated. IC50 indicates the concentration of cisplatin at which cell viability is inhibited by 50%. (G,H) Xenograft mice were divided into four groups: vehicle control, baicalein, cisplatin and baicalein combined with cisplatin. Average radiance of each mouse was observed weekly, and tumour weights were recorded at week 4. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Baicalein inhibits cell growth and increases cisplatin sensitivity of A549 and H460 cells via miR‐424‐3p and targeting PTEN/PI3K/Akt pathway. Baicalein inhibits cell growth and increases cisplatin sensitivity of A549 and H460 cells via miR‐424‐3p and targeting PTEN/PI3K/Akt pathway

    doi: 10.1111/jcmm.13556

    Figure Lengend Snippet: Baicalein inhibits cell proliferation, promotes apoptosis and increases cisplatin sensitivity in A549 and H460 cells via up‐regulation of PTEN and suppression of the PI3K/Akt pathway. (A) A549 and H460 cells were treated with 0 or 40 μmol/L baicalein for 0‐72 h, and CCK‐8 was performed to measure cell proliferation. (B) Clone formation assay was used to detect number of colonies 24 h after baicalein treatment. (C) Annexin V‐FITC/PI double staining and flow cytometry were used to detect apoptosis in A549 and H460 cells treated with 0, 40, 80 μmol/L baicalein for 24 h. (D) Caspase‐3/7 activity assay kit was used to detect caspase‐3/7 activity in A549 and H460 cells. (E) Western blotting was performed to detect expression levels of survivin, Bcl‐xL and proteins involved in the PTEN/PI3K/Akt pathway 48 h after baicalein treatment. (F) Cells treated with 0 or 40 μmol/L baicalein were cotreated with different concentrations of cisplatin for 24 h, CCK‐8 was used to detect cell viability, and the IC50 was calculated. IC50 indicates the concentration of cisplatin at which cell viability is inhibited by 50%. (G,H) Xenograft mice were divided into four groups: vehicle control, baicalein, cisplatin and baicalein combined with cisplatin. Average radiance of each mouse was observed weekly, and tumour weights were recorded at week 4. * P

    Article Snippet: Primary antibodies for Western blotting against to PTEN (ab32199), survivin (ab469), Bcl‐xL (ab32370) and β‐actin (ab8227) was purchased from Abcam (Cambridge, UK); antibodies for PI3K (SAB5500162), total Akt (t‐Akt, SAB4500797) and phosphor‐forms (p‐Akt, SAB4301414) were purchased from Sigma‐Aldrich; the secondary antibody (ab205718) for Western blotting was purchased from Abcam (Cambridge, UK).

    Techniques: CCK-8 Assay, Tube Formation Assay, Double Staining, Flow Cytometry, Cytometry, Activity Assay, Western Blot, Expressing, Concentration Assay, Mouse Assay

    Dihydromyricetin (DHM) promoted autophagy by inhibiting phosphatidylinositol 3’-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway. The pathway-related proteins were detected by western blotting in NRK-52E cells. ** p

    Journal: Bosnian Journal of Basic Medical Sciences

    Article Title: Dihydromyricetin promotes autophagy and attenuates renal interstitial fibrosis by regulating miR-155-5p/PTEN signaling in diabetic nephropathy

    doi: 10.17305/bjbms.2019.4410

    Figure Lengend Snippet: Dihydromyricetin (DHM) promoted autophagy by inhibiting phosphatidylinositol 3’-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway. The pathway-related proteins were detected by western blotting in NRK-52E cells. ** p

    Article Snippet: Then, the membranes were placed in 5% milk for 2 h, incubated with 1000 times diluted antibodies of Col IV (ab6566), α-smooth muscle actin (α-SMA, ab32575), p62 (ab109012), microtubule-associated protein 1A/1B-light chain 3 (LC3)-II/I (ab128025), Beclin 1 (ab62557), phosphatase and tensin homolog deleted on chromosome ten (PTEN, ab32199), phosphatidylinositol 3-kinase (PI3K, ab189403), phosphorylated (p)-PI3K (ab182651), total (t)-protein kinase B (t-AKT, ab8805), p-AKT (ab38449), p-mTOR (p-mTOR, ab109268), and t-mTOR (ab63552, Abcam, Cambridge, USA) at 4°C overnight; then anti-mouse immunoglobulin G (IgG) antibody (1:2000; ab150113, Abcam, Cambridge, USA) was added and the membranes were incubated for 1 h. The bands were analyzed by an imaging system (Bio-Rad, Hercules, USA) and ImageJ software (NIH Image, Bethesda, USA).

    Techniques: Digital Holographic Microscopy, Western Blot