gsk-3β activity assays Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore gsk 3β activity assay kit
    Transient increase in <t>GSK-3β</t> activity induced by MDMA in the mouse hippocampus. GSK-3β activity ( A ) and immunoblot analysis of GSK-3β and P-GSK-3β levels ( B ) in the hippocampus of mice treated with MDMA (acute treatment;
    Gsk 3β Activity Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gsk 3β activity assay kit/product/Millipore
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    gsk 3β activity assay kit - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    90
    Genmed gsk 3β activity assay kit
    Stimulation of EphB2 up-regulates PI3K and Akt with <t>GSK-3β</t> inhibition. HEK293-tau cells were untreated (Con) or transfected with pcDNA3.0 (Vector) or transfected with wild type EphB2 alone (EphB2wt, inactive) for 24 h, or transfected with EphB2wt for 24 h and then stimulated with ephrinB1/Fc for 45 min. The activity-dependent phosphorylation of GSK-3β at Ser9 (inactive form) and Tyr216 (active form) ( a, b ), Akt at Ser473 and Thr308 (active form) ( c, d ), and PI3K at Tyr458/199 (active form) (e, f) was measured respectively by Western blotting and quantitative analysis. The phosphorylation level of the kinases was normalized against the total level. Data were representative of at least three independent experiments with cells from 9~12 culture wells for each group and presented as the mean ± SD. * P
    Gsk 3β Activity Assay Kit, supplied by Genmed, 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/result/gsk 3β activity assay kit/product/Genmed
    Average 90 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    gsk 3β activity assay kit - by Bioz Stars, 2020-04
    90/100 stars
      Buy from Supplier

    91
    Genmed gsk 3β activity assay
    <t>GSK-3β</t> deletion in DG decreases several synapse-associated proteins. ( a,b ) GSK-3β deletion decreased the levels of multiple pre- and post-synaptic proteins in DG extracts measured by Western blotting and the quantitative analyses (n = 4 each group) (* by unpaired t test, # by multiple t-tests using Sidak-Bonferroin method of GraphPad Prism 6.0). ( c,d ) GSK-3β deletion in DG excitatory neurons did not change the synapse-associated protein in prefrontal cortex (n = 3 each group) (* by unpaired t test, # by multiple t-tests using Sidak-Bonferroin method of GraphPad Prism 6.0). ( e,f ) The representative images of the hippocampus by Nissl staining and the densitometric analyses (n = 5–6 each group, unpaired t test). Scale bars, 100 μm. Data were presented as mean ± s.e.m. * P
    Gsk 3β Activity Assay, supplied by Genmed, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gsk 3β activity assay/product/Genmed
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gsk 3β activity assay - by Bioz Stars, 2020-04
    91/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc glycogen synthase kinase 3β gsk 3β
    Downregulation of MYC is involved in resistance to polo‐like kinase inhibitors ( PLK is). (a) Expression levels of MYC , AKT s, AKT downstream proteins, and polo‐like kinase 1 ( PLK 1) in BI 2536‐resistant cell lines. (b) Sensitivity to PLK is in MYC ‐knockdown cells. HCT 116 cells were transfected with MYC ‐targeting si RNA , and MYC protein expression was examined by Western blot analysis (upper left panels). The normalized intensity ratio ( MYC / GAPDH ) is indicated at the bottom of the blots. At 48 h after transfection, the cells were treated with PLK is for an additional 48 h and subjected to WST ‐8 assay. (c) PLK i‐induced caspase activation in MYC ‐knockdown cells. HCT 116 cells were transfected with MYC ‐targeting si RNA . At 48 h after transfection, the cells were treated with BI 6727 (100 nmol/L) for an additional 24 h. The levels of cleaved caspase ( CASP )‐3, ‐8, and ‐9 and MYC were examined by Western blot analysis. <t>GSK</t> <t>‐3β,</t> glycogen <t>synthase</t> kinase 3β; p‐, phosphorylated.
    Glycogen Synthase Kinase 3β Gsk 3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glycogen synthase kinase 3β gsk 3β/product/Cell Signaling Technology Inc
    Average 99 stars, based on 72 article reviews
    Price from $9.99 to $1999.99
    glycogen synthase kinase 3β gsk 3β - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    86
    Cell Signaling Technology Inc glycogen synthase kinase 3β gsk 3β fusion protein
    RNA-bound HuD interacts with active Akt1 and retains its kinase activity. ( A ) RNA-bound HuD interacts with active Akt1. Active or inactive Akt1 was incubated with GST, GST-HuD or GST-HuR, pulled down with poly(U)-sepharose beads and analyzed by IB and for pull-down efficiency (lower panel) by IB. ( B ) Akt1 retains its protein kinase activity in the Akt1–HuD–RNA complex. Active Akt1 was incubated with GST-HuD, GST-HuR or GST and then pulled down with poly(U)-sepharose beads. Protein kinase assays were performed using a <t>GSK-3β</t> peptide, which is a specific substrate for Akt1. Controls are standard assays with ‘no enzyme’, ‘inactive Akt1’ or ‘active Akt1’. In the pull-down lanes approximately 10% of Akt1 has been used for the kinase assay as estimated by western blot analysis (data not shown).
    Glycogen Synthase Kinase 3β Gsk 3β Fusion Protein, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glycogen synthase kinase 3β gsk 3β fusion protein/product/Cell Signaling Technology Inc
    Average 86 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    glycogen synthase kinase 3β gsk 3β fusion protein - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    88
    Santa Cruz Biotechnology anti glycogen synthase kinase gsk 3β
    β-Catenin suppression and <t>GSK-3β</t> activation in lungs of IAV-infected mice. A1, change in body weight of infected and uninfected mice (five mice in each group). A2, change in viral NP levels in the lungs monitored by western immunoblotting (B). One infected animal died on day 6 postinfection. The values represent the mean ± SD of five or four mice. B and C, Data represent the expression levels (B) and relative quantified data (C) of β-catenin, VE-cadherin, GSK-3β, and phospho-GSK-3β (Ser9) in lung extracts (20 μg protein/lane) of control (no-infection) and IAV-infected mice from day 1 to day 6 postinfection. C.S., calibration standard (mixture of uninfected sample except for NP analysis) to normalize the intensity of protein bands in different gels. As the C.S. for NP, a sample from one animal after infection for 4 days was used. β-Actin was used as an internal control. Data are representative of three separate experiments. Multiple comparison (Dunnett test) after ANOVA were used for statistical analysis. P -values are relative to the control (no infection)
    Anti Glycogen Synthase Kinase Gsk 3β, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti glycogen synthase kinase gsk 3β/product/Santa Cruz Biotechnology
    Average 88 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    anti glycogen synthase kinase gsk 3β - by Bioz Stars, 2020-04
    88/100 stars
      Buy from Supplier

    99
    Millipore glycogen synthase kinase 3β gsk3β activity
    <t>GSK-3β-mediated</t> GR phosphorylation redirects gene transcription. ANOVA results from microarray analysis were loaded into Ingenuity Pathways analysis software to compare the biological pathway regulation in WT- and S404A-GR microarray samples. The most statistically significant biological pathways regulated by WT-GR (A) and S404A-GR (B) are shown. The shade of red (induction) or green (repression), lighter to darker, signifies the least to the greatest degree of gene induction or repression, respectively.
    Glycogen Synthase Kinase 3β Gsk3β Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glycogen synthase kinase 3β gsk3β activity/product/Millipore
    Average 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    glycogen synthase kinase 3β gsk3β activity - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology glycogen synthase kinase 3β gsk 3β sirna
    HSP90 increases PKM2 phosphorylation through <t>GSK-3β.</t> a Co-IP was performed for HSP90, PKM2 and GSK-3β to confirm whether these protein formed protein complex in Huh7 cells. b Huh7 cells transfected with HSP90 vector or control vector were treated with GSK3i IX, a GSK-3β inhibitor. Phosphorylation of PKM2 was examined by western blot after inhibiting GSK-3β activity. c Huh7 cells transfected with HSP90 vector or control vector were treated with GSK-3β <t>siRNA</t> to knockdown GSK-3β. Phosphorylation of PKM2 was examined by western blot after GSK-3β knockdown. d In vitro kinase assay to determine the effects of recombinant GSK3β on threonine phosphorylation of PKM2-WT or T328A
    Glycogen Synthase Kinase 3β Gsk 3β Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glycogen synthase kinase 3β gsk 3β sirna/product/Santa Cruz Biotechnology
    Average 93 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    glycogen synthase kinase 3β gsk 3β sirna - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    84
    Upstate Biotechnology Inc active gsk 3β
    Cotransfection of CGNs with GFP-Bax α and a constitutively active mutant of <t>GSK-3β(S9A)</t> induces translocation of the Bax fusion protein to mitochondria and triggers CGN apoptosis. A , CGNs were cotransfected with GFP-Bax α and either empty vector or HA-tagged GSK-3βS9A using the Helios Gene-Gun system, as described in Materials and Methods. At 48 hr after transfection, cotransfected cells were maintained in control medium and were visualized by staining for the epitope tag using an HA polyclonal antibody and a Cy3-conjugated secondary antibody. The images shown are composites of four to six fields captured with a 63× oil objective to give a representative view of the localization of the Bax fusion protein in the absence or presence of constitutively active GSK-3β. B , The areas demarcated by the boxes in A were enlarged 300% to show fine structure. Scale bars, 10 μm.
    Active Gsk 3β, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/active gsk 3β/product/Upstate Biotechnology Inc
    Average 84 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    active gsk 3β - by Bioz Stars, 2020-04
    84/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc phosphorylated p glycogen synthase kinase 3β
    Fisetin suppresses <t>PI3K-Akt-GSK-3β</t> signal pathway but upregulates PTEN expression in vitro . TNBC cell lines MDA-MB-231 and BT549 were treated with vehicle or 30 μM fisetin for immunofluorescence assay and with vehicle or various concentrations of fisetin (10, 30, and 100 μM) for western blot and qRT-PCR. (A) The expression of p-AKT was evaluated by immunofluorescence. (B) The expression of PTEN was evaluated by immunofluorescence. (C) The expression of PTEN protein as well as p-AKT and p-GSK-3β was determined by western blot. (D) The expression of PTEN mRNA was determined by qRT-PCR. The results are shown as the mean ± SD of three experiments, ∗ P
    Phosphorylated P Glycogen Synthase Kinase 3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phosphorylated p glycogen synthase kinase 3β/product/Cell Signaling Technology Inc
    Average 99 stars, based on 51 article reviews
    Price from $9.99 to $1999.99
    phosphorylated p glycogen synthase kinase 3β - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    85
    Millipore active gsk 3β
    Perfusion of active <t>GSK-3β</t> or filamentous tau induces kinesin-1 release from squid vesicle fractions. A: Purified vesicle fractions from individual axoplasms perfused with buffer control or active GSK-3β and with soluble tau or filamentous
    Active Gsk 3β, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/active gsk 3β/product/Millipore
    Average 85 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    active gsk 3β - by Bioz Stars, 2020-04
    85/100 stars
      Buy from Supplier

    90
    Millipore glycogen synthase kinase 3β gsk3β activity inhibitor lithium chloride
    Perfusion of active <t>GSK-3β</t> or filamentous tau induces kinesin-1 release from squid vesicle fractions. A: Purified vesicle fractions from individual axoplasms perfused with buffer control or active GSK-3β and with soluble tau or filamentous
    Glycogen Synthase Kinase 3β Gsk3β Activity Inhibitor Lithium Chloride, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glycogen synthase kinase 3β gsk3β activity inhibitor lithium chloride/product/Millipore
    Average 90 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    glycogen synthase kinase 3β gsk3β activity inhibitor lithium chloride - by Bioz Stars, 2020-04
    90/100 stars
      Buy from Supplier

    99
    Millipore anti gsk 3β
    Effect of signaling components on the actin cytoskeleton, focal adhesions, and transformation. (A) Anchorage-independent growth of wild-type, N ras −/− , and K ras −/− MEFs following knockdown of Akt or Raf (shRNAs directed to the genes encoding Akt and Raf are indicated by shAkt and shRaf, respectively) or infected with empty vector (V) or vector directing the expression of an shRNA to green fluorescent protein (shGFP). Results are means ± standard deviations (SDs) of three independent experiments. Protein knockdown was confirmed by immunoblotting, with tubulin as a loading control (bottom panels). (B) Shown is FITC-phalloidin staining (green) for stress fibers, vinculin staining (red) for focal adhesions, and DAPI staining (blue) for nuclei in wild-type and N ras -deficient cells following knockdown of Akt or Cdc42. Bar, 10 μm. (C) Same experiment as in panel A, except cells of the indicated genotype following knockdown of small GTPases Rac, RhoA, or Cdc42 were analyzed (shRNAs directed to the genes encoding these Rho GTPases are denoted shRac, shRhoA, and shCdc42, respectively). Results are means ± SDs of three independent experiments. Depletion of proteins was confirmed by immunoblotting, with tubulin as a loading control (bottom panels). (D) Same experiment as in panel B, except wild-type and K ras -deficient cells following knockdown of Raf or RhoA were analyzed. (E) Immunoblotting of whole-cell lysates prepared from cells of the indicated genotype was performed with antibodies to phosphorylated <t>GSK-3β</t> and total GSK-3β, phosphorylated ERK1 and ERK2, and total ERK1 and ERK2. (F) In the indicated cells, the presence of activated, GTP-bound Rac, Cdc42, and RhoA was assayed as described in Materials and Methods. Whole-cell lysates were analyzed for total Rac, Cdc42, and RhoA as loading controls. Panels showing activated Rho GTPases are representative of three independent experiments, and loading controls are from the same lysates used for activation assays.
    Anti Gsk 3β, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gsk 3β/product/Millipore
    Average 99 stars, based on 50 article reviews
    Price from $9.99 to $1999.99
    anti gsk 3β - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    86
    Cell Signaling Technology Inc ps9 glycogen synthase kinase 3β
    TRIP6 regulates growth factor-induced AKT activation and T157 phosphorylation of p27 KIP1 . (A) Knockdown of TRIP6 specifically eliminates serum-induced T157 phosphorylation of p27 KIP1 . Confluent U373-MG cells, as indicated, were starved overnight. After pretreatment with MG-132 for 1 h, cells were stimulated with serum for 15 min. Immunoblotting was performed to detect phosphorylated p27 KIP1 (pT157, pT198, and pS10), total p27 KIP1 , or TRIP6 in the lysates. (B to D) Knockdown of TRIP6 eliminates growth factor-induced T157 phosphorylation; however, this effect can be reversed by reconstitution with TRIP6. Confluent U373-MG cells (B) or SKOV-3 cells (C and D), as indicated, were starved overnight, followed by stimulation with the indicated growth factors for 15 min (B) or 5 min (C and D). Immunoblotting was performed to detect pT157-p27 KIP1 or total p27 KIP1 . The expression of EGFP-TRIP6 and TRIP6 in the whole-cell lysates was detected by using anti-TRIP6 antibody. (E) TRIP6 promotes cytosolic mislocalization of p27 KIP1 through the regulation of its phosphorylation at T157. WT or T157D EGFP-p27 KIP1 was expressed in SKOV-3 cells (siScr and siTRIP6). Subcellular fractionation was performed to determine the expression level of EGFP-p27 KIP1 or EGFP-T157D p27 KIP1 in the nucleus (N) or cytosol (C). The intensity of EGFP-p27 KIP1 or EGFP-T157D p27 KIP1 was quantified by NIH IMAGE J software to determine the relative levels of nuclear versus cytosolic p27 KIP1 . Histone H3 and GAPDH served as nuclear and cytosolic markers, respectively. (F and G) Knockdown of TRIP6 attenuates EGF-induced AKT activation; however, this effect can be reversed by TRIP6 overexpression. U373-MG cells (F) or SKOV-3 cells (G), as indicated, were starved overnight, followed by stimulation with EGF for 15 min (F or G) or 30 min (F). Immunoblotting was performed to detect the levels of pT308-AKT, AKT1, <t>pS9–GSK-3β,</t> GSK-3β, and TRIP6. (H) TRIP6 enhances AKT-mediated in vitro phosphorylation of p27 KIP1 at T157 but not at T198. An in vitro kinase assay was performed by incubating purified recombinant p27 KIP1 with active AKT1 in the presence or absence of TRIP6 at 30°C for 30 min. Immunoblotting (IB) was performed to detect pT157-p27 KIP1 , pT198-p27 KIP1 , pT308-AKT1, p27 KIP1 , or TRIP6. The relative intensity of pT157-p27 KIP1 or pT198-p27 KIP1 was quantified by using NIH IMAGE J software.
    Ps9 Glycogen Synthase Kinase 3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ps9 glycogen synthase kinase 3β/product/Cell Signaling Technology Inc
    Average 86 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    ps9 glycogen synthase kinase 3β - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc phospho gsk 3β
    Aspirin impedes the Wnt/β-catenin pathway by promoting β-catenin acetylation, phosphorylation and cytoplasmic degradation, and inhibiting its nuclear accumulation and transcriptional activity. (A) Western blot of p-β-catenin, β-cateninm, <t>p-GSK-3β,</t> GSK-3β and acetylated β-catenin in MDA-MB-231 cells incubated with or without 10 mM LiCl for 6 h prior to treatment with or without 5 mM Aspirin for 24 h. (B) Western blot analysis of β-catenin protein distribution in nuclear extract (NE) and cytoplasmic fractions (CE) of MDA-MB-231 cells incubated with or without 10 mM LiCl for 6 h prior to treatment with 5 mM Aspirin for 24 h. (C) Immunofluorescent staining of MDA-MB-231 cells that were treated with or without Aspirin or LiCl. Cells were fixed and stained with a β-catenin antibody (green) and DAPI (blue), and imaged by confocal microscopy. (D) Same procedure performed as in (B) with the use of 4T1 cells. (E) Same procedure performed as in (C) with the use of 4T1 cells. (F) Western blot analysis of β-catenin and ubiquitinated β-catenin in MDA-MB-231 cells that were incubated with the indicated reagents for 24 h and subsequently treated with proteasome inhibitor benzyloxycarbonyl-leu-leu-leu-aldehyde (MG132) 25 µM for 5 h before harvest. Endogenous β-cat was immunoprecipitated with anti-β-catenin antibodies and Western blot was performed with anti-β-catenin or anti-ubiquitin antibodies.
    Phospho Gsk 3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 538 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho gsk 3β/product/Cell Signaling Technology Inc
    Average 99 stars, based on 538 article reviews
    Price from $9.99 to $1999.99
    phospho gsk 3β - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    86
    Cell Signaling Technology Inc active gst gsk 3β
    Phosphorylation of Cdc25A by <t>GSK-3β</t> promotes β-TrCP binding and ubiquitin-mediated proteolysis
    Active Gst Gsk 3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/active gst gsk 3β/product/Cell Signaling Technology Inc
    Average 86 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    active gst gsk 3β - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    93
    Becton Dickinson gsk 3β
    Functional effects of canonical Wnt/β-catenin signaling activation in bone sarcoma cells A. β-catenin accumulation and nuclear translocation in bone sarcoma cells in response to Wnt3a. SW1353 and U2OS cells were incubated for 12 hr in the absence or presence of 200ng/ml Wnt3a. To assess β-catenin nuclear accumulation, cells were lysed and were immunoblotted by using a monoclonal anti-β-catenin antibody (Top). The middle and bottom part of the blot was stained with anti-β-actin or hnRNP to verify equal loading. B. Activation of the TCF-reporter transcriptional activity in bone sarcoma cells in response to Wnt3a. SW1353 and U2OS cells were incubated for 12 hr in the absence or presence of 200ng/ml Wnt3a, and then the TCF-reporter transcriptional activity was determined by luciferase activity as indicated in Materials and Methods. C. Western blot analysis of Wnt3a effects on total β-catenin, active dephosphorylated β-catenin, Axin2, <t>GSK-3β,</t> c-Myc, Cyclin-D1, and β-actin protein expression. SW1353 and U2OS cells were incubated for 12 hr in the absence or presence of 200ng/ml Wnt3a. The whole-cell lysates were immunoblotted with the indicated antibodies. D. Effect of Wnt3a on the cell proliferation by Brdu incorporation assay. SW1353 and U2OS cells were incubated for 48 hr in the absence or presence of 200ng/ml Wnt3a. Cell proliferation was determined by Brdu incorporation assay as indicated in Material and Methods. E , F. Effect of Wnt3a on the cell survival by MTS assay. SW1353 E. and U2OS F. cells were incubated for 24, 48, 72 hr in the absence or presence of 200ng/ml Wnt3a. Cell survival was determined by MTS assay as indicated in Materials and Methods # P
    Gsk 3β, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gsk 3β/product/Becton Dickinson
    Average 93 stars, based on 219 article reviews
    Price from $9.99 to $1999.99
    gsk 3β - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    92
    Epitomics gsk 3β
    Proposed model of <t>GSK-3β-mediated</t> regulation of the NFATc2 metabolism in pancreatic cancer cells Activation of the phosphatase calcineurin following an intracellular calcium influx dephosphorylates NFAT proteins and stimulates their nuclear import. There, GSK-3β-mediated SP2 phosphorylation protects NFATc2 from degradation and further stimulates DNA binding and transcriptional activation of target genes (left side). In addition, GSK-3β stabilizes NFATc2/STAT3 complex formation independent of and dominant to SP2 phosphorylation resulting in target gene expression (right side).
    Gsk 3β, supplied by Epitomics, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gsk 3β/product/Epitomics
    Average 92 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    gsk 3β - by Bioz Stars, 2020-04
    92/100 stars
      Buy from Supplier

    99
    Abcam p glycogen synthase kinase 3β gsk 3β ab75745
    The activation of AKT/glycogen <t>synthase</t> <t>kinase-3β</t> <t>(GSK-3β)</t> and extracellular signal-regulated kinase (ERK) contributes to Grifola frondosa polysaccharide (GFP)-mediated apoptotic cell death. GFPs (200 μ g/ml) reduced the expression levels of phosphorylated (p)-AKT, p-GSK-3β and p-ERK from 0.5 to 3 h. The average fold of band intensity compared to the related controls was marked respectively (n=6 repeats in each group).
    P Glycogen Synthase Kinase 3β Gsk 3β Ab75745, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p glycogen synthase kinase 3β gsk 3β ab75745/product/Abcam
    Average 99 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    p glycogen synthase kinase 3β gsk 3β ab75745 - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    94
    Abcam gsk 3β
    Activation of the Wnt/β-catenin signalling pathway via lithium released from Li/CPC. ( A – E ) Gene expression of Col1a1, Bglap, OPG, Runx2 and β-catenin were better in Li/CPC-50 and Li/CPC-100 than in CPC and Li/CPC-200 (n = 3). ( F ) β-catenin accumulation in the cytosol and translocation to the nucleus in Li/CPC-100 (n = 3). ( G , H ) Representative Western blot analysis of <t>p-GSK-3β,</t> t-GSK-3β, p-β-catenin, t-β-catenin and Runx2. Li/CPC-50 and Li/CPC-100 increased the amount of p-GSK-3β significantly and decreased the amount of p-β-catenin compared with Li/CPC-200 and CPC, expression of Runx2 was increased significantly in Li/CPC-50 and Li/CPC-100 compared with Li/CPC-200 and CPC. The band density was quantified using ImageJ software and data from three independent experiments were presented (CPC as the control group, the values were expressed as the mean ± SD, n = 3). Statistical analyses were done using Students’t-test. *p
    Gsk 3β, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 289 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gsk 3β/product/Abcam
    Average 94 stars, based on 289 article reviews
    Price from $9.99 to $1999.99
    gsk 3β - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    97
    Santa Cruz Biotechnology gsk 3β
    Activation of the mTORC1 and β-catenin signaling pathways by OIP5 A . Activation of mTORC1 in HLK3 cells transiently infected with Ad-OIP5 compared to Ad-LacZ control cells on Western blot analysis. B . Phosphorylation of <t>GSK-3β</t> by OIP5 in HLK3 and SH-J1 cells transiently infected with Ad-OIP5 or Ad-LacZ adenovirus (n = 3). C . Phosphorylation of β-catenin at the S552 site by OIP5 in HLK3 cells transiently infected with Ad-OIP5 or Ad-LacZ adenovirus (n = 3). D . Immunoblot analysis of nuclear and cytoplasmic levels of β-catenin in HLK3 cells transiently infected with Ad-OIP5 or Ad-LacZ adenovirus (n = 3). E . TCF/LEF-dependent transcriptional activity of β-catenin in HEK293T cells transfected with TOP/FOP flash reporter plasmids. Assays of relative luciferase activity in cells were performed (n = 3). Each bar represents mean ± SD. ** P
    Gsk 3β, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 565 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gsk 3β/product/Santa Cruz Biotechnology
    Average 97 stars, based on 565 article reviews
    Price from $9.99 to $1999.99
    gsk 3β - by Bioz Stars, 2020-04
    97/100 stars
      Buy from Supplier

    89
    OriGene gsk 3β
    MiR-632 activates Wnt/β-catenin pathway by directly targeting <t>GSK-3β.</t> a Data from bioinformatics tools (microRNA.org, TargetScan and miRDB) showed that there were putative binding sites between 3′-UTR of GSK-3β-wt and miR-632. GSK-3β-mut means mutation of binding sites in 3′-UTR of GSK-3β. b Luciferase reporter gene assays illustrated that miR-632 negatively regulated the luciferase activity of GSK-3β-wt-3′-UTR, rather than of GSK-3β-mut-3′-UTR. n = three independent experiments. ** P
    Gsk 3β, supplied by OriGene, used in various techniques. Bioz Stars score: 89/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gsk 3β/product/OriGene
    Average 89 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    gsk 3β - by Bioz Stars, 2020-04
    89/100 stars
      Buy from Supplier

    85
    Serono activity against human gsk 3β
    MiR-632 activates Wnt/β-catenin pathway by directly targeting <t>GSK-3β.</t> a Data from bioinformatics tools (microRNA.org, TargetScan and miRDB) showed that there were putative binding sites between 3′-UTR of GSK-3β-wt and miR-632. GSK-3β-mut means mutation of binding sites in 3′-UTR of GSK-3β. b Luciferase reporter gene assays illustrated that miR-632 negatively regulated the luciferase activity of GSK-3β-wt-3′-UTR, rather than of GSK-3β-mut-3′-UTR. n = three independent experiments. ** P
    Activity Against Human Gsk 3β, supplied by Serono, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/activity against human gsk 3β/product/Serono
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    activity against human gsk 3β - by Bioz Stars, 2020-04
    85/100 stars
      Buy from Supplier

    99
    Abcam glycogen synthase kinase gsk 3β
    Astragaloside IV (AS IV) attenuated isoflurane-induced NF-κB activation and apoptotic pathway activation in hippocampus tissues. Lane 1: Sham group; 2: Isoflurane group; 3: Isoflurane + AS IV (10 mg/kg); 4: Isoflurane + AS IV (40 mg/kg); and 5: Isoflurane + AS IV (100 mg/kg). (A) Cytoplasmic and nuclear NF-κB levels from rat hippocampus tissues. Histone H3 and GAPDH were used as loading control for nuclear protein and cytoplasmic protein, respectively. (B) Expression levels of apoptosis-related proteins in total cell lysates from rat hippocampus tissues were determined by western blot assay. GAPDH levels were used for equal loading control. NF-κB, nuclear factor-κB; Bcl-2, B-cell lymphoma 2; <t>GSK-3β,</t> glycogen <t>synthase</t> kinase 3β; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; AKT, protein kinase B; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
    Glycogen Synthase Kinase Gsk 3β, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glycogen synthase kinase gsk 3β/product/Abcam
    Average 99 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    glycogen synthase kinase gsk 3β - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    90
    Millipore gsk 3β recombinant gsk 3β
    <t>GSK-3β</t> is truncated selectively by calcium-mediated truncation/activation of calpain I. (A) Western blots show that truncation of GSK-3β coincides with truncation/activation of calpain I in a calcium-dependent manner in human brain extracts. Normal human brain extract was incubated at 30°C for 10 min in the presence of 2.0 mM EDTA and various concentrations (0.00–2.14 mM) of CaCl 2 . Then the incubated extracts were analyzed by Western blots developed with specific antibodies to calpain I or GSK-3β. (B) Calcium-activated truncations of calpain I and GSK-3β are selectively inhibited by calpain inhibitors. Normal human brain extract was incubated at 30°C for 10 min in the presence of 2.0 mM each of EDTA and CaCl 2 plus various selective protease inhibitors, as indicated above the blots, followed by Western blots probed with anti-calpain I or anti-GSK-3β to detect the proteolysis. Apr, aprotinin (a serine protease inhibitor); Pep, pepstatin (an aspartic protease inhibitor); Leu, leupeptin (cysteine and serine protease inhibitor that also inhibits calpain); ALLN, N-acetyl-Leu-Leu-Nle-CHO (a cysteine protease inhibitor that also inhibits calpain); Calp, calpastatin peptide (a specific calpain inhibitor). (C, D) Western blots show calcium concentration-dependent truncation of recombinant GSK-3β from bacteria (C) or from mammalian cells (D) by calpain I. Recombinant GSK-3β was incubated with various concentration of calpain I in the presence of CaCl 2 for 10 min at 30°C and the reaction products were subjected to Western blots.
    Gsk 3β Recombinant Gsk 3β, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gsk 3β recombinant gsk 3β/product/Millipore
    Average 90 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    gsk 3β recombinant gsk 3β - by Bioz Stars, 2020-04
    90/100 stars
      Buy from Supplier

    93
    Cell Signaling Technology Inc glycogen synthase kinase 3β cell signal
    <t>GSK-3β</t> is truncated selectively by calcium-mediated truncation/activation of calpain I. (A) Western blots show that truncation of GSK-3β coincides with truncation/activation of calpain I in a calcium-dependent manner in human brain extracts. Normal human brain extract was incubated at 30°C for 10 min in the presence of 2.0 mM EDTA and various concentrations (0.00–2.14 mM) of CaCl 2 . Then the incubated extracts were analyzed by Western blots developed with specific antibodies to calpain I or GSK-3β. (B) Calcium-activated truncations of calpain I and GSK-3β are selectively inhibited by calpain inhibitors. Normal human brain extract was incubated at 30°C for 10 min in the presence of 2.0 mM each of EDTA and CaCl 2 plus various selective protease inhibitors, as indicated above the blots, followed by Western blots probed with anti-calpain I or anti-GSK-3β to detect the proteolysis. Apr, aprotinin (a serine protease inhibitor); Pep, pepstatin (an aspartic protease inhibitor); Leu, leupeptin (cysteine and serine protease inhibitor that also inhibits calpain); ALLN, N-acetyl-Leu-Leu-Nle-CHO (a cysteine protease inhibitor that also inhibits calpain); Calp, calpastatin peptide (a specific calpain inhibitor). (C, D) Western blots show calcium concentration-dependent truncation of recombinant GSK-3β from bacteria (C) or from mammalian cells (D) by calpain I. Recombinant GSK-3β was incubated with various concentration of calpain I in the presence of CaCl 2 for 10 min at 30°C and the reaction products were subjected to Western blots.
    Glycogen Synthase Kinase 3β Cell Signal, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glycogen synthase kinase 3β cell signal/product/Cell Signaling Technology Inc
    Average 93 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    glycogen synthase kinase 3β cell signal - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    93
    Millipore gsk 3β activator
    Up-regulation of Nrf2 by SFN is partially achieved through the <t>AKT/GSK-3β/Fyn</t> pathway. Cardiac tissues were collected as described in Fig. 1 . Cardiac Akt ( A ) and GSK-3β ( B ) phosphorylation was examined by Western blot. The nuclear translocation of Fyn was determined by immunofluorescent staining of Fyn nuclear accumulation (red) on heart tissue sections ( C , bar = 100μm) and Western blot assay of Fyn expression in cardiac nuclear fraction ( D ). Data are presented as the mean ± SD (n = 7). *, p
    Gsk 3β Activator, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gsk 3β activator/product/Millipore
    Average 93 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    gsk 3β activator - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    86
    Millipore gst gsk 3β
    Up-regulation of Nrf2 by SFN is partially achieved through the <t>AKT/GSK-3β/Fyn</t> pathway. Cardiac tissues were collected as described in Fig. 1 . Cardiac Akt ( A ) and GSK-3β ( B ) phosphorylation was examined by Western blot. The nuclear translocation of Fyn was determined by immunofluorescent staining of Fyn nuclear accumulation (red) on heart tissue sections ( C , bar = 100μm) and Western blot assay of Fyn expression in cardiac nuclear fraction ( D ). Data are presented as the mean ± SD (n = 7). *, p
    Gst Gsk 3β, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gst gsk 3β/product/Millipore
    Average 86 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    gst gsk 3β - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    92
    Bioworld Antibodies gsk 3β ser
    A simplified model of the relationship between Wnt signaling and AD pathology. Chronic noise exposure and aging could induce activation of Dkk-1, a Wnt pathway inhibitor. In the one hand, in absence of Wnt ligands, Dvl protein expression decreased and both β-catenin and tau are phosphorylated by <t>GSK-3β,</t> in the other hand, without the inhibition of Wnt, the expression of Aβ significantly increased, which further exacerbates the downregulation of wnt. All of this indicates that the AD-like neuropathology observed in mice of accelerated aging after chronic noise is likely to be the result of dysregulation of Wnt signaling. Dkk-1, Dickkopf-related protein 1; Dvl, disheveled; p, phosphorylation.
    Gsk 3β Ser, supplied by Bioworld Antibodies, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gsk 3β ser/product/Bioworld Antibodies
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    gsk 3β ser - by Bioz Stars, 2020-04
    92/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc phospho gsk 3β ser9
    Western blots (WB) performed on frontal cortex of mice treated with haloperidol decanoate (N = 11) or vehicle (N = 9) for 2 weeks. (A) No differences were observed in total <t>GSK-3β.</t> (B) Inactivated pGSK-3β was reduced in haloperidol treated mice. (C) The ratio of inactive/total pGSK-3β/total GSK-3β was reduced in haloperidol treated mice, suggesting an activation of the kinase. The insert displays typical gel lanes from immunoblots used for quantitative analyses with ImageJ.
    Phospho Gsk 3β Ser9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 415 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho gsk 3β ser9/product/Cell Signaling Technology Inc
    Average 99 stars, based on 415 article reviews
    Price from $9.99 to $1999.99
    phospho gsk 3β ser9 - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    Abcam anti gsk 3β
    KLK11 affected the expression of the related factors in Wnt/β-catenin pathway and cell cycle-mediated factors. A. The expression of <t>p-GSK-3β/GSK-3β</t> in EC18 and TE-1 cells after lentivirus vectors delivery in each group as measured by western blot. B. The TCF/LEF luciferase ratio reported the activity of Wnt/β-catenin pathway in the indicated cells. C. Expression of CDK4, cyclin D1, CDK6, p-Rb, Rb, β-catenin (cytosol), and β-catenin (nucleus) were determined in TE-1 and EC18 cells transfected with KLK11. KLK11 inhibited the proliferation of ESCC by regulating the expression of the above proteins. D. Analysis of Ki67, cyclin D1, CDK4, CDK6, and c-myc expression in KLK11-overexpression or KLK11-knockdown ESCC cells by qPCR. Data were representative of three independent experiments (mean and SEM). *P
    Anti Gsk 3β, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gsk 3β/product/Abcam
    Average 99 stars, based on 154 article reviews
    Price from $9.99 to $1999.99
    anti gsk 3β - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    Transient increase in GSK-3β activity induced by MDMA in the mouse hippocampus. GSK-3β activity ( A ) and immunoblot analysis of GSK-3β and P-GSK-3β levels ( B ) in the hippocampus of mice treated with MDMA (acute treatment;

    Journal: The Journal of Neuroscience

    Article Title: Enhanced Tau Phosphorylation in the Hippocampus of Mice Treated with 3,4-Methylenedioxymethamphetamine (“Ecstasy”)

    doi: 10.1523/JNEUROSCI.0159-08.2008

    Figure Lengend Snippet: Transient increase in GSK-3β activity induced by MDMA in the mouse hippocampus. GSK-3β activity ( A ) and immunoblot analysis of GSK-3β and P-GSK-3β levels ( B ) in the hippocampus of mice treated with MDMA (acute treatment;

    Article Snippet: GSK-3β activity was measured as 32 P transfer from [γ-32 P]ATP to a peptide substrate by using the GSK-3β Activity Assay kit (Sigma).

    Techniques: Activity Assay, Mouse Assay

    Stimulation of EphB2 up-regulates PI3K and Akt with GSK-3β inhibition. HEK293-tau cells were untreated (Con) or transfected with pcDNA3.0 (Vector) or transfected with wild type EphB2 alone (EphB2wt, inactive) for 24 h, or transfected with EphB2wt for 24 h and then stimulated with ephrinB1/Fc for 45 min. The activity-dependent phosphorylation of GSK-3β at Ser9 (inactive form) and Tyr216 (active form) ( a, b ), Akt at Ser473 and Thr308 (active form) ( c, d ), and PI3K at Tyr458/199 (active form) (e, f) was measured respectively by Western blotting and quantitative analysis. The phosphorylation level of the kinases was normalized against the total level. Data were representative of at least three independent experiments with cells from 9~12 culture wells for each group and presented as the mean ± SD. * P

    Journal: Scientific Reports

    Article Title: Stimulation of EphB2 attenuates tau phosphorylation through PI3K/Akt-mediated inactivation of glycogen synthase kinase-3β

    doi: 10.1038/srep11765

    Figure Lengend Snippet: Stimulation of EphB2 up-regulates PI3K and Akt with GSK-3β inhibition. HEK293-tau cells were untreated (Con) or transfected with pcDNA3.0 (Vector) or transfected with wild type EphB2 alone (EphB2wt, inactive) for 24 h, or transfected with EphB2wt for 24 h and then stimulated with ephrinB1/Fc for 45 min. The activity-dependent phosphorylation of GSK-3β at Ser9 (inactive form) and Tyr216 (active form) ( a, b ), Akt at Ser473 and Thr308 (active form) ( c, d ), and PI3K at Tyr458/199 (active form) (e, f) was measured respectively by Western blotting and quantitative analysis. The phosphorylation level of the kinases was normalized against the total level. Data were representative of at least three independent experiments with cells from 9~12 culture wells for each group and presented as the mean ± SD. * P

    Article Snippet: GSK-3β activity assay Activity of GSK-3β was measured using GSK-3β Activity Assay Kit (GENMED Scientifics INC., ARLINGTON, MA) by following the manufacturer’s manufacture’s instructions.

    Techniques: Inhibition, Transfection, Plasmid Preparation, Activity Assay, Western Blot

    Stimulation of EphB2 inhibits GSK-3β. ( a, b ) SK-N-SH cells were treated with ephrinB1/Fc or Fc or DMEM (vehicle control) for 45 min to activate EphB2 and th en the activity-dependent phosphorylation of GSK-3β at Ser9 (inactivation) and Tyr216 (activation) was measured by Western blotting ( a ) and quantitative analysis ( b ). ( c ) The inactivation of GSK-3β was also observed by biochemical assay of the kinase activity. Data were representative of at least three independent experiments with cells from 9~12 culture wells for each group and presented as the mean±SD. * P

    Journal: Scientific Reports

    Article Title: Stimulation of EphB2 attenuates tau phosphorylation through PI3K/Akt-mediated inactivation of glycogen synthase kinase-3β

    doi: 10.1038/srep11765

    Figure Lengend Snippet: Stimulation of EphB2 inhibits GSK-3β. ( a, b ) SK-N-SH cells were treated with ephrinB1/Fc or Fc or DMEM (vehicle control) for 45 min to activate EphB2 and th en the activity-dependent phosphorylation of GSK-3β at Ser9 (inactivation) and Tyr216 (activation) was measured by Western blotting ( a ) and quantitative analysis ( b ). ( c ) The inactivation of GSK-3β was also observed by biochemical assay of the kinase activity. Data were representative of at least three independent experiments with cells from 9~12 culture wells for each group and presented as the mean±SD. * P

    Article Snippet: GSK-3β activity assay Activity of GSK-3β was measured using GSK-3β Activity Assay Kit (GENMED Scientifics INC., ARLINGTON, MA) by following the manufacturer’s manufacture’s instructions.

    Techniques: Activity Assay, Activation Assay, Western Blot

    Simultaneous inhibition of PI3K or overexpression of GSK-3β abolishes the EphB2 activation-induced tau dephosphorylation. ( a-c ) SK-N-SH cells were treated with ephrinB1/Fc alone or ephrinB1/Fc plus wortmannin (an inhibitor of PI3K) for 2 h, and then phosphorylation level of GSK-3β at Ser9 and tau at Thr231 was measured by Western blotting. * P

    Journal: Scientific Reports

    Article Title: Stimulation of EphB2 attenuates tau phosphorylation through PI3K/Akt-mediated inactivation of glycogen synthase kinase-3β

    doi: 10.1038/srep11765

    Figure Lengend Snippet: Simultaneous inhibition of PI3K or overexpression of GSK-3β abolishes the EphB2 activation-induced tau dephosphorylation. ( a-c ) SK-N-SH cells were treated with ephrinB1/Fc alone or ephrinB1/Fc plus wortmannin (an inhibitor of PI3K) for 2 h, and then phosphorylation level of GSK-3β at Ser9 and tau at Thr231 was measured by Western blotting. * P

    Article Snippet: GSK-3β activity assay Activity of GSK-3β was measured using GSK-3β Activity Assay Kit (GENMED Scientifics INC., ARLINGTON, MA) by following the manufacturer’s manufacture’s instructions.

    Techniques: Inhibition, Over Expression, Activation Assay, De-Phosphorylation Assay, Western Blot

    EphrinB1/Fc stimulates tyrosine phosphorylation of EphB2 and deletion of the kinase domain (VM) but not the PDZ binding domain eliminates EphB2-induced tau dephosphorylation. HEK293-tau cells were treated with ephrinB1/Fc for 45 min, and then the phosphorylation level of EphB2 was measured by immunoprecipitation with anti-EphB2 antibody and Western blotting probed by PY99 and anti-EphB2 antibody ( a, b ). HEK293-tau cells were transfected with deletion of PDZ ( c, d ) or VM ( e, f ) domain of EphB2 or the control Vector for 24 h and then treated with ephrinB1/Fc for 45 min. The phosphorylation levels of tau at Thr231 and Ser396, or GSK-3β at Ser9, or PI3K at Tyr458/Tyr199 were measured by Western blotting. The phosphorylation levels of tau and the kinases were normalized against the total levels. Data were representative of at least three independent experiments from 9 ~ 12 culture wells for each group and presented as the mean ± SD. * P

    Journal: Scientific Reports

    Article Title: Stimulation of EphB2 attenuates tau phosphorylation through PI3K/Akt-mediated inactivation of glycogen synthase kinase-3β

    doi: 10.1038/srep11765

    Figure Lengend Snippet: EphrinB1/Fc stimulates tyrosine phosphorylation of EphB2 and deletion of the kinase domain (VM) but not the PDZ binding domain eliminates EphB2-induced tau dephosphorylation. HEK293-tau cells were treated with ephrinB1/Fc for 45 min, and then the phosphorylation level of EphB2 was measured by immunoprecipitation with anti-EphB2 antibody and Western blotting probed by PY99 and anti-EphB2 antibody ( a, b ). HEK293-tau cells were transfected with deletion of PDZ ( c, d ) or VM ( e, f ) domain of EphB2 or the control Vector for 24 h and then treated with ephrinB1/Fc for 45 min. The phosphorylation levels of tau at Thr231 and Ser396, or GSK-3β at Ser9, or PI3K at Tyr458/Tyr199 were measured by Western blotting. The phosphorylation levels of tau and the kinases were normalized against the total levels. Data were representative of at least three independent experiments from 9 ~ 12 culture wells for each group and presented as the mean ± SD. * P

    Article Snippet: GSK-3β activity assay Activity of GSK-3β was measured using GSK-3β Activity Assay Kit (GENMED Scientifics INC., ARLINGTON, MA) by following the manufacturer’s manufacture’s instructions.

    Techniques: Binding Assay, De-Phosphorylation Assay, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation

    GSK-3β deletion in DG decreases several synapse-associated proteins. ( a,b ) GSK-3β deletion decreased the levels of multiple pre- and post-synaptic proteins in DG extracts measured by Western blotting and the quantitative analyses (n = 4 each group) (* by unpaired t test, # by multiple t-tests using Sidak-Bonferroin method of GraphPad Prism 6.0). ( c,d ) GSK-3β deletion in DG excitatory neurons did not change the synapse-associated protein in prefrontal cortex (n = 3 each group) (* by unpaired t test, # by multiple t-tests using Sidak-Bonferroin method of GraphPad Prism 6.0). ( e,f ) The representative images of the hippocampus by Nissl staining and the densitometric analyses (n = 5–6 each group, unpaired t test). Scale bars, 100 μm. Data were presented as mean ± s.e.m. * P

    Journal: Scientific Reports

    Article Title: GSK-3β deletion in dentate gyrus excitatory neuron impairs synaptic plasticity and memory

    doi: 10.1038/s41598-017-06173-4

    Figure Lengend Snippet: GSK-3β deletion in DG decreases several synapse-associated proteins. ( a,b ) GSK-3β deletion decreased the levels of multiple pre- and post-synaptic proteins in DG extracts measured by Western blotting and the quantitative analyses (n = 4 each group) (* by unpaired t test, # by multiple t-tests using Sidak-Bonferroin method of GraphPad Prism 6.0). ( c,d ) GSK-3β deletion in DG excitatory neurons did not change the synapse-associated protein in prefrontal cortex (n = 3 each group) (* by unpaired t test, # by multiple t-tests using Sidak-Bonferroin method of GraphPad Prism 6.0). ( e,f ) The representative images of the hippocampus by Nissl staining and the densitometric analyses (n = 5–6 each group, unpaired t test). Scale bars, 100 μm. Data were presented as mean ± s.e.m. * P

    Article Snippet: GSK-3β activity assay The activity of GSK-3β was assayed using a kit (GMS50161.6, Genmed) by following the manufacturer’s instructions.

    Techniques: Western Blot, Staining

    GSK-3β deletion in DG induces spatial and fear memory deficits. ( a ) GSK-3β deletion in DG excitatory neurons does not change the latency to find the hidden platform during 6 days Morris water maze (MWM) training. ( b,c ) GSK-3β deletion induces memory deficits shown by the decreased time in previous target quadrant and the crossings in the platform region measured at day 8 by removed the platform (Vector, n = 10; Cre, n = 8). ( d ) The schematic representation of contextual fear memory test. ( e ) No change of freezing time was detected in GSK-3β deletion during the contextual fear training test. ( f ) The percentage time of freezing was remarkably decreased during the contextual memory test carried out in the next day (Vector, n = 10; Cre, n = 8). Data were presented as mean ± s.e.m. Two–way repeated-measures ANOVA with Huynh-Feldt-Lecoutre correction for panels (a) and (e); unpaired t test for panels (b, c, and f). * P

    Journal: Scientific Reports

    Article Title: GSK-3β deletion in dentate gyrus excitatory neuron impairs synaptic plasticity and memory

    doi: 10.1038/s41598-017-06173-4

    Figure Lengend Snippet: GSK-3β deletion in DG induces spatial and fear memory deficits. ( a ) GSK-3β deletion in DG excitatory neurons does not change the latency to find the hidden platform during 6 days Morris water maze (MWM) training. ( b,c ) GSK-3β deletion induces memory deficits shown by the decreased time in previous target quadrant and the crossings in the platform region measured at day 8 by removed the platform (Vector, n = 10; Cre, n = 8). ( d ) The schematic representation of contextual fear memory test. ( e ) No change of freezing time was detected in GSK-3β deletion during the contextual fear training test. ( f ) The percentage time of freezing was remarkably decreased during the contextual memory test carried out in the next day (Vector, n = 10; Cre, n = 8). Data were presented as mean ± s.e.m. Two–way repeated-measures ANOVA with Huynh-Feldt-Lecoutre correction for panels (a) and (e); unpaired t test for panels (b, c, and f). * P

    Article Snippet: GSK-3β activity assay The activity of GSK-3β was assayed using a kit (GMS50161.6, Genmed) by following the manufacturer’s instructions.

    Techniques: Plasmid Preparation

    GSK-3β deletion in DG induces an anti-anxiety behavior. ( a ) The representative route recorded in open field test. ( b–d ) GSK-3β deletion reduced the percentage time spent in corner without changing the time spent in the center and the total distance moved in the open field test.(Vector, n = 10; Cre, n = 8, unpaired t test) ( e ) The representative route in the elevated maze test. ( f–h ) GSK-3β deletion increased the percentage of open arm entries, the percentage distance and total time spent in the open arm(Vector, n = 10; Cre, n = 8, unpaired t test). Data were presented as mean ± s.e.m. * P

    Journal: Scientific Reports

    Article Title: GSK-3β deletion in dentate gyrus excitatory neuron impairs synaptic plasticity and memory

    doi: 10.1038/s41598-017-06173-4

    Figure Lengend Snippet: GSK-3β deletion in DG induces an anti-anxiety behavior. ( a ) The representative route recorded in open field test. ( b–d ) GSK-3β deletion reduced the percentage time spent in corner without changing the time spent in the center and the total distance moved in the open field test.(Vector, n = 10; Cre, n = 8, unpaired t test) ( e ) The representative route in the elevated maze test. ( f–h ) GSK-3β deletion increased the percentage of open arm entries, the percentage distance and total time spent in the open arm(Vector, n = 10; Cre, n = 8, unpaired t test). Data were presented as mean ± s.e.m. * P

    Article Snippet: GSK-3β activity assay The activity of GSK-3β was assayed using a kit (GMS50161.6, Genmed) by following the manufacturer’s instructions.

    Techniques: Plasmid Preparation

    GSK-3β deletion inhibits activity-dependent neural activation. ( a ) GSK-3β deletion significantly suppressed basal synaptic transmission shown by the reduced input/output (I/O) curve. ( b,c ) GSK-3β deletion induced LTP impairment shown by a decreased slop of the evoked fEPSP, and the decrease was still significant at 60 min after high frequency stimulation (HFS). n = 8–10 hippocampal slices from 6 mice in each group. ( d,e ) The representative micrographs of spine morphology and the decreased spine density in GFP-positive neurons (6 mice were analyzed in each group). ( f,g ) The representative micrographs of spine morphology and the spine density in prefrontal cortex neurons (6 mice were analyzed in each group). ( h–k ) The representative immunohistochemical images of c-fos, and the densitometric analyses in DG (dentate gyrus), BLA (basal lateral amygdala) and PV (paraventricular thalamic nucleus) subsets (Scale bars, 20 μm; n = 5–6 each group). Data were presented as mean ± s.e.m. Two–way repeated-measures ANOVA with Huynh-Feldt-Lecoutre correction for panels ( a ), unpaired t test for panels ( c , e , i – k ). * P

    Journal: Scientific Reports

    Article Title: GSK-3β deletion in dentate gyrus excitatory neuron impairs synaptic plasticity and memory

    doi: 10.1038/s41598-017-06173-4

    Figure Lengend Snippet: GSK-3β deletion inhibits activity-dependent neural activation. ( a ) GSK-3β deletion significantly suppressed basal synaptic transmission shown by the reduced input/output (I/O) curve. ( b,c ) GSK-3β deletion induced LTP impairment shown by a decreased slop of the evoked fEPSP, and the decrease was still significant at 60 min after high frequency stimulation (HFS). n = 8–10 hippocampal slices from 6 mice in each group. ( d,e ) The representative micrographs of spine morphology and the decreased spine density in GFP-positive neurons (6 mice were analyzed in each group). ( f,g ) The representative micrographs of spine morphology and the spine density in prefrontal cortex neurons (6 mice were analyzed in each group). ( h–k ) The representative immunohistochemical images of c-fos, and the densitometric analyses in DG (dentate gyrus), BLA (basal lateral amygdala) and PV (paraventricular thalamic nucleus) subsets (Scale bars, 20 μm; n = 5–6 each group). Data were presented as mean ± s.e.m. Two–way repeated-measures ANOVA with Huynh-Feldt-Lecoutre correction for panels ( a ), unpaired t test for panels ( c , e , i – k ). * P

    Article Snippet: GSK-3β activity assay The activity of GSK-3β was assayed using a kit (GMS50161.6, Genmed) by following the manufacturer’s instructions.

    Techniques: Activity Assay, Activation Assay, Transmission Assay, Mouse Assay, Immunohistochemistry

    Expression of Cre recombinase selectively deletes GSK-3β in DG excitatory neurons of GSK-3β loxp mice. ( a ) Schematic representation of pAAV-CaMKII-Cre-2A-eGFP and the empty pAAV-CaMKII-eGFP vector. ( b ) A representative image showing efficient virus infection in DG and the mossy fibers. ( c,d ) Injection of Cre recombinase into hippocampal DG of GSK-3β loxp mice for 1-m efficiently downregulated GSK-3β protein level measured by Western blotting (n = 3 each group). ( e ) The reduced GSK-3β activity in DG extracts after Cre recombinase injection (n = 3 each group). ( f ) Deletion of GSK-3β in DG neurons by Cre recombinase injection measured by immunohistochemistry, scale bars, 100 μm. ( g,h ) Specific deletion of GSK-3β in DG excitatory neurons measured by co-immunofluorescence of GFP with CaMKII but not with GAD67 antibody, scale bars, 10 μm. ( i , j ) Deletion of GSK-3β did not significantly affect GSK-3α in DG subset (n = 3 each group). Data were presented as mean ± s.e.m. unpaired t test, ** P

    Journal: Scientific Reports

    Article Title: GSK-3β deletion in dentate gyrus excitatory neuron impairs synaptic plasticity and memory

    doi: 10.1038/s41598-017-06173-4

    Figure Lengend Snippet: Expression of Cre recombinase selectively deletes GSK-3β in DG excitatory neurons of GSK-3β loxp mice. ( a ) Schematic representation of pAAV-CaMKII-Cre-2A-eGFP and the empty pAAV-CaMKII-eGFP vector. ( b ) A representative image showing efficient virus infection in DG and the mossy fibers. ( c,d ) Injection of Cre recombinase into hippocampal DG of GSK-3β loxp mice for 1-m efficiently downregulated GSK-3β protein level measured by Western blotting (n = 3 each group). ( e ) The reduced GSK-3β activity in DG extracts after Cre recombinase injection (n = 3 each group). ( f ) Deletion of GSK-3β in DG neurons by Cre recombinase injection measured by immunohistochemistry, scale bars, 100 μm. ( g,h ) Specific deletion of GSK-3β in DG excitatory neurons measured by co-immunofluorescence of GFP with CaMKII but not with GAD67 antibody, scale bars, 10 μm. ( i , j ) Deletion of GSK-3β did not significantly affect GSK-3α in DG subset (n = 3 each group). Data were presented as mean ± s.e.m. unpaired t test, ** P

    Article Snippet: GSK-3β activity assay The activity of GSK-3β was assayed using a kit (GMS50161.6, Genmed) by following the manufacturer’s instructions.

    Techniques: Expressing, Mouse Assay, Plasmid Preparation, Infection, Injection, Western Blot, Activity Assay, Immunohistochemistry, Immunofluorescence

    GSK-3β deletion inhibits CaMKII/IV-CREB signaling. ( a,b ) GSK-3β deletion decreased total and/or the phosphorylated levels of CaMKII, CaMKIV and CREB in DG extracts measured by Western blotting and the quantitative analyses. ( c ) The reduced intensity of pCREB in DG excitatory neurons was also shown by co-immunofluorescent staining. Scale bars, 100 μm. Data were presented as mean ± s.e.m. n = 4 each group, analyzed by unpaired t-test (*) or multiple t-tests (#). * P

    Journal: Scientific Reports

    Article Title: GSK-3β deletion in dentate gyrus excitatory neuron impairs synaptic plasticity and memory

    doi: 10.1038/s41598-017-06173-4

    Figure Lengend Snippet: GSK-3β deletion inhibits CaMKII/IV-CREB signaling. ( a,b ) GSK-3β deletion decreased total and/or the phosphorylated levels of CaMKII, CaMKIV and CREB in DG extracts measured by Western blotting and the quantitative analyses. ( c ) The reduced intensity of pCREB in DG excitatory neurons was also shown by co-immunofluorescent staining. Scale bars, 100 μm. Data were presented as mean ± s.e.m. n = 4 each group, analyzed by unpaired t-test (*) or multiple t-tests (#). * P

    Article Snippet: GSK-3β activity assay The activity of GSK-3β was assayed using a kit (GMS50161.6, Genmed) by following the manufacturer’s instructions.

    Techniques: Western Blot, Staining

    Downregulation of MYC is involved in resistance to polo‐like kinase inhibitors ( PLK is). (a) Expression levels of MYC , AKT s, AKT downstream proteins, and polo‐like kinase 1 ( PLK 1) in BI 2536‐resistant cell lines. (b) Sensitivity to PLK is in MYC ‐knockdown cells. HCT 116 cells were transfected with MYC ‐targeting si RNA , and MYC protein expression was examined by Western blot analysis (upper left panels). The normalized intensity ratio ( MYC / GAPDH ) is indicated at the bottom of the blots. At 48 h after transfection, the cells were treated with PLK is for an additional 48 h and subjected to WST ‐8 assay. (c) PLK i‐induced caspase activation in MYC ‐knockdown cells. HCT 116 cells were transfected with MYC ‐targeting si RNA . At 48 h after transfection, the cells were treated with BI 6727 (100 nmol/L) for an additional 24 h. The levels of cleaved caspase ( CASP )‐3, ‐8, and ‐9 and MYC were examined by Western blot analysis. GSK ‐3β, glycogen synthase kinase 3β; p‐, phosphorylated.

    Journal: Cancer Science

    Article Title: Effect of AKT3 expression on MYC‐ and caspase‐8‐dependent apoptosis caused by polo‐like kinase inhibitors in HCT 116 cells

    doi: 10.1111/cas.13093

    Figure Lengend Snippet: Downregulation of MYC is involved in resistance to polo‐like kinase inhibitors ( PLK is). (a) Expression levels of MYC , AKT s, AKT downstream proteins, and polo‐like kinase 1 ( PLK 1) in BI 2536‐resistant cell lines. (b) Sensitivity to PLK is in MYC ‐knockdown cells. HCT 116 cells were transfected with MYC ‐targeting si RNA , and MYC protein expression was examined by Western blot analysis (upper left panels). The normalized intensity ratio ( MYC / GAPDH ) is indicated at the bottom of the blots. At 48 h after transfection, the cells were treated with PLK is for an additional 48 h and subjected to WST ‐8 assay. (c) PLK i‐induced caspase activation in MYC ‐knockdown cells. HCT 116 cells were transfected with MYC ‐targeting si RNA . At 48 h after transfection, the cells were treated with BI 6727 (100 nmol/L) for an additional 24 h. The levels of cleaved caspase ( CASP )‐3, ‐8, and ‐9 and MYC were examined by Western blot analysis. GSK ‐3β, glycogen synthase kinase 3β; p‐, phosphorylated.

    Article Snippet: The antibodies used were directed against: p53 (DO‐1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); MDR1 + 3 (C219) (Abcam, Cambridge, MA, USA); GAPDH (6C5) (Millipore); cleaved caspase‐3 (Asp175; 5A1E), caspase‐8 (1C12), cleaved caspase‐9 (Asp330; #9501), AKT1 (C73H10), AKT2 (D6G4), AKT3 (L47B1), phospho‐AKT (Ser473; D9E), glycogen synthase kinase 3β (GSK‐3β) (D5C5Z), phospho‐GSK‐3β (Ser‐9; D85E12), PLK1 (208G4) (Cell Signaling Technology, Danvers, MA, USA); and HA (3F10) and MYC (9E10) (Roche Diagnostics, Indianapolis, IN, USA).

    Techniques: Expressing, Transfection, Western Blot, Activation Assay

    Western blots (WB) performed on frontal cortex of mice treated with haloperidol decanoate (N = 11) or vehicle (N = 9) for 2 weeks. (A) No differences were observed in total GSK-3β. (B) Inactivated pGSK-3β was reduced in haloperidol treated mice. (C) The ratio of inactive/total pGSK-3β/total GSK-3β was reduced in haloperidol treated mice, suggesting an activation of the kinase. The insert displays typical gel lanes from immunoblots used for quantitative analyses with ImageJ.

    Journal: Alzheimer's & Dementia : Translational Research & Clinical Interventions

    Article Title: Haloperidol inactivates AMPK and reduces tau phosphorylation in a tau mouse model of Alzheimer's disease

    doi: 10.1016/j.trci.2016.05.003

    Figure Lengend Snippet: Western blots (WB) performed on frontal cortex of mice treated with haloperidol decanoate (N = 11) or vehicle (N = 9) for 2 weeks. (A) No differences were observed in total GSK-3β. (B) Inactivated pGSK-3β was reduced in haloperidol treated mice. (C) The ratio of inactive/total pGSK-3β/total GSK-3β was reduced in haloperidol treated mice, suggesting an activation of the kinase. The insert displays typical gel lanes from immunoblots used for quantitative analyses with ImageJ.

    Article Snippet: Once transferred, membranes were probed with antibodies to glycogen synthase kinase (GSK)-3β (cell signaling #9315); Phospho GSK-3β (Ser9) (cell signaling #5558); Phospho-AMPKα (Thr172); and AMPKα (cell signaling #2603).

    Techniques: Western Blot, Mouse Assay, Activation Assay

    Attenuation of isoflurane post-treatment-induced Akt signaling by inhibition of Akt activation in RD-fed mouse hippocampus. Hippocampal slices were freshly prepared from 16-week old CD-1 mice that fed RD. The slices were subjected to a 20-min OGD and then exposed to 3% isoflurane (Iso) for 30 min. LY 294002 (LY) was presented from the beginning of OGD to the end of isoflurane exposure. Slices were harvested at 5 h after the OGD for Western blotting. A representative Western blotting image is presented at the top panel and the pooled results are presented in the bottom panel for each protein. A: CTMP. B: Akt and phospho-Akt (P-Akt). C: GSK-3β and phospho-GSK-3β (P-GSK-3β). D: FoxO3a and phospho-FoxO3a (P-FoxO3a). Results are mean ± S.E.M. (n = 8). * P

    Journal: Obesity (Silver Spring, Md.)

    Article Title: High fat diet reduces neuroprotection of isoflurane post-treatment: role of carboxyl-terminal modulator protein-Akt signaling

    doi: 10.1002/oby.20879

    Figure Lengend Snippet: Attenuation of isoflurane post-treatment-induced Akt signaling by inhibition of Akt activation in RD-fed mouse hippocampus. Hippocampal slices were freshly prepared from 16-week old CD-1 mice that fed RD. The slices were subjected to a 20-min OGD and then exposed to 3% isoflurane (Iso) for 30 min. LY 294002 (LY) was presented from the beginning of OGD to the end of isoflurane exposure. Slices were harvested at 5 h after the OGD for Western blotting. A representative Western blotting image is presented at the top panel and the pooled results are presented in the bottom panel for each protein. A: CTMP. B: Akt and phospho-Akt (P-Akt). C: GSK-3β and phospho-GSK-3β (P-GSK-3β). D: FoxO3a and phospho-FoxO3a (P-FoxO3a). Results are mean ± S.E.M. (n = 8). * P

    Article Snippet: Membranes were incubated with the following primary antibodies overnight at 4°C: the anti-CTMP antibody (1:1000, catalog number: SAB3500057; Sigma-Aldrich, St. Louis, MO), anti-Akt antibody (1:1000, catalog number: 9272; Cell Signaling Technology, Danvers, MA), anti-phospho-Akt (Ser473) antibody (1:1000, catalog number: 9271; Cell Signaling Technology), anti-glycogen synthase kinase 3β (GSK-3β) antibody (1:1000, catalog number: 9315; Cell Signaling Technology), anti-phospho-GSK-3β (Ser9) antibody (1:1000, catalog number: 9336, Cell Signaling Technology), anti-Forkhead box O3 (FoxO3a) antibody (1:1000, catalog number: 2497; Cell Signaling Technology), anti-phospho-FoxO3a (Ser253) antibody (1:1000, catalog number: 9466; Cell Signaling Technology), anti-β-actin polyclonal antibody (1:2000; catalog number: A2228; Sigma-Aldrich), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:5000, catalog number: G9545; Sigma-Aldrich).

    Techniques: Inhibition, Activation Assay, Mouse Assay, Western Blot

    HFD-induced reduction of Akt signaling. Hippocampus was harvested from 11- or 16-week old CD-1 mice that fed HFD or RD for 5 or 10 weeks, respectively. The hippocampus was used for Western blotting. A representative Western blotting image is presented at the top panel and the pooled results are presented in the bottom panel for each protein. A: CTMP. B: Akt and phospho-Akt (P-Akt). C: GSK-3β and phospho-GSK-3β (P-GSK-3β). D: FoxO3a and phospho-FoxO3a (P-FoxO3a). Results are mean ± S.E.M. (n = 6 – 8). * P

    Journal: Obesity (Silver Spring, Md.)

    Article Title: High fat diet reduces neuroprotection of isoflurane post-treatment: role of carboxyl-terminal modulator protein-Akt signaling

    doi: 10.1002/oby.20879

    Figure Lengend Snippet: HFD-induced reduction of Akt signaling. Hippocampus was harvested from 11- or 16-week old CD-1 mice that fed HFD or RD for 5 or 10 weeks, respectively. The hippocampus was used for Western blotting. A representative Western blotting image is presented at the top panel and the pooled results are presented in the bottom panel for each protein. A: CTMP. B: Akt and phospho-Akt (P-Akt). C: GSK-3β and phospho-GSK-3β (P-GSK-3β). D: FoxO3a and phospho-FoxO3a (P-FoxO3a). Results are mean ± S.E.M. (n = 6 – 8). * P

    Article Snippet: Membranes were incubated with the following primary antibodies overnight at 4°C: the anti-CTMP antibody (1:1000, catalog number: SAB3500057; Sigma-Aldrich, St. Louis, MO), anti-Akt antibody (1:1000, catalog number: 9272; Cell Signaling Technology, Danvers, MA), anti-phospho-Akt (Ser473) antibody (1:1000, catalog number: 9271; Cell Signaling Technology), anti-glycogen synthase kinase 3β (GSK-3β) antibody (1:1000, catalog number: 9315; Cell Signaling Technology), anti-phospho-GSK-3β (Ser9) antibody (1:1000, catalog number: 9336, Cell Signaling Technology), anti-Forkhead box O3 (FoxO3a) antibody (1:1000, catalog number: 2497; Cell Signaling Technology), anti-phospho-FoxO3a (Ser253) antibody (1:1000, catalog number: 9466; Cell Signaling Technology), anti-β-actin polyclonal antibody (1:2000; catalog number: A2228; Sigma-Aldrich), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:5000, catalog number: G9545; Sigma-Aldrich).

    Techniques: Mouse Assay, Western Blot

    Attenuation of isoflurane post-treatment-induced Akt signaling by inhibition of Akt activation in HFD-fed mouse hippocampus. Hippocampal slices were freshly prepared from 16-week old CD-1 mice that fed HFD for 10 weeks. The slices were subjected to a 20-min OGD and then exposed to 3% isoflurane (Iso) for 30 min. LY 294002 (LY) was presented from the beginning of OGD to the end of isoflurane exposure. Slices were harvested at 5 h after the OGD for Western blotting. A representative Western blotting image is presented at the top panel and the pooled results are presented in the bottom panel for each protein. A: CTMP. B: Akt and phospho-Akt (P-Akt). C: GSK-3β and phospho-GSK-3β (P-GSK-3β). D: FoxO3a and phospho-FoxO3a (P-FoxO3a). Results are mean ± S.E.M. (n = 8). * P

    Journal: Obesity (Silver Spring, Md.)

    Article Title: High fat diet reduces neuroprotection of isoflurane post-treatment: role of carboxyl-terminal modulator protein-Akt signaling

    doi: 10.1002/oby.20879

    Figure Lengend Snippet: Attenuation of isoflurane post-treatment-induced Akt signaling by inhibition of Akt activation in HFD-fed mouse hippocampus. Hippocampal slices were freshly prepared from 16-week old CD-1 mice that fed HFD for 10 weeks. The slices were subjected to a 20-min OGD and then exposed to 3% isoflurane (Iso) for 30 min. LY 294002 (LY) was presented from the beginning of OGD to the end of isoflurane exposure. Slices were harvested at 5 h after the OGD for Western blotting. A representative Western blotting image is presented at the top panel and the pooled results are presented in the bottom panel for each protein. A: CTMP. B: Akt and phospho-Akt (P-Akt). C: GSK-3β and phospho-GSK-3β (P-GSK-3β). D: FoxO3a and phospho-FoxO3a (P-FoxO3a). Results are mean ± S.E.M. (n = 8). * P

    Article Snippet: Membranes were incubated with the following primary antibodies overnight at 4°C: the anti-CTMP antibody (1:1000, catalog number: SAB3500057; Sigma-Aldrich, St. Louis, MO), anti-Akt antibody (1:1000, catalog number: 9272; Cell Signaling Technology, Danvers, MA), anti-phospho-Akt (Ser473) antibody (1:1000, catalog number: 9271; Cell Signaling Technology), anti-glycogen synthase kinase 3β (GSK-3β) antibody (1:1000, catalog number: 9315; Cell Signaling Technology), anti-phospho-GSK-3β (Ser9) antibody (1:1000, catalog number: 9336, Cell Signaling Technology), anti-Forkhead box O3 (FoxO3a) antibody (1:1000, catalog number: 2497; Cell Signaling Technology), anti-phospho-FoxO3a (Ser253) antibody (1:1000, catalog number: 9466; Cell Signaling Technology), anti-β-actin polyclonal antibody (1:2000; catalog number: A2228; Sigma-Aldrich), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:5000, catalog number: G9545; Sigma-Aldrich).

    Techniques: Inhibition, Activation Assay, Mouse Assay, Western Blot

    Effect of paclitaxel stimulation and EphA2 overexpression on activation of the PI3K/Akt signalling pathway in NPC 5-8F cells. (A) NPC 5-8F cells were treated with varying concentrations of paclitaxel and PI3K/Akt pathway signalling molecules (total Akt, p-Akt) were measured by western blot analysis. (B) EphA2 over-expression stimulates the PI3K/Akt pathway. The total quantity Akt, p-Akt, GSK-3β and p-GSK-3β was determined by western blot analysis. EphA2, ephrin type-A receptor 2; PI3K, phosphoinositide 3-kinase; p-Akt, phosphorylated Akt; GSK-3β, glycogen synthase kinase-3β; p-GSK-3β, phosphorylated-GSK-3β.

    Journal: Molecular Medicine Reports

    Article Title: Ephrin type-A receptor 2 regulates sensitivity to paclitaxel in nasopharyngeal carcinoma via the phosphoinositide 3-kinase/Akt signalling pathway

    doi: 10.3892/mmr.2014.2799

    Figure Lengend Snippet: Effect of paclitaxel stimulation and EphA2 overexpression on activation of the PI3K/Akt signalling pathway in NPC 5-8F cells. (A) NPC 5-8F cells were treated with varying concentrations of paclitaxel and PI3K/Akt pathway signalling molecules (total Akt, p-Akt) were measured by western blot analysis. (B) EphA2 over-expression stimulates the PI3K/Akt pathway. The total quantity Akt, p-Akt, GSK-3β and p-GSK-3β was determined by western blot analysis. EphA2, ephrin type-A receptor 2; PI3K, phosphoinositide 3-kinase; p-Akt, phosphorylated Akt; GSK-3β, glycogen synthase kinase-3β; p-GSK-3β, phosphorylated-GSK-3β.

    Article Snippet: Rabbit Akt, phosphor-Akt (p-Akt), p21, cyclin-dependent kinase 2 (CDK2) and Cyclin E monoclonal antibodies, and mouse p27, retinoblastoma protein (Rb), phosphor-Rb, glycogen synthase kinase-3β (GSK-3β) and phosphor-GSK-3β monoclonal antibodies, and the PI3K/Akt signalling pathway small molecule inhibitor, LY294002, were obtained from Cell Signaling Technology, Inc., (Danvers, MA, USA).

    Techniques: Over Expression, Activation Assay, Western Blot

    PARP inhibitors interfere with EndoMT, EMT and vasculogenic mimicry in melanoma cells. Vimentin down-regulation is pivotal in driving this effect of PARP inhibitors, acting through the ILK/GSk-3β (see the text). While VE-cadherin is upregulated by PARP inhibitors in endothelial cells, contributing to vascular normalisation, its levels are down-regulated in malignant melanoma cells ( Figure 5C ). The ultimate reason for this cell-specific regulation of VE-cadherin expression by PARP is being studying currently in our laboratory.

    Journal: PLoS Genetics

    Article Title: PARP-1 Regulates Metastatic Melanoma through Modulation of Vimentin-induced Malignant Transformation

    doi: 10.1371/journal.pgen.1003531

    Figure Lengend Snippet: PARP inhibitors interfere with EndoMT, EMT and vasculogenic mimicry in melanoma cells. Vimentin down-regulation is pivotal in driving this effect of PARP inhibitors, acting through the ILK/GSk-3β (see the text). While VE-cadherin is upregulated by PARP inhibitors in endothelial cells, contributing to vascular normalisation, its levels are down-regulated in malignant melanoma cells ( Figure 5C ). The ultimate reason for this cell-specific regulation of VE-cadherin expression by PARP is being studying currently in our laboratory.

    Article Snippet: Primary antibodies used in these studies consisted of vimentin and VE-cadherin (mouse monoclonal), E-cadherin (rabbit polyclonal) (Santa Cruz Biotechnology), Snail1 and pVE-cadherin (rabbit polyclonal) (Abcam), ILK (rabbit monoclonal) (Millipore), Axl (rabbit polyclonal), total-GSK-3β (mouse monoclonal) and pGSK-3β (rabbit monoclonal) (Cell Signaling), β-catenin (mouse monoclonal) (BD Transduction Laboratories), PARP-1 (monoclonal) (Alexis) as well as β-actin (Sigma Aldrich).

    Techniques: Expressing

    PARP-1 or vimentin is sufficient to reverse EMT and confer increased cell motility. ( A ) Melanoma (G361) and endothelial (HUVEC) ( B ) cells were silenced for PARP-1 or vimentin and the expression levels of Axl, E-/VE-cadherin, Snail1, ILK, β-catenin, GSK-3β, PARP-1, and vimentin were determined by immunoblot. ( C ) HUVEC were silenced for vimentin and wound healing was measured. After over-expression of vimentin wound healing closure was measured in HUVEC cells ( D ) or B16-F10 ( E ). ( F ) Cell migration was analyzed in epithelial cell line Madin Darby canine kidney (MDCK) cells transfected with either GFP or GFP-vimentin using video-microscopy and MetaMorph Image Analysis software. While vimentin was able to increase the length of the trajectories in the absence or presence of hepatocyte growth factor (HGF), treatment with PARP inhibitor resulted in a sustained reduction in cell motility (* P

    Journal: PLoS Genetics

    Article Title: PARP-1 Regulates Metastatic Melanoma through Modulation of Vimentin-induced Malignant Transformation

    doi: 10.1371/journal.pgen.1003531

    Figure Lengend Snippet: PARP-1 or vimentin is sufficient to reverse EMT and confer increased cell motility. ( A ) Melanoma (G361) and endothelial (HUVEC) ( B ) cells were silenced for PARP-1 or vimentin and the expression levels of Axl, E-/VE-cadherin, Snail1, ILK, β-catenin, GSK-3β, PARP-1, and vimentin were determined by immunoblot. ( C ) HUVEC were silenced for vimentin and wound healing was measured. After over-expression of vimentin wound healing closure was measured in HUVEC cells ( D ) or B16-F10 ( E ). ( F ) Cell migration was analyzed in epithelial cell line Madin Darby canine kidney (MDCK) cells transfected with either GFP or GFP-vimentin using video-microscopy and MetaMorph Image Analysis software. While vimentin was able to increase the length of the trajectories in the absence or presence of hepatocyte growth factor (HGF), treatment with PARP inhibitor resulted in a sustained reduction in cell motility (* P

    Article Snippet: Primary antibodies used in these studies consisted of vimentin and VE-cadherin (mouse monoclonal), E-cadherin (rabbit polyclonal) (Santa Cruz Biotechnology), Snail1 and pVE-cadherin (rabbit polyclonal) (Abcam), ILK (rabbit monoclonal) (Millipore), Axl (rabbit polyclonal), total-GSK-3β (mouse monoclonal) and pGSK-3β (rabbit monoclonal) (Cell Signaling), β-catenin (mouse monoclonal) (BD Transduction Laboratories), PARP-1 (monoclonal) (Alexis) as well as β-actin (Sigma Aldrich).

    Techniques: Expressing, Over Expression, Migration, Transfection, Microscopy, Software

    Interaction between vimentin over-expression and the activation of EMT signaling pathway. ( A ) Over-expression of vimentin in G361 cells. ( B ) Forced expression of vimentin drives human breast tumor epithelial cells (MCF7) to a mesenchymal phenotype through the integrin-linked-kinase/GSK-3β axis. 5 mM LiCl was used to inhibit GSK-3β, as detected by the accumulation of beta-catenin. ( C ) ILK was knocked down to analyze the significance of the interaction between vimentin and ILK in promoting the transition to a mesenchymal phenotype.

    Journal: PLoS Genetics

    Article Title: PARP-1 Regulates Metastatic Melanoma through Modulation of Vimentin-induced Malignant Transformation

    doi: 10.1371/journal.pgen.1003531

    Figure Lengend Snippet: Interaction between vimentin over-expression and the activation of EMT signaling pathway. ( A ) Over-expression of vimentin in G361 cells. ( B ) Forced expression of vimentin drives human breast tumor epithelial cells (MCF7) to a mesenchymal phenotype through the integrin-linked-kinase/GSK-3β axis. 5 mM LiCl was used to inhibit GSK-3β, as detected by the accumulation of beta-catenin. ( C ) ILK was knocked down to analyze the significance of the interaction between vimentin and ILK in promoting the transition to a mesenchymal phenotype.

    Article Snippet: Primary antibodies used in these studies consisted of vimentin and VE-cadherin (mouse monoclonal), E-cadherin (rabbit polyclonal) (Santa Cruz Biotechnology), Snail1 and pVE-cadherin (rabbit polyclonal) (Abcam), ILK (rabbit monoclonal) (Millipore), Axl (rabbit polyclonal), total-GSK-3β (mouse monoclonal) and pGSK-3β (rabbit monoclonal) (Cell Signaling), β-catenin (mouse monoclonal) (BD Transduction Laboratories), PARP-1 (monoclonal) (Alexis) as well as β-actin (Sigma Aldrich).

    Techniques: Over Expression, Activation Assay, Expressing

    RNA-bound HuD interacts with active Akt1 and retains its kinase activity. ( A ) RNA-bound HuD interacts with active Akt1. Active or inactive Akt1 was incubated with GST, GST-HuD or GST-HuR, pulled down with poly(U)-sepharose beads and analyzed by IB and for pull-down efficiency (lower panel) by IB. ( B ) Akt1 retains its protein kinase activity in the Akt1–HuD–RNA complex. Active Akt1 was incubated with GST-HuD, GST-HuR or GST and then pulled down with poly(U)-sepharose beads. Protein kinase assays were performed using a GSK-3β peptide, which is a specific substrate for Akt1. Controls are standard assays with ‘no enzyme’, ‘inactive Akt1’ or ‘active Akt1’. In the pull-down lanes approximately 10% of Akt1 has been used for the kinase assay as estimated by western blot analysis (data not shown).

    Journal: Nucleic Acids Research

    Article Title: Functional and direct interaction between the RNA binding protein HuD and active Akt1

    doi: 10.1093/nar/gkr979

    Figure Lengend Snippet: RNA-bound HuD interacts with active Akt1 and retains its kinase activity. ( A ) RNA-bound HuD interacts with active Akt1. Active or inactive Akt1 was incubated with GST, GST-HuD or GST-HuR, pulled down with poly(U)-sepharose beads and analyzed by IB and for pull-down efficiency (lower panel) by IB. ( B ) Akt1 retains its protein kinase activity in the Akt1–HuD–RNA complex. Active Akt1 was incubated with GST-HuD, GST-HuR or GST and then pulled down with poly(U)-sepharose beads. Protein kinase assays were performed using a GSK-3β peptide, which is a specific substrate for Akt1. Controls are standard assays with ‘no enzyme’, ‘inactive Akt1’ or ‘active Akt1’. In the pull-down lanes approximately 10% of Akt1 has been used for the kinase assay as estimated by western blot analysis (data not shown).

    Article Snippet: GSK-3β protein kinase assay The enzyme activity of Akt1 was assayed by measuring the incorporation of radioactivity from [γ-32 P] ATP to glycogen synthase kinase 3β (GSK-3β) fusion protein (cell signaling), a synthetic substrate specific to Akt.

    Techniques: Activity Assay, Incubation, Kinase Assay, Western Blot

    β-Catenin suppression and GSK-3β activation in lungs of IAV-infected mice. A1, change in body weight of infected and uninfected mice (five mice in each group). A2, change in viral NP levels in the lungs monitored by western immunoblotting (B). One infected animal died on day 6 postinfection. The values represent the mean ± SD of five or four mice. B and C, Data represent the expression levels (B) and relative quantified data (C) of β-catenin, VE-cadherin, GSK-3β, and phospho-GSK-3β (Ser9) in lung extracts (20 μg protein/lane) of control (no-infection) and IAV-infected mice from day 1 to day 6 postinfection. C.S., calibration standard (mixture of uninfected sample except for NP analysis) to normalize the intensity of protein bands in different gels. As the C.S. for NP, a sample from one animal after infection for 4 days was used. β-Actin was used as an internal control. Data are representative of three separate experiments. Multiple comparison (Dunnett test) after ANOVA were used for statistical analysis. P -values are relative to the control (no infection)

    Journal: Archives of Virology

    Article Title: Influenza A virus infection of vascular endothelial cells induces GSK-3β-mediated β-catenin degradation in adherens junctions, with a resultant increase in membrane permeability

    doi: 10.1007/s00705-014-2270-5

    Figure Lengend Snippet: β-Catenin suppression and GSK-3β activation in lungs of IAV-infected mice. A1, change in body weight of infected and uninfected mice (five mice in each group). A2, change in viral NP levels in the lungs monitored by western immunoblotting (B). One infected animal died on day 6 postinfection. The values represent the mean ± SD of five or four mice. B and C, Data represent the expression levels (B) and relative quantified data (C) of β-catenin, VE-cadherin, GSK-3β, and phospho-GSK-3β (Ser9) in lung extracts (20 μg protein/lane) of control (no-infection) and IAV-infected mice from day 1 to day 6 postinfection. C.S., calibration standard (mixture of uninfected sample except for NP analysis) to normalize the intensity of protein bands in different gels. As the C.S. for NP, a sample from one animal after infection for 4 days was used. β-Actin was used as an internal control. Data are representative of three separate experiments. Multiple comparison (Dunnett test) after ANOVA were used for statistical analysis. P -values are relative to the control (no infection)

    Article Snippet: After blocking with 5 % non-fat milk in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.05 % Tween 20, the membranes were incubated with optimal primary antibodies for 2 h. The primary antibodies included anti-β-catenin, anti-VE-cadherin, anti-β-actin and anti-glycogen synthase kinase (GSK)-3β (all from Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-GSK-3β (Ser9) (Cell Signaling Technology, Beverly, MA) and anti-IAV (Takara).

    Techniques: Activation Assay, Infection, Mouse Assay, Western Blot, Expressing

    Diagram illustrating the proposed mechanism of disruption of the adherens junctional complexes of vascular endothelial cells after severe IAV infection. Infection of vascular endothelial cells with IAV decreases β-catenin in the VE-cadherin-β-catenin adhesive complex by activation of GSK-3β and the phosphorylation-dependent ubiquitin-proteasome degradation pathway. TLR, Toll-like receptor; dsDNA, double-stranded DNA; ssRNA, single-stranded RNA. The question mark indicates that signal transductions between TLRs and GSK-3β are unidentified

    Journal: Archives of Virology

    Article Title: Influenza A virus infection of vascular endothelial cells induces GSK-3β-mediated β-catenin degradation in adherens junctions, with a resultant increase in membrane permeability

    doi: 10.1007/s00705-014-2270-5

    Figure Lengend Snippet: Diagram illustrating the proposed mechanism of disruption of the adherens junctional complexes of vascular endothelial cells after severe IAV infection. Infection of vascular endothelial cells with IAV decreases β-catenin in the VE-cadherin-β-catenin adhesive complex by activation of GSK-3β and the phosphorylation-dependent ubiquitin-proteasome degradation pathway. TLR, Toll-like receptor; dsDNA, double-stranded DNA; ssRNA, single-stranded RNA. The question mark indicates that signal transductions between TLRs and GSK-3β are unidentified

    Article Snippet: After blocking with 5 % non-fat milk in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.05 % Tween 20, the membranes were incubated with optimal primary antibodies for 2 h. The primary antibodies included anti-β-catenin, anti-VE-cadherin, anti-β-actin and anti-glycogen synthase kinase (GSK)-3β (all from Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-GSK-3β (Ser9) (Cell Signaling Technology, Beverly, MA) and anti-IAV (Takara).

    Techniques: Infection, Activation Assay

    GSK-3β knockdown protects against IAV-induced suppression of β-catenin. A. Representative immunoblots (from three separate experiments) of GSK-3β and β-catenin in cell lysates (20 μg protein/lane) of HUVECs pre-treated with or without GSK-3β knockdown under control (no-infection) and IAV-infection conditions. B. Relative levels of GSK-3β and β-catenin in the bolt in panel A (n = 3). Differences between groups were analyzed using the unpaired t -test

    Journal: Archives of Virology

    Article Title: Influenza A virus infection of vascular endothelial cells induces GSK-3β-mediated β-catenin degradation in adherens junctions, with a resultant increase in membrane permeability

    doi: 10.1007/s00705-014-2270-5

    Figure Lengend Snippet: GSK-3β knockdown protects against IAV-induced suppression of β-catenin. A. Representative immunoblots (from three separate experiments) of GSK-3β and β-catenin in cell lysates (20 μg protein/lane) of HUVECs pre-treated with or without GSK-3β knockdown under control (no-infection) and IAV-infection conditions. B. Relative levels of GSK-3β and β-catenin in the bolt in panel A (n = 3). Differences between groups were analyzed using the unpaired t -test

    Article Snippet: After blocking with 5 % non-fat milk in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.05 % Tween 20, the membranes were incubated with optimal primary antibodies for 2 h. The primary antibodies included anti-β-catenin, anti-VE-cadherin, anti-β-actin and anti-glycogen synthase kinase (GSK)-3β (all from Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-GSK-3β (Ser9) (Cell Signaling Technology, Beverly, MA) and anti-IAV (Takara).

    Techniques: Western Blot, Infection

    IAV infection induces suppression of phosphorylated GSK-3β in HUVECs. A. Representative immunoblots (from three separate experiments) of total GSK-3β and phosphor-GSK-3β (Ser9) in cell lysates (20 μg protein/lane) of control (uninfected) or IAV-infected HUVECs. B. Relative levels of total GSK-3β and phosphor-GSK-3β (Ser9) in the blot in panel A (n = 3). Data are mean ± SD. Statistical analyses were conducted using the unpaired t -test

    Journal: Archives of Virology

    Article Title: Influenza A virus infection of vascular endothelial cells induces GSK-3β-mediated β-catenin degradation in adherens junctions, with a resultant increase in membrane permeability

    doi: 10.1007/s00705-014-2270-5

    Figure Lengend Snippet: IAV infection induces suppression of phosphorylated GSK-3β in HUVECs. A. Representative immunoblots (from three separate experiments) of total GSK-3β and phosphor-GSK-3β (Ser9) in cell lysates (20 μg protein/lane) of control (uninfected) or IAV-infected HUVECs. B. Relative levels of total GSK-3β and phosphor-GSK-3β (Ser9) in the blot in panel A (n = 3). Data are mean ± SD. Statistical analyses were conducted using the unpaired t -test

    Article Snippet: After blocking with 5 % non-fat milk in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.05 % Tween 20, the membranes were incubated with optimal primary antibodies for 2 h. The primary antibodies included anti-β-catenin, anti-VE-cadherin, anti-β-actin and anti-glycogen synthase kinase (GSK)-3β (all from Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-GSK-3β (Ser9) (Cell Signaling Technology, Beverly, MA) and anti-IAV (Takara).

    Techniques: Infection, Western Blot

    GSK-3β-mediated GR phosphorylation redirects gene transcription. ANOVA results from microarray analysis were loaded into Ingenuity Pathways analysis software to compare the biological pathway regulation in WT- and S404A-GR microarray samples. The most statistically significant biological pathways regulated by WT-GR (A) and S404A-GR (B) are shown. The shade of red (induction) or green (repression), lighter to darker, signifies the least to the greatest degree of gene induction or repression, respectively.

    Journal: Molecular and Cellular Biology

    Article Title: Glycogen Synthase Kinase 3?-Mediated Serine Phosphorylation of the Human Glucocorticoid Receptor Redirects Gene Expression Profiles ▿

    doi: 10.1128/MCB.00808-08

    Figure Lengend Snippet: GSK-3β-mediated GR phosphorylation redirects gene transcription. ANOVA results from microarray analysis were loaded into Ingenuity Pathways analysis software to compare the biological pathway regulation in WT- and S404A-GR microarray samples. The most statistically significant biological pathways regulated by WT-GR (A) and S404A-GR (B) are shown. The shade of red (induction) or green (repression), lighter to darker, signifies the least to the greatest degree of gene induction or repression, respectively.

    Article Snippet: For in vitro kinase assays, GR immunocomplexes were incubated with active GSK-3β (3 ng; Chemicon), 20 mM MOPS (morpholinepropanesulfonic acid), 10 mM magnesium acetate, 100 μM ATP, and 1 μCi of γ-32 P for 10 min at 30°C.

    Techniques: Microarray, Software

    GSK-3β phosphorylates GR on Ser404. (A) Alignment of human GR sequence with the consensus site for GSK-3 kinase substrates. (B and C) U-2 OS cells stably expressing WT-GR or S404A-GR were pretreated with 0 to 5 μM concentrations of the GSK3α/β inhibitor BIO for 1 h before incubation with 100 nM Dex for 1 h. Afterward, cell extracts were analyzed by Western blot analysis to determine the amount of Ser211 and Ser404 phosphorylation of the GR. (D) U-2 OS cells stably expressing WT-GR or A549 cells that endogenously express GR were treated with 100 nM Dex for 1 h. Cell lysates were immunoprecipitated using anti-GR antibodies, and immunoblots were probed with anti-GSK-3α and anti-GSK-3β antibodies. (E) WT- and S404A-GR were immunoprecipitated from the stable U-2 OS cell lines. Immunoprecipitates were incubated with active recombinant GSK-3β, and GSK-3β-mediated phosphorylation of GR was visualized by autoradiography. (F) WT-GR-expressing U-2 OS cells transiently overexpressing WT, constitutively active (S9A), or kinase-dead (Y216F) versions of GSK-3β were lysed and assayed by Western blotting to determine pSer404-GR levels (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Glycogen Synthase Kinase 3?-Mediated Serine Phosphorylation of the Human Glucocorticoid Receptor Redirects Gene Expression Profiles ▿

    doi: 10.1128/MCB.00808-08

    Figure Lengend Snippet: GSK-3β phosphorylates GR on Ser404. (A) Alignment of human GR sequence with the consensus site for GSK-3 kinase substrates. (B and C) U-2 OS cells stably expressing WT-GR or S404A-GR were pretreated with 0 to 5 μM concentrations of the GSK3α/β inhibitor BIO for 1 h before incubation with 100 nM Dex for 1 h. Afterward, cell extracts were analyzed by Western blot analysis to determine the amount of Ser211 and Ser404 phosphorylation of the GR. (D) U-2 OS cells stably expressing WT-GR or A549 cells that endogenously express GR were treated with 100 nM Dex for 1 h. Cell lysates were immunoprecipitated using anti-GR antibodies, and immunoblots were probed with anti-GSK-3α and anti-GSK-3β antibodies. (E) WT- and S404A-GR were immunoprecipitated from the stable U-2 OS cell lines. Immunoprecipitates were incubated with active recombinant GSK-3β, and GSK-3β-mediated phosphorylation of GR was visualized by autoradiography. (F) WT-GR-expressing U-2 OS cells transiently overexpressing WT, constitutively active (S9A), or kinase-dead (Y216F) versions of GSK-3β were lysed and assayed by Western blotting to determine pSer404-GR levels (*, P

    Article Snippet: For in vitro kinase assays, GR immunocomplexes were incubated with active GSK-3β (3 ng; Chemicon), 20 mM MOPS (morpholinepropanesulfonic acid), 10 mM magnesium acetate, 100 μM ATP, and 1 μCi of γ-32 P for 10 min at 30°C.

    Techniques: Sequencing, Stable Transfection, Expressing, Incubation, Western Blot, Immunoprecipitation, Recombinant, Autoradiography

    Phosphorylation of Ser404 of GR protects against Dex-dependent death in U-2 OS cells. (A) U-2 OS cells expressing WT-, S404A-, or S404D-GR were treated with Dex (100 nM) for 48 h. (B) Parental, WT-, or S404A-GR-expressing U-2 OS cells were treated with the GSK-3α/β inhibitor BIO (5 μM) and/or Dex (100 nM) for 24 h. (C and D) WT- or S404A-GR-expressing U-2 OS cells were transfected with control or shGSK-3 knockdown plasmids for 3 days. Cells were subsequently treated with Dex (100 nM) for 30 h. (A, B, and C) After the indicated treatments, the cells were resuspended in 10 mg of PI/ml. Dead cells were analyzed for PI incorporation on a flow cytometer. Western blot analysis was performed in parallel with PI staining to confirm GSK-3β protein knockdown and the level of Ser404 phosphorylation of GR (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Glycogen Synthase Kinase 3?-Mediated Serine Phosphorylation of the Human Glucocorticoid Receptor Redirects Gene Expression Profiles ▿

    doi: 10.1128/MCB.00808-08

    Figure Lengend Snippet: Phosphorylation of Ser404 of GR protects against Dex-dependent death in U-2 OS cells. (A) U-2 OS cells expressing WT-, S404A-, or S404D-GR were treated with Dex (100 nM) for 48 h. (B) Parental, WT-, or S404A-GR-expressing U-2 OS cells were treated with the GSK-3α/β inhibitor BIO (5 μM) and/or Dex (100 nM) for 24 h. (C and D) WT- or S404A-GR-expressing U-2 OS cells were transfected with control or shGSK-3 knockdown plasmids for 3 days. Cells were subsequently treated with Dex (100 nM) for 30 h. (A, B, and C) After the indicated treatments, the cells were resuspended in 10 mg of PI/ml. Dead cells were analyzed for PI incorporation on a flow cytometer. Western blot analysis was performed in parallel with PI staining to confirm GSK-3β protein knockdown and the level of Ser404 phosphorylation of GR (*, P

    Article Snippet: For in vitro kinase assays, GR immunocomplexes were incubated with active GSK-3β (3 ng; Chemicon), 20 mM MOPS (morpholinepropanesulfonic acid), 10 mM magnesium acetate, 100 μM ATP, and 1 μCi of γ-32 P for 10 min at 30°C.

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Western Blot, Staining

    The binding of hormone to the GR allows for DNA binding and the subsequent alteration of gene transcription. The phosphorylation of the GR on Ser404 by GSK-3β significant ly alters the GR-mediated transcriptional response. Shown is a cluster analysis of the microarray data to include the GC-regulated genes involved in the cell death pathway of WT GR (805 genes) and phospho-deficient GR (991 genes).

    Journal: Molecular and Cellular Biology

    Article Title: Glycogen Synthase Kinase 3?-Mediated Serine Phosphorylation of the Human Glucocorticoid Receptor Redirects Gene Expression Profiles ▿

    doi: 10.1128/MCB.00808-08

    Figure Lengend Snippet: The binding of hormone to the GR allows for DNA binding and the subsequent alteration of gene transcription. The phosphorylation of the GR on Ser404 by GSK-3β significant ly alters the GR-mediated transcriptional response. Shown is a cluster analysis of the microarray data to include the GC-regulated genes involved in the cell death pathway of WT GR (805 genes) and phospho-deficient GR (991 genes).

    Article Snippet: For in vitro kinase assays, GR immunocomplexes were incubated with active GSK-3β (3 ng; Chemicon), 20 mM MOPS (morpholinepropanesulfonic acid), 10 mM magnesium acetate, 100 μM ATP, and 1 μCi of γ-32 P for 10 min at 30°C.

    Techniques: Binding Assay, Microarray

    Repression of eIF2B guanine nucleotide exchange activity in response to simvastatin treatment is associated with altered phosphorylation of Akt, GSK-3β, and eIF2Bϵ. C 2 C 12 myoblasts were treated with vehicle (control) or 10 μM simvastatin for 24 h. A :lysates of control and simvastatin-treated myoblasts were subjected to eIF2B guanine nucleotide exchange activity assay. Results represent means ± SE of 3 experiments, each performed at least in triplicate, and are expressed as %mean control values. * P

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Simvastatin represses protein synthesis in the muscle-derived C2C12 cell line with a concomitant reduction in eukaryotic initiation factor 2B expression

    doi: 10.1152/ajpendo.00383.2010

    Figure Lengend Snippet: Repression of eIF2B guanine nucleotide exchange activity in response to simvastatin treatment is associated with altered phosphorylation of Akt, GSK-3β, and eIF2Bϵ. C 2 C 12 myoblasts were treated with vehicle (control) or 10 μM simvastatin for 24 h. A :lysates of control and simvastatin-treated myoblasts were subjected to eIF2B guanine nucleotide exchange activity assay. Results represent means ± SE of 3 experiments, each performed at least in triplicate, and are expressed as %mean control values. * P

    Article Snippet: Membranes were incubated overnight at 4°C with one of the following primary antibodies diluted in TBS-T: eIF2Bα (rabbit polyclonal generously provided by Glen N. Barber, University of Miami); eIF2Bβ, -γ, -δ, and -ϵ (mouse monoclonal); eIF2α [mouse monoclonal ( )]; eIF2Bϵ phospho-Ser535 and eIF2α phospho-Ser51 (rabbit polyclonal; Biosource-Invitrogen); Akt phospho-Ser473 and total (rabbit polyclonal; Cell Signaling Technology, Danvers, MA); GSK-3β total (rabbit polyclonal, Calbiochem-EMD Biosciences, La Jolla, CA); GSK-3β phospho-Ser9 (rabbit polyclonal, Cell Signaling); and GAPDH (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Activity Assay

    HSP90 increases PKM2 phosphorylation through GSK-3β. a Co-IP was performed for HSP90, PKM2 and GSK-3β to confirm whether these protein formed protein complex in Huh7 cells. b Huh7 cells transfected with HSP90 vector or control vector were treated with GSK3i IX, a GSK-3β inhibitor. Phosphorylation of PKM2 was examined by western blot after inhibiting GSK-3β activity. c Huh7 cells transfected with HSP90 vector or control vector were treated with GSK-3β siRNA to knockdown GSK-3β. Phosphorylation of PKM2 was examined by western blot after GSK-3β knockdown. d In vitro kinase assay to determine the effects of recombinant GSK3β on threonine phosphorylation of PKM2-WT or T328A

    Journal: Molecular Cancer

    Article Title: HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma

    doi: 10.1186/s12943-017-0748-y

    Figure Lengend Snippet: HSP90 increases PKM2 phosphorylation through GSK-3β. a Co-IP was performed for HSP90, PKM2 and GSK-3β to confirm whether these protein formed protein complex in Huh7 cells. b Huh7 cells transfected with HSP90 vector or control vector were treated with GSK3i IX, a GSK-3β inhibitor. Phosphorylation of PKM2 was examined by western blot after inhibiting GSK-3β activity. c Huh7 cells transfected with HSP90 vector or control vector were treated with GSK-3β siRNA to knockdown GSK-3β. Phosphorylation of PKM2 was examined by western blot after GSK-3β knockdown. d In vitro kinase assay to determine the effects of recombinant GSK3β on threonine phosphorylation of PKM2-WT or T328A

    Article Snippet: Glycogen synthase kinase-3β (GSK-3β) siRNA (sc-35,527) and control siRNA (sc-37,007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and were transfected into Huh7 cells using Lipofectamine® RNAiMAX (Thermo Fisher Scientific).

    Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Western Blot, Activity Assay, In Vitro, Kinase Assay, Recombinant

    Cotransfection of CGNs with GFP-Bax α and a constitutively active mutant of GSK-3β(S9A) induces translocation of the Bax fusion protein to mitochondria and triggers CGN apoptosis. A , CGNs were cotransfected with GFP-Bax α and either empty vector or HA-tagged GSK-3βS9A using the Helios Gene-Gun system, as described in Materials and Methods. At 48 hr after transfection, cotransfected cells were maintained in control medium and were visualized by staining for the epitope tag using an HA polyclonal antibody and a Cy3-conjugated secondary antibody. The images shown are composites of four to six fields captured with a 63× oil objective to give a representative view of the localization of the Bax fusion protein in the absence or presence of constitutively active GSK-3β. B , The areas demarcated by the boxes in A were enlarged 300% to show fine structure. Scale bars, 10 μm.

    Journal: The Journal of Neuroscience

    Article Title: Glycogen Synthase Kinase-3β Phosphorylates Bax and Promotes Its Mitochondrial Localization during Neuronal Apoptosis

    doi: 10.1523/JNEUROSCI.2057-04.2004

    Figure Lengend Snippet: Cotransfection of CGNs with GFP-Bax α and a constitutively active mutant of GSK-3β(S9A) induces translocation of the Bax fusion protein to mitochondria and triggers CGN apoptosis. A , CGNs were cotransfected with GFP-Bax α and either empty vector or HA-tagged GSK-3βS9A using the Helios Gene-Gun system, as described in Materials and Methods. At 48 hr after transfection, cotransfected cells were maintained in control medium and were visualized by staining for the epitope tag using an HA polyclonal antibody and a Cy3-conjugated secondary antibody. The images shown are composites of four to six fields captured with a 63× oil objective to give a representative view of the localization of the Bax fusion protein in the absence or presence of constitutively active GSK-3β. B , The areas demarcated by the boxes in A were enlarged 300% to show fine structure. Scale bars, 10 μm.

    Article Snippet: Recombinant, active GSK-3β was from Upstate Biotechnology (Charlottesville, VA).

    Techniques: Cotransfection, Mutagenesis, Translocation Assay, Plasmid Preparation, Transfection, Staining

    Fisetin suppresses PI3K-Akt-GSK-3β signal pathway but upregulates PTEN expression in vitro . TNBC cell lines MDA-MB-231 and BT549 were treated with vehicle or 30 μM fisetin for immunofluorescence assay and with vehicle or various concentrations of fisetin (10, 30, and 100 μM) for western blot and qRT-PCR. (A) The expression of p-AKT was evaluated by immunofluorescence. (B) The expression of PTEN was evaluated by immunofluorescence. (C) The expression of PTEN protein as well as p-AKT and p-GSK-3β was determined by western blot. (D) The expression of PTEN mRNA was determined by qRT-PCR. The results are shown as the mean ± SD of three experiments, ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Fisetin Inhibited Growth and Metastasis of Triple-Negative Breast Cancer by Reversing Epithelial-to-Mesenchymal Transition via PTEN/Akt/GSK3β Signal Pathway

    doi: 10.3389/fphar.2018.00772

    Figure Lengend Snippet: Fisetin suppresses PI3K-Akt-GSK-3β signal pathway but upregulates PTEN expression in vitro . TNBC cell lines MDA-MB-231 and BT549 were treated with vehicle or 30 μM fisetin for immunofluorescence assay and with vehicle or various concentrations of fisetin (10, 30, and 100 μM) for western blot and qRT-PCR. (A) The expression of p-AKT was evaluated by immunofluorescence. (B) The expression of PTEN was evaluated by immunofluorescence. (C) The expression of PTEN protein as well as p-AKT and p-GSK-3β was determined by western blot. (D) The expression of PTEN mRNA was determined by qRT-PCR. The results are shown as the mean ± SD of three experiments, ∗ P

    Article Snippet: Primary antibodies used in this study including anti-Vimentin, anti-E-cadherin, anti-Claudin, anti-N-cadherin, anti-Slug, anti-Snail, anti-PTEN, anti-p-Akt, anti-Akt, anti-p-GSK-3β were obtained from Cell Signaling Technology (New England Biolabs, Ipswich, MA, United States), anti-Ki67 was from Abcam (Cambridge, United Kingdom).

    Techniques: Expressing, In Vitro, Multiple Displacement Amplification, Immunofluorescence, Western Blot, Quantitative RT-PCR

    Silencing of PTEN abrogates the effects of fisetin on TNBC cells proliferation and metastasis as well as EMT. TNBC cell line MDA-MB-231 cells were transfected with Ad-RFP or Ad-siPTEN, and subsequently treated with fisetin (100 μM). (A) The expression of PTEN and p-AKT and p-GSK-3β protein was determined by western blot. (B) EMT molecule markers were determined by western blot. (C) The cell proliferation was determined by MTT assay. (D) The cell migration was determined by wound-healing assay. (E) The cell invasion was determined by transwell invasion assay. The results are shown as the mean ± SD of three experiments, ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Fisetin Inhibited Growth and Metastasis of Triple-Negative Breast Cancer by Reversing Epithelial-to-Mesenchymal Transition via PTEN/Akt/GSK3β Signal Pathway

    doi: 10.3389/fphar.2018.00772

    Figure Lengend Snippet: Silencing of PTEN abrogates the effects of fisetin on TNBC cells proliferation and metastasis as well as EMT. TNBC cell line MDA-MB-231 cells were transfected with Ad-RFP or Ad-siPTEN, and subsequently treated with fisetin (100 μM). (A) The expression of PTEN and p-AKT and p-GSK-3β protein was determined by western blot. (B) EMT molecule markers were determined by western blot. (C) The cell proliferation was determined by MTT assay. (D) The cell migration was determined by wound-healing assay. (E) The cell invasion was determined by transwell invasion assay. The results are shown as the mean ± SD of three experiments, ∗ P

    Article Snippet: Primary antibodies used in this study including anti-Vimentin, anti-E-cadherin, anti-Claudin, anti-N-cadherin, anti-Slug, anti-Snail, anti-PTEN, anti-p-Akt, anti-Akt, anti-p-GSK-3β were obtained from Cell Signaling Technology (New England Biolabs, Ipswich, MA, United States), anti-Ki67 was from Abcam (Cambridge, United Kingdom).

    Techniques: Multiple Displacement Amplification, Transfection, Expressing, Western Blot, MTT Assay, Migration, Wound Healing Assay, Transwell Invasion Assay

    Fisetin inhibits PI3K/AKT/GSK-3β signaling pathway and reverses EMT in vivo . Xenograft tumor metastasis was established by subcutaneous injection of MDA-MB-231 cells in the presence or absence of fisetin (100 mg/kg). (A) AKT activation was evaluated by immunohistochemistry staining. (B) Representative images of PTEN stained section of tumor tissues isolated from nude mice bearing breast cancer. (C) Vimentin and Snail in primary tumor tissues were examined by immunofluorescence assay. (D) PTEN and p-Akt and p-GSK-3β protein in the primary tumor tissues was examined by western blot. (E) EMT molecule markers were determined by western blot. The results are shown as the mean ± SD of six experiments, ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Fisetin Inhibited Growth and Metastasis of Triple-Negative Breast Cancer by Reversing Epithelial-to-Mesenchymal Transition via PTEN/Akt/GSK3β Signal Pathway

    doi: 10.3389/fphar.2018.00772

    Figure Lengend Snippet: Fisetin inhibits PI3K/AKT/GSK-3β signaling pathway and reverses EMT in vivo . Xenograft tumor metastasis was established by subcutaneous injection of MDA-MB-231 cells in the presence or absence of fisetin (100 mg/kg). (A) AKT activation was evaluated by immunohistochemistry staining. (B) Representative images of PTEN stained section of tumor tissues isolated from nude mice bearing breast cancer. (C) Vimentin and Snail in primary tumor tissues were examined by immunofluorescence assay. (D) PTEN and p-Akt and p-GSK-3β protein in the primary tumor tissues was examined by western blot. (E) EMT molecule markers were determined by western blot. The results are shown as the mean ± SD of six experiments, ∗ P

    Article Snippet: Primary antibodies used in this study including anti-Vimentin, anti-E-cadherin, anti-Claudin, anti-N-cadherin, anti-Slug, anti-Snail, anti-PTEN, anti-p-Akt, anti-Akt, anti-p-GSK-3β were obtained from Cell Signaling Technology (New England Biolabs, Ipswich, MA, United States), anti-Ki67 was from Abcam (Cambridge, United Kingdom).

    Techniques: In Vivo, Injection, Multiple Displacement Amplification, Activation Assay, Immunohistochemistry, Staining, Isolation, Mouse Assay, Immunofluorescence, Western Blot

    PRL-3 promotes MMP7 expression by activating the ERK pathway in diffuse glioma cells. A. LN229 cells were transfected with pcDNA-PRL-3, and the levels of Erk1/2 and phosphorylated Erk1/2 (p-Erk1/2) were detected by western blotting. B. MMP7 protein expression was increased following Erk1/2 activation. C and D. Both Erk1/2 activation and MMP7 expression were inhibited compared with the group without the inhibitor SCH772984 ( lane 2 ). E and F. Phosphorylated Akt and GSK-3β were analyzed by western blotting as described above. G. The transfected cells pretreated with the PI3K inhibitor LY294002 ( lane 3 ) were analyzed by western blotting to detect levels of Akt, p-Akt, GSK-3β and p-GSK-3β.

    Journal: Theranostics

    Article Title: PRL-3 is a potential glioblastoma prognostic marker and promotes glioblastoma progression by enhancing MMP7 through the ERK and JNK pathways

    doi: 10.7150/thno.22699

    Figure Lengend Snippet: PRL-3 promotes MMP7 expression by activating the ERK pathway in diffuse glioma cells. A. LN229 cells were transfected with pcDNA-PRL-3, and the levels of Erk1/2 and phosphorylated Erk1/2 (p-Erk1/2) were detected by western blotting. B. MMP7 protein expression was increased following Erk1/2 activation. C and D. Both Erk1/2 activation and MMP7 expression were inhibited compared with the group without the inhibitor SCH772984 ( lane 2 ). E and F. Phosphorylated Akt and GSK-3β were analyzed by western blotting as described above. G. The transfected cells pretreated with the PI3K inhibitor LY294002 ( lane 3 ) were analyzed by western blotting to detect levels of Akt, p-Akt, GSK-3β and p-GSK-3β.

    Article Snippet: Antibodies against PRL-3 (1:1000, Abcam, USA); Ki-67 (1:500, Abcam, USA); MMP7 (1:1000, Abcam, USA); p-Akt (Ser473), Akt, GSK-3β, p-GSK-3β (Ser9), Erk1/2, p-Erk1/2 (1:1000, Cell Signaling Technology, USA); and β-actin (1:5000, CWBIO, China) were applied for immunoblotting.

    Techniques: Expressing, Transfection, Western Blot, Activation Assay

    Acute stimulation of insulin signaling in mouse heart inhibits UCP3 expression. A , time course analysis of insulin signaling activation in the heart of overnight fasted mice ( n = 37). Representative immunoblots for the phosphorylation of Akt at Thr-308 and Ser-473, the phosphorylation of the direct Akt target GSK-3β at Ser-9, and the phosphorylation of the p44/42 MAPK at both Thr-202 and Tyr-204 are shown. β-Tubulin levels are shown as a loading control. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Chronic Hyperinsulinemia Causes Selective Insulin Resistance and Down-regulates Uncoupling Protein 3 (UCP3) through the Activation of Sterol Regulatory Element-binding Protein (SREBP)-1 Transcription Factor in the Mouse Heart *

    doi: 10.1074/jbc.M115.673988

    Figure Lengend Snippet: Acute stimulation of insulin signaling in mouse heart inhibits UCP3 expression. A , time course analysis of insulin signaling activation in the heart of overnight fasted mice ( n = 37). Representative immunoblots for the phosphorylation of Akt at Thr-308 and Ser-473, the phosphorylation of the direct Akt target GSK-3β at Ser-9, and the phosphorylation of the p44/42 MAPK at both Thr-202 and Tyr-204 are shown. β-Tubulin levels are shown as a loading control. *, p

    Article Snippet: The phospho-Akt Thr-308 (catalog no. 4056), phospho-Akt Ser-473 (catalog no. 4058), phospho-GSK-3β Ser-9 (catalog no. 9323), GSK-3β (catalog no. 9315), phospho-p44/42 MAPK Thr-202/Tyr-204 (catalog no. 4377), p44/42 MAPK (catalog no. 4695), phosphotuberin/TSC2 Ser-939 (catalog no. 3615), tuberin/TSC2 (catalog no. 3990), and voltage-dependent anion channel (VDAC) (catalog no. 4661) antibodies were from Cell Signaling Technology.

    Techniques: Expressing, Activation Assay, Mouse Assay, Western Blot

    Schematic diagram of the role of glycogen synthase kinase ( GSK )‐3β in the signaling pathways of the antitumor effect of nafamostat mesilate. The antitumor effect of nafamostat mesilate is in proportion to the level of phosphorylated GSK ‐3β (inactive form of GSK ‐3β) and nafamostat mesilate may enhance its antitumor effect by increasing phosphorylated GSK ‐3β. PP 2A inhibitor increases phosphorylated GSK ‐3β as a result of inhibition of activation of GSK ‐3β by PP 2A, and enhances the antitumor effect of nafamostat mesilate. There is an interaction between nuclear factor‐kappa B ( NF ‐κB) and GSK ‐3β

    Journal: Annals of Gastroenterological Surgery

    Article Title: Glycogen synthase kinase‐3β activity plays a key role in the antitumor effect of nafamostat mesilate in pancreatic cancer cells, et al. Glycogen synthase kinase‐3β activity plays a key role in the antitumor effect of nafamostat mesilate in pancreatic cancer cells

    doi: 10.1002/ags3.12025

    Figure Lengend Snippet: Schematic diagram of the role of glycogen synthase kinase ( GSK )‐3β in the signaling pathways of the antitumor effect of nafamostat mesilate. The antitumor effect of nafamostat mesilate is in proportion to the level of phosphorylated GSK ‐3β (inactive form of GSK ‐3β) and nafamostat mesilate may enhance its antitumor effect by increasing phosphorylated GSK ‐3β. PP 2A inhibitor increases phosphorylated GSK ‐3β as a result of inhibition of activation of GSK ‐3β by PP 2A, and enhances the antitumor effect of nafamostat mesilate. There is an interaction between nuclear factor‐kappa B ( NF ‐κB) and GSK ‐3β

    Article Snippet: 2.3 Antibodies Antibodies specific to phosphorylated AKT, phosphorylated GSK‐3β and cleaved caspase‐8 were obtained from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Inhibition, Activation Assay

    Levels of phosphorylated AKT and phosphorylated DVL in PANC ‐1 were greater than in MIAP aCa‐2. However, the level of phosphorylated GSK ‐3β was significantly greater in MIAP aCa‐2 than in PANC ‐1. Phosphorylated AKT was increased by FUT ‐175 in both cell lines, whereas phosphorylated DVL was decreased by FUT ‐175 in both cell lines

    Journal: Annals of Gastroenterological Surgery

    Article Title: Glycogen synthase kinase‐3β activity plays a key role in the antitumor effect of nafamostat mesilate in pancreatic cancer cells, et al. Glycogen synthase kinase‐3β activity plays a key role in the antitumor effect of nafamostat mesilate in pancreatic cancer cells

    doi: 10.1002/ags3.12025

    Figure Lengend Snippet: Levels of phosphorylated AKT and phosphorylated DVL in PANC ‐1 were greater than in MIAP aCa‐2. However, the level of phosphorylated GSK ‐3β was significantly greater in MIAP aCa‐2 than in PANC ‐1. Phosphorylated AKT was increased by FUT ‐175 in both cell lines, whereas phosphorylated DVL was decreased by FUT ‐175 in both cell lines

    Article Snippet: 2.3 Antibodies Antibodies specific to phosphorylated AKT, phosphorylated GSK‐3β and cleaved caspase‐8 were obtained from Cell Signaling Technology (Beverly, MA, USA).

    Techniques:

    Potential mechanism by D206008-mediated melanogenesis in B16 cells. Notes: D206008 activates Akt and GSK-3β via phosphorylation and decreases the phosphorylation level of β-catenin, which increases its accumulation in cytoplasm, and subsequent nuclear translocation. This leads to the upregulation of the melanogenic genes TYR , TRP-1 , and TRP-2 via MITF. D206008 promoted phosphorylation of Akt and led to increased intracellular p-GSK-3β expression, resulting in the accumulation of the β-catenin in the cytoplasm by inhibiting the phosphorylation of β-catenin, which translocated into the nucleus and activated the transcription of melanogenic genes TYR , TRP-1 , and TRP-2 via MITF. D206008 activates Akt and GSK-3β via phosphorylation and decreases the phosphorylation level of β-catenin, which increases its accumulation in cytoplasm, and subsequent nuclear translocation. This leads to the upregulation of the melanogenic genes TYR , TRP-1 , and TRP-2 via MITF. D206008-induced melanin synthesis was completely blocked by GSK-3β and Akt inhibitors. D206008-induced melanin synthesis was completely blocked by GSK-3β and Akt inhibitors. Abbreviations: BIO, 6-bromoindirubin-3 ′-oxime; D206008, 5-(morpholino methyl)- 3-phenyl-7 H -furo[3,2- g ]chromen-7-one; GSK-3β, glycogen synthase kinase-3β; MITF, microphthalmia-associated transcription factor; TYR, tyrosinase.

    Journal: Drug Design, Development and Therapy

    Article Title: Amine derivatives of furocoumarin induce melanogenesis by activating Akt/GSK-3β/β-catenin signal pathway

    doi: 10.2147/DDDT.S180960

    Figure Lengend Snippet: Potential mechanism by D206008-mediated melanogenesis in B16 cells. Notes: D206008 activates Akt and GSK-3β via phosphorylation and decreases the phosphorylation level of β-catenin, which increases its accumulation in cytoplasm, and subsequent nuclear translocation. This leads to the upregulation of the melanogenic genes TYR , TRP-1 , and TRP-2 via MITF. D206008 promoted phosphorylation of Akt and led to increased intracellular p-GSK-3β expression, resulting in the accumulation of the β-catenin in the cytoplasm by inhibiting the phosphorylation of β-catenin, which translocated into the nucleus and activated the transcription of melanogenic genes TYR , TRP-1 , and TRP-2 via MITF. D206008 activates Akt and GSK-3β via phosphorylation and decreases the phosphorylation level of β-catenin, which increases its accumulation in cytoplasm, and subsequent nuclear translocation. This leads to the upregulation of the melanogenic genes TYR , TRP-1 , and TRP-2 via MITF. D206008-induced melanin synthesis was completely blocked by GSK-3β and Akt inhibitors. D206008-induced melanin synthesis was completely blocked by GSK-3β and Akt inhibitors. Abbreviations: BIO, 6-bromoindirubin-3 ′-oxime; D206008, 5-(morpholino methyl)- 3-phenyl-7 H -furo[3,2- g ]chromen-7-one; GSK-3β, glycogen synthase kinase-3β; MITF, microphthalmia-associated transcription factor; TYR, tyrosinase.

    Article Snippet: Phosphor-Akt (Thr308, #5106), Akt (#5373), phosphor-GSK-3β (Ser9, #9323), GSK-3β (#9832), phosphor β-catenin (Ser 33,37,41#9561), phospho-β-catenin (Ser675, #4176), β-catenin (#8480), and NUP98 (#2598) were obtained from Cell Signaling Technology (Beverly, MA, USA), and the antibodies against β-actin and tyrosinase (C-19), TRP-1 (H-90), and TRP-2 (H-150) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

    Techniques: Translocation Assay, Expressing

    D206008 activates β-catenin signaling pathway via the phosphorylation of GSK-3β and Akt. Notes: ( A ) Cells were treated with different concentrations (0–100 µM) of D206008 for 48 hours, and the levels of p-Akt, p-GSK-3β, p-β-catenin, and β-catenin proteins were analyzed relative to β-actin expression. ( B ) B16 cells treated with either vehicle (0.1% DMSO) or D206008 (100 µM) for 48 hours were analyzed for the levels of nuclear and cytoplasmic β-catenin. Cytoplasmic protein levels were normalized against GAPDH and nuclear protein against nup98. The band densities of proteins were measured by the Quantity One program. The data are shown as mean ± SD and analyzed by one-way ANOVA followed by Tukey’s test. ** P

    Journal: Drug Design, Development and Therapy

    Article Title: Amine derivatives of furocoumarin induce melanogenesis by activating Akt/GSK-3β/β-catenin signal pathway

    doi: 10.2147/DDDT.S180960

    Figure Lengend Snippet: D206008 activates β-catenin signaling pathway via the phosphorylation of GSK-3β and Akt. Notes: ( A ) Cells were treated with different concentrations (0–100 µM) of D206008 for 48 hours, and the levels of p-Akt, p-GSK-3β, p-β-catenin, and β-catenin proteins were analyzed relative to β-actin expression. ( B ) B16 cells treated with either vehicle (0.1% DMSO) or D206008 (100 µM) for 48 hours were analyzed for the levels of nuclear and cytoplasmic β-catenin. Cytoplasmic protein levels were normalized against GAPDH and nuclear protein against nup98. The band densities of proteins were measured by the Quantity One program. The data are shown as mean ± SD and analyzed by one-way ANOVA followed by Tukey’s test. ** P

    Article Snippet: Phosphor-Akt (Thr308, #5106), Akt (#5373), phosphor-GSK-3β (Ser9, #9323), GSK-3β (#9832), phosphor β-catenin (Ser 33,37,41#9561), phospho-β-catenin (Ser675, #4176), β-catenin (#8480), and NUP98 (#2598) were obtained from Cell Signaling Technology (Beverly, MA, USA), and the antibodies against β-actin and tyrosinase (C-19), TRP-1 (H-90), and TRP-2 (H-150) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

    Techniques: Expressing

    Effect of Akt and GSK-3β inhibitors on D206008-induced melanogenesis. Notes: B16 cells were pretreated with 6-bromoindirubin-3′-oxime (BIO) (5 µM) or Akt inhibitor IV (1 µM) for 2 hours and then incubated with D206008 (100 µM) for ( A ) 48 hours to measure the melanin content or ( B ) 24 hours to measure TYR activity. Result shows that pretreating the cells with the specific GSK-3β inhibitor BIO increased the tyrosinase activity and melanin content. ## P

    Journal: Drug Design, Development and Therapy

    Article Title: Amine derivatives of furocoumarin induce melanogenesis by activating Akt/GSK-3β/β-catenin signal pathway

    doi: 10.2147/DDDT.S180960

    Figure Lengend Snippet: Effect of Akt and GSK-3β inhibitors on D206008-induced melanogenesis. Notes: B16 cells were pretreated with 6-bromoindirubin-3′-oxime (BIO) (5 µM) or Akt inhibitor IV (1 µM) for 2 hours and then incubated with D206008 (100 µM) for ( A ) 48 hours to measure the melanin content or ( B ) 24 hours to measure TYR activity. Result shows that pretreating the cells with the specific GSK-3β inhibitor BIO increased the tyrosinase activity and melanin content. ## P

    Article Snippet: Phosphor-Akt (Thr308, #5106), Akt (#5373), phosphor-GSK-3β (Ser9, #9323), GSK-3β (#9832), phosphor β-catenin (Ser 33,37,41#9561), phospho-β-catenin (Ser675, #4176), β-catenin (#8480), and NUP98 (#2598) were obtained from Cell Signaling Technology (Beverly, MA, USA), and the antibodies against β-actin and tyrosinase (C-19), TRP-1 (H-90), and TRP-2 (H-150) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

    Techniques: Incubation, Activity Assay

    (A–H) A significant increase in GKS-3β mediated inhibitory phosphorylation at serine 129 of CREB and concordant significant decreases in the amount of c-fos and ARC were observed in brain extracts from 15 month-old APP sw –tau vlw mice in comparison to similarly aged wt mice. These changes were fully rescued by pharmacological inhibition of GSK-3β in APP sw –tau vlw mice. Data are derived from n=3 animals per group. *p

    Journal: Neurobiology of disease

    Article Title: Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease , ☆☆Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease , ☆☆,

    doi: 10.1016/j.nbd.2011.09.002

    Figure Lengend Snippet: (A–H) A significant increase in GKS-3β mediated inhibitory phosphorylation at serine 129 of CREB and concordant significant decreases in the amount of c-fos and ARC were observed in brain extracts from 15 month-old APP sw –tau vlw mice in comparison to similarly aged wt mice. These changes were fully rescued by pharmacological inhibition of GSK-3β in APP sw –tau vlw mice. Data are derived from n=3 animals per group. *p

    Article Snippet: Primary antibodies for GSK-3α, GSK-3β, GSK-3α-pSer21, GSK-3β-pSer9 and CREB (CST, Danvers, MA), 129-pCREB and c-fos (Santa Cruz Biotechnology, Santa Cruz, CA), BDNF (Abcam, Cambridge, UK; Millipore, Billerica, MA; Santa Cruz Biotechnology, Santa Cruz, CA) and ARC (Santa Cruz Biotechnology, Santa Cruz, CA) were used.

    Techniques: Mouse Assay, Inhibition, Derivative Assay

    (A–H) Increased activity of GSK-3β triggered by exposure of neurons to Tg2576 CM media resulted in a significant increase in the inhibitory phosphorylation at serine 129 of CREB promoted by this enzyme and a significant decrease in the amount of BDNF. Treatment with TDZD-8 at 1 μm concentration significantly decreased the inhibitory phosphorylation at Ser 129 of CREB mediated by GSK-3β and restored the normal level of BDNF in neuronal cultures. Data are derived from 24 dishes per condition. *p

    Journal: Neurobiology of disease

    Article Title: Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease , ☆☆Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease , ☆☆,

    doi: 10.1016/j.nbd.2011.09.002

    Figure Lengend Snippet: (A–H) Increased activity of GSK-3β triggered by exposure of neurons to Tg2576 CM media resulted in a significant increase in the inhibitory phosphorylation at serine 129 of CREB promoted by this enzyme and a significant decrease in the amount of BDNF. Treatment with TDZD-8 at 1 μm concentration significantly decreased the inhibitory phosphorylation at Ser 129 of CREB mediated by GSK-3β and restored the normal level of BDNF in neuronal cultures. Data are derived from 24 dishes per condition. *p

    Article Snippet: Primary antibodies for GSK-3α, GSK-3β, GSK-3α-pSer21, GSK-3β-pSer9 and CREB (CST, Danvers, MA), 129-pCREB and c-fos (Santa Cruz Biotechnology, Santa Cruz, CA), BDNF (Abcam, Cambridge, UK; Millipore, Billerica, MA; Santa Cruz Biotechnology, Santa Cruz, CA) and ARC (Santa Cruz Biotechnology, Santa Cruz, CA) were used.

    Techniques: Activity Assay, Concentration Assay, Derivative Assay

    (A–D) GSK-3β levels in total homogenates (A, B) and nuclear fractions (C, D) prepared from the temporal cortex of AD samples were markedly decreased compared to those seen in control samples, as expected due to neuronal cell loss in AD. The ratio GSK-3β/pSer9GSK-3β was increased in the total homogenates from AD brains by 260%, and by 450% in the nuclear fraction when compared to controls indicating that GSK-3β is more active in AD brains (n=12 brains per group). *p

    Journal: Neurobiology of disease

    Article Title: Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease , ☆☆Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease , ☆☆,

    doi: 10.1016/j.nbd.2011.09.002

    Figure Lengend Snippet: (A–D) GSK-3β levels in total homogenates (A, B) and nuclear fractions (C, D) prepared from the temporal cortex of AD samples were markedly decreased compared to those seen in control samples, as expected due to neuronal cell loss in AD. The ratio GSK-3β/pSer9GSK-3β was increased in the total homogenates from AD brains by 260%, and by 450% in the nuclear fraction when compared to controls indicating that GSK-3β is more active in AD brains (n=12 brains per group). *p

    Article Snippet: Primary antibodies for GSK-3α, GSK-3β, GSK-3α-pSer21, GSK-3β-pSer9 and CREB (CST, Danvers, MA), 129-pCREB and c-fos (Santa Cruz Biotechnology, Santa Cruz, CA), BDNF (Abcam, Cambridge, UK; Millipore, Billerica, MA; Santa Cruz Biotechnology, Santa Cruz, CA) and ARC (Santa Cruz Biotechnology, Santa Cruz, CA) were used.

    Techniques:

    (A, B). Total level of GSK-3β in nuclear extracts did not significantly change in primary cultured neurons after 24 hour exposure to Tg2576 CM compared to wt CM. GSK-3β/pSer9GSK-3β ratio was increased by 30% indicating that oligomeric Aβ exposure triggers a significant increase in GSK-3β activity. 6E10 immunodepletion completely prevented the elevation of GSK-3β activity. Treatment of cultured neurons with TDZD-8 at 1 μM significantly decreased GSK-3β/pSer9GSK-3β ratio indicating that the enzyme is inhibited by this treatment. Data are derived from 24 dishes per condition. *p

    Journal: Neurobiology of disease

    Article Title: Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease , ☆☆Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease , ☆☆,

    doi: 10.1016/j.nbd.2011.09.002

    Figure Lengend Snippet: (A, B). Total level of GSK-3β in nuclear extracts did not significantly change in primary cultured neurons after 24 hour exposure to Tg2576 CM compared to wt CM. GSK-3β/pSer9GSK-3β ratio was increased by 30% indicating that oligomeric Aβ exposure triggers a significant increase in GSK-3β activity. 6E10 immunodepletion completely prevented the elevation of GSK-3β activity. Treatment of cultured neurons with TDZD-8 at 1 μM significantly decreased GSK-3β/pSer9GSK-3β ratio indicating that the enzyme is inhibited by this treatment. Data are derived from 24 dishes per condition. *p

    Article Snippet: Primary antibodies for GSK-3α, GSK-3β, GSK-3α-pSer21, GSK-3β-pSer9 and CREB (CST, Danvers, MA), 129-pCREB and c-fos (Santa Cruz Biotechnology, Santa Cruz, CA), BDNF (Abcam, Cambridge, UK; Millipore, Billerica, MA; Santa Cruz Biotechnology, Santa Cruz, CA) and ARC (Santa Cruz Biotechnology, Santa Cruz, CA) were used.

    Techniques: Cell Culture, Activity Assay, Derivative Assay

    (A, B) Treatment of neurons with TDZD-8 at 1 μM increased the overall amount of dendritic spines by over 25% in comparison to neurons exposed to wt CM. Concentrations of TDZD-8 higher than 1 μM were toxic and resulted in significant reduction in the number of spines suggesting that excessive inhibition of GSK-3β endogenous activity might not be safe. Data are derived from 20 neurons per condition. Calibration bars 10 μm. *p

    Journal: Neurobiology of disease

    Article Title: Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease , ☆☆Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease , ☆☆,

    doi: 10.1016/j.nbd.2011.09.002

    Figure Lengend Snippet: (A, B) Treatment of neurons with TDZD-8 at 1 μM increased the overall amount of dendritic spines by over 25% in comparison to neurons exposed to wt CM. Concentrations of TDZD-8 higher than 1 μM were toxic and resulted in significant reduction in the number of spines suggesting that excessive inhibition of GSK-3β endogenous activity might not be safe. Data are derived from 20 neurons per condition. Calibration bars 10 μm. *p

    Article Snippet: Primary antibodies for GSK-3α, GSK-3β, GSK-3α-pSer21, GSK-3β-pSer9 and CREB (CST, Danvers, MA), 129-pCREB and c-fos (Santa Cruz Biotechnology, Santa Cruz, CA), BDNF (Abcam, Cambridge, UK; Millipore, Billerica, MA; Santa Cruz Biotechnology, Santa Cruz, CA) and ARC (Santa Cruz Biotechnology, Santa Cruz, CA) were used.

    Techniques: Inhibition, Activity Assay, Derivative Assay

    (A, B) TDZD-8 at 1 μM fully rescued spine loss triggered by exposure to oligomeric Aβ containing Tg2576 CM. 14 day transfection of wt GSK-3β into neurons led to a very robust spine loss recapitulating that observed after exposure to Tg2576 CM. The phenotype is even more dramatic after transfection of a constitutively active mutant form of GSK-3β (GSK-3β mut (S9A)) that leads to a complete loss of all dendritic spines in cultured neurons. Data are derived from 20 neurons per condition. Calibration bars 10 μm. *p

    Journal: Neurobiology of disease

    Article Title: Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease , ☆☆Activation of glycogen synthase kinase-3 beta mediates ?-amyloid induced neuritic damage in Alzheimer's disease , ☆☆,

    doi: 10.1016/j.nbd.2011.09.002

    Figure Lengend Snippet: (A, B) TDZD-8 at 1 μM fully rescued spine loss triggered by exposure to oligomeric Aβ containing Tg2576 CM. 14 day transfection of wt GSK-3β into neurons led to a very robust spine loss recapitulating that observed after exposure to Tg2576 CM. The phenotype is even more dramatic after transfection of a constitutively active mutant form of GSK-3β (GSK-3β mut (S9A)) that leads to a complete loss of all dendritic spines in cultured neurons. Data are derived from 20 neurons per condition. Calibration bars 10 μm. *p

    Article Snippet: Primary antibodies for GSK-3α, GSK-3β, GSK-3α-pSer21, GSK-3β-pSer9 and CREB (CST, Danvers, MA), 129-pCREB and c-fos (Santa Cruz Biotechnology, Santa Cruz, CA), BDNF (Abcam, Cambridge, UK; Millipore, Billerica, MA; Santa Cruz Biotechnology, Santa Cruz, CA) and ARC (Santa Cruz Biotechnology, Santa Cruz, CA) were used.

    Techniques: Transfection, Mutagenesis, Cell Culture, Derivative Assay

    Effect of GSK-3β inhibition on the expression, phosphorylation, subcellular localization and co-transcriptional activity of β-catenin in osteosarcoma and osteoblast cells ( A ) Western-blotting analysis was used to compare the expression and phosphorylation of β-catenin between cells treated with DMSO and either GSK-3β inhibitor. Expression of β-actin was monitored as a loading control. ( B ) The left panels show representative immunofluorescence microscopic findings of expression and subcellular localization of β-catenin in osteosarcoma (143B, MG-63) and osteoblast (hFOB1.19) cells. The scale bar in each panel indicates 25 μm. The number shown below each panel indicates the percentage of nuclear β-catenin-positive cells among the total number of cells. The bar graphs on the right shows the effects of DMSO and AR-A014418 on the incidence of nuclear localization of β-catenin in osteosarcoma and osteoblast cells. In each assay, the mean percentage of nuclear β-catenin-positive cells in 3 microscopic fields was evaluated with standard deviation. ( C ) Relative co-transcriptional activity of β-catenin was measured by the TOP/FOP flash assay and compared between cells treated with DMSO, AR-A014418 and SB-216763, respectively. (B, C) Asterisks denote a statistically-significant difference between the data after administration of vehicle and GSK-3β inhibitors.

    Journal: Oncotarget

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    doi: 10.18632/oncotarget.12781

    Figure Lengend Snippet: Effect of GSK-3β inhibition on the expression, phosphorylation, subcellular localization and co-transcriptional activity of β-catenin in osteosarcoma and osteoblast cells ( A ) Western-blotting analysis was used to compare the expression and phosphorylation of β-catenin between cells treated with DMSO and either GSK-3β inhibitor. Expression of β-actin was monitored as a loading control. ( B ) The left panels show representative immunofluorescence microscopic findings of expression and subcellular localization of β-catenin in osteosarcoma (143B, MG-63) and osteoblast (hFOB1.19) cells. The scale bar in each panel indicates 25 μm. The number shown below each panel indicates the percentage of nuclear β-catenin-positive cells among the total number of cells. The bar graphs on the right shows the effects of DMSO and AR-A014418 on the incidence of nuclear localization of β-catenin in osteosarcoma and osteoblast cells. In each assay, the mean percentage of nuclear β-catenin-positive cells in 3 microscopic fields was evaluated with standard deviation. ( C ) Relative co-transcriptional activity of β-catenin was measured by the TOP/FOP flash assay and compared between cells treated with DMSO, AR-A014418 and SB-216763, respectively. (B, C) Asterisks denote a statistically-significant difference between the data after administration of vehicle and GSK-3β inhibitors.

    Article Snippet: The following primary antibodies were used at the dilutions shown against the indicated proteins: both GSK-3 isoforms (GSK-3α and β) (1:1,000, Millipore, Billerica, MA); GSK-3β (1:1,000, BD Biosciences, Lexington, KY); GSK-3β fractions phosphorylated at the serine (S) 9 residue (pGSK-3βS9) , (1:1,000, Cell Signaling Technology, Beverly, MA); and at the tyrosine (Y) 216 residue (pGSK-3βY216 ) (1:1,000, BD Biosciences); β-catenin (1:1,000; BD Biosciences); β-catenin phosphorylated at S33, S37 and/or threonine (T) 41 residues (p-β-cateninS33/37/T41 ) (Cell Signaling Technology); AKT (1:1,000) and its fractions phosphorylated at T308 residue (pAKTT308 ) (1:1,000) and at S473 residue (pAKTS473 ) (1:1,000) (Cell Signaling Technology); and β-actin (1:4,000, Ambion, Austin, TX).

    Techniques: Inhibition, Expressing, Activity Assay, Western Blot, Immunofluorescence, Standard Deviation

    Effect of small-molecule GSK-3β inhibitors on the survival of osteosarcoma cells The osteosarcoma cells were treated with DMSO or the indicated concentrations of AR-A014418 or SB-216763 for the designated times. The relative number of viable cells at each time point was measured by the WST-8 assay. Values shown are the means ± SD of six separate experiments.

    Journal: Oncotarget

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    doi: 10.18632/oncotarget.12781

    Figure Lengend Snippet: Effect of small-molecule GSK-3β inhibitors on the survival of osteosarcoma cells The osteosarcoma cells were treated with DMSO or the indicated concentrations of AR-A014418 or SB-216763 for the designated times. The relative number of viable cells at each time point was measured by the WST-8 assay. Values shown are the means ± SD of six separate experiments.

    Article Snippet: The following primary antibodies were used at the dilutions shown against the indicated proteins: both GSK-3 isoforms (GSK-3α and β) (1:1,000, Millipore, Billerica, MA); GSK-3β (1:1,000, BD Biosciences, Lexington, KY); GSK-3β fractions phosphorylated at the serine (S) 9 residue (pGSK-3βS9) , (1:1,000, Cell Signaling Technology, Beverly, MA); and at the tyrosine (Y) 216 residue (pGSK-3βY216 ) (1:1,000, BD Biosciences); β-catenin (1:1,000; BD Biosciences); β-catenin phosphorylated at S33, S37 and/or threonine (T) 41 residues (p-β-cateninS33/37/T41 ) (Cell Signaling Technology); AKT (1:1,000) and its fractions phosphorylated at T308 residue (pAKTT308 ) (1:1,000) and at S473 residue (pAKTS473 ) (1:1,000) (Cell Signaling Technology); and β-actin (1:4,000, Ambion, Austin, TX).

    Techniques:

    Effects of GSK-3β inhibitors on the proliferation and apoptosis of osteosarcoma cells and osteoblasts ( A ) The indicated cells were treated with DMSO or 25 μmol/L each of either AR-A014418 or SB-216763 for 72 hours. The relative number of proliferating cells was evaluated by measuring the amount of BrdU incorporation. ( B ) Relative numbers of TUNEL-positive apoptotic cells were scored for the indicated cells at 12 hours after treatment with DMSO or 25 μmol/L each of either GSK-3β inhibitor. (A, B) Values shown are the means ± SD of six separate experiments. Asterisks denote a statistically-significant difference ( p

    Journal: Oncotarget

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    doi: 10.18632/oncotarget.12781

    Figure Lengend Snippet: Effects of GSK-3β inhibitors on the proliferation and apoptosis of osteosarcoma cells and osteoblasts ( A ) The indicated cells were treated with DMSO or 25 μmol/L each of either AR-A014418 or SB-216763 for 72 hours. The relative number of proliferating cells was evaluated by measuring the amount of BrdU incorporation. ( B ) Relative numbers of TUNEL-positive apoptotic cells were scored for the indicated cells at 12 hours after treatment with DMSO or 25 μmol/L each of either GSK-3β inhibitor. (A, B) Values shown are the means ± SD of six separate experiments. Asterisks denote a statistically-significant difference ( p

    Article Snippet: The following primary antibodies were used at the dilutions shown against the indicated proteins: both GSK-3 isoforms (GSK-3α and β) (1:1,000, Millipore, Billerica, MA); GSK-3β (1:1,000, BD Biosciences, Lexington, KY); GSK-3β fractions phosphorylated at the serine (S) 9 residue (pGSK-3βS9) , (1:1,000, Cell Signaling Technology, Beverly, MA); and at the tyrosine (Y) 216 residue (pGSK-3βY216 ) (1:1,000, BD Biosciences); β-catenin (1:1,000; BD Biosciences); β-catenin phosphorylated at S33, S37 and/or threonine (T) 41 residues (p-β-cateninS33/37/T41 ) (Cell Signaling Technology); AKT (1:1,000) and its fractions phosphorylated at T308 residue (pAKTT308 ) (1:1,000) and at S473 residue (pAKTS473 ) (1:1,000) (Cell Signaling Technology); and β-actin (1:4,000, Ambion, Austin, TX).

    Techniques: BrdU Incorporation Assay, TUNEL Assay

    Effect of RNA interference on the expression of GSK-3β, cell viability, proliferation and apoptosis in osteosarcoma and osteoblast cells ( A ) Western-blotting analysis compared the level of expression of GSK-3α and GSK-3β between cells treated with non-specific (N) and GSK-3β-specific (S) siRNA (20 nmol/L each), respectively. Expression of β-actin was monitored as a loading control. ( B – D ) Relative number of surviving cells, BrdU-positive proliferating cells and TUNEL-positive apoptotic cells were counted and compared between cell types 96 hours after transfection of non-specific and GSK3β-specific siRNA. Values shown are the mean ± SD of six separate experiments. Asterisks denote a statistically-significant difference between cells transfected with non-specific and GSK- 3β-specific siRNA.

    Journal: Oncotarget

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    doi: 10.18632/oncotarget.12781

    Figure Lengend Snippet: Effect of RNA interference on the expression of GSK-3β, cell viability, proliferation and apoptosis in osteosarcoma and osteoblast cells ( A ) Western-blotting analysis compared the level of expression of GSK-3α and GSK-3β between cells treated with non-specific (N) and GSK-3β-specific (S) siRNA (20 nmol/L each), respectively. Expression of β-actin was monitored as a loading control. ( B – D ) Relative number of surviving cells, BrdU-positive proliferating cells and TUNEL-positive apoptotic cells were counted and compared between cell types 96 hours after transfection of non-specific and GSK3β-specific siRNA. Values shown are the mean ± SD of six separate experiments. Asterisks denote a statistically-significant difference between cells transfected with non-specific and GSK- 3β-specific siRNA.

    Article Snippet: The following primary antibodies were used at the dilutions shown against the indicated proteins: both GSK-3 isoforms (GSK-3α and β) (1:1,000, Millipore, Billerica, MA); GSK-3β (1:1,000, BD Biosciences, Lexington, KY); GSK-3β fractions phosphorylated at the serine (S) 9 residue (pGSK-3βS9) , (1:1,000, Cell Signaling Technology, Beverly, MA); and at the tyrosine (Y) 216 residue (pGSK-3βY216 ) (1:1,000, BD Biosciences); β-catenin (1:1,000; BD Biosciences); β-catenin phosphorylated at S33, S37 and/or threonine (T) 41 residues (p-β-cateninS33/37/T41 ) (Cell Signaling Technology); AKT (1:1,000) and its fractions phosphorylated at T308 residue (pAKTT308 ) (1:1,000) and at S473 residue (pAKTS473 ) (1:1,000) (Cell Signaling Technology); and β-actin (1:4,000, Ambion, Austin, TX).

    Techniques: Expressing, Western Blot, TUNEL Assay, Transfection

    ( A, B ) Efficacy of GSK-3β inhibitors on the size and weight of orthotopic 143B tumors (A) Tumor size was measured weekly and the volume calculated. (B) Mean weight of the tumors removed at necropsy. Asterisks denote a statistically-significant difference in tumor volume and weight compared to mice treated with DMSO. The scatter plots corresponding to the data in (A) and (B) are shown in Supplementary Figure S7 . ( C ) Histological and immunohistochemical findings for orthotopic tumors in mice treated with DMSO or with GSK-3β inhibitors. Representative paraffin-embedded sections of tumors were stained with H E or immunostained for β-catenin. Magnified images of the sections (upper three panels) are shown in Supplementary Figure S8 . Higher resolution versions of the lower six panels are shown in Supplementary Figure S9 . Scale bar in each of the upper three panels indicates 5 mm and that of the middle and lower six panels indicates 100 μm.

    Journal: Oncotarget

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    doi: 10.18632/oncotarget.12781

    Figure Lengend Snippet: ( A, B ) Efficacy of GSK-3β inhibitors on the size and weight of orthotopic 143B tumors (A) Tumor size was measured weekly and the volume calculated. (B) Mean weight of the tumors removed at necropsy. Asterisks denote a statistically-significant difference in tumor volume and weight compared to mice treated with DMSO. The scatter plots corresponding to the data in (A) and (B) are shown in Supplementary Figure S7 . ( C ) Histological and immunohistochemical findings for orthotopic tumors in mice treated with DMSO or with GSK-3β inhibitors. Representative paraffin-embedded sections of tumors were stained with H E or immunostained for β-catenin. Magnified images of the sections (upper three panels) are shown in Supplementary Figure S8 . Higher resolution versions of the lower six panels are shown in Supplementary Figure S9 . Scale bar in each of the upper three panels indicates 5 mm and that of the middle and lower six panels indicates 100 μm.

    Article Snippet: The following primary antibodies were used at the dilutions shown against the indicated proteins: both GSK-3 isoforms (GSK-3α and β) (1:1,000, Millipore, Billerica, MA); GSK-3β (1:1,000, BD Biosciences, Lexington, KY); GSK-3β fractions phosphorylated at the serine (S) 9 residue (pGSK-3βS9) , (1:1,000, Cell Signaling Technology, Beverly, MA); and at the tyrosine (Y) 216 residue (pGSK-3βY216 ) (1:1,000, BD Biosciences); β-catenin (1:1,000; BD Biosciences); β-catenin phosphorylated at S33, S37 and/or threonine (T) 41 residues (p-β-cateninS33/37/T41 ) (Cell Signaling Technology); AKT (1:1,000) and its fractions phosphorylated at T308 residue (pAKTT308 ) (1:1,000) and at S473 residue (pAKTS473 ) (1:1,000) (Cell Signaling Technology); and β-actin (1:4,000, Ambion, Austin, TX).

    Techniques: Mouse Assay, Immunohistochemistry, Staining

    Expression and phosphorylation of GSK3β in osteosarcoma cells and in untransformed osteoblasts Fractions of phosphorylated GSK-3β (pGSK-3β Y216 : active form; pGSK-3β S9 : inactive form) and total GSK-3β were detected in protein extracts from osteosarcoma and hFOB1.19 cells by Western immunoblotting analysis. The amount of protein extract from each sample was compared to the expression of β-actin.

    Journal: Oncotarget

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    doi: 10.18632/oncotarget.12781

    Figure Lengend Snippet: Expression and phosphorylation of GSK3β in osteosarcoma cells and in untransformed osteoblasts Fractions of phosphorylated GSK-3β (pGSK-3β Y216 : active form; pGSK-3β S9 : inactive form) and total GSK-3β were detected in protein extracts from osteosarcoma and hFOB1.19 cells by Western immunoblotting analysis. The amount of protein extract from each sample was compared to the expression of β-actin.

    Article Snippet: The following primary antibodies were used at the dilutions shown against the indicated proteins: both GSK-3 isoforms (GSK-3α and β) (1:1,000, Millipore, Billerica, MA); GSK-3β (1:1,000, BD Biosciences, Lexington, KY); GSK-3β fractions phosphorylated at the serine (S) 9 residue (pGSK-3βS9) , (1:1,000, Cell Signaling Technology, Beverly, MA); and at the tyrosine (Y) 216 residue (pGSK-3βY216 ) (1:1,000, BD Biosciences); β-catenin (1:1,000; BD Biosciences); β-catenin phosphorylated at S33, S37 and/or threonine (T) 41 residues (p-β-cateninS33/37/T41 ) (Cell Signaling Technology); AKT (1:1,000) and its fractions phosphorylated at T308 residue (pAKTT308 ) (1:1,000) and at S473 residue (pAKTS473 ) (1:1,000) (Cell Signaling Technology); and β-actin (1:4,000, Ambion, Austin, TX).

    Techniques: Expressing, Western Blot

    Perfusion of active GSK-3β or filamentous tau induces kinesin-1 release from squid vesicle fractions. A: Purified vesicle fractions from individual axoplasms perfused with buffer control or active GSK-3β and with soluble tau or filamentous

    Journal: Journal of neuroscience research

    Article Title: The Amino Terminus of Tau Inhibits Kinesin-Dependent Axonal Transport: Implications for Filament Toxicity

    doi: 10.1002/jnr.21850

    Figure Lengend Snippet: Perfusion of active GSK-3β or filamentous tau induces kinesin-1 release from squid vesicle fractions. A: Purified vesicle fractions from individual axoplasms perfused with buffer control or active GSK-3β and with soluble tau or filamentous

    Article Snippet: Active GSK-3β was from Sigma (No. G1663).

    Techniques: Purification

    Effect of signaling components on the actin cytoskeleton, focal adhesions, and transformation. (A) Anchorage-independent growth of wild-type, N ras −/− , and K ras −/− MEFs following knockdown of Akt or Raf (shRNAs directed to the genes encoding Akt and Raf are indicated by shAkt and shRaf, respectively) or infected with empty vector (V) or vector directing the expression of an shRNA to green fluorescent protein (shGFP). Results are means ± standard deviations (SDs) of three independent experiments. Protein knockdown was confirmed by immunoblotting, with tubulin as a loading control (bottom panels). (B) Shown is FITC-phalloidin staining (green) for stress fibers, vinculin staining (red) for focal adhesions, and DAPI staining (blue) for nuclei in wild-type and N ras -deficient cells following knockdown of Akt or Cdc42. Bar, 10 μm. (C) Same experiment as in panel A, except cells of the indicated genotype following knockdown of small GTPases Rac, RhoA, or Cdc42 were analyzed (shRNAs directed to the genes encoding these Rho GTPases are denoted shRac, shRhoA, and shCdc42, respectively). Results are means ± SDs of three independent experiments. Depletion of proteins was confirmed by immunoblotting, with tubulin as a loading control (bottom panels). (D) Same experiment as in panel B, except wild-type and K ras -deficient cells following knockdown of Raf or RhoA were analyzed. (E) Immunoblotting of whole-cell lysates prepared from cells of the indicated genotype was performed with antibodies to phosphorylated GSK-3β and total GSK-3β, phosphorylated ERK1 and ERK2, and total ERK1 and ERK2. (F) In the indicated cells, the presence of activated, GTP-bound Rac, Cdc42, and RhoA was assayed as described in Materials and Methods. Whole-cell lysates were analyzed for total Rac, Cdc42, and RhoA as loading controls. Panels showing activated Rho GTPases are representative of three independent experiments, and loading controls are from the same lysates used for activation assays.

    Journal: Molecular and Cellular Biology

    Article Title: Wild-Type NRas and KRas Perform Distinct Functions during Transformation ▿

    doi: 10.1128/MCB.00234-07

    Figure Lengend Snippet: Effect of signaling components on the actin cytoskeleton, focal adhesions, and transformation. (A) Anchorage-independent growth of wild-type, N ras −/− , and K ras −/− MEFs following knockdown of Akt or Raf (shRNAs directed to the genes encoding Akt and Raf are indicated by shAkt and shRaf, respectively) or infected with empty vector (V) or vector directing the expression of an shRNA to green fluorescent protein (shGFP). Results are means ± standard deviations (SDs) of three independent experiments. Protein knockdown was confirmed by immunoblotting, with tubulin as a loading control (bottom panels). (B) Shown is FITC-phalloidin staining (green) for stress fibers, vinculin staining (red) for focal adhesions, and DAPI staining (blue) for nuclei in wild-type and N ras -deficient cells following knockdown of Akt or Cdc42. Bar, 10 μm. (C) Same experiment as in panel A, except cells of the indicated genotype following knockdown of small GTPases Rac, RhoA, or Cdc42 were analyzed (shRNAs directed to the genes encoding these Rho GTPases are denoted shRac, shRhoA, and shCdc42, respectively). Results are means ± SDs of three independent experiments. Depletion of proteins was confirmed by immunoblotting, with tubulin as a loading control (bottom panels). (D) Same experiment as in panel B, except wild-type and K ras -deficient cells following knockdown of Raf or RhoA were analyzed. (E) Immunoblotting of whole-cell lysates prepared from cells of the indicated genotype was performed with antibodies to phosphorylated GSK-3β and total GSK-3β, phosphorylated ERK1 and ERK2, and total ERK1 and ERK2. (F) In the indicated cells, the presence of activated, GTP-bound Rac, Cdc42, and RhoA was assayed as described in Materials and Methods. Whole-cell lysates were analyzed for total Rac, Cdc42, and RhoA as loading controls. Panels showing activated Rho GTPases are representative of three independent experiments, and loading controls are from the same lysates used for activation assays.

    Article Snippet: Total cell lysates (40 μg) were resolved by SDS-polyacrylamide gel electrophoresis, transferred to Immobilon membranes, and immunoblotted with the following primary antibodies: anti-simian virus 40 (SV40) TAg (Pab 101), anti-KRas (F234), anti-HRas (F235), anti-NRas (F155), anti-Cdc42 (B-8), anti-RhoA (26C4), and anti-phospho-cofilin (sc-21867-R) were from Santa Cruz Biotechnology; anti-c-Raf (9422), anti-phospho-Akt (Ser473; 9271), anti-Akt (9272), anti-phospho-p44/42 MAPK (ERK1/2) (9101), anti-p44/42 MAPK (ERK1/2) (9102), anti-phospho-MEK1/2 (MEK1 and -2; 9121), anti-MEK1/2 (9122), anti-phosho-GSK-3β (9336S), anti-FAK (3285), and anti-PTEN (9552) were from Cell Signaling; anti-pan-Ras (Calbiochem), anti-Rac1 (clone 23A8; Upstate/Millipore), anti-GSK-3β (clone 4G; Upstate/Millipore), anti-FAK-Y397 (44-624G; BioSource), anti-Glu tubulin (Chemicon), and antitubulin (Sigma) were also used.

    Techniques: Transformation Assay, Infection, Plasmid Preparation, Expressing, shRNA, Staining, Activation Assay

    TRIP6 regulates growth factor-induced AKT activation and T157 phosphorylation of p27 KIP1 . (A) Knockdown of TRIP6 specifically eliminates serum-induced T157 phosphorylation of p27 KIP1 . Confluent U373-MG cells, as indicated, were starved overnight. After pretreatment with MG-132 for 1 h, cells were stimulated with serum for 15 min. Immunoblotting was performed to detect phosphorylated p27 KIP1 (pT157, pT198, and pS10), total p27 KIP1 , or TRIP6 in the lysates. (B to D) Knockdown of TRIP6 eliminates growth factor-induced T157 phosphorylation; however, this effect can be reversed by reconstitution with TRIP6. Confluent U373-MG cells (B) or SKOV-3 cells (C and D), as indicated, were starved overnight, followed by stimulation with the indicated growth factors for 15 min (B) or 5 min (C and D). Immunoblotting was performed to detect pT157-p27 KIP1 or total p27 KIP1 . The expression of EGFP-TRIP6 and TRIP6 in the whole-cell lysates was detected by using anti-TRIP6 antibody. (E) TRIP6 promotes cytosolic mislocalization of p27 KIP1 through the regulation of its phosphorylation at T157. WT or T157D EGFP-p27 KIP1 was expressed in SKOV-3 cells (siScr and siTRIP6). Subcellular fractionation was performed to determine the expression level of EGFP-p27 KIP1 or EGFP-T157D p27 KIP1 in the nucleus (N) or cytosol (C). The intensity of EGFP-p27 KIP1 or EGFP-T157D p27 KIP1 was quantified by NIH IMAGE J software to determine the relative levels of nuclear versus cytosolic p27 KIP1 . Histone H3 and GAPDH served as nuclear and cytosolic markers, respectively. (F and G) Knockdown of TRIP6 attenuates EGF-induced AKT activation; however, this effect can be reversed by TRIP6 overexpression. U373-MG cells (F) or SKOV-3 cells (G), as indicated, were starved overnight, followed by stimulation with EGF for 15 min (F or G) or 30 min (F). Immunoblotting was performed to detect the levels of pT308-AKT, AKT1, pS9–GSK-3β, GSK-3β, and TRIP6. (H) TRIP6 enhances AKT-mediated in vitro phosphorylation of p27 KIP1 at T157 but not at T198. An in vitro kinase assay was performed by incubating purified recombinant p27 KIP1 with active AKT1 in the presence or absence of TRIP6 at 30°C for 30 min. Immunoblotting (IB) was performed to detect pT157-p27 KIP1 , pT198-p27 KIP1 , pT308-AKT1, p27 KIP1 , or TRIP6. The relative intensity of pT157-p27 KIP1 or pT198-p27 KIP1 was quantified by using NIH IMAGE J software.

    Journal: Molecular and Cellular Biology

    Article Title: TRIP6 Regulates p27KIP1 To Promote Tumorigenesis

    doi: 10.1128/MCB.01149-12

    Figure Lengend Snippet: TRIP6 regulates growth factor-induced AKT activation and T157 phosphorylation of p27 KIP1 . (A) Knockdown of TRIP6 specifically eliminates serum-induced T157 phosphorylation of p27 KIP1 . Confluent U373-MG cells, as indicated, were starved overnight. After pretreatment with MG-132 for 1 h, cells were stimulated with serum for 15 min. Immunoblotting was performed to detect phosphorylated p27 KIP1 (pT157, pT198, and pS10), total p27 KIP1 , or TRIP6 in the lysates. (B to D) Knockdown of TRIP6 eliminates growth factor-induced T157 phosphorylation; however, this effect can be reversed by reconstitution with TRIP6. Confluent U373-MG cells (B) or SKOV-3 cells (C and D), as indicated, were starved overnight, followed by stimulation with the indicated growth factors for 15 min (B) or 5 min (C and D). Immunoblotting was performed to detect pT157-p27 KIP1 or total p27 KIP1 . The expression of EGFP-TRIP6 and TRIP6 in the whole-cell lysates was detected by using anti-TRIP6 antibody. (E) TRIP6 promotes cytosolic mislocalization of p27 KIP1 through the regulation of its phosphorylation at T157. WT or T157D EGFP-p27 KIP1 was expressed in SKOV-3 cells (siScr and siTRIP6). Subcellular fractionation was performed to determine the expression level of EGFP-p27 KIP1 or EGFP-T157D p27 KIP1 in the nucleus (N) or cytosol (C). The intensity of EGFP-p27 KIP1 or EGFP-T157D p27 KIP1 was quantified by NIH IMAGE J software to determine the relative levels of nuclear versus cytosolic p27 KIP1 . Histone H3 and GAPDH served as nuclear and cytosolic markers, respectively. (F and G) Knockdown of TRIP6 attenuates EGF-induced AKT activation; however, this effect can be reversed by TRIP6 overexpression. U373-MG cells (F) or SKOV-3 cells (G), as indicated, were starved overnight, followed by stimulation with EGF for 15 min (F or G) or 30 min (F). Immunoblotting was performed to detect the levels of pT308-AKT, AKT1, pS9–GSK-3β, GSK-3β, and TRIP6. (H) TRIP6 enhances AKT-mediated in vitro phosphorylation of p27 KIP1 at T157 but not at T198. An in vitro kinase assay was performed by incubating purified recombinant p27 KIP1 with active AKT1 in the presence or absence of TRIP6 at 30°C for 30 min. Immunoblotting (IB) was performed to detect pT157-p27 KIP1 , pT198-p27 KIP1 , pT308-AKT1, p27 KIP1 , or TRIP6. The relative intensity of pT157-p27 KIP1 or pT198-p27 KIP1 was quantified by using NIH IMAGE J software.

    Article Snippet: An in vitro kinase assay was performed by incubating purified recombinant p27KIP1 with active AKT1 (Biosources) in the presence or absence of recombinant TRIP6 at 30°C for 30 min. Immunoblotting was performed by using antibodies specific to human TRIP6 (Bethyl Laboratories), pT157-p27KIP1 , pT198-p27KIP1 (R & D Systems), pS10-p27KIP1 (Epitomics), pS473-AKT, pT308-AKT, pS9-glycogen synthase kinase 3β (GSK-3β), AKT1 (Cell Signaling), GSK-3β, mouse TRIP6 (BD Biosciences), Skp2, cyclin D1, PCNA, green fluorescent protein (GFP), hemagglutinin (HA) (Santa Cruz Biotechnology), or FLAG (Sigma-Aldrich).

    Techniques: Activation Assay, Expressing, Fractionation, Software, Over Expression, In Vitro, Kinase Assay, Purification, Recombinant

    Aspirin impedes the Wnt/β-catenin pathway by promoting β-catenin acetylation, phosphorylation and cytoplasmic degradation, and inhibiting its nuclear accumulation and transcriptional activity. (A) Western blot of p-β-catenin, β-cateninm, p-GSK-3β, GSK-3β and acetylated β-catenin in MDA-MB-231 cells incubated with or without 10 mM LiCl for 6 h prior to treatment with or without 5 mM Aspirin for 24 h. (B) Western blot analysis of β-catenin protein distribution in nuclear extract (NE) and cytoplasmic fractions (CE) of MDA-MB-231 cells incubated with or without 10 mM LiCl for 6 h prior to treatment with 5 mM Aspirin for 24 h. (C) Immunofluorescent staining of MDA-MB-231 cells that were treated with or without Aspirin or LiCl. Cells were fixed and stained with a β-catenin antibody (green) and DAPI (blue), and imaged by confocal microscopy. (D) Same procedure performed as in (B) with the use of 4T1 cells. (E) Same procedure performed as in (C) with the use of 4T1 cells. (F) Western blot analysis of β-catenin and ubiquitinated β-catenin in MDA-MB-231 cells that were incubated with the indicated reagents for 24 h and subsequently treated with proteasome inhibitor benzyloxycarbonyl-leu-leu-leu-aldehyde (MG132) 25 µM for 5 h before harvest. Endogenous β-cat was immunoprecipitated with anti-β-catenin antibodies and Western blot was performed with anti-β-catenin or anti-ubiquitin antibodies.

    Journal: Frontiers in Pharmacology

    Article Title: Wnt/β-Catenin Pathway-Regulated Fibromodulin Expression Is Crucial for Breast Cancer Metastasis and Inhibited by Aspirin

    doi: 10.3389/fphar.2019.01308

    Figure Lengend Snippet: Aspirin impedes the Wnt/β-catenin pathway by promoting β-catenin acetylation, phosphorylation and cytoplasmic degradation, and inhibiting its nuclear accumulation and transcriptional activity. (A) Western blot of p-β-catenin, β-cateninm, p-GSK-3β, GSK-3β and acetylated β-catenin in MDA-MB-231 cells incubated with or without 10 mM LiCl for 6 h prior to treatment with or without 5 mM Aspirin for 24 h. (B) Western blot analysis of β-catenin protein distribution in nuclear extract (NE) and cytoplasmic fractions (CE) of MDA-MB-231 cells incubated with or without 10 mM LiCl for 6 h prior to treatment with 5 mM Aspirin for 24 h. (C) Immunofluorescent staining of MDA-MB-231 cells that were treated with or without Aspirin or LiCl. Cells were fixed and stained with a β-catenin antibody (green) and DAPI (blue), and imaged by confocal microscopy. (D) Same procedure performed as in (B) with the use of 4T1 cells. (E) Same procedure performed as in (C) with the use of 4T1 cells. (F) Western blot analysis of β-catenin and ubiquitinated β-catenin in MDA-MB-231 cells that were incubated with the indicated reagents for 24 h and subsequently treated with proteasome inhibitor benzyloxycarbonyl-leu-leu-leu-aldehyde (MG132) 25 µM for 5 h before harvest. Endogenous β-cat was immunoprecipitated with anti-β-catenin antibodies and Western blot was performed with anti-β-catenin or anti-ubiquitin antibodies.

    Article Snippet: After blocking the membrane with 5% non-fat dry milk for 1 h at room temperature, the membrane was washed 3 times for 7 min intervals with 1XTBST and then incubated with primary antibodies against FMOD (Polyclonal 2621108 Temecula California ABT 124), GSK-3β (27C10) Rabbit Mab, Phospho GSK-3β (Ser 9) (5b3) Rabbit Mab, β-catenin Rabbit primary antibody (D10A8), Phospho β-catenin (Ser 33/37/Thr 41) antibody, anti-ERK 1/2, phospho-ERK1/2 (Thr202/Tyr204) (9102S, 4370S CST); TCF4 (CST#2569), LEF1 (CST), Acetyl-β-catenin (Lys 49) (CST), GAPDH (Santa Cruz), Lamin B (Santa Cruz), and anti-Ubiquitin (Santa Cruz) overnight at 4 °C.

    Techniques: Activity Assay, Western Blot, Multiple Displacement Amplification, Incubation, Staining, Confocal Microscopy, Immunoprecipitation

    Phosphorylation of Cdc25A by GSK-3β promotes β-TrCP binding and ubiquitin-mediated proteolysis

    Journal:

    Article Title: GSK-3? Targets Cdc25A for Ubiquitin-Mediated Proteolysis and GSK-3? Inactivation Correlates with Cdc25A Overproduction in Human Cancers

    doi: 10.1016/j.ccr.2007.12.002

    Figure Lengend Snippet: Phosphorylation of Cdc25A by GSK-3β promotes β-TrCP binding and ubiquitin-mediated proteolysis

    Article Snippet: Active GST-GSK-3β was purchased from Cell Signaling.

    Techniques: Binding Assay

    Inactivation of GSK-3β correlates with overexpression of Cdc25A in multiple cancerous tissues

    Journal:

    Article Title: GSK-3? Targets Cdc25A for Ubiquitin-Mediated Proteolysis and GSK-3? Inactivation Correlates with Cdc25A Overproduction in Human Cancers

    doi: 10.1016/j.ccr.2007.12.002

    Figure Lengend Snippet: Inactivation of GSK-3β correlates with overexpression of Cdc25A in multiple cancerous tissues

    Article Snippet: Active GST-GSK-3β was purchased from Cell Signaling.

    Techniques: Over Expression

    GSK-3β promotes Cdc25A turnover during an unperturbed cell cycle and in response to IR

    Journal:

    Article Title: GSK-3? Targets Cdc25A for Ubiquitin-Mediated Proteolysis and GSK-3? Inactivation Correlates with Cdc25A Overproduction in Human Cancers

    doi: 10.1016/j.ccr.2007.12.002

    Figure Lengend Snippet: GSK-3β promotes Cdc25A turnover during an unperturbed cell cycle and in response to IR

    Article Snippet: Active GST-GSK-3β was purchased from Cell Signaling.

    Techniques:

    Interactions between Cdc25A and GSK-3β

    Journal:

    Article Title: GSK-3? Targets Cdc25A for Ubiquitin-Mediated Proteolysis and GSK-3? Inactivation Correlates with Cdc25A Overproduction in Human Cancers

    doi: 10.1016/j.ccr.2007.12.002

    Figure Lengend Snippet: Interactions between Cdc25A and GSK-3β

    Article Snippet: Active GST-GSK-3β was purchased from Cell Signaling.

    Techniques:

    Regulation of Cdc25A by GSK-3β and Plk-3

    Journal:

    Article Title: GSK-3? Targets Cdc25A for Ubiquitin-Mediated Proteolysis and GSK-3? Inactivation Correlates with Cdc25A Overproduction in Human Cancers

    doi: 10.1016/j.ccr.2007.12.002

    Figure Lengend Snippet: Regulation of Cdc25A by GSK-3β and Plk-3

    Article Snippet: Active GST-GSK-3β was purchased from Cell Signaling.

    Techniques:

    Cell Cycle Regulation of Cdc25A by GSK-3β and Chk1

    Journal:

    Article Title: GSK-3? Targets Cdc25A for Ubiquitin-Mediated Proteolysis and GSK-3? Inactivation Correlates with Cdc25A Overproduction in Human Cancers

    doi: 10.1016/j.ccr.2007.12.002

    Figure Lengend Snippet: Cell Cycle Regulation of Cdc25A by GSK-3β and Chk1

    Article Snippet: Active GST-GSK-3β was purchased from Cell Signaling.

    Techniques:

    Cdc25A protein levels are negatively regulated by GSK-3β in vivo

    Journal:

    Article Title: GSK-3? Targets Cdc25A for Ubiquitin-Mediated Proteolysis and GSK-3? Inactivation Correlates with Cdc25A Overproduction in Human Cancers

    doi: 10.1016/j.ccr.2007.12.002

    Figure Lengend Snippet: Cdc25A protein levels are negatively regulated by GSK-3β in vivo

    Article Snippet: Active GST-GSK-3β was purchased from Cell Signaling.

    Techniques: In Vivo

    Functional effects of canonical Wnt/β-catenin signaling activation in bone sarcoma cells A. β-catenin accumulation and nuclear translocation in bone sarcoma cells in response to Wnt3a. SW1353 and U2OS cells were incubated for 12 hr in the absence or presence of 200ng/ml Wnt3a. To assess β-catenin nuclear accumulation, cells were lysed and were immunoblotted by using a monoclonal anti-β-catenin antibody (Top). The middle and bottom part of the blot was stained with anti-β-actin or hnRNP to verify equal loading. B. Activation of the TCF-reporter transcriptional activity in bone sarcoma cells in response to Wnt3a. SW1353 and U2OS cells were incubated for 12 hr in the absence or presence of 200ng/ml Wnt3a, and then the TCF-reporter transcriptional activity was determined by luciferase activity as indicated in Materials and Methods. C. Western blot analysis of Wnt3a effects on total β-catenin, active dephosphorylated β-catenin, Axin2, GSK-3β, c-Myc, Cyclin-D1, and β-actin protein expression. SW1353 and U2OS cells were incubated for 12 hr in the absence or presence of 200ng/ml Wnt3a. The whole-cell lysates were immunoblotted with the indicated antibodies. D. Effect of Wnt3a on the cell proliferation by Brdu incorporation assay. SW1353 and U2OS cells were incubated for 48 hr in the absence or presence of 200ng/ml Wnt3a. Cell proliferation was determined by Brdu incorporation assay as indicated in Material and Methods. E , F. Effect of Wnt3a on the cell survival by MTS assay. SW1353 E. and U2OS F. cells were incubated for 24, 48, 72 hr in the absence or presence of 200ng/ml Wnt3a. Cell survival was determined by MTS assay as indicated in Materials and Methods # P

    Journal: Oncotarget

    Article Title: Aberrant activation of Wnt/β-catenin signaling drives proliferation of bone sarcoma cells

    doi:

    Figure Lengend Snippet: Functional effects of canonical Wnt/β-catenin signaling activation in bone sarcoma cells A. β-catenin accumulation and nuclear translocation in bone sarcoma cells in response to Wnt3a. SW1353 and U2OS cells were incubated for 12 hr in the absence or presence of 200ng/ml Wnt3a. To assess β-catenin nuclear accumulation, cells were lysed and were immunoblotted by using a monoclonal anti-β-catenin antibody (Top). The middle and bottom part of the blot was stained with anti-β-actin or hnRNP to verify equal loading. B. Activation of the TCF-reporter transcriptional activity in bone sarcoma cells in response to Wnt3a. SW1353 and U2OS cells were incubated for 12 hr in the absence or presence of 200ng/ml Wnt3a, and then the TCF-reporter transcriptional activity was determined by luciferase activity as indicated in Materials and Methods. C. Western blot analysis of Wnt3a effects on total β-catenin, active dephosphorylated β-catenin, Axin2, GSK-3β, c-Myc, Cyclin-D1, and β-actin protein expression. SW1353 and U2OS cells were incubated for 12 hr in the absence or presence of 200ng/ml Wnt3a. The whole-cell lysates were immunoblotted with the indicated antibodies. D. Effect of Wnt3a on the cell proliferation by Brdu incorporation assay. SW1353 and U2OS cells were incubated for 48 hr in the absence or presence of 200ng/ml Wnt3a. Cell proliferation was determined by Brdu incorporation assay as indicated in Material and Methods. E , F. Effect of Wnt3a on the cell survival by MTS assay. SW1353 E. and U2OS F. cells were incubated for 24, 48, 72 hr in the absence or presence of 200ng/ml Wnt3a. Cell survival was determined by MTS assay as indicated in Materials and Methods # P

    Article Snippet: Antibodies for detection of the following targets were purchased as indicated: total β-catenin and GSK-3β from BD Biosciences, active β-catenin (anti-ABC, clone 8E7) dephosphorylated on Ser37 or Thr41 from Millipore Corporation (Billerica, MA, USA), Cyclin D1 and c-Myc from Santa Cruz Technology, Axin2, TCF4 and LEF1 from Cell Signaling Technology, LRP6 and hnRNP from Abcam, and β-actin from Sigma.

    Techniques: Functional Assay, Activation Assay, Translocation Assay, Incubation, Staining, Activity Assay, Luciferase, Western Blot, Expressing, BrdU Incorporation Assay, MTS Assay

    Activation of canonical Wnt/β-catenin signaling and enhancement of bone sarcoma cell survival by a GSK-3β inhibitor A. GSK-3β inhibitor SB-216763 increased active β-catenin levels by inhibiting GSK-3β activity in SW1353 and U2OS cells. Bone Sarcoma cells were incubated with 10ng/ml SB-216763 or the DMSO vehicle for 24 hr. The whole-cell lysates were immunoblotted with the indicated antibodies. B. Activation of the TCF-response elements by SB-216763. SW1353 and U2OS cells were treated with the GSK-3β inhibitor SB-216763 (10ng/ml) or the DMSO vehicle for 24 hr, and then the TCF-reporter transcriptional activity was determined by luciferase activity as indicated in Materials and Methods. C. The prosurvival activity of SB-216763 in SW1353 and U2OS cells. Bone sarcoma cells were incubated with 10ng/ml SB-216763 or the DMSO vehicle for 24 hr before determinations of cell proliferation by relative Brdu incorporation assay. The mean incremental survivals measured in triplicate and the SD were shown. D. SB-216763 protected bone sarcoma cells from apoptosis induced by cisplatin. SW1353 and U2OS cells were incubated with 10ng/ml SB-216763 or the DMSO vehicle for 24 h, and then treated with 1μg/ml cisplatin for 24 h. The apoptotic cells were measured by flow cytometry by using the FITC-conjugated Annexin V and PI. Three independent experiments were analyzed. E. Two representative bone sarcoma cells of the anti-apoptotic effect of SB-216763 were shown by the FITC-conjugated Annexin V and PI # P

    Journal: Oncotarget

    Article Title: Aberrant activation of Wnt/β-catenin signaling drives proliferation of bone sarcoma cells

    doi:

    Figure Lengend Snippet: Activation of canonical Wnt/β-catenin signaling and enhancement of bone sarcoma cell survival by a GSK-3β inhibitor A. GSK-3β inhibitor SB-216763 increased active β-catenin levels by inhibiting GSK-3β activity in SW1353 and U2OS cells. Bone Sarcoma cells were incubated with 10ng/ml SB-216763 or the DMSO vehicle for 24 hr. The whole-cell lysates were immunoblotted with the indicated antibodies. B. Activation of the TCF-response elements by SB-216763. SW1353 and U2OS cells were treated with the GSK-3β inhibitor SB-216763 (10ng/ml) or the DMSO vehicle for 24 hr, and then the TCF-reporter transcriptional activity was determined by luciferase activity as indicated in Materials and Methods. C. The prosurvival activity of SB-216763 in SW1353 and U2OS cells. Bone sarcoma cells were incubated with 10ng/ml SB-216763 or the DMSO vehicle for 24 hr before determinations of cell proliferation by relative Brdu incorporation assay. The mean incremental survivals measured in triplicate and the SD were shown. D. SB-216763 protected bone sarcoma cells from apoptosis induced by cisplatin. SW1353 and U2OS cells were incubated with 10ng/ml SB-216763 or the DMSO vehicle for 24 h, and then treated with 1μg/ml cisplatin for 24 h. The apoptotic cells were measured by flow cytometry by using the FITC-conjugated Annexin V and PI. Three independent experiments were analyzed. E. Two representative bone sarcoma cells of the anti-apoptotic effect of SB-216763 were shown by the FITC-conjugated Annexin V and PI # P

    Article Snippet: Antibodies for detection of the following targets were purchased as indicated: total β-catenin and GSK-3β from BD Biosciences, active β-catenin (anti-ABC, clone 8E7) dephosphorylated on Ser37 or Thr41 from Millipore Corporation (Billerica, MA, USA), Cyclin D1 and c-Myc from Santa Cruz Technology, Axin2, TCF4 and LEF1 from Cell Signaling Technology, LRP6 and hnRNP from Abcam, and β-actin from Sigma.

    Techniques: Activation Assay, Activity Assay, Incubation, Luciferase, BrdU Incorporation Assay, Flow Cytometry, Cytometry

    Proliferation and Apoptosis in Islets of the Irs2 −/− and Gsk-3β +/− Irs2 −/− Mice (A) Pancreatic sections from 8-wk-old mice of the indicated genotypes were stained with antibodies to Ki67 (red) and insulin (green). Arrowheads indicate proliferating cells. Scale bar represents 50 μm. (B) The number of cells that are positive for both Ki67 and insulin have been quantified as a percentage of total number of insulin-positive cells in the sections. (WT: n = 3, Gsk-3β +/− : n = 3, Irs2 −/− : n = 5, and Gsk-3β +/− Irs2 −/− : n = 5). Results are represented as mean ± S.E.M. Double asterisks (**) indicate p

    Journal: PLoS Biology

    Article Title: Genetic Deficiency of Glycogen Synthase Kinase-3? Corrects Diabetes in Mouse Models of Insulin Resistance

    doi: 10.1371/journal.pbio.0060037

    Figure Lengend Snippet: Proliferation and Apoptosis in Islets of the Irs2 −/− and Gsk-3β +/− Irs2 −/− Mice (A) Pancreatic sections from 8-wk-old mice of the indicated genotypes were stained with antibodies to Ki67 (red) and insulin (green). Arrowheads indicate proliferating cells. Scale bar represents 50 μm. (B) The number of cells that are positive for both Ki67 and insulin have been quantified as a percentage of total number of insulin-positive cells in the sections. (WT: n = 3, Gsk-3β +/− : n = 3, Irs2 −/− : n = 5, and Gsk-3β +/− Irs2 −/− : n = 5). Results are represented as mean ± S.E.M. Double asterisks (**) indicate p

    Article Snippet: The extracts (50 μg of total protein) were subjected to immunoblot analysis with antibodies to Gsk-3β phosphorylated on Ser9, Akt phosphorylated on Ser473, Akt phosphorylated on Thr308, total Akt, total Gsk-3α/β (Cell Signaling), total Gsk-3β (BD Transduction Laboratories), and glycogen synthase phosphorylated on Ser 641 and 645 (Biosource/Invitrogen), Irs2 (Upstate), Pdx1 (Santa Cruz SC14664), p27kip1 (BD Transduction Laboratories), α-tubulin, and β-actin (Sigma-Aldrich).

    Techniques: Mouse Assay, Staining

    Effects of Inhibition of Gsk-3 Activity on Protein Stability of p27 kip1 (A) MIN6 cells were pretreated with either 40 mM lithium chloride (LiCl) or 40 mM NaCl in DMEM with 15% FBS for 1 h and were cotreated with 25 μg/ml cyclohexamide and lithium or NaCl for 4 h. The lysates were subjected to western blot analysis with anti-p27 kip1 , phospho-glycogen synthase, total Gsk-3β, and α-tubulin. Densitometry of p27 kip1 was measured and normalized over α-tubulin. (B) Islets isolated from 16-wk-old WT mice were deprived of serum for 4 h and were incubated with no addition, or with addition of 100 nM IGF-1, or 40 mM LiCl, or both for 3 h. Lysates were then prepared from islets and were subjected to western blot analysis with anti-p27 kip1 , anti-phospho glycogen synthase, anti-total Gsk-3β, and β-actin. Representative results of three independent experiments are presented. Densitometry of p27 kip1 was measured and normalized over β-actin. Mean protein levels ± S.E.M. are summarized on the graph. A single asterisk (*) indicates p

    Journal: PLoS Biology

    Article Title: Genetic Deficiency of Glycogen Synthase Kinase-3? Corrects Diabetes in Mouse Models of Insulin Resistance

    doi: 10.1371/journal.pbio.0060037

    Figure Lengend Snippet: Effects of Inhibition of Gsk-3 Activity on Protein Stability of p27 kip1 (A) MIN6 cells were pretreated with either 40 mM lithium chloride (LiCl) or 40 mM NaCl in DMEM with 15% FBS for 1 h and were cotreated with 25 μg/ml cyclohexamide and lithium or NaCl for 4 h. The lysates were subjected to western blot analysis with anti-p27 kip1 , phospho-glycogen synthase, total Gsk-3β, and α-tubulin. Densitometry of p27 kip1 was measured and normalized over α-tubulin. (B) Islets isolated from 16-wk-old WT mice were deprived of serum for 4 h and were incubated with no addition, or with addition of 100 nM IGF-1, or 40 mM LiCl, or both for 3 h. Lysates were then prepared from islets and were subjected to western blot analysis with anti-p27 kip1 , anti-phospho glycogen synthase, anti-total Gsk-3β, and β-actin. Representative results of three independent experiments are presented. Densitometry of p27 kip1 was measured and normalized over β-actin. Mean protein levels ± S.E.M. are summarized on the graph. A single asterisk (*) indicates p

    Article Snippet: The extracts (50 μg of total protein) were subjected to immunoblot analysis with antibodies to Gsk-3β phosphorylated on Ser9, Akt phosphorylated on Ser473, Akt phosphorylated on Thr308, total Akt, total Gsk-3α/β (Cell Signaling), total Gsk-3β (BD Transduction Laboratories), and glycogen synthase phosphorylated on Ser 641 and 645 (Biosource/Invitrogen), Irs2 (Upstate), Pdx1 (Santa Cruz SC14664), p27kip1 (BD Transduction Laboratories), α-tubulin, and β-actin (Sigma-Aldrich).

    Techniques: Inhibition, Activity Assay, Western Blot, Isolation, Mouse Assay, Incubation

    Morphologic Phenotypes of Irs2 −/− and Gsk-3β +/− Irs2 −/− Mice (A–C) Pancreatic sections from 8-wk-old mice of the indicated genotypes were stained with antibodies to insulin and then were counterstained with hematoxylin. Scale bar represents 100 μm. (D–F) Pancreatic sections from 8-wk-old mice of the indicated genotypes at 8 wk were stained with antibodies to insulin (red) and glucagon (green). Scale bar represents 50 μm. (G) Random sections of the entire pancreas from 8-wk-old mice of the indicated genotypes were stained with hematoxylin and were measured to determine the β-cell area and quantification of the β cell mass. Results are expressed as the mg of total weight containing insulin-immunoreactive cells. (WT: n = 3, Gsk-3β +/− : n = 4, Irs2 −/− : n = 6, and Gsk-3β +/− Irs2 −/− : n = 5). (H) Quantification of the β/α cell ratio is shown. (WT: n = 3, Gsk-3β +/− : n = 4, Irs2 −/− : n = 5, and Gsk-3β +/− Irs2 −/− : n = 5). Results are expressed as the mean ± S.E.M.. A single asterisk (*) indicates p

    Journal: PLoS Biology

    Article Title: Genetic Deficiency of Glycogen Synthase Kinase-3? Corrects Diabetes in Mouse Models of Insulin Resistance

    doi: 10.1371/journal.pbio.0060037

    Figure Lengend Snippet: Morphologic Phenotypes of Irs2 −/− and Gsk-3β +/− Irs2 −/− Mice (A–C) Pancreatic sections from 8-wk-old mice of the indicated genotypes were stained with antibodies to insulin and then were counterstained with hematoxylin. Scale bar represents 100 μm. (D–F) Pancreatic sections from 8-wk-old mice of the indicated genotypes at 8 wk were stained with antibodies to insulin (red) and glucagon (green). Scale bar represents 50 μm. (G) Random sections of the entire pancreas from 8-wk-old mice of the indicated genotypes were stained with hematoxylin and were measured to determine the β-cell area and quantification of the β cell mass. Results are expressed as the mg of total weight containing insulin-immunoreactive cells. (WT: n = 3, Gsk-3β +/− : n = 4, Irs2 −/− : n = 6, and Gsk-3β +/− Irs2 −/− : n = 5). (H) Quantification of the β/α cell ratio is shown. (WT: n = 3, Gsk-3β +/− : n = 4, Irs2 −/− : n = 5, and Gsk-3β +/− Irs2 −/− : n = 5). Results are expressed as the mean ± S.E.M.. A single asterisk (*) indicates p

    Article Snippet: The extracts (50 μg of total protein) were subjected to immunoblot analysis with antibodies to Gsk-3β phosphorylated on Ser9, Akt phosphorylated on Ser473, Akt phosphorylated on Thr308, total Akt, total Gsk-3α/β (Cell Signaling), total Gsk-3β (BD Transduction Laboratories), and glycogen synthase phosphorylated on Ser 641 and 645 (Biosource/Invitrogen), Irs2 (Upstate), Pdx1 (Santa Cruz SC14664), p27kip1 (BD Transduction Laboratories), α-tubulin, and β-actin (Sigma-Aldrich).

    Techniques: Mouse Assay, Staining

    Glucose and Insulin Values in Wild Type (WT), Gsk-3β +/− , Ir +/− , or Gsk-3β +/− Ir +/− Mice and Hyperinsulinemic-Euglycemic Clamp in Male Ir +/− and Gsk-3β +/− Ir +/− Mice Fasting and fed glucose and insulin values in male mice either WT ( n ≥ 6), haploinsufficient for Gsk-3β ( Gsk-3β +/− ; n ≥ 6) or the insulin receptor ( Ir +/− ; n ≥ 6), or combined ( Gsk-3β +/− Ir +/ ; n ≥ 6), as indicated. (A) Fasting and fed glucose (26 wk of age). (B) Fasting and fed insulin (26 wk of age). (C) Glucose disposal rate (mg/kg/min). (D) Glucose infusion rate (mg/kg/min). (E) Hepatic glucose output (mg/kg/min) determined on fasted, conscious 28-wk-old mice of indicated genotypes using the hyperinsulinemic-euglycemic clamp ( n = 4 per each genotype). Results are presented as the mean values ± S.E.M. in the graph. A single asterisk (*) indicates p

    Journal: PLoS Biology

    Article Title: Genetic Deficiency of Glycogen Synthase Kinase-3? Corrects Diabetes in Mouse Models of Insulin Resistance

    doi: 10.1371/journal.pbio.0060037

    Figure Lengend Snippet: Glucose and Insulin Values in Wild Type (WT), Gsk-3β +/− , Ir +/− , or Gsk-3β +/− Ir +/− Mice and Hyperinsulinemic-Euglycemic Clamp in Male Ir +/− and Gsk-3β +/− Ir +/− Mice Fasting and fed glucose and insulin values in male mice either WT ( n ≥ 6), haploinsufficient for Gsk-3β ( Gsk-3β +/− ; n ≥ 6) or the insulin receptor ( Ir +/− ; n ≥ 6), or combined ( Gsk-3β +/− Ir +/ ; n ≥ 6), as indicated. (A) Fasting and fed glucose (26 wk of age). (B) Fasting and fed insulin (26 wk of age). (C) Glucose disposal rate (mg/kg/min). (D) Glucose infusion rate (mg/kg/min). (E) Hepatic glucose output (mg/kg/min) determined on fasted, conscious 28-wk-old mice of indicated genotypes using the hyperinsulinemic-euglycemic clamp ( n = 4 per each genotype). Results are presented as the mean values ± S.E.M. in the graph. A single asterisk (*) indicates p

    Article Snippet: The extracts (50 μg of total protein) were subjected to immunoblot analysis with antibodies to Gsk-3β phosphorylated on Ser9, Akt phosphorylated on Ser473, Akt phosphorylated on Thr308, total Akt, total Gsk-3α/β (Cell Signaling), total Gsk-3β (BD Transduction Laboratories), and glycogen synthase phosphorylated on Ser 641 and 645 (Biosource/Invitrogen), Irs2 (Upstate), Pdx1 (Santa Cruz SC14664), p27kip1 (BD Transduction Laboratories), α-tubulin, and β-actin (Sigma-Aldrich).

    Techniques: Mouse Assay

    Effects of Inhibition of Gsk-3β Activity on Pdx1 and p27 kip1 Expression in Islets from Irs2 −/− Mice Islets were isolated from 6 wk-old WT, Gsk-3β +/− Irs2 −/− , and Irs2 −/− mice. (A) Western blot analysis with anti-total Gsk-3, anti-phospho glycogen synthase, anti-phospho Akt (S473), anti-total Akt, and β-actin. Representative results of four independent experiments are presented. Densitometry of phosphorylation of Akt (S473) was measured and normalized over total Akt. Mean values ± S.E.M. are summarized on the graph. A single asterisk (*) indicates p

    Journal: PLoS Biology

    Article Title: Genetic Deficiency of Glycogen Synthase Kinase-3? Corrects Diabetes in Mouse Models of Insulin Resistance

    doi: 10.1371/journal.pbio.0060037

    Figure Lengend Snippet: Effects of Inhibition of Gsk-3β Activity on Pdx1 and p27 kip1 Expression in Islets from Irs2 −/− Mice Islets were isolated from 6 wk-old WT, Gsk-3β +/− Irs2 −/− , and Irs2 −/− mice. (A) Western blot analysis with anti-total Gsk-3, anti-phospho glycogen synthase, anti-phospho Akt (S473), anti-total Akt, and β-actin. Representative results of four independent experiments are presented. Densitometry of phosphorylation of Akt (S473) was measured and normalized over total Akt. Mean values ± S.E.M. are summarized on the graph. A single asterisk (*) indicates p

    Article Snippet: The extracts (50 μg of total protein) were subjected to immunoblot analysis with antibodies to Gsk-3β phosphorylated on Ser9, Akt phosphorylated on Ser473, Akt phosphorylated on Thr308, total Akt, total Gsk-3α/β (Cell Signaling), total Gsk-3β (BD Transduction Laboratories), and glycogen synthase phosphorylated on Ser 641 and 645 (Biosource/Invitrogen), Irs2 (Upstate), Pdx1 (Santa Cruz SC14664), p27kip1 (BD Transduction Laboratories), α-tubulin, and β-actin (Sigma-Aldrich).

    Techniques: Inhibition, Activity Assay, Expressing, Mouse Assay, Isolation, Western Blot

    The Effects of β-Cell–Specific Gsk-3β Deficiency in Irs2 −/− Mice (A) Islets were isolated from 6-wk-old control mice, Irs2 −/− mice, and βGsk-3β −/− Irs2 −/− mice were lysed. Lysates were subjected to western blot analysis using anti-Irs2, anti-total Gsk-3, anti-phosho glycogen synthase, and α-tubulin. One of two independent experiments is shown. (B) Fed glucose levels were assessed in control mice (filled triangles; n = 11), Irs2 −/− mice (filled diamonds; n = 10), and βGsk-3β −/− Irs2 −/− mice (filled squares; n = 5) at the indicated ages. (C) Fed plasma insulin levels were assessed in control mice (filled triangles; n = 11), Irs2 −/− mice (filled diamonds; n = 10), and βGsk-3β −/− Irs2 −/− mice (filled squares; n = 5) at the indicated ages. Results are presented as the mean ± S.E.M. in the graph. A single asterisk (*) indicates p

    Journal: PLoS Biology

    Article Title: Genetic Deficiency of Glycogen Synthase Kinase-3? Corrects Diabetes in Mouse Models of Insulin Resistance

    doi: 10.1371/journal.pbio.0060037

    Figure Lengend Snippet: The Effects of β-Cell–Specific Gsk-3β Deficiency in Irs2 −/− Mice (A) Islets were isolated from 6-wk-old control mice, Irs2 −/− mice, and βGsk-3β −/− Irs2 −/− mice were lysed. Lysates were subjected to western blot analysis using anti-Irs2, anti-total Gsk-3, anti-phosho glycogen synthase, and α-tubulin. One of two independent experiments is shown. (B) Fed glucose levels were assessed in control mice (filled triangles; n = 11), Irs2 −/− mice (filled diamonds; n = 10), and βGsk-3β −/− Irs2 −/− mice (filled squares; n = 5) at the indicated ages. (C) Fed plasma insulin levels were assessed in control mice (filled triangles; n = 11), Irs2 −/− mice (filled diamonds; n = 10), and βGsk-3β −/− Irs2 −/− mice (filled squares; n = 5) at the indicated ages. Results are presented as the mean ± S.E.M. in the graph. A single asterisk (*) indicates p

    Article Snippet: The extracts (50 μg of total protein) were subjected to immunoblot analysis with antibodies to Gsk-3β phosphorylated on Ser9, Akt phosphorylated on Ser473, Akt phosphorylated on Thr308, total Akt, total Gsk-3α/β (Cell Signaling), total Gsk-3β (BD Transduction Laboratories), and glycogen synthase phosphorylated on Ser 641 and 645 (Biosource/Invitrogen), Irs2 (Upstate), Pdx1 (Santa Cruz SC14664), p27kip1 (BD Transduction Laboratories), α-tubulin, and β-actin (Sigma-Aldrich).

    Techniques: Mouse Assay, Isolation, Western Blot

    Metabolic Phenotypes of Irs2 −/− and Gsk-3β +/− Irs2 −/− Mice (A) Islets were isolated from 6-wk-old WT and Irs2 −/− mice, and were lysed. Lysates were subjected to western blot analysis using anti-phospho Akt (S473), anti-phospho Gsk-3β (S9), anti-total Gsk-3β, anti-phosho glycogen synthase, anti-total Akt, and β-actin. One of four independent experiments is shown. (B) Densitometry of phospho-Gsk-3β (S9) and phospho-glycogen synthase was measured and normalized over total Gsk-3β and β-actin, respectively. Mean phosphorylation ± S.E.M. is represented in the graph. (C) Body weights at indicated age (WT: n = 12, Gsk-3β +/− : n = 12, Irs2 −/− : n = 29, and Gsk-3β +/− Irs2 −/− : n = 14). (D) Fed glucose concentration in blood derived from mouse tail vein at indicated age. (WT: n = 12, Gsk-3β +/− : n = 12, Irs2 −/− : n = 29, and Gsk-3β +/− Irs2 −/− : n = 14). (E) Fasting and fed plasma insulin levels at 8 wk of age and fed insulin levels at 12 wk of age in mice either WT ( n = 10), Gsk-3β +/− ( n = 9), Irs2 −/− ( n = 15), or Gsk-3β +/− Irs2 −/− ( n = 12) were determined. (F) Insulin tolerance tests were performed on 4-h fasted male WT ( n = 4), Irs2 −/− ( n = 6), and Gsk-3β +/− Irs2 −/− ( n = 4) mice at 6 wk of age. Glucose levels were assessed at the indicated time intervals after intraperitoneal injection of human regular insulin (0.5 U/kg). Results are presented as the mean ± S.E.M. in the graph. A single asterisk (*) indicates p

    Journal: PLoS Biology

    Article Title: Genetic Deficiency of Glycogen Synthase Kinase-3? Corrects Diabetes in Mouse Models of Insulin Resistance

    doi: 10.1371/journal.pbio.0060037

    Figure Lengend Snippet: Metabolic Phenotypes of Irs2 −/− and Gsk-3β +/− Irs2 −/− Mice (A) Islets were isolated from 6-wk-old WT and Irs2 −/− mice, and were lysed. Lysates were subjected to western blot analysis using anti-phospho Akt (S473), anti-phospho Gsk-3β (S9), anti-total Gsk-3β, anti-phosho glycogen synthase, anti-total Akt, and β-actin. One of four independent experiments is shown. (B) Densitometry of phospho-Gsk-3β (S9) and phospho-glycogen synthase was measured and normalized over total Gsk-3β and β-actin, respectively. Mean phosphorylation ± S.E.M. is represented in the graph. (C) Body weights at indicated age (WT: n = 12, Gsk-3β +/− : n = 12, Irs2 −/− : n = 29, and Gsk-3β +/− Irs2 −/− : n = 14). (D) Fed glucose concentration in blood derived from mouse tail vein at indicated age. (WT: n = 12, Gsk-3β +/− : n = 12, Irs2 −/− : n = 29, and Gsk-3β +/− Irs2 −/− : n = 14). (E) Fasting and fed plasma insulin levels at 8 wk of age and fed insulin levels at 12 wk of age in mice either WT ( n = 10), Gsk-3β +/− ( n = 9), Irs2 −/− ( n = 15), or Gsk-3β +/− Irs2 −/− ( n = 12) were determined. (F) Insulin tolerance tests were performed on 4-h fasted male WT ( n = 4), Irs2 −/− ( n = 6), and Gsk-3β +/− Irs2 −/− ( n = 4) mice at 6 wk of age. Glucose levels were assessed at the indicated time intervals after intraperitoneal injection of human regular insulin (0.5 U/kg). Results are presented as the mean ± S.E.M. in the graph. A single asterisk (*) indicates p

    Article Snippet: The extracts (50 μg of total protein) were subjected to immunoblot analysis with antibodies to Gsk-3β phosphorylated on Ser9, Akt phosphorylated on Ser473, Akt phosphorylated on Thr308, total Akt, total Gsk-3α/β (Cell Signaling), total Gsk-3β (BD Transduction Laboratories), and glycogen synthase phosphorylated on Ser 641 and 645 (Biosource/Invitrogen), Irs2 (Upstate), Pdx1 (Santa Cruz SC14664), p27kip1 (BD Transduction Laboratories), α-tubulin, and β-actin (Sigma-Aldrich).

    Techniques: Mouse Assay, Isolation, Western Blot, Concentration Assay, Derivative Assay, Injection

    β-Cell Mass in WT, Gsk-3β +/− , Ir +/− , or Gsk-3β +/− Ir +/− (A–D) Pancreatic sections from 10-mo-old mice of the indicated genotypes were stained with antibodies to insulin and then were counterstained with hematoxylin. Scale bar represents 100 μm. (E) Random sections of the entire pancreas from 10-mo-old mice of the indicated genotypes were stained with hematoxylin and were measured to determine the β-cell mass (WT: n = 3, Gsk-3β +/− : n = 3, Ir +/− : n = 4, and Gsk-3β +/− Ir +/− : n = 4). Results are expressed as β-cell mass (mg). All values are represented as the mean ± S.E.M. A single asterisk (*) indicates p

    Journal: PLoS Biology

    Article Title: Genetic Deficiency of Glycogen Synthase Kinase-3? Corrects Diabetes in Mouse Models of Insulin Resistance

    doi: 10.1371/journal.pbio.0060037

    Figure Lengend Snippet: β-Cell Mass in WT, Gsk-3β +/− , Ir +/− , or Gsk-3β +/− Ir +/− (A–D) Pancreatic sections from 10-mo-old mice of the indicated genotypes were stained with antibodies to insulin and then were counterstained with hematoxylin. Scale bar represents 100 μm. (E) Random sections of the entire pancreas from 10-mo-old mice of the indicated genotypes were stained with hematoxylin and were measured to determine the β-cell mass (WT: n = 3, Gsk-3β +/− : n = 3, Ir +/− : n = 4, and Gsk-3β +/− Ir +/− : n = 4). Results are expressed as β-cell mass (mg). All values are represented as the mean ± S.E.M. A single asterisk (*) indicates p

    Article Snippet: The extracts (50 μg of total protein) were subjected to immunoblot analysis with antibodies to Gsk-3β phosphorylated on Ser9, Akt phosphorylated on Ser473, Akt phosphorylated on Thr308, total Akt, total Gsk-3α/β (Cell Signaling), total Gsk-3β (BD Transduction Laboratories), and glycogen synthase phosphorylated on Ser 641 and 645 (Biosource/Invitrogen), Irs2 (Upstate), Pdx1 (Santa Cruz SC14664), p27kip1 (BD Transduction Laboratories), α-tubulin, and β-actin (Sigma-Aldrich).

    Techniques: Mouse Assay, Staining

    Enhanced CD28-induced phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3β (GSK-3β) in lamina propria T lymphocytes (LPT). (a) CD4 + peripheral blood T lymphocytes (PBT) and LPT were cultured in the presence or absence of the CD28 monoclonal antibody (mAb) 248 (5 µg/ml) for the indicated time-periods. The phosphorylation state of GSK-3β (Ser9) and AKT (Ser473) was determined by immunoblotting of whole cell extracts. Results are representative of three to four independent experiments performed with cells from three to four different patients. *Non-specific band. (b) Densitometric determination of the phosphorylation index of AKT (Ser473 and Thr 308; upper panels) and GSK-3β (Ser9; lower panel) in CD4 + PBT and LPT in response to CD28 activation. Data are expressed as mean ± standard deviation (s.d.) ( n = 3–7; four independent experiments with cells from four different patients were performed for the medium control and the time-points 15 min, 30 min and 1 h; three separate experiments with cells from three different patients were performed for medium control and the time-points 1 h and 2 h; hence, there are n = 7 for medium control and the time-point 1 h, while for all other conditions there are n = 4 and n = 3, respectively). (c) CD4 + PBT and CD4 + CD45RO + PBT were cultured in the presence or absence of the CD28 mAb 248 (5 µg/ml) for the indicated time-periods. The phosphorylation state of GSK-3β (Ser9) and AKT (Ser473) was analysed by immunoblotting of whole cell extracts. Shown is the phosphorylation index of AKT (Ser473; upper panel) and GSK-3β (Ser9; lower panel) as determined by densitometry. Data are represented as the mean ± s.d. (AKT phosphorylation: n = 3 for all conditions using cells from three different individuals; GSK-3 phosphorylation: n = 4 for all conditions using cells from four different individuals).

    Journal: Clinical and Experimental Immunology

    Article Title: Human mucosal CD4+ T cells but not blood CD4+ T cells respond vigorously towards CD28 engagement

    doi: 10.1111/j.1365-2249.2011.04539.x

    Figure Lengend Snippet: Enhanced CD28-induced phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3β (GSK-3β) in lamina propria T lymphocytes (LPT). (a) CD4 + peripheral blood T lymphocytes (PBT) and LPT were cultured in the presence or absence of the CD28 monoclonal antibody (mAb) 248 (5 µg/ml) for the indicated time-periods. The phosphorylation state of GSK-3β (Ser9) and AKT (Ser473) was determined by immunoblotting of whole cell extracts. Results are representative of three to four independent experiments performed with cells from three to four different patients. *Non-specific band. (b) Densitometric determination of the phosphorylation index of AKT (Ser473 and Thr 308; upper panels) and GSK-3β (Ser9; lower panel) in CD4 + PBT and LPT in response to CD28 activation. Data are expressed as mean ± standard deviation (s.d.) ( n = 3–7; four independent experiments with cells from four different patients were performed for the medium control and the time-points 15 min, 30 min and 1 h; three separate experiments with cells from three different patients were performed for medium control and the time-points 1 h and 2 h; hence, there are n = 7 for medium control and the time-point 1 h, while for all other conditions there are n = 4 and n = 3, respectively). (c) CD4 + PBT and CD4 + CD45RO + PBT were cultured in the presence or absence of the CD28 mAb 248 (5 µg/ml) for the indicated time-periods. The phosphorylation state of GSK-3β (Ser9) and AKT (Ser473) was analysed by immunoblotting of whole cell extracts. Shown is the phosphorylation index of AKT (Ser473; upper panel) and GSK-3β (Ser9; lower panel) as determined by densitometry. Data are represented as the mean ± s.d. (AKT phosphorylation: n = 3 for all conditions using cells from three different individuals; GSK-3 phosphorylation: n = 4 for all conditions using cells from four different individuals).

    Article Snippet: GSK-3β mAb was obtained from BD Bioscience.

    Techniques: Cell Culture, Activation Assay, Standard Deviation

    Effect of GSNO treatment on GSK-3β activation under chronic cerebral hypoperfusion

    Journal: Brain research

    Article Title: S-nitrosoglutathione Reduces Tau Hyper-phosphorylation and Provides Neuroprotection in Rat Model of Chronic Cerebral Hypoperfusion

    doi: 10.1016/j.brainres.2015.07.057

    Figure Lengend Snippet: Effect of GSNO treatment on GSK-3β activation under chronic cerebral hypoperfusion

    Article Snippet: Western immunoblot analysis was performed using antibodies against phospho-tau (p-tau) S396 (Abcam, Cambridge, MA), p-tau S404 (Abcam), p-tau S202 /T205 (Pierce, Rockford, IL), pan-tau (Cell Signaling Technology), β-actin (Abcam), p35/p25 (Cell Signaling Technology, Danvers, MA), phospho-GSK-3β (p-GSK-3β) Y216 /Y279 (Abcam), p-GSK-3β S9 (Cell Signaling Technology), pan-GSK-3β (BD Transduction Laboratory, San Diego, CA).

    Techniques: Activation Assay

    Proposed model of GSK-3β-mediated regulation of the NFATc2 metabolism in pancreatic cancer cells Activation of the phosphatase calcineurin following an intracellular calcium influx dephosphorylates NFAT proteins and stimulates their nuclear import. There, GSK-3β-mediated SP2 phosphorylation protects NFATc2 from degradation and further stimulates DNA binding and transcriptional activation of target genes (left side). In addition, GSK-3β stabilizes NFATc2/STAT3 complex formation independent of and dominant to SP2 phosphorylation resulting in target gene expression (right side).

    Journal: Molecular cancer therapeutics

    Article Title: GSK-3β governs inflammation-induced NFATc2 signaling hubs to promote pancreatic cancer progression

    doi: 10.1158/1535-7163.MCT-15-0309

    Figure Lengend Snippet: Proposed model of GSK-3β-mediated regulation of the NFATc2 metabolism in pancreatic cancer cells Activation of the phosphatase calcineurin following an intracellular calcium influx dephosphorylates NFAT proteins and stimulates their nuclear import. There, GSK-3β-mediated SP2 phosphorylation protects NFATc2 from degradation and further stimulates DNA binding and transcriptional activation of target genes (left side). In addition, GSK-3β stabilizes NFATc2/STAT3 complex formation independent of and dominant to SP2 phosphorylation resulting in target gene expression (right side).

    Article Snippet: The following antibodies were used for immunofluorescence: HA (1:1000) and STAT3 (1:200) (all Cell signaling, Danvers, USA) and for immunohistochemistry: NFATc2 (1:200, Abcam, Cambridge, UK), p-NFATc2 (S213/217/221) (1:50, Santa Cruz Biotechnology, Santa Cruz, USA), STAT3 (1:100, Cell Signaling), p-STAT3 (Y705) (1:50, Cell signaling), p-GS (S641/645) (1:25, Cell Signaling), Ki67 (1:600, Neomarker, Fremont, USA) and GSK-3β (1:200, Epitomics, Hamburg, Germany), CD45 (BD Pharmingen, 1:100).

    Techniques: Activation Assay, Binding Assay, Expressing

    Stimulation of euchromatin formation on CDK-6 target promoter through NFATc2-STAT3 complexes (A) Transcription start site (TSS) binding motives for NFAT and STAT3. (B) Human CDK-6 promoter with putative NFATc2 and STAT3 binding sites. (C) ChIP analysis determines NFATc2, STAT3, RNA-Pol II and H3K4me3 binding to the CDK-6 promoter with and without AR-A014418 (GSK-3i) treatment (25 μM for 12h). Means ± SD. (D) Quantitative real-time PCR illustrating reduced CDK-6 mRNA expression in PaTu8988t cells following GSK-3β inhibition with GSK-3i (25 μM for 3h). (E) Lysates from PaTu8988t cells with stable expression of GSK-3β S9A or following transfection with GSK-3β siRNA were subjected to immunoblotting with indicated antibodies.

    Journal: Molecular cancer therapeutics

    Article Title: GSK-3β governs inflammation-induced NFATc2 signaling hubs to promote pancreatic cancer progression

    doi: 10.1158/1535-7163.MCT-15-0309

    Figure Lengend Snippet: Stimulation of euchromatin formation on CDK-6 target promoter through NFATc2-STAT3 complexes (A) Transcription start site (TSS) binding motives for NFAT and STAT3. (B) Human CDK-6 promoter with putative NFATc2 and STAT3 binding sites. (C) ChIP analysis determines NFATc2, STAT3, RNA-Pol II and H3K4me3 binding to the CDK-6 promoter with and without AR-A014418 (GSK-3i) treatment (25 μM for 12h). Means ± SD. (D) Quantitative real-time PCR illustrating reduced CDK-6 mRNA expression in PaTu8988t cells following GSK-3β inhibition with GSK-3i (25 μM for 3h). (E) Lysates from PaTu8988t cells with stable expression of GSK-3β S9A or following transfection with GSK-3β siRNA were subjected to immunoblotting with indicated antibodies.

    Article Snippet: The following antibodies were used for immunofluorescence: HA (1:1000) and STAT3 (1:200) (all Cell signaling, Danvers, USA) and for immunohistochemistry: NFATc2 (1:200, Abcam, Cambridge, UK), p-NFATc2 (S213/217/221) (1:50, Santa Cruz Biotechnology, Santa Cruz, USA), STAT3 (1:100, Cell Signaling), p-STAT3 (Y705) (1:50, Cell signaling), p-GS (S641/645) (1:25, Cell Signaling), Ki67 (1:600, Neomarker, Fremont, USA) and GSK-3β (1:200, Epitomics, Hamburg, Germany), CD45 (BD Pharmingen, 1:100).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Inhibition, Transfection

    GSK-3β stabilizes NFATc2 in vitro and in vivo (A,B) PaTu8988t (A) and IMIM-PC1 (B) cells were treated with indicated concentrations of GSK-3β inhibitor AR-A014418 (hereafter referred to as GSK-3i) and analyzed for BrdU incorporation after 24h and 48h. (C) Nude mice bearing IMIM-PC1 xenograft tumors were treated 3x weekly with 10 mg/kg BW GSK-3i (n=5) or 10% DMSO (n=5) intraperitoneally. Tumors were extracted after 4 weeks. The tumor growth was normalized to the respective starting volumes. Means ± SE. *p

    Journal: Molecular cancer therapeutics

    Article Title: GSK-3β governs inflammation-induced NFATc2 signaling hubs to promote pancreatic cancer progression

    doi: 10.1158/1535-7163.MCT-15-0309

    Figure Lengend Snippet: GSK-3β stabilizes NFATc2 in vitro and in vivo (A,B) PaTu8988t (A) and IMIM-PC1 (B) cells were treated with indicated concentrations of GSK-3β inhibitor AR-A014418 (hereafter referred to as GSK-3i) and analyzed for BrdU incorporation after 24h and 48h. (C) Nude mice bearing IMIM-PC1 xenograft tumors were treated 3x weekly with 10 mg/kg BW GSK-3i (n=5) or 10% DMSO (n=5) intraperitoneally. Tumors were extracted after 4 weeks. The tumor growth was normalized to the respective starting volumes. Means ± SE. *p

    Article Snippet: The following antibodies were used for immunofluorescence: HA (1:1000) and STAT3 (1:200) (all Cell signaling, Danvers, USA) and for immunohistochemistry: NFATc2 (1:200, Abcam, Cambridge, UK), p-NFATc2 (S213/217/221) (1:50, Santa Cruz Biotechnology, Santa Cruz, USA), STAT3 (1:100, Cell Signaling), p-STAT3 (Y705) (1:50, Cell signaling), p-GS (S641/645) (1:25, Cell Signaling), Ki67 (1:600, Neomarker, Fremont, USA) and GSK-3β (1:200, Epitomics, Hamburg, Germany), CD45 (BD Pharmingen, 1:100).

    Techniques: In Vitro, In Vivo, BrdU Incorporation Assay, Mouse Assay

    SP2-independent regulation of NFATc2 activity by GSK-3β (A) Luciferase reporter gene assay demonstrating regulation of the cisNFAT promoter in PaTu8988t cells with stable wt NFATc2 and NFATc2 pSP2 expression or control cells following transfection with GSK-3β siRNA or GSK-3β S9A (B). Means ± SD. (C) PaTu8988t cells with stable expression of indicated constructs were transfected with wt STAT3 and cisNFAT and subjected to reporter gene assay. Shown are means ± SD normalized to controls. (D) PaTu8988t cells with stable expression of indicated constructs were treated with 25 μM GSK-3i and after 3h nuclear lysates were subjected to co-immunoprecipitation and subsequent immunoblotting as indicated. (E) Reporter gene assay of Suit-007 cells with stable expression of indicated constructs after transfection with wt STAT3 and cisNFAT and subsequent administration of 25 μM GSK-3i for 6h. Means ± SD. Successful knock-down of STAT3 is shown in the right panel.

    Journal: Molecular cancer therapeutics

    Article Title: GSK-3β governs inflammation-induced NFATc2 signaling hubs to promote pancreatic cancer progression

    doi: 10.1158/1535-7163.MCT-15-0309

    Figure Lengend Snippet: SP2-independent regulation of NFATc2 activity by GSK-3β (A) Luciferase reporter gene assay demonstrating regulation of the cisNFAT promoter in PaTu8988t cells with stable wt NFATc2 and NFATc2 pSP2 expression or control cells following transfection with GSK-3β siRNA or GSK-3β S9A (B). Means ± SD. (C) PaTu8988t cells with stable expression of indicated constructs were transfected with wt STAT3 and cisNFAT and subjected to reporter gene assay. Shown are means ± SD normalized to controls. (D) PaTu8988t cells with stable expression of indicated constructs were treated with 25 μM GSK-3i and after 3h nuclear lysates were subjected to co-immunoprecipitation and subsequent immunoblotting as indicated. (E) Reporter gene assay of Suit-007 cells with stable expression of indicated constructs after transfection with wt STAT3 and cisNFAT and subsequent administration of 25 μM GSK-3i for 6h. Means ± SD. Successful knock-down of STAT3 is shown in the right panel.

    Article Snippet: The following antibodies were used for immunofluorescence: HA (1:1000) and STAT3 (1:200) (all Cell signaling, Danvers, USA) and for immunohistochemistry: NFATc2 (1:200, Abcam, Cambridge, UK), p-NFATc2 (S213/217/221) (1:50, Santa Cruz Biotechnology, Santa Cruz, USA), STAT3 (1:100, Cell Signaling), p-STAT3 (Y705) (1:50, Cell signaling), p-GS (S641/645) (1:25, Cell Signaling), Ki67 (1:600, Neomarker, Fremont, USA) and GSK-3β (1:200, Epitomics, Hamburg, Germany), CD45 (BD Pharmingen, 1:100).

    Techniques: Activity Assay, Luciferase, Reporter Gene Assay, Expressing, Transfection, Construct, Immunoprecipitation

    The activation of AKT/glycogen synthase kinase-3β (GSK-3β) and extracellular signal-regulated kinase (ERK) contributes to Grifola frondosa polysaccharide (GFP)-mediated apoptotic cell death. GFPs (200 μ g/ml) reduced the expression levels of phosphorylated (p)-AKT, p-GSK-3β and p-ERK from 0.5 to 3 h. The average fold of band intensity compared to the related controls was marked respectively (n=6 repeats in each group).

    Journal: International Journal of Molecular Medicine

    Article Title: Grifola frondosa polysaccharides induce breast cancer cell apoptosis via the mitochondrial-dependent apoptotic pathway

    doi: 10.3892/ijmm.2017.3081

    Figure Lengend Snippet: The activation of AKT/glycogen synthase kinase-3β (GSK-3β) and extracellular signal-regulated kinase (ERK) contributes to Grifola frondosa polysaccharide (GFP)-mediated apoptotic cell death. GFPs (200 μ g/ml) reduced the expression levels of phosphorylated (p)-AKT, p-GSK-3β and p-ERK from 0.5 to 3 h. The average fold of band intensity compared to the related controls was marked respectively (n=6 repeats in each group).

    Article Snippet: The membranes were incubated at 4°C overnight with Bcl-2 (MABC573), Bcl-extra large (Bcl-xL; MAB4625), Bax (AB2915), cleaved caspase-3 (AB3623), cleaved caspase-8 (AB1879), and phosphorylated (p)-AKT (05–1003) (all from Merck Millipore, Darmstadt, Germany), total (t)-AKT (ab126811) and p-glycogen synthase kinase-3β (GSK-3β) (ab75745) (both from Abcam, Cambrige, UK), T-GSK-3β (PK1111) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ABS16) (both from Merck Millipore) at dilution of 1:1,000.

    Techniques: Activation Assay, Expressing

    Activation of the Wnt/β-catenin signalling pathway via lithium released from Li/CPC. ( A – E ) Gene expression of Col1a1, Bglap, OPG, Runx2 and β-catenin were better in Li/CPC-50 and Li/CPC-100 than in CPC and Li/CPC-200 (n = 3). ( F ) β-catenin accumulation in the cytosol and translocation to the nucleus in Li/CPC-100 (n = 3). ( G , H ) Representative Western blot analysis of p-GSK-3β, t-GSK-3β, p-β-catenin, t-β-catenin and Runx2. Li/CPC-50 and Li/CPC-100 increased the amount of p-GSK-3β significantly and decreased the amount of p-β-catenin compared with Li/CPC-200 and CPC, expression of Runx2 was increased significantly in Li/CPC-50 and Li/CPC-100 compared with Li/CPC-200 and CPC. The band density was quantified using ImageJ software and data from three independent experiments were presented (CPC as the control group, the values were expressed as the mean ± SD, n = 3). Statistical analyses were done using Students’t-test. *p

    Journal: Scientific Reports

    Article Title: Acceleration of bone regeneration by activating Wnt/β-catenin signalling pathway via lithium released from lithium chloride/calcium phosphate cement in osteoporosis

    doi: 10.1038/srep45204

    Figure Lengend Snippet: Activation of the Wnt/β-catenin signalling pathway via lithium released from Li/CPC. ( A – E ) Gene expression of Col1a1, Bglap, OPG, Runx2 and β-catenin were better in Li/CPC-50 and Li/CPC-100 than in CPC and Li/CPC-200 (n = 3). ( F ) β-catenin accumulation in the cytosol and translocation to the nucleus in Li/CPC-100 (n = 3). ( G , H ) Representative Western blot analysis of p-GSK-3β, t-GSK-3β, p-β-catenin, t-β-catenin and Runx2. Li/CPC-50 and Li/CPC-100 increased the amount of p-GSK-3β significantly and decreased the amount of p-β-catenin compared with Li/CPC-200 and CPC, expression of Runx2 was increased significantly in Li/CPC-50 and Li/CPC-100 compared with Li/CPC-200 and CPC. The band density was quantified using ImageJ software and data from three independent experiments were presented (CPC as the control group, the values were expressed as the mean ± SD, n = 3). Statistical analyses were done using Students’t-test. *p

    Article Snippet: Membranes were blocked for 2 h at room temperature in 5% non-fat powdered milk in Tris-buffer, followed by incubation at 4 °C with primary antibodies to β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin, p-β-catenin, Runx2 (Cell Signaling Technology, Danvers, MA, USA), GSK-3β, and p-GSK-3β (Abcam, UK).

    Techniques: Activation Assay, Expressing, Translocation Assay, Western Blot, Software

    Activation of the mTORC1 and β-catenin signaling pathways by OIP5 A . Activation of mTORC1 in HLK3 cells transiently infected with Ad-OIP5 compared to Ad-LacZ control cells on Western blot analysis. B . Phosphorylation of GSK-3β by OIP5 in HLK3 and SH-J1 cells transiently infected with Ad-OIP5 or Ad-LacZ adenovirus (n = 3). C . Phosphorylation of β-catenin at the S552 site by OIP5 in HLK3 cells transiently infected with Ad-OIP5 or Ad-LacZ adenovirus (n = 3). D . Immunoblot analysis of nuclear and cytoplasmic levels of β-catenin in HLK3 cells transiently infected with Ad-OIP5 or Ad-LacZ adenovirus (n = 3). E . TCF/LEF-dependent transcriptional activity of β-catenin in HEK293T cells transfected with TOP/FOP flash reporter plasmids. Assays of relative luciferase activity in cells were performed (n = 3). Each bar represents mean ± SD. ** P

    Journal: Oncotarget

    Article Title: OIP5, a target of miR-15b-5p, regulates hepatocellular carcinoma growth and metastasis through the AKT/mTORC1 and β-catenin signaling pathways

    doi: 10.18632/oncotarget.15185

    Figure Lengend Snippet: Activation of the mTORC1 and β-catenin signaling pathways by OIP5 A . Activation of mTORC1 in HLK3 cells transiently infected with Ad-OIP5 compared to Ad-LacZ control cells on Western blot analysis. B . Phosphorylation of GSK-3β by OIP5 in HLK3 and SH-J1 cells transiently infected with Ad-OIP5 or Ad-LacZ adenovirus (n = 3). C . Phosphorylation of β-catenin at the S552 site by OIP5 in HLK3 cells transiently infected with Ad-OIP5 or Ad-LacZ adenovirus (n = 3). D . Immunoblot analysis of nuclear and cytoplasmic levels of β-catenin in HLK3 cells transiently infected with Ad-OIP5 or Ad-LacZ adenovirus (n = 3). E . TCF/LEF-dependent transcriptional activity of β-catenin in HEK293T cells transfected with TOP/FOP flash reporter plasmids. Assays of relative luciferase activity in cells were performed (n = 3). Each bar represents mean ± SD. ** P

    Article Snippet: Polyclonal anti-human Cdc25C (C-20), Cdc2, cyclin A (H-432), cyclin B1 (H-433), cyclin D1 (C-20), cyclin E (C-19), GSK-3β (L-17), β-catenin (H-102), Lamin B (C-20), Cytokeratin 18 (C-04), Cytokeratin 8 (M20), N-cadherin (H69), Desmoplakin I/II (G20), c-Myc (9E10), GFP (FL), P21 (C-19), CDK2 (M2), CDK4 (H-22) and CDK6 (C-21) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

    Techniques: Activation Assay, Infection, Western Blot, Activity Assay, Transfection, Luciferase

    MiR-632 activates Wnt/β-catenin pathway by directly targeting GSK-3β. a Data from bioinformatics tools (microRNA.org, TargetScan and miRDB) showed that there were putative binding sites between 3′-UTR of GSK-3β-wt and miR-632. GSK-3β-mut means mutation of binding sites in 3′-UTR of GSK-3β. b Luciferase reporter gene assays illustrated that miR-632 negatively regulated the luciferase activity of GSK-3β-wt-3′-UTR, rather than of GSK-3β-mut-3′-UTR. n = three independent experiments. ** P

    Journal: Cell Death & Disease

    Article Title: LncRNA RUNX1-IT1 which is downregulated by hypoxia-driven histone deacetylase 3 represses proliferation and cancer stem-like properties in hepatocellular carcinoma cells

    doi: 10.1038/s41419-020-2274-x

    Figure Lengend Snippet: MiR-632 activates Wnt/β-catenin pathway by directly targeting GSK-3β. a Data from bioinformatics tools (microRNA.org, TargetScan and miRDB) showed that there were putative binding sites between 3′-UTR of GSK-3β-wt and miR-632. GSK-3β-mut means mutation of binding sites in 3′-UTR of GSK-3β. b Luciferase reporter gene assays illustrated that miR-632 negatively regulated the luciferase activity of GSK-3β-wt-3′-UTR, rather than of GSK-3β-mut-3′-UTR. n = three independent experiments. ** P

    Article Snippet: The GSK-3β Human cDNA ORF Clone (GSK-3β) was purchased from OriGene (OriGene Technologies, Inc., USA).

    Techniques: Binding Assay, Mutagenesis, Luciferase, Activity Assay

    RUNX1-IT1 suppresses Wnt/β-catenin pathway through RUNX1-IT1/miR-632/ GSK-3β cascades. Rescue experiments revealed that RUNX1-IT1 negatively regulated the miR-632 expression a , b , whereas positively regulated both the mRNA a , b and protein e , f expression of GSK-3β by RUNX1-IT1/miR-632/GSK-3β cascades. n = three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: LncRNA RUNX1-IT1 which is downregulated by hypoxia-driven histone deacetylase 3 represses proliferation and cancer stem-like properties in hepatocellular carcinoma cells

    doi: 10.1038/s41419-020-2274-x

    Figure Lengend Snippet: RUNX1-IT1 suppresses Wnt/β-catenin pathway through RUNX1-IT1/miR-632/ GSK-3β cascades. Rescue experiments revealed that RUNX1-IT1 negatively regulated the miR-632 expression a , b , whereas positively regulated both the mRNA a , b and protein e , f expression of GSK-3β by RUNX1-IT1/miR-632/GSK-3β cascades. n = three independent experiments. * P

    Article Snippet: The GSK-3β Human cDNA ORF Clone (GSK-3β) was purchased from OriGene (OriGene Technologies, Inc., USA).

    Techniques: Expressing

    Astragaloside IV (AS IV) attenuated isoflurane-induced NF-κB activation and apoptotic pathway activation in hippocampus tissues. Lane 1: Sham group; 2: Isoflurane group; 3: Isoflurane + AS IV (10 mg/kg); 4: Isoflurane + AS IV (40 mg/kg); and 5: Isoflurane + AS IV (100 mg/kg). (A) Cytoplasmic and nuclear NF-κB levels from rat hippocampus tissues. Histone H3 and GAPDH were used as loading control for nuclear protein and cytoplasmic protein, respectively. (B) Expression levels of apoptosis-related proteins in total cell lysates from rat hippocampus tissues were determined by western blot assay. GAPDH levels were used for equal loading control. NF-κB, nuclear factor-κB; Bcl-2, B-cell lymphoma 2; GSK-3β, glycogen synthase kinase 3β; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; AKT, protein kinase B; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Astragaloside IV protects new born rats from anesthesia-induced apoptosis in the developing brain

    doi: 10.3892/etm.2016.3519

    Figure Lengend Snippet: Astragaloside IV (AS IV) attenuated isoflurane-induced NF-κB activation and apoptotic pathway activation in hippocampus tissues. Lane 1: Sham group; 2: Isoflurane group; 3: Isoflurane + AS IV (10 mg/kg); 4: Isoflurane + AS IV (40 mg/kg); and 5: Isoflurane + AS IV (100 mg/kg). (A) Cytoplasmic and nuclear NF-κB levels from rat hippocampus tissues. Histone H3 and GAPDH were used as loading control for nuclear protein and cytoplasmic protein, respectively. (B) Expression levels of apoptosis-related proteins in total cell lysates from rat hippocampus tissues were determined by western blot assay. GAPDH levels were used for equal loading control. NF-κB, nuclear factor-κB; Bcl-2, B-cell lymphoma 2; GSK-3β, glycogen synthase kinase 3β; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; AKT, protein kinase B; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Article Snippet: Antibodies against caspase-3 (cat. no. ab2171), glycogen synthase kinase (GSK)-3β (cat. no. ab32391), p-GSK-3β (cat. no. ab75814) and Klotho (cat. no. ab181373) were purchased from Abcam, Inc. Anti-BCL2 was from Santa Cruz Biotechnology, Inc. (La Jolla, CA, USA).

    Techniques: Activation Assay, Expressing, Western Blot

    GSK-3β is truncated selectively by calcium-mediated truncation/activation of calpain I. (A) Western blots show that truncation of GSK-3β coincides with truncation/activation of calpain I in a calcium-dependent manner in human brain extracts. Normal human brain extract was incubated at 30°C for 10 min in the presence of 2.0 mM EDTA and various concentrations (0.00–2.14 mM) of CaCl 2 . Then the incubated extracts were analyzed by Western blots developed with specific antibodies to calpain I or GSK-3β. (B) Calcium-activated truncations of calpain I and GSK-3β are selectively inhibited by calpain inhibitors. Normal human brain extract was incubated at 30°C for 10 min in the presence of 2.0 mM each of EDTA and CaCl 2 plus various selective protease inhibitors, as indicated above the blots, followed by Western blots probed with anti-calpain I or anti-GSK-3β to detect the proteolysis. Apr, aprotinin (a serine protease inhibitor); Pep, pepstatin (an aspartic protease inhibitor); Leu, leupeptin (cysteine and serine protease inhibitor that also inhibits calpain); ALLN, N-acetyl-Leu-Leu-Nle-CHO (a cysteine protease inhibitor that also inhibits calpain); Calp, calpastatin peptide (a specific calpain inhibitor). (C, D) Western blots show calcium concentration-dependent truncation of recombinant GSK-3β from bacteria (C) or from mammalian cells (D) by calpain I. Recombinant GSK-3β was incubated with various concentration of calpain I in the presence of CaCl 2 for 10 min at 30°C and the reaction products were subjected to Western blots.

    Journal: Scientific Reports

    Article Title: Truncation and activation of GSK-3β by calpain I: a molecular mechanism links to tau hyperphosphorylation in Alzheimer's disease

    doi: 10.1038/srep08187

    Figure Lengend Snippet: GSK-3β is truncated selectively by calcium-mediated truncation/activation of calpain I. (A) Western blots show that truncation of GSK-3β coincides with truncation/activation of calpain I in a calcium-dependent manner in human brain extracts. Normal human brain extract was incubated at 30°C for 10 min in the presence of 2.0 mM EDTA and various concentrations (0.00–2.14 mM) of CaCl 2 . Then the incubated extracts were analyzed by Western blots developed with specific antibodies to calpain I or GSK-3β. (B) Calcium-activated truncations of calpain I and GSK-3β are selectively inhibited by calpain inhibitors. Normal human brain extract was incubated at 30°C for 10 min in the presence of 2.0 mM each of EDTA and CaCl 2 plus various selective protease inhibitors, as indicated above the blots, followed by Western blots probed with anti-calpain I or anti-GSK-3β to detect the proteolysis. Apr, aprotinin (a serine protease inhibitor); Pep, pepstatin (an aspartic protease inhibitor); Leu, leupeptin (cysteine and serine protease inhibitor that also inhibits calpain); ALLN, N-acetyl-Leu-Leu-Nle-CHO (a cysteine protease inhibitor that also inhibits calpain); Calp, calpastatin peptide (a specific calpain inhibitor). (C, D) Western blots show calcium concentration-dependent truncation of recombinant GSK-3β from bacteria (C) or from mammalian cells (D) by calpain I. Recombinant GSK-3β was incubated with various concentration of calpain I in the presence of CaCl 2 for 10 min at 30°C and the reaction products were subjected to Western blots.

    Article Snippet: In vitro proteolysis of GSK-3β Recombinant GSK-3β (Calbiochem, San Diego, CA) or affinity purified GSK-3β, as described above, was proteolyzed in vitro by incubating with calpain I (Sigma) or calpain II (Calbiochem) in proteolysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM CaCl2 , 10 mM β-mercaptoethanol) for 10 min at 30°C.

    Techniques: Activation Assay, Western Blot, Incubation, Protease Inhibitor, Concentration Assay, Recombinant

    Kainic acid (KA)-induced excitotoxicity causes truncation of GSK-3β and an increase in tau phosphorylation in mouse brain. (A, B) Western blots show C-terminal truncation of GSK-3β (A) and hyperphosphorylation of tau (B) in brains of mice intraperitoneally injected with KA. Homogenates of forebrains of mice sacrificed at the indicated time points after single intraperitoneal KA injection were analyzed by Western blots developed with GSK-3β antibodies indicated at right side of each blot. (C, D) KA-induced excitotoxicity increased phosphorylation of tau at Ser 199 and Ser 396. Forebrain homogenates of KA-injected mice were analyzed by Western blots (B) developed with anti-pSer199, PHF-1 (pS396), anti-total-tau, anti-NeuN and anti-GAPDH. The levels of total tau (C) were normalized with NeuN or GAPDH, and Phosphorylation of tau at Ser 199 and Ser 396 (D) were normalized with total tau level and plotted against the time after KA-injection. The data are presented as mean of two mice. (E, F) Inhibition of calpain prevents kainic acid (KA)-induced truncation and phosphorylation of GSK-3β in mouse brains. Calpain inhibitor calpeptin was intracerebroventricularly injected 3 hr before KA intraperitoneal injection. Homogenates of forebrains of mice sacrificed after 12 hr KA injection were analyzed by Western blots developed with GSK-3β and quantified densitometrically. The level of pSer9-GSK-3β (normalized with GSK-3β) (F) is shown as mean ± S.D. (n = 4–6). (G, H) Inhibition of calpain suppressed tau phosphorylation at Ser396 caused by KA even though inhibitory phosphorylation of GSK-3β at Ser 9 was decreased. Total tau and phosphorylated tau at Ser 396 in above samples were analyzed by Western blots (G) and quantified as described above. The level of phosphorylated tau at Ser 396 normalized with total tau (H) is shown as mean ± S.D. (n = 4–6); *, KA Via Con and p

    Journal: Scientific Reports

    Article Title: Truncation and activation of GSK-3β by calpain I: a molecular mechanism links to tau hyperphosphorylation in Alzheimer's disease

    doi: 10.1038/srep08187

    Figure Lengend Snippet: Kainic acid (KA)-induced excitotoxicity causes truncation of GSK-3β and an increase in tau phosphorylation in mouse brain. (A, B) Western blots show C-terminal truncation of GSK-3β (A) and hyperphosphorylation of tau (B) in brains of mice intraperitoneally injected with KA. Homogenates of forebrains of mice sacrificed at the indicated time points after single intraperitoneal KA injection were analyzed by Western blots developed with GSK-3β antibodies indicated at right side of each blot. (C, D) KA-induced excitotoxicity increased phosphorylation of tau at Ser 199 and Ser 396. Forebrain homogenates of KA-injected mice were analyzed by Western blots (B) developed with anti-pSer199, PHF-1 (pS396), anti-total-tau, anti-NeuN and anti-GAPDH. The levels of total tau (C) were normalized with NeuN or GAPDH, and Phosphorylation of tau at Ser 199 and Ser 396 (D) were normalized with total tau level and plotted against the time after KA-injection. The data are presented as mean of two mice. (E, F) Inhibition of calpain prevents kainic acid (KA)-induced truncation and phosphorylation of GSK-3β in mouse brains. Calpain inhibitor calpeptin was intracerebroventricularly injected 3 hr before KA intraperitoneal injection. Homogenates of forebrains of mice sacrificed after 12 hr KA injection were analyzed by Western blots developed with GSK-3β and quantified densitometrically. The level of pSer9-GSK-3β (normalized with GSK-3β) (F) is shown as mean ± S.D. (n = 4–6). (G, H) Inhibition of calpain suppressed tau phosphorylation at Ser396 caused by KA even though inhibitory phosphorylation of GSK-3β at Ser 9 was decreased. Total tau and phosphorylated tau at Ser 396 in above samples were analyzed by Western blots (G) and quantified as described above. The level of phosphorylated tau at Ser 396 normalized with total tau (H) is shown as mean ± S.D. (n = 4–6); *, KA Via Con and p

    Article Snippet: In vitro proteolysis of GSK-3β Recombinant GSK-3β (Calbiochem, San Diego, CA) or affinity purified GSK-3β, as described above, was proteolyzed in vitro by incubating with calpain I (Sigma) or calpain II (Calbiochem) in proteolysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM CaCl2 , 10 mM β-mercaptoethanol) for 10 min at 30°C.

    Techniques: Western Blot, Mouse Assay, Injection, Inhibition

    Truncation of GSK-3β is positively correlated with site-specific phosphorylation of tau, Tangle Score and Braak State in human brain. (A) Quantitation of immune-dot-blots (not shown) by densitometry shows hyperphosphorylation of tau at each site in AD brain. Tau phosphorylation at individual phosphorylation sites in the frontal cortical crude extracts from 7 AD and 7 control cases were determined by quantitative immuno-dot-blots and relative signal obtained by densitrometry is shown as mean ± SD (n = 7). **, p

    Journal: Scientific Reports

    Article Title: Truncation and activation of GSK-3β by calpain I: a molecular mechanism links to tau hyperphosphorylation in Alzheimer's disease

    doi: 10.1038/srep08187

    Figure Lengend Snippet: Truncation of GSK-3β is positively correlated with site-specific phosphorylation of tau, Tangle Score and Braak State in human brain. (A) Quantitation of immune-dot-blots (not shown) by densitometry shows hyperphosphorylation of tau at each site in AD brain. Tau phosphorylation at individual phosphorylation sites in the frontal cortical crude extracts from 7 AD and 7 control cases were determined by quantitative immuno-dot-blots and relative signal obtained by densitrometry is shown as mean ± SD (n = 7). **, p

    Article Snippet: In vitro proteolysis of GSK-3β Recombinant GSK-3β (Calbiochem, San Diego, CA) or affinity purified GSK-3β, as described above, was proteolyzed in vitro by incubating with calpain I (Sigma) or calpain II (Calbiochem) in proteolysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM CaCl2 , 10 mM β-mercaptoethanol) for 10 min at 30°C.

    Techniques: Quantitation Assay

    Truncation of GSK-3β affects its ability to phosphorylate PKA-primed tau in site-specific manner. Recombinant GSK-3β was first incubated without or with 0.2 μg/ml calpain I in the presence of 1 mM CaCl 2 for 10 min at 30°C. The GSK-3β truncation by calpain I was inhibited by adding ALLN, followed by incubation with tau 441 or PKA-prephosphorylated tau 441 for various times. The phosphorylation of tau at individual sites was measured by immuno-dot blots developed with phospho-dependent antibodies to various specific phosphorylation sites of tau (A) and the relative levels of tau phosphorylation at individual sites detected in panel A were quantitated by densitometry and plotted against tau phosphorylation reaction times (B). Truncation of GSK-3β by calpain I enhanced its kinase activity toward tau Ser 199, Thr 205, Thr 217 and Ser 396, but like full length GSK-3β (see Fig. S5 ) the ability of the truncated kinase to phosphorylate PKA-phosphorylated tau at Thr 212 is reduced.

    Journal: Scientific Reports

    Article Title: Truncation and activation of GSK-3β by calpain I: a molecular mechanism links to tau hyperphosphorylation in Alzheimer's disease

    doi: 10.1038/srep08187

    Figure Lengend Snippet: Truncation of GSK-3β affects its ability to phosphorylate PKA-primed tau in site-specific manner. Recombinant GSK-3β was first incubated without or with 0.2 μg/ml calpain I in the presence of 1 mM CaCl 2 for 10 min at 30°C. The GSK-3β truncation by calpain I was inhibited by adding ALLN, followed by incubation with tau 441 or PKA-prephosphorylated tau 441 for various times. The phosphorylation of tau at individual sites was measured by immuno-dot blots developed with phospho-dependent antibodies to various specific phosphorylation sites of tau (A) and the relative levels of tau phosphorylation at individual sites detected in panel A were quantitated by densitometry and plotted against tau phosphorylation reaction times (B). Truncation of GSK-3β by calpain I enhanced its kinase activity toward tau Ser 199, Thr 205, Thr 217 and Ser 396, but like full length GSK-3β (see Fig. S5 ) the ability of the truncated kinase to phosphorylate PKA-phosphorylated tau at Thr 212 is reduced.

    Article Snippet: In vitro proteolysis of GSK-3β Recombinant GSK-3β (Calbiochem, San Diego, CA) or affinity purified GSK-3β, as described above, was proteolyzed in vitro by incubating with calpain I (Sigma) or calpain II (Calbiochem) in proteolysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM CaCl2 , 10 mM β-mercaptoethanol) for 10 min at 30°C.

    Techniques: Recombinant, Incubation, Activity Assay

    Proteolysis of GSK-3β by calpain I increases its tau kinase activity. (A) Proteolysis of GSK-3β by calpain I elevates its kinase activity toward tau in a time dependent manner. Recombinant GSK-3β was incubated with 0.1 μg/ml calpain I for various times at 30°C. The reaction products were subjected to kinase activity assay toward tau 441 in presence of a calpain inhibitor, ALLN. (B, C) Immuno-dot-blots developed with phospho-dependent antibodies to various phosphorylation sites of tau and anti-total tau show that proteolysis of GKS-3β by calpain I increases its kinase activity. Recombinant GSK-3β from mammalian cells was incubated without or with 0.2 μg/ml calpain I in presence of 1 mM CaCl 2 for 10 min at 30°C. The reaction products were incubated with tau 441 for various times (0.5–60 min) in the presence of ALLN. The phosphorylation of tau at individual sites was assayed by immune-dot blots (B) and relative level of tau phosphorylation at individual sites detected in panel B was quantitated by densitometry and plotted against the reaction time (0–60 min) (C).

    Journal: Scientific Reports

    Article Title: Truncation and activation of GSK-3β by calpain I: a molecular mechanism links to tau hyperphosphorylation in Alzheimer's disease

    doi: 10.1038/srep08187

    Figure Lengend Snippet: Proteolysis of GSK-3β by calpain I increases its tau kinase activity. (A) Proteolysis of GSK-3β by calpain I elevates its kinase activity toward tau in a time dependent manner. Recombinant GSK-3β was incubated with 0.1 μg/ml calpain I for various times at 30°C. The reaction products were subjected to kinase activity assay toward tau 441 in presence of a calpain inhibitor, ALLN. (B, C) Immuno-dot-blots developed with phospho-dependent antibodies to various phosphorylation sites of tau and anti-total tau show that proteolysis of GKS-3β by calpain I increases its kinase activity. Recombinant GSK-3β from mammalian cells was incubated without or with 0.2 μg/ml calpain I in presence of 1 mM CaCl 2 for 10 min at 30°C. The reaction products were incubated with tau 441 for various times (0.5–60 min) in the presence of ALLN. The phosphorylation of tau at individual sites was assayed by immune-dot blots (B) and relative level of tau phosphorylation at individual sites detected in panel B was quantitated by densitometry and plotted against the reaction time (0–60 min) (C).

    Article Snippet: In vitro proteolysis of GSK-3β Recombinant GSK-3β (Calbiochem, San Diego, CA) or affinity purified GSK-3β, as described above, was proteolyzed in vitro by incubating with calpain I (Sigma) or calpain II (Calbiochem) in proteolysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM CaCl2 , 10 mM β-mercaptoethanol) for 10 min at 30°C.

    Techniques: Activity Assay, Recombinant, Incubation, Kinase Assay

    GSK-3β is truncated at the C-terminus between Ser381 and Ser382 by calpain I in vitro and in AD brain. (A) Schematic of GSK-3β domain structures and the epitopes of antibodies used to map the truncation. (B) Western blots of GSK-3β proteolysis by calpain I and of control (no calpain I) developed with various antibodies to N-terminal and C-terminal of GSK-3β. Recombinant GSK-3β was incubated without or with 0.2 μg/ml calpain I in the presence of 1 mM CaCl 2 for 10 min at 30°C. The proteolyzed products were analyzed by Western blots with a series of antibodies against different epitopes of GSK-3β. NT, N-terminal region; CT, C-terminal region; HC, Ig G heavy chain; LC, Ig G light chain. (C) Western blots of AD and control brain homogenates developed with various N-terminal and C-terminal anti-GSK-3β. Western blots of frontal cortical homogenates from AD and control cases developed with antibodies against different epitopes of GSK-3β, as labeled in panel A. Arrow indicates truncated GSK-3β; arrow head indicates a non-AD-related truncation of GSK-3β. (D) Western blots of AD and control brain homogenates and of HA-GSK-3β proteolyzed by calpain I in vitro and its control developed with R127. Western blots of frontal cortical homogenates from AD, control cases, and immunpuified GSK-3β proteolyzed with or without calpain I as described above developed with antibody R127 to GSK-3β. (E) Amino acid sequences of regions of GSK-3β (β) and GSK-3α (α) where calpain I cleaves GSK-3β (between Ser 381 and Ser 382). GSK-3β-GST recombinant fusion protein was proteolyzed with calpain I in vitro , then subjected to SDS-PAGE and the truncated GSK-3β band recognized by anti-GST was cut out and subjected to N-terminal sequencing. Dotted arrow shows the truncation site between Ile 397-Gln 398 reported in the literature for calpain II 29 .

    Journal: Scientific Reports

    Article Title: Truncation and activation of GSK-3β by calpain I: a molecular mechanism links to tau hyperphosphorylation in Alzheimer's disease

    doi: 10.1038/srep08187

    Figure Lengend Snippet: GSK-3β is truncated at the C-terminus between Ser381 and Ser382 by calpain I in vitro and in AD brain. (A) Schematic of GSK-3β domain structures and the epitopes of antibodies used to map the truncation. (B) Western blots of GSK-3β proteolysis by calpain I and of control (no calpain I) developed with various antibodies to N-terminal and C-terminal of GSK-3β. Recombinant GSK-3β was incubated without or with 0.2 μg/ml calpain I in the presence of 1 mM CaCl 2 for 10 min at 30°C. The proteolyzed products were analyzed by Western blots with a series of antibodies against different epitopes of GSK-3β. NT, N-terminal region; CT, C-terminal region; HC, Ig G heavy chain; LC, Ig G light chain. (C) Western blots of AD and control brain homogenates developed with various N-terminal and C-terminal anti-GSK-3β. Western blots of frontal cortical homogenates from AD and control cases developed with antibodies against different epitopes of GSK-3β, as labeled in panel A. Arrow indicates truncated GSK-3β; arrow head indicates a non-AD-related truncation of GSK-3β. (D) Western blots of AD and control brain homogenates and of HA-GSK-3β proteolyzed by calpain I in vitro and its control developed with R127. Western blots of frontal cortical homogenates from AD, control cases, and immunpuified GSK-3β proteolyzed with or without calpain I as described above developed with antibody R127 to GSK-3β. (E) Amino acid sequences of regions of GSK-3β (β) and GSK-3α (α) where calpain I cleaves GSK-3β (between Ser 381 and Ser 382). GSK-3β-GST recombinant fusion protein was proteolyzed with calpain I in vitro , then subjected to SDS-PAGE and the truncated GSK-3β band recognized by anti-GST was cut out and subjected to N-terminal sequencing. Dotted arrow shows the truncation site between Ile 397-Gln 398 reported in the literature for calpain II 29 .

    Article Snippet: In vitro proteolysis of GSK-3β Recombinant GSK-3β (Calbiochem, San Diego, CA) or affinity purified GSK-3β, as described above, was proteolyzed in vitro by incubating with calpain I (Sigma) or calpain II (Calbiochem) in proteolysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM CaCl2 , 10 mM β-mercaptoethanol) for 10 min at 30°C.

    Techniques: In Vitro, Western Blot, Recombinant, Incubation, Labeling, SDS Page, Sequencing

    Activation of calpain I and truncation of GSK-3β are elevated in AD brain and truncation of GSK-3β is correlated with the activation of calpain I in human brain. (A) Western blots of frontal cortical homogenates from AD and control cases show an increase in truncation of GSK-3β and calpain I in AD. Arrow indicates the full-length GSK-3β or calpain I and vertical bars indicate the truncated forms of these proteins. (B) The levels of full length and truncated GSK-3β were decreased and increased, respectively, in AD brains. Blots from panel A were quantitated by densitometry and the relative levels of total, full-length and truncated GSK-3β are presented as mean ± S.D. (n = 3). ***, p

    Journal: Scientific Reports

    Article Title: Truncation and activation of GSK-3β by calpain I: a molecular mechanism links to tau hyperphosphorylation in Alzheimer's disease

    doi: 10.1038/srep08187

    Figure Lengend Snippet: Activation of calpain I and truncation of GSK-3β are elevated in AD brain and truncation of GSK-3β is correlated with the activation of calpain I in human brain. (A) Western blots of frontal cortical homogenates from AD and control cases show an increase in truncation of GSK-3β and calpain I in AD. Arrow indicates the full-length GSK-3β or calpain I and vertical bars indicate the truncated forms of these proteins. (B) The levels of full length and truncated GSK-3β were decreased and increased, respectively, in AD brains. Blots from panel A were quantitated by densitometry and the relative levels of total, full-length and truncated GSK-3β are presented as mean ± S.D. (n = 3). ***, p

    Article Snippet: In vitro proteolysis of GSK-3β Recombinant GSK-3β (Calbiochem, San Diego, CA) or affinity purified GSK-3β, as described above, was proteolyzed in vitro by incubating with calpain I (Sigma) or calpain II (Calbiochem) in proteolysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM CaCl2 , 10 mM β-mercaptoethanol) for 10 min at 30°C.

    Techniques: Activation Assay, Western Blot

    Truncation of GSK-3β at Ser381 enhances its tau kinase activity. (A) Schematics of GSK-3β full legth and C-terminally truncated at Ile 397 or Ser 381. (B) Western blots of full length and truncated GSK-3β overexpressed in HEK-293FT cells. pCI/HA-GSK-3β, pCI/HA-GSK-3β 1-397 or pCI/HA-GSK-3β 1-381 was co-transfected with pCI/tau 441 into HEK-293FT cells. The levels of GSK-3β and actin were analyzed by Western blots, quantified by densitometry and normalized by GAPDH. The data are presented as mean ± S.D. (n = 3). **, p

    Journal: Scientific Reports

    Article Title: Truncation and activation of GSK-3β by calpain I: a molecular mechanism links to tau hyperphosphorylation in Alzheimer's disease

    doi: 10.1038/srep08187

    Figure Lengend Snippet: Truncation of GSK-3β at Ser381 enhances its tau kinase activity. (A) Schematics of GSK-3β full legth and C-terminally truncated at Ile 397 or Ser 381. (B) Western blots of full length and truncated GSK-3β overexpressed in HEK-293FT cells. pCI/HA-GSK-3β, pCI/HA-GSK-3β 1-397 or pCI/HA-GSK-3β 1-381 was co-transfected with pCI/tau 441 into HEK-293FT cells. The levels of GSK-3β and actin were analyzed by Western blots, quantified by densitometry and normalized by GAPDH. The data are presented as mean ± S.D. (n = 3). **, p

    Article Snippet: In vitro proteolysis of GSK-3β Recombinant GSK-3β (Calbiochem, San Diego, CA) or affinity purified GSK-3β, as described above, was proteolyzed in vitro by incubating with calpain I (Sigma) or calpain II (Calbiochem) in proteolysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM CaCl2 , 10 mM β-mercaptoethanol) for 10 min at 30°C.

    Techniques: Activity Assay, Western Blot, Transfection

    Identification of critical residues for Nrf2 docking onto β-TrCP. (A) HEK293T cells were cotransfected with Flag-tagged β-TrCP, HA-tagged GSK-3β Y216F (plus incubation with SB216763), and GSK-3β Δ9 , as indicated,

    Journal: Molecular and Cellular Biology

    Article Title: Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/?-TrCP Axis

    doi: 10.1128/MCB.00180-12

    Figure Lengend Snippet: Identification of critical residues for Nrf2 docking onto β-TrCP. (A) HEK293T cells were cotransfected with Flag-tagged β-TrCP, HA-tagged GSK-3β Y216F (plus incubation with SB216763), and GSK-3β Δ9 , as indicated,

    Article Snippet: In all cases, the kinase assays were performed using 5 ng of active recombinant GSK-3β (Millipore) per reaction and 5 μCi of [γ-32 P]ATP in 25 μl of reaction buffer (10 mM MgCl2 , 100 μM ATP in 40 mM MOPS [pH 7.0], and 1 mM EDTA) for 20 min at 30°C with continuous shaking.

    Techniques: Incubation

    The Neh6 domain is phosphorylated by GSK-3β. (A) (Left) HEK293T cells were transfected with expression vectors for EYFP-mNrf2 (317–372) -V5 and mutant EYFP-mNrf2 (317–372)6S/6A -V5. Cell lysates were incubated with λPPase as

    Journal: Molecular and Cellular Biology

    Article Title: Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/?-TrCP Axis

    doi: 10.1128/MCB.00180-12

    Figure Lengend Snippet: The Neh6 domain is phosphorylated by GSK-3β. (A) (Left) HEK293T cells were transfected with expression vectors for EYFP-mNrf2 (317–372) -V5 and mutant EYFP-mNrf2 (317–372)6S/6A -V5. Cell lysates were incubated with λPPase as

    Article Snippet: In all cases, the kinase assays were performed using 5 ng of active recombinant GSK-3β (Millipore) per reaction and 5 μCi of [γ-32 P]ATP in 25 μl of reaction buffer (10 mM MgCl2 , 100 μM ATP in 40 mM MOPS [pH 7.0], and 1 mM EDTA) for 20 min at 30°C with continuous shaking.

    Techniques: Transfection, Expressing, Mutagenesis, Incubation

    GSK-3β deficiency results in increased expression of Nrf2-target genes and antioxidant capacity. (A) Immunoblots showing protein levels of five enzymes regulated by Nrf2 (heme oxygenase 1 [HO-1], glutathione S -transferase M5 [GSTM5], γ-glutamyl

    Journal: Molecular and Cellular Biology

    Article Title: Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/?-TrCP Axis

    doi: 10.1128/MCB.00180-12

    Figure Lengend Snippet: GSK-3β deficiency results in increased expression of Nrf2-target genes and antioxidant capacity. (A) Immunoblots showing protein levels of five enzymes regulated by Nrf2 (heme oxygenase 1 [HO-1], glutathione S -transferase M5 [GSTM5], γ-glutamyl

    Article Snippet: In all cases, the kinase assays were performed using 5 ng of active recombinant GSK-3β (Millipore) per reaction and 5 μCi of [γ-32 P]ATP in 25 μl of reaction buffer (10 mM MgCl2 , 100 μM ATP in 40 mM MOPS [pH 7.0], and 1 mM EDTA) for 20 min at 30°C with continuous shaking.

    Techniques: Expressing, Western Blot

    Up-regulation of Nrf2 by SFN is partially achieved through the AKT/GSK-3β/Fyn pathway. Cardiac tissues were collected as described in Fig. 1 . Cardiac Akt ( A ) and GSK-3β ( B ) phosphorylation was examined by Western blot. The nuclear translocation of Fyn was determined by immunofluorescent staining of Fyn nuclear accumulation (red) on heart tissue sections ( C , bar = 100μm) and Western blot assay of Fyn expression in cardiac nuclear fraction ( D ). Data are presented as the mean ± SD (n = 7). *, p

    Journal: Redox Biology

    Article Title: Sulforaphane prevents angiotensin II-induced cardiomyopathy by activation of Nrf2 via stimulating the Akt/GSK-3ß/Fyn pathway

    doi: 10.1016/j.redox.2017.12.016

    Figure Lengend Snippet: Up-regulation of Nrf2 by SFN is partially achieved through the AKT/GSK-3β/Fyn pathway. Cardiac tissues were collected as described in Fig. 1 . Cardiac Akt ( A ) and GSK-3β ( B ) phosphorylation was examined by Western blot. The nuclear translocation of Fyn was determined by immunofluorescent staining of Fyn nuclear accumulation (red) on heart tissue sections ( C , bar = 100μm) and Western blot assay of Fyn expression in cardiac nuclear fraction ( D ). Data are presented as the mean ± SD (n = 7). *, p

    Article Snippet: In the mechanistic study, cells were exposed to a PI3K inhibitor (Ly294002 at 10 µmol/l, Sigma-Aldrich) or a GSK-3β activator [sodium nitroprusside (SNP) at 2 mmol/l, Sigma-Aldrich, based on a pilot study] with or without the presence of SFN (100 nmol/l, Sigma-Aldrich) for 24 h .

    Techniques: Western Blot, Translocation Assay, Staining, Expressing

    A simplified model of the relationship between Wnt signaling and AD pathology. Chronic noise exposure and aging could induce activation of Dkk-1, a Wnt pathway inhibitor. In the one hand, in absence of Wnt ligands, Dvl protein expression decreased and both β-catenin and tau are phosphorylated by GSK-3β, in the other hand, without the inhibition of Wnt, the expression of Aβ significantly increased, which further exacerbates the downregulation of wnt. All of this indicates that the AD-like neuropathology observed in mice of accelerated aging after chronic noise is likely to be the result of dysregulation of Wnt signaling. Dkk-1, Dickkopf-related protein 1; Dvl, disheveled; p, phosphorylation.

    Journal: Scientific Reports

    Article Title: Chronic noise exposure exacerbates AD-like neuropathology in SAMP8 mice in relation to Wnt signaling in the PFC and hippocampus

    doi: 10.1038/s41598-018-32948-4

    Figure Lengend Snippet: A simplified model of the relationship between Wnt signaling and AD pathology. Chronic noise exposure and aging could induce activation of Dkk-1, a Wnt pathway inhibitor. In the one hand, in absence of Wnt ligands, Dvl protein expression decreased and both β-catenin and tau are phosphorylated by GSK-3β, in the other hand, without the inhibition of Wnt, the expression of Aβ significantly increased, which further exacerbates the downregulation of wnt. All of this indicates that the AD-like neuropathology observed in mice of accelerated aging after chronic noise is likely to be the result of dysregulation of Wnt signaling. Dkk-1, Dickkopf-related protein 1; Dvl, disheveled; p, phosphorylation.

    Article Snippet: GSK-3β (polyclonal, 1:1000; Bioworld Technology, China), GSK-3β Ser (polyclonal, 1:1000; Bioworld Technology, China), Dvl (polyclonal, 1:100; Santa Cruz Biotechnology, USA), β-catenin (polyclonal, 1:1000; Boster Technology, USA), β-catenin (S33/37, polyclonal, 1:1000; Boster Technology, USA), Dkk-1 (polyclonal, 1:100; Bioworld Technology), and p53 (polyclonal, 1:100; Santa Cruz Biotechnology, USA) antibodies were used to detect endogenous levels of Wnt.

    Techniques: Activation Assay, Expressing, Inhibition, Mouse Assay

    A simplified model of the relationship between Wnt signaling and AD pathology. Chronic noise exposure and aging could induce activation of Dkk-1, a Wnt pathway inhibitor. In the one hand, in absence of Wnt ligands, Dvl protein expression decreased and both β-catenin and tau are phosphorylated by GSK-3β, in the other hand, without the inhibition of Wnt, the expression of Aβ significantly increased, which further exacerbates the downregulation of wnt. All of this indicates that the AD-like neuropathology observed in mice of accelerated aging after chronic noise is likely to be the result of dysregulation of Wnt signaling. Dkk-1, Dickkopf-related protein 1; Dvl, disheveled; p, phosphorylation.

    Journal: Scientific Reports

    Article Title: Chronic noise exposure exacerbates AD-like neuropathology in SAMP8 mice in relation to Wnt signaling in the PFC and hippocampus

    doi: 10.1038/s41598-018-32948-4

    Figure Lengend Snippet: A simplified model of the relationship between Wnt signaling and AD pathology. Chronic noise exposure and aging could induce activation of Dkk-1, a Wnt pathway inhibitor. In the one hand, in absence of Wnt ligands, Dvl protein expression decreased and both β-catenin and tau are phosphorylated by GSK-3β, in the other hand, without the inhibition of Wnt, the expression of Aβ significantly increased, which further exacerbates the downregulation of wnt. All of this indicates that the AD-like neuropathology observed in mice of accelerated aging after chronic noise is likely to be the result of dysregulation of Wnt signaling. Dkk-1, Dickkopf-related protein 1; Dvl, disheveled; p, phosphorylation.

    Article Snippet: GSK-3β (polyclonal, 1:1000; Bioworld Technology, China), GSK-3β Ser (polyclonal, 1:1000; Bioworld Technology, China), Dvl (polyclonal, 1:100; Santa Cruz Biotechnology, USA), β-catenin (polyclonal, 1:1000; Boster Technology, USA), β-catenin (S33/37, polyclonal, 1:1000; Boster Technology, USA), Dkk-1 (polyclonal, 1:100; Bioworld Technology), and p53 (polyclonal, 1:100; Santa Cruz Biotechnology, USA) antibodies were used to detect endogenous levels of Wnt.

    Techniques: Activation Assay, Expressing, Inhibition, Mouse Assay

    Western blots (WB) performed on frontal cortex of mice treated with haloperidol decanoate (N = 11) or vehicle (N = 9) for 2 weeks. (A) No differences were observed in total GSK-3β. (B) Inactivated pGSK-3β was reduced in haloperidol treated mice. (C) The ratio of inactive/total pGSK-3β/total GSK-3β was reduced in haloperidol treated mice, suggesting an activation of the kinase. The insert displays typical gel lanes from immunoblots used for quantitative analyses with ImageJ.

    Journal: Alzheimer's & Dementia : Translational Research & Clinical Interventions

    Article Title: Haloperidol inactivates AMPK and reduces tau phosphorylation in a tau mouse model of Alzheimer's disease

    doi: 10.1016/j.trci.2016.05.003

    Figure Lengend Snippet: Western blots (WB) performed on frontal cortex of mice treated with haloperidol decanoate (N = 11) or vehicle (N = 9) for 2 weeks. (A) No differences were observed in total GSK-3β. (B) Inactivated pGSK-3β was reduced in haloperidol treated mice. (C) The ratio of inactive/total pGSK-3β/total GSK-3β was reduced in haloperidol treated mice, suggesting an activation of the kinase. The insert displays typical gel lanes from immunoblots used for quantitative analyses with ImageJ.

    Article Snippet: Once transferred, membranes were probed with antibodies to glycogen synthase kinase (GSK)-3β (cell signaling #9315); Phospho GSK-3β (Ser9) (cell signaling #5558); Phospho-AMPKα (Thr172); and AMPKα (cell signaling #2603).

    Techniques: Western Blot, Mouse Assay, Activation Assay

    KLK11 affected the expression of the related factors in Wnt/β-catenin pathway and cell cycle-mediated factors. A. The expression of p-GSK-3β/GSK-3β in EC18 and TE-1 cells after lentivirus vectors delivery in each group as measured by western blot. B. The TCF/LEF luciferase ratio reported the activity of Wnt/β-catenin pathway in the indicated cells. C. Expression of CDK4, cyclin D1, CDK6, p-Rb, Rb, β-catenin (cytosol), and β-catenin (nucleus) were determined in TE-1 and EC18 cells transfected with KLK11. KLK11 inhibited the proliferation of ESCC by regulating the expression of the above proteins. D. Analysis of Ki67, cyclin D1, CDK4, CDK6, and c-myc expression in KLK11-overexpression or KLK11-knockdown ESCC cells by qPCR. Data were representative of three independent experiments (mean and SEM). *P

    Journal: American Journal of Cancer Research

    Article Title: KLK11 suppresses cellular proliferation via inhibition of Wnt/β-catenin signaling pathway in esophageal squamous cell carcinoma

    doi:

    Figure Lengend Snippet: KLK11 affected the expression of the related factors in Wnt/β-catenin pathway and cell cycle-mediated factors. A. The expression of p-GSK-3β/GSK-3β in EC18 and TE-1 cells after lentivirus vectors delivery in each group as measured by western blot. B. The TCF/LEF luciferase ratio reported the activity of Wnt/β-catenin pathway in the indicated cells. C. Expression of CDK4, cyclin D1, CDK6, p-Rb, Rb, β-catenin (cytosol), and β-catenin (nucleus) were determined in TE-1 and EC18 cells transfected with KLK11. KLK11 inhibited the proliferation of ESCC by regulating the expression of the above proteins. D. Analysis of Ki67, cyclin D1, CDK4, CDK6, and c-myc expression in KLK11-overexpression or KLK11-knockdown ESCC cells by qPCR. Data were representative of three independent experiments (mean and SEM). *P

    Article Snippet: Western blotting was performed according to the standard protocol with the following antibodies: anti-KLK11 antibody (Abcam, Cambridge, MA), anti-Ki67 (Abcam, Cambridge, MA), anti-GSK-3β (Abcam, Cambridge, MA), anti-Cyclin D1 antibody (Cell Signaling, Danvers, MA), anti-Rb antibody (Thermo, Waltham, MA), anti-p-Rb antibody (Bioworld Technology, Louis Park, MN, USA), anti-β-Catenin antibody (Cell Signaling, Danvers, MA), anti-CDK6 antibody (Bioworld Technology, Louis Park, MN, USA), anti-CDK4 antibody (Bioworld Technology, Louis Park, MN, USA), anti-GAPDH antibody (Bioworld Technology, Louis Park, MN, USA), anti-p84 antibody (Abcam, Cambridge, MA).

    Techniques: Expressing, Western Blot, Luciferase, Activity Assay, Transfection, Over Expression, Real-time Polymerase Chain Reaction