recombinant human gsk-3β Search Results


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  • 99
    Thermo Fisher gsk3β protein
    <t>GSK3β</t> regulates poly I:C-mediated c-Fos induction through the ERK and p38 pathways. ( a ) RAW264.7 cells stably expressing control shRNA (Con) or GSK3β-specific shRNA (sh-GSK3β) were stimulated with 10 μg ml −1 poly I:C for the indicated time points. Cells were separated into cytosolic and nuclear fractions, and the protein levels of p65, ATF2, c-Jun and c-Fos were determined by western blotting. α-tubulin and TBP served as markers for the cytosolic and nuclear fractions, respectively. ( b ) As in a , except that the cells were preincubated with dimethylsulphoxide or 10 μM SB216763 for 1 h and then stimulated with 10 μg ml −1 poly I:C for 90 min. Cytosolic and nuclear c-Fos levels were determined by western blotting. ( c ) BMDMs transfected with 20 nM control siRNAs (Con) or GSK3α- or GSK3β-specific siRNAs were stimulated with 10 μg ml −1 poly I:C for 1 h. Levels of c-Fos mRNA were determined by real-time PCR. ( d ) As in c , except that Gsk3b +/+ and Gsk3b −/− MEFs were transiently transfected with V5-GSK3α, HA-GSK3β (WT) or GSK3β (K85A) plasmids. ( e – h ) As in b , except that the cells were preincubated with 10 μM PD98059 ( e ), 10 μM SB203580 ( f ), 25 μM SP600125 ( g ) or 10 μM BAY 11-7085 ( h ) for 1 h and then stimulated with 10 μg ml −1 poly I:C for 90 min. Cytosolic and nuclear c-Fos and p65 protein levels were determined by western blotting. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test ( ** P
    Gsk3β Protein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore glycogen synthase kinase 3β gsk3β
    <t>GSK3β</t> regulates poly I:C-mediated c-Fos induction through the ERK and p38 pathways. ( a ) RAW264.7 cells stably expressing control shRNA (Con) or GSK3β-specific shRNA (sh-GSK3β) were stimulated with 10 μg ml −1 poly I:C for the indicated time points. Cells were separated into cytosolic and nuclear fractions, and the protein levels of p65, ATF2, c-Jun and c-Fos were determined by western blotting. α-tubulin and TBP served as markers for the cytosolic and nuclear fractions, respectively. ( b ) As in a , except that the cells were preincubated with dimethylsulphoxide or 10 μM SB216763 for 1 h and then stimulated with 10 μg ml −1 poly I:C for 90 min. Cytosolic and nuclear c-Fos levels were determined by western blotting. ( c ) BMDMs transfected with 20 nM control siRNAs (Con) or GSK3α- or GSK3β-specific siRNAs were stimulated with 10 μg ml −1 poly I:C for 1 h. Levels of c-Fos mRNA were determined by real-time PCR. ( d ) As in c , except that Gsk3b +/+ and Gsk3b −/− MEFs were transiently transfected with V5-GSK3α, HA-GSK3β (WT) or GSK3β (K85A) plasmids. ( e – h ) As in b , except that the cells were preincubated with 10 μM PD98059 ( e ), 10 μM SB203580 ( f ), 25 μM SP600125 ( g ) or 10 μM BAY 11-7085 ( h ) for 1 h and then stimulated with 10 μg ml −1 poly I:C for 90 min. Cytosolic and nuclear c-Fos and p65 protein levels were determined by western blotting. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test ( ** P
    Glycogen Synthase Kinase 3β Gsk3β, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Prospec human recombinant gsk 3β
    ( A ) Molecular docking of <t>GSK-3β</t> binding with salvianolic acid B and with positive controls. The chemical structures of phosphoaminophosphonic acid-adenylate ester (AMP-PNP), andrographolide, VP0.7, and salvianolic acid B are shown using red, black, blue, and green colored sticks, respectively. Magnesium ions are shown as gray spheres. Amino acid residues of enzyme are shown in yellow sticks. ( B – E ) Closer views of the salvianolic acid B binding pose in ( B , C ), the ATP binding site and ( D , E ) is the substrate binding site.
    Human Recombinant Gsk 3β, supplied by Prospec, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore recombinant human gsk 3β
    Expression of <t>GSK-3β</t> in Gsk-3 DKO ESCs rescues m 6 A. A , Western blotting of WT and Gsk-3 DKO ESCs stably expressing GSK-3β. ESCs were grown in the absence of LIF for 14 days. GSK-3α/β Western blotting demonstrates the expression of GSK-3β in Gsk-3 DKO ESCs (DKO:GSK-3β). Note the absence of GSK-3α protein. B , qPCR analysis of the expression of pluripotency-related genes in DKO:GSK-3β ESCs after 14 days in culture in the absence of LIF. Relative mRNA levels of pluripotency markers Nanog and Esrrb are shown with respect to Gapdh mRNA. Each experiment shown was performed in triplicate. Error bars , S.D. *, statistical significance between the indicated groups ( p
    Recombinant Human Gsk 3β, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher recombinant human gsk 3β
    A proposed model illustrating the mechanism underlying the efficiency of LX2343 in the amelioration of cognitive impairment in AD. LX2343 rescued neuronal cells from apoptosis by maintaining the integrity of mitochondrial function and morphology, alleviating the OS and JNK/p38 pathway and regulating anti- and pro-apoptotic proteins. In addition, LX2343 also inhibited tau hyperphosphorylation by functioning as a non-ATP competitive inhibitor of <t>GSK-3β.</t>
    Recombinant Human Gsk 3β, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc human recombinant gsk 3β
    A proposed model illustrating the mechanism underlying the efficiency of LX2343 in the amelioration of cognitive impairment in AD. LX2343 rescued neuronal cells from apoptosis by maintaining the integrity of mitochondrial function and morphology, alleviating the OS and JNK/p38 pathway and regulating anti- and pro-apoptotic proteins. In addition, LX2343 also inhibited tau hyperphosphorylation by functioning as a non-ATP competitive inhibitor of <t>GSK-3β.</t>
    Human Recombinant Gsk 3β, 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
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    SignalChem recombinant human gsk 3β
    (Phyto)ceramide binds to pYGSK3 and sustains its phosphorylation level and localization in flagella and cilia. (A) Immunoblot of pYGSK, total GSK3, and acetylated tubulin from Chlamydomonas treated for 24 h with myriocin (5 or 10 nM) ± 1 μM DHS. Bottom, Ponceau S staining for loading control. (B) Immunocytochemistry using antibodies against (phyto)ceramide rabbit (green), pYGSK (red), and acetylated tubulin (blue) with Chlamydomonas incubated for 60 min with GSK3β inhibitor BIO (2 μM) or lithium chloride (25 mM). (C) Higher magnification of flagella in left side of B. (D) Same as in B and C with ependymal cells ( Z -scan projection). (E) Z -scan orthogonal view of ependymal cells labeled with DAPI (blue) and antibodies against acetylated tubulin (green), pYGSK (green; left), and pSGSK (green; right). (F) C18 sphingomyelin and indicated ceramides were coated on ELISA plates and bound to recombinant <t>GSK-3β;</t> binding was quantified by development with anti-GSK-3α/β IgG and anti-mouse IgG-HRP. Mean ± SEM, n = 3, * p
    Recombinant Human Gsk 3β, supplied by SignalChem, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc human recombinant gsk 3β kinase
    Inhibition of <t>GSK-3β</t> Activity Modulates AR Transcriptional Activity
    Human Recombinant Gsk 3β Kinase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 157 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gsk 3β
    Effects of DQP on regulating glucose metabolism in ischemic heart tissue. (A) Western blot bands of GLUT4 and PFK and their quantitative results in heart tissues of rats, DQP could promote glucose intake and glycolysis to provide more energy for the ischemic heart. (B) Immunohistochemistry images of GLUT4 and quantitative results in the heart tissues of rats in different groups. (C) Myocardial glycogen levels in different groups. (D) Western blot bands of <t>GSK-3β,</t> pGSK-3β and their quantitative results in heart tissues of rats. (E) Western blot bands of GS, pGS and their quantitative results in heart tissues of rats. DQP could promote glycogen synthesis by increasing glycogen synthase. The raw date were listed in Supplementary Figure 2 . # P
    Gsk 3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2518 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho gsk 3β ser9
    14-3-3σ interacts with <t>GSK-3β.</t> (A) CCE, R1 and D3 cells transfected with control or 14-3-3σ vectors were lysed and 14-3-3σ complex was pulled down by immunoprecipitation (IP) with a Flag antibody. Proteins in the complex were analyzed by Western blotting (WB). (B) CCE cells were transfected with each isoform vector. 14-3-3 complex was pulled down and GSK-3β was determined by Western blotting. (C) Association of 14-3-3σ with WT or mutant GSK-3β was determined by immunoprecipitation (IP) with a Flag Antibody followed by Western blotting (WB) with GSK-3β and p-GSK-3β antibodies. (D) CCE cells transfected with Flag-tagged 14-3-3σ (σ) or control (CTR) vectors were treated with wortmannin. 14-3-3σ complex was immunoprecipitated with a Flag antibody and GSK-3β was analyzed by Western blotting. (E) 14-3-3σ overexpression reduced GSK-3β activity. Data represents mean ± s.d. (n = 3). *, p
    Phospho Gsk 3β Ser9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti gsk 3β
    Proposed mechanism of neuroprotective action of MMP-9 in preventing Aβ-mediated impairment of the insulin survival pathway. Aβ oligomers generated during early stages of AD stimulate pro-apoptotic pathways, such as TNFR signaling, which activates JNK kinase, resulting in neuronal insulin resistance via IRS1-Ser636 phosphorylation and <t>GSK-3β</t> activation. MMP-9 protects, in part, by reducing Aβ amyloid peptide accumulation, thus blocking Aβ oligomeric-induced defects in insulin signaling. MMP-9 also provides an additional stimulus to insulin, by increasing BDNF levels. The latter binds to the TrkB receptor, inducing IRS1-Tyr465 phosphorylation and Akt activation, hence contributing to normal neuronal function.
    Anti Gsk 3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 764 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gsk 3β d5c5z rabbit mab
    Reserved DSG(X) n S motif provides a binding site for β-transduction repeat-containing protein (β-TrCP), and activated glycogen synthase <t>kinase-3β</t> <t>(GSK-3β)</t> mediates this process. (A) Sequence alignment of human, mouse, rat and coturnix vascular endothelial growth factor receptor-2 (VEGFR-2) cDNA showed a conserved DSG(X) n S binding motif. (B) Reduced GSK-3β phosphorylation with almost unaltered total GSK-3β in human umbilical vein endothelial cells (HUVECs) exposed to glucose oxidase (GO). (C) Pretreatment for 30 min with LiCl (20 µ M) or SB216763 (20 µ M) enhanced the protein expression of VEGFR-2 in HUVECs cultured with GO. The results are expressed as the means ± SD. * P
    Gsk 3β D5c5z Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc total gsk 3β
    The induction of apoptosis by TAT-CTMP4 occurs via inhibition of the AKT pathway (A) Model human pancreatic adenocarcinoma (CFPAC-1) were treated with escalating doses of TAT-CTMP4. Samples were treated for 1 hour prior to preparation of cell lysates. Western blots were subsequently prepared and stained with anti-total AKT and anti-phospho-AKT (Ser 473 ). (B) Panc-1 lysates were generated after exposure to PBS (no treatment control), DMSO (vehicle control for wortmannin), wortmannin (1 µM), TAT-CTMP4-Inactive (10 µM), and TAT-CTMP4 (10 µM) for 18 hours. Western blots were probed with anti-total <t>GSK-3β</t> and phospho-GSK-3α/β (Ser 21/9 ). (C) Human model pancreatic adenocarcinoma (CFPAC-1) were treated with PBS (no treatment), gemcitabine (30 nM), recombinant human TNF-α (10 ng/ml), wortmannin (1 µM), TAT-CTMP4-Inactive (10 µM), and TAT-CTMP4 (10 µM). One hour later, cell lysates were generated and phospho-proteins were quantitated by bioplex analysis. Each experimental group represents an n=4. Results are expressed as the mean, with bars representing standard error of the mean.
    Total Gsk 3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 345 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho gsk 3β antibody
    <t>GSK-3β</t> phosphorylates the C-terminal domain of p190A in vitro . A , p190A protein sequences from human, mouse, and rat were analyzed using Scansite software (high stringency), and GSK-3 kinase consensus sites were identified corresponding to amino acids Ser-1472, Ser-1476, and Thr-1480. Amino acid Ser-1483 was also identified as a potential p38 MAPK site. The percentile scores correspond to the probability that these putative sites are related to documented consensus sites by chance. B , full-length p190A baculovirus-expressed protein purified from Sf9 insect cells can be phosphorylated by GSK-3β in vitro . The GSK-3β kinase assay was performed in vitro as described under “Experimental Procedures,” and reaction products were resolved by SDS-PAGE and visualized by autoradiography. C , pEBG, pEBG-50AA-WT, pEBG-50AA-1472A, pEBG-50AA-1476A, pEBG-50AA-1480A, and pEBG-50AA-1483A were transfected into COS-7 cells, and the corresponding GST fusion proteins were purified on glutathione beads. In vitro kinase assays, with or without GSK-3β, were performed as indicated. The left panel corresponds to an immunoblot demonstrating levels of the various fusion protein proteins captured from the cell lysates. The right panel corresponds to the kinase reaction products following SDS-PAGE and autoradiography. D , phosphorylation of a domain containing the C-terminal 50 amino acids of p190A in vivo . The GST-50AA fusion proteins derived from p190A were purified either from E. coli (pGEX-KG-encoded) or from COS-7 cells (pEBG-encoded). The proteins on the glutathione beads were mock-treated, treated with λ-phosphatase, or treated with λ-phosphatase in the presence of phosphatase inhibitors ( PPI ). Then the beads were washed and boiled in sample buffer, and the proteins were resolved by SDS-PAGE and stained with Coomassie Blue. The faster migrating species are presumed to reflect degradation products derived from the GST fusion proteins. E , mass spectrometry analysis identified sites of phosphorylation within the C-terminal 50 amino acids of p190A. pEBG-50 AA-WT was transfected into COS-7 cells and purified as described under “Experimental Procedures.” The proteins were gel-purified, and proteolytically cleaved peptides were analyzed by mass spectrometry. The phosphorylation sites detected in vivo are underscored in three different peptide species that were detected, and these correspond to amino acids 1472, 1476, and 1483 in p190A.
    Phospho Gsk 3β Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gsk3β antibody
    Both <t>GSK3β</t> and hnRNPK antagonized TRAIL-induced apoptosis. H1299 cells were transfected with siRNA against GSK3β ( a ), Flag-GSK3β ( b ), siRNA against hnRNPK ( c ), or Flag-hnRNPK ( d ), and treated with TRAIL (20 ng/ml, 8 hours), then subjected to Western blot analysis with the indicated antibodies. ( e,f ) Analysis of the cell death rate by Flow cytometry. H1299 cells transfected with GSK3β siRNA or hnRNPK siRNA ( e ), or transfected with Flag-GSK3β or Flag-hnRNPK ( f ) were treated with TRAIL (20 ng/ml, 8 hours) and analyzed using Annexin V/PI double staining with a FACS Calibure flow cytometer. ( g,h ) Statistical analyses of the experiments shown in ( e , f ) respectively. The analyses were performed with the results of three independent replicates for each experiment. *p
    Gsk3β Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH recombinant human gsk3β
    PDA-66 does not inhibit <t>GSK3β</t> kinase activity. Recombinant human GSK3β was incubated with pGS2, ATP and different concentrations of PDA-66 and SB-216763. While treatment with the known GSK3β inhibitor SB-216763 lead to a stable reduction of enzyme activity with significant alterations at 5 μM PDA-66 showed only a slight inhibitory potential. Results are displayed as the mean ± SD of five independent experiments. In each experiment the concentrations of PDA-66 and the control were tested with 4 replicates. RLU = relative luminescence units. *Significant treatment effect vs. DMSO control, α = 0.05.
    Recombinant Human Gsk3β, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam recombinant human gsk3β
    Increased PLK2 expression and activation of GSK-NRF2 pathway are associated with mitochondrial dysfunction in SCO2 deficient cells ( A ) Protein levels of SCO2, PLK2, and serine phosphorylated GSK3 (GSK3-S-P) (comprised of GSK3A and GSK3B, commonly known as α and β, respectively) in whole cell lysates, and NRF2 in the nuclear fraction (nuc) of the indicated cell lines. Tubulin and lamin B1 (LMNB1) serve as whole cell and nuclear fraction protein loading controls, respectively. ( B ) In vitro phosphorylation of GSK3B by PLK2 using purified recombinant proteins. Note the reciprocal relationship between the levels of the inactivating Ser-9 phosphorylation by PLK2 and the activating Tyr-216 autophosphorylation of GSK3B <t>(GSK3β-S9-P</t> and GSK3β-Y216-P, respectively). ( C ) Subcellular localization of NRF2 by confocal immunofluorescence (upper and middle panels) with DAPI staining of the nuclei (lower panel). The upper panels show enlarged images of the cells enclosed by white rectangles in the middle panels. At least 300 cells were counted in three independent experiments to quantify the fraction of cells with nuclear NRF2 localization. * P
    Recombinant Human Gsk3β, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore glycogensynthase kinase 3β gsk 3β stock
    Tau phosphorylation in vitro . One microgram recombinant human tau (left, A) or mouse tau (right, A) was incubated with <t>glycogensynthase-kinase-3β</t> <t>(GSK-3ß)</t> and ATP in kinase buffer and analyzed by Western blot using antibodies against total tau (tau-5) phospho-tau-S199, phospho-tau-T231 and phospho-tau-S396. Lanes B and C served as a control omitting either the enzyme GSK-3β (Lane B) or tau protein (Lane C). Size markers are given as kDa.
    Glycogensynthase Kinase 3β Gsk 3β Stock, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human gsk3β
    AMPK phosphorylates tau in vitro. WB analysis of recombinant tau incubated in vitro with AMPK or <t>GSK3β</t> using antibodies directed against total tau (CP27), or tau phosphorylated on Ser-396/404 (PHF1), Thr-231 (2E12), Ser-202 (CP13), Ser-409 (PG5),
    Recombinant Human Gsk3β, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam gsk3β
    mTORC2 regulates <t>GSK3β</t> in response to glucose limitation. a Schematic showing the mTORC1 and mTORC2 complexes and downstream targets. b – d Western blots using indicated antibodies of HT29 cells grown under indicated conditions. Unless otherwise indicated cells were placed in HBSS for 2 h, or treated with Torin 1 (250 nM) or starved of glucose for 2 h and where indicted refed glucose for 1 h. e Western blot using indicated antibodies of HT29 cells grown in DMEM or starved of glucose for 4 h and treated with DMSO, MK2206 (3 μM), or LY333531 (5 μM). f Western blot using indicated antibodies of HT29 cells. 48 h after transfection with RICTOR-specific or control siRNAs cells were glucose starved for 2 h, as indicated. g Western blot using indicated antibodies of HT29 cells transfected with control or RICTOR-specific siRNA for 48 h prior to glucose starvation for indicated times. h Western blot using indicated antibodies of HT29 cells grown in the presence of absence of glucose (4 h) of cells treated with Rapamycin (1 μM) or Torin 1 (250 nM). i Western blot using indicated antibodies of HT29 cells following glucose starvation for indicated times
    Gsk3β, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 368 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gsk 3β inhibitors
    Schematic of the possible regulation of AREG on the <t>EGFR/GSK-3β/Foxp3</t> axis.
    Gsk 3β Inhibitors, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SignalChem recombinant full length human gsk3β
    The half-life of USF2 is induced upon <t>GSK3β-dependent</t> phosphorylation. (A) GSK3β +/+ and GSK3β −/− cells were treated with 10 µg/ml CHX for the indicated time periods. Proteins were isolated, separated by SDS-PAGE and detected by Western blotting. Protein levels were quantified and the relative protein level of USF2 was blotted against the duration of CHX treatment for estimation of the half-life. The dashed line indicates the USF2 half-life where 50% of the USF2 protein level was reached. (B) Representative Western Blot. 50 µg of protein from were probed with an antibody against USF2 and α-tubulin.
    Recombinant Full Length Human Gsk3β, supplied by SignalChem, used in various techniques. Bioz Stars score: 87/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems full length human recombinant n gst tagged gsk3β
    The half-life of USF2 is induced upon <t>GSK3β-dependent</t> phosphorylation. (A) GSK3β +/+ and GSK3β −/− cells were treated with 10 µg/ml CHX for the indicated time periods. Proteins were isolated, separated by SDS-PAGE and detected by Western blotting. Protein levels were quantified and the relative protein level of USF2 was blotted against the duration of CHX treatment for estimation of the half-life. The dashed line indicates the USF2 half-life where 50% of the USF2 protein level was reached. (B) Representative Western Blot. 50 µg of protein from were probed with an antibody against USF2 and α-tubulin.
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    Wnt/β-catenin reduces MKX and TNMD expressions in SCX -programmed tendon progenitors (hMSC-Scx cells). hMSC-Scx cells were treated with either <t>Wnt3a,</t> <t>BIO</t> (an activator of β-catenin), IWR (an inhibitor of β-catenin), or their combination, as in Fig 2 . Relative expressions of AXIN2 , MKX , and TNMD in hMSC-Scx cells treated with 50 ng/ml Wnt3a with or without 5 μM IWR (A) , 0 to 2 μM BIO (B) , or 0 to 20 μM IWR (C) are indicated. As BIO was dissolved in DMSO, all samples in B were incubated under 0.004% DMSO. (B) Increasing concentrations (0.5, 1, and 2 μM) of BIO increased AXIN2 expression to 130%, 240%, and 700% of that without BIO, respectively. (C) Increasing concentrations (5, 10, and 20 μM) of IWR decreased AXIN2 expression to 94%, 84%, and 84% of that without IWR, respectively. Each mRNA expression is normalized by GAPDH mRNA. Mean and SD are indicated ( n = 3 wells each). p
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    Wnt/β-catenin reduces MKX and TNMD expressions in SCX -programmed tendon progenitors (hMSC-Scx cells). hMSC-Scx cells were treated with either <t>Wnt3a,</t> <t>BIO</t> (an activator of β-catenin), IWR (an inhibitor of β-catenin), or their combination, as in Fig 2 . Relative expressions of AXIN2 , MKX , and TNMD in hMSC-Scx cells treated with 50 ng/ml Wnt3a with or without 5 μM IWR (A) , 0 to 2 μM BIO (B) , or 0 to 20 μM IWR (C) are indicated. As BIO was dissolved in DMSO, all samples in B were incubated under 0.004% DMSO. (B) Increasing concentrations (0.5, 1, and 2 μM) of BIO increased AXIN2 expression to 130%, 240%, and 700% of that without BIO, respectively. (C) Increasing concentrations (5, 10, and 20 μM) of IWR decreased AXIN2 expression to 94%, 84%, and 84% of that without IWR, respectively. Each mRNA expression is normalized by GAPDH mRNA. Mean and SD are indicated ( n = 3 wells each). p
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    Thermo Fisher human gsk3β
    a) Western blots of total glycogen synthase kinase (GSK) 3β and phosphorylated (p-) <t>GSK3β</t> to verify expression of recombinant GSK3β proteins. Expression of transfected (transf.) GSK3β was differentiated from that of endogenous (End.) GSK3β by a shift in molecular size. Non-transfected myocytes served as controls. Recombinant proteins of wild-type (wtGSK), constitutively active (caGSK) or dominant-negative (dnGSK) GSK3β plasmids were detected 72 h after adenoviral infection. Corresponding samples were incubated with phosphospecific GSK3β antibody. Myocytes transfected with wtGSK and dnGSK showed clear phosphorylation. In contrast, myocytes transfected with caGSK showed no phosphorylation of the recombinant protein. (b) Cardiomyocytes stimulated with 1 μmol/L SB216763 (SB21) or [d-Ala 2 -d-Leu 5 ]-enkephalin (DADLE) exhibited GSK3β phosphorylation compared with untreated control cells, suggesting a role for GSK3β within the protective signalling of DADLE.
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    Thermo Fisher human gsk3β stealth primer set
    12B2 and 15C2 are specific for nonphospho-S GSK3 in brain lysates of human, mouse, and rat, and both effectively immunoprecipitate GSK3 from cell lysates. (A) Protein sequence alignments for <t>GSK3β</t> (amino acids 1–25) and GSK3α (amino acids 13–37) from human, mouse and rat (Uniprot IDs in parentheses). (B,C) Blots of lysates from human, mouse and rat cortical tissue and GAPDH was used as a loading control (40 μg/lane total protein loaded; experiment repeated three times). (B) 12B2 (red) specifically labeled GSK3β, not GSK3α, in lysates and total GSK3α/β (green) was used to identify both isoforms. (C) 15C2 (red) labeled both GSK3β and GSK3α in lysates and total GSK3α/β (green) was used to identify both isoforms. (D–F) The 12B2 (D) , 15C2 (E) , or control mouse IgG ( F , Ms IgG) were used to immunoprecipitate GSK3 enzymes from HEK293T cell lysates. The starting lysate (Input) was incubated with magnetic beads coated with 12B2 (D) , 15C2 (E) , or Ms IgG control (F) antibodies. 12B2 pulled down only GSK3β (12B2-IP), 15C2 pulled down both GSK3α and β (15C2-IP) and Ms IgG did not pull down GSK3α or β (MsIgG-IP). The post-IP lysates were also run for comparisons to the input samples. These experiments were performed three independent times.
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    Induction of gene expression in <t>Gsk3β</t> +/+ (wild-type) and Gsk3β −/− (knockout) mouse embryonic fibroblasts (MEFs) by IL-17 family cytokines ( A – D ) MEFs were treated with 20 ng/ml mouse recombinant IL-17A (A), IL-17F (B), IL-17C (C), and IL-17E (D) for 2 h. Gene expression was determined using qRT-PCR analysis. The levels of the control group (treated with phosphate-buffered saline) were taken as the basal levels. Data represent the mean ± standard deviation of three independent experiments ( n = 3); * P
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    Biaffin human gsk3β
    T 613 is phosphorylated by GSK3. ( A–D ) Expression and localization of Pf GSK3 and its PKA dependent phosphorylation of AMA1. ( A ) Schematic drawing of the GSK3-GFP 3′replacement approach in 3D7 parasites and diagnostic PCR revealing plasmid integration. The gsk3 gene has a six exon structure and an open reading frame of 2472 base pairs. Approximately 1 kb of the 3′ end was fused with the coding sequence of GFP (black) and cloned into a pARL derivate (pARL-gsk3-3’repl-gfp). The human dihydrofolate reductase (hDHFR, grey box) of the plasmid allowed selection of transgenic parasites. Position of oligonucleotides used for diagnostic PCR are shown with blue and red arrows. Sizes are indicated in kilo bases (kb). ( B ) Expression of Pf GSK3-GFP in late stage parasites was analyzed by Western blot analysis using anti-GFP specific antibodies. Anti-Aldolase specific antibodies were used as a loading control. ( C , D ) Epifluorescence ( C ) and confocal ( D ) localization of Pf GSK3-GFP in late trophozoites (LT) schizonts (S) and merozoites (M) revealed perinuclear and cytosolic distribution. Nuclei are stained with DAPI (blue). Scale bars, 2 μm. E. SDS-PAGE and autoradiograph of in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) of AMA1 WT and AMA1 PM incubated with human <t>GSK3β</t> (hGSK3β). ( F ) Differential in vitro phosphorylation of AMA1 variants with single phosphorylation sites (AMA1 S588 , AMA1 S601 , AMA1 S610 , AMA1 T612 , AMA1 T613 ) by hGSK3β. SDS-PAGE and autoradiograph of the in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) are shown.
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    R&D Systems anti gsk3β rat
    BSSM activates <t>AKT/GSK3β</t> pathway, promotes tau hyperphosphorylation and PHF accumulation and increases the expression of active asparagine endopeptidase (AEP) and tau cleavage. (A,B) Western blotting showed higher expressions of p-AKT and the downstream p-GSK in hippocampus in BSSM treated group. (C,D) Phosphorylation of tau at S396 was higher in BSSM group than in sham and USSM group. PHF accumulation also showed significant differences between sham and the two shear stress modifier groups. (E–G) Expression of active AEP and tau N368 truncation were higher in BSSM group. (H,I) Immunofluorescent staining further reconfirmed the finding that BSSM group had more tau N368 fragments in hippocampus than the other two groups. L, left hippocampus; R, right hippocampus. * p
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    Image Search Results


    GSK3β regulates poly I:C-mediated c-Fos induction through the ERK and p38 pathways. ( a ) RAW264.7 cells stably expressing control shRNA (Con) or GSK3β-specific shRNA (sh-GSK3β) were stimulated with 10 μg ml −1 poly I:C for the indicated time points. Cells were separated into cytosolic and nuclear fractions, and the protein levels of p65, ATF2, c-Jun and c-Fos were determined by western blotting. α-tubulin and TBP served as markers for the cytosolic and nuclear fractions, respectively. ( b ) As in a , except that the cells were preincubated with dimethylsulphoxide or 10 μM SB216763 for 1 h and then stimulated with 10 μg ml −1 poly I:C for 90 min. Cytosolic and nuclear c-Fos levels were determined by western blotting. ( c ) BMDMs transfected with 20 nM control siRNAs (Con) or GSK3α- or GSK3β-specific siRNAs were stimulated with 10 μg ml −1 poly I:C for 1 h. Levels of c-Fos mRNA were determined by real-time PCR. ( d ) As in c , except that Gsk3b +/+ and Gsk3b −/− MEFs were transiently transfected with V5-GSK3α, HA-GSK3β (WT) or GSK3β (K85A) plasmids. ( e – h ) As in b , except that the cells were preincubated with 10 μM PD98059 ( e ), 10 μM SB203580 ( f ), 25 μM SP600125 ( g ) or 10 μM BAY 11-7085 ( h ) for 1 h and then stimulated with 10 μg ml −1 poly I:C for 90 min. Cytosolic and nuclear c-Fos and p65 protein levels were determined by western blotting. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test ( ** P

    Journal: Nature Communications

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    doi: 10.1038/ncomms7765

    Figure Lengend Snippet: GSK3β regulates poly I:C-mediated c-Fos induction through the ERK and p38 pathways. ( a ) RAW264.7 cells stably expressing control shRNA (Con) or GSK3β-specific shRNA (sh-GSK3β) were stimulated with 10 μg ml −1 poly I:C for the indicated time points. Cells were separated into cytosolic and nuclear fractions, and the protein levels of p65, ATF2, c-Jun and c-Fos were determined by western blotting. α-tubulin and TBP served as markers for the cytosolic and nuclear fractions, respectively. ( b ) As in a , except that the cells were preincubated with dimethylsulphoxide or 10 μM SB216763 for 1 h and then stimulated with 10 μg ml −1 poly I:C for 90 min. Cytosolic and nuclear c-Fos levels were determined by western blotting. ( c ) BMDMs transfected with 20 nM control siRNAs (Con) or GSK3α- or GSK3β-specific siRNAs were stimulated with 10 μg ml −1 poly I:C for 1 h. Levels of c-Fos mRNA were determined by real-time PCR. ( d ) As in c , except that Gsk3b +/+ and Gsk3b −/− MEFs were transiently transfected with V5-GSK3α, HA-GSK3β (WT) or GSK3β (K85A) plasmids. ( e – h ) As in b , except that the cells were preincubated with 10 μM PD98059 ( e ), 10 μM SB203580 ( f ), 25 μM SP600125 ( g ) or 10 μM BAY 11-7085 ( h ) for 1 h and then stimulated with 10 μg ml −1 poly I:C for 90 min. Cytosolic and nuclear c-Fos and p65 protein levels were determined by western blotting. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test ( ** P

    Article Snippet: In vitro ubiquitination assay The GSK3β protein was obtained from Invitrogen (cat # PV3365) and the Flag-TRAF6 protein was purified as previously described .

    Techniques: Stable Transfection, Expressing, shRNA, Western Blot, Transfection, Real-time Polymerase Chain Reaction

    GSK3β regulates poly I:C-mediated ERK and p38 activation. ( a ) Western blotting of the phosphorylation of the MAPKs ERK, p38 and JNK and NF-κB p65 in poly I:C-stimulated RAW264.7 cells stably expressing control shRNA (Con) or GSK3β-specific shRNA (sh-GSK3β). ( b ) Western blotting of ERK, p38 and JNK phosphorylation in Gsk3b +/+ and Gsk3b −/− MEFs stimulated with 10 μg ml −1 poly I:C. ( c , d ) As in a , except that the cells were stimulated with 100 ng ml −1 LPS ( c ) or 100 ng ml −1 Pam3CSK4 ( d ) as indicated. Data are representative of two or three independent experiments.

    Journal: Nature Communications

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    doi: 10.1038/ncomms7765

    Figure Lengend Snippet: GSK3β regulates poly I:C-mediated ERK and p38 activation. ( a ) Western blotting of the phosphorylation of the MAPKs ERK, p38 and JNK and NF-κB p65 in poly I:C-stimulated RAW264.7 cells stably expressing control shRNA (Con) or GSK3β-specific shRNA (sh-GSK3β). ( b ) Western blotting of ERK, p38 and JNK phosphorylation in Gsk3b +/+ and Gsk3b −/− MEFs stimulated with 10 μg ml −1 poly I:C. ( c , d ) As in a , except that the cells were stimulated with 100 ng ml −1 LPS ( c ) or 100 ng ml −1 Pam3CSK4 ( d ) as indicated. Data are representative of two or three independent experiments.

    Article Snippet: In vitro ubiquitination assay The GSK3β protein was obtained from Invitrogen (cat # PV3365) and the Flag-TRAF6 protein was purified as previously described .

    Techniques: Activation Assay, Western Blot, Stable Transfection, Expressing, shRNA

    GSK3β associates with TRAF6, TAK1 and TRIF. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-GSK3β antibody. TRAF6 and TAK1 protein levels from whole-cell lysates (WCL) and TLR3 immunocomplexes (IP: α-GSK3β) were determined by western blotting. ( b ) GSK3β formed a ternary complex with TRAF6 and TAK1. HEK293T cells were transfected with the indicated combinations of expression plasmids. Co-imunoprecipitations were performed with anti-Flag antibody followed by western blotting with the indicated antibodies. The expression levels of the transfected plasmids were confirmed by western blot analysis of whole-cell lysates. ( c ) As in Fig. 4b , except that whole-cell lysates were immunoprecipitated with anti-TRAF6 antibody followed by western blotting as indicated. ( d ) GSK3β associated with TAB1 and TAB2 through TAK1. These experiments were performed as described in b . ( e ) TRAF6 associated with TAB1, TAB2, TAK1 and GSK3β. The experiments were performed as described in b . ( f ) GSK3β associates with TRIF, TRAF6 and TAK1. The experiments were performed as described in b . Data are representative of two or three independent experiments.

    Journal: Nature Communications

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    doi: 10.1038/ncomms7765

    Figure Lengend Snippet: GSK3β associates with TRAF6, TAK1 and TRIF. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-GSK3β antibody. TRAF6 and TAK1 protein levels from whole-cell lysates (WCL) and TLR3 immunocomplexes (IP: α-GSK3β) were determined by western blotting. ( b ) GSK3β formed a ternary complex with TRAF6 and TAK1. HEK293T cells were transfected with the indicated combinations of expression plasmids. Co-imunoprecipitations were performed with anti-Flag antibody followed by western blotting with the indicated antibodies. The expression levels of the transfected plasmids were confirmed by western blot analysis of whole-cell lysates. ( c ) As in Fig. 4b , except that whole-cell lysates were immunoprecipitated with anti-TRAF6 antibody followed by western blotting as indicated. ( d ) GSK3β associated with TAB1 and TAB2 through TAK1. These experiments were performed as described in b . ( e ) TRAF6 associated with TAB1, TAB2, TAK1 and GSK3β. The experiments were performed as described in b . ( f ) GSK3β associates with TRIF, TRAF6 and TAK1. The experiments were performed as described in b . Data are representative of two or three independent experiments.

    Article Snippet: In vitro ubiquitination assay The GSK3β protein was obtained from Invitrogen (cat # PV3365) and the Flag-TRAF6 protein was purified as previously described .

    Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing

    GSK3β is required for the recruitment of the TRAF6–TAK1–TAB1–TAB2 complex to TLR3. Gsk3b +/+ and Gsk3b −/− MEFs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-TLR3 antibody. TRIF, RIP1, TRAF6, TAK1, TAB1, TAB2 and GSK3β protein levels from whole-cell lysates (WCL) and TLR3 immunocomplexes (IP: α-TLR3) were determined by western blotting. Data are representative of two independent experiments.

    Journal: Nature Communications

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    doi: 10.1038/ncomms7765

    Figure Lengend Snippet: GSK3β is required for the recruitment of the TRAF6–TAK1–TAB1–TAB2 complex to TLR3. Gsk3b +/+ and Gsk3b −/− MEFs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-TLR3 antibody. TRIF, RIP1, TRAF6, TAK1, TAB1, TAB2 and GSK3β protein levels from whole-cell lysates (WCL) and TLR3 immunocomplexes (IP: α-TLR3) were determined by western blotting. Data are representative of two independent experiments.

    Article Snippet: In vitro ubiquitination assay The GSK3β protein was obtained from Invitrogen (cat # PV3365) and the Flag-TRAF6 protein was purified as previously described .

    Techniques: Immunoprecipitation, Western Blot

    TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P

    Journal: Nature Communications

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    doi: 10.1038/ncomms7765

    Figure Lengend Snippet: TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P

    Article Snippet: In vitro ubiquitination assay The GSK3β protein was obtained from Invitrogen (cat # PV3365) and the Flag-TRAF6 protein was purified as previously described .

    Techniques: Immunoprecipitation, Western Blot, Transfection, Incubation, In Vitro, Real-time Polymerase Chain Reaction, Expressing

    GSK3β but not GSK3α is involved in TLR3-mediated pro-inflammatory cytokine production. ( a ) RAW264.7 cells stably expressing control shRNA (Con) or GSK3β-specific shRNA (sh-GSK3β) were stimulated with 10 μg ml −1 poly I:C for 1 h. Levels of IL-6, TNF-α, IP-10, IL-12 and IL-10 mRNA were determined by real-time PCR analysis, and the values were normalized to β-actin mRNA expression. GSK3β knockdown was confirmed by reverse transcription-PCR and western blotting. ( b ) BMDMs were treated with 10 μg ml −1 poly I:C for the indicated time points. Whole-cell lysates were immunoblotted with antibodies to the molecules along the right margin. ( c ) HEK293-TLR3 cells were transiently transfected with V5-GSK3α or HA-GSK3β plasmids. Levels of IL-6 and TNF-α mRNA were determined as described in a . Expression of the transduced proteins was detected by western blotting with anti-V5 (for GSK3α) and anti-HA (for GSK3β). ( d ) BMDMs were preincubated for 1 h with or without 10 μM SB216763 and stimulated with or without 10 μg ml −1 poly I:C for 20 h. Levels of IL-6, TNF-α and IL-10 in culture supernatants were determined by enzyme-linked immunosorbent assay. ( e ) Gsk3b −/− MEFs were transiently transfected with V5-GSK3α, HA-GSK3β (WT) or GSK3β (K85A) plasmids, and cells were stimulated with 10 μg ml −1 poly I:C for 1 h. Levels of IL-6, TNF-α and IL-10 mRNA were determined as described in a . Expression of the transduced proteins was detected by western blotting as described in c . Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (* P

    Journal: Nature Communications

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    doi: 10.1038/ncomms7765

    Figure Lengend Snippet: GSK3β but not GSK3α is involved in TLR3-mediated pro-inflammatory cytokine production. ( a ) RAW264.7 cells stably expressing control shRNA (Con) or GSK3β-specific shRNA (sh-GSK3β) were stimulated with 10 μg ml −1 poly I:C for 1 h. Levels of IL-6, TNF-α, IP-10, IL-12 and IL-10 mRNA were determined by real-time PCR analysis, and the values were normalized to β-actin mRNA expression. GSK3β knockdown was confirmed by reverse transcription-PCR and western blotting. ( b ) BMDMs were treated with 10 μg ml −1 poly I:C for the indicated time points. Whole-cell lysates were immunoblotted with antibodies to the molecules along the right margin. ( c ) HEK293-TLR3 cells were transiently transfected with V5-GSK3α or HA-GSK3β plasmids. Levels of IL-6 and TNF-α mRNA were determined as described in a . Expression of the transduced proteins was detected by western blotting with anti-V5 (for GSK3α) and anti-HA (for GSK3β). ( d ) BMDMs were preincubated for 1 h with or without 10 μM SB216763 and stimulated with or without 10 μg ml −1 poly I:C for 20 h. Levels of IL-6, TNF-α and IL-10 in culture supernatants were determined by enzyme-linked immunosorbent assay. ( e ) Gsk3b −/− MEFs were transiently transfected with V5-GSK3α, HA-GSK3β (WT) or GSK3β (K85A) plasmids, and cells were stimulated with 10 μg ml −1 poly I:C for 1 h. Levels of IL-6, TNF-α and IL-10 mRNA were determined as described in a . Expression of the transduced proteins was detected by western blotting as described in c . Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (* P

    Article Snippet: In vitro ubiquitination assay The GSK3β protein was obtained from Invitrogen (cat # PV3365) and the Flag-TRAF6 protein was purified as previously described .

    Techniques: Stable Transfection, Expressing, shRNA, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay

    Ubiquitination of GSK3β is required for the formation of a signalling complex containing TRIF, TRAF6, TAK1 and GSK3β. ( a – c ) The GSK3β K183R mutant abrogates the interaction of GSK3β with TRAF6 but not with TRIF or TAK1. HEK293T cells were transfected with the indicated combinations of expression plasmids. Co-imunoprecipitations were performed with an anti-HA antibody followed by western blotting with the indicated antibodies. The expression levels of the transfected plasmids were confirmed by western blotting of whole-cell lysates. ( d , e ) The GSK3β K183R mutation disrupts the TRIF–GSK3β–TRAF6–TAK1 complex. HEK293T cells were transfected with the indicated combinations of expression plasmids. Co-imunoprecipitations were performed with anti-HA ( d ) or with anti-Flag ( e ) antibodies followed by western blotting with the indicated antibodies. The expression levels of the transfected plasmids were confirmed by western blotting of whole-cell lysates. Data are representative of two or three independent experiments.

    Journal: Nature Communications

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    doi: 10.1038/ncomms7765

    Figure Lengend Snippet: Ubiquitination of GSK3β is required for the formation of a signalling complex containing TRIF, TRAF6, TAK1 and GSK3β. ( a – c ) The GSK3β K183R mutant abrogates the interaction of GSK3β with TRAF6 but not with TRIF or TAK1. HEK293T cells were transfected with the indicated combinations of expression plasmids. Co-imunoprecipitations were performed with an anti-HA antibody followed by western blotting with the indicated antibodies. The expression levels of the transfected plasmids were confirmed by western blotting of whole-cell lysates. ( d , e ) The GSK3β K183R mutation disrupts the TRIF–GSK3β–TRAF6–TAK1 complex. HEK293T cells were transfected with the indicated combinations of expression plasmids. Co-imunoprecipitations were performed with anti-HA ( d ) or with anti-Flag ( e ) antibodies followed by western blotting with the indicated antibodies. The expression levels of the transfected plasmids were confirmed by western blotting of whole-cell lysates. Data are representative of two or three independent experiments.

    Article Snippet: In vitro ubiquitination assay The GSK3β protein was obtained from Invitrogen (cat # PV3365) and the Flag-TRAF6 protein was purified as previously described .

    Techniques: Mutagenesis, Transfection, Expressing, Western Blot

    ( A ) Molecular docking of GSK-3β binding with salvianolic acid B and with positive controls. The chemical structures of phosphoaminophosphonic acid-adenylate ester (AMP-PNP), andrographolide, VP0.7, and salvianolic acid B are shown using red, black, blue, and green colored sticks, respectively. Magnesium ions are shown as gray spheres. Amino acid residues of enzyme are shown in yellow sticks. ( B – E ) Closer views of the salvianolic acid B binding pose in ( B , C ), the ATP binding site and ( D , E ) is the substrate binding site.

    Journal: Molecules

    Article Title: Rosmarinic Acid Derivatives’ Inhibition of Glycogen Synthase Kinase-3β Is the Pharmacological Basis of Kangen-Karyu in Alzheimer’s Disease

    doi: 10.3390/molecules23112919

    Figure Lengend Snippet: ( A ) Molecular docking of GSK-3β binding with salvianolic acid B and with positive controls. The chemical structures of phosphoaminophosphonic acid-adenylate ester (AMP-PNP), andrographolide, VP0.7, and salvianolic acid B are shown using red, black, blue, and green colored sticks, respectively. Magnesium ions are shown as gray spheres. Amino acid residues of enzyme are shown in yellow sticks. ( B – E ) Closer views of the salvianolic acid B binding pose in ( B , C ), the ATP binding site and ( D , E ) is the substrate binding site.

    Article Snippet: Human recombinant GSK-3β was obtained from Prospec (ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel).

    Techniques: Binding Assay

    2D Diagram showing the binding of reference controls, AMP-PNP ( A ), indirubin ( B ), andrographolide ( C ), and VP0.7 ( D ), to GSK-3β active site.

    Journal: Molecules

    Article Title: Rosmarinic Acid Derivatives’ Inhibition of Glycogen Synthase Kinase-3β Is the Pharmacological Basis of Kangen-Karyu in Alzheimer’s Disease

    doi: 10.3390/molecules23112919

    Figure Lengend Snippet: 2D Diagram showing the binding of reference controls, AMP-PNP ( A ), indirubin ( B ), andrographolide ( C ), and VP0.7 ( D ), to GSK-3β active site.

    Article Snippet: Human recombinant GSK-3β was obtained from Prospec (ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel).

    Techniques: Binding Assay

    Lineweaver–Burk plots for GSK-3β inhibition by Sal B in the presence of various concentrations of ( A ) ATP and ( B ) GSM. Graphs below each Lineweaver-Burk plots represents secondary plots.

    Journal: Molecules

    Article Title: Rosmarinic Acid Derivatives’ Inhibition of Glycogen Synthase Kinase-3β Is the Pharmacological Basis of Kangen-Karyu in Alzheimer’s Disease

    doi: 10.3390/molecules23112919

    Figure Lengend Snippet: Lineweaver–Burk plots for GSK-3β inhibition by Sal B in the presence of various concentrations of ( A ) ATP and ( B ) GSM. Graphs below each Lineweaver-Burk plots represents secondary plots.

    Article Snippet: Human recombinant GSK-3β was obtained from Prospec (ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel).

    Techniques: Inhibition

    Expression of GSK-3β in Gsk-3 DKO ESCs rescues m 6 A. A , Western blotting of WT and Gsk-3 DKO ESCs stably expressing GSK-3β. ESCs were grown in the absence of LIF for 14 days. GSK-3α/β Western blotting demonstrates the expression of GSK-3β in Gsk-3 DKO ESCs (DKO:GSK-3β). Note the absence of GSK-3α protein. B , qPCR analysis of the expression of pluripotency-related genes in DKO:GSK-3β ESCs after 14 days in culture in the absence of LIF. Relative mRNA levels of pluripotency markers Nanog and Esrrb are shown with respect to Gapdh mRNA. Each experiment shown was performed in triplicate. Error bars , S.D. *, statistical significance between the indicated groups ( p

    Journal: The Journal of Biological Chemistry

    Article Title: Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells

    doi: 10.1074/jbc.RA117.001298

    Figure Lengend Snippet: Expression of GSK-3β in Gsk-3 DKO ESCs rescues m 6 A. A , Western blotting of WT and Gsk-3 DKO ESCs stably expressing GSK-3β. ESCs were grown in the absence of LIF for 14 days. GSK-3α/β Western blotting demonstrates the expression of GSK-3β in Gsk-3 DKO ESCs (DKO:GSK-3β). Note the absence of GSK-3α protein. B , qPCR analysis of the expression of pluripotency-related genes in DKO:GSK-3β ESCs after 14 days in culture in the absence of LIF. Relative mRNA levels of pluripotency markers Nanog and Esrrb are shown with respect to Gapdh mRNA. Each experiment shown was performed in triplicate. Error bars , S.D. *, statistical significance between the indicated groups ( p

    Article Snippet: Recombinant human GSK-3β (Sigma-Aldrich) was incubated with recombinant mouse FTO (Sigma-Aldrich) or recombinant 2N4R His-tagged Tau in the presence of fresh ATP for 1 h at 30 °C.

    Techniques: Expressing, Western Blot, Stable Transfection, Real-time Polymerase Chain Reaction

    Analysis of FTO phosphorylation in ESCs. A , consensus GSK-3 phosphorylation motif in mouse FTO. The numbering indicates amino acid position within FTO protein; serines are highlighted in red. B , Western blotting of overexpressed V5-tagged FTO (V5-FTO) in WT and Gsk-3 DKO ESCs before ( lanes 1–3 ) and after enrichment for phosphorylated proteins ( lanes 4–6 ). C , identification of FTO phosphorylation sites. Mass spectrometry was performed on FLAG-FTO overexpressed in WT ESCs. Black bars located within the sequence represent singly and doubly charged nonphosphorylated b- or y-ions. Red bars represent singly and doubly charged phosphorylated b- or y-ions. Asterisks represent neutral water losses from nonphosphorylated fragment ions ( black , −18 Da) and neutral phosphate losses from phosphorylated fragment ions ( red , −98 Da). c , a carbamidomethylated Cys residue. FTO is phosphorylated on serines 249 and 253 in mouse ESCs, within the GSK-3 consensus sequence. D , FLAG Western blotting of overexpressed WT FLAG-tagged FTO (FLAG-FTO-WT) and FLAG-FTO mutated at the GSK-3 phosphorylation site (FLAG-FTO-S249A/S253A) in WT ESCs after enrichment for phosphorylated proteins. Protein lysates before phospho-enrichment ( Input ) show total expression of FLAG-FTO proteins. E , in vitro kinase assay. Recombinant GSK-3β ( rGsk-3 β) was incubated with recombinant mouse FTO ( rFTO ) for 1 h at 30 °C. Proteins were then separated on 7.5% Tris/Tricine acrylamide gels and stained with Pro-Q Diamond stain to detect phosphorylated proteins ( top ). The arrow shows that the recombinant GSK-3β is phosphorylated. Phosphorylated FTO is denoted by the bracket. Middle panel , Western blotting showing the amount of recombinant FTO added to each reaction; bottom panel , Western blotting showing the amount of recombinant GSK-3β added to each reaction.

    Journal: The Journal of Biological Chemistry

    Article Title: Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells

    doi: 10.1074/jbc.RA117.001298

    Figure Lengend Snippet: Analysis of FTO phosphorylation in ESCs. A , consensus GSK-3 phosphorylation motif in mouse FTO. The numbering indicates amino acid position within FTO protein; serines are highlighted in red. B , Western blotting of overexpressed V5-tagged FTO (V5-FTO) in WT and Gsk-3 DKO ESCs before ( lanes 1–3 ) and after enrichment for phosphorylated proteins ( lanes 4–6 ). C , identification of FTO phosphorylation sites. Mass spectrometry was performed on FLAG-FTO overexpressed in WT ESCs. Black bars located within the sequence represent singly and doubly charged nonphosphorylated b- or y-ions. Red bars represent singly and doubly charged phosphorylated b- or y-ions. Asterisks represent neutral water losses from nonphosphorylated fragment ions ( black , −18 Da) and neutral phosphate losses from phosphorylated fragment ions ( red , −98 Da). c , a carbamidomethylated Cys residue. FTO is phosphorylated on serines 249 and 253 in mouse ESCs, within the GSK-3 consensus sequence. D , FLAG Western blotting of overexpressed WT FLAG-tagged FTO (FLAG-FTO-WT) and FLAG-FTO mutated at the GSK-3 phosphorylation site (FLAG-FTO-S249A/S253A) in WT ESCs after enrichment for phosphorylated proteins. Protein lysates before phospho-enrichment ( Input ) show total expression of FLAG-FTO proteins. E , in vitro kinase assay. Recombinant GSK-3β ( rGsk-3 β) was incubated with recombinant mouse FTO ( rFTO ) for 1 h at 30 °C. Proteins were then separated on 7.5% Tris/Tricine acrylamide gels and stained with Pro-Q Diamond stain to detect phosphorylated proteins ( top ). The arrow shows that the recombinant GSK-3β is phosphorylated. Phosphorylated FTO is denoted by the bracket. Middle panel , Western blotting showing the amount of recombinant FTO added to each reaction; bottom panel , Western blotting showing the amount of recombinant GSK-3β added to each reaction.

    Article Snippet: Recombinant human GSK-3β (Sigma-Aldrich) was incubated with recombinant mouse FTO (Sigma-Aldrich) or recombinant 2N4R His-tagged Tau in the presence of fresh ATP for 1 h at 30 °C.

    Techniques: Western Blot, Mass Spectrometry, Sequencing, Expressing, In Vitro, Kinase Assay, Recombinant, Incubation, Staining

    Neuroprotection of cellular L-BMAA model by diverse GSK-3 inhibitors. A) Diverse GSK-3 inhibitors selected showing their inhibition kind. B) Cell viability measured in the experiment with or without inhibitors.

    Journal: PLoS ONE

    Article Title: Small GSK-3 Inhibitor Shows Efficacy in a Motor Neuron Disease Murine Model Modulating Autophagy

    doi: 10.1371/journal.pone.0162723

    Figure Lengend Snippet: Neuroprotection of cellular L-BMAA model by diverse GSK-3 inhibitors. A) Diverse GSK-3 inhibitors selected showing their inhibition kind. B) Cell viability measured in the experiment with or without inhibitors.

    Article Snippet: Inhibition of GSK-3 Human recombinant GSK-3β was purchased from Millipore (Millipore Iberica S.A.U.).

    Techniques: Inhibition

    Biological activity of VP2.51 on GSK-3 and other protein kinases. A) Time-dependent GSK-3 inhibition of VP2.51 at 1 and 2 μM. B) Kinetic GSK-3 inhibition data determined for VP2.51. ATP concentrations in the reaction mixture varied from 1 to 50 μM. Compound concentrations used are depicted in the plot, and the concentration of GS-2 was kept constant at 12.5 μM. Each point is the mean of two different experiments, each one analyzed in duplicate. C) Kinase profiling of VP2.51 on human recombinant kinases. The percentage (%) of kinase activity after the treatment with a fixed concentration (10 μM) of the compound is shown.

    Journal: PLoS ONE

    Article Title: Small GSK-3 Inhibitor Shows Efficacy in a Motor Neuron Disease Murine Model Modulating Autophagy

    doi: 10.1371/journal.pone.0162723

    Figure Lengend Snippet: Biological activity of VP2.51 on GSK-3 and other protein kinases. A) Time-dependent GSK-3 inhibition of VP2.51 at 1 and 2 μM. B) Kinetic GSK-3 inhibition data determined for VP2.51. ATP concentrations in the reaction mixture varied from 1 to 50 μM. Compound concentrations used are depicted in the plot, and the concentration of GS-2 was kept constant at 12.5 μM. Each point is the mean of two different experiments, each one analyzed in duplicate. C) Kinase profiling of VP2.51 on human recombinant kinases. The percentage (%) of kinase activity after the treatment with a fixed concentration (10 μM) of the compound is shown.

    Article Snippet: Inhibition of GSK-3 Human recombinant GSK-3β was purchased from Millipore (Millipore Iberica S.A.U.).

    Techniques: Activity Assay, Inhibition, Concentration Assay, Recombinant

    A proposed model illustrating the mechanism underlying the efficiency of LX2343 in the amelioration of cognitive impairment in AD. LX2343 rescued neuronal cells from apoptosis by maintaining the integrity of mitochondrial function and morphology, alleviating the OS and JNK/p38 pathway and regulating anti- and pro-apoptotic proteins. In addition, LX2343 also inhibited tau hyperphosphorylation by functioning as a non-ATP competitive inhibitor of GSK-3β.

    Journal: Acta Pharmacologica Sinica

    Article Title: LX2343 alleviates cognitive impairments in AD model rats by inhibiting oxidative stress-induced neuronal apoptosis and tauopathy

    doi: 10.1038/aps.2016.128

    Figure Lengend Snippet: A proposed model illustrating the mechanism underlying the efficiency of LX2343 in the amelioration of cognitive impairment in AD. LX2343 rescued neuronal cells from apoptosis by maintaining the integrity of mitochondrial function and morphology, alleviating the OS and JNK/p38 pathway and regulating anti- and pro-apoptotic proteins. In addition, LX2343 also inhibited tau hyperphosphorylation by functioning as a non-ATP competitive inhibitor of GSK-3β.

    Article Snippet: Briefly, recombinant human GSK-3β (Invitrogen, Grand Island, NY, USA) was added to the reactions with the substrate-recombinant human TAU-441 (Merck Millipore, Darmstadt, Germany) in a 5:1 ratio.

    Techniques:

    LX2343 ameliorated tau pathology involving GSK-3β inhibition. (A–D) Western blot and quantification results demonstrated that LX2343 reduced tau phosphorylation at sites of serine 396/199 and threonine 231 in SH-SY5Y and primary neuronal cells (one-way ANOVA, Dunnett's multiple comparison test, * P

    Journal: Acta Pharmacologica Sinica

    Article Title: LX2343 alleviates cognitive impairments in AD model rats by inhibiting oxidative stress-induced neuronal apoptosis and tauopathy

    doi: 10.1038/aps.2016.128

    Figure Lengend Snippet: LX2343 ameliorated tau pathology involving GSK-3β inhibition. (A–D) Western blot and quantification results demonstrated that LX2343 reduced tau phosphorylation at sites of serine 396/199 and threonine 231 in SH-SY5Y and primary neuronal cells (one-way ANOVA, Dunnett's multiple comparison test, * P

    Article Snippet: Briefly, recombinant human GSK-3β (Invitrogen, Grand Island, NY, USA) was added to the reactions with the substrate-recombinant human TAU-441 (Merck Millipore, Darmstadt, Germany) in a 5:1 ratio.

    Techniques: Inhibition, Western Blot

    (Phyto)ceramide binds to pYGSK3 and sustains its phosphorylation level and localization in flagella and cilia. (A) Immunoblot of pYGSK, total GSK3, and acetylated tubulin from Chlamydomonas treated for 24 h with myriocin (5 or 10 nM) ± 1 μM DHS. Bottom, Ponceau S staining for loading control. (B) Immunocytochemistry using antibodies against (phyto)ceramide rabbit (green), pYGSK (red), and acetylated tubulin (blue) with Chlamydomonas incubated for 60 min with GSK3β inhibitor BIO (2 μM) or lithium chloride (25 mM). (C) Higher magnification of flagella in left side of B. (D) Same as in B and C with ependymal cells ( Z -scan projection). (E) Z -scan orthogonal view of ependymal cells labeled with DAPI (blue) and antibodies against acetylated tubulin (green), pYGSK (green; left), and pSGSK (green; right). (F) C18 sphingomyelin and indicated ceramides were coated on ELISA plates and bound to recombinant GSK-3β; binding was quantified by development with anti-GSK-3α/β IgG and anti-mouse IgG-HRP. Mean ± SEM, n = 3, * p

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of Chlamydomonas flagella and ependymal cell motile cilia by ceramide-mediated translocation of GSK3

    doi: 10.1091/mbc.E15-06-0371

    Figure Lengend Snippet: (Phyto)ceramide binds to pYGSK3 and sustains its phosphorylation level and localization in flagella and cilia. (A) Immunoblot of pYGSK, total GSK3, and acetylated tubulin from Chlamydomonas treated for 24 h with myriocin (5 or 10 nM) ± 1 μM DHS. Bottom, Ponceau S staining for loading control. (B) Immunocytochemistry using antibodies against (phyto)ceramide rabbit (green), pYGSK (red), and acetylated tubulin (blue) with Chlamydomonas incubated for 60 min with GSK3β inhibitor BIO (2 μM) or lithium chloride (25 mM). (C) Higher magnification of flagella in left side of B. (D) Same as in B and C with ependymal cells ( Z -scan projection). (E) Z -scan orthogonal view of ependymal cells labeled with DAPI (blue) and antibodies against acetylated tubulin (green), pYGSK (green; left), and pSGSK (green; right). (F) C18 sphingomyelin and indicated ceramides were coated on ELISA plates and bound to recombinant GSK-3β; binding was quantified by development with anti-GSK-3α/β IgG and anti-mouse IgG-HRP. Mean ± SEM, n = 3, * p

    Article Snippet: Recombinant human aPKCζ (50 ng/50 μl; Enzo Life Sciences) or recombinant human GSK-3β (50 ng/50 μl; SignalChem, Richmond, Canada) in 0.1% BSA/PBS was incubated for 2 h at 37°C.

    Techniques: Staining, Immunocytochemistry, Incubation, Labeling, Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay

    Model for flagella/cilia length regulation by (phyto)ceramide. (A) Flux equilibrium model. Lipid vesicles from the ceramide compartment (red) are incorporated into the ciliary membrane at the cilium base. Lipid–cargo protein (green) cotransport ensures the stoichiometry required for simultaneous cilium elongation and membrane expansion. (Phyto)ceramide may serve as a membrane anchor for cotransported cargo proteins, or, alternatively, it may activate a loading/unloading mechanism at the base or tip of the cilium. The cilium length is regulated by the size or ceramide content of the compartment at the base and its lipid flux to the cilium: more ceramide favors cilium assembly and less favors disassembly, until flux rates in both directions are equal and the cilium length is maintained. (B) Regulation of GSK3 by (phyto)ceramide. In Chlamydomonas and ependymal cells, (phyto)ceramide binds to pYGSK3 at the ceramide-enriched compartment (ACEC) and transports it to flagella/cilia. In ependymal cells, ACEC-resident ceramide (red) binding and sequestration of aPKCζ prevent inactivation of GSK-3β by aPKCζ-mediated phosphorylation of Ser-9, which complements activation and transport of pYGSK3. In the cilium tip, pYGSK3 down-regulates HDAC6 or induces cargo release from the motor protein complex, most likely regulated by its interaction with (phyto)ceramide.

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of Chlamydomonas flagella and ependymal cell motile cilia by ceramide-mediated translocation of GSK3

    doi: 10.1091/mbc.E15-06-0371

    Figure Lengend Snippet: Model for flagella/cilia length regulation by (phyto)ceramide. (A) Flux equilibrium model. Lipid vesicles from the ceramide compartment (red) are incorporated into the ciliary membrane at the cilium base. Lipid–cargo protein (green) cotransport ensures the stoichiometry required for simultaneous cilium elongation and membrane expansion. (Phyto)ceramide may serve as a membrane anchor for cotransported cargo proteins, or, alternatively, it may activate a loading/unloading mechanism at the base or tip of the cilium. The cilium length is regulated by the size or ceramide content of the compartment at the base and its lipid flux to the cilium: more ceramide favors cilium assembly and less favors disassembly, until flux rates in both directions are equal and the cilium length is maintained. (B) Regulation of GSK3 by (phyto)ceramide. In Chlamydomonas and ependymal cells, (phyto)ceramide binds to pYGSK3 at the ceramide-enriched compartment (ACEC) and transports it to flagella/cilia. In ependymal cells, ACEC-resident ceramide (red) binding and sequestration of aPKCζ prevent inactivation of GSK-3β by aPKCζ-mediated phosphorylation of Ser-9, which complements activation and transport of pYGSK3. In the cilium tip, pYGSK3 down-regulates HDAC6 or induces cargo release from the motor protein complex, most likely regulated by its interaction with (phyto)ceramide.

    Article Snippet: Recombinant human aPKCζ (50 ng/50 μl; Enzo Life Sciences) or recombinant human GSK-3β (50 ng/50 μl; SignalChem, Richmond, Canada) in 0.1% BSA/PBS was incubated for 2 h at 37°C.

    Techniques: Binding Assay, Activation Assay

    Ceramide binds to aPKCζ, whose inhibition leads to cilium length extension. (A) pacFACer was UV cross-linked to protein in primary cultured ependymal cells, linked to Alexa Fluor 594 azide (red), and labeled with anti-aPKCζ (magenta) and anti–acetylated tubulin (green) antibodies. Scale bar, 2 μm. (B) pacFACer was coated on ELISA plates and recombinant aPKCζ cross-linked by UV (365 nm) radiation. Presence/absence of pacFACer (pCer) and recombinant aPKCζ along with primary antibody used. (C) Indicated ceramides were coated on ELISA plates and bound to recombinant aPKCζ, and binding was quantified by development with anti-aPKCζ IgG and anti-rabbit IgG-HRP. Mean ± SEM, n = 3. (D) Incubation of ependymal cells (24 h) with FB1 (10 μM) reduces cilium length, whereas addition of aPKCζ inhibitor PZI (10 μM) leads to extension of untreated or (partial) restoration of FB1-treated cilia. The GSK-3β inhibitor BIO (2 μM) obliterates cilia. Data presented show mean ± SEM, n = 5, * p

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of Chlamydomonas flagella and ependymal cell motile cilia by ceramide-mediated translocation of GSK3

    doi: 10.1091/mbc.E15-06-0371

    Figure Lengend Snippet: Ceramide binds to aPKCζ, whose inhibition leads to cilium length extension. (A) pacFACer was UV cross-linked to protein in primary cultured ependymal cells, linked to Alexa Fluor 594 azide (red), and labeled with anti-aPKCζ (magenta) and anti–acetylated tubulin (green) antibodies. Scale bar, 2 μm. (B) pacFACer was coated on ELISA plates and recombinant aPKCζ cross-linked by UV (365 nm) radiation. Presence/absence of pacFACer (pCer) and recombinant aPKCζ along with primary antibody used. (C) Indicated ceramides were coated on ELISA plates and bound to recombinant aPKCζ, and binding was quantified by development with anti-aPKCζ IgG and anti-rabbit IgG-HRP. Mean ± SEM, n = 3. (D) Incubation of ependymal cells (24 h) with FB1 (10 μM) reduces cilium length, whereas addition of aPKCζ inhibitor PZI (10 μM) leads to extension of untreated or (partial) restoration of FB1-treated cilia. The GSK-3β inhibitor BIO (2 μM) obliterates cilia. Data presented show mean ± SEM, n = 5, * p

    Article Snippet: Recombinant human aPKCζ (50 ng/50 μl; Enzo Life Sciences) or recombinant human GSK-3β (50 ng/50 μl; SignalChem, Richmond, Canada) in 0.1% BSA/PBS was incubated for 2 h at 37°C.

    Techniques: Inhibition, Cell Culture, Labeling, Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay, Incubation

    Inhibition of GSK-3β Activity Modulates AR Transcriptional Activity

    Journal:

    Article Title: Inhibition of Glycogen Synthase Kinase-3 in Androgen-Responsive Prostate Cancer Cell Lines: Are GSK Inhibitors Therapeutically Useful? 1Inhibition of Glycogen Synthase Kinase-3 in Androgen-Responsive Prostate Cancer Cell Lines: Are GSK Inhibitors Therapeutically Useful? 1 2

    doi:

    Figure Lengend Snippet: Inhibition of GSK-3β Activity Modulates AR Transcriptional Activity

    Article Snippet: Human recombinant GSK-3β-kinase was provided by Cell Signaling Technology (New England Biolabs GmbH, Frankfurt am Main, Germany), the GSK-3 substrate representing a part of the hydrophilic loop domain of presenilin 1 was a product of Calbiochem (Merck Biosciences).

    Techniques: Inhibition, Activity Assay

    Inhibition of GSK-3β Activity Modulates AR Transcriptional Activity

    Journal:

    Article Title: Inhibition of Glycogen Synthase Kinase-3 in Androgen-Responsive Prostate Cancer Cell Lines: Are GSK Inhibitors Therapeutically Useful? 1Inhibition of Glycogen Synthase Kinase-3 in Androgen-Responsive Prostate Cancer Cell Lines: Are GSK Inhibitors Therapeutically Useful? 1 2

    doi:

    Figure Lengend Snippet: Inhibition of GSK-3β Activity Modulates AR Transcriptional Activity

    Article Snippet: Human recombinant GSK-3β-kinase was provided by Cell Signaling Technology (New England Biolabs GmbH, Frankfurt am Main, Germany), the GSK-3 substrate representing a part of the hydrophilic loop domain of presenilin 1 was a product of Calbiochem (Merck Biosciences).

    Techniques: Inhibition, Activity Assay

    Silencing of GSK-3 in 22Rv1 cells. (A) GSK-3-directed shRNA diminishes GSK-3 protein expression: 22Rv1 cells were transfected with shRNA directed either against GSK-3 (pKD-GSK-3β-v1) or control shRNA (pKD-NegCon-v1). A total of 30 µ g of

    Journal:

    Article Title: Inhibition of Glycogen Synthase Kinase-3 in Androgen-Responsive Prostate Cancer Cell Lines: Are GSK Inhibitors Therapeutically Useful? 1Inhibition of Glycogen Synthase Kinase-3 in Androgen-Responsive Prostate Cancer Cell Lines: Are GSK Inhibitors Therapeutically Useful? 1 2

    doi:

    Figure Lengend Snippet: Silencing of GSK-3 in 22Rv1 cells. (A) GSK-3-directed shRNA diminishes GSK-3 protein expression: 22Rv1 cells were transfected with shRNA directed either against GSK-3 (pKD-GSK-3β-v1) or control shRNA (pKD-NegCon-v1). A total of 30 µ g of

    Article Snippet: Human recombinant GSK-3β-kinase was provided by Cell Signaling Technology (New England Biolabs GmbH, Frankfurt am Main, Germany), the GSK-3 substrate representing a part of the hydrophilic loop domain of presenilin 1 was a product of Calbiochem (Merck Biosciences).

    Techniques: shRNA, Expressing, Transfection

    Proposed SCE antidepressant-like mechanisms for the interaction among BDNF, TrkB, CREB, ERK, PI3K, AKT and GSK-3β. SCE regulates TrkB/CREB/ERK pathway to increase the BDNF to alleviate the depression and cognitive decline. On the other aspect, BDNF upregulates PI3K/AKT/ GSK-3β pathway to ameliorate the depressive symptoms and cognition disability induced by CUMS.

    Journal: Scientific Reports

    Article Title: Antidepressant-like effects and cognitive enhancement of Schisandra chinensis in chronic unpredictable mild stress mice and its related mechanism

    doi: 10.1038/s41598-017-07407-1

    Figure Lengend Snippet: Proposed SCE antidepressant-like mechanisms for the interaction among BDNF, TrkB, CREB, ERK, PI3K, AKT and GSK-3β. SCE regulates TrkB/CREB/ERK pathway to increase the BDNF to alleviate the depression and cognitive decline. On the other aspect, BDNF upregulates PI3K/AKT/ GSK-3β pathway to ameliorate the depressive symptoms and cognition disability induced by CUMS.

    Article Snippet: The membranes were saturated and blocked with 5% fat-free powdered milk at 37 °C for 1.5 h and incubated overnight at 4 °C in one of the following primary antibodies, which were diluted in 5% fat-free powdered milk in TBS: PI3 Kinase p85 (19H8) (1:1000, CST, USA), Akt (pan) (C67E7) (1:1000, CST, USA), GSK-3β (D5C5Z) (1:1000, CST, USA), GADPH Rabbit mAb (1:1000, CST, USA), β-actin Rabbit mAb (1:1000, CST, USA).

    Techniques:

    Effects of SCE administration on pPI3K/PI3K ( A ), pAKT/AKT ( B ) and p GSK-3β/ GSK-3β ( C ) levels in hippocampus. The data represented the values of mean ± S.E.M. from 10 mice/group. *p

    Journal: Scientific Reports

    Article Title: Antidepressant-like effects and cognitive enhancement of Schisandra chinensis in chronic unpredictable mild stress mice and its related mechanism

    doi: 10.1038/s41598-017-07407-1

    Figure Lengend Snippet: Effects of SCE administration on pPI3K/PI3K ( A ), pAKT/AKT ( B ) and p GSK-3β/ GSK-3β ( C ) levels in hippocampus. The data represented the values of mean ± S.E.M. from 10 mice/group. *p

    Article Snippet: The membranes were saturated and blocked with 5% fat-free powdered milk at 37 °C for 1.5 h and incubated overnight at 4 °C in one of the following primary antibodies, which were diluted in 5% fat-free powdered milk in TBS: PI3 Kinase p85 (19H8) (1:1000, CST, USA), Akt (pan) (C67E7) (1:1000, CST, USA), GSK-3β (D5C5Z) (1:1000, CST, USA), GADPH Rabbit mAb (1:1000, CST, USA), β-actin Rabbit mAb (1:1000, CST, USA).

    Techniques: Mouse Assay

    PEDF regulated osteocyte genes in LTD cultures through PEDF-R and induction of Erk/GSK-3β/β-catenin signalling. A ) PEDF suppressed expression of Sost, MEPE and DMP-1; Erk inhibition reversed PEDF inhibitory activity in LTD osteoblast cultures. B ) Blocking PEDF-R with neutralizing antibodies had similar effects as inhibiting Erk activation on Sost, MEPE and DMP-1 mRNA expression. C ) Inhibition of LRP5/6 activity by Dkk-1 had minimal effects on PEDF inhibitory activity of Sost, MEPE and DMP-1 expression. These data indicate that PEDF inhibitory activity of osteocyte gene expression may not involve LRP5/6 although binding of PEDF to LRP6 cannot be ruled out.

    Journal: Biochimica et biophysica acta. Molecular basis of disease

    Article Title: Pigment epithelium derived factor regulates human Sost/Sclerostin and other osteocyte gene expression via the receptor and induction of Erk/GSK-3beta/beta-catenin signaling

    doi: 10.1016/j.bbadis.2018.07.034

    Figure Lengend Snippet: PEDF regulated osteocyte genes in LTD cultures through PEDF-R and induction of Erk/GSK-3β/β-catenin signalling. A ) PEDF suppressed expression of Sost, MEPE and DMP-1; Erk inhibition reversed PEDF inhibitory activity in LTD osteoblast cultures. B ) Blocking PEDF-R with neutralizing antibodies had similar effects as inhibiting Erk activation on Sost, MEPE and DMP-1 mRNA expression. C ) Inhibition of LRP5/6 activity by Dkk-1 had minimal effects on PEDF inhibitory activity of Sost, MEPE and DMP-1 expression. These data indicate that PEDF inhibitory activity of osteocyte gene expression may not involve LRP5/6 although binding of PEDF to LRP6 cannot be ruled out.

    Article Snippet: Aliquots of LTD cells established at 4 months time point as described above were seeded onto collagen coated 6-well plates at 1×104 / cm2 and pretreated with 10 mM Erk docking domain inhibitor (294675–79-9, Sigma-Aldrich, CA),or 4 ug/ml of PEDF-receptor antibody (Santa Cruz, SC-365278), or 20 ng/ml Dkk-1 (R and D, or control solution (DMSO) for 4h. β-catenin signaling was determined by using antibodies against total β-catenin and non-phosphorylated β-catenin; non-phosphorylated and phosphorylated GSK-3β antibodies (Cell signaling catalog numbers 9315 and 9336 respectively), and non-phosphorylated and phosphorylated Erk1/2 antibodies purchased from Cell Signaling.

    Techniques: Expressing, Inhibition, Activity Assay, Blocking Assay, Activation Assay, Binding Assay

    PEDF-regulated synthesis of osteocyte proteins via PEDF-R and induction of Erk/GSK-3β/β-catenin signaling pathway. A, B ) Western blot and quantitative analysis indicating reduction of Scl, MEPE and DMP-1 synthesis by PEDF; Erk inhibition reversed PEDF inhibitory activity in LTD osteoblasts. C, D ) Blocking PEDF-R with neutralizing antibodies showed similar effects as inhibiting Erk activation on Scl, MEPE and DMP-1 protein synthesis by osteocytes in LTD osteoblasts. E, F ) Inhibition of LRP5/6 activity by Dkk-1 had no effect on PEDF inhibitory activity on Scl, MEPE and DMP-1 synthesis. These data indicate that PEDF inhibitory activity of osteocyte protein synthesis may not involve LRP5/6.

    Journal: Biochimica et biophysica acta. Molecular basis of disease

    Article Title: Pigment epithelium derived factor regulates human Sost/Sclerostin and other osteocyte gene expression via the receptor and induction of Erk/GSK-3beta/beta-catenin signaling

    doi: 10.1016/j.bbadis.2018.07.034

    Figure Lengend Snippet: PEDF-regulated synthesis of osteocyte proteins via PEDF-R and induction of Erk/GSK-3β/β-catenin signaling pathway. A, B ) Western blot and quantitative analysis indicating reduction of Scl, MEPE and DMP-1 synthesis by PEDF; Erk inhibition reversed PEDF inhibitory activity in LTD osteoblasts. C, D ) Blocking PEDF-R with neutralizing antibodies showed similar effects as inhibiting Erk activation on Scl, MEPE and DMP-1 protein synthesis by osteocytes in LTD osteoblasts. E, F ) Inhibition of LRP5/6 activity by Dkk-1 had no effect on PEDF inhibitory activity on Scl, MEPE and DMP-1 synthesis. These data indicate that PEDF inhibitory activity of osteocyte protein synthesis may not involve LRP5/6.

    Article Snippet: Aliquots of LTD cells established at 4 months time point as described above were seeded onto collagen coated 6-well plates at 1×104 / cm2 and pretreated with 10 mM Erk docking domain inhibitor (294675–79-9, Sigma-Aldrich, CA),or 4 ug/ml of PEDF-receptor antibody (Santa Cruz, SC-365278), or 20 ng/ml Dkk-1 (R and D, or control solution (DMSO) for 4h. β-catenin signaling was determined by using antibodies against total β-catenin and non-phosphorylated β-catenin; non-phosphorylated and phosphorylated GSK-3β antibodies (Cell signaling catalog numbers 9315 and 9336 respectively), and non-phosphorylated and phosphorylated Erk1/2 antibodies purchased from Cell Signaling.

    Techniques: Western Blot, Inhibition, Activity Assay, Blocking Assay, Activation Assay

    PEDF induced Erk/GSK-3β/β-catenin signaling in LTD osteoblasts via PEDF-R. A ) Osteocytes in LTD culture treated with PEDF induced Erk and Gsk-3β phosphorylation, and accumulation of nonphosphorylated β-catenin. B ) Erk inhibition eliminated Gsk-3β phosphorylation and accumulation of β-catenin. C ) Neutralizing antibodies to PEDF-R induced similar effects as inhibiting Erk activation. D ) Addition of Dkk-1 to the cultures had minimal effects suggesting that PEDF may not act through LRP5/6 but through PEDF-R to induce Erk/GSK-3β/β-catenin signaling. E, F, G) Topflash assays demonstrated that PEDF activated luciferase reporter activity and this activity was inhibited by treatment with Erk inhibitor and neutralizing antibodies to PEDF-R suggesting their role in stabilizing β-catenin.. Dkk-1 treatment did not have effect on luciferase activity induced by PEDF treatment, supporting the data that β-catenin stabilization may not involve LRP5/6.

    Journal: Biochimica et biophysica acta. Molecular basis of disease

    Article Title: Pigment epithelium derived factor regulates human Sost/Sclerostin and other osteocyte gene expression via the receptor and induction of Erk/GSK-3beta/beta-catenin signaling

    doi: 10.1016/j.bbadis.2018.07.034

    Figure Lengend Snippet: PEDF induced Erk/GSK-3β/β-catenin signaling in LTD osteoblasts via PEDF-R. A ) Osteocytes in LTD culture treated with PEDF induced Erk and Gsk-3β phosphorylation, and accumulation of nonphosphorylated β-catenin. B ) Erk inhibition eliminated Gsk-3β phosphorylation and accumulation of β-catenin. C ) Neutralizing antibodies to PEDF-R induced similar effects as inhibiting Erk activation. D ) Addition of Dkk-1 to the cultures had minimal effects suggesting that PEDF may not act through LRP5/6 but through PEDF-R to induce Erk/GSK-3β/β-catenin signaling. E, F, G) Topflash assays demonstrated that PEDF activated luciferase reporter activity and this activity was inhibited by treatment with Erk inhibitor and neutralizing antibodies to PEDF-R suggesting their role in stabilizing β-catenin.. Dkk-1 treatment did not have effect on luciferase activity induced by PEDF treatment, supporting the data that β-catenin stabilization may not involve LRP5/6.

    Article Snippet: Aliquots of LTD cells established at 4 months time point as described above were seeded onto collagen coated 6-well plates at 1×104 / cm2 and pretreated with 10 mM Erk docking domain inhibitor (294675–79-9, Sigma-Aldrich, CA),or 4 ug/ml of PEDF-receptor antibody (Santa Cruz, SC-365278), or 20 ng/ml Dkk-1 (R and D, or control solution (DMSO) for 4h. β-catenin signaling was determined by using antibodies against total β-catenin and non-phosphorylated β-catenin; non-phosphorylated and phosphorylated GSK-3β antibodies (Cell signaling catalog numbers 9315 and 9336 respectively), and non-phosphorylated and phosphorylated Erk1/2 antibodies purchased from Cell Signaling.

    Techniques: Inhibition, Activation Assay, Activated Clotting Time Assay, Luciferase, Activity Assay

    Mean ± SEM values (n=5) for the ratio of phosphorylated protein p-GSk-3β/GSk-3β (A) and p-β-catenin/β-catenin (B) in preglomerular arterioles from control mice (open boxes), diabetic mice (blue filled boxes) and Sulindac treated diabetic mice (grey filled boxes). Comparing groups: * P

    Journal: Kidney & blood pressure research

    Article Title: Enhanced Renal Afferent Arteriolar Reactive Oxygen Species and Contractility to Endothelin-1 Are Associated with Canonical Wnt Signaling in Diabetic Mice

    doi: 10.1159/000490334

    Figure Lengend Snippet: Mean ± SEM values (n=5) for the ratio of phosphorylated protein p-GSk-3β/GSk-3β (A) and p-β-catenin/β-catenin (B) in preglomerular arterioles from control mice (open boxes), diabetic mice (blue filled boxes) and Sulindac treated diabetic mice (grey filled boxes). Comparing groups: * P

    Article Snippet: Antibody sources were as follows: rabbit anti-GSK-3β, rabbit anti-phospho-GSK-3β (Ser9), rabbit anti-β-catenin, rabbit anti-phospho-β-catenin (Ser33/37/Thr41), rabbit anti-SODl (#2770), rabbit anti-SOD2 (#13194), rabbit anti-catalase (#14097), horseradish peroxidase-labeled IgG anti-rabbit (or mouse) antibodies (Cell Signaling Technology, Beverly, CA, USA) and mouse anti-β-actin (Abeam, Cambridge, MA, USA).

    Techniques: Mouse Assay

    Anti-growth effects of PGZ in LNCaP cells. LNCaP cells were incubated with PGZ for 72 h, and then, cytotoxicity was assessed by the WST-1 assay ( A ); Representative immunoblots of phospho-p38 MAPK (p-P38 MAPK), p38 MAPK, phospho-NF-κB (p-NFκB), NFκB, GSK-3β and cyclin D1 in LNCaP cells ( B ); The effect of a specific p38 MAPK inhibitor (p38-In) and an NFκB activation inhibitor (NFκB-In) on LNCaP cell proliferation ( C ). Results are expressed as a proportion of untreated control ( A , C ). Data are expressed as the mean ± SD and are from more than three independent experiments ( A , C ). ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Pioglitazone, a Peroxisome Proliferator-Activated Receptor γ Agonist, Suppresses Rat Prostate Carcinogenesis

    doi: 10.3390/ijms17122071

    Figure Lengend Snippet: Anti-growth effects of PGZ in LNCaP cells. LNCaP cells were incubated with PGZ for 72 h, and then, cytotoxicity was assessed by the WST-1 assay ( A ); Representative immunoblots of phospho-p38 MAPK (p-P38 MAPK), p38 MAPK, phospho-NF-κB (p-NFκB), NFκB, GSK-3β and cyclin D1 in LNCaP cells ( B ); The effect of a specific p38 MAPK inhibitor (p38-In) and an NFκB activation inhibitor (NFκB-In) on LNCaP cell proliferation ( C ). Results are expressed as a proportion of untreated control ( A , C ). Data are expressed as the mean ± SD and are from more than three independent experiments ( A , C ). ** p

    Article Snippet: Membranes were probed with antibodies for rabbit polyclonal PPARγ (Thermo Fisher Scientific Inc.), rabbit polyclonal cyclin D1, rabbit polyclonal p38 MAPK, rabbit monoclonal phospho-p38 (p-p38) MAPK, rabbit polyclonal NFκB-p65 (NFκB), rabbit polyclonal phospho-NFκB-p65 (p-NFκB), rabbit monoclonal GSK-3β, rabbit polyclonal p44/42 MAPK (ERK1/2) or rabbit polyclonal phospho-ERK1/2 (Cell Signaling Technology, Inc., Danvers, MA, USA) in 1× TBS with 0.1% Tween 20 at 4 °C overnight, followed by exposure to peroxidase-conjugated appropriate secondary antibodies and visualization with an enhanced chemiluminescence detection system (GE Healthcare Bio-Sciences, Buckinghamshire, UK).

    Techniques: Incubation, WST-1 Assay, Western Blot, Activation Assay

    Immunoblot analysis in ventral prostate. Representative immunoblots of PPARγ, phospho-ERK (p-ERK), ERK, phospho-p38 MAPK (p-p38 MAPK), p38 MAPK, phospho-NFκB (p-NFκB), NFκB, GSK-3β and cyclin D1 in ventral prostate samples of TRAP rats treated with PGZ.

    Journal: International Journal of Molecular Sciences

    Article Title: Pioglitazone, a Peroxisome Proliferator-Activated Receptor γ Agonist, Suppresses Rat Prostate Carcinogenesis

    doi: 10.3390/ijms17122071

    Figure Lengend Snippet: Immunoblot analysis in ventral prostate. Representative immunoblots of PPARγ, phospho-ERK (p-ERK), ERK, phospho-p38 MAPK (p-p38 MAPK), p38 MAPK, phospho-NFκB (p-NFκB), NFκB, GSK-3β and cyclin D1 in ventral prostate samples of TRAP rats treated with PGZ.

    Article Snippet: Membranes were probed with antibodies for rabbit polyclonal PPARγ (Thermo Fisher Scientific Inc.), rabbit polyclonal cyclin D1, rabbit polyclonal p38 MAPK, rabbit monoclonal phospho-p38 (p-p38) MAPK, rabbit polyclonal NFκB-p65 (NFκB), rabbit polyclonal phospho-NFκB-p65 (p-NFκB), rabbit monoclonal GSK-3β, rabbit polyclonal p44/42 MAPK (ERK1/2) or rabbit polyclonal phospho-ERK1/2 (Cell Signaling Technology, Inc., Danvers, MA, USA) in 1× TBS with 0.1% Tween 20 at 4 °C overnight, followed by exposure to peroxidase-conjugated appropriate secondary antibodies and visualization with an enhanced chemiluminescence detection system (GE Healthcare Bio-Sciences, Buckinghamshire, UK).

    Techniques: Western Blot

    Effect of CGE on the PI3K/AKT/ GSK-3β pathway. (A) CGE inhibited the constitutively active PI3K/AKT/glycogen synthase kinase-3β (GSK-3β) signaling pathway in skin cancer cell lines in a dose-dependent manner. B16F1 and SKMEL-5 cells were treated with CGE at the indicated doses for 48 h. Equal amounts of protein from each sample were separated by SDS-PAGE and immunoblotted with p-AKT (Ser 473), AKT, and GAPDH. (B) B16F1 and SKMEL-5 cells were treated with 50 μg·mL -1 CGE for 8, 24, and 48 h. Proteins from each sample were separated by SDS-PAGE and immunoblotted with p-PI3K, p-AKT (Thr 308), p-AKT (Ser 473), p-GSK-3β (Ser 9), GSK-3β, p-BAD (Ser 136), BAD, and GAPDH.

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Molecular mechanism of apoptosis induction in skin cancer cells by the centipedegrass extract

    doi: 10.1186/1472-6882-13-350

    Figure Lengend Snippet: Effect of CGE on the PI3K/AKT/ GSK-3β pathway. (A) CGE inhibited the constitutively active PI3K/AKT/glycogen synthase kinase-3β (GSK-3β) signaling pathway in skin cancer cell lines in a dose-dependent manner. B16F1 and SKMEL-5 cells were treated with CGE at the indicated doses for 48 h. Equal amounts of protein from each sample were separated by SDS-PAGE and immunoblotted with p-AKT (Ser 473), AKT, and GAPDH. (B) B16F1 and SKMEL-5 cells were treated with 50 μg·mL -1 CGE for 8, 24, and 48 h. Proteins from each sample were separated by SDS-PAGE and immunoblotted with p-PI3K, p-AKT (Thr 308), p-AKT (Ser 473), p-GSK-3β (Ser 9), GSK-3β, p-BAD (Ser 136), BAD, and GAPDH.

    Article Snippet: Antibodies for p-PI3K, p-AKT (Ser 473), p-AKT (Thr 308), AKT, p-GSK-3β (Ser 9), GSK-3β, p-BAD (Ser 136), BAD, procaspase-3, cleaved caspase-3, cytochrome-c, poly ADP-ribose polymerase (PARP), GAPDH, horseradish peroxidase (HRP)-conjugated secondary antibody, and the PI3K inhibitor LY294002 were obtained from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: SDS Page

    Apoptosis induced by the combined treatment of CGE with the PI3K inhibitor LY294002. (A) Flow cytometry analysis was used to detect the cell cycle distribution of CGE-treated skin cancer cells for 48 h in the presence or absence of LY294002 (PI3K inhibitor). (B) The protein levels of p-PI3K, p-AKT, p-GSK-3β, AKT, and GAPDH were examined in cells with or without CGE and LY294002 treatment. (C) Flow cytometry analysis of annexin V-FITC and PI double-stained cells. Data are presented as mean ± SD values for at least three independent experiments. *** P

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Molecular mechanism of apoptosis induction in skin cancer cells by the centipedegrass extract

    doi: 10.1186/1472-6882-13-350

    Figure Lengend Snippet: Apoptosis induced by the combined treatment of CGE with the PI3K inhibitor LY294002. (A) Flow cytometry analysis was used to detect the cell cycle distribution of CGE-treated skin cancer cells for 48 h in the presence or absence of LY294002 (PI3K inhibitor). (B) The protein levels of p-PI3K, p-AKT, p-GSK-3β, AKT, and GAPDH were examined in cells with or without CGE and LY294002 treatment. (C) Flow cytometry analysis of annexin V-FITC and PI double-stained cells. Data are presented as mean ± SD values for at least three independent experiments. *** P

    Article Snippet: Antibodies for p-PI3K, p-AKT (Ser 473), p-AKT (Thr 308), AKT, p-GSK-3β (Ser 9), GSK-3β, p-BAD (Ser 136), BAD, procaspase-3, cleaved caspase-3, cytochrome-c, poly ADP-ribose polymerase (PARP), GAPDH, horseradish peroxidase (HRP)-conjugated secondary antibody, and the PI3K inhibitor LY294002 were obtained from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Flow Cytometry, Cytometry, Staining

    Remifentanil infusion increases the GSK-3β activity in spinal dorsal horn. The total GSK-3β and phosphorylated GSK-3β in spinal dorsal horn were tested by Western blot. β-actin was used as the internal standard (a). The band intensity of C group was assigned a value of 1. Remifentanil resulted in significant decreases of pGSK-3β (ser9) and p-GSK-3β (ser9)/GSK-3β ratio, but had no effect on the total protein level of GSK-3β (b). GSK-3β inhibitor TDZD-8 prevented the changes of pGSK-3β (ser9) and pGSK-3β (ser9)/GSK-3β ratio. n = 5 for each group. Compaired with C group, * P

    Journal: PLoS ONE

    Article Title: Inhibition of Glycogen Synthase Kinase-3? Prevents Remifentanil-Induced Hyperalgesia via Regulating the Expression and Function of Spinal N-Methyl-D-Aspartate Receptors In Vivo and Vitro

    doi: 10.1371/journal.pone.0077790

    Figure Lengend Snippet: Remifentanil infusion increases the GSK-3β activity in spinal dorsal horn. The total GSK-3β and phosphorylated GSK-3β in spinal dorsal horn were tested by Western blot. β-actin was used as the internal standard (a). The band intensity of C group was assigned a value of 1. Remifentanil resulted in significant decreases of pGSK-3β (ser9) and p-GSK-3β (ser9)/GSK-3β ratio, but had no effect on the total protein level of GSK-3β (b). GSK-3β inhibitor TDZD-8 prevented the changes of pGSK-3β (ser9) and pGSK-3β (ser9)/GSK-3β ratio. n = 5 for each group. Compaired with C group, * P

    Article Snippet: The membranes were blocked with 5% nonfat milk in Tris-Tween buffer saline for 1 h (TBST; 50 mM Tris-HCl, 154 mM NaCl, and 0.05% Tween 20, pH 7.4), incubated overnight at 4 °C with polyclonal rabbit antibodies against rat NR1, NR2A, NR2B (all 1:300 dilution in 5% nonfat milk in TBST, Chemicon, USA) or rabbit anti-rat GSK-3β and phosphorylated (ser9) GSK-3β antibodies (all 1:1000 dilution in 5% nonfat milk in TBST, Cell Signaling Technology, USA), then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (1:2, 000 in 5% nonfat milk in TBST, Jackson Immuno Research, USA) for 1 h. Membrane bound secondary antibodies were detected using Chemiluminescence plus reagent (Perkin Elmer Life and Analytical Sciences, USA) and visualized using a chemiluminescence imaging system (Syngene, Cambridge, UK).

    Techniques: Activity Assay, Western Blot

    GSK-3β inhibition prevents the enhancement effect of remifentanil on NMDA receptor-mediated mEPSCs in dorsal horn neurons NMDA receptor-mediated mEPSCs in dorsal horn neurons were recorded at the holding potential of -70mV in the presence of TTX (10 μM), GABA receptor antagonist bicuculline (BIM, 20 μM) and AMPA receptor antagonist CNQX (20 μM). Representative traces of mEPSCs under control conditions (C group) and Remifentanil (R group) and Remifentainil+TDZD-8 treatment (RT group) were showed in A. Scale bar , 100 pA, 30 s. Cumulative probability plots of mEPSCs amplitude distribution showed significant shift after remifentanil treatment in spinal dorsal horn neurons (Kolmogorov-Smirnov test, P

    Journal: PLoS ONE

    Article Title: Inhibition of Glycogen Synthase Kinase-3? Prevents Remifentanil-Induced Hyperalgesia via Regulating the Expression and Function of Spinal N-Methyl-D-Aspartate Receptors In Vivo and Vitro

    doi: 10.1371/journal.pone.0077790

    Figure Lengend Snippet: GSK-3β inhibition prevents the enhancement effect of remifentanil on NMDA receptor-mediated mEPSCs in dorsal horn neurons NMDA receptor-mediated mEPSCs in dorsal horn neurons were recorded at the holding potential of -70mV in the presence of TTX (10 μM), GABA receptor antagonist bicuculline (BIM, 20 μM) and AMPA receptor antagonist CNQX (20 μM). Representative traces of mEPSCs under control conditions (C group) and Remifentanil (R group) and Remifentainil+TDZD-8 treatment (RT group) were showed in A. Scale bar , 100 pA, 30 s. Cumulative probability plots of mEPSCs amplitude distribution showed significant shift after remifentanil treatment in spinal dorsal horn neurons (Kolmogorov-Smirnov test, P

    Article Snippet: The membranes were blocked with 5% nonfat milk in Tris-Tween buffer saline for 1 h (TBST; 50 mM Tris-HCl, 154 mM NaCl, and 0.05% Tween 20, pH 7.4), incubated overnight at 4 °C with polyclonal rabbit antibodies against rat NR1, NR2A, NR2B (all 1:300 dilution in 5% nonfat milk in TBST, Chemicon, USA) or rabbit anti-rat GSK-3β and phosphorylated (ser9) GSK-3β antibodies (all 1:1000 dilution in 5% nonfat milk in TBST, Cell Signaling Technology, USA), then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (1:2, 000 in 5% nonfat milk in TBST, Jackson Immuno Research, USA) for 1 h. Membrane bound secondary antibodies were detected using Chemiluminescence plus reagent (Perkin Elmer Life and Analytical Sciences, USA) and visualized using a chemiluminescence imaging system (Syngene, Cambridge, UK).

    Techniques: Inhibition

    GSK-3β regulates the expression of NMDA receptors in spinal dorsal horn. Western blot for membrane NR1, NR2A and NR2B subunit was performed on rat spinal cord dorsal horn neuron (A). Epidermal growth factor receptor (EGFR) was used as the loading control. Pooled densitometric results for NR1, NR2A and NR2B, with the band intensity of C group assigned the value of 1. Remifentanil induced significant increases of both membrane NR1 and NR2B, but had no effect on membrane protein level of NR2A. GSK-3β inhibitor TDZD-8 prevented the changes of membrane NR1 and NR2B. n = 5 for each group, compared with C group, * P

    Journal: PLoS ONE

    Article Title: Inhibition of Glycogen Synthase Kinase-3? Prevents Remifentanil-Induced Hyperalgesia via Regulating the Expression and Function of Spinal N-Methyl-D-Aspartate Receptors In Vivo and Vitro

    doi: 10.1371/journal.pone.0077790

    Figure Lengend Snippet: GSK-3β regulates the expression of NMDA receptors in spinal dorsal horn. Western blot for membrane NR1, NR2A and NR2B subunit was performed on rat spinal cord dorsal horn neuron (A). Epidermal growth factor receptor (EGFR) was used as the loading control. Pooled densitometric results for NR1, NR2A and NR2B, with the band intensity of C group assigned the value of 1. Remifentanil induced significant increases of both membrane NR1 and NR2B, but had no effect on membrane protein level of NR2A. GSK-3β inhibitor TDZD-8 prevented the changes of membrane NR1 and NR2B. n = 5 for each group, compared with C group, * P

    Article Snippet: The membranes were blocked with 5% nonfat milk in Tris-Tween buffer saline for 1 h (TBST; 50 mM Tris-HCl, 154 mM NaCl, and 0.05% Tween 20, pH 7.4), incubated overnight at 4 °C with polyclonal rabbit antibodies against rat NR1, NR2A, NR2B (all 1:300 dilution in 5% nonfat milk in TBST, Chemicon, USA) or rabbit anti-rat GSK-3β and phosphorylated (ser9) GSK-3β antibodies (all 1:1000 dilution in 5% nonfat milk in TBST, Cell Signaling Technology, USA), then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (1:2, 000 in 5% nonfat milk in TBST, Jackson Immuno Research, USA) for 1 h. Membrane bound secondary antibodies were detected using Chemiluminescence plus reagent (Perkin Elmer Life and Analytical Sciences, USA) and visualized using a chemiluminescence imaging system (Syngene, Cambridge, UK).

    Techniques: Expressing, Western Blot

    GSK-3β inhibition prevents remifentanil-induced thermal (A) and mechanical (B) hyperalgesia. Sixty adult rats were randomly divided into 5 groups (n = 12 in each group): saline group (C group, 0.1 ml·kg -1 ·min -1 , 60 min, iv), Glycine group (G group, 15 μg·kg -1 ·min -1 , 60 min, iv; Glycine is an accessory in the pharmaceutical preparation of remifenanil), remifentanil group (R group, 1.0 μg·kg -1 ·min -1 , 60 min, iv), remifentanil plus TDZD-8 (a GSK-3β inhibitor, Sigma, Canada) group (RT group, remifentanil: 1.0 μg·kg -1 ·min -1 and TDZD-8: 1.0 μg·kg -1 , 60 min, iv) and TDZD-8 group (T group, normal saline: 0.1 ml·kg -1 ·min -1 and TDZD-8: 1.0 μg·kg -1 , 60 min, iv). Thermal latency to noxious heat and mechanical paw withdraw threshold were recorded at baseline (-24 h) and 2 h, 6 h, 24 h and 48 h after infusion. The antihyperalgesic effect of TDZD-8 was shown in remifentanil-induced hyperalgesia rats. Compaired with baseline (-24 h), ## P

    Journal: PLoS ONE

    Article Title: Inhibition of Glycogen Synthase Kinase-3? Prevents Remifentanil-Induced Hyperalgesia via Regulating the Expression and Function of Spinal N-Methyl-D-Aspartate Receptors In Vivo and Vitro

    doi: 10.1371/journal.pone.0077790

    Figure Lengend Snippet: GSK-3β inhibition prevents remifentanil-induced thermal (A) and mechanical (B) hyperalgesia. Sixty adult rats were randomly divided into 5 groups (n = 12 in each group): saline group (C group, 0.1 ml·kg -1 ·min -1 , 60 min, iv), Glycine group (G group, 15 μg·kg -1 ·min -1 , 60 min, iv; Glycine is an accessory in the pharmaceutical preparation of remifenanil), remifentanil group (R group, 1.0 μg·kg -1 ·min -1 , 60 min, iv), remifentanil plus TDZD-8 (a GSK-3β inhibitor, Sigma, Canada) group (RT group, remifentanil: 1.0 μg·kg -1 ·min -1 and TDZD-8: 1.0 μg·kg -1 , 60 min, iv) and TDZD-8 group (T group, normal saline: 0.1 ml·kg -1 ·min -1 and TDZD-8: 1.0 μg·kg -1 , 60 min, iv). Thermal latency to noxious heat and mechanical paw withdraw threshold were recorded at baseline (-24 h) and 2 h, 6 h, 24 h and 48 h after infusion. The antihyperalgesic effect of TDZD-8 was shown in remifentanil-induced hyperalgesia rats. Compaired with baseline (-24 h), ## P

    Article Snippet: The membranes were blocked with 5% nonfat milk in Tris-Tween buffer saline for 1 h (TBST; 50 mM Tris-HCl, 154 mM NaCl, and 0.05% Tween 20, pH 7.4), incubated overnight at 4 °C with polyclonal rabbit antibodies against rat NR1, NR2A, NR2B (all 1:300 dilution in 5% nonfat milk in TBST, Chemicon, USA) or rabbit anti-rat GSK-3β and phosphorylated (ser9) GSK-3β antibodies (all 1:1000 dilution in 5% nonfat milk in TBST, Cell Signaling Technology, USA), then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (1:2, 000 in 5% nonfat milk in TBST, Jackson Immuno Research, USA) for 1 h. Membrane bound secondary antibodies were detected using Chemiluminescence plus reagent (Perkin Elmer Life and Analytical Sciences, USA) and visualized using a chemiluminescence imaging system (Syngene, Cambridge, UK).

    Techniques: Inhibition

    GSK3β inhibition suppresses ESCC cell viability and migration in vitro A. The mRNA expression of GSK3β was detected by qPCR in KYSE-30, KYSE-70, EC-9706 and SHEE cells. The viability of the ESCC cell lines KYSE-30 and KYSE-70 was assessed using a (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A wound healing assay was used to study cell migration. Cells were pre-treated with various concentration of LiCl (25 mM) or SB216763 (25 μM) for 2 h, and the anti-proliferative effects of LiCl (B) and SB216763 (C) on KYSE-30 and KYSE-70 cells were assessed at 48 h. (D, E) After KYSE-30 and KYSE-70 cells were transfected with either a GSK3β-specific siRNA or a scramble control siRNA, MTT cell viability assays were performed at different time points on days 1 and 2. siRNA-mediated GSK3β silencing (D) significantly decreased the viability of KYSE-30 and KYSE-70 cells (E). Using the same treatments as above, a wound healing assay was performed to examine the effects of LiCl (25 mM), SB216763 (25 μM), and GSK3β-specific siRNA on the migration of KYSE-30 (F-I) and KYSE-70 (J-M) cells. All results are the average of at least three independent experiments. Error bars represent standard deviation; n =3. * P

    Journal: Molecular carcinogenesis

    Article Title: Inhibition of Glycogen Synthase Kinase 3 beta (GSK3β) Suppresses the Progression of Esophageal Squamous Cell Carcinoma by Modifying STAT3 Activity

    doi: 10.1002/mc.22685

    Figure Lengend Snippet: GSK3β inhibition suppresses ESCC cell viability and migration in vitro A. The mRNA expression of GSK3β was detected by qPCR in KYSE-30, KYSE-70, EC-9706 and SHEE cells. The viability of the ESCC cell lines KYSE-30 and KYSE-70 was assessed using a (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A wound healing assay was used to study cell migration. Cells were pre-treated with various concentration of LiCl (25 mM) or SB216763 (25 μM) for 2 h, and the anti-proliferative effects of LiCl (B) and SB216763 (C) on KYSE-30 and KYSE-70 cells were assessed at 48 h. (D, E) After KYSE-30 and KYSE-70 cells were transfected with either a GSK3β-specific siRNA or a scramble control siRNA, MTT cell viability assays were performed at different time points on days 1 and 2. siRNA-mediated GSK3β silencing (D) significantly decreased the viability of KYSE-30 and KYSE-70 cells (E). Using the same treatments as above, a wound healing assay was performed to examine the effects of LiCl (25 mM), SB216763 (25 μM), and GSK3β-specific siRNA on the migration of KYSE-30 (F-I) and KYSE-70 (J-M) cells. All results are the average of at least three independent experiments. Error bars represent standard deviation; n =3. * P

    Article Snippet: Antibodies against phosphorylated and total GSK3β, phosphorylated and total STAT3, HA, FLAG, and tubulin and the horseradish peroxidase (HRP)–conjugated secondary antibody were obtained from Cell Signaling Technology.

    Techniques: Inhibition, Migration, In Vitro, Expressing, Real-time Polymerase Chain Reaction, MTT Assay, Wound Healing Assay, Concentration Assay, Transfection, Standard Deviation

    Model of how GSK3β affects ESCC cell survival and cancer progression Pro-tumorigenic factors led to the higher expression of total- and lower expression of phospho-GSK3β in cancerous tissue of ESCC, which enhanced the activity of GSK3β. Higher levels of GSK3β activation increased the phosphorylation of STAT3 and by which promoted cancer cell survival and progression of ESCC (Green arrows). In contrast, inhibition of GSK3β by LiCl or SB216763 reduced phosphorylation of STAT3 and consequently suppressed the growth of cancer cell (Pink arrows). Direct association between GSK3β and STAT3 was not observed in this study. Other factors, i.e., IL-6, might be involved in this process. GSK3β activation could enhance the production of IL-6, and through which facilitate the phosphorylation of STAT3 and promote the growth of ESCC cells.

    Journal: Molecular carcinogenesis

    Article Title: Inhibition of Glycogen Synthase Kinase 3 beta (GSK3β) Suppresses the Progression of Esophageal Squamous Cell Carcinoma by Modifying STAT3 Activity

    doi: 10.1002/mc.22685

    Figure Lengend Snippet: Model of how GSK3β affects ESCC cell survival and cancer progression Pro-tumorigenic factors led to the higher expression of total- and lower expression of phospho-GSK3β in cancerous tissue of ESCC, which enhanced the activity of GSK3β. Higher levels of GSK3β activation increased the phosphorylation of STAT3 and by which promoted cancer cell survival and progression of ESCC (Green arrows). In contrast, inhibition of GSK3β by LiCl or SB216763 reduced phosphorylation of STAT3 and consequently suppressed the growth of cancer cell (Pink arrows). Direct association between GSK3β and STAT3 was not observed in this study. Other factors, i.e., IL-6, might be involved in this process. GSK3β activation could enhance the production of IL-6, and through which facilitate the phosphorylation of STAT3 and promote the growth of ESCC cells.

    Article Snippet: Antibodies against phosphorylated and total GSK3β, phosphorylated and total STAT3, HA, FLAG, and tubulin and the horseradish peroxidase (HRP)–conjugated secondary antibody were obtained from Cell Signaling Technology.

    Techniques: Expressing, Activity Assay, Activation Assay, Inhibition

    Inhibition of GSK3β-STAT3 signaling suppresses the initiation and progression of ESCC in SCID tumor-bearing mice ESCC was induced in SCID mice by implanting KYSE-70 cells in the flanks of the mice ( n ). Then, LiCl (100 mg/kg), WP-1066 (20 mg/kg), or both were injected intraperitoneally beginning on day 10 and were administered every two days for a total of 7 treatments. (A) Representative tumor specimens dissected from the SCID mice xenografted with KYSE-70 cells at the end of the study. The average tumor weight (B) and volume (C) were calculated at the time indicated. Both LiCl and WP-1066 suppressed tumor growth, and the combined use of LiCl and WP-1066 decreased tumor growth more significantly than either treatment alone. However, no significantly synergistic effects were observed between the treatment by WP-1066 or LiCl only and their combination. The data represent the mean ± standard deviation of twelve mice per group. (D) Phospho-STAT3 expression in xenograft tissues from mice treated with different inhibitor(s) was detected by immunohistochemistry, and the representative images are shown here. “*” indicates P

    Journal: Molecular carcinogenesis

    Article Title: Inhibition of Glycogen Synthase Kinase 3 beta (GSK3β) Suppresses the Progression of Esophageal Squamous Cell Carcinoma by Modifying STAT3 Activity

    doi: 10.1002/mc.22685

    Figure Lengend Snippet: Inhibition of GSK3β-STAT3 signaling suppresses the initiation and progression of ESCC in SCID tumor-bearing mice ESCC was induced in SCID mice by implanting KYSE-70 cells in the flanks of the mice ( n ). Then, LiCl (100 mg/kg), WP-1066 (20 mg/kg), or both were injected intraperitoneally beginning on day 10 and were administered every two days for a total of 7 treatments. (A) Representative tumor specimens dissected from the SCID mice xenografted with KYSE-70 cells at the end of the study. The average tumor weight (B) and volume (C) were calculated at the time indicated. Both LiCl and WP-1066 suppressed tumor growth, and the combined use of LiCl and WP-1066 decreased tumor growth more significantly than either treatment alone. However, no significantly synergistic effects were observed between the treatment by WP-1066 or LiCl only and their combination. The data represent the mean ± standard deviation of twelve mice per group. (D) Phospho-STAT3 expression in xenograft tissues from mice treated with different inhibitor(s) was detected by immunohistochemistry, and the representative images are shown here. “*” indicates P

    Article Snippet: Antibodies against phosphorylated and total GSK3β, phosphorylated and total STAT3, HA, FLAG, and tubulin and the horseradish peroxidase (HRP)–conjugated secondary antibody were obtained from Cell Signaling Technology.

    Techniques: Inhibition, Mouse Assay, Injection, Standard Deviation, Expressing, Immunohistochemistry

    GSK3β inhibition-mediated suppression of cancer cell viability and migration is dependent on STAT3 activation KYSE-70 cells were transfected with a GSK3β-specific siRNA (A), a constitutively active mutant of GSK3β (S9A) (C) or a mutant plasmid STAT3 (Y705F) (E), or the relevant control including a scramble siRNA and empty control plasmids for 48 h, and whole-cell lysates were then collected and analyzed by immunoblot using antibodies against phospho-STAT3/total STAT3, HA tag, FLAG tag, and tubulin (A, C, and E). Densitometric quantification was performed by calculating the ratio of target proteins to tubulin (B, D and F). Direct association between GSK3β and STAT3 was determined by immunoprecipitation of GSK3β followed by Western blot for STAT3 and total GSK3β in KYSE-70 cells(G). Cells transfected with a STAT3 (Y705F) mutant or the empty control vector (F) were pre-treated with LiCl, and the viability and migration of the cells were assessed by MTT and wound healing assays, respectively. (H, I) Viability analysis of cells transfected with overexpression of STAT3 mutant indicates that STAT3 enhances cell viability and abrogates the effects of GSK3β inhibition on KYSE-70 cells (H). By contrast, a STAT3 inhibitor (WP-1066; 20 μM) enhances the effect of LiCl (I). (J to M) In a wound healing assay, overexpression of active STAT3 enhances cell migration and offsets the suppressive effect of LiCl on the migration of KYSE-70 cells (J, L). By contrast, a STAT3 inhibitor (WP-1066) substantially enhances the suppressive effect of LiCl (K, M). All results are the average of at least three independent experiments. Error bars represent standard deviation; n =3. * P

    Journal: Molecular carcinogenesis

    Article Title: Inhibition of Glycogen Synthase Kinase 3 beta (GSK3β) Suppresses the Progression of Esophageal Squamous Cell Carcinoma by Modifying STAT3 Activity

    doi: 10.1002/mc.22685

    Figure Lengend Snippet: GSK3β inhibition-mediated suppression of cancer cell viability and migration is dependent on STAT3 activation KYSE-70 cells were transfected with a GSK3β-specific siRNA (A), a constitutively active mutant of GSK3β (S9A) (C) or a mutant plasmid STAT3 (Y705F) (E), or the relevant control including a scramble siRNA and empty control plasmids for 48 h, and whole-cell lysates were then collected and analyzed by immunoblot using antibodies against phospho-STAT3/total STAT3, HA tag, FLAG tag, and tubulin (A, C, and E). Densitometric quantification was performed by calculating the ratio of target proteins to tubulin (B, D and F). Direct association between GSK3β and STAT3 was determined by immunoprecipitation of GSK3β followed by Western blot for STAT3 and total GSK3β in KYSE-70 cells(G). Cells transfected with a STAT3 (Y705F) mutant or the empty control vector (F) were pre-treated with LiCl, and the viability and migration of the cells were assessed by MTT and wound healing assays, respectively. (H, I) Viability analysis of cells transfected with overexpression of STAT3 mutant indicates that STAT3 enhances cell viability and abrogates the effects of GSK3β inhibition on KYSE-70 cells (H). By contrast, a STAT3 inhibitor (WP-1066; 20 μM) enhances the effect of LiCl (I). (J to M) In a wound healing assay, overexpression of active STAT3 enhances cell migration and offsets the suppressive effect of LiCl on the migration of KYSE-70 cells (J, L). By contrast, a STAT3 inhibitor (WP-1066) substantially enhances the suppressive effect of LiCl (K, M). All results are the average of at least three independent experiments. Error bars represent standard deviation; n =3. * P

    Article Snippet: Antibodies against phosphorylated and total GSK3β, phosphorylated and total STAT3, HA, FLAG, and tubulin and the horseradish peroxidase (HRP)–conjugated secondary antibody were obtained from Cell Signaling Technology.

    Techniques: Inhibition, Migration, Activation Assay, Transfection, Mutagenesis, Plasmid Preparation, FLAG-tag, Immunoprecipitation, Western Blot, MTT Assay, Over Expression, Wound Healing Assay, Standard Deviation

    Blast induces GSK3β activation in ApoE4 mice and synj1 knockdown reduces pTau through inactivation of GSK3β. Levels of ( A ) active and ( B ) inactive pGSK3β after blast. Results are presented as % of controls ± SEM (ApoE3 sham). Levels of ( C ) active pGSK3β and ( D ) pTau in N2a cells with synj1 siRNA, GSK inhibitors, or combination. Results are presented as % of controls ± SEM.

    Journal: Scientific Reports

    Article Title: ApoE4-associated phospholipid dysregulation contributes to development of Tau hyper-phosphorylation after traumatic brain injury

    doi: 10.1038/s41598-017-11654-7

    Figure Lengend Snippet: Blast induces GSK3β activation in ApoE4 mice and synj1 knockdown reduces pTau through inactivation of GSK3β. Levels of ( A ) active and ( B ) inactive pGSK3β after blast. Results are presented as % of controls ± SEM (ApoE3 sham). Levels of ( C ) active pGSK3β and ( D ) pTau in N2a cells with synj1 siRNA, GSK inhibitors, or combination. Results are presented as % of controls ± SEM.

    Article Snippet: Antibodies and reagents The following commercially available antibodies were used: anti-pTau (AT8; pSer202, pThr205) and total Tau (Tau 5) from ThermoFisher (RRID:AB_223647 and 10980631), anti-synj1 (rabbit polyclonal Ab, Novus; RRID:AB_11047653), anti-β actin and tubulin (Santa Cruz; RRID:AB_476697 and 477498), anti-holoAPP (MAB348, Millipore; RRID:AB_94882), anti-GSK3β (rabbit monoclonal Ab, Cell Signaling; RRID:AB_490890), anti-pGSK3β (Ser9, inactive form of GSK3β, rabbit polyclonal Ab, Cell Signaling; RRID:AB_331405), anti-pGSK3β (Y216, active form of GSK3β, rabbit polyclonal Ab, Abcam; RRID:AB_1310290), anti-mouse and rabbit horse radish peroxidase, Texas-Red or Alexa488 conjugated anti-mouse IgG (ThermoFisher; RRID:AB_2556542, 2540618, 10374713, 10983944, 2535987 and 1090271), were purchased.

    Techniques: Activation Assay, Mouse Assay

    Ratio of phosphorylated GSK3β to total GSK3β in fetal St muscle of C (open bar) and OB (closed bar) sheep. Data are means ± SE; n = 5. * P

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Maternal obesity downregulates myogenesis and β-catenin signaling in fetal skeletal muscle

    doi: 10.1152/ajpendo.90924.2008

    Figure Lengend Snippet: Ratio of phosphorylated GSK3β to total GSK3β in fetal St muscle of C (open bar) and OB (closed bar) sheep. Data are means ± SE; n = 5. * P

    Article Snippet: Antibodies against GSK3β, phospho-GSK3β at Ser9, β-catenin, phospho-IKKα/β at Ser176/180, phospho-IκBα at Ser32, phospho-NF-κB p65 at Ser536, FOXO3a, and horseradish peroxidase linked secondary antibody were purchased from Cell Signaling (Danvers, MA).

    Techniques:

    Effects of dexamethasone (DEX) on Ostf1 expression and Akt-GSK3β signaling in Japanese eel primary gill cell cultures. ( A ) A representative Western blot shows that DEX treatment for 6 hours induced the Ostf1 protein expression level. A transient increase of Akt and GSK3β phosphorylation was observed followed by a decrease of the phosphorylated levels. ( B ) Real-time PCR analysis of time-dependent Ostf1 mRNA expression levels in the DEX-treated cells. * P

    Journal: Biology Open

    Article Title: Dexamethasone (DEX) induces Osmotic stress transcription factor 1 (Ostf1) through the Akt-GSK3? pathway in freshwater Japanese eel gill cell cultures

    doi: 10.1242/bio.20134135

    Figure Lengend Snippet: Effects of dexamethasone (DEX) on Ostf1 expression and Akt-GSK3β signaling in Japanese eel primary gill cell cultures. ( A ) A representative Western blot shows that DEX treatment for 6 hours induced the Ostf1 protein expression level. A transient increase of Akt and GSK3β phosphorylation was observed followed by a decrease of the phosphorylated levels. ( B ) Real-time PCR analysis of time-dependent Ostf1 mRNA expression levels in the DEX-treated cells. * P

    Article Snippet: Western blotting was conducted using rabbit polyclonal anti-GilZ/TilZ antibody (1:1000) (Abcam) , anti-phospho-Akt, anti-total Akt, anti-phospho-GSK3β or anti-total GSK3β (1:1000) (Cell signaling), followed by an incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:4000) (Bio-Rad).

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

    Effects of GSK3β inhibitor, LiCl, on Ostf1 expression in Japanese eel primary gill culture. ( A ) A representative Western blot shows the inhibitory effects of LiCl on GSK3β activity (as shown by an increase in the level of GSK3β phosphorylation) and Ostf1 expression level. ( B ) Real-time PCR analysis revealed the inhibitory effect of LiCl on Ostf1 mRNA expression level. * P

    Journal: Biology Open

    Article Title: Dexamethasone (DEX) induces Osmotic stress transcription factor 1 (Ostf1) through the Akt-GSK3? pathway in freshwater Japanese eel gill cell cultures

    doi: 10.1242/bio.20134135

    Figure Lengend Snippet: Effects of GSK3β inhibitor, LiCl, on Ostf1 expression in Japanese eel primary gill culture. ( A ) A representative Western blot shows the inhibitory effects of LiCl on GSK3β activity (as shown by an increase in the level of GSK3β phosphorylation) and Ostf1 expression level. ( B ) Real-time PCR analysis revealed the inhibitory effect of LiCl on Ostf1 mRNA expression level. * P

    Article Snippet: Western blotting was conducted using rabbit polyclonal anti-GilZ/TilZ antibody (1:1000) (Abcam) , anti-phospho-Akt, anti-total Akt, anti-phospho-GSK3β or anti-total GSK3β (1:1000) (Cell signaling), followed by an incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:4000) (Bio-Rad).

    Techniques: Expressing, Western Blot, Activity Assay, Real-time Polymerase Chain Reaction

    Hyperosmolality (500 mosmol l −1 ) activated Akt-GSK3β signaling and Ostf1 expression in Japanese eel primary gill cell culture. ( A ) A representative Western blot shows the effects of hyperosmolality on a transient increase of Akt and GSK3β phosphorylation, followed by a decrease of the phosphorylated levels. Ostf1 expression level was increased in a time-dependent manner after hyperosmotic treatment. ( B ) Real-time PCR analysis revealed the hyperosmolality induced Ostf1 mRNA expression. * P

    Journal: Biology Open

    Article Title: Dexamethasone (DEX) induces Osmotic stress transcription factor 1 (Ostf1) through the Akt-GSK3? pathway in freshwater Japanese eel gill cell cultures

    doi: 10.1242/bio.20134135

    Figure Lengend Snippet: Hyperosmolality (500 mosmol l −1 ) activated Akt-GSK3β signaling and Ostf1 expression in Japanese eel primary gill cell culture. ( A ) A representative Western blot shows the effects of hyperosmolality on a transient increase of Akt and GSK3β phosphorylation, followed by a decrease of the phosphorylated levels. Ostf1 expression level was increased in a time-dependent manner after hyperosmotic treatment. ( B ) Real-time PCR analysis revealed the hyperosmolality induced Ostf1 mRNA expression. * P

    Article Snippet: Western blotting was conducted using rabbit polyclonal anti-GilZ/TilZ antibody (1:1000) (Abcam) , anti-phospho-Akt, anti-total Akt, anti-phospho-GSK3β or anti-total GSK3β (1:1000) (Cell signaling), followed by an incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:4000) (Bio-Rad).

    Techniques: Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction

    The effect of insulin on Akt and Gsk3β phosphorylation in TIRKO and their corresponding WT controls. (A) Representative western-blots of Akt phosphorylation on Ser473 and total Akt expression and Gsk3β phosphorylation on Ser9 and total

    Journal: Journal of molecular and cellular cardiology

    Article Title: INSULIN SUPPRESSES ISCHEMIC PRECONDITIONING-MEDIATED CARDIOPROTECTION THROUGH AKT-DEPENDENT MECHANISMS

    doi: 10.1016/j.yjmcc.2013.08.005

    Figure Lengend Snippet: The effect of insulin on Akt and Gsk3β phosphorylation in TIRKO and their corresponding WT controls. (A) Representative western-blots of Akt phosphorylation on Ser473 and total Akt expression and Gsk3β phosphorylation on Ser9 and total

    Article Snippet: Membranes were probed with the following primary antibodies: mouse anti-Akt1, rabbit anti-p-Akt (Ser 473) and rabbit anti-p-Akt (Thr 308) (1/1000; 1/750; 1/1000 respectively, Cell Signaling Technology Inc. Danvers, MA), mouse anti-Gsk3α/β and rabbit anti-Phospho-Gsk3β (Ser 9) (1/1000 and 1/2000 respectively, Cell Signaling Technology Inc., Danvers, MA), rabbit-anti LC3 (1/1000, Sigma-Aldrich, St. Louis, MO), rabbit anti-p38MAPK, rabbit anti-Phosphop38MAPK, rabbit anti-MEK and rabbit anti-PhosphoMEK (1/1000, Cell Signaling Technology Inc., Danvers MA) and mouse anti-actin (1/1000, Sigma-Aldrich, St. Louis, MO).

    Techniques: Western Blot, Expressing

    Effects of DQP on regulating glucose metabolism in ischemic heart tissue. (A) Western blot bands of GLUT4 and PFK and their quantitative results in heart tissues of rats, DQP could promote glucose intake and glycolysis to provide more energy for the ischemic heart. (B) Immunohistochemistry images of GLUT4 and quantitative results in the heart tissues of rats in different groups. (C) Myocardial glycogen levels in different groups. (D) Western blot bands of GSK-3β, pGSK-3β and their quantitative results in heart tissues of rats. (E) Western blot bands of GS, pGS and their quantitative results in heart tissues of rats. DQP could promote glycogen synthesis by increasing glycogen synthase. The raw date were listed in Supplementary Figure 2 . # P

    Journal: Frontiers in Pharmacology

    Article Title: The Effect of Chinese Medicine on Lipid and Glucose Metabolism in Acute Myocardial Infarction Through PPARγ Pathway

    doi: 10.3389/fphar.2018.01209

    Figure Lengend Snippet: Effects of DQP on regulating glucose metabolism in ischemic heart tissue. (A) Western blot bands of GLUT4 and PFK and their quantitative results in heart tissues of rats, DQP could promote glucose intake and glycolysis to provide more energy for the ischemic heart. (B) Immunohistochemistry images of GLUT4 and quantitative results in the heart tissues of rats in different groups. (C) Myocardial glycogen levels in different groups. (D) Western blot bands of GSK-3β, pGSK-3β and their quantitative results in heart tissues of rats. (E) Western blot bands of GS, pGS and their quantitative results in heart tissues of rats. DQP could promote glycogen synthesis by increasing glycogen synthase. The raw date were listed in Supplementary Figure 2 . # P

    Article Snippet: The membranes were incubated with skimmed milk for 1 h and subsequently incubated with different first antibodies, including ACADL (ab196655, Abcam, 1:4000), SCP2 (ab140126, Abcam, 1:4000), GLUT4 (#2213, CST, 1:2000), PFK (#13123, CST, 1:2000), pGSK-3β (#5558, CST, 1:2000), GSK-3β (#9832, CST, 1:2000), pGS (#3891, CST, 1:4000), GS (#3893, CST, 1:4000), PPARγ (ab45036, Abcam, 1:2000), GAPDH (ab8245, Abcam, 1:5000) was used as a loading control.

    Techniques: Western Blot, Immunohistochemistry

    Detection of GSK ‐ 3β‐targeted residues in rat IC‐1B . CID ‐based LC / MS / MS analyses of phosphorylation in full‐length rat IC‐1B . A) An MS / MS spectrum of a phosphorylated peptide ( SVS * TPSEAGSQDDLGPLTR ) was acquired on a doubly charged ion at m/z 998.95 Da. The precursor mass (1996.89299 Da) is consistent with one phosphate in the peptide. MS / MS fragmentation of this peptide indicates a single phosphorylation in fragments y17 and b4, thus placing the modification unambiguously on S3 , corresponding to S87 in IC‐1B . B) Another MS / MS spectrum of a phosphorylated peptide ( SVST * PSEAGSQDDLGPLTR ) was acquired on a doubly charged ion at m/z 998.95117 Da. Single phosphorylation of fragments y16 and b4 indicates that T4 , corresponding to T88 in IC‐1B , is phosphorylated. MS / MS fragmentation eliminates four C‐terminal S/T sites ( S6 , S10 , S13 and S20 ) by showing that y4–y15 fragments have no phosphorylation. C) Two‐point mutations, S87A and T88V , were introduced into GFP‐IC‐1B [ EGFP‐IC1B ( S87A / T87V )]. WT and mutant constructs were expressed in Cos‐7 cells, immunoprecipitated with a GFP antibody and then exposed to GSK ‐3β in an in vitro kinase assay. Less 32 P was incorporated into the mutant construct.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: GSK‐3β Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility

    doi: 10.1111/tra.12304

    Figure Lengend Snippet: Detection of GSK ‐ 3β‐targeted residues in rat IC‐1B . CID ‐based LC / MS / MS analyses of phosphorylation in full‐length rat IC‐1B . A) An MS / MS spectrum of a phosphorylated peptide ( SVS * TPSEAGSQDDLGPLTR ) was acquired on a doubly charged ion at m/z 998.95 Da. The precursor mass (1996.89299 Da) is consistent with one phosphate in the peptide. MS / MS fragmentation of this peptide indicates a single phosphorylation in fragments y17 and b4, thus placing the modification unambiguously on S3 , corresponding to S87 in IC‐1B . B) Another MS / MS spectrum of a phosphorylated peptide ( SVST * PSEAGSQDDLGPLTR ) was acquired on a doubly charged ion at m/z 998.95117 Da. Single phosphorylation of fragments y16 and b4 indicates that T4 , corresponding to T88 in IC‐1B , is phosphorylated. MS / MS fragmentation eliminates four C‐terminal S/T sites ( S6 , S10 , S13 and S20 ) by showing that y4–y15 fragments have no phosphorylation. C) Two‐point mutations, S87A and T88V , were introduced into GFP‐IC‐1B [ EGFP‐IC1B ( S87A / T87V )]. WT and mutant constructs were expressed in Cos‐7 cells, immunoprecipitated with a GFP antibody and then exposed to GSK ‐3β in an in vitro kinase assay. Less 32 P was incorporated into the mutant construct.

    Article Snippet: The 3D10 GSK‐3β mouse mAb, 5B3 S9 Phospho‐GSK‐3β rabbit mAb, 11E7 Akt rabbit mAb, D25E6 T308 pAkt rabbit mAb and D9E S473 p‐Akt rabbit mAb were from Cell Signaling, Inc.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Modification, Mutagenesis, Construct, Immunoprecipitation, In Vitro, Kinase Assay

    An insulin‐sensitizing drug, ROZ , causes centrosomal dynein accumulation via GSK ‐ 3β inactivation. A) ROZ exposure resulted in IC accumulation at centrosomes ( N = 150 cells for each treatment, mean ± 95% CI , ***p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: GSK‐3β Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility

    doi: 10.1111/tra.12304

    Figure Lengend Snippet: An insulin‐sensitizing drug, ROZ , causes centrosomal dynein accumulation via GSK ‐ 3β inactivation. A) ROZ exposure resulted in IC accumulation at centrosomes ( N = 150 cells for each treatment, mean ± 95% CI , ***p

    Article Snippet: The 3D10 GSK‐3β mouse mAb, 5B3 S9 Phospho‐GSK‐3β rabbit mAb, 11E7 Akt rabbit mAb, D25E6 T308 pAkt rabbit mAb and D9E S473 p‐Akt rabbit mAb were from Cell Signaling, Inc.

    Techniques:

    Dynein is released from peripheral/cortical sites in response to GSK ‐ 3β inhibition. A) YAMC cells were treated with or without CT99021 for 12 h after serum starvation. Cells were fixed and immunostained with IC antibodies (red) and Hoechst dye to label nuclei. B) Thirty images of lamella of DMSO control and CT99021 ‐treated interphase cells were acquired for each condition using the same exposure times. All of the images were adjusted together in image j using the image j threshold controls to select pixels between 150 and 200 afu, a good fit for the data. image j was used to quantify the number and size of dynein puncta. C) The average number of dynein puncta did not change (∼60 per lamella). D) The average size of dynein puncta in CT99021 ‐treated cells was significantly reduced compared to control cells (mean ± 95% CI , ***p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: GSK‐3β Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility

    doi: 10.1111/tra.12304

    Figure Lengend Snippet: Dynein is released from peripheral/cortical sites in response to GSK ‐ 3β inhibition. A) YAMC cells were treated with or without CT99021 for 12 h after serum starvation. Cells were fixed and immunostained with IC antibodies (red) and Hoechst dye to label nuclei. B) Thirty images of lamella of DMSO control and CT99021 ‐treated interphase cells were acquired for each condition using the same exposure times. All of the images were adjusted together in image j using the image j threshold controls to select pixels between 150 and 200 afu, a good fit for the data. image j was used to quantify the number and size of dynein puncta. C) The average number of dynein puncta did not change (∼60 per lamella). D) The average size of dynein puncta in CT99021 ‐treated cells was significantly reduced compared to control cells (mean ± 95% CI , ***p

    Article Snippet: The 3D10 GSK‐3β mouse mAb, 5B3 S9 Phospho‐GSK‐3β rabbit mAb, 11E7 Akt rabbit mAb, D25E6 T308 pAkt rabbit mAb and D9E S473 p‐Akt rabbit mAb were from Cell Signaling, Inc.

    Techniques: Inhibition

    GSK ‐ 3β inhibition is responsible for the dynein accumulation at centrosomes. A and B) IC intensity (red) was determined within a circle drawn around the centrosome (green). C) Cells with IC intensity equal to or greater than 60 afu were considered to have dynein accumulation. N = 400 cells measured for each condition. CT99021 exposure for 12 h caused a significant increase in the percentage of HCT116 or YAMC cells with dynein accumulation (mean ± 95% CI , ***p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: GSK‐3β Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility

    doi: 10.1111/tra.12304

    Figure Lengend Snippet: GSK ‐ 3β inhibition is responsible for the dynein accumulation at centrosomes. A and B) IC intensity (red) was determined within a circle drawn around the centrosome (green). C) Cells with IC intensity equal to or greater than 60 afu were considered to have dynein accumulation. N = 400 cells measured for each condition. CT99021 exposure for 12 h caused a significant increase in the percentage of HCT116 or YAMC cells with dynein accumulation (mean ± 95% CI , ***p

    Article Snippet: The 3D10 GSK‐3β mouse mAb, 5B3 S9 Phospho‐GSK‐3β rabbit mAb, 11E7 Akt rabbit mAb, D25E6 T308 pAkt rabbit mAb and D9E S473 p‐Akt rabbit mAb were from Cell Signaling, Inc.

    Techniques: Inhibition

    Dynein phosphorylation by GSK ‐ 3β affects Ndel1 interaction. A) Dynein and Ndel1 distribution were evaluated in YAMC cells by IC (red) and Ndel1 (green) immunofluorescence. Cells were serum starved for 12 h and then exposed to CT99021 or vehicle alone ( DMSO ) for 12 h in medium supplemented with FBS and ITS . Representative images for each treatment are shown. B) The percentage of cells with Ndel1 accumulation at apparent centrosomes increased with exposure to CT99021 ( N = 225 cells for each treatment, mean ± 95% CI , ***p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: GSK‐3β Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility

    doi: 10.1111/tra.12304

    Figure Lengend Snippet: Dynein phosphorylation by GSK ‐ 3β affects Ndel1 interaction. A) Dynein and Ndel1 distribution were evaluated in YAMC cells by IC (red) and Ndel1 (green) immunofluorescence. Cells were serum starved for 12 h and then exposed to CT99021 or vehicle alone ( DMSO ) for 12 h in medium supplemented with FBS and ITS . Representative images for each treatment are shown. B) The percentage of cells with Ndel1 accumulation at apparent centrosomes increased with exposure to CT99021 ( N = 225 cells for each treatment, mean ± 95% CI , ***p

    Article Snippet: The 3D10 GSK‐3β mouse mAb, 5B3 S9 Phospho‐GSK‐3β rabbit mAb, 11E7 Akt rabbit mAb, D25E6 T308 pAkt rabbit mAb and D9E S473 p‐Akt rabbit mAb were from Cell Signaling, Inc.

    Techniques: Immunofluorescence

    Direct phosphorylation of dynein by GSK ‐ 3β. A) Cytoplasmic dynein was immunoprecipitated from mouse brain using an IC antibody (α‐ IC IP ). A No 1° antibody pulldown served as a control. GSK ‐3β was specifically pulled down by dynein (arrow). FT = 10% of flow through, W = 10% of first wash, PD = pull down. B) Purified bovine dynein was incubated with GST‐GSK ‐3β (left panel) or His‐ GSK ‐3β (right panel). HC , IC and LIC subunits were phosphorylated. Auto‐phosphorylated GST‐GSK ‐3β (∼73 kDa ) and His‐ GSK ‐3β (∼48 kDa ) were also detected (red asterisks). C) An anti‐ GFP antibody was used to isolate EGFP‐IC‐2C and EGFP‐IC‐1B expressed in Cos‐7 cells. Both proteins were phosphorylated by GSK ‐3β. The phosphorylated bands in the red box are likely endogenous ICs in the complex. Bands in the blue box may be endogenous LICs interacting with IC‐1B . D) Mouse brain ICs that had been stripped of endogenous phosphates by lambda phosphatase could still be phosphorylated by GSK ‐3β, indicating that one or more sites do not require priming. E and F) Bacterially expressed full‐length His‐tagged IC‐2C ( FL‐IC‐2C ) and an N‐terminal fragment ( N237 IC‐2C ) were phosphorylated by GSK ‐3β in vitro and phosphorylation was blocked by the specific GSK ‐3β inhibitor, CT990221 , confirming the presence of sites that do not require priming phosphorylation.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: GSK‐3β Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility

    doi: 10.1111/tra.12304

    Figure Lengend Snippet: Direct phosphorylation of dynein by GSK ‐ 3β. A) Cytoplasmic dynein was immunoprecipitated from mouse brain using an IC antibody (α‐ IC IP ). A No 1° antibody pulldown served as a control. GSK ‐3β was specifically pulled down by dynein (arrow). FT = 10% of flow through, W = 10% of first wash, PD = pull down. B) Purified bovine dynein was incubated with GST‐GSK ‐3β (left panel) or His‐ GSK ‐3β (right panel). HC , IC and LIC subunits were phosphorylated. Auto‐phosphorylated GST‐GSK ‐3β (∼73 kDa ) and His‐ GSK ‐3β (∼48 kDa ) were also detected (red asterisks). C) An anti‐ GFP antibody was used to isolate EGFP‐IC‐2C and EGFP‐IC‐1B expressed in Cos‐7 cells. Both proteins were phosphorylated by GSK ‐3β. The phosphorylated bands in the red box are likely endogenous ICs in the complex. Bands in the blue box may be endogenous LICs interacting with IC‐1B . D) Mouse brain ICs that had been stripped of endogenous phosphates by lambda phosphatase could still be phosphorylated by GSK ‐3β, indicating that one or more sites do not require priming. E and F) Bacterially expressed full‐length His‐tagged IC‐2C ( FL‐IC‐2C ) and an N‐terminal fragment ( N237 IC‐2C ) were phosphorylated by GSK ‐3β in vitro and phosphorylation was blocked by the specific GSK ‐3β inhibitor, CT990221 , confirming the presence of sites that do not require priming phosphorylation.

    Article Snippet: The 3D10 GSK‐3β mouse mAb, 5B3 S9 Phospho‐GSK‐3β rabbit mAb, 11E7 Akt rabbit mAb, D25E6 T308 pAkt rabbit mAb and D9E S473 p‐Akt rabbit mAb were from Cell Signaling, Inc.

    Techniques: Immunoprecipitation, Flow Cytometry, Purification, Incubation, In Vitro

    A model for GSK ‐ 3β regulation of dynein‐dependent retrograde organelle transport. A) In control cells, GSK ‐3β can phosphorylate IC at S87 / T88 , decreasing its interaction with Ndel1 and reducing dynein motility toward microtubule minus ends. B) When the cell is stimulated by insulin, PI3K / AKT signaling is initiated and GSK ‐3β is shut off. As cortical dynein loses phosphate at S87 / T88 on ICs , it interacts more efficiently with Ndel1. Ndel1 can increase dynein force production, leading to increased retrograde transport of cargo and accumulation of dynein and cargo at minus ends near the centrosome. C) Inhibition of GSK ‐3β by pharmacological inhibitors such as CT99021 ( CT ) or LiCl bypasses the need for insulin signaling and stimulates transport.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: GSK‐3β Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility

    doi: 10.1111/tra.12304

    Figure Lengend Snippet: A model for GSK ‐ 3β regulation of dynein‐dependent retrograde organelle transport. A) In control cells, GSK ‐3β can phosphorylate IC at S87 / T88 , decreasing its interaction with Ndel1 and reducing dynein motility toward microtubule minus ends. B) When the cell is stimulated by insulin, PI3K / AKT signaling is initiated and GSK ‐3β is shut off. As cortical dynein loses phosphate at S87 / T88 on ICs , it interacts more efficiently with Ndel1. Ndel1 can increase dynein force production, leading to increased retrograde transport of cargo and accumulation of dynein and cargo at minus ends near the centrosome. C) Inhibition of GSK ‐3β by pharmacological inhibitors such as CT99021 ( CT ) or LiCl bypasses the need for insulin signaling and stimulates transport.

    Article Snippet: The 3D10 GSK‐3β mouse mAb, 5B3 S9 Phospho‐GSK‐3β rabbit mAb, 11E7 Akt rabbit mAb, D25E6 T308 pAkt rabbit mAb and D9E S473 p‐Akt rabbit mAb were from Cell Signaling, Inc.

    Techniques: Inhibition

    Inhibition of GSK ‐ 3β stimulates retrograde transport in adult rat DRG neurons. Time‐lapse movies of Lysotracker‐labeled organelles moving in living DRG axons exposed to GSK ‐3 inhibitors were converted to kymographs using NIH image J software. A and B) Representative kymographs for axons exposed to DMSO or CT99021 for 12 h are shown. The horizontal arrow indicates the retrograde direction (toward the cell body for the 100‐µm axon segment). The vertical arrow indicates time (2‐min total recording time). Sixteen axons in cultures from two different rats were analyzed for each condition. Organelle movement was categorized as anterograde (ant), retrograde (retro), both or static and the average percentage of organelles per axon in each category was determined. C and D) A significantly higher percentage of organelles moved retrogradely, and fewer were static following CT99021 exposure (mean ± 95% CI , n.s., p > 0.05, ***p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: GSK‐3β Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility

    doi: 10.1111/tra.12304

    Figure Lengend Snippet: Inhibition of GSK ‐ 3β stimulates retrograde transport in adult rat DRG neurons. Time‐lapse movies of Lysotracker‐labeled organelles moving in living DRG axons exposed to GSK ‐3 inhibitors were converted to kymographs using NIH image J software. A and B) Representative kymographs for axons exposed to DMSO or CT99021 for 12 h are shown. The horizontal arrow indicates the retrograde direction (toward the cell body for the 100‐µm axon segment). The vertical arrow indicates time (2‐min total recording time). Sixteen axons in cultures from two different rats were analyzed for each condition. Organelle movement was categorized as anterograde (ant), retrograde (retro), both or static and the average percentage of organelles per axon in each category was determined. C and D) A significantly higher percentage of organelles moved retrogradely, and fewer were static following CT99021 exposure (mean ± 95% CI , n.s., p > 0.05, ***p

    Article Snippet: The 3D10 GSK‐3β mouse mAb, 5B3 S9 Phospho‐GSK‐3β rabbit mAb, 11E7 Akt rabbit mAb, D25E6 T308 pAkt rabbit mAb and D9E S473 p‐Akt rabbit mAb were from Cell Signaling, Inc.

    Techniques: Inhibition, Labeling, Software

    Pharmacological inhibition of GSK ‐ 3β causes dynein to accumulate at centrosomes in colon cell lines. A and B) HCT116 human colon cancer cells were serum starved for 12 h and then exposed to DMSO or CT99021 in medium with FBS and ITS for an additional 12 h. Dynein distribution was assessed by IC immunofluorescence (red, and middle panels). Centrosomes were labeled with a CDK5RAP2 antibody (green, and right panels). Nuclei are stained with Hoechst dye (blue). Insets show digitally enlarged images of centrosomes indicated by the pink arrows. C and D) The same response was observed in YAMC cells derived from adult mouse colon.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: GSK‐3β Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility

    doi: 10.1111/tra.12304

    Figure Lengend Snippet: Pharmacological inhibition of GSK ‐ 3β causes dynein to accumulate at centrosomes in colon cell lines. A and B) HCT116 human colon cancer cells were serum starved for 12 h and then exposed to DMSO or CT99021 in medium with FBS and ITS for an additional 12 h. Dynein distribution was assessed by IC immunofluorescence (red, and middle panels). Centrosomes were labeled with a CDK5RAP2 antibody (green, and right panels). Nuclei are stained with Hoechst dye (blue). Insets show digitally enlarged images of centrosomes indicated by the pink arrows. C and D) The same response was observed in YAMC cells derived from adult mouse colon.

    Article Snippet: The 3D10 GSK‐3β mouse mAb, 5B3 S9 Phospho‐GSK‐3β rabbit mAb, 11E7 Akt rabbit mAb, D25E6 T308 pAkt rabbit mAb and D9E S473 p‐Akt rabbit mAb were from Cell Signaling, Inc.

    Techniques: Inhibition, Immunofluorescence, Labeling, Staining, Derivative Assay

    Identification of GSK ‐ 3β‐targeted residues in mouse IC‐2C . A–C) CID ‐based LC / MS / MS analyses of phosphorylation in a 237 amino acid, N‐terminal fragment of IC‐2C . A) An MS / MS spectrum of a phosphorylated peptide ( SVS * TPSEAGSQDSGDGAVGSR ) was acquired on a doubly charged ion at m/z 1015.9. The precursor mass (2030.82268 Da) is consistent with one phosphate in the peptide. MS / MS fragmentation of this peptide indicates a single phosphorylation of fragments y19 and b4, thus placing the modification unambiguously on S3 , corresponding to S88 in IC‐2C . Furthermore, MS / MS fragmentation eliminates all five C‐terminal S/T sites ( T4 , S6 , S10 , S13 and S20 ) by showing that y3–y18 fragments are not phosphorylated. B) Another MS / MS spectrum of a phosphorylated peptide ( SVST * PSEAGSQDSGDGAVGSR ) was acquired on a doubly charged ion at m/z 1015.9. MS / MS fragmentation of this peptide indicates a single phosphorylation of fragment y18 and b4 indicates that T4 , corresponding to T89 in IC‐2C , is phosphorylated. MS / MS fragmentation eliminates four C‐terminal S/T sites ( S6 , S10 , S13 and S20 ) because y3–y17 fragments have no phosphate. C) MS / MS spectrum of a phosphorylated peptide ( EDEEEEDDVAT * PKPPVEPEEEK ) was acquired on a triply charged ion at m/z 874.0. The precursor mass, 2620.07279 Da, indicates one phosphate in the peptide. The b11/y12 and b10/y11 fragments enable the conclusive assignment of T11 ( T154 in IC‐2C ) as the phosphorylated residue. D) S88 and T89 are conserved across mammalian species (mouse‐ MM , rat‐ RN , human‐ HS , bovine‐ BT and horse‐ EC ) and isoforms of IC ‐2, and both are found in mouse IC ‐1. T154 is not present in human or rat IC ‐2 or in mouse IC ‐1. E) Double site‐directed mutagenesis ( S88A and T89V ) in a 106 amino acid N‐terminal fragment of IC‐2C significantly reduced phosphorylation by GSK ‐3β in vitro . F) Full‐length mouse EGFP‐IC‐2C and two mutants ( T154V and S88A / T89V / T154V ) were expressed in Cos7 cells, immunopurified using an EGFP antibody and exposed to GSK ‐3β. Less phosphate was incorporated into the triple mutant, indicating that S88 / T89 sites are targeted in the full‐length protein. G) Schematic of IC showing previously identified interaction domains. S88 and T89 (black bar, asterisks) are near Nde1/Ndel1 and P150 Glued interacting regions. T154 (pink bar, asterisk) is near the dimerization and LC7 ‐binding domains.

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: GSK‐3β Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility

    doi: 10.1111/tra.12304

    Figure Lengend Snippet: Identification of GSK ‐ 3β‐targeted residues in mouse IC‐2C . A–C) CID ‐based LC / MS / MS analyses of phosphorylation in a 237 amino acid, N‐terminal fragment of IC‐2C . A) An MS / MS spectrum of a phosphorylated peptide ( SVS * TPSEAGSQDSGDGAVGSR ) was acquired on a doubly charged ion at m/z 1015.9. The precursor mass (2030.82268 Da) is consistent with one phosphate in the peptide. MS / MS fragmentation of this peptide indicates a single phosphorylation of fragments y19 and b4, thus placing the modification unambiguously on S3 , corresponding to S88 in IC‐2C . Furthermore, MS / MS fragmentation eliminates all five C‐terminal S/T sites ( T4 , S6 , S10 , S13 and S20 ) by showing that y3–y18 fragments are not phosphorylated. B) Another MS / MS spectrum of a phosphorylated peptide ( SVST * PSEAGSQDSGDGAVGSR ) was acquired on a doubly charged ion at m/z 1015.9. MS / MS fragmentation of this peptide indicates a single phosphorylation of fragment y18 and b4 indicates that T4 , corresponding to T89 in IC‐2C , is phosphorylated. MS / MS fragmentation eliminates four C‐terminal S/T sites ( S6 , S10 , S13 and S20 ) because y3–y17 fragments have no phosphate. C) MS / MS spectrum of a phosphorylated peptide ( EDEEEEDDVAT * PKPPVEPEEEK ) was acquired on a triply charged ion at m/z 874.0. The precursor mass, 2620.07279 Da, indicates one phosphate in the peptide. The b11/y12 and b10/y11 fragments enable the conclusive assignment of T11 ( T154 in IC‐2C ) as the phosphorylated residue. D) S88 and T89 are conserved across mammalian species (mouse‐ MM , rat‐ RN , human‐ HS , bovine‐ BT and horse‐ EC ) and isoforms of IC ‐2, and both are found in mouse IC ‐1. T154 is not present in human or rat IC ‐2 or in mouse IC ‐1. E) Double site‐directed mutagenesis ( S88A and T89V ) in a 106 amino acid N‐terminal fragment of IC‐2C significantly reduced phosphorylation by GSK ‐3β in vitro . F) Full‐length mouse EGFP‐IC‐2C and two mutants ( T154V and S88A / T89V / T154V ) were expressed in Cos7 cells, immunopurified using an EGFP antibody and exposed to GSK ‐3β. Less phosphate was incorporated into the triple mutant, indicating that S88 / T89 sites are targeted in the full‐length protein. G) Schematic of IC showing previously identified interaction domains. S88 and T89 (black bar, asterisks) are near Nde1/Ndel1 and P150 Glued interacting regions. T154 (pink bar, asterisk) is near the dimerization and LC7 ‐binding domains.

    Article Snippet: The 3D10 GSK‐3β mouse mAb, 5B3 S9 Phospho‐GSK‐3β rabbit mAb, 11E7 Akt rabbit mAb, D25E6 T308 pAkt rabbit mAb and D9E S473 p‐Akt rabbit mAb were from Cell Signaling, Inc.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Modification, Mutagenesis, In Vitro, Binding Assay

    The interaction of dynein with the C-terminus of APC is modulated by GSK-3β activity and dynein phosphorylation. (A) A GSK-3β inhibitor CT99021 (CT) reduced the amount of endogenous APC that coprecipitated with endogenous dynein. (B) Similarly, less EGFP-cAPC coprecipitated with dynein from cells exposed to CT. (C) An EGFP antibody was used to pull down transiently expressed, EGFP-tagged dynein subunit IC1B. More endogenous APC coprecipitated with a WT construct than with an IC1B construct engineered with nonphosphorylatable S87A and T88V mutations in key GSK-3β sites, EGFP-IC1B (M). (D) Left, Coomassie-stained gel of histidine-tagged cAPC (cAPC) or purified bovine brain dynein (dynein). Both were exposed to purified GSK-3β in an in vitro kinase assay. Right, autoradiogram (autorad) shows γ- 32 P-ATP incorporation in several dynein subunits but not in cAPC. HC, heavy chain; IC, intermediate chain; LIC, light intermediate chain. (E) His-tagged cAPC preferentially coprecipitated with dynein that had been previously phosphorylated by GSK-3β. (F) Quantitation of three separate IPs, average ± 95% CI. Significance was determined by paired two-tailed Student’s t test, * p

    Journal: Molecular Biology of the Cell

    Article Title: Insulin signaling regulates a functional interaction between adenomatous polyposis coli and cytoplasmic dynein

    doi: 10.1091/mbc.E16-07-0555

    Figure Lengend Snippet: The interaction of dynein with the C-terminus of APC is modulated by GSK-3β activity and dynein phosphorylation. (A) A GSK-3β inhibitor CT99021 (CT) reduced the amount of endogenous APC that coprecipitated with endogenous dynein. (B) Similarly, less EGFP-cAPC coprecipitated with dynein from cells exposed to CT. (C) An EGFP antibody was used to pull down transiently expressed, EGFP-tagged dynein subunit IC1B. More endogenous APC coprecipitated with a WT construct than with an IC1B construct engineered with nonphosphorylatable S87A and T88V mutations in key GSK-3β sites, EGFP-IC1B (M). (D) Left, Coomassie-stained gel of histidine-tagged cAPC (cAPC) or purified bovine brain dynein (dynein). Both were exposed to purified GSK-3β in an in vitro kinase assay. Right, autoradiogram (autorad) shows γ- 32 P-ATP incorporation in several dynein subunits but not in cAPC. HC, heavy chain; IC, intermediate chain; LIC, light intermediate chain. (E) His-tagged cAPC preferentially coprecipitated with dynein that had been previously phosphorylated by GSK-3β. (F) Quantitation of three separate IPs, average ± 95% CI. Significance was determined by paired two-tailed Student’s t test, * p

    Article Snippet: Antibodies We used the following antibodies: CDK5RAP2 rabbit polyclonal antibody (pAb; Millipore), DIC mouse monoclonal antibody (mAb; 74.1; Santa Cruz Biotechnology), APC-M2 rabbit pAb (raised against the 15–amino acid repeat region; described in ), DIC mouse mAb (74.1; Santa Cruz Biotechnology), GSK-3β mouse mAb (3D10; Cell Signaling Technology), phospho–GSK-3β (Ser-9) rabbit pAb (5B3; Cell Signaling Technology), GFP rabbit pAb (Ab290; Abcam), His-probe rabbit pAb (H-3; Santa Cruz Biotechnology), PPARγ1 rabbit pAb (H100; Santa Cruz Biotechnology), detyro­sinated α-tubulin rabbit pAb (ab48389; Abcam), tyrosinated tubulin mouse mAb (TUB1A2; Sigma-Aldrich); acetylated tubulin mouse mAb (Clone 6-11-B1; Sigma-Aldrich); and α-tubulin mAb (T5168; Sigma-Aldrich).

    Techniques: Activity Assay, Construct, Staining, Purification, In Vitro, Kinase Assay, Quantitation Assay, Two Tailed Test

    The MIN isoform interferes with an interaction between dynein and APC. (A) Domains in full-length APC and MIN APC. olig, oligomerization domain; Arm (armadillo) repeats interact with AMER membrane-associated proteins; 15–amino acid repeats are β-catenin–binding domains; MCR, mutation cluster region; SAMP and 20–amino acid repeats are important for Wnt signaling; MT, a basic region that confers microtubule binding; EB1, EB1-binding domain; PDZ, binding domain for PDZ-containing proteins. (B) Dynein was immunoprecipitated from WT mouse brain extract using the DIC 74.1 antibody. Both APC and GSK-3β were present in the immunoprecipitate but not in the no-antibody control (No 1˚ antibody). FT, flowthrough, that is, material that did not bind in the IP. W, material that was released from the IP in the first wash. (C) DIC and GSK-3β also coimmunoprecipitated specifically with APC pulled down by the APC-M2 antibody. (D) The dynein IP was repeated using mouse IgG as a nonspecific antibody control. Endogenous APC and GSK-3β coprecipitated specifically with the DIC immunoprecipitate but not the IgG immunoprecipitate. (E) By Western blotting, MIN brain extracts contain ∼50% of the full-length APC in WT extracts, as quantified from four separate experiments. Significance was determined by paired two-tailed Student’s t test, ** p

    Journal: Molecular Biology of the Cell

    Article Title: Insulin signaling regulates a functional interaction between adenomatous polyposis coli and cytoplasmic dynein

    doi: 10.1091/mbc.E16-07-0555

    Figure Lengend Snippet: The MIN isoform interferes with an interaction between dynein and APC. (A) Domains in full-length APC and MIN APC. olig, oligomerization domain; Arm (armadillo) repeats interact with AMER membrane-associated proteins; 15–amino acid repeats are β-catenin–binding domains; MCR, mutation cluster region; SAMP and 20–amino acid repeats are important for Wnt signaling; MT, a basic region that confers microtubule binding; EB1, EB1-binding domain; PDZ, binding domain for PDZ-containing proteins. (B) Dynein was immunoprecipitated from WT mouse brain extract using the DIC 74.1 antibody. Both APC and GSK-3β were present in the immunoprecipitate but not in the no-antibody control (No 1˚ antibody). FT, flowthrough, that is, material that did not bind in the IP. W, material that was released from the IP in the first wash. (C) DIC and GSK-3β also coimmunoprecipitated specifically with APC pulled down by the APC-M2 antibody. (D) The dynein IP was repeated using mouse IgG as a nonspecific antibody control. Endogenous APC and GSK-3β coprecipitated specifically with the DIC immunoprecipitate but not the IgG immunoprecipitate. (E) By Western blotting, MIN brain extracts contain ∼50% of the full-length APC in WT extracts, as quantified from four separate experiments. Significance was determined by paired two-tailed Student’s t test, ** p

    Article Snippet: Antibodies We used the following antibodies: CDK5RAP2 rabbit polyclonal antibody (pAb; Millipore), DIC mouse monoclonal antibody (mAb; 74.1; Santa Cruz Biotechnology), APC-M2 rabbit pAb (raised against the 15–amino acid repeat region; described in ), DIC mouse mAb (74.1; Santa Cruz Biotechnology), GSK-3β mouse mAb (3D10; Cell Signaling Technology), phospho–GSK-3β (Ser-9) rabbit pAb (5B3; Cell Signaling Technology), GFP rabbit pAb (Ab290; Abcam), His-probe rabbit pAb (H-3; Santa Cruz Biotechnology), PPARγ1 rabbit pAb (H100; Santa Cruz Biotechnology), detyro­sinated α-tubulin rabbit pAb (ab48389; Abcam), tyrosinated tubulin mouse mAb (TUB1A2; Sigma-Aldrich); acetylated tubulin mouse mAb (Clone 6-11-B1; Sigma-Aldrich); and α-tubulin mAb (T5168; Sigma-Aldrich).

    Techniques: Binding Assay, Mutagenesis, Immunoprecipitation, Western Blot, Two Tailed Test

    MIN cells respond differently to acute insulin exposure or GSK-3β inhibition after 12 h in starvation medium. (A) Primers for genotyping APC +/MIN mice were used to determine that the cell line derived from APC +/min mouse colon (MIN), but not the cell line derived from WT mice, expresses the truncated MIN isoform of APC. Sequencing indicated that the MIN cells retain one copy of the WT APC gene (unpublished data). (B) The ratio of inactivated GSK-3β (GSK-3β-pS9) to total GSK-3β (pan–GSK-3β) for WT and MIN cells exposed to 0, 1, or 6 h of insulin was determined by Western blot. Phospho-S9 antibody band, red; pan–GSK-3β band, green. (C) Li-Cor densitometry analysis of three Western blots shows mean ± 95% CI. * p

    Journal: Molecular Biology of the Cell

    Article Title: Insulin signaling regulates a functional interaction between adenomatous polyposis coli and cytoplasmic dynein

    doi: 10.1091/mbc.E16-07-0555

    Figure Lengend Snippet: MIN cells respond differently to acute insulin exposure or GSK-3β inhibition after 12 h in starvation medium. (A) Primers for genotyping APC +/MIN mice were used to determine that the cell line derived from APC +/min mouse colon (MIN), but not the cell line derived from WT mice, expresses the truncated MIN isoform of APC. Sequencing indicated that the MIN cells retain one copy of the WT APC gene (unpublished data). (B) The ratio of inactivated GSK-3β (GSK-3β-pS9) to total GSK-3β (pan–GSK-3β) for WT and MIN cells exposed to 0, 1, or 6 h of insulin was determined by Western blot. Phospho-S9 antibody band, red; pan–GSK-3β band, green. (C) Li-Cor densitometry analysis of three Western blots shows mean ± 95% CI. * p

    Article Snippet: Antibodies We used the following antibodies: CDK5RAP2 rabbit polyclonal antibody (pAb; Millipore), DIC mouse monoclonal antibody (mAb; 74.1; Santa Cruz Biotechnology), APC-M2 rabbit pAb (raised against the 15–amino acid repeat region; described in ), DIC mouse mAb (74.1; Santa Cruz Biotechnology), GSK-3β mouse mAb (3D10; Cell Signaling Technology), phospho–GSK-3β (Ser-9) rabbit pAb (5B3; Cell Signaling Technology), GFP rabbit pAb (Ab290; Abcam), His-probe rabbit pAb (H-3; Santa Cruz Biotechnology), PPARγ1 rabbit pAb (H100; Santa Cruz Biotechnology), detyro­sinated α-tubulin rabbit pAb (ab48389; Abcam), tyrosinated tubulin mouse mAb (TUB1A2; Sigma-Aldrich); acetylated tubulin mouse mAb (Clone 6-11-B1; Sigma-Aldrich); and α-tubulin mAb (T5168; Sigma-Aldrich).

    Techniques: Inhibition, Mouse Assay, Derivative Assay, Sequencing, Western Blot

    Dynein is reduced at the cell periphery in WT cells. (A) A GSK-3β inhibitor, CT99021 (CT), prevents an activating tyrosine (auto) phosphorylation (Y216). (B) Acute GSK-3β inhibition with CT also increased CEI in WT but not MIN cells. Significance determined by ANOVA from four independent experiments, ∼500 cells/condition. * p

    Journal: Molecular Biology of the Cell

    Article Title: Insulin signaling regulates a functional interaction between adenomatous polyposis coli and cytoplasmic dynein

    doi: 10.1091/mbc.E16-07-0555

    Figure Lengend Snippet: Dynein is reduced at the cell periphery in WT cells. (A) A GSK-3β inhibitor, CT99021 (CT), prevents an activating tyrosine (auto) phosphorylation (Y216). (B) Acute GSK-3β inhibition with CT also increased CEI in WT but not MIN cells. Significance determined by ANOVA from four independent experiments, ∼500 cells/condition. * p

    Article Snippet: Antibodies We used the following antibodies: CDK5RAP2 rabbit polyclonal antibody (pAb; Millipore), DIC mouse monoclonal antibody (mAb; 74.1; Santa Cruz Biotechnology), APC-M2 rabbit pAb (raised against the 15–amino acid repeat region; described in ), DIC mouse mAb (74.1; Santa Cruz Biotechnology), GSK-3β mouse mAb (3D10; Cell Signaling Technology), phospho–GSK-3β (Ser-9) rabbit pAb (5B3; Cell Signaling Technology), GFP rabbit pAb (Ab290; Abcam), His-probe rabbit pAb (H-3; Santa Cruz Biotechnology), PPARγ1 rabbit pAb (H100; Santa Cruz Biotechnology), detyro­sinated α-tubulin rabbit pAb (ab48389; Abcam), tyrosinated tubulin mouse mAb (TUB1A2; Sigma-Aldrich); acetylated tubulin mouse mAb (Clone 6-11-B1; Sigma-Aldrich); and α-tubulin mAb (T5168; Sigma-Aldrich).

    Techniques: Inhibition

    Model for how APC mutations affect insulin-induced dynein movement toward MT minus ends. (A) WT cells in the absence of insulin or serum factors: GSK-3β (orange star) near the membrane (gray) is phosphorylated on Y216 and active. Nearby dynein motors are phosphorylated, rendering them more likely to bind to APC than to Ndel1. Here phosphorylated dynein (pDyn, red oval) is shown bound to an APC homodimer (purple ovals) at sites of MT (green line) capture at the plasma membrane. Unphosphorylated dynein (Dyn) may be bound to Ndel1 (yellow triangle) and actively translocating (arrow). (B) Insulin (dark orange circle) binds to receptors, activating a signaling pathway that locally and transiently inactivates GSK-3β by S9 phosphorylation. Dynein becomes locally and transiently dephosphorylated, is released from APC, binds to Ndel1, and then moves toward MT minus ends. The increase in number of processive dyneins results in minus-end accumulation at centrosomes (not shown). (C) MIN cells in the absence of insulin or serum factors: GSK-3β is active, but there is a reduced pool of dynein associated with FL-APC because MIN APC blocks the interaction. MIN APC also forms aggregates in the cytoplasm. (D) Normal inhibition of GSK3β occurs in response to insulin signaling but as the APC-bound, insulin-sensitive pool of dynein is reduced, the net effect of insulin on motor distribution is minimal.

    Journal: Molecular Biology of the Cell

    Article Title: Insulin signaling regulates a functional interaction between adenomatous polyposis coli and cytoplasmic dynein

    doi: 10.1091/mbc.E16-07-0555

    Figure Lengend Snippet: Model for how APC mutations affect insulin-induced dynein movement toward MT minus ends. (A) WT cells in the absence of insulin or serum factors: GSK-3β (orange star) near the membrane (gray) is phosphorylated on Y216 and active. Nearby dynein motors are phosphorylated, rendering them more likely to bind to APC than to Ndel1. Here phosphorylated dynein (pDyn, red oval) is shown bound to an APC homodimer (purple ovals) at sites of MT (green line) capture at the plasma membrane. Unphosphorylated dynein (Dyn) may be bound to Ndel1 (yellow triangle) and actively translocating (arrow). (B) Insulin (dark orange circle) binds to receptors, activating a signaling pathway that locally and transiently inactivates GSK-3β by S9 phosphorylation. Dynein becomes locally and transiently dephosphorylated, is released from APC, binds to Ndel1, and then moves toward MT minus ends. The increase in number of processive dyneins results in minus-end accumulation at centrosomes (not shown). (C) MIN cells in the absence of insulin or serum factors: GSK-3β is active, but there is a reduced pool of dynein associated with FL-APC because MIN APC blocks the interaction. MIN APC also forms aggregates in the cytoplasm. (D) Normal inhibition of GSK3β occurs in response to insulin signaling but as the APC-bound, insulin-sensitive pool of dynein is reduced, the net effect of insulin on motor distribution is minimal.

    Article Snippet: Antibodies We used the following antibodies: CDK5RAP2 rabbit polyclonal antibody (pAb; Millipore), DIC mouse monoclonal antibody (mAb; 74.1; Santa Cruz Biotechnology), APC-M2 rabbit pAb (raised against the 15–amino acid repeat region; described in ), DIC mouse mAb (74.1; Santa Cruz Biotechnology), GSK-3β mouse mAb (3D10; Cell Signaling Technology), phospho–GSK-3β (Ser-9) rabbit pAb (5B3; Cell Signaling Technology), GFP rabbit pAb (Ab290; Abcam), His-probe rabbit pAb (H-3; Santa Cruz Biotechnology), PPARγ1 rabbit pAb (H100; Santa Cruz Biotechnology), detyro­sinated α-tubulin rabbit pAb (ab48389; Abcam), tyrosinated tubulin mouse mAb (TUB1A2; Sigma-Aldrich); acetylated tubulin mouse mAb (Clone 6-11-B1; Sigma-Aldrich); and α-tubulin mAb (T5168; Sigma-Aldrich).

    Techniques: Inhibition

    The MIN mutation alters the dynein distribution response to rosiglitazone (ROZ) in vivo. (A, B) A 12-h exposure of starved cells to serum plus ITS and ROZ causes DIC (green) to accumulate at centrosomes in WT but not MIN cells. Treatment of WT or MIN cells with ROZ does not lead to an increase in PPARγ expression (top, Western blots). (C) ROZ-induced dynein accumulation is also defective if EGFP-nAPC isoform is transiently expressed in HCT116 cells. (D) Inhibition of GSK-3β is detected after 12 h of ROZ treatment in both WT and MIN cells. (E) Phospho-AKT immunoreactivity is observed at the cell periphery (arrows) in both cell types after ROZ treatment. (F) Colonic crypts from WT but not MIN mice showed an alteration in DIC distribution (red) after 6 d of gavage-administered rosiglitazone. Significant differences in B and C were determined by ANOVA from three separate experiments, 75 cells/treatment; *** p

    Journal: Molecular Biology of the Cell

    Article Title: Insulin signaling regulates a functional interaction between adenomatous polyposis coli and cytoplasmic dynein

    doi: 10.1091/mbc.E16-07-0555

    Figure Lengend Snippet: The MIN mutation alters the dynein distribution response to rosiglitazone (ROZ) in vivo. (A, B) A 12-h exposure of starved cells to serum plus ITS and ROZ causes DIC (green) to accumulate at centrosomes in WT but not MIN cells. Treatment of WT or MIN cells with ROZ does not lead to an increase in PPARγ expression (top, Western blots). (C) ROZ-induced dynein accumulation is also defective if EGFP-nAPC isoform is transiently expressed in HCT116 cells. (D) Inhibition of GSK-3β is detected after 12 h of ROZ treatment in both WT and MIN cells. (E) Phospho-AKT immunoreactivity is observed at the cell periphery (arrows) in both cell types after ROZ treatment. (F) Colonic crypts from WT but not MIN mice showed an alteration in DIC distribution (red) after 6 d of gavage-administered rosiglitazone. Significant differences in B and C were determined by ANOVA from three separate experiments, 75 cells/treatment; *** p

    Article Snippet: Antibodies We used the following antibodies: CDK5RAP2 rabbit polyclonal antibody (pAb; Millipore), DIC mouse monoclonal antibody (mAb; 74.1; Santa Cruz Biotechnology), APC-M2 rabbit pAb (raised against the 15–amino acid repeat region; described in ), DIC mouse mAb (74.1; Santa Cruz Biotechnology), GSK-3β mouse mAb (3D10; Cell Signaling Technology), phospho–GSK-3β (Ser-9) rabbit pAb (5B3; Cell Signaling Technology), GFP rabbit pAb (Ab290; Abcam), His-probe rabbit pAb (H-3; Santa Cruz Biotechnology), PPARγ1 rabbit pAb (H100; Santa Cruz Biotechnology), detyro­sinated α-tubulin rabbit pAb (ab48389; Abcam), tyrosinated tubulin mouse mAb (TUB1A2; Sigma-Aldrich); acetylated tubulin mouse mAb (Clone 6-11-B1; Sigma-Aldrich); and α-tubulin mAb (T5168; Sigma-Aldrich).

    Techniques: Mutagenesis, In Vivo, Expressing, Western Blot, Inhibition, Mouse Assay

    Distribution of GSK-3β immunofluorescence in adult DG of control and PAE mice Confocal images through DG of dorsal hippocampus from control (A) and PAE (B) mice. GSK-3β immunofluorescence is depicted in green, whereas tdTomato fluorescence in adult-generated DGCs is depicted in red. Cell nuclei are depicted in blue (DAPI nuclear stain). tdTomato+ mossy fibers can also be seen in hilar region at this magnification. Boxed areas in A and B are shown at higher magnification in A 1 and B 1 , respectively. Intense GSK-3β expression is observed in scattered cells throughout the hilus, SGZ and granule cell layer (arrows) with little expression apparent within tdTomato + adult-generated DGCs. Note that the overall distribution of GSK-3β appears unchanged by PAE, whereas the number of adult-generated DGCs appears less in PAE mice. Abbreviations: GCL, dentate granule cell layer; SGZ, subgranular zone; H, hilus.

    Journal: Alcoholism, clinical and experimental research

    Article Title: Prenatal alcohol exposure leads to enhanced serine 9 phosphorylation of glycogen synthase kinase-3β (GSK-3β) in the hippocampal dentate gyrus of adult mouse

    doi: 10.1111/acer.13489

    Figure Lengend Snippet: Distribution of GSK-3β immunofluorescence in adult DG of control and PAE mice Confocal images through DG of dorsal hippocampus from control (A) and PAE (B) mice. GSK-3β immunofluorescence is depicted in green, whereas tdTomato fluorescence in adult-generated DGCs is depicted in red. Cell nuclei are depicted in blue (DAPI nuclear stain). tdTomato+ mossy fibers can also be seen in hilar region at this magnification. Boxed areas in A and B are shown at higher magnification in A 1 and B 1 , respectively. Intense GSK-3β expression is observed in scattered cells throughout the hilus, SGZ and granule cell layer (arrows) with little expression apparent within tdTomato + adult-generated DGCs. Note that the overall distribution of GSK-3β appears unchanged by PAE, whereas the number of adult-generated DGCs appears less in PAE mice. Abbreviations: GCL, dentate granule cell layer; SGZ, subgranular zone; H, hilus.

    Article Snippet: The following primary antibodies were used in this study: Anti-GSK-3β (3D10) mouse mAB (1:1000 dilution; #9832, Cell Signaling, Danvers, MA), Anti-phospho-GSK-3β (Ser9) (D85E12) XP rabbit mAb (1:1000 dilution; #5558, Cell Signaling, Danvers, MA).

    Techniques: Immunofluorescence, Mouse Assay, Fluorescence, Generated, Staining, Expressing

    Hippocampal expression pattern of GSK-3β Montage confocal image of coronal section from tamoxifen-treated nestin-CreER T2 :tdTomato mouse immunofluorescently labeled for GSK-3β (green). Adult-generated DGCs are tdTomato + (red). Note pattern of intense GSK-3β expression within hippocampal CA pyramidal cell layers, within processes of stratum radiatum and stratum lacunosum-moleculare, and within interneurons scattered throughout all regions of hippocampus. Intense GSK-3β immunofluorescence is also observed within pyramidal neurons of the cerebral cortex. GSK-3β immunofluorescence was only dimly visable within the granule cell layer of the dentate gyrus. Note the presence of tdTomato+ mossy fibers from adult-generated DGCs within the hilar/CA3 regions of DG. Abbreviations: DG, dentate gyrus; O, stratum oriens; P, CA pyramidal layer; R, stratum radiatum; L-M, stratum lacunosum-moleculare.

    Journal: Alcoholism, clinical and experimental research

    Article Title: Prenatal alcohol exposure leads to enhanced serine 9 phosphorylation of glycogen synthase kinase-3β (GSK-3β) in the hippocampal dentate gyrus of adult mouse

    doi: 10.1111/acer.13489

    Figure Lengend Snippet: Hippocampal expression pattern of GSK-3β Montage confocal image of coronal section from tamoxifen-treated nestin-CreER T2 :tdTomato mouse immunofluorescently labeled for GSK-3β (green). Adult-generated DGCs are tdTomato + (red). Note pattern of intense GSK-3β expression within hippocampal CA pyramidal cell layers, within processes of stratum radiatum and stratum lacunosum-moleculare, and within interneurons scattered throughout all regions of hippocampus. Intense GSK-3β immunofluorescence is also observed within pyramidal neurons of the cerebral cortex. GSK-3β immunofluorescence was only dimly visable within the granule cell layer of the dentate gyrus. Note the presence of tdTomato+ mossy fibers from adult-generated DGCs within the hilar/CA3 regions of DG. Abbreviations: DG, dentate gyrus; O, stratum oriens; P, CA pyramidal layer; R, stratum radiatum; L-M, stratum lacunosum-moleculare.

    Article Snippet: The following primary antibodies were used in this study: Anti-GSK-3β (3D10) mouse mAB (1:1000 dilution; #9832, Cell Signaling, Danvers, MA), Anti-phospho-GSK-3β (Ser9) (D85E12) XP rabbit mAb (1:1000 dilution; #5558, Cell Signaling, Danvers, MA).

    Techniques: Expressing, Labeling, Generated, Immunofluorescence

    Increased p(Ser9) GSK-3β in adult hippocampal dentate gyrus from PAE mice Comparison of total GSK-3β (A) and p(Ser9)GSK-3β (B) as assayed by Western immunoblot analysis of DG homogenates from control vs. PAE mice. Representative immunoblot obtained from 3 separate mice per treatment group is depicted below each graph. (C) Ratio of p(Ser9)GSK-3β/total GSK-3β in control vs. PAE adult DG homogenates. *p=0.04, **p=0.003. Pups from individual litters are color coded for each treatment group (n=11 PAE across 5 litters; n=10 SAC across 4 litters).

    Journal: Alcoholism, clinical and experimental research

    Article Title: Prenatal alcohol exposure leads to enhanced serine 9 phosphorylation of glycogen synthase kinase-3β (GSK-3β) in the hippocampal dentate gyrus of adult mouse

    doi: 10.1111/acer.13489

    Figure Lengend Snippet: Increased p(Ser9) GSK-3β in adult hippocampal dentate gyrus from PAE mice Comparison of total GSK-3β (A) and p(Ser9)GSK-3β (B) as assayed by Western immunoblot analysis of DG homogenates from control vs. PAE mice. Representative immunoblot obtained from 3 separate mice per treatment group is depicted below each graph. (C) Ratio of p(Ser9)GSK-3β/total GSK-3β in control vs. PAE adult DG homogenates. *p=0.04, **p=0.003. Pups from individual litters are color coded for each treatment group (n=11 PAE across 5 litters; n=10 SAC across 4 litters).

    Article Snippet: The following primary antibodies were used in this study: Anti-GSK-3β (3D10) mouse mAB (1:1000 dilution; #9832, Cell Signaling, Danvers, MA), Anti-phospho-GSK-3β (Ser9) (D85E12) XP rabbit mAb (1:1000 dilution; #5558, Cell Signaling, Danvers, MA).

    Techniques: Mouse Assay, Western Blot

    GSK-3β is expressed within mossy cells a subset of DG hilar interneurons Confocal image through DG of dorsal hippocampus from adult naïve VGAT-Venus mouse. GSK-3β immunofluorescence is depicted in red, whereas GABAergic inhibitory interneurons are depicted by green (Venus reporter expression under transcriptional control of endogenous vesicular GABA transporter (VGAT) promotor sequences). Higher power images of boxed area are depicted in lower panels. Note that intense GSK-3β immunofluorescence is observed within CA4 pyramidal neurons (CA4, upper panel), and in scattered cells throughout all regions of the dentate gyrus. Lower panels demonstrate GSK-3β expression in mossy cell interneurons (large Venus − neurons indicated by filled arrowheads) and within a subset of Venus + GABAergic interneurons within the hilus (hatched arrowheads). Not all Venus + GABAergic interneurons express GSK3β (open arrowhead depicts Venus + /GSK3β − interneuron). Less intense, but detectable, GSK-3β immunoreactivity is also observed in granule neurons throughout the dentate granule cell layer (DG).

    Journal: Alcoholism, clinical and experimental research

    Article Title: Prenatal alcohol exposure leads to enhanced serine 9 phosphorylation of glycogen synthase kinase-3β (GSK-3β) in the hippocampal dentate gyrus of adult mouse

    doi: 10.1111/acer.13489

    Figure Lengend Snippet: GSK-3β is expressed within mossy cells a subset of DG hilar interneurons Confocal image through DG of dorsal hippocampus from adult naïve VGAT-Venus mouse. GSK-3β immunofluorescence is depicted in red, whereas GABAergic inhibitory interneurons are depicted by green (Venus reporter expression under transcriptional control of endogenous vesicular GABA transporter (VGAT) promotor sequences). Higher power images of boxed area are depicted in lower panels. Note that intense GSK-3β immunofluorescence is observed within CA4 pyramidal neurons (CA4, upper panel), and in scattered cells throughout all regions of the dentate gyrus. Lower panels demonstrate GSK-3β expression in mossy cell interneurons (large Venus − neurons indicated by filled arrowheads) and within a subset of Venus + GABAergic interneurons within the hilus (hatched arrowheads). Not all Venus + GABAergic interneurons express GSK3β (open arrowhead depicts Venus + /GSK3β − interneuron). Less intense, but detectable, GSK-3β immunoreactivity is also observed in granule neurons throughout the dentate granule cell layer (DG).

    Article Snippet: The following primary antibodies were used in this study: Anti-GSK-3β (3D10) mouse mAB (1:1000 dilution; #9832, Cell Signaling, Danvers, MA), Anti-phospho-GSK-3β (Ser9) (D85E12) XP rabbit mAb (1:1000 dilution; #5558, Cell Signaling, Danvers, MA).

    Techniques: Immunofluorescence, Expressing

    14-3-3σ interacts with GSK-3β. (A) CCE, R1 and D3 cells transfected with control or 14-3-3σ vectors were lysed and 14-3-3σ complex was pulled down by immunoprecipitation (IP) with a Flag antibody. Proteins in the complex were analyzed by Western blotting (WB). (B) CCE cells were transfected with each isoform vector. 14-3-3 complex was pulled down and GSK-3β was determined by Western blotting. (C) Association of 14-3-3σ with WT or mutant GSK-3β was determined by immunoprecipitation (IP) with a Flag Antibody followed by Western blotting (WB) with GSK-3β and p-GSK-3β antibodies. (D) CCE cells transfected with Flag-tagged 14-3-3σ (σ) or control (CTR) vectors were treated with wortmannin. 14-3-3σ complex was immunoprecipitated with a Flag antibody and GSK-3β was analyzed by Western blotting. (E) 14-3-3σ overexpression reduced GSK-3β activity. Data represents mean ± s.d. (n = 3). *, p

    Journal: PLoS ONE

    Article Title: 14-3-3? Regulates ?-Catenin-Mediated Mouse Embryonic Stem Cell Proliferation by Sequestering GSK-3?

    doi: 10.1371/journal.pone.0040193

    Figure Lengend Snippet: 14-3-3σ interacts with GSK-3β. (A) CCE, R1 and D3 cells transfected with control or 14-3-3σ vectors were lysed and 14-3-3σ complex was pulled down by immunoprecipitation (IP) with a Flag antibody. Proteins in the complex were analyzed by Western blotting (WB). (B) CCE cells were transfected with each isoform vector. 14-3-3 complex was pulled down and GSK-3β was determined by Western blotting. (C) Association of 14-3-3σ with WT or mutant GSK-3β was determined by immunoprecipitation (IP) with a Flag Antibody followed by Western blotting (WB) with GSK-3β and p-GSK-3β antibodies. (D) CCE cells transfected with Flag-tagged 14-3-3σ (σ) or control (CTR) vectors were treated with wortmannin. 14-3-3σ complex was immunoprecipitated with a Flag antibody and GSK-3β was analyzed by Western blotting. (E) 14-3-3σ overexpression reduced GSK-3β activity. Data represents mean ± s.d. (n = 3). *, p

    Article Snippet: Rabbit polyclonal antibodies against β-catenin, phosphor-β-catenin, GSK-3β, phospho-GSK-3β (Ser9), Akt, phospho-Akt, APC, axin, ubiquitin and cyclin D1 were from Cell Signaling.

    Techniques: Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Mutagenesis, Over Expression, Activity Assay

    14-3-3σ reduces β-catenin association with the APC/axin/GSK-3β complex and increases β-catenin. (A) Association of HA-tagged (left panels) and endogenous (right panels) β-catenin with the APC complex was determined by IP with HA antibodies and Western blotting (WB) with the indicated antibodies. (B) CCE cells were transfected with GSK-3β WT or the indicated mutants and β-catenin, GSK-3β, APC and Axin proteins were analyzed by Western blotting. (C) CCE cells with or without 14-3-3σ overexpression were treated with RA. The indicated GSK-3β, β-catenin and Oct3/4 proteins were analyzed by Western blotting. (D) Cells were treated with or without RA for 48 h, the association of endogenous 14-3-3σ with GSK-3β was determined by IP with GSK-3β antibody and Western blotting of 14-3-3σ.

    Journal: PLoS ONE

    Article Title: 14-3-3? Regulates ?-Catenin-Mediated Mouse Embryonic Stem Cell Proliferation by Sequestering GSK-3?

    doi: 10.1371/journal.pone.0040193

    Figure Lengend Snippet: 14-3-3σ reduces β-catenin association with the APC/axin/GSK-3β complex and increases β-catenin. (A) Association of HA-tagged (left panels) and endogenous (right panels) β-catenin with the APC complex was determined by IP with HA antibodies and Western blotting (WB) with the indicated antibodies. (B) CCE cells were transfected with GSK-3β WT or the indicated mutants and β-catenin, GSK-3β, APC and Axin proteins were analyzed by Western blotting. (C) CCE cells with or without 14-3-3σ overexpression were treated with RA. The indicated GSK-3β, β-catenin and Oct3/4 proteins were analyzed by Western blotting. (D) Cells were treated with or without RA for 48 h, the association of endogenous 14-3-3σ with GSK-3β was determined by IP with GSK-3β antibody and Western blotting of 14-3-3σ.

    Article Snippet: Rabbit polyclonal antibodies against β-catenin, phosphor-β-catenin, GSK-3β, phospho-GSK-3β (Ser9), Akt, phospho-Akt, APC, axin, ubiquitin and cyclin D1 were from Cell Signaling.

    Techniques: Western Blot, Transfection, Over Expression

    14-3-3σ enhances Wnt/β-catenin signaling. (A) CCE cells were transfected with control or 14-3-3σ vectors for 30 h followed by treatment with recombinant mouse Wnt3a (10 ng/mL) for 18 h. Recombinant mouse DKK-1 (1 ng/mL) was added 2 h before Wnt3a. Protein levels of β-catenin, GSK-3β and phosphorylated GSK-3β were detected by Western blot analysis. (B) 14-3-3σ/GSK-3β complex was determined by IP with a GSK-3β antibody, and Flag and GSK-3β proteins were analyzed by Western blotting.

    Journal: PLoS ONE

    Article Title: 14-3-3? Regulates ?-Catenin-Mediated Mouse Embryonic Stem Cell Proliferation by Sequestering GSK-3?

    doi: 10.1371/journal.pone.0040193

    Figure Lengend Snippet: 14-3-3σ enhances Wnt/β-catenin signaling. (A) CCE cells were transfected with control or 14-3-3σ vectors for 30 h followed by treatment with recombinant mouse Wnt3a (10 ng/mL) for 18 h. Recombinant mouse DKK-1 (1 ng/mL) was added 2 h before Wnt3a. Protein levels of β-catenin, GSK-3β and phosphorylated GSK-3β were detected by Western blot analysis. (B) 14-3-3σ/GSK-3β complex was determined by IP with a GSK-3β antibody, and Flag and GSK-3β proteins were analyzed by Western blotting.

    Article Snippet: Rabbit polyclonal antibodies against β-catenin, phosphor-β-catenin, GSK-3β, phospho-GSK-3β (Ser9), Akt, phospho-Akt, APC, axin, ubiquitin and cyclin D1 were from Cell Signaling.

    Techniques: Transfection, Recombinant, Western Blot

    A schematic model illustrating the role of 14-3-3σ in regulating ES cell proliferation via GSK-3β/β-catenin pathway.

    Journal: PLoS ONE

    Article Title: 14-3-3? Regulates ?-Catenin-Mediated Mouse Embryonic Stem Cell Proliferation by Sequestering GSK-3?

    doi: 10.1371/journal.pone.0040193

    Figure Lengend Snippet: A schematic model illustrating the role of 14-3-3σ in regulating ES cell proliferation via GSK-3β/β-catenin pathway.

    Article Snippet: Rabbit polyclonal antibodies against β-catenin, phosphor-β-catenin, GSK-3β, phospho-GSK-3β (Ser9), Akt, phospho-Akt, APC, axin, ubiquitin and cyclin D1 were from Cell Signaling.

    Techniques:

    sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Expressing

    Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Immunostaining

    GSK-3β/Rac-1 pathway involved in sFRP-1-induced EC spreading. a: HUVECs were either infected with adenovirus expressing β-galactosidase, GSK-3β, GSK-3β-S9A, or GSK-3β-KM or treated with SB216763. After the treatment,

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: GSK-3β/Rac-1 pathway involved in sFRP-1-induced EC spreading. a: HUVECs were either infected with adenovirus expressing β-galactosidase, GSK-3β, GSK-3β-S9A, or GSK-3β-KM or treated with SB216763. After the treatment,

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Infection, Expressing

    Effects of NO donor on IRS-2 expression in mouse islets and IRS-2 protein expression in islets from diabetic mice. A and B , treatment with GSNO (400 μ m ) for 5 h resulted in decreases in IRS-2 protein expression and phosphorylation of GSK-3β

    Journal: The Journal of Biological Chemistry

    Article Title: Inducible Nitric-oxide Synthase and Nitric Oxide Donor Decrease Insulin Receptor Substrate-2 Protein Expression by Promoting Proteasome-dependent Degradation in Pancreatic ?-Cells

    doi: 10.1074/jbc.M110.192732

    Figure Lengend Snippet: Effects of NO donor on IRS-2 expression in mouse islets and IRS-2 protein expression in islets from diabetic mice. A and B , treatment with GSNO (400 μ m ) for 5 h resulted in decreases in IRS-2 protein expression and phosphorylation of GSK-3β

    Article Snippet: S -Nitroso- l -glutathione (GSNO), S -nitroso- N -acetyl- dl -penicillamine (SNAP), l -NIL, and carboxy-PTIO (Cayman Chemical, Ann Arbor, MI); MG132, reduced glutathione (GSH), and oxidized glutathione (GSSG) (Sigma); cycloheximide (Calbiochem); lactacystin (Boston Biochem, Cambridge, MA); SB216763 and SB415268 (Tocris Bioscience, Ellisville, MO); recombinant mouse IFN-γ (R & D Systems, Minneapolis, MN); mouse IL-1β (Roche Applied Science); human tumor necrosis factor-α (TNF-α; Cell Signaling, Beverly, MA); anti-IRS-2, anti-iNOS, and anti-phosphotyrosine (Millipore, Billerica, MA); anti-ubiquitin (Affiniti Research Products, Golden, CO); anti-phospho-glycogen synthase (Novus Biologicals, Littleton, CO); anti-GSK-3β (BD Transduction Laboratories); anti-IRS-2 (EMD Chemicals, Gibbstown, NJ); anti-GAPDH (Trevigen, Gaithersburg, MD); anti-Akt, anti-phospho-Akt (Ser-473); and anti-phospho-GSK-3β (Ser-9), anti-glycogen synthase, anti-c-Jun, anti-phospho-c-Jun (Ser-63), anti-p70 S6 kinase, and anti-phospho-p70 S6 kinase (Thr-389) antibodies (Cell Signaling) were purchased commercially. siRNA oligonucleotides for rat iNOS and GSK-3β were purchased from Invitrogen (iNOS: RSS302394, RSS302395, and RSS351448; GSK-3β: r(CGAUUACACGUCUAGUAUA)dTdT (sense) and r(UAUACUAGACGUGUAAUCG)dTdT (antisense)).

    Techniques: Expressing, Mouse Assay

    The effects of inhibitors for GSK-3β, JNK/SAPK, and mTOR on IRS-2 protein expression in NO donor-treated β-cells. INS-1/832 cells were treated with and without GSNO (200 μ m ) for 5 h in the presence and absence of inhibitors for

    Journal: The Journal of Biological Chemistry

    Article Title: Inducible Nitric-oxide Synthase and Nitric Oxide Donor Decrease Insulin Receptor Substrate-2 Protein Expression by Promoting Proteasome-dependent Degradation in Pancreatic ?-Cells

    doi: 10.1074/jbc.M110.192732

    Figure Lengend Snippet: The effects of inhibitors for GSK-3β, JNK/SAPK, and mTOR on IRS-2 protein expression in NO donor-treated β-cells. INS-1/832 cells were treated with and without GSNO (200 μ m ) for 5 h in the presence and absence of inhibitors for

    Article Snippet: S -Nitroso- l -glutathione (GSNO), S -nitroso- N -acetyl- dl -penicillamine (SNAP), l -NIL, and carboxy-PTIO (Cayman Chemical, Ann Arbor, MI); MG132, reduced glutathione (GSH), and oxidized glutathione (GSSG) (Sigma); cycloheximide (Calbiochem); lactacystin (Boston Biochem, Cambridge, MA); SB216763 and SB415268 (Tocris Bioscience, Ellisville, MO); recombinant mouse IFN-γ (R & D Systems, Minneapolis, MN); mouse IL-1β (Roche Applied Science); human tumor necrosis factor-α (TNF-α; Cell Signaling, Beverly, MA); anti-IRS-2, anti-iNOS, and anti-phosphotyrosine (Millipore, Billerica, MA); anti-ubiquitin (Affiniti Research Products, Golden, CO); anti-phospho-glycogen synthase (Novus Biologicals, Littleton, CO); anti-GSK-3β (BD Transduction Laboratories); anti-IRS-2 (EMD Chemicals, Gibbstown, NJ); anti-GAPDH (Trevigen, Gaithersburg, MD); anti-Akt, anti-phospho-Akt (Ser-473); and anti-phospho-GSK-3β (Ser-9), anti-glycogen synthase, anti-c-Jun, anti-phospho-c-Jun (Ser-63), anti-p70 S6 kinase, and anti-phospho-p70 S6 kinase (Thr-389) antibodies (Cell Signaling) were purchased commercially. siRNA oligonucleotides for rat iNOS and GSK-3β were purchased from Invitrogen (iNOS: RSS302394, RSS302395, and RSS351448; GSK-3β: r(CGAUUACACGUCUAGUAUA)dTdT (sense) and r(UAUACUAGACGUGUAAUCG)dTdT (antisense)).

    Techniques: Expressing

    Inhibition of GSK-3β significantly blocked NO donor-induced decrease in IRS-2 expression in β-cells. We evaluated the effects of GSK-3β inhibitors, SB216763 and SB415268, and siRNA-mediated knockdown of GSK-3β on IRS-2

    Journal: The Journal of Biological Chemistry

    Article Title: Inducible Nitric-oxide Synthase and Nitric Oxide Donor Decrease Insulin Receptor Substrate-2 Protein Expression by Promoting Proteasome-dependent Degradation in Pancreatic ?-Cells

    doi: 10.1074/jbc.M110.192732

    Figure Lengend Snippet: Inhibition of GSK-3β significantly blocked NO donor-induced decrease in IRS-2 expression in β-cells. We evaluated the effects of GSK-3β inhibitors, SB216763 and SB415268, and siRNA-mediated knockdown of GSK-3β on IRS-2

    Article Snippet: S -Nitroso- l -glutathione (GSNO), S -nitroso- N -acetyl- dl -penicillamine (SNAP), l -NIL, and carboxy-PTIO (Cayman Chemical, Ann Arbor, MI); MG132, reduced glutathione (GSH), and oxidized glutathione (GSSG) (Sigma); cycloheximide (Calbiochem); lactacystin (Boston Biochem, Cambridge, MA); SB216763 and SB415268 (Tocris Bioscience, Ellisville, MO); recombinant mouse IFN-γ (R & D Systems, Minneapolis, MN); mouse IL-1β (Roche Applied Science); human tumor necrosis factor-α (TNF-α; Cell Signaling, Beverly, MA); anti-IRS-2, anti-iNOS, and anti-phosphotyrosine (Millipore, Billerica, MA); anti-ubiquitin (Affiniti Research Products, Golden, CO); anti-phospho-glycogen synthase (Novus Biologicals, Littleton, CO); anti-GSK-3β (BD Transduction Laboratories); anti-IRS-2 (EMD Chemicals, Gibbstown, NJ); anti-GAPDH (Trevigen, Gaithersburg, MD); anti-Akt, anti-phospho-Akt (Ser-473); and anti-phospho-GSK-3β (Ser-9), anti-glycogen synthase, anti-c-Jun, anti-phospho-c-Jun (Ser-63), anti-p70 S6 kinase, and anti-phospho-p70 S6 kinase (Thr-389) antibodies (Cell Signaling) were purchased commercially. siRNA oligonucleotides for rat iNOS and GSK-3β were purchased from Invitrogen (iNOS: RSS302394, RSS302395, and RSS351448; GSK-3β: r(CGAUUACACGUCUAGUAUA)dTdT (sense) and r(UAUACUAGACGUGUAAUCG)dTdT (antisense)).

    Techniques: Inhibition, Expressing

    Proposed mechanism of neuroprotective action of MMP-9 in preventing Aβ-mediated impairment of the insulin survival pathway. Aβ oligomers generated during early stages of AD stimulate pro-apoptotic pathways, such as TNFR signaling, which activates JNK kinase, resulting in neuronal insulin resistance via IRS1-Ser636 phosphorylation and GSK-3β activation. MMP-9 protects, in part, by reducing Aβ amyloid peptide accumulation, thus blocking Aβ oligomeric-induced defects in insulin signaling. MMP-9 also provides an additional stimulus to insulin, by increasing BDNF levels. The latter binds to the TrkB receptor, inducing IRS1-Tyr465 phosphorylation and Akt activation, hence contributing to normal neuronal function.

    Journal: Scientific Reports

    Article Title: Overexpression of matrix metalloproteinase-9 (MMP-9) rescues insulin-mediated impairment in the 5XFAD model of Alzheimer’s disease

    doi: 10.1038/s41598-017-00794-5

    Figure Lengend Snippet: Proposed mechanism of neuroprotective action of MMP-9 in preventing Aβ-mediated impairment of the insulin survival pathway. Aβ oligomers generated during early stages of AD stimulate pro-apoptotic pathways, such as TNFR signaling, which activates JNK kinase, resulting in neuronal insulin resistance via IRS1-Ser636 phosphorylation and GSK-3β activation. MMP-9 protects, in part, by reducing Aβ amyloid peptide accumulation, thus blocking Aβ oligomeric-induced defects in insulin signaling. MMP-9 also provides an additional stimulus to insulin, by increasing BDNF levels. The latter binds to the TrkB receptor, inducing IRS1-Tyr465 phosphorylation and Akt activation, hence contributing to normal neuronal function.

    Article Snippet: Membranes were blocked with 5% non-fat milk, 0.1% Tween-20 in TBS for 1 hour at room temperature and incubated overnight at 4 °C with primary antibodies, anti-pIRS1-Ser636, anti-IRS1, anti-IR, anti-BDNF in dilution 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pAkt, anti-Akt, anti-pGSK-3β, anti-GSK-3β, anti-pJNK, anti-JNK in dilution 1:1000 (Cell signalling Technology, Danvers, MA, USA) and anti-6E10 in dilution 1:1000 (BioLegend, San Diego, CA, USA), according to manufacturers’ instructions.

    Techniques: Generated, Activation Assay, Blocking Assay

    Representative Western blots of protein samples from primary hippocampal cell lysates ( A , C ) and hippocampal homogenates from 3 month-old mice ( B , D ) of WT, 5XFAD, 5XFAD/TgMMP-9 and TgMMP-9 mice. Equal amounts of total protein were analysed on 7.5% SDS-PAGE gels and immunoblotted with primary antibodies against pAkt and Akt ( A , B ), pGSK-3β and GSK-3β ( C , D ). To ensure equal loading, membranes were re-probed against β-Actin. Graphs depict densitometric quantification of phosphorylated proteins normalized to their respective total protein levels. n = 15–20 ( A ), n = 8–11 ( C ), n = 6–15 ( B ), n = 6–14 ( D ). All data are presented as mean ± SEM (*p

    Journal: Scientific Reports

    Article Title: Overexpression of matrix metalloproteinase-9 (MMP-9) rescues insulin-mediated impairment in the 5XFAD model of Alzheimer’s disease

    doi: 10.1038/s41598-017-00794-5

    Figure Lengend Snippet: Representative Western blots of protein samples from primary hippocampal cell lysates ( A , C ) and hippocampal homogenates from 3 month-old mice ( B , D ) of WT, 5XFAD, 5XFAD/TgMMP-9 and TgMMP-9 mice. Equal amounts of total protein were analysed on 7.5% SDS-PAGE gels and immunoblotted with primary antibodies against pAkt and Akt ( A , B ), pGSK-3β and GSK-3β ( C , D ). To ensure equal loading, membranes were re-probed against β-Actin. Graphs depict densitometric quantification of phosphorylated proteins normalized to their respective total protein levels. n = 15–20 ( A ), n = 8–11 ( C ), n = 6–15 ( B ), n = 6–14 ( D ). All data are presented as mean ± SEM (*p

    Article Snippet: Membranes were blocked with 5% non-fat milk, 0.1% Tween-20 in TBS for 1 hour at room temperature and incubated overnight at 4 °C with primary antibodies, anti-pIRS1-Ser636, anti-IRS1, anti-IR, anti-BDNF in dilution 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pAkt, anti-Akt, anti-pGSK-3β, anti-GSK-3β, anti-pJNK, anti-JNK in dilution 1:1000 (Cell signalling Technology, Danvers, MA, USA) and anti-6E10 in dilution 1:1000 (BioLegend, San Diego, CA, USA), according to manufacturers’ instructions.

    Techniques: Western Blot, Mouse Assay, SDS Page

    Primary hippocampal cultures of 5XFAD mice were treated with 100 ng/ml of recombinant BDNF for 30 min. ( A ) Cells labelled against pIRS1-Tyr465 antibody are depicted in red and cell nuclei labelled with DAPI are depicted in blue. Graph depicts quantification by the integrated density of total pIRS1-Tyr465. Magnification 63x, Scale bar: 20 μm. ( B ) Equal amounts of total protein from treated cells were analyzed on 10% SDS-PAGE gels and immunoblotted with primary antibodies against pGSK-3β and GSK-3β. To ensure equal loading, membranes were re-probed against β-Actin. Graphs depict densitometric quantification of phosphorylated proteins normalized to their respective total protein levels. n = 3 ( A ) and n = 4 ( B ). All data are presented as mean ± SEM (*p

    Journal: Scientific Reports

    Article Title: Overexpression of matrix metalloproteinase-9 (MMP-9) rescues insulin-mediated impairment in the 5XFAD model of Alzheimer’s disease

    doi: 10.1038/s41598-017-00794-5

    Figure Lengend Snippet: Primary hippocampal cultures of 5XFAD mice were treated with 100 ng/ml of recombinant BDNF for 30 min. ( A ) Cells labelled against pIRS1-Tyr465 antibody are depicted in red and cell nuclei labelled with DAPI are depicted in blue. Graph depicts quantification by the integrated density of total pIRS1-Tyr465. Magnification 63x, Scale bar: 20 μm. ( B ) Equal amounts of total protein from treated cells were analyzed on 10% SDS-PAGE gels and immunoblotted with primary antibodies against pGSK-3β and GSK-3β. To ensure equal loading, membranes were re-probed against β-Actin. Graphs depict densitometric quantification of phosphorylated proteins normalized to their respective total protein levels. n = 3 ( A ) and n = 4 ( B ). All data are presented as mean ± SEM (*p

    Article Snippet: Membranes were blocked with 5% non-fat milk, 0.1% Tween-20 in TBS for 1 hour at room temperature and incubated overnight at 4 °C with primary antibodies, anti-pIRS1-Ser636, anti-IRS1, anti-IR, anti-BDNF in dilution 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pAkt, anti-Akt, anti-pGSK-3β, anti-GSK-3β, anti-pJNK, anti-JNK in dilution 1:1000 (Cell signalling Technology, Danvers, MA, USA) and anti-6E10 in dilution 1:1000 (BioLegend, San Diego, CA, USA), according to manufacturers’ instructions.

    Techniques: Mouse Assay, Recombinant, SDS Page

    Generation of GSK-3α fx mice, confirmation and characterization of GSK-3α cko mice Conditional KO GSK-3a mice generation. (A) Restriction maps (from top to bottom) of the genomic locus, targeting vector, homologous alleles, conditional knock out allele, and a constitutive KO allele. After proper homologous recombination, exons 2–4 of targeted allele were flanked by loxP sites with the addition of a neomycin (neo) resistant marker flanked by FRT sites and in the absence of Zs green. Non-fluorescent selected clones were analyzed by Southern blot. Probes (blue rectangles) were located at the 5′ and 3′ ends and in the neo fragment. Genomic DNA was digested with either AflIII for the 5′ (B) and Neo probe (D), or KpnI for the 3′ probe (C). All four clones were positive for proper homologous recombination and ES clone BC-09 was selected. After Flp-mediated deletion, a conditional knock out (KO) allele was generated without Neo through mating with a Cre-deleter mouse line. Using this Cre-loxP ystem, wt or cko genotypes (n=10–15 mice/group) were generated (GSK-3α wt, GSK-3α cko, GSK-3α wt;PDAPP and GSK-3α cko;PDAPP) and analyzed at 17 moths of age. GSK-3α cko was confirmed by IHC using GSK-3αβ-specific antibodies. (A) Representative images counter-stained with hematoxylin show that in the CA1 region of hippocampus, GSK-3α cko produced a mosaic pattern of neuronal GSK-3α expression and significantly reduced GSK-3α but not GSK-3β immunoreactivity (scale bar: 100 μm). Neurons with reduced GSK-3 expression are highlighted by black circles and neurons expressing normal GSK-3 levels are highlighted by dashed black circles. (B) GSK-3α cko was also confirmed by western blot from hippocampal lysates (n=4 mice/group) and densitometric analysis showing a significant reduction in GSK-3α (C) (67%, t (5) =9, p=0.0003) but not in GSK-3β levels (D) (t (3) =0.4912, p=0.6570). Data show mean ± SEM. ***p

    Journal: The Journal of Neuroscience

    Article Title: Selectively Silencing GSK-3 Isoforms Reduces Plaques and Tangles in Mouse Models of Alzheimer's disease

    doi: 10.1523/JNEUROSCI.0889-12.2012

    Figure Lengend Snippet: Generation of GSK-3α fx mice, confirmation and characterization of GSK-3α cko mice Conditional KO GSK-3a mice generation. (A) Restriction maps (from top to bottom) of the genomic locus, targeting vector, homologous alleles, conditional knock out allele, and a constitutive KO allele. After proper homologous recombination, exons 2–4 of targeted allele were flanked by loxP sites with the addition of a neomycin (neo) resistant marker flanked by FRT sites and in the absence of Zs green. Non-fluorescent selected clones were analyzed by Southern blot. Probes (blue rectangles) were located at the 5′ and 3′ ends and in the neo fragment. Genomic DNA was digested with either AflIII for the 5′ (B) and Neo probe (D), or KpnI for the 3′ probe (C). All four clones were positive for proper homologous recombination and ES clone BC-09 was selected. After Flp-mediated deletion, a conditional knock out (KO) allele was generated without Neo through mating with a Cre-deleter mouse line. Using this Cre-loxP ystem, wt or cko genotypes (n=10–15 mice/group) were generated (GSK-3α wt, GSK-3α cko, GSK-3α wt;PDAPP and GSK-3α cko;PDAPP) and analyzed at 17 moths of age. GSK-3α cko was confirmed by IHC using GSK-3αβ-specific antibodies. (A) Representative images counter-stained with hematoxylin show that in the CA1 region of hippocampus, GSK-3α cko produced a mosaic pattern of neuronal GSK-3α expression and significantly reduced GSK-3α but not GSK-3β immunoreactivity (scale bar: 100 μm). Neurons with reduced GSK-3 expression are highlighted by black circles and neurons expressing normal GSK-3 levels are highlighted by dashed black circles. (B) GSK-3α cko was also confirmed by western blot from hippocampal lysates (n=4 mice/group) and densitometric analysis showing a significant reduction in GSK-3α (C) (67%, t (5) =9, p=0.0003) but not in GSK-3β levels (D) (t (3) =0.4912, p=0.6570). Data show mean ± SEM. ***p

    Article Snippet: IP was performed using 35 μl of Protein A/G PLUS-Agarose slurry (Santa Cruz Biotechnology), 50 μg of lysate, and 2 μl of either GSK-3α or GSK-3β specific antibodies (Cell Signaling Technology).

    Techniques: Mouse Assay, Plasmid Preparation, Knock-Out, Homologous Recombination, Marker, Clone Assay, Southern Blot, Generated, Immunohistochemistry, Staining, Produced, Expressing, Western Blot

    Confirmation of GSK-3 KD ShRNA treated and non-injected wt mice were evaluated at 4 months of age to confirm targeted-gene KD. (A) Representative IHC images of the CA1 region of hippocampus counter-stained with hematoxylin reveal normal, baseline GSK-3αβ expression levels in untreated and shRNA-scr treated mice. ShRNA-α treatment significantly reduced GSK-3α expression without altering GSK-3β expression and vice versa for shRNA-β treated mice. Neurons with reduced GSK-3 expression are highlighted by black circles and neurons expressing normal GSK-3 levels are highlighted by dashed black circles. (scale bar: 100 μm). (B) Immunoblot analyses of hippocampal lysates probed with GSK-3 isoform-specific antibodies. (C) Corresponding quantification showing that shRNA-α treatment significantly reduced GSK-3α expression without altering GSK-3β expression (F (3,12) =68.47, p

    Journal: The Journal of Neuroscience

    Article Title: Selectively Silencing GSK-3 Isoforms Reduces Plaques and Tangles in Mouse Models of Alzheimer's disease

    doi: 10.1523/JNEUROSCI.0889-12.2012

    Figure Lengend Snippet: Confirmation of GSK-3 KD ShRNA treated and non-injected wt mice were evaluated at 4 months of age to confirm targeted-gene KD. (A) Representative IHC images of the CA1 region of hippocampus counter-stained with hematoxylin reveal normal, baseline GSK-3αβ expression levels in untreated and shRNA-scr treated mice. ShRNA-α treatment significantly reduced GSK-3α expression without altering GSK-3β expression and vice versa for shRNA-β treated mice. Neurons with reduced GSK-3 expression are highlighted by black circles and neurons expressing normal GSK-3 levels are highlighted by dashed black circles. (scale bar: 100 μm). (B) Immunoblot analyses of hippocampal lysates probed with GSK-3 isoform-specific antibodies. (C) Corresponding quantification showing that shRNA-α treatment significantly reduced GSK-3α expression without altering GSK-3β expression (F (3,12) =68.47, p

    Article Snippet: IP was performed using 35 μl of Protein A/G PLUS-Agarose slurry (Santa Cruz Biotechnology), 50 μg of lysate, and 2 μl of either GSK-3α or GSK-3β specific antibodies (Cell Signaling Technology).

    Techniques: shRNA, Injection, Mouse Assay, Immunohistochemistry, Staining, Expressing

    GSK-3α or GSK-3β KD reduces tau phosphorylation and conformational changes in PS19 +/− ;PDAPP +/− mice PS19 +/− ;PDAPP +/− neonates were uninjected (un) or injected with shRNA-α, -β or –scr and evaluated at 11 months of age (n=15 per group). (A) Representative IHC images counter-stained with hematoxylin show CA3 regions of posterior hippocampus stained with antibodies to total tau (T14), phospho-tau Ser202/Thr205 (AT8), phospho-tau Thr231 (AT180), phospho-tau Ser262 (12E8), or conformational specific antibodies Alz50, MC1 or ThS as indicated (scale bar: 100 μm). PS19 +/− ;PDAPP +/− mice treated with either shRNA-α, or -β showed a significant reduction in phospho-tau, Alz50, MC1 and ThS staining, but not in total tau load compared to shRNA-scr and non-injected controls. The phospho-tau burden in the CA3 region was quantified using ImageJ for mAb AT180 (B) and 12E8 (C) in n=3–4 mice/group, to demonstrate the quantification of > 50% reduction with shRNA-α and β treatment compared to controls (F (3,8) =8.283, p=0.0078 and F (3,9) =15.28, p=0.0007, respectively). Data show mean ± SEM. **p

    Journal: The Journal of Neuroscience

    Article Title: Selectively Silencing GSK-3 Isoforms Reduces Plaques and Tangles in Mouse Models of Alzheimer's disease

    doi: 10.1523/JNEUROSCI.0889-12.2012

    Figure Lengend Snippet: GSK-3α or GSK-3β KD reduces tau phosphorylation and conformational changes in PS19 +/− ;PDAPP +/− mice PS19 +/− ;PDAPP +/− neonates were uninjected (un) or injected with shRNA-α, -β or –scr and evaluated at 11 months of age (n=15 per group). (A) Representative IHC images counter-stained with hematoxylin show CA3 regions of posterior hippocampus stained with antibodies to total tau (T14), phospho-tau Ser202/Thr205 (AT8), phospho-tau Thr231 (AT180), phospho-tau Ser262 (12E8), or conformational specific antibodies Alz50, MC1 or ThS as indicated (scale bar: 100 μm). PS19 +/− ;PDAPP +/− mice treated with either shRNA-α, or -β showed a significant reduction in phospho-tau, Alz50, MC1 and ThS staining, but not in total tau load compared to shRNA-scr and non-injected controls. The phospho-tau burden in the CA3 region was quantified using ImageJ for mAb AT180 (B) and 12E8 (C) in n=3–4 mice/group, to demonstrate the quantification of > 50% reduction with shRNA-α and β treatment compared to controls (F (3,8) =8.283, p=0.0078 and F (3,9) =15.28, p=0.0007, respectively). Data show mean ± SEM. **p

    Article Snippet: IP was performed using 35 μl of Protein A/G PLUS-Agarose slurry (Santa Cruz Biotechnology), 50 μg of lysate, and 2 μl of either GSK-3α or GSK-3β specific antibodies (Cell Signaling Technology).

    Techniques: Mouse Assay, Injection, shRNA, Immunohistochemistry, Staining

    shRNA selection, rAAV distribution and expression, and lack of evidence of off-target effects N2a cells were transfected with hairpin constructs and selected with puromycin for seven days. As controls, N2a cells were transfected with an empty vector (VEC) or a scrambled hairpin control (scr). (A) Representative western blot of GSK-3α shRNA #1-#4 and (B) GSK-3β shRNA #1-#4 demonstrates that shRNA GSK-3α #2 showed the highest KD level (F (2,6) = 54.82, p

    Journal: The Journal of Neuroscience

    Article Title: Selectively Silencing GSK-3 Isoforms Reduces Plaques and Tangles in Mouse Models of Alzheimer's disease

    doi: 10.1523/JNEUROSCI.0889-12.2012

    Figure Lengend Snippet: shRNA selection, rAAV distribution and expression, and lack of evidence of off-target effects N2a cells were transfected with hairpin constructs and selected with puromycin for seven days. As controls, N2a cells were transfected with an empty vector (VEC) or a scrambled hairpin control (scr). (A) Representative western blot of GSK-3α shRNA #1-#4 and (B) GSK-3β shRNA #1-#4 demonstrates that shRNA GSK-3α #2 showed the highest KD level (F (2,6) = 54.82, p

    Article Snippet: IP was performed using 35 μl of Protein A/G PLUS-Agarose slurry (Santa Cruz Biotechnology), 50 μg of lysate, and 2 μl of either GSK-3α or GSK-3β specific antibodies (Cell Signaling Technology).

    Techniques: shRNA, Selection, Expressing, Transfection, Construct, Plasmid Preparation, 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: All proteins were electrophoretically immobilized to nitrocellulose membranes, which were subsequently probed with anti-GR antibodies (1:1,000; BD Biosciences), anti-GR 57 antibodies (1:500), anti-pSer404 GR antibodies (1:1,000), anti-pSer211 GR antibodies (1:2,000), anti-β-actin antibodies (1:2,500; Chemicon, Billerica, MA), anti-Bcl-xL antibodies (1:1,000; Chemicon), anti-GSK-3β antibodies (1:500; Cell Signaling, Danvers, MA), anti-GSK-3α antibodies (1:500; Chemicon), anti-p65 (RelA) antibodies (1:500; Cell Signaling), anti-CBP/p300 antibodies (1:500; Santa Cruz Biotechnologies, Santa Cruz, CA), anti-GRIP1 antibodies (1:500; Chemicon), anti-cIAP antibodies (1:1,000; Cell Signaling), anti-GAPDH antibodies (1:500; Cell Signaling), anti-nucleolin antibodies (1:1,000; Santa Cruz Biotechnologies), or anti-survivin antibodies (1:1,000; Stressgen, Ann Arbor, MI).

    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: All proteins were electrophoretically immobilized to nitrocellulose membranes, which were subsequently probed with anti-GR antibodies (1:1,000; BD Biosciences), anti-GR 57 antibodies (1:500), anti-pSer404 GR antibodies (1:1,000), anti-pSer211 GR antibodies (1:2,000), anti-β-actin antibodies (1:2,500; Chemicon, Billerica, MA), anti-Bcl-xL antibodies (1:1,000; Chemicon), anti-GSK-3β antibodies (1:500; Cell Signaling, Danvers, MA), anti-GSK-3α antibodies (1:500; Chemicon), anti-p65 (RelA) antibodies (1:500; Cell Signaling), anti-CBP/p300 antibodies (1:500; Santa Cruz Biotechnologies, Santa Cruz, CA), anti-GRIP1 antibodies (1:500; Chemicon), anti-cIAP antibodies (1:1,000; Cell Signaling), anti-GAPDH antibodies (1:500; Cell Signaling), anti-nucleolin antibodies (1:1,000; Santa Cruz Biotechnologies), or anti-survivin antibodies (1:1,000; Stressgen, Ann Arbor, MI).

    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: All proteins were electrophoretically immobilized to nitrocellulose membranes, which were subsequently probed with anti-GR antibodies (1:1,000; BD Biosciences), anti-GR 57 antibodies (1:500), anti-pSer404 GR antibodies (1:1,000), anti-pSer211 GR antibodies (1:2,000), anti-β-actin antibodies (1:2,500; Chemicon, Billerica, MA), anti-Bcl-xL antibodies (1:1,000; Chemicon), anti-GSK-3β antibodies (1:500; Cell Signaling, Danvers, MA), anti-GSK-3α antibodies (1:500; Chemicon), anti-p65 (RelA) antibodies (1:500; Cell Signaling), anti-CBP/p300 antibodies (1:500; Santa Cruz Biotechnologies, Santa Cruz, CA), anti-GRIP1 antibodies (1:500; Chemicon), anti-cIAP antibodies (1:1,000; Cell Signaling), anti-GAPDH antibodies (1:500; Cell Signaling), anti-nucleolin antibodies (1:1,000; Santa Cruz Biotechnologies), or anti-survivin antibodies (1:1,000; Stressgen, Ann Arbor, MI).

    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: All proteins were electrophoretically immobilized to nitrocellulose membranes, which were subsequently probed with anti-GR antibodies (1:1,000; BD Biosciences), anti-GR 57 antibodies (1:500), anti-pSer404 GR antibodies (1:1,000), anti-pSer211 GR antibodies (1:2,000), anti-β-actin antibodies (1:2,500; Chemicon, Billerica, MA), anti-Bcl-xL antibodies (1:1,000; Chemicon), anti-GSK-3β antibodies (1:500; Cell Signaling, Danvers, MA), anti-GSK-3α antibodies (1:500; Chemicon), anti-p65 (RelA) antibodies (1:500; Cell Signaling), anti-CBP/p300 antibodies (1:500; Santa Cruz Biotechnologies, Santa Cruz, CA), anti-GRIP1 antibodies (1:500; Chemicon), anti-cIAP antibodies (1:1,000; Cell Signaling), anti-GAPDH antibodies (1:500; Cell Signaling), anti-nucleolin antibodies (1:1,000; Santa Cruz Biotechnologies), or anti-survivin antibodies (1:1,000; Stressgen, Ann Arbor, MI).

    Techniques: Binding Assay, Microarray

    sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Expressing

    Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Immunostaining

    GSK-3β/Rac-1 pathway involved in sFRP-1-induced EC spreading. a: HUVECs were either infected with adenovirus expressing β-galactosidase, GSK-3β, GSK-3β-S9A, or GSK-3β-KM or treated with SB216763. After the treatment,

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: GSK-3β/Rac-1 pathway involved in sFRP-1-induced EC spreading. a: HUVECs were either infected with adenovirus expressing β-galactosidase, GSK-3β, GSK-3β-S9A, or GSK-3β-KM or treated with SB216763. After the treatment,

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Infection, Expressing

    The effects of GMF, GSK-3β, and caspase-3 inhibitors on GMF-induced loss of cell viability. N18 cells (1 × 10 5 ) were plated on 24-well plates. After 24 h, the cells were infected with mock, LacZ or GMF-V at 20 MOI. Selective inhibitors of GMF, GsiRNA (20 nM), cell-permeable peptide GSK-3 β inhibitor (20 μM), or cell-permeable CPP32 caspase-3 inhibitor (0.25 nM) were added 4 h before infection. Cell viability was assessed after 96 h by MTT assay as described in methods. The data shown are mean ± SD of three separate experiments. p

    Journal: Brain research

    Article Title: Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3? and caspase-3

    doi: 10.1016/j.brainres.2007.11.011

    Figure Lengend Snippet: The effects of GMF, GSK-3β, and caspase-3 inhibitors on GMF-induced loss of cell viability. N18 cells (1 × 10 5 ) were plated on 24-well plates. After 24 h, the cells were infected with mock, LacZ or GMF-V at 20 MOI. Selective inhibitors of GMF, GsiRNA (20 nM), cell-permeable peptide GSK-3 β inhibitor (20 μM), or cell-permeable CPP32 caspase-3 inhibitor (0.25 nM) were added 4 h before infection. Cell viability was assessed after 96 h by MTT assay as described in methods. The data shown are mean ± SD of three separate experiments. p

    Article Snippet: Affinity purified polyclonal antibody against GSK-3β, caspase-3, and β-actin were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Infection, MTT Assay

    Inhibitors of GSK-3β prevented GMF-dependent activation of caspase-3. Cells were infected with GMF-V or LacZ at 20 MOI in the presence of a specific cell-permeable peptide (CPP) inhibitor of GSK-3 β at 10, 20, and 40 μM concentration (A) , or with LiCl at 2, 5, and 10 mM (B) . Inhibitors were added 4 h before infection. Cells were harvested after 48 h of incubation and caspase-3 activity was measured as described in Methods. The data shown are mean ± SD of three separate experiments. p

    Journal: Brain research

    Article Title: Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3? and caspase-3

    doi: 10.1016/j.brainres.2007.11.011

    Figure Lengend Snippet: Inhibitors of GSK-3β prevented GMF-dependent activation of caspase-3. Cells were infected with GMF-V or LacZ at 20 MOI in the presence of a specific cell-permeable peptide (CPP) inhibitor of GSK-3 β at 10, 20, and 40 μM concentration (A) , or with LiCl at 2, 5, and 10 mM (B) . Inhibitors were added 4 h before infection. Cells were harvested after 48 h of incubation and caspase-3 activity was measured as described in Methods. The data shown are mean ± SD of three separate experiments. p

    Article Snippet: Affinity purified polyclonal antibody against GSK-3β, caspase-3, and β-actin were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Activation Assay, Infection, Conditioned Place Preference, Concentration Assay, Incubation, Activity Assay

    Overexpression of GMF activates GSK-3β kinase in N18 cells. Cells were infected with LacZ (20 MOI), GMF-V (5, 10, and 20 MOI), and GMV-V (20 MOI) in the presence of GMF-specific siRNA (GsiRNA, 20 nM) or control scrambled siRNA (CsiRNA, 20 nM) and harvested at 48 h. GSK-3β kinase was immunoprecipitated from cell lysates and subjected to immune-complex kinase assay using glycogen synthase peptide-2 as the substrate, as described in Methods. The results show a significant dose-dependent increase in activation of GSK-3β kinase in GMF-V transfected cells and significant blocking of GMF-induced GSK-3β activation by GMF-specific siRNA. (siRNA was added 4 h before infection). The figure is representing three independent experiments. p

    Journal: Brain research

    Article Title: Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3? and caspase-3

    doi: 10.1016/j.brainres.2007.11.011

    Figure Lengend Snippet: Overexpression of GMF activates GSK-3β kinase in N18 cells. Cells were infected with LacZ (20 MOI), GMF-V (5, 10, and 20 MOI), and GMV-V (20 MOI) in the presence of GMF-specific siRNA (GsiRNA, 20 nM) or control scrambled siRNA (CsiRNA, 20 nM) and harvested at 48 h. GSK-3β kinase was immunoprecipitated from cell lysates and subjected to immune-complex kinase assay using glycogen synthase peptide-2 as the substrate, as described in Methods. The results show a significant dose-dependent increase in activation of GSK-3β kinase in GMF-V transfected cells and significant blocking of GMF-induced GSK-3β activation by GMF-specific siRNA. (siRNA was added 4 h before infection). The figure is representing three independent experiments. p

    Article Snippet: Affinity purified polyclonal antibody against GSK-3β, caspase-3, and β-actin were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Over Expression, Infection, Immunoprecipitation, Immune Complex Kinase Assay, Activation Assay, Transfection, Blocking Assay

    GMF overexpression alters GSK-3β and Akt phosphorylation. Immunoblots show reduced phospho-GSK-3β ( A ) and reduced phospho-Akt ( C ) levels in GMF-V ( lane 3 ) compared to control mock ( lane 1 ) and LacZ ( lane 2 ) treated N18 cells. Note no changes in total Akt levels ( D ). Actin served as a loading control ( B ).

    Journal: Brain research

    Article Title: Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3? and caspase-3

    doi: 10.1016/j.brainres.2007.11.011

    Figure Lengend Snippet: GMF overexpression alters GSK-3β and Akt phosphorylation. Immunoblots show reduced phospho-GSK-3β ( A ) and reduced phospho-Akt ( C ) levels in GMF-V ( lane 3 ) compared to control mock ( lane 1 ) and LacZ ( lane 2 ) treated N18 cells. Note no changes in total Akt levels ( D ). Actin served as a loading control ( B ).

    Article Snippet: Affinity purified polyclonal antibody against GSK-3β, caspase-3, and β-actin were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Over Expression, Western Blot

    Reserved DSG(X) n S motif provides a binding site for β-transduction repeat-containing protein (β-TrCP), and activated glycogen synthase kinase-3β (GSK-3β) mediates this process. (A) Sequence alignment of human, mouse, rat and coturnix vascular endothelial growth factor receptor-2 (VEGFR-2) cDNA showed a conserved DSG(X) n S binding motif. (B) Reduced GSK-3β phosphorylation with almost unaltered total GSK-3β in human umbilical vein endothelial cells (HUVECs) exposed to glucose oxidase (GO). (C) Pretreatment for 30 min with LiCl (20 µ M) or SB216763 (20 µ M) enhanced the protein expression of VEGFR-2 in HUVECs cultured with GO. The results are expressed as the means ± SD. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Downregulation of vascular endothelial growth factor receptor-2 under oxidative stress conditions is mediated by β-transduction repeat-containing protein via glycogen synthase kinase-3β signaling

    doi: 10.3892/ijmm.2016.2493

    Figure Lengend Snippet: Reserved DSG(X) n S motif provides a binding site for β-transduction repeat-containing protein (β-TrCP), and activated glycogen synthase kinase-3β (GSK-3β) mediates this process. (A) Sequence alignment of human, mouse, rat and coturnix vascular endothelial growth factor receptor-2 (VEGFR-2) cDNA showed a conserved DSG(X) n S binding motif. (B) Reduced GSK-3β phosphorylation with almost unaltered total GSK-3β in human umbilical vein endothelial cells (HUVECs) exposed to glucose oxidase (GO). (C) Pretreatment for 30 min with LiCl (20 µ M) or SB216763 (20 µ M) enhanced the protein expression of VEGFR-2 in HUVECs cultured with GO. The results are expressed as the means ± SD. * P

    Article Snippet: All the primary antibodies [phospho-VEGFR-2 (Tyr1175) (19A10) rabbit monoclonal antibody (mAb) (#2478); VEGFR-2 (D5B1) rabbit mAb (#9698); β-TrCP (D13F10) rabbit mAb (#4394); ubiquitin antibody (#3933); phospho-GSK-3β (Ser9) (D85E12) rabbit mAb (#5558); GSK-3β (D5C5Z) rabbit mAb (#12456)], secondary antibodies [anti-rabbit IgG, HRP-linked antibody (#7074)], human vascular endothelial growth factor 165 (hVEGF165 ) and MG132 (a potent proteasome inhibitor) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Binding Assay, Transduction, Sequencing, Expressing, Cell Culture

    Regulation of PD-L1 protein expression (A) Western blot and semiquanititative analysis of GSK-3beta and its inactivated state represented as phosphorylated-GSK-3beta (P-GSK-3beta) at Ser09 24h and 96h after irradiation with 8Gy. Non-IRR cells (0Gy) were used as controls. RR HNSCC cell lines did not show any significant change in GSK-3beta expression (lower left panel). However, inactivation of GSK-3beta occured 96h after irradiation. Phosphorylation at Ser09 was on average 10x higher compared to the level of non-IRR cells. Beta-Actin was used as loading control. n=3, Student’s t-Test ** = p

    Journal: Oncotarget

    Article Title: Increased PD-L1 expression in radioresistant HNSCC cell lines after irradiation affects cell proliferation due to inactivation of GSK-3beta

    doi: 10.18632/oncotarget.26542

    Figure Lengend Snippet: Regulation of PD-L1 protein expression (A) Western blot and semiquanititative analysis of GSK-3beta and its inactivated state represented as phosphorylated-GSK-3beta (P-GSK-3beta) at Ser09 24h and 96h after irradiation with 8Gy. Non-IRR cells (0Gy) were used as controls. RR HNSCC cell lines did not show any significant change in GSK-3beta expression (lower left panel). However, inactivation of GSK-3beta occured 96h after irradiation. Phosphorylation at Ser09 was on average 10x higher compared to the level of non-IRR cells. Beta-Actin was used as loading control. n=3, Student’s t-Test ** = p

    Article Snippet: Antibodies used for western blot analysis were Phospho-GSK-3beta (Ser09) (D85E12) rabbit mAb #5558 Cell Signaling, GSK-3beta (D5C5Z) XP® rabbit mAb #12456 Cell Signaling, PD L1 (E1L3N) rabbit mAb #13684 Cell Signaling, E-cadherin mouse mAb 610182 BD Bioscience and vimentin rabbit mAB #5741 Cell Signaling.

    Techniques: Expressing, Western Blot, Irradiation

    Balance between the PAR‐3/aPKCλ complex versus the aPKCλ/GSK3β complex modulates GSK3β activity Western blotting of ECs transfected with control scrambled siRNA (indicated as “‐” in the figure) or siPAR‐3#1 and exposed to 18 dyn/cm 2 laminar flow. HEK293 cells were transfected with different amounts of GFP‐PAR‐3 cDNA with HA‐GSK3β and myc‐aPKCλ and were analyzed by Western blotting. Model for PAR‐3‐mediated GSK3β inactivation. The balance between PAR‐3/aPKCλ complex versus the aPKCλ/GSK3β complex controls GSK3β activity. HUVECs were transfected with indicated cDNAs and were treated with or without Rho‐kinase inhibitor (Y‐27632, 20 μM). After immunoprecipitation with indicated antibodies, samples were analyzed by Western blotting. HUVECs were transfected with indicated cDNAs, seeded in the flow chamber, and exposed to 18 dyn/cm 2 laminar flow for 60 min. After immunoprecipitation with anti‐myc antibody, samples were analyzed with indicated antibodies by Western blotting. Relative intensity of each signal was statistically analyzed. RhoA/Rho‐kinase pathway controls GSK3β activity by modulating balance between the PAR‐3/aPKCλ complex versus the aPKCλ/GSK3β complex. Data information: In (A, B, D, and E), data are presented as mean ± SEM (A and B, n = 3; D and E, n = 5). ns: not significant; P ≥ 0.05; differences * P

    Journal: EMBO Reports

    Article Title: PAR‐3 controls endothelial planar polarity and vascular inflammation under laminar flow

    doi: 10.15252/embr.201745253

    Figure Lengend Snippet: Balance between the PAR‐3/aPKCλ complex versus the aPKCλ/GSK3β complex modulates GSK3β activity Western blotting of ECs transfected with control scrambled siRNA (indicated as “‐” in the figure) or siPAR‐3#1 and exposed to 18 dyn/cm 2 laminar flow. HEK293 cells were transfected with different amounts of GFP‐PAR‐3 cDNA with HA‐GSK3β and myc‐aPKCλ and were analyzed by Western blotting. Model for PAR‐3‐mediated GSK3β inactivation. The balance between PAR‐3/aPKCλ complex versus the aPKCλ/GSK3β complex controls GSK3β activity. HUVECs were transfected with indicated cDNAs and were treated with or without Rho‐kinase inhibitor (Y‐27632, 20 μM). After immunoprecipitation with indicated antibodies, samples were analyzed by Western blotting. HUVECs were transfected with indicated cDNAs, seeded in the flow chamber, and exposed to 18 dyn/cm 2 laminar flow for 60 min. After immunoprecipitation with anti‐myc antibody, samples were analyzed with indicated antibodies by Western blotting. Relative intensity of each signal was statistically analyzed. RhoA/Rho‐kinase pathway controls GSK3β activity by modulating balance between the PAR‐3/aPKCλ complex versus the aPKCλ/GSK3β complex. Data information: In (A, B, D, and E), data are presented as mean ± SEM (A and B, n = 3; D and E, n = 5). ns: not significant; P ≥ 0.05; differences * P

    Article Snippet: GSK3β/aPKCλ complex formation was analyzed with anti‐GSK3β antibody (rabbit monoclonal, clone D5C5Z, Cell Signaling #12456) and anti‐aPKCλ antibody (mouse monoclonal, 610207).

    Techniques: Activity Assay, Western Blot, Transfection, Flow Cytometry, Immunoprecipitation

    Spatio‐temporal antagonism of the PAR‐3/aPKCλ complex versus the aPKCλ/GSK3β complex controls microtubule stabilization under flow Representative time‐lapse images of ECs transfected with RhoA biosensor. FRET/CFP ratio is represented in IMD mode. Cells were subjected to laminar flow of 12 dyn/cm 2 flow for 2 h. Scale bar, 15 μm. PLA in ECs expressing GFP‐PAR‐3 and myc‐PKCλ in static and under 18 dyn/cm 2 flow for 1 h. Arrowheads indicate PAR‐3/aPKCλ PLA signal colocalized with EC junction. Scale bar, 20 μm. Quantification of the images shown in (B). Representative images of PLA in ECs expressing GFP‐GSK3β and myc‐PKCλ in static (0 h) and after 1 h subjected to 18 dyn/cm 2 flow. EC junction (VE‐cadherin) is green, nuclear stain (DAPI) is blue, and GFP‐GSK3β is gray. Right panels show higher magnification images of the indicated areas. Yellow signals indicate PLA signal colocalized with EC junction. Scale bars, 10 μm (left panel) and 20 μm (right panel). Quantification of percentile of the PLA signals in the front and rear region of the ECs under static and 1‐h treatment with flow. Data information: Direction of the flow is indicated in the pictures with arrows. In (C and E), data are means ± SEM ( n = 3 experiments, n = 30 cells from each experiment); statistical significance (* P

    Journal: EMBO Reports

    Article Title: PAR‐3 controls endothelial planar polarity and vascular inflammation under laminar flow

    doi: 10.15252/embr.201745253

    Figure Lengend Snippet: Spatio‐temporal antagonism of the PAR‐3/aPKCλ complex versus the aPKCλ/GSK3β complex controls microtubule stabilization under flow Representative time‐lapse images of ECs transfected with RhoA biosensor. FRET/CFP ratio is represented in IMD mode. Cells were subjected to laminar flow of 12 dyn/cm 2 flow for 2 h. Scale bar, 15 μm. PLA in ECs expressing GFP‐PAR‐3 and myc‐PKCλ in static and under 18 dyn/cm 2 flow for 1 h. Arrowheads indicate PAR‐3/aPKCλ PLA signal colocalized with EC junction. Scale bar, 20 μm. Quantification of the images shown in (B). Representative images of PLA in ECs expressing GFP‐GSK3β and myc‐PKCλ in static (0 h) and after 1 h subjected to 18 dyn/cm 2 flow. EC junction (VE‐cadherin) is green, nuclear stain (DAPI) is blue, and GFP‐GSK3β is gray. Right panels show higher magnification images of the indicated areas. Yellow signals indicate PLA signal colocalized with EC junction. Scale bars, 10 μm (left panel) and 20 μm (right panel). Quantification of percentile of the PLA signals in the front and rear region of the ECs under static and 1‐h treatment with flow. Data information: Direction of the flow is indicated in the pictures with arrows. In (C and E), data are means ± SEM ( n = 3 experiments, n = 30 cells from each experiment); statistical significance (* P

    Article Snippet: GSK3β/aPKCλ complex formation was analyzed with anti‐GSK3β antibody (rabbit monoclonal, clone D5C5Z, Cell Signaling #12456) and anti‐aPKCλ antibody (mouse monoclonal, 610207).

    Techniques: Flow Cytometry, Transfection, Radial Immuno Diffusion, Proximity Ligation Assay, Expressing, Staining

    Discrete regulation of axial cell polarity and inflammation by the balance between the PAR‐3/aPKCλ and aPKCλ/GSK3β complexes Laminar flow discretely controls endothelial polarity toward the flow axis and vascular inflammatory responses. In the aortic arch, EC Golgi at the sidewall and marginal regions are polarized toward the flow direction. On the other hand, ECs at the inner curvature are not polarized toward the flow direction and have a high risk for atherosclerotic plaque formation. ECs at the marginal region and the inner curvature in Pard3 iΔEC mice showed misoriented Golgi, while those at the sidewall do not. Middle flow restricted RhoA activation to the rear region of ECs, resulting in the non‐uniform distribution of the PAR‐3/aPKCλ and aPKCλ/GSK3β complexes along the flow direction. In the front region, aPKCλ forms a complex with PAR‐3, and hence, the free inactive form of GSK3β is increased. This GSK3β inactivation mediates both EC planar cell polarization and pro‐inflammatory responses. While spatial distribution of GSK3β activation would be important of EC polarity toward the flow axis, local activation of GSK3β is not essential for vascular inflammation.

    Journal: EMBO Reports

    Article Title: PAR‐3 controls endothelial planar polarity and vascular inflammation under laminar flow

    doi: 10.15252/embr.201745253

    Figure Lengend Snippet: Discrete regulation of axial cell polarity and inflammation by the balance between the PAR‐3/aPKCλ and aPKCλ/GSK3β complexes Laminar flow discretely controls endothelial polarity toward the flow axis and vascular inflammatory responses. In the aortic arch, EC Golgi at the sidewall and marginal regions are polarized toward the flow direction. On the other hand, ECs at the inner curvature are not polarized toward the flow direction and have a high risk for atherosclerotic plaque formation. ECs at the marginal region and the inner curvature in Pard3 iΔEC mice showed misoriented Golgi, while those at the sidewall do not. Middle flow restricted RhoA activation to the rear region of ECs, resulting in the non‐uniform distribution of the PAR‐3/aPKCλ and aPKCλ/GSK3β complexes along the flow direction. In the front region, aPKCλ forms a complex with PAR‐3, and hence, the free inactive form of GSK3β is increased. This GSK3β inactivation mediates both EC planar cell polarization and pro‐inflammatory responses. While spatial distribution of GSK3β activation would be important of EC polarity toward the flow axis, local activation of GSK3β is not essential for vascular inflammation.

    Article Snippet: GSK3β/aPKCλ complex formation was analyzed with anti‐GSK3β antibody (rabbit monoclonal, clone D5C5Z, Cell Signaling #12456) and anti‐aPKCλ antibody (mouse monoclonal, 610207).

    Techniques: Flow Cytometry, Mouse Assay, Activation Assay

    PAR‐3 controls nuclear localization of p65 in aortic endothelial cells in a GSK3β activity‐dependent manner Representative images of control and Pard3KO ECs treated with control (DMSO) or GSK3β inhibitor (6‐BIO, 1 μM) under static conditions or after subjected to 12 dyn/cm 2 flow for the indicated times. EC junction (VE‐cadherin) is gray, NF‐κB p65 subunit (p65) is green, and nuclear stain (DAPI) is blue. Scale bar, 50 μm. White arrowheads indicate p65‐positive nuclei. Quantitative analysis of the percentile of p65‐positive ECs. Data are shown as box and whisker plots. The box spans the interquartile range, horizontal central values are median, and the whiskers extend to the highest and lowest observations ( n = 50 cells); statistical significance (* P

    Journal: EMBO Reports

    Article Title: PAR‐3 controls endothelial planar polarity and vascular inflammation under laminar flow

    doi: 10.15252/embr.201745253

    Figure Lengend Snippet: PAR‐3 controls nuclear localization of p65 in aortic endothelial cells in a GSK3β activity‐dependent manner Representative images of control and Pard3KO ECs treated with control (DMSO) or GSK3β inhibitor (6‐BIO, 1 μM) under static conditions or after subjected to 12 dyn/cm 2 flow for the indicated times. EC junction (VE‐cadherin) is gray, NF‐κB p65 subunit (p65) is green, and nuclear stain (DAPI) is blue. Scale bar, 50 μm. White arrowheads indicate p65‐positive nuclei. Quantitative analysis of the percentile of p65‐positive ECs. Data are shown as box and whisker plots. The box spans the interquartile range, horizontal central values are median, and the whiskers extend to the highest and lowest observations ( n = 50 cells); statistical significance (* P

    Article Snippet: GSK3β/aPKCλ complex formation was analyzed with anti‐GSK3β antibody (rabbit monoclonal, clone D5C5Z, Cell Signaling #12456) and anti‐aPKCλ antibody (mouse monoclonal, 610207).

    Techniques: Activity Assay, Flow Cytometry, Staining, Whisker Assay

    GSK3β indirectly controls EC polarity toward the flow axis and anti‐inflammatory effects in vivo A En face confocal microscopy of the aortic arch from the inner curvature to sidewall of control and mutant mice treated with GSK3β inhibitor. Scale bar: 50 μm. B, C Regional quantification of Golgi orientation (Polarity Index, B) and nuclear p65 intensity (C) of ECs. Data are presented as mean ± SEM ( n = 5 mice, n = 50 cells from each region). Differences: * P

    Journal: EMBO Reports

    Article Title: PAR‐3 controls endothelial planar polarity and vascular inflammation under laminar flow

    doi: 10.15252/embr.201745253

    Figure Lengend Snippet: GSK3β indirectly controls EC polarity toward the flow axis and anti‐inflammatory effects in vivo A En face confocal microscopy of the aortic arch from the inner curvature to sidewall of control and mutant mice treated with GSK3β inhibitor. Scale bar: 50 μm. B, C Regional quantification of Golgi orientation (Polarity Index, B) and nuclear p65 intensity (C) of ECs. Data are presented as mean ± SEM ( n = 5 mice, n = 50 cells from each region). Differences: * P

    Article Snippet: GSK3β/aPKCλ complex formation was analyzed with anti‐GSK3β antibody (rabbit monoclonal, clone D5C5Z, Cell Signaling #12456) and anti‐aPKCλ antibody (mouse monoclonal, 610207).

    Techniques: Flow Cytometry, In Vivo, Confocal Microscopy, Mutagenesis, Mouse Assay

    Spatio‐temporal antagonism of the PAR‐3/aPKCλ complex versus the aPKCλ/GSK3β complex controls microtubule stabilization under flow A Representative time‐lapse images of ECs transfected with RhoA biosensor under 18 dyn/cm 2 laminar flow. FRET/CFP ratio is represented in IMD mode. B Statistical analysis of FRET intensity of EC front and rear region. C–F PLA analysis of Par3 and aPKCλ (C, D) or GSK3β and aPKCλ (E, F) in EC in static and treated with 18 dyn/cm 2 laminar flow for 60 min. Arrowheads indicate PLA signal. (D, F) Quantification of the PLA images. Percentile of the PLA signal in the front half of the ECs toward flow was calculated. G Acetylated and total α‐tubulin in ECs under 18 dyn/cm 2 flow, with or without Rho‐kinase inhibitor (Y‐27632, 20 μM). H Quantification of regional Ac/total α‐tubulin ratio in the front and rear half of each cells. Data information: In (D, F, and H), data are presented as mean ± SEM (D and F, n = 3 experiments, n = 50 cells from each experiment; H, n = 5 experiments, n = 50 cells from each experiment). Differences: * P

    Journal: EMBO Reports

    Article Title: PAR‐3 controls endothelial planar polarity and vascular inflammation under laminar flow

    doi: 10.15252/embr.201745253

    Figure Lengend Snippet: Spatio‐temporal antagonism of the PAR‐3/aPKCλ complex versus the aPKCλ/GSK3β complex controls microtubule stabilization under flow A Representative time‐lapse images of ECs transfected with RhoA biosensor under 18 dyn/cm 2 laminar flow. FRET/CFP ratio is represented in IMD mode. B Statistical analysis of FRET intensity of EC front and rear region. C–F PLA analysis of Par3 and aPKCλ (C, D) or GSK3β and aPKCλ (E, F) in EC in static and treated with 18 dyn/cm 2 laminar flow for 60 min. Arrowheads indicate PLA signal. (D, F) Quantification of the PLA images. Percentile of the PLA signal in the front half of the ECs toward flow was calculated. G Acetylated and total α‐tubulin in ECs under 18 dyn/cm 2 flow, with or without Rho‐kinase inhibitor (Y‐27632, 20 μM). H Quantification of regional Ac/total α‐tubulin ratio in the front and rear half of each cells. Data information: In (D, F, and H), data are presented as mean ± SEM (D and F, n = 3 experiments, n = 50 cells from each experiment; H, n = 5 experiments, n = 50 cells from each experiment). Differences: * P

    Article Snippet: GSK3β/aPKCλ complex formation was analyzed with anti‐GSK3β antibody (rabbit monoclonal, clone D5C5Z, Cell Signaling #12456) and anti‐aPKCλ antibody (mouse monoclonal, 610207).

    Techniques: Flow Cytometry, Transfection, Radial Immuno Diffusion, Proximity Ligation Assay

    The induction of apoptosis by TAT-CTMP4 occurs via inhibition of the AKT pathway (A) Model human pancreatic adenocarcinoma (CFPAC-1) were treated with escalating doses of TAT-CTMP4. Samples were treated for 1 hour prior to preparation of cell lysates. Western blots were subsequently prepared and stained with anti-total AKT and anti-phospho-AKT (Ser 473 ). (B) Panc-1 lysates were generated after exposure to PBS (no treatment control), DMSO (vehicle control for wortmannin), wortmannin (1 µM), TAT-CTMP4-Inactive (10 µM), and TAT-CTMP4 (10 µM) for 18 hours. Western blots were probed with anti-total GSK-3β and phospho-GSK-3α/β (Ser 21/9 ). (C) Human model pancreatic adenocarcinoma (CFPAC-1) were treated with PBS (no treatment), gemcitabine (30 nM), recombinant human TNF-α (10 ng/ml), wortmannin (1 µM), TAT-CTMP4-Inactive (10 µM), and TAT-CTMP4 (10 µM). One hour later, cell lysates were generated and phospho-proteins were quantitated by bioplex analysis. Each experimental group represents an n=4. Results are expressed as the mean, with bars representing standard error of the mean.

    Journal: International journal of cancer. Journal international du cancer

    Article Title: Targeting AKT with the Pro-apoptotic Peptide, TAT-CTMP: a Novel Strategy for the Treatment of Human Pancreatic Adenocarcinoma

    doi: 10.1002/ijc.24424

    Figure Lengend Snippet: The induction of apoptosis by TAT-CTMP4 occurs via inhibition of the AKT pathway (A) Model human pancreatic adenocarcinoma (CFPAC-1) were treated with escalating doses of TAT-CTMP4. Samples were treated for 1 hour prior to preparation of cell lysates. Western blots were subsequently prepared and stained with anti-total AKT and anti-phospho-AKT (Ser 473 ). (B) Panc-1 lysates were generated after exposure to PBS (no treatment control), DMSO (vehicle control for wortmannin), wortmannin (1 µM), TAT-CTMP4-Inactive (10 µM), and TAT-CTMP4 (10 µM) for 18 hours. Western blots were probed with anti-total GSK-3β and phospho-GSK-3α/β (Ser 21/9 ). (C) Human model pancreatic adenocarcinoma (CFPAC-1) were treated with PBS (no treatment), gemcitabine (30 nM), recombinant human TNF-α (10 ng/ml), wortmannin (1 µM), TAT-CTMP4-Inactive (10 µM), and TAT-CTMP4 (10 µM). One hour later, cell lysates were generated and phospho-proteins were quantitated by bioplex analysis. Each experimental group represents an n=4. Results are expressed as the mean, with bars representing standard error of the mean.

    Article Snippet: Blots were blocked with nonfat dry milk (5% in TBS) and incubated overnight with anti-total AKT or anti-phospho-AKT (Ser473 ), total GSK-3β, or phospho-GSK-3α/β (Ser21/9 ) antibodies (Cell Signal Technologies, Inc. Boston, MA) at 4 °C.

    Techniques: Inhibition, Western Blot, Staining, Generated, Recombinant

    Generation of GSK-3α fx mice, confirmation and characterization of GSK-3α cko mice Conditional KO GSK-3a mice generation. (A) Restriction maps (from top to bottom) of the genomic locus, targeting vector, homologous alleles, conditional knock out allele, and a constitutive KO allele. After proper homologous recombination, exons 2–4 of targeted allele were flanked by loxP sites with the addition of a neomycin (neo) resistant marker flanked by FRT sites and in the absence of Zs green. Non-fluorescent selected clones were analyzed by Southern blot. Probes (blue rectangles) were located at the 5′ and 3′ ends and in the neo fragment. Genomic DNA was digested with either AflIII for the 5′ (B) and Neo probe (D), or KpnI for the 3′ probe (C). All four clones were positive for proper homologous recombination and ES clone BC-09 was selected. After Flp-mediated deletion, a conditional knock out (KO) allele was generated without Neo through mating with a Cre-deleter mouse line. Using this Cre-loxP ystem, wt or cko genotypes (n=10–15 mice/group) were generated (GSK-3α wt, GSK-3α cko, GSK-3α wt;PDAPP and GSK-3α cko;PDAPP) and analyzed at 17 moths of age. GSK-3α cko was confirmed by IHC using GSK-3αβ-specific antibodies. (A) Representative images counter-stained with hematoxylin show that in the CA1 region of hippocampus, GSK-3α cko produced a mosaic pattern of neuronal GSK-3α expression and significantly reduced GSK-3α but not GSK-3β immunoreactivity (scale bar: 100 μm). Neurons with reduced GSK-3 expression are highlighted by black circles and neurons expressing normal GSK-3 levels are highlighted by dashed black circles. (B) GSK-3α cko was also confirmed by western blot from hippocampal lysates (n=4 mice/group) and densitometric analysis showing a significant reduction in GSK-3α (C) (67%, t (5) =9, p=0.0003) but not in GSK-3β levels (D) (t (3) =0.4912, p=0.6570). Data show mean ± SEM. ***p

    Journal: The Journal of Neuroscience

    Article Title: Selectively Silencing GSK-3 Isoforms Reduces Plaques and Tangles in Mouse Models of Alzheimer's disease

    doi: 10.1523/JNEUROSCI.0889-12.2012

    Figure Lengend Snippet: Generation of GSK-3α fx mice, confirmation and characterization of GSK-3α cko mice Conditional KO GSK-3a mice generation. (A) Restriction maps (from top to bottom) of the genomic locus, targeting vector, homologous alleles, conditional knock out allele, and a constitutive KO allele. After proper homologous recombination, exons 2–4 of targeted allele were flanked by loxP sites with the addition of a neomycin (neo) resistant marker flanked by FRT sites and in the absence of Zs green. Non-fluorescent selected clones were analyzed by Southern blot. Probes (blue rectangles) were located at the 5′ and 3′ ends and in the neo fragment. Genomic DNA was digested with either AflIII for the 5′ (B) and Neo probe (D), or KpnI for the 3′ probe (C). All four clones were positive for proper homologous recombination and ES clone BC-09 was selected. After Flp-mediated deletion, a conditional knock out (KO) allele was generated without Neo through mating with a Cre-deleter mouse line. Using this Cre-loxP ystem, wt or cko genotypes (n=10–15 mice/group) were generated (GSK-3α wt, GSK-3α cko, GSK-3α wt;PDAPP and GSK-3α cko;PDAPP) and analyzed at 17 moths of age. GSK-3α cko was confirmed by IHC using GSK-3αβ-specific antibodies. (A) Representative images counter-stained with hematoxylin show that in the CA1 region of hippocampus, GSK-3α cko produced a mosaic pattern of neuronal GSK-3α expression and significantly reduced GSK-3α but not GSK-3β immunoreactivity (scale bar: 100 μm). Neurons with reduced GSK-3 expression are highlighted by black circles and neurons expressing normal GSK-3 levels are highlighted by dashed black circles. (B) GSK-3α cko was also confirmed by western blot from hippocampal lysates (n=4 mice/group) and densitometric analysis showing a significant reduction in GSK-3α (C) (67%, t (5) =9, p=0.0003) but not in GSK-3β levels (D) (t (3) =0.4912, p=0.6570). Data show mean ± SEM. ***p

    Article Snippet: GSK-3 expression was monitored through the use of a GSK-3α rabbit pAb (9338) and GSK-3β rabbit mouse monoclonal antibody (mAb) (27C10) (Cell Signaling Technology).

    Techniques: Mouse Assay, Plasmid Preparation, Knock-Out, Homologous Recombination, Marker, Clone Assay, Southern Blot, Generated, Immunohistochemistry, Staining, Produced, Expressing, Western Blot

    Confirmation of GSK-3 KD ShRNA treated and non-injected wt mice were evaluated at 4 months of age to confirm targeted-gene KD. (A) Representative IHC images of the CA1 region of hippocampus counter-stained with hematoxylin reveal normal, baseline GSK-3αβ expression levels in untreated and shRNA-scr treated mice. ShRNA-α treatment significantly reduced GSK-3α expression without altering GSK-3β expression and vice versa for shRNA-β treated mice. Neurons with reduced GSK-3 expression are highlighted by black circles and neurons expressing normal GSK-3 levels are highlighted by dashed black circles. (scale bar: 100 μm). (B) Immunoblot analyses of hippocampal lysates probed with GSK-3 isoform-specific antibodies. (C) Corresponding quantification showing that shRNA-α treatment significantly reduced GSK-3α expression without altering GSK-3β expression (F (3,12) =68.47, p

    Journal: The Journal of Neuroscience

    Article Title: Selectively Silencing GSK-3 Isoforms Reduces Plaques and Tangles in Mouse Models of Alzheimer's disease

    doi: 10.1523/JNEUROSCI.0889-12.2012

    Figure Lengend Snippet: Confirmation of GSK-3 KD ShRNA treated and non-injected wt mice were evaluated at 4 months of age to confirm targeted-gene KD. (A) Representative IHC images of the CA1 region of hippocampus counter-stained with hematoxylin reveal normal, baseline GSK-3αβ expression levels in untreated and shRNA-scr treated mice. ShRNA-α treatment significantly reduced GSK-3α expression without altering GSK-3β expression and vice versa for shRNA-β treated mice. Neurons with reduced GSK-3 expression are highlighted by black circles and neurons expressing normal GSK-3 levels are highlighted by dashed black circles. (scale bar: 100 μm). (B) Immunoblot analyses of hippocampal lysates probed with GSK-3 isoform-specific antibodies. (C) Corresponding quantification showing that shRNA-α treatment significantly reduced GSK-3α expression without altering GSK-3β expression (F (3,12) =68.47, p

    Article Snippet: GSK-3 expression was monitored through the use of a GSK-3α rabbit pAb (9338) and GSK-3β rabbit mouse monoclonal antibody (mAb) (27C10) (Cell Signaling Technology).

    Techniques: shRNA, Injection, Mouse Assay, Immunohistochemistry, Staining, Expressing

    GSK-3α or GSK-3β KD reduces tau phosphorylation and conformational changes in PS19 +/− ;PDAPP +/− mice PS19 +/− ;PDAPP +/− neonates were uninjected (un) or injected with shRNA-α, -β or –scr and evaluated at 11 months of age (n=15 per group). (A) Representative IHC images counter-stained with hematoxylin show CA3 regions of posterior hippocampus stained with antibodies to total tau (T14), phospho-tau Ser202/Thr205 (AT8), phospho-tau Thr231 (AT180), phospho-tau Ser262 (12E8), or conformational specific antibodies Alz50, MC1 or ThS as indicated (scale bar: 100 μm). PS19 +/− ;PDAPP +/− mice treated with either shRNA-α, or -β showed a significant reduction in phospho-tau, Alz50, MC1 and ThS staining, but not in total tau load compared to shRNA-scr and non-injected controls. The phospho-tau burden in the CA3 region was quantified using ImageJ for mAb AT180 (B) and 12E8 (C) in n=3–4 mice/group, to demonstrate the quantification of > 50% reduction with shRNA-α and β treatment compared to controls (F (3,8) =8.283, p=0.0078 and F (3,9) =15.28, p=0.0007, respectively). Data show mean ± SEM. **p

    Journal: The Journal of Neuroscience

    Article Title: Selectively Silencing GSK-3 Isoforms Reduces Plaques and Tangles in Mouse Models of Alzheimer's disease

    doi: 10.1523/JNEUROSCI.0889-12.2012

    Figure Lengend Snippet: GSK-3α or GSK-3β KD reduces tau phosphorylation and conformational changes in PS19 +/− ;PDAPP +/− mice PS19 +/− ;PDAPP +/− neonates were uninjected (un) or injected with shRNA-α, -β or –scr and evaluated at 11 months of age (n=15 per group). (A) Representative IHC images counter-stained with hematoxylin show CA3 regions of posterior hippocampus stained with antibodies to total tau (T14), phospho-tau Ser202/Thr205 (AT8), phospho-tau Thr231 (AT180), phospho-tau Ser262 (12E8), or conformational specific antibodies Alz50, MC1 or ThS as indicated (scale bar: 100 μm). PS19 +/− ;PDAPP +/− mice treated with either shRNA-α, or -β showed a significant reduction in phospho-tau, Alz50, MC1 and ThS staining, but not in total tau load compared to shRNA-scr and non-injected controls. The phospho-tau burden in the CA3 region was quantified using ImageJ for mAb AT180 (B) and 12E8 (C) in n=3–4 mice/group, to demonstrate the quantification of > 50% reduction with shRNA-α and β treatment compared to controls (F (3,8) =8.283, p=0.0078 and F (3,9) =15.28, p=0.0007, respectively). Data show mean ± SEM. **p

    Article Snippet: GSK-3 expression was monitored through the use of a GSK-3α rabbit pAb (9338) and GSK-3β rabbit mouse monoclonal antibody (mAb) (27C10) (Cell Signaling Technology).

    Techniques: Mouse Assay, Injection, shRNA, Immunohistochemistry, Staining

    shRNA selection, rAAV distribution and expression, and lack of evidence of off-target effects N2a cells were transfected with hairpin constructs and selected with puromycin for seven days. As controls, N2a cells were transfected with an empty vector (VEC) or a scrambled hairpin control (scr). (A) Representative western blot of GSK-3α shRNA #1-#4 and (B) GSK-3β shRNA #1-#4 demonstrates that shRNA GSK-3α #2 showed the highest KD level (F (2,6) = 54.82, p

    Journal: The Journal of Neuroscience

    Article Title: Selectively Silencing GSK-3 Isoforms Reduces Plaques and Tangles in Mouse Models of Alzheimer's disease

    doi: 10.1523/JNEUROSCI.0889-12.2012

    Figure Lengend Snippet: shRNA selection, rAAV distribution and expression, and lack of evidence of off-target effects N2a cells were transfected with hairpin constructs and selected with puromycin for seven days. As controls, N2a cells were transfected with an empty vector (VEC) or a scrambled hairpin control (scr). (A) Representative western blot of GSK-3α shRNA #1-#4 and (B) GSK-3β shRNA #1-#4 demonstrates that shRNA GSK-3α #2 showed the highest KD level (F (2,6) = 54.82, p

    Article Snippet: GSK-3 expression was monitored through the use of a GSK-3α rabbit pAb (9338) and GSK-3β rabbit mouse monoclonal antibody (mAb) (27C10) (Cell Signaling Technology).

    Techniques: shRNA, Selection, Expressing, Transfection, Construct, Plasmid Preparation, Western Blot

    A schematic drawing of Integrin αvβ3/PI3K/Akt/GSK3β/β-catenin axis for microenvironment mediated TKI insensitivity in FLT3-ITD AML cells

    Journal: Oncotarget

    Article Title: Integrin alphavbeta3 enhances β-catenin signaling in acute myeloid leukemia harboring Fms-like tyrosine kinase-3 internal tandem duplication mutations: implications for microenvironment influence on sorafenib sensitivity

    doi: 10.18632/oncotarget.9617

    Figure Lengend Snippet: A schematic drawing of Integrin αvβ3/PI3K/Akt/GSK3β/β-catenin axis for microenvironment mediated TKI insensitivity in FLT3-ITD AML cells

    Article Snippet: Primary rabbit anti-human antibodies against β-catenin, phospho-Akt (Ser473), Akt, phospho-GSK3β (Ser9), GSK3β were purchased from Cell Signaling Technology, Inc (Beverley, MA).

    Techniques:

    OPN/αvβ3 activated β-catenin via PI3K/Akt/GSK3β signaling pathway A. MV4-11 was treated by various amount of recombinant human OPN for 24 hours, then β-catenin was detected by Western blot assay. B. MV4-11 was pretreated by αvβ3 blocking antibody (1μg/ml) for 2 hours, then treated by OPN (1.2 μg/ml) for another 24 hours. The phosphorylation level of Akt and GSK3β and the expression of β-catenin were measured by Western blot analysis. C. MV4-11 was pretreated by PI3K inhibitor LY294002 (25μM) for 2 hours, then treated by OPN (1.2 μg/ml) for another 24 hours. The phosphorylation level of Akt and GSK3β and the expression of β-catenin were measured by Western blotting analysis.

    Journal: Oncotarget

    Article Title: Integrin alphavbeta3 enhances β-catenin signaling in acute myeloid leukemia harboring Fms-like tyrosine kinase-3 internal tandem duplication mutations: implications for microenvironment influence on sorafenib sensitivity

    doi: 10.18632/oncotarget.9617

    Figure Lengend Snippet: OPN/αvβ3 activated β-catenin via PI3K/Akt/GSK3β signaling pathway A. MV4-11 was treated by various amount of recombinant human OPN for 24 hours, then β-catenin was detected by Western blot assay. B. MV4-11 was pretreated by αvβ3 blocking antibody (1μg/ml) for 2 hours, then treated by OPN (1.2 μg/ml) for another 24 hours. The phosphorylation level of Akt and GSK3β and the expression of β-catenin were measured by Western blot analysis. C. MV4-11 was pretreated by PI3K inhibitor LY294002 (25μM) for 2 hours, then treated by OPN (1.2 μg/ml) for another 24 hours. The phosphorylation level of Akt and GSK3β and the expression of β-catenin were measured by Western blotting analysis.

    Article Snippet: Primary rabbit anti-human antibodies against β-catenin, phospho-Akt (Ser473), Akt, phospho-GSK3β (Ser9), GSK3β were purchased from Cell Signaling Technology, Inc (Beverley, MA).

    Techniques: Recombinant, Western Blot, Blocking Assay, Expressing

    GSK-3β phosphorylates the C-terminal domain of p190A in vitro . A , p190A protein sequences from human, mouse, and rat were analyzed using Scansite software (high stringency), and GSK-3 kinase consensus sites were identified corresponding to amino acids Ser-1472, Ser-1476, and Thr-1480. Amino acid Ser-1483 was also identified as a potential p38 MAPK site. The percentile scores correspond to the probability that these putative sites are related to documented consensus sites by chance. B , full-length p190A baculovirus-expressed protein purified from Sf9 insect cells can be phosphorylated by GSK-3β in vitro . The GSK-3β kinase assay was performed in vitro as described under “Experimental Procedures,” and reaction products were resolved by SDS-PAGE and visualized by autoradiography. C , pEBG, pEBG-50AA-WT, pEBG-50AA-1472A, pEBG-50AA-1476A, pEBG-50AA-1480A, and pEBG-50AA-1483A were transfected into COS-7 cells, and the corresponding GST fusion proteins were purified on glutathione beads. In vitro kinase assays, with or without GSK-3β, were performed as indicated. The left panel corresponds to an immunoblot demonstrating levels of the various fusion protein proteins captured from the cell lysates. The right panel corresponds to the kinase reaction products following SDS-PAGE and autoradiography. D , phosphorylation of a domain containing the C-terminal 50 amino acids of p190A in vivo . The GST-50AA fusion proteins derived from p190A were purified either from E. coli (pGEX-KG-encoded) or from COS-7 cells (pEBG-encoded). The proteins on the glutathione beads were mock-treated, treated with λ-phosphatase, or treated with λ-phosphatase in the presence of phosphatase inhibitors ( PPI ). Then the beads were washed and boiled in sample buffer, and the proteins were resolved by SDS-PAGE and stained with Coomassie Blue. The faster migrating species are presumed to reflect degradation products derived from the GST fusion proteins. E , mass spectrometry analysis identified sites of phosphorylation within the C-terminal 50 amino acids of p190A. pEBG-50 AA-WT was transfected into COS-7 cells and purified as described under “Experimental Procedures.” The proteins were gel-purified, and proteolytically cleaved peptides were analyzed by mass spectrometry. The phosphorylation sites detected in vivo are underscored in three different peptide species that were detected, and these correspond to amino acids 1472, 1476, and 1483 in p190A.

    Journal: The Journal of Biological Chemistry

    Article Title: p190A RhoGAP Is a Glycogen Synthase Kinase-3-? Substrate Required for Polarized Cell Migration *p190A RhoGAP Is a Glycogen Synthase Kinase-3-? Substrate Required for Polarized Cell Migration * S⃞

    doi: 10.1074/jbc.M802588200

    Figure Lengend Snippet: GSK-3β phosphorylates the C-terminal domain of p190A in vitro . A , p190A protein sequences from human, mouse, and rat were analyzed using Scansite software (high stringency), and GSK-3 kinase consensus sites were identified corresponding to amino acids Ser-1472, Ser-1476, and Thr-1480. Amino acid Ser-1483 was also identified as a potential p38 MAPK site. The percentile scores correspond to the probability that these putative sites are related to documented consensus sites by chance. B , full-length p190A baculovirus-expressed protein purified from Sf9 insect cells can be phosphorylated by GSK-3β in vitro . The GSK-3β kinase assay was performed in vitro as described under “Experimental Procedures,” and reaction products were resolved by SDS-PAGE and visualized by autoradiography. C , pEBG, pEBG-50AA-WT, pEBG-50AA-1472A, pEBG-50AA-1476A, pEBG-50AA-1480A, and pEBG-50AA-1483A were transfected into COS-7 cells, and the corresponding GST fusion proteins were purified on glutathione beads. In vitro kinase assays, with or without GSK-3β, were performed as indicated. The left panel corresponds to an immunoblot demonstrating levels of the various fusion protein proteins captured from the cell lysates. The right panel corresponds to the kinase reaction products following SDS-PAGE and autoradiography. D , phosphorylation of a domain containing the C-terminal 50 amino acids of p190A in vivo . The GST-50AA fusion proteins derived from p190A were purified either from E. coli (pGEX-KG-encoded) or from COS-7 cells (pEBG-encoded). The proteins on the glutathione beads were mock-treated, treated with λ-phosphatase, or treated with λ-phosphatase in the presence of phosphatase inhibitors ( PPI ). Then the beads were washed and boiled in sample buffer, and the proteins were resolved by SDS-PAGE and stained with Coomassie Blue. The faster migrating species are presumed to reflect degradation products derived from the GST fusion proteins. E , mass spectrometry analysis identified sites of phosphorylation within the C-terminal 50 amino acids of p190A. pEBG-50 AA-WT was transfected into COS-7 cells and purified as described under “Experimental Procedures.” The proteins were gel-purified, and proteolytically cleaved peptides were analyzed by mass spectrometry. The phosphorylation sites detected in vivo are underscored in three different peptide species that were detected, and these correspond to amino acids 1472, 1476, and 1483 in p190A.

    Article Snippet: For immunoblotting, p190A RhoGAP antibody (BD Transduction Laboratories) was used at 1:1000; 12CA5 anti-hemagglutinin antibody, an ascites, was used at 1:1000; p120 RasGAP antibody B4F8 (Upstate) was used at 1:1000; GSK-3β antibody (BD Transduction laboratories) was used at 1:1000; and phospho-GSK-3β antibody (Cell Signaling Technology) was used at 1:1000.

    Techniques: In Vitro, Software, Purification, Kinase Assay, SDS Page, Autoradiography, Transfection, In Vivo, Derivative Assay, Staining, Mass Spectrometry

    GSK-3β phosphorylation inhibits p190A GAP activity. A, in vitro assay of p190A GAP activity toward the Rho GTPase, demonstrating that MAPK priming-dependent phosphorylation by GSK-3β reduced GAP activity as reflected in an increase in the percentage of [ 32 P]GTP-loaded GTPase retained on filters. Error bars reflect S.D. from four independent assays. B, in vitro phosphorylation reactions using [ 32 P]ATP, demonstrating the kinetics of priming and subsequent phosphorylation of p190A by GSK-3β following SDS-PAGE and autoradiography ( lower panel ). The upper panel demonstrates equivalent levels of p190A as shown by Coomassie Blue staining. Similar conditions (leading to maximal phosphorylation; 60 min priming and 60 min GSK-3β phosphorylation) were used to prepare the material for use in the assays presented in A. C , two distinct types of morphologies are seen in COS-7 cells transiently transfected with pEGFP-p190A-WT or the various indicated mutants. The left panel exemplifies a cell demonstrating strong RhoGAP activity, as evidenced by the rounded morphology and thin cellular processes. The fluorescent signal is derived from the GFP portion of the p190A fusion protein. D , the indicated p190A constructs were transiently transfected into COS-7 cells. Immunoblots demonstrate the protein levels of p190A in transfected cells. GSK-3β was immunoblotted as a loading control. E , quantification of the percentage of transfected (GFP-positive) cells exhibiting the morphology characteristic of strong RhoGAP activity for each of the various p190A constructs. Error bars indicate S.D. ( n = 200 for each construct). F , COS-7 cells were transiently transfected with either GFP or pEGFP-p190A-WT. At 40 h post-transfection, the cells were treated with DMSO, GSK-3β inhibitor XII, SB203580, U0126, or roscovitine for 4 h as indicated. The cells were then fixed and counted. The graph illustrates the average percentage of transfected cells exhibiting the strong RhoGAP phenotype (“GAP cells”) from three independent experiments, with the error bars representing S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: p190A RhoGAP Is a Glycogen Synthase Kinase-3-? Substrate Required for Polarized Cell Migration *p190A RhoGAP Is a Glycogen Synthase Kinase-3-? Substrate Required for Polarized Cell Migration * S⃞

    doi: 10.1074/jbc.M802588200

    Figure Lengend Snippet: GSK-3β phosphorylation inhibits p190A GAP activity. A, in vitro assay of p190A GAP activity toward the Rho GTPase, demonstrating that MAPK priming-dependent phosphorylation by GSK-3β reduced GAP activity as reflected in an increase in the percentage of [ 32 P]GTP-loaded GTPase retained on filters. Error bars reflect S.D. from four independent assays. B, in vitro phosphorylation reactions using [ 32 P]ATP, demonstrating the kinetics of priming and subsequent phosphorylation of p190A by GSK-3β following SDS-PAGE and autoradiography ( lower panel ). The upper panel demonstrates equivalent levels of p190A as shown by Coomassie Blue staining. Similar conditions (leading to maximal phosphorylation; 60 min priming and 60 min GSK-3β phosphorylation) were used to prepare the material for use in the assays presented in A. C , two distinct types of morphologies are seen in COS-7 cells transiently transfected with pEGFP-p190A-WT or the various indicated mutants. The left panel exemplifies a cell demonstrating strong RhoGAP activity, as evidenced by the rounded morphology and thin cellular processes. The fluorescent signal is derived from the GFP portion of the p190A fusion protein. D , the indicated p190A constructs were transiently transfected into COS-7 cells. Immunoblots demonstrate the protein levels of p190A in transfected cells. GSK-3β was immunoblotted as a loading control. E , quantification of the percentage of transfected (GFP-positive) cells exhibiting the morphology characteristic of strong RhoGAP activity for each of the various p190A constructs. Error bars indicate S.D. ( n = 200 for each construct). F , COS-7 cells were transiently transfected with either GFP or pEGFP-p190A-WT. At 40 h post-transfection, the cells were treated with DMSO, GSK-3β inhibitor XII, SB203580, U0126, or roscovitine for 4 h as indicated. The cells were then fixed and counted. The graph illustrates the average percentage of transfected cells exhibiting the strong RhoGAP phenotype (“GAP cells”) from three independent experiments, with the error bars representing S.D.

    Article Snippet: For immunoblotting, p190A RhoGAP antibody (BD Transduction Laboratories) was used at 1:1000; 12CA5 anti-hemagglutinin antibody, an ascites, was used at 1:1000; p120 RasGAP antibody B4F8 (Upstate) was used at 1:1000; GSK-3β antibody (BD Transduction laboratories) was used at 1:1000; and phospho-GSK-3β antibody (Cell Signaling Technology) was used at 1:1000.

    Techniques: Activity Assay, In Vitro, SDS Page, Autoradiography, Staining, Transfection, Derivative Assay, Construct, Western Blot

    Mapping the p190A priming sites. A , mapping the sites of p38 and p42 MAPK phosphorylation on p190A. pEBG (vector) or pEBG-50AA-WT and the indicated phosphorylation site mutant proteins were captured from transfected COS-7 cells, and the proteins on beads were incubated with either p38MAPK or p42MAPK and [γ- 32 P]ATP. The leftmost lane corresponds to the pEBG-WT protein with buffer only. Reaction products were analyzed by SDS-PAGE and autoradiography ( upper panels ) and Coomassie Blue staining ( lower panels ). B , mapping the priming sites on p190A in vivo . pEBG-50AA-WT and the various phosphorylation site mutant proteins were purified from transfected COS-7 cells as indicated. The proteins on beads were then either mock-treated or prephosphorylated in vitro by either p38 or p42 MAPK in the presence of cold ATP. They were then incubated with GSK-3β in the presence of [γ- 32 P]ATP. Reaction products were analyzed by SDS-PAGE and autoradiography. C , inhibiting p38MAPK in the priming lysate disrupts priming activity. pEBG-50AA-WT proteins were purified from COS-7 cells as described under “Experimental Procedures.” For priming, following cell lysis the lysates were preincubated with either DMSO (vehicle control) or the p38 MAPK inhibitor SB203580. The lysates were then used to prime the pEBG-50AA-WT proteins on beads. The beads were then processed for GSK-3β kinase assays using [γ- 32 P]ATP. Reaction products were analyzed by SDS-PAGE and autoradiography ( upper panel ) and Coomassie Blue staining ( lower panel ).

    Journal: The Journal of Biological Chemistry

    Article Title: p190A RhoGAP Is a Glycogen Synthase Kinase-3-? Substrate Required for Polarized Cell Migration *p190A RhoGAP Is a Glycogen Synthase Kinase-3-? Substrate Required for Polarized Cell Migration * S⃞

    doi: 10.1074/jbc.M802588200

    Figure Lengend Snippet: Mapping the p190A priming sites. A , mapping the sites of p38 and p42 MAPK phosphorylation on p190A. pEBG (vector) or pEBG-50AA-WT and the indicated phosphorylation site mutant proteins were captured from transfected COS-7 cells, and the proteins on beads were incubated with either p38MAPK or p42MAPK and [γ- 32 P]ATP. The leftmost lane corresponds to the pEBG-WT protein with buffer only. Reaction products were analyzed by SDS-PAGE and autoradiography ( upper panels ) and Coomassie Blue staining ( lower panels ). B , mapping the priming sites on p190A in vivo . pEBG-50AA-WT and the various phosphorylation site mutant proteins were purified from transfected COS-7 cells as indicated. The proteins on beads were then either mock-treated or prephosphorylated in vitro by either p38 or p42 MAPK in the presence of cold ATP. They were then incubated with GSK-3β in the presence of [γ- 32 P]ATP. Reaction products were analyzed by SDS-PAGE and autoradiography. C , inhibiting p38MAPK in the priming lysate disrupts priming activity. pEBG-50AA-WT proteins were purified from COS-7 cells as described under “Experimental Procedures.” For priming, following cell lysis the lysates were preincubated with either DMSO (vehicle control) or the p38 MAPK inhibitor SB203580. The lysates were then used to prime the pEBG-50AA-WT proteins on beads. The beads were then processed for GSK-3β kinase assays using [γ- 32 P]ATP. Reaction products were analyzed by SDS-PAGE and autoradiography ( upper panel ) and Coomassie Blue staining ( lower panel ).

    Article Snippet: For immunoblotting, p190A RhoGAP antibody (BD Transduction Laboratories) was used at 1:1000; 12CA5 anti-hemagglutinin antibody, an ascites, was used at 1:1000; p120 RasGAP antibody B4F8 (Upstate) was used at 1:1000; GSK-3β antibody (BD Transduction laboratories) was used at 1:1000; and phospho-GSK-3β antibody (Cell Signaling Technology) was used at 1:1000.

    Techniques: Plasmid Preparation, Mutagenesis, Transfection, Incubation, SDS Page, Autoradiography, Staining, In Vivo, Purification, In Vitro, Activity Assay, Lysis

    Priming-dependent phosphorylation of p190A by GSK-3 β. A , priming is required for p190A phosphorylation by GSK-3β. Bacterially purified p190A-50AA (pGEX-KG-50AA-WT) on beads was treated either with or without priming by COS-7 lysate. The beads were washed extensively and then subjected to in vitro phosphorylation by GSK-3β. p190A-50AA (pEBG-50AA-WT) expressed in transfected COS-7 cells was purified on glutathione beads, pretreated with either λ-phosphatase, λ-phosphatase plus phosphatase inhibitors ( PPI ), or phosphatase inhibitors alone. The beads were washed extensively and then subjected to in vitro phosphorylation by GSK-3β. Reaction products were resolved by SDS-PAGE and autoradiography. B , p38 or p42 MAPK, but not CKI, can prime the C-terminal domain of p190A for subsequent phosphorylation by GSK-3β in vitro . Bacterially purified p190A-50AA (pGEX-KG-50AA-WT) was first phosphorylated in vitro by purified recombinant p38MAPK, p42 MAPK, or CKI in the presence of cold ATP. Proteins were then subjected to GSK-3β kinase assays using [γ- 32 P]ATP, and products were resolved by SDS-PAGE and visualized by autoradiography. Note the absolute requirement for priming by MAPKs.

    Journal: The Journal of Biological Chemistry

    Article Title: p190A RhoGAP Is a Glycogen Synthase Kinase-3-? Substrate Required for Polarized Cell Migration *p190A RhoGAP Is a Glycogen Synthase Kinase-3-? Substrate Required for Polarized Cell Migration * S⃞

    doi: 10.1074/jbc.M802588200

    Figure Lengend Snippet: Priming-dependent phosphorylation of p190A by GSK-3 β. A , priming is required for p190A phosphorylation by GSK-3β. Bacterially purified p190A-50AA (pGEX-KG-50AA-WT) on beads was treated either with or without priming by COS-7 lysate. The beads were washed extensively and then subjected to in vitro phosphorylation by GSK-3β. p190A-50AA (pEBG-50AA-WT) expressed in transfected COS-7 cells was purified on glutathione beads, pretreated with either λ-phosphatase, λ-phosphatase plus phosphatase inhibitors ( PPI ), or phosphatase inhibitors alone. The beads were washed extensively and then subjected to in vitro phosphorylation by GSK-3β. Reaction products were resolved by SDS-PAGE and autoradiography. B , p38 or p42 MAPK, but not CKI, can prime the C-terminal domain of p190A for subsequent phosphorylation by GSK-3β in vitro . Bacterially purified p190A-50AA (pGEX-KG-50AA-WT) was first phosphorylated in vitro by purified recombinant p38MAPK, p42 MAPK, or CKI in the presence of cold ATP. Proteins were then subjected to GSK-3β kinase assays using [γ- 32 P]ATP, and products were resolved by SDS-PAGE and visualized by autoradiography. Note the absolute requirement for priming by MAPKs.

    Article Snippet: For immunoblotting, p190A RhoGAP antibody (BD Transduction Laboratories) was used at 1:1000; 12CA5 anti-hemagglutinin antibody, an ascites, was used at 1:1000; p120 RasGAP antibody B4F8 (Upstate) was used at 1:1000; GSK-3β antibody (BD Transduction laboratories) was used at 1:1000; and phospho-GSK-3β antibody (Cell Signaling Technology) was used at 1:1000.

    Techniques: Purification, In Vitro, Transfection, SDS Page, Autoradiography, Recombinant

    GSK-3β phosphorylation-defective p190A mutants fail to rescue the directional migration defects in p190A-deficient cells. A , stable cell lines derived from p190A -/- fibroblasts, expressing either pEGFP-C1 vector alone or with pEGFP-C1-p190A-WT (full-length) or the various phosphorylation site mutants, were established. Immunoblots demonstrate expression of p190A. p120 RasGAP was also detected as a loading control. B , wound closure assays of the various stable cell lines as indicated. Photomicrographs were taken immediately after and 15 h after wounding. The dashed lines correspond to the boundaries of cell monolayers along the wound edge. Note that the wound closure defect can be rescued by wild-type p190A and the 1472A and 1483A mutants but not by the 1476A and 1480A mutants. C , Golgi reorientation 6 h post-wounding with the indicated p190A-expressing cell lines as indicated. Cells were seeded on coverslips and grown to confluence. A single wound was induced, and the cells were fixed at 6 h post-wounding. They were then double-stained with anti-GM130 and DAPI, and immunofluorescence microscopy was performed. For each genotype, 200 cells were counted for two individual lines. Graphs depict the mean from three independent experiments, and S.D. are indicated by the error bars . The dashed horizontal line corresponds to the 33% value, which would reflect random polarization. Note that the 1476A and 1480A mutants are defective for Golgi reorientation. D , F-actin staining (phalloidin) at 6 h post-wounding of the indicated p190A-expressing cell lines. White arrows highlight the oriented protrusions along the leading edge.

    Journal: The Journal of Biological Chemistry

    Article Title: p190A RhoGAP Is a Glycogen Synthase Kinase-3-? Substrate Required for Polarized Cell Migration *p190A RhoGAP Is a Glycogen Synthase Kinase-3-? Substrate Required for Polarized Cell Migration * S⃞

    doi: 10.1074/jbc.M802588200

    Figure Lengend Snippet: GSK-3β phosphorylation-defective p190A mutants fail to rescue the directional migration defects in p190A-deficient cells. A , stable cell lines derived from p190A -/- fibroblasts, expressing either pEGFP-C1 vector alone or with pEGFP-C1-p190A-WT (full-length) or the various phosphorylation site mutants, were established. Immunoblots demonstrate expression of p190A. p120 RasGAP was also detected as a loading control. B , wound closure assays of the various stable cell lines as indicated. Photomicrographs were taken immediately after and 15 h after wounding. The dashed lines correspond to the boundaries of cell monolayers along the wound edge. Note that the wound closure defect can be rescued by wild-type p190A and the 1472A and 1483A mutants but not by the 1476A and 1480A mutants. C , Golgi reorientation 6 h post-wounding with the indicated p190A-expressing cell lines as indicated. Cells were seeded on coverslips and grown to confluence. A single wound was induced, and the cells were fixed at 6 h post-wounding. They were then double-stained with anti-GM130 and DAPI, and immunofluorescence microscopy was performed. For each genotype, 200 cells were counted for two individual lines. Graphs depict the mean from three independent experiments, and S.D. are indicated by the error bars . The dashed horizontal line corresponds to the 33% value, which would reflect random polarization. Note that the 1476A and 1480A mutants are defective for Golgi reorientation. D , F-actin staining (phalloidin) at 6 h post-wounding of the indicated p190A-expressing cell lines. White arrows highlight the oriented protrusions along the leading edge.

    Article Snippet: For immunoblotting, p190A RhoGAP antibody (BD Transduction Laboratories) was used at 1:1000; 12CA5 anti-hemagglutinin antibody, an ascites, was used at 1:1000; p120 RasGAP antibody B4F8 (Upstate) was used at 1:1000; GSK-3β antibody (BD Transduction laboratories) was used at 1:1000; and phospho-GSK-3β antibody (Cell Signaling Technology) was used at 1:1000.

    Techniques: Migration, Stable Transfection, Derivative Assay, Expressing, Plasmid Preparation, Western Blot, Staining, Immunofluorescence, Microscopy

    Both GSK3β and hnRNPK antagonized TRAIL-induced apoptosis. H1299 cells were transfected with siRNA against GSK3β ( a ), Flag-GSK3β ( b ), siRNA against hnRNPK ( c ), or Flag-hnRNPK ( d ), and treated with TRAIL (20 ng/ml, 8 hours), then subjected to Western blot analysis with the indicated antibodies. ( e,f ) Analysis of the cell death rate by Flow cytometry. H1299 cells transfected with GSK3β siRNA or hnRNPK siRNA ( e ), or transfected with Flag-GSK3β or Flag-hnRNPK ( f ) were treated with TRAIL (20 ng/ml, 8 hours) and analyzed using Annexin V/PI double staining with a FACS Calibure flow cytometer. ( g,h ) Statistical analyses of the experiments shown in ( e , f ) respectively. The analyses were performed with the results of three independent replicates for each experiment. *p

    Journal: Scientific Reports

    Article Title: hnRNPK inhibits GSK3β Ser9 phosphorylation, thereby stabilizing c-FLIP and contributes to TRAIL resistance in H1299 lung adenocarcinoma cells

    doi: 10.1038/srep22999

    Figure Lengend Snippet: Both GSK3β and hnRNPK antagonized TRAIL-induced apoptosis. H1299 cells were transfected with siRNA against GSK3β ( a ), Flag-GSK3β ( b ), siRNA against hnRNPK ( c ), or Flag-hnRNPK ( d ), and treated with TRAIL (20 ng/ml, 8 hours), then subjected to Western blot analysis with the indicated antibodies. ( e,f ) Analysis of the cell death rate by Flow cytometry. H1299 cells transfected with GSK3β siRNA or hnRNPK siRNA ( e ), or transfected with Flag-GSK3β or Flag-hnRNPK ( f ) were treated with TRAIL (20 ng/ml, 8 hours) and analyzed using Annexin V/PI double staining with a FACS Calibure flow cytometer. ( g,h ) Statistical analyses of the experiments shown in ( e , f ) respectively. The analyses were performed with the results of three independent replicates for each experiment. *p

    Article Snippet: The co-immunoprecipitation assay were carried out with the protocols described previously using the GSK3β antibody (27C10, Cell Signaling Technology).

    Techniques: Transfection, Western Blot, Flow Cytometry, Cytometry, Double Staining, FACS

    Both GSK3β and hnRNPK antagonized TRAIL-induced apoptosis. ( a ) MTT assays of cellular viability with TRAIL treatment at different concentrations. H1299 cells were seed into 96-well plate (4000 cells per well) and treated with TRAIL at increasing concentrations as indicated for 24 hours. MTT assays were then performed as described in Method section. The cellular viability was monitored by the absorbance of formazan crystals at 570 nm. The absorbance of the control group was set as 100%. *p

    Journal: Scientific Reports

    Article Title: hnRNPK inhibits GSK3β Ser9 phosphorylation, thereby stabilizing c-FLIP and contributes to TRAIL resistance in H1299 lung adenocarcinoma cells

    doi: 10.1038/srep22999

    Figure Lengend Snippet: Both GSK3β and hnRNPK antagonized TRAIL-induced apoptosis. ( a ) MTT assays of cellular viability with TRAIL treatment at different concentrations. H1299 cells were seed into 96-well plate (4000 cells per well) and treated with TRAIL at increasing concentrations as indicated for 24 hours. MTT assays were then performed as described in Method section. The cellular viability was monitored by the absorbance of formazan crystals at 570 nm. The absorbance of the control group was set as 100%. *p

    Article Snippet: The co-immunoprecipitation assay were carried out with the protocols described previously using the GSK3β antibody (27C10, Cell Signaling Technology).

    Techniques: MTT Assay

    hnRNPK stabilized of c-FLIP protein through inhibition of GSK3β Ser9 phosphorylation during the TRAIL-induced apoptosis. ( a ) GSK3β upregulated the protein level of c-FLIP in H1299 cells treated with TRAIL. H1299 cells treated with LiCl (20 mM, 8 hours) and/or TRAIL (20 ng/ml, 8 hours) were harvested for Western blot analysis with the indicated antibodies. ( b ) Effect of hnRNPK overexpression on c-FLIP protein stability by Cycloheximide (CHX) chase experiments. H1299 cells transfected with either 2 μg Flag-hnRNPK or Flag-vector plasmids were treated with 10 μg/ml CHX for the indicated durations, then subjected to Western blot analysis with the indicated antibodies. ( c ) Effect of hnRNPK overexpression on c-FLIP protein stability by dose-response experiment. H1299 cells were transfected with Flag-hnRNPK or Flag-vector plasmids at the indicated doses and stimulated with CHX (10 μg/ml, 2 h), then subjected to Western blot analysis with the indicated antibodies. ( d ) Effect of hnRNPK on c-FLIP protein stability was dependent on the Ser9 phosphorylation state of GSK3β. H1299 cells transfected with Flag-hnRNPK or Flag-vector plasmids were stimulated with CHX (10 μg/ml, 2 h), TRAIL (20 ng/ml, 8 hours), or LiCl (20 mM, 8 hours) as indicated, then subjected to Western blot analysis with the indicated antibodies.

    Journal: Scientific Reports

    Article Title: hnRNPK inhibits GSK3β Ser9 phosphorylation, thereby stabilizing c-FLIP and contributes to TRAIL resistance in H1299 lung adenocarcinoma cells

    doi: 10.1038/srep22999

    Figure Lengend Snippet: hnRNPK stabilized of c-FLIP protein through inhibition of GSK3β Ser9 phosphorylation during the TRAIL-induced apoptosis. ( a ) GSK3β upregulated the protein level of c-FLIP in H1299 cells treated with TRAIL. H1299 cells treated with LiCl (20 mM, 8 hours) and/or TRAIL (20 ng/ml, 8 hours) were harvested for Western blot analysis with the indicated antibodies. ( b ) Effect of hnRNPK overexpression on c-FLIP protein stability by Cycloheximide (CHX) chase experiments. H1299 cells transfected with either 2 μg Flag-hnRNPK or Flag-vector plasmids were treated with 10 μg/ml CHX for the indicated durations, then subjected to Western blot analysis with the indicated antibodies. ( c ) Effect of hnRNPK overexpression on c-FLIP protein stability by dose-response experiment. H1299 cells were transfected with Flag-hnRNPK or Flag-vector plasmids at the indicated doses and stimulated with CHX (10 μg/ml, 2 h), then subjected to Western blot analysis with the indicated antibodies. ( d ) Effect of hnRNPK on c-FLIP protein stability was dependent on the Ser9 phosphorylation state of GSK3β. H1299 cells transfected with Flag-hnRNPK or Flag-vector plasmids were stimulated with CHX (10 μg/ml, 2 h), TRAIL (20 ng/ml, 8 hours), or LiCl (20 mM, 8 hours) as indicated, then subjected to Western blot analysis with the indicated antibodies.

    Article Snippet: The co-immunoprecipitation assay were carried out with the protocols described previously using the GSK3β antibody (27C10, Cell Signaling Technology).

    Techniques: Inhibition, Western Blot, Over Expression, Transfection, Plasmid Preparation

    hnRNPK inhibited GSK3β Ser9 phosphorylation by PKC. H1299 cells respectively transfected with Flag-hnRNPK ( a ) or hnRNPK siRNA ( b ), and treated with or without TRAIL (20 ng/ml, 8 hours) were harvested for Western blot analysis with the indicated antibodies. ( c ) PKC inhibitor Gö6983 cancelled the regulatory effect of hnRNPK on GSK3β Ser9 phosphorylation. H1299 cells transfected with hnRNPK siRNA or/and treated with Gö6983 (1 μM) were analyzed by Western blotting with the indicated antibodies. ( d ) PKC inhibitor Rottlerin had no obvious effect on the regulation of GSK3β Ser9 phosphorylation by hnRNPK. H1299 cells transfected with hnRNPK siRNA or/and treated with Rottlerin (6 μM) were analyzed by Western blotting with the indicated antibodies. ( e ) PP1 inhibitor Okadaic acid did not affect the regulation of GSK3β Ser9 phosphorylation by hnRNPK. H1299 cells transfected with Flag-hnRNPK or/and treated with okadaic acid (100 nM) were analyzed by Western blotting with the indicated antibodies.

    Journal: Scientific Reports

    Article Title: hnRNPK inhibits GSK3β Ser9 phosphorylation, thereby stabilizing c-FLIP and contributes to TRAIL resistance in H1299 lung adenocarcinoma cells

    doi: 10.1038/srep22999

    Figure Lengend Snippet: hnRNPK inhibited GSK3β Ser9 phosphorylation by PKC. H1299 cells respectively transfected with Flag-hnRNPK ( a ) or hnRNPK siRNA ( b ), and treated with or without TRAIL (20 ng/ml, 8 hours) were harvested for Western blot analysis with the indicated antibodies. ( c ) PKC inhibitor Gö6983 cancelled the regulatory effect of hnRNPK on GSK3β Ser9 phosphorylation. H1299 cells transfected with hnRNPK siRNA or/and treated with Gö6983 (1 μM) were analyzed by Western blotting with the indicated antibodies. ( d ) PKC inhibitor Rottlerin had no obvious effect on the regulation of GSK3β Ser9 phosphorylation by hnRNPK. H1299 cells transfected with hnRNPK siRNA or/and treated with Rottlerin (6 μM) were analyzed by Western blotting with the indicated antibodies. ( e ) PP1 inhibitor Okadaic acid did not affect the regulation of GSK3β Ser9 phosphorylation by hnRNPK. H1299 cells transfected with Flag-hnRNPK or/and treated with okadaic acid (100 nM) were analyzed by Western blotting with the indicated antibodies.

    Article Snippet: The co-immunoprecipitation assay were carried out with the protocols described previously using the GSK3β antibody (27C10, Cell Signaling Technology).

    Techniques: Transfection, Western Blot

    hnRNPK expression negatively correlates with Ser9 phosphorylated GSK3β in tissue microarrays (TMAs). TMAs of lung adenocarcinoma (n = 52) were immunohistochemically scored and statistically analyzed for the cytoplasmic hnRNPK ( a ), nuclear hnRNPK ( b ), total hnRNPK ( c ), and total phospho-GSK3β ( d ). **p

    Journal: Scientific Reports

    Article Title: hnRNPK inhibits GSK3β Ser9 phosphorylation, thereby stabilizing c-FLIP and contributes to TRAIL resistance in H1299 lung adenocarcinoma cells

    doi: 10.1038/srep22999

    Figure Lengend Snippet: hnRNPK expression negatively correlates with Ser9 phosphorylated GSK3β in tissue microarrays (TMAs). TMAs of lung adenocarcinoma (n = 52) were immunohistochemically scored and statistically analyzed for the cytoplasmic hnRNPK ( a ), nuclear hnRNPK ( b ), total hnRNPK ( c ), and total phospho-GSK3β ( d ). **p

    Article Snippet: The co-immunoprecipitation assay were carried out with the protocols described previously using the GSK3β antibody (27C10, Cell Signaling Technology).

    Techniques: Expressing

    Interaction and co-localization of GSK3β with hnRNPK in H1299 cells. ( a ) GST-hnRNPK pull-down assay. H1299 cell lysates were incubated with GST or GST-hnRNPK fusion proteins immobilized on glutathione sepharose beads as described. Western blotting was then performed with GSK3β and phospho-GSK3β (Ser9) antibodies to detect the presence of GSK3β and phospho-GSK3β (Ser9) in the bound fractions. The amounts of recombinant proteins GST and GST-hnRNPK were estimated by CBB staining. ( b ) Co-immunoprecipitation assay of the interaction of GSK3β with hnRNPK. H1299 cells were transfected with Flag-hnRNPK or empty Flag-vector as indicated and treated with TRAIL (20 ng/ml, 8 hours). The existence of Flag-hnRNPK and GSK3β in the Sepharose-beads bound fractions were analyzed by immunoblot (IB) with Flag and GSK3β antibodies. WCL: whole cell lysates. ( c ) Subcellular localization of GSK3β and hnRNPK in H1299 cells after TRAIL treatment. H1299 cells treated with or without TRAIL (20 ng/ml, 8 hours) were fixed and stained with both GSK3β and hnRNPK antibodies. The Alexa Fluor 488 and Alexa Fluor 594 conjugated secondary antibody were used. Arrows: the co-localization of the two proteins. Bar: 5 μm. ( d ) Subcellular localization of GSK3β and ectopically expressed hnRNPK in H1299 cells after TRAIL treatment. H1299 cells transfected with Flag-hnRNPK were treated with TRAIL (20 ng/ml, 8 hours), then fixed and stained with both GSK3β and Flag antibodies. The same secondary antibodies used in ( c ) were used. Arrows: the co-localization of the two proteins. Bar: 10 μm.

    Journal: Scientific Reports

    Article Title: hnRNPK inhibits GSK3β Ser9 phosphorylation, thereby stabilizing c-FLIP and contributes to TRAIL resistance in H1299 lung adenocarcinoma cells

    doi: 10.1038/srep22999

    Figure Lengend Snippet: Interaction and co-localization of GSK3β with hnRNPK in H1299 cells. ( a ) GST-hnRNPK pull-down assay. H1299 cell lysates were incubated with GST or GST-hnRNPK fusion proteins immobilized on glutathione sepharose beads as described. Western blotting was then performed with GSK3β and phospho-GSK3β (Ser9) antibodies to detect the presence of GSK3β and phospho-GSK3β (Ser9) in the bound fractions. The amounts of recombinant proteins GST and GST-hnRNPK were estimated by CBB staining. ( b ) Co-immunoprecipitation assay of the interaction of GSK3β with hnRNPK. H1299 cells were transfected with Flag-hnRNPK or empty Flag-vector as indicated and treated with TRAIL (20 ng/ml, 8 hours). The existence of Flag-hnRNPK and GSK3β in the Sepharose-beads bound fractions were analyzed by immunoblot (IB) with Flag and GSK3β antibodies. WCL: whole cell lysates. ( c ) Subcellular localization of GSK3β and hnRNPK in H1299 cells after TRAIL treatment. H1299 cells treated with or without TRAIL (20 ng/ml, 8 hours) were fixed and stained with both GSK3β and hnRNPK antibodies. The Alexa Fluor 488 and Alexa Fluor 594 conjugated secondary antibody were used. Arrows: the co-localization of the two proteins. Bar: 5 μm. ( d ) Subcellular localization of GSK3β and ectopically expressed hnRNPK in H1299 cells after TRAIL treatment. H1299 cells transfected with Flag-hnRNPK were treated with TRAIL (20 ng/ml, 8 hours), then fixed and stained with both GSK3β and Flag antibodies. The same secondary antibodies used in ( c ) were used. Arrows: the co-localization of the two proteins. Bar: 10 μm.

    Article Snippet: The co-immunoprecipitation assay were carried out with the protocols described previously using the GSK3β antibody (27C10, Cell Signaling Technology).

    Techniques: Pull Down Assay, Incubation, Western Blot, Recombinant, Staining, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation

    PDA-66 does not inhibit GSK3β kinase activity. Recombinant human GSK3β was incubated with pGS2, ATP and different concentrations of PDA-66 and SB-216763. While treatment with the known GSK3β inhibitor SB-216763 lead to a stable reduction of enzyme activity with significant alterations at 5 μM PDA-66 showed only a slight inhibitory potential. Results are displayed as the mean ± SD of five independent experiments. In each experiment the concentrations of PDA-66 and the control were tested with 4 replicates. RLU = relative luminescence units. *Significant treatment effect vs. DMSO control, α = 0.05.

    Journal: BMC Cancer

    Article Title: The novel arylindolylmaleimide PDA-66 displays pronounced antiproliferative effects in acute lymphoblastic leukemia cells

    doi: 10.1186/1471-2407-14-71

    Figure Lengend Snippet: PDA-66 does not inhibit GSK3β kinase activity. Recombinant human GSK3β was incubated with pGS2, ATP and different concentrations of PDA-66 and SB-216763. While treatment with the known GSK3β inhibitor SB-216763 lead to a stable reduction of enzyme activity with significant alterations at 5 μM PDA-66 showed only a slight inhibitory potential. Results are displayed as the mean ± SD of five independent experiments. In each experiment the concentrations of PDA-66 and the control were tested with 4 replicates. RLU = relative luminescence units. *Significant treatment effect vs. DMSO control, α = 0.05.

    Article Snippet: Briefly, 20 ng of recombinant human GSK3β (Biomol, Hamburg, Germany) were incubated with the substrate phospho glycogen synthase peptide 2 (pGS2, 25 μM) (Millipore, Billerica, USA), ATP (1 μM) (Cell Signaling, Frankfurt am Main, Germany) and different concentrations of PDA-66 and SB-216763 for 30 min at 30°C.

    Techniques: Activity Assay, Recombinant, Incubation

    PDA-66 does not influence expression of proteins of Wnt/β-catenin pathway but alters expression of 4EBP-1. After treatment with PDA-66 and DMSO, respectively, cells were lyzed and protein expression analyzed with Western blot. (A) Exemplary results of PDA-66 treated SEM cells after 4 and 24 h are displayed. No influence on expression of total GSK3β and the total form of Akt could be noticed. PhosphoGSK3βSer9 seemed decreased at higher PDA-66 concentrations after 4 h. No influence on the amount of β-catenin was observed. (B) Exemplary results of SEM, RS4;11 and MOLT4 cells are displayed. In SEM and RS4;11 cells a decrease of 4EBP-1 and p4EBP-1Ser65 was detectable, in contrast MOLT4 cells showed an increased expression of p4EBP-1Ser65 after PDA-66 treatment.

    Journal: BMC Cancer

    Article Title: The novel arylindolylmaleimide PDA-66 displays pronounced antiproliferative effects in acute lymphoblastic leukemia cells

    doi: 10.1186/1471-2407-14-71

    Figure Lengend Snippet: PDA-66 does not influence expression of proteins of Wnt/β-catenin pathway but alters expression of 4EBP-1. After treatment with PDA-66 and DMSO, respectively, cells were lyzed and protein expression analyzed with Western blot. (A) Exemplary results of PDA-66 treated SEM cells after 4 and 24 h are displayed. No influence on expression of total GSK3β and the total form of Akt could be noticed. PhosphoGSK3βSer9 seemed decreased at higher PDA-66 concentrations after 4 h. No influence on the amount of β-catenin was observed. (B) Exemplary results of SEM, RS4;11 and MOLT4 cells are displayed. In SEM and RS4;11 cells a decrease of 4EBP-1 and p4EBP-1Ser65 was detectable, in contrast MOLT4 cells showed an increased expression of p4EBP-1Ser65 after PDA-66 treatment.

    Article Snippet: Briefly, 20 ng of recombinant human GSK3β (Biomol, Hamburg, Germany) were incubated with the substrate phospho glycogen synthase peptide 2 (pGS2, 25 μM) (Millipore, Billerica, USA), ATP (1 μM) (Cell Signaling, Frankfurt am Main, Germany) and different concentrations of PDA-66 and SB-216763 for 30 min at 30°C.

    Techniques: Expressing, Western Blot

    Increased PLK2 expression and activation of GSK-NRF2 pathway are associated with mitochondrial dysfunction in SCO2 deficient cells ( A ) Protein levels of SCO2, PLK2, and serine phosphorylated GSK3 (GSK3-S-P) (comprised of GSK3A and GSK3B, commonly known as α and β, respectively) in whole cell lysates, and NRF2 in the nuclear fraction (nuc) of the indicated cell lines. Tubulin and lamin B1 (LMNB1) serve as whole cell and nuclear fraction protein loading controls, respectively. ( B ) In vitro phosphorylation of GSK3B by PLK2 using purified recombinant proteins. Note the reciprocal relationship between the levels of the inactivating Ser-9 phosphorylation by PLK2 and the activating Tyr-216 autophosphorylation of GSK3B (GSK3β-S9-P and GSK3β-Y216-P, respectively). ( C ) Subcellular localization of NRF2 by confocal immunofluorescence (upper and middle panels) with DAPI staining of the nuclei (lower panel). The upper panels show enlarged images of the cells enclosed by white rectangles in the middle panels. At least 300 cells were counted in three independent experiments to quantify the fraction of cells with nuclear NRF2 localization. * P

    Journal: Free radical biology & medicine

    Article Title: Polo-like kinase 2 activates an antioxidant pathway to promote the survival of cells with mitochondrial dysfunction

    doi: 10.1016/j.freeradbiomed.2014.05.022

    Figure Lengend Snippet: Increased PLK2 expression and activation of GSK-NRF2 pathway are associated with mitochondrial dysfunction in SCO2 deficient cells ( A ) Protein levels of SCO2, PLK2, and serine phosphorylated GSK3 (GSK3-S-P) (comprised of GSK3A and GSK3B, commonly known as α and β, respectively) in whole cell lysates, and NRF2 in the nuclear fraction (nuc) of the indicated cell lines. Tubulin and lamin B1 (LMNB1) serve as whole cell and nuclear fraction protein loading controls, respectively. ( B ) In vitro phosphorylation of GSK3B by PLK2 using purified recombinant proteins. Note the reciprocal relationship between the levels of the inactivating Ser-9 phosphorylation by PLK2 and the activating Tyr-216 autophosphorylation of GSK3B (GSK3β-S9-P and GSK3β-Y216-P, respectively). ( C ) Subcellular localization of NRF2 by confocal immunofluorescence (upper and middle panels) with DAPI staining of the nuclei (lower panel). The upper panels show enlarged images of the cells enclosed by white rectangles in the middle panels. At least 300 cells were counted in three independent experiments to quantify the fraction of cells with nuclear NRF2 localization. * P

    Article Snippet: Recombinant GSK3B is known to be highly phosphorylated, therefore 400 ng of the purified recombinant human GSK3β (Abcam 43626) was pretreated with 16 units of lambda phosphatase (Santa Cruz) for 1 h at 30 °C [ ].

    Techniques: Expressing, Activation Assay, In Vitro, Purification, Recombinant, Immunofluorescence, Staining

    Tau phosphorylation in vitro . One microgram recombinant human tau (left, A) or mouse tau (right, A) was incubated with glycogensynthase-kinase-3β (GSK-3ß) and ATP in kinase buffer and analyzed by Western blot using antibodies against total tau (tau-5) phospho-tau-S199, phospho-tau-T231 and phospho-tau-S396. Lanes B and C served as a control omitting either the enzyme GSK-3β (Lane B) or tau protein (Lane C). Size markers are given as kDa.

    Journal: Frontiers in Aging Neuroscience

    Article Title: Differential Hyperphosphorylation of Tau-S199, -T231 and -S396 in Organotypic Brain Slices of Alzheimer Mice. A Model to Study Early Tau Hyperphosphorylation Using Okadaic Acid

    doi: 10.3389/fnagi.2018.00113

    Figure Lengend Snippet: Tau phosphorylation in vitro . One microgram recombinant human tau (left, A) or mouse tau (right, A) was incubated with glycogensynthase-kinase-3β (GSK-3ß) and ATP in kinase buffer and analyzed by Western blot using antibodies against total tau (tau-5) phospho-tau-S199, phospho-tau-T231 and phospho-tau-S396. Lanes B and C served as a control omitting either the enzyme GSK-3β (Lane B) or tau protein (Lane C). Size markers are given as kDa.

    Article Snippet: RPB983Mu01) was incubated with 2 μl glycogensynthase-kinase-3β (GSK-3β) stock (170–200 nmol min/mg, Sigma G4296) in 25 μl tau kinase buffer (40 mM HEPES, 5 mM EGTA, 3 mM MgCl2, pH 7.6) including 2 mM ATP overnight at 37°C.

    Techniques: In Vitro, Recombinant, Incubation, Western Blot

    AMPK phosphorylates tau in vitro. WB analysis of recombinant tau incubated in vitro with AMPK or GSK3β using antibodies directed against total tau (CP27), or tau phosphorylated on Ser-396/404 (PHF1), Thr-231 (2E12), Ser-202 (CP13), Ser-409 (PG5),

    Journal: Acta neuropathologica

    Article Title: AMPK is abnormally activated in tangle- and pre-tangle-bearing neurons in Alzheimer's disease and other tauopathies

    doi: 10.1007/s00401-010-0759-x

    Figure Lengend Snippet: AMPK phosphorylates tau in vitro. WB analysis of recombinant tau incubated in vitro with AMPK or GSK3β using antibodies directed against total tau (CP27), or tau phosphorylated on Ser-396/404 (PHF1), Thr-231 (2E12), Ser-202 (CP13), Ser-409 (PG5),

    Article Snippet: Recombinant human GSK3β (5 ng, R & D Systems, Minneapolis, MN, USA) was used as a positive control.

    Techniques: In Vitro, Western Blot, Recombinant, Incubation

    mTORC2 regulates GSK3β in response to glucose limitation. a Schematic showing the mTORC1 and mTORC2 complexes and downstream targets. b – d Western blots using indicated antibodies of HT29 cells grown under indicated conditions. Unless otherwise indicated cells were placed in HBSS for 2 h, or treated with Torin 1 (250 nM) or starved of glucose for 2 h and where indicted refed glucose for 1 h. e Western blot using indicated antibodies of HT29 cells grown in DMEM or starved of glucose for 4 h and treated with DMSO, MK2206 (3 μM), or LY333531 (5 μM). f Western blot using indicated antibodies of HT29 cells. 48 h after transfection with RICTOR-specific or control siRNAs cells were glucose starved for 2 h, as indicated. g Western blot using indicated antibodies of HT29 cells transfected with control or RICTOR-specific siRNA for 48 h prior to glucose starvation for indicated times. h Western blot using indicated antibodies of HT29 cells grown in the presence of absence of glucose (4 h) of cells treated with Rapamycin (1 μM) or Torin 1 (250 nM). i Western blot using indicated antibodies of HT29 cells following glucose starvation for indicated times

    Journal: Nature Communications

    Article Title: A TFEB nuclear export signal integrates amino acid supply and glucose availability

    doi: 10.1038/s41467-018-04849-7

    Figure Lengend Snippet: mTORC2 regulates GSK3β in response to glucose limitation. a Schematic showing the mTORC1 and mTORC2 complexes and downstream targets. b – d Western blots using indicated antibodies of HT29 cells grown under indicated conditions. Unless otherwise indicated cells were placed in HBSS for 2 h, or treated with Torin 1 (250 nM) or starved of glucose for 2 h and where indicted refed glucose for 1 h. e Western blot using indicated antibodies of HT29 cells grown in DMEM or starved of glucose for 4 h and treated with DMSO, MK2206 (3 μM), or LY333531 (5 μM). f Western blot using indicated antibodies of HT29 cells. 48 h after transfection with RICTOR-specific or control siRNAs cells were glucose starved for 2 h, as indicated. g Western blot using indicated antibodies of HT29 cells transfected with control or RICTOR-specific siRNA for 48 h prior to glucose starvation for indicated times. h Western blot using indicated antibodies of HT29 cells grown in the presence of absence of glucose (4 h) of cells treated with Rapamycin (1 μM) or Torin 1 (250 nM). i Western blot using indicated antibodies of HT29 cells following glucose starvation for indicated times

    Article Snippet: For in vitro kinase assays, reaction mixes were prepared using 2 μg of recombinant TFEB (1–200 6xnHis), 0.3 mM cold ATP and 100 ng of ERK2 (Sigma Aldrich, Cat No. E1283-10UG) and/or 100 ng of GSK3β (Abcam, Cat No. ab60863).

    Techniques: Western Blot, Transfection

    Phosphorylation at S142 primes for GSK3β phosphorylation at S138. a Fluorescence images of cells expressing indicated WT and mutant cargo vectors in the presence and absence of BIO (10 μM). n > 50 cells per condition. Error bars = SD. Scale bar = 20 μM. **** p

    Journal: Nature Communications

    Article Title: A TFEB nuclear export signal integrates amino acid supply and glucose availability

    doi: 10.1038/s41467-018-04849-7

    Figure Lengend Snippet: Phosphorylation at S142 primes for GSK3β phosphorylation at S138. a Fluorescence images of cells expressing indicated WT and mutant cargo vectors in the presence and absence of BIO (10 μM). n > 50 cells per condition. Error bars = SD. Scale bar = 20 μM. **** p

    Article Snippet: For in vitro kinase assays, reaction mixes were prepared using 2 μg of recombinant TFEB (1–200 6xnHis), 0.3 mM cold ATP and 100 ng of ERK2 (Sigma Aldrich, Cat No. E1283-10UG) and/or 100 ng of GSK3β (Abcam, Cat No. ab60863).

    Techniques: Fluorescence, Expressing, Mutagenesis

    FBW 7α promotes SOX 9 protein turnover in a GSK 3‐dependent manner through ubiquitin–proteasome system Endogenous SOX9 protein turnover in the presence of cycloheximide following RNAi depletion of FBW7α, FBW7β, or FBW7γ. HEK293 cells were transfected with 20 nM of scrambled siRNA or siRNAs specifically targeting FBW7α, FBW7β , or FBW7γ for 72 h prior to chase with the addition of 100 ng/ml cycloheximide. Immunoblot of cyclin E, an established SCF FBW7 substrate, was used to assess the efficacy of RNAi‐mediated depletion of FBW7 protein. GAPDH served as a protein loading control. Immunofluorescence staining of D324MED medulloblastoma cells showing nuclear co‐localization of HA‐SOX9 WT (Alexa Fluor 568; red) and FLAG‐FBW7α (Alexa Fluor 488; green). The cell nuclei were counterstained with DAPI (blue). Images are representative of multiple fields taken at 40× objective magnification. Scale bar indicates 20 μm. Endogenous SOX9 protein levels following transfection (24 h) of 100, 250, 500, and 1,000 ng of plasmid expressing FLAG‐FBW7α. Changes in SOX9 protein levels were analyzed relative to GAPDH levels using ImageJ. The blots shown are representative of three independent experiments. Expression of FLAG‐FBW7α enhance HA‐SOX9‐WT protein turnover but not HA‐SOX9‐T236A or HA‐SOX9‐T236A/T240A protein turnover over a 4‐h cycloheximide (100 ng/ml) chase in HEK293 cells. β‐actin protein served as a loading control. The blots shown are representative of four independent experiments. Treatment of HEK293 cells with increasing concentration of the GSK3α/β inhibitor BIO, increase HA‐SOX9‐WT protein level in a dose‐dependent manner. HA‐SOX9‐WT and FLAG‐FBW7α were co‐expressed in HEK293 cells prior to treatment with different concentrations of BIO for 4 h. Whole‐cell lysates were collected for Western blotting with anti‐HA (SOX9) and anti‐FLAG (FBW7) antibodies. GAPDH served as a protein loading control. The blots shown are representative of two independent experiments. RNAi depletion of GSK3β attenuate FBW7α‐induced HA‐SOX9‐WT turnover in HEK293 cells. HA‐SOX9‐WT protein turnover was examined following 48 h depletion of siGSK3b (20 nM) in the presence of cycloheximide (100 ng/ml). Immunoblots with GSK3β antibodies demonstrated depletion of GSK3β protein with the siRNA. Changes in SOX9 protein levels were analyzed relative to GAPDH levels using ImageJ. The blots shown are representative of three independent experiments. FLAG‐FBW7α‐WT expression promote poly‐ubiquitylation of endogenous SOX9 in HEK293 cells. Expression of FBW7α lacking the F‐box domain (ΔF), or containing R465A mutation did not induce SOX9 poly‐ubiquitylation in vivo . Ubiquitylation assay was performed under denaturing condition to disrupt non‐covalently linked ubiquitin as described in the Materials and Methods. Expression of different FLAG‐FBW7α constructs and endogenous SOX9 protein were examined in the whole‐cell lysates. GAPDH protein was used as a loading control. The blots shown are representative of four independent experiments. Reconstitution of SOX9 poly‐ubiquitylation by FLAG‐FBW7α in vitro . Immobilized IVT HA‐SOX9 WT was incubated with FLAG‐FBW7α‐WT or the ΔF mutant for 60 min at 37°C. Reaction mixture lacking the UbE1, an E1 ubiquitin‐activating enzyme, or UbcH3, an E2 ubiquitin‐conjugating enzyme, served as a negative control for the in vitro reaction. SOX9 poly‐ubiquitylation was assessed following elution of the protein from the immobilized beads under denaturing condition as described in Materials and Methods . The blots shown are representative of three independent experiments.

    Journal: The EMBO Journal

    Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

    doi: 10.15252/embj.201693889

    Figure Lengend Snippet: FBW 7α promotes SOX 9 protein turnover in a GSK 3‐dependent manner through ubiquitin–proteasome system Endogenous SOX9 protein turnover in the presence of cycloheximide following RNAi depletion of FBW7α, FBW7β, or FBW7γ. HEK293 cells were transfected with 20 nM of scrambled siRNA or siRNAs specifically targeting FBW7α, FBW7β , or FBW7γ for 72 h prior to chase with the addition of 100 ng/ml cycloheximide. Immunoblot of cyclin E, an established SCF FBW7 substrate, was used to assess the efficacy of RNAi‐mediated depletion of FBW7 protein. GAPDH served as a protein loading control. Immunofluorescence staining of D324MED medulloblastoma cells showing nuclear co‐localization of HA‐SOX9 WT (Alexa Fluor 568; red) and FLAG‐FBW7α (Alexa Fluor 488; green). The cell nuclei were counterstained with DAPI (blue). Images are representative of multiple fields taken at 40× objective magnification. Scale bar indicates 20 μm. Endogenous SOX9 protein levels following transfection (24 h) of 100, 250, 500, and 1,000 ng of plasmid expressing FLAG‐FBW7α. Changes in SOX9 protein levels were analyzed relative to GAPDH levels using ImageJ. The blots shown are representative of three independent experiments. Expression of FLAG‐FBW7α enhance HA‐SOX9‐WT protein turnover but not HA‐SOX9‐T236A or HA‐SOX9‐T236A/T240A protein turnover over a 4‐h cycloheximide (100 ng/ml) chase in HEK293 cells. β‐actin protein served as a loading control. The blots shown are representative of four independent experiments. Treatment of HEK293 cells with increasing concentration of the GSK3α/β inhibitor BIO, increase HA‐SOX9‐WT protein level in a dose‐dependent manner. HA‐SOX9‐WT and FLAG‐FBW7α were co‐expressed in HEK293 cells prior to treatment with different concentrations of BIO for 4 h. Whole‐cell lysates were collected for Western blotting with anti‐HA (SOX9) and anti‐FLAG (FBW7) antibodies. GAPDH served as a protein loading control. The blots shown are representative of two independent experiments. RNAi depletion of GSK3β attenuate FBW7α‐induced HA‐SOX9‐WT turnover in HEK293 cells. HA‐SOX9‐WT protein turnover was examined following 48 h depletion of siGSK3b (20 nM) in the presence of cycloheximide (100 ng/ml). Immunoblots with GSK3β antibodies demonstrated depletion of GSK3β protein with the siRNA. Changes in SOX9 protein levels were analyzed relative to GAPDH levels using ImageJ. The blots shown are representative of three independent experiments. FLAG‐FBW7α‐WT expression promote poly‐ubiquitylation of endogenous SOX9 in HEK293 cells. Expression of FBW7α lacking the F‐box domain (ΔF), or containing R465A mutation did not induce SOX9 poly‐ubiquitylation in vivo . Ubiquitylation assay was performed under denaturing condition to disrupt non‐covalently linked ubiquitin as described in the Materials and Methods. Expression of different FLAG‐FBW7α constructs and endogenous SOX9 protein were examined in the whole‐cell lysates. GAPDH protein was used as a loading control. The blots shown are representative of four independent experiments. Reconstitution of SOX9 poly‐ubiquitylation by FLAG‐FBW7α in vitro . Immobilized IVT HA‐SOX9 WT was incubated with FLAG‐FBW7α‐WT or the ΔF mutant for 60 min at 37°C. Reaction mixture lacking the UbE1, an E1 ubiquitin‐activating enzyme, or UbcH3, an E2 ubiquitin‐conjugating enzyme, served as a negative control for the in vitro reaction. SOX9 poly‐ubiquitylation was assessed following elution of the protein from the immobilized beads under denaturing condition as described in Materials and Methods . The blots shown are representative of three independent experiments.

    Article Snippet: Complete GSK3 reaction buffer consists of 50 mM Tris–HCl pH 7.4, 1 mM EGTA, 150 mM NaCl, 0.1% β‐mercaptoethanol, 2 mM ATP, and 1 U of recombinant, active human GSK3β (Abcam, AB60863).

    Techniques: Transfection, Immunofluorescence, Staining, Plasmid Preparation, Expressing, Concentration Assay, Western Blot, Mutagenesis, In Vivo, Ubiquitin Assay, Construct, In Vitro, Incubation, Negative Control

    SOX 9 interacts with FBW 7α through its conserved degron motif phosphorylated by GSK 3 Western blotting of total SOX9 protein levels in the cytoplasmic and nuclear fractions of HCT116‐FBW7 WT versus KO cells. The β‐actin was used as a loading control. General evolutionary conservation for SOX9 amino acid sequence surrounding the human CPD motif (highlighted in red) of threonine 236–240 across species. Western blotting of FLAG‐FBW7α eluted from the immunoprecipitated HA‐SOX9 wild‐type (WT) or CPD mutants (‐T236/240A and ‐T240A). The HA‐SOX9‐WT and the CPD mutant constructs were transiently co‐expressed for 24 h with FLAG‐FBW7α in HEK293 prior to immunoprecipitation with anti‐HA antibody. Equal protein expression of FBW7α across the HEK293 cells transfected with different SOX9 constructs was assessed by immunoblotting of the whole‐cell extract. Co‐expression of FBW7α with HA‐SOX9 WT or various other CPD mutant constructs (‐T236/240A, ‐T236A, or ‐T240A) in HEK293 cells. Whole‐cell lysates were collected 24 h following transfection for Western blotting of the total exogenous and the phosphorylated SOX9 proteins using anti‐HA and our pT236‐SOX9 antibody, respectively. Immunoblot of GAPDH protein was used to indicate protein loading in each lane. Detection of both exogenous and endogenous phosphorylated SOX9 protein from SOX9 immunoprecipitates. HA‐SOX9‐transfected or non‐transfected HEK293 cells were used as sources for exogenous and endogenous SOX9 protein, respectively. Following SOX9 immunoprecipitation with either anti‐HA (for exogenous) or anti‐SOX9 (for endogenous) antibody, the resulting immunoprecipitates were divided and either treated with λ‐phosphatase or left untreated prior to gel electrophoresis and immunoblotting with pT236‐SOX9 antibody. The SOX9 protein blot shows the total protein level present in each sample. Treatment of HEK293 with proteasome inhibitor MG132 (10 μM) increased the level of phosphorylated SOX9. Immunoblots of endogenous pT236 and total SOX9 protein 24 h following transfection of D324MED medulloblastoma cell line with either non‐targeting scramble RNA (siScr) or increasing concentrations of siRNA against SOX9. GAPDH protein was used to indicated protein loading for each sample Representative immunofluorescence staining depicting high intensity of pT236‐SOX9 (Alexa Fluor 488; green) staining in the nucleus (counterstained with DAPI; blue) in Daoy medulloblastoma cells. Transfection of Daoy cells with 20 nM siSOX9 depleted the nuclear staining of pT236‐SOX9. Images were taken using a 40× objective. Scale bar: 20 μm. Bead‐immobilized IVT HA‐SOX9 WT were subjected to in vitro kinase reaction with 1 unit of recombinant active GSK3α, GSK3β, or their combination (i.e., 0.5 unit for each isoform) for 90 min at 37°C prior to elution and gel electrophoresis. The SOX9 blot shows total SOX9 protein eluted from the beads from each in vitro kinase reaction.

    Journal: The EMBO Journal

    Article Title: FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

    doi: 10.15252/embj.201693889

    Figure Lengend Snippet: SOX 9 interacts with FBW 7α through its conserved degron motif phosphorylated by GSK 3 Western blotting of total SOX9 protein levels in the cytoplasmic and nuclear fractions of HCT116‐FBW7 WT versus KO cells. The β‐actin was used as a loading control. General evolutionary conservation for SOX9 amino acid sequence surrounding the human CPD motif (highlighted in red) of threonine 236–240 across species. Western blotting of FLAG‐FBW7α eluted from the immunoprecipitated HA‐SOX9 wild‐type (WT) or CPD mutants (‐T236/240A and ‐T240A). The HA‐SOX9‐WT and the CPD mutant constructs were transiently co‐expressed for 24 h with FLAG‐FBW7α in HEK293 prior to immunoprecipitation with anti‐HA antibody. Equal protein expression of FBW7α across the HEK293 cells transfected with different SOX9 constructs was assessed by immunoblotting of the whole‐cell extract. Co‐expression of FBW7α with HA‐SOX9 WT or various other CPD mutant constructs (‐T236/240A, ‐T236A, or ‐T240A) in HEK293 cells. Whole‐cell lysates were collected 24 h following transfection for Western blotting of the total exogenous and the phosphorylated SOX9 proteins using anti‐HA and our pT236‐SOX9 antibody, respectively. Immunoblot of GAPDH protein was used to indicate protein loading in each lane. Detection of both exogenous and endogenous phosphorylated SOX9 protein from SOX9 immunoprecipitates. HA‐SOX9‐transfected or non‐transfected HEK293 cells were used as sources for exogenous and endogenous SOX9 protein, respectively. Following SOX9 immunoprecipitation with either anti‐HA (for exogenous) or anti‐SOX9 (for endogenous) antibody, the resulting immunoprecipitates were divided and either treated with λ‐phosphatase or left untreated prior to gel electrophoresis and immunoblotting with pT236‐SOX9 antibody. The SOX9 protein blot shows the total protein level present in each sample. Treatment of HEK293 with proteasome inhibitor MG132 (10 μM) increased the level of phosphorylated SOX9. Immunoblots of endogenous pT236 and total SOX9 protein 24 h following transfection of D324MED medulloblastoma cell line with either non‐targeting scramble RNA (siScr) or increasing concentrations of siRNA against SOX9. GAPDH protein was used to indicated protein loading for each sample Representative immunofluorescence staining depicting high intensity of pT236‐SOX9 (Alexa Fluor 488; green) staining in the nucleus (counterstained with DAPI; blue) in Daoy medulloblastoma cells. Transfection of Daoy cells with 20 nM siSOX9 depleted the nuclear staining of pT236‐SOX9. Images were taken using a 40× objective. Scale bar: 20 μm. Bead‐immobilized IVT HA‐SOX9 WT were subjected to in vitro kinase reaction with 1 unit of recombinant active GSK3α, GSK3β, or their combination (i.e., 0.5 unit for each isoform) for 90 min at 37°C prior to elution and gel electrophoresis. The SOX9 blot shows total SOX9 protein eluted from the beads from each in vitro kinase reaction.

    Article Snippet: Complete GSK3 reaction buffer consists of 50 mM Tris–HCl pH 7.4, 1 mM EGTA, 150 mM NaCl, 0.1% β‐mercaptoethanol, 2 mM ATP, and 1 U of recombinant, active human GSK3β (Abcam, AB60863).

    Techniques: Western Blot, Sequencing, Immunoprecipitation, Mutagenesis, Construct, Expressing, Transfection, Nucleic Acid Electrophoresis, Immunofluorescence, Staining, In Vitro, Recombinant

    Schematic of the possible regulation of AREG on the EGFR/GSK-3β/Foxp3 axis.

    Journal: The Journal of Biological Chemistry

    Article Title: Amphiregulin Confers Regulatory T Cell Suppressive Function and Tumor Invasion via the EGFR/GSK-3β/Foxp3 Axis *

    doi: 10.1074/jbc.M116.717892

    Figure Lengend Snippet: Schematic of the possible regulation of AREG on the EGFR/GSK-3β/Foxp3 axis.

    Article Snippet: GSK-3β activator (staurosporine, 50 μ m ) or GSK-3β inhibitors (SB216763, 5 μ m or GSK-3β siRNA, Sigma-Aldrich) were added to the culture.

    Techniques:

    AREG modulates the GSK-3β/Foxp3 axis in Treg cells. A , interaction of GSK-3β and Foxp3 in CD4 + CD25 hi Treg cells. IP , immunoprecipitation. B , GSK-3β siRNA reduced phosphorylated Foxp3 (p270 and p274). Right panel , semiquantitative analysis of Ser 270 and Ser 274 phosphorylation of Foxp3 and protein expression of FLAG-Foxp3 and GSK-3β. C , measurement of Foxp3 protein half-life in control or GSK-3β siRNA knockdown CD4 + CD25 hi Treg cells. The half-life of Foxp3 was significantly increased in GSK-3β knockdown CD4 + CD25 hi Treg cells. CHX , cycloheximide. D and E , expression levels of GSK-3β, p-GSK-3β, and Foxp3 in CD4 + CD25 hi Treg cells in response to recombinant AREG (100 ng/ml). AREG significantly increased phosphorylation of GSK-3β, but not total GSK-3β protein level, in CD4 + CD25 hi Treg cells (isolated from LC- and GC-PBMC). F and G , AREG significantly increased the Foxp3 protein level, but not the Foxp3 mRNA level, in CD4 + CD25 hi Treg cells (isolated from LC- and GC-PBMC). H , the AREG-stimulated Foxp3 protein level in CD4 + CD25 hi Treg cells was decreased in response to gefitinib or anti-AREG administration. Data are mean ± S.D. of five independent experiments. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Amphiregulin Confers Regulatory T Cell Suppressive Function and Tumor Invasion via the EGFR/GSK-3β/Foxp3 Axis *

    doi: 10.1074/jbc.M116.717892

    Figure Lengend Snippet: AREG modulates the GSK-3β/Foxp3 axis in Treg cells. A , interaction of GSK-3β and Foxp3 in CD4 + CD25 hi Treg cells. IP , immunoprecipitation. B , GSK-3β siRNA reduced phosphorylated Foxp3 (p270 and p274). Right panel , semiquantitative analysis of Ser 270 and Ser 274 phosphorylation of Foxp3 and protein expression of FLAG-Foxp3 and GSK-3β. C , measurement of Foxp3 protein half-life in control or GSK-3β siRNA knockdown CD4 + CD25 hi Treg cells. The half-life of Foxp3 was significantly increased in GSK-3β knockdown CD4 + CD25 hi Treg cells. CHX , cycloheximide. D and E , expression levels of GSK-3β, p-GSK-3β, and Foxp3 in CD4 + CD25 hi Treg cells in response to recombinant AREG (100 ng/ml). AREG significantly increased phosphorylation of GSK-3β, but not total GSK-3β protein level, in CD4 + CD25 hi Treg cells (isolated from LC- and GC-PBMC). F and G , AREG significantly increased the Foxp3 protein level, but not the Foxp3 mRNA level, in CD4 + CD25 hi Treg cells (isolated from LC- and GC-PBMC). H , the AREG-stimulated Foxp3 protein level in CD4 + CD25 hi Treg cells was decreased in response to gefitinib or anti-AREG administration. Data are mean ± S.D. of five independent experiments. *, p

    Article Snippet: GSK-3β activator (staurosporine, 50 μ m ) or GSK-3β inhibitors (SB216763, 5 μ m or GSK-3β siRNA, Sigma-Aldrich) were added to the culture.

    Techniques: Immunoprecipitation, Expressing, Recombinant, Isolation

    β-TrCP is involved in GSK-3β-mediated Foxp3 degradation. A , blockage of GSK-3β with siRNA decreased Foxp3 ubiquitination in HEK293T cells. IP , immunoprecipitation. B , interaction of β-TrCP with Foxp3 in HeLa and HEK293T cells. C , β-TrCP increased Foxp3 ubiquitination in the presence of GSK-3β in HEK293T cells. Ub , ubiquitin.

    Journal: The Journal of Biological Chemistry

    Article Title: Amphiregulin Confers Regulatory T Cell Suppressive Function and Tumor Invasion via the EGFR/GSK-3β/Foxp3 Axis *

    doi: 10.1074/jbc.M116.717892

    Figure Lengend Snippet: β-TrCP is involved in GSK-3β-mediated Foxp3 degradation. A , blockage of GSK-3β with siRNA decreased Foxp3 ubiquitination in HEK293T cells. IP , immunoprecipitation. B , interaction of β-TrCP with Foxp3 in HeLa and HEK293T cells. C , β-TrCP increased Foxp3 ubiquitination in the presence of GSK-3β in HEK293T cells. Ub , ubiquitin.

    Article Snippet: GSK-3β activator (staurosporine, 50 μ m ) or GSK-3β inhibitors (SB216763, 5 μ m or GSK-3β siRNA, Sigma-Aldrich) were added to the culture.

    Techniques: Immunoprecipitation

    The ubiquitination site of Foxp3 is Lys 356 . A , mass spectrometry identifies Lys 356 as the ubiquitination site of Foxp3 by β-TrCP in HEK293T cells. B , the Foxp3 mutant (Lys 356 ) cannot be ubiquitinated in the presence of GSK-3β. IP , immunoprecipitation.

    Journal: The Journal of Biological Chemistry

    Article Title: Amphiregulin Confers Regulatory T Cell Suppressive Function and Tumor Invasion via the EGFR/GSK-3β/Foxp3 Axis *

    doi: 10.1074/jbc.M116.717892

    Figure Lengend Snippet: The ubiquitination site of Foxp3 is Lys 356 . A , mass spectrometry identifies Lys 356 as the ubiquitination site of Foxp3 by β-TrCP in HEK293T cells. B , the Foxp3 mutant (Lys 356 ) cannot be ubiquitinated in the presence of GSK-3β. IP , immunoprecipitation.

    Article Snippet: GSK-3β activator (staurosporine, 50 μ m ) or GSK-3β inhibitors (SB216763, 5 μ m or GSK-3β siRNA, Sigma-Aldrich) were added to the culture.

    Techniques: Mass Spectrometry, Mutagenesis, Immunoprecipitation

    A , blockage of EGFR with gefitinib-inhibited Foxp3 expression in CD4 + CD25 hi Treg cells derived from LC-PBMC. B , blockage of EGFR with gefitinib-inhibited GSK-3β phosphorylation in HCC827 and PC-9 cells.

    Journal: The Journal of Biological Chemistry

    Article Title: Amphiregulin Confers Regulatory T Cell Suppressive Function and Tumor Invasion via the EGFR/GSK-3β/Foxp3 Axis *

    doi: 10.1074/jbc.M116.717892

    Figure Lengend Snippet: A , blockage of EGFR with gefitinib-inhibited Foxp3 expression in CD4 + CD25 hi Treg cells derived from LC-PBMC. B , blockage of EGFR with gefitinib-inhibited GSK-3β phosphorylation in HCC827 and PC-9 cells.

    Article Snippet: GSK-3β activator (staurosporine, 50 μ m ) or GSK-3β inhibitors (SB216763, 5 μ m or GSK-3β siRNA, Sigma-Aldrich) were added to the culture.

    Techniques: Expressing, Derivative Assay

    β-Catenin is partially necessary for KRT9 expression in keratinocytes. A: β-Catenin knockdown (βCat-siR) in keratinocytes inhibits β-catenin protein and KRT9, but not KRT6a, mRNA compared with Scr-siR. B: rhDKK-1 inhibits KRT9 expression in either solo keratinocyte cultures or co-cultures with sole FBs. C: Effects of SB216763 to increase KRT9 and AXIN2 are reversible by rhDKK-1 in keratinocytes. ∗ P

    Journal: The American Journal of Pathology

    Article Title: To Control Site-Specific Skin Gene Expression, Autocrine Mimics Paracrine Canonical Wnt Signaling and Is Activated Ectopically in Skin Disease

    doi: 10.1016/j.ajpath.2015.12.030

    Figure Lengend Snippet: β-Catenin is partially necessary for KRT9 expression in keratinocytes. A: β-Catenin knockdown (βCat-siR) in keratinocytes inhibits β-catenin protein and KRT9, but not KRT6a, mRNA compared with Scr-siR. B: rhDKK-1 inhibits KRT9 expression in either solo keratinocyte cultures or co-cultures with sole FBs. C: Effects of SB216763 to increase KRT9 and AXIN2 are reversible by rhDKK-1 in keratinocytes. ∗ P

    Article Snippet: Recombinant human (rh) DKK-1 protein (R & D Systems, Minneapolis, MN) and SB216763 (Sigma-Aldrich) were purchased.

    Techniques: Expressing

    Keratinocytes induce KRT9 through canonical activation of β-catenin. A: WNT5a and AXIN2 mRNA are elevated in co-culture with foreskin keratinocytes and plantar (sole) fibroblasts analyzed by quantitative RT-PCR. B: WNT5a and AXIN2 mRNA are induced in keratinocytes alone at higher cell densities and culture media conditions promoting differentiation in time-course experiments. C: mRNA of WNT5a is elevated in volar fibroblasts comparing with nonvolar fibroblasts. D: Active β-catenin protein, AXIN2 mRNA, and KRT9 mRNA and protein are induced with 5 μmol/L of SB216763, but not KRT6a mRNA. ∗ P

    Journal: The American Journal of Pathology

    Article Title: To Control Site-Specific Skin Gene Expression, Autocrine Mimics Paracrine Canonical Wnt Signaling and Is Activated Ectopically in Skin Disease

    doi: 10.1016/j.ajpath.2015.12.030

    Figure Lengend Snippet: Keratinocytes induce KRT9 through canonical activation of β-catenin. A: WNT5a and AXIN2 mRNA are elevated in co-culture with foreskin keratinocytes and plantar (sole) fibroblasts analyzed by quantitative RT-PCR. B: WNT5a and AXIN2 mRNA are induced in keratinocytes alone at higher cell densities and culture media conditions promoting differentiation in time-course experiments. C: mRNA of WNT5a is elevated in volar fibroblasts comparing with nonvolar fibroblasts. D: Active β-catenin protein, AXIN2 mRNA, and KRT9 mRNA and protein are induced with 5 μmol/L of SB216763, but not KRT6a mRNA. ∗ P

    Article Snippet: Recombinant human (rh) DKK-1 protein (R & D Systems, Minneapolis, MN) and SB216763 (Sigma-Aldrich) were purchased.

    Techniques: Activation Assay, Co-Culture Assay, Quantitative RT-PCR

    The half-life of USF2 is induced upon GSK3β-dependent phosphorylation. (A) GSK3β +/+ and GSK3β −/− cells were treated with 10 µg/ml CHX for the indicated time periods. Proteins were isolated, separated by SDS-PAGE and detected by Western blotting. Protein levels were quantified and the relative protein level of USF2 was blotted against the duration of CHX treatment for estimation of the half-life. The dashed line indicates the USF2 half-life where 50% of the USF2 protein level was reached. (B) Representative Western Blot. 50 µg of protein from were probed with an antibody against USF2 and α-tubulin.

    Journal: PLoS ONE

    Article Title: GSK3β-Dependent Phosphorylation Alters DNA Binding, Transactivity and Half-Life of the Transcription Factor USF2

    doi: 10.1371/journal.pone.0107914

    Figure Lengend Snippet: The half-life of USF2 is induced upon GSK3β-dependent phosphorylation. (A) GSK3β +/+ and GSK3β −/− cells were treated with 10 µg/ml CHX for the indicated time periods. Proteins were isolated, separated by SDS-PAGE and detected by Western blotting. Protein levels were quantified and the relative protein level of USF2 was blotted against the duration of CHX treatment for estimation of the half-life. The dashed line indicates the USF2 half-life where 50% of the USF2 protein level was reached. (B) Representative Western Blot. 50 µg of protein from were probed with an antibody against USF2 and α-tubulin.

    Article Snippet: Purified GST fusion proteins (1.5 µg) or proteins immunoprecipitated from whole cell extracts (350 µg) were incubated in kinase assay buffer (15 mM MOPS pH 7.2, 15 mM MgCl2 , 3 mM EGTA, 1.2 mM EDTA, 150 µM DTT, 6 mM β-glycerophosphate) containing 20 ng of active, recombinant full-length human GSK3β (SignalChem), 50 µM unlabeled ATP and 1.5 µCi [γ−32 P] ATP at 30°C for 20 min.

    Techniques: Isolation, SDS Page, Western Blot

    The phosphorylation of USF2 by GSK3β affects cell migration. (A, B, C, D) GSK3β −/− cells were transfected with vectors allowing expression of USF2-S155A/T230A or USF2-S155D/T230D or an empty vector. Cellular viability, proliferation and cell migration were monitored by MTT, BrdU (A) and cell migration (B, C) assays. (B) Photographs from a representative Transwell chamber experiment. (C) Data represent the absorbance of crystal violet at 595 nm relative to the control. *, significant difference between GSK3β −/− cells and GSK3β −/− cells + USF2-S155D/T230D. (D) The expression of USF2 was controlled by Western blotting. 50 µg of protein from transfected cells were probed with an antibody against USF2 and α-tubulin.

    Journal: PLoS ONE

    Article Title: GSK3β-Dependent Phosphorylation Alters DNA Binding, Transactivity and Half-Life of the Transcription Factor USF2

    doi: 10.1371/journal.pone.0107914

    Figure Lengend Snippet: The phosphorylation of USF2 by GSK3β affects cell migration. (A, B, C, D) GSK3β −/− cells were transfected with vectors allowing expression of USF2-S155A/T230A or USF2-S155D/T230D or an empty vector. Cellular viability, proliferation and cell migration were monitored by MTT, BrdU (A) and cell migration (B, C) assays. (B) Photographs from a representative Transwell chamber experiment. (C) Data represent the absorbance of crystal violet at 595 nm relative to the control. *, significant difference between GSK3β −/− cells and GSK3β −/− cells + USF2-S155D/T230D. (D) The expression of USF2 was controlled by Western blotting. 50 µg of protein from transfected cells were probed with an antibody against USF2 and α-tubulin.

    Article Snippet: Purified GST fusion proteins (1.5 µg) or proteins immunoprecipitated from whole cell extracts (350 µg) were incubated in kinase assay buffer (15 mM MOPS pH 7.2, 15 mM MgCl2 , 3 mM EGTA, 1.2 mM EDTA, 150 µM DTT, 6 mM β-glycerophosphate) containing 20 ng of active, recombinant full-length human GSK3β (SignalChem), 50 µM unlabeled ATP and 1.5 µCi [γ−32 P] ATP at 30°C for 20 min.

    Techniques: Migration, Transfection, Expressing, Plasmid Preparation, MTT Assay, Western Blot

    USF2 is phosphorylated by GSK3β. (A) USF2 was immunoprecipitated from GSK3β +/+ and GSK3β −/− cells and phospho-USF2 protein levels were detected with phospho-threonine or phospho-serine antibodies. (B) USF2 was immunoprecipitated from HeLa cells transfected with pcDNA3-GSK3β-WT-HA. Following SDS-PAGE phosphoproteins were visualized with the Pro-Q Diamond Phosphoprotein Gel Stain. The total protein amount was detected by silver staining and GSK3β expression was verified by Western blotting. (C) Where indicated, HeLa cells transfected as above were treated with the GSK3 selective inhibitors BIO (1 µM), LiCl (10 mM), 1-Azakenpaullone (Aza, 7.5 µM) for 1 h. Proteins were isolated 24 h after transfection and detected by Western blotting. (D) Cells were transfected with expression vectors for USF2 and GSK3β and the cell extract was incubated with calf intestinal phosphatase (CIP) or only with buffer (Mock). Proteins were detected by Western blotting.

    Journal: PLoS ONE

    Article Title: GSK3β-Dependent Phosphorylation Alters DNA Binding, Transactivity and Half-Life of the Transcription Factor USF2

    doi: 10.1371/journal.pone.0107914

    Figure Lengend Snippet: USF2 is phosphorylated by GSK3β. (A) USF2 was immunoprecipitated from GSK3β +/+ and GSK3β −/− cells and phospho-USF2 protein levels were detected with phospho-threonine or phospho-serine antibodies. (B) USF2 was immunoprecipitated from HeLa cells transfected with pcDNA3-GSK3β-WT-HA. Following SDS-PAGE phosphoproteins were visualized with the Pro-Q Diamond Phosphoprotein Gel Stain. The total protein amount was detected by silver staining and GSK3β expression was verified by Western blotting. (C) Where indicated, HeLa cells transfected as above were treated with the GSK3 selective inhibitors BIO (1 µM), LiCl (10 mM), 1-Azakenpaullone (Aza, 7.5 µM) for 1 h. Proteins were isolated 24 h after transfection and detected by Western blotting. (D) Cells were transfected with expression vectors for USF2 and GSK3β and the cell extract was incubated with calf intestinal phosphatase (CIP) or only with buffer (Mock). Proteins were detected by Western blotting.

    Article Snippet: Purified GST fusion proteins (1.5 µg) or proteins immunoprecipitated from whole cell extracts (350 µg) were incubated in kinase assay buffer (15 mM MOPS pH 7.2, 15 mM MgCl2 , 3 mM EGTA, 1.2 mM EDTA, 150 µM DTT, 6 mM β-glycerophosphate) containing 20 ng of active, recombinant full-length human GSK3β (SignalChem), 50 µM unlabeled ATP and 1.5 µCi [γ−32 P] ATP at 30°C for 20 min.

    Techniques: Immunoprecipitation, Transfection, SDS Page, Staining, Silver Staining, Expressing, Western Blot, Isolation, Incubation

    GSK3β-mediated phosphorylation of USF2 affects its transactivity, DNA binding and target gene expression. (A) HeLa cells were cotransfected with pFR-5Gal4-RE-Luc, an expression vector for constitutively active GSK3β-S9A and WT or mutant pcDNA6-Gal4-USF2 (1-231) or the appropriate empty Gal4 vector. The luciferase activity was calculated as fold induction compared to the Gal4-USF2 (1-231)-WT luciferase activity after subtracting the values from the empty Gal4 expression vector. *, significant differences control vs. GSK3β. (B) Representative Western blot of the transfected constructs. 50 µg of protein from transfected cells were probed with an antibody against Gal4, HA-tag and α-tubulin. (C) Quantitative RT-PCR analyses of FAS, HO-1, PAI-1 and USF2 mRNA levels in GSK3β +/+ and GSK3β −/− cells. *, significant differences WT vs. GSK3β −/− . (D) Western Blot analyses of FAS, HO-1 and PAI-1 expression in GSK3β +/+ and GSK3β −/− cells . 50 µg of protein were subjected to Western analysis with antibodies against FAS, HO-1, PAI-1 or β-catenin, c-Myc or α-tubulin; the latter served as a loading control. (E) ChIP was performed in GSK3β +/+ and GSK3β −/− cells with either USF2 antibody, control IgG or RNA Pol II antibody. The quantitative PCR was performed with primers amplifying the FAS, HO-1, and PAI-1 promoter containing the USF2 binding sites, and with primers amplifying the β-actin promoter binding RNA Pol II as outlined in Materials and Methods . *, significant differences WT vs. GSK3β −/− .

    Journal: PLoS ONE

    Article Title: GSK3β-Dependent Phosphorylation Alters DNA Binding, Transactivity and Half-Life of the Transcription Factor USF2

    doi: 10.1371/journal.pone.0107914

    Figure Lengend Snippet: GSK3β-mediated phosphorylation of USF2 affects its transactivity, DNA binding and target gene expression. (A) HeLa cells were cotransfected with pFR-5Gal4-RE-Luc, an expression vector for constitutively active GSK3β-S9A and WT or mutant pcDNA6-Gal4-USF2 (1-231) or the appropriate empty Gal4 vector. The luciferase activity was calculated as fold induction compared to the Gal4-USF2 (1-231)-WT luciferase activity after subtracting the values from the empty Gal4 expression vector. *, significant differences control vs. GSK3β. (B) Representative Western blot of the transfected constructs. 50 µg of protein from transfected cells were probed with an antibody against Gal4, HA-tag and α-tubulin. (C) Quantitative RT-PCR analyses of FAS, HO-1, PAI-1 and USF2 mRNA levels in GSK3β +/+ and GSK3β −/− cells. *, significant differences WT vs. GSK3β −/− . (D) Western Blot analyses of FAS, HO-1 and PAI-1 expression in GSK3β +/+ and GSK3β −/− cells . 50 µg of protein were subjected to Western analysis with antibodies against FAS, HO-1, PAI-1 or β-catenin, c-Myc or α-tubulin; the latter served as a loading control. (E) ChIP was performed in GSK3β +/+ and GSK3β −/− cells with either USF2 antibody, control IgG or RNA Pol II antibody. The quantitative PCR was performed with primers amplifying the FAS, HO-1, and PAI-1 promoter containing the USF2 binding sites, and with primers amplifying the β-actin promoter binding RNA Pol II as outlined in Materials and Methods . *, significant differences WT vs. GSK3β −/− .

    Article Snippet: Purified GST fusion proteins (1.5 µg) or proteins immunoprecipitated from whole cell extracts (350 µg) were incubated in kinase assay buffer (15 mM MOPS pH 7.2, 15 mM MgCl2 , 3 mM EGTA, 1.2 mM EDTA, 150 µM DTT, 6 mM β-glycerophosphate) containing 20 ng of active, recombinant full-length human GSK3β (SignalChem), 50 µM unlabeled ATP and 1.5 µCi [γ−32 P] ATP at 30°C for 20 min.

    Techniques: Binding Assay, Expressing, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Western Blot, Transfection, Construct, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    GSK3β-mediated phosphorylation occurs in two USF2 domains. (A) Schematic representation of the USF2 deletion mutants used to identify the domains that are phosphorylated by GSK3β. (B–E) Purified GST-tagged USF2 proteins were incubated with recombinant human GSK3β in the presence of [γ −32 P] ATP. Proteins were separated by SDS-PAGE and incorporated radioactivity was detected by autoradiography. The total amount of proteins was detected by Coomassie staining. (F) HeLa cells were transfected with expression vectors for USF2 or the empty vector. USF2 was immunoprecipitated from the total cell extract and then incubated with recombinant human GSK3β in the presence of [γ −32 P] ATP. Proteins were separated by SDS-PAGE and incorporated radioactivity was detected by autoradiography. The total amount of proteins was detected by silver staining.

    Journal: PLoS ONE

    Article Title: GSK3β-Dependent Phosphorylation Alters DNA Binding, Transactivity and Half-Life of the Transcription Factor USF2

    doi: 10.1371/journal.pone.0107914

    Figure Lengend Snippet: GSK3β-mediated phosphorylation occurs in two USF2 domains. (A) Schematic representation of the USF2 deletion mutants used to identify the domains that are phosphorylated by GSK3β. (B–E) Purified GST-tagged USF2 proteins were incubated with recombinant human GSK3β in the presence of [γ −32 P] ATP. Proteins were separated by SDS-PAGE and incorporated radioactivity was detected by autoradiography. The total amount of proteins was detected by Coomassie staining. (F) HeLa cells were transfected with expression vectors for USF2 or the empty vector. USF2 was immunoprecipitated from the total cell extract and then incubated with recombinant human GSK3β in the presence of [γ −32 P] ATP. Proteins were separated by SDS-PAGE and incorporated radioactivity was detected by autoradiography. The total amount of proteins was detected by silver staining.

    Article Snippet: Purified GST fusion proteins (1.5 µg) or proteins immunoprecipitated from whole cell extracts (350 µg) were incubated in kinase assay buffer (15 mM MOPS pH 7.2, 15 mM MgCl2 , 3 mM EGTA, 1.2 mM EDTA, 150 µM DTT, 6 mM β-glycerophosphate) containing 20 ng of active, recombinant full-length human GSK3β (SignalChem), 50 µM unlabeled ATP and 1.5 µCi [γ−32 P] ATP at 30°C for 20 min.

    Techniques: Purification, Incubation, Recombinant, SDS Page, Radioactivity, Autoradiography, Staining, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Silver Staining

    Phosphorylation of USF2 by GSK3β increases domain (residue) distance. (A–C) Simulated structures of wild type (A), S155 phosphorylated (B), and T230 phosphorylated (C) USF2 from the MD simulation trajectories. The side chains of Ser155 and Thr230 are shown in stick representation. Phosphorylated amino acids are labeled with an asterisk (*). The distance between side chain oxygen of S155 and T230 were analyzed (D). Phosphorylation of S155 (red) or T230 (green) increases the distance compared to non-phosphorylated USF-2 (black).

    Journal: PLoS ONE

    Article Title: GSK3β-Dependent Phosphorylation Alters DNA Binding, Transactivity and Half-Life of the Transcription Factor USF2

    doi: 10.1371/journal.pone.0107914

    Figure Lengend Snippet: Phosphorylation of USF2 by GSK3β increases domain (residue) distance. (A–C) Simulated structures of wild type (A), S155 phosphorylated (B), and T230 phosphorylated (C) USF2 from the MD simulation trajectories. The side chains of Ser155 and Thr230 are shown in stick representation. Phosphorylated amino acids are labeled with an asterisk (*). The distance between side chain oxygen of S155 and T230 were analyzed (D). Phosphorylation of S155 (red) or T230 (green) increases the distance compared to non-phosphorylated USF-2 (black).

    Article Snippet: Purified GST fusion proteins (1.5 µg) or proteins immunoprecipitated from whole cell extracts (350 µg) were incubated in kinase assay buffer (15 mM MOPS pH 7.2, 15 mM MgCl2 , 3 mM EGTA, 1.2 mM EDTA, 150 µM DTT, 6 mM β-glycerophosphate) containing 20 ng of active, recombinant full-length human GSK3β (SignalChem), 50 µM unlabeled ATP and 1.5 µCi [γ−32 P] ATP at 30°C for 20 min.

    Techniques: Labeling

    GSK3β-mediated phosphorylation of USF2 affects activation of target gene promoters. (A–C) HeLa cells were cotransfected with the indicated luciferase gene construct and with WT or mutant p3xFLAG-USF2-myc-CMV24 or the appropriate empty vector. The measured luciferase activity is plotted as fold induction compared to the luciferase activity measured in the control transfected with the empty expression vector. *, significant difference WT vs. mutant. (D) Representative Western blot of the transfected constructs. 50 µg of protein from transfected cells were probed with an antibody against myc-tag and α-tubulin.

    Journal: PLoS ONE

    Article Title: GSK3β-Dependent Phosphorylation Alters DNA Binding, Transactivity and Half-Life of the Transcription Factor USF2

    doi: 10.1371/journal.pone.0107914

    Figure Lengend Snippet: GSK3β-mediated phosphorylation of USF2 affects activation of target gene promoters. (A–C) HeLa cells were cotransfected with the indicated luciferase gene construct and with WT or mutant p3xFLAG-USF2-myc-CMV24 or the appropriate empty vector. The measured luciferase activity is plotted as fold induction compared to the luciferase activity measured in the control transfected with the empty expression vector. *, significant difference WT vs. mutant. (D) Representative Western blot of the transfected constructs. 50 µg of protein from transfected cells were probed with an antibody against myc-tag and α-tubulin.

    Article Snippet: Purified GST fusion proteins (1.5 µg) or proteins immunoprecipitated from whole cell extracts (350 µg) were incubated in kinase assay buffer (15 mM MOPS pH 7.2, 15 mM MgCl2 , 3 mM EGTA, 1.2 mM EDTA, 150 µM DTT, 6 mM β-glycerophosphate) containing 20 ng of active, recombinant full-length human GSK3β (SignalChem), 50 µM unlabeled ATP and 1.5 µCi [γ−32 P] ATP at 30°C for 20 min.

    Techniques: Activation Assay, Luciferase, Construct, Mutagenesis, Plasmid Preparation, Activity Assay, Transfection, Expressing, Western Blot

    Wnt/β-catenin reduces MKX and TNMD expressions in SCX -programmed tendon progenitors (hMSC-Scx cells). hMSC-Scx cells were treated with either Wnt3a, BIO (an activator of β-catenin), IWR (an inhibitor of β-catenin), or their combination, as in Fig 2 . Relative expressions of AXIN2 , MKX , and TNMD in hMSC-Scx cells treated with 50 ng/ml Wnt3a with or without 5 μM IWR (A) , 0 to 2 μM BIO (B) , or 0 to 20 μM IWR (C) are indicated. As BIO was dissolved in DMSO, all samples in B were incubated under 0.004% DMSO. (B) Increasing concentrations (0.5, 1, and 2 μM) of BIO increased AXIN2 expression to 130%, 240%, and 700% of that without BIO, respectively. (C) Increasing concentrations (5, 10, and 20 μM) of IWR decreased AXIN2 expression to 94%, 84%, and 84% of that without IWR, respectively. Each mRNA expression is normalized by GAPDH mRNA. Mean and SD are indicated ( n = 3 wells each). p

    Journal: PLoS ONE

    Article Title: Wnt/β-catenin signaling suppresses expressions of Scx, Mkx, and Tnmd in tendon-derived cells

    doi: 10.1371/journal.pone.0182051

    Figure Lengend Snippet: Wnt/β-catenin reduces MKX and TNMD expressions in SCX -programmed tendon progenitors (hMSC-Scx cells). hMSC-Scx cells were treated with either Wnt3a, BIO (an activator of β-catenin), IWR (an inhibitor of β-catenin), or their combination, as in Fig 2 . Relative expressions of AXIN2 , MKX , and TNMD in hMSC-Scx cells treated with 50 ng/ml Wnt3a with or without 5 μM IWR (A) , 0 to 2 μM BIO (B) , or 0 to 20 μM IWR (C) are indicated. As BIO was dissolved in DMSO, all samples in B were incubated under 0.004% DMSO. (B) Increasing concentrations (0.5, 1, and 2 μM) of BIO increased AXIN2 expression to 130%, 240%, and 700% of that without BIO, respectively. (C) Increasing concentrations (5, 10, and 20 μM) of IWR decreased AXIN2 expression to 94%, 84%, and 84% of that without IWR, respectively. Each mRNA expression is normalized by GAPDH mRNA. Mean and SD are indicated ( n = 3 wells each). p

    Article Snippet: Treatment of cultured cells with chemical compounds and recombinant proteins TDCs and hMSC-Scx cells were cultured in the medium stated above, and were supplemented with 0.5–4 μM BIO (Sigma, #B1686), 50 ng/ml human recombinant Wnt3a protein (R & D Systems, #5036-WN), 5–20 μM IWR1-endo (IWR, Tocris #3532), 0.5–8 ng/ml human recombinant TGF-β1 protein (R & D Systems, #100-21C), and/or 0.5–8 μM SD208 (Wako, 193–16331).

    Techniques: Incubation, Expressing

    Activation of Wnt/β-catenin signaling decreases mRNA expressions of Scx , Mkx and Tnmd in rat TDCs. Relative expressions of Axin2 , Scx , Mkx , and Tnmd in TDCs treated with 50 ng/ml Wnt3a with or without 5 μM IWR (an inhibitor of β-catenin) (A) , 0 to 4 μM BIO (an activator of β-catenin) (B) , or 0 to 20 μM IWR (C) for 72 hrs. (B) Increasing concentrations (1, 2, and, 4 μM) of BIO increased Axin2 expression to 150%, 220%, and 730% of that without BIO, respectively. (C) Increasing concentrations (5, 10, and, 20 μM) of IWR decreased Axin2 expression to 73%, 71%, and 66% of that without IWR, respectively. As BIO was dissolved in DMSO, all samples in B were incubated under 0.008% DMSO. Each mRNA expression is normalized by Gapdh mRNA. Mean and SD are indicated ( n = 3 wells each). Tukey-Kramer post-hoc test (* p

    Journal: PLoS ONE

    Article Title: Wnt/β-catenin signaling suppresses expressions of Scx, Mkx, and Tnmd in tendon-derived cells

    doi: 10.1371/journal.pone.0182051

    Figure Lengend Snippet: Activation of Wnt/β-catenin signaling decreases mRNA expressions of Scx , Mkx and Tnmd in rat TDCs. Relative expressions of Axin2 , Scx , Mkx , and Tnmd in TDCs treated with 50 ng/ml Wnt3a with or without 5 μM IWR (an inhibitor of β-catenin) (A) , 0 to 4 μM BIO (an activator of β-catenin) (B) , or 0 to 20 μM IWR (C) for 72 hrs. (B) Increasing concentrations (1, 2, and, 4 μM) of BIO increased Axin2 expression to 150%, 220%, and 730% of that without BIO, respectively. (C) Increasing concentrations (5, 10, and, 20 μM) of IWR decreased Axin2 expression to 73%, 71%, and 66% of that without IWR, respectively. As BIO was dissolved in DMSO, all samples in B were incubated under 0.008% DMSO. Each mRNA expression is normalized by Gapdh mRNA. Mean and SD are indicated ( n = 3 wells each). Tukey-Kramer post-hoc test (* p

    Article Snippet: Treatment of cultured cells with chemical compounds and recombinant proteins TDCs and hMSC-Scx cells were cultured in the medium stated above, and were supplemented with 0.5–4 μM BIO (Sigma, #B1686), 50 ng/ml human recombinant Wnt3a protein (R & D Systems, #5036-WN), 5–20 μM IWR1-endo (IWR, Tocris #3532), 0.5–8 ng/ml human recombinant TGF-β1 protein (R & D Systems, #100-21C), and/or 0.5–8 μM SD208 (Wako, 193–16331).

    Techniques: Activation Assay, Expressing, Incubation

    a) Western blots of total glycogen synthase kinase (GSK) 3β and phosphorylated (p-) GSK3β to verify expression of recombinant GSK3β proteins. Expression of transfected (transf.) GSK3β was differentiated from that of endogenous (End.) GSK3β by a shift in molecular size. Non-transfected myocytes served as controls. Recombinant proteins of wild-type (wtGSK), constitutively active (caGSK) or dominant-negative (dnGSK) GSK3β plasmids were detected 72 h after adenoviral infection. Corresponding samples were incubated with phosphospecific GSK3β antibody. Myocytes transfected with wtGSK and dnGSK showed clear phosphorylation. In contrast, myocytes transfected with caGSK showed no phosphorylation of the recombinant protein. (b) Cardiomyocytes stimulated with 1 μmol/L SB216763 (SB21) or [d-Ala 2 -d-Leu 5 ]-enkephalin (DADLE) exhibited GSK3β phosphorylation compared with untreated control cells, suggesting a role for GSK3β within the protective signalling of DADLE.

    Journal: Clinical and experimental pharmacology & physiology

    Article Title: Inhibition of glycogen synthase kinase 3? prevents peroxide-induced collapse of mitochondrial membrane potential in rat ventricular myocytes

    doi: 10.1111/j.1440-1681.2010.05372.x

    Figure Lengend Snippet: a) Western blots of total glycogen synthase kinase (GSK) 3β and phosphorylated (p-) GSK3β to verify expression of recombinant GSK3β proteins. Expression of transfected (transf.) GSK3β was differentiated from that of endogenous (End.) GSK3β by a shift in molecular size. Non-transfected myocytes served as controls. Recombinant proteins of wild-type (wtGSK), constitutively active (caGSK) or dominant-negative (dnGSK) GSK3β plasmids were detected 72 h after adenoviral infection. Corresponding samples were incubated with phosphospecific GSK3β antibody. Myocytes transfected with wtGSK and dnGSK showed clear phosphorylation. In contrast, myocytes transfected with caGSK showed no phosphorylation of the recombinant protein. (b) Cardiomyocytes stimulated with 1 μmol/L SB216763 (SB21) or [d-Ala 2 -d-Leu 5 ]-enkephalin (DADLE) exhibited GSK3β phosphorylation compared with untreated control cells, suggesting a role for GSK3β within the protective signalling of DADLE.

    Article Snippet: The wild-type cDNA of human GSK3β (NCBI reference: ) was obtained from Open Biosystems (Huntsville, AL, USA).

    Techniques: Western Blot, Expressing, Recombinant, Transfection, Dominant Negative Mutation, Infection, Incubation

    Rat isolated cardiomyocytes stained with tetramethylrhodamine ethyl ester (TMRE) and stressed for 40 min by exposure to 100 μmol/L H 2 O 2 (control) exhibited a significant reduction in mean fluorescence compared with untreated cells (no peroxide). Treatment of cells with SB216763 (SB) and the other GSK3β inhibitor SB415286 (SB41) significantly increased mean fluorescence, indicating that glycogen synthase kinase (GSK) 3β inhibition preserves mitochondrial membrane potential in the face of oxidative stress. A similar protective effect was seen after treatment of cells with the Δ-opioid receptor agonist [d-Ala 2 -d-Leu 5 ]-enkephalin (DADLE). Myocytes transfected with wild-type (wtGSK) GSK3β plasmid showed no changes in response to either H 2 O 2 , SB216763 or DADLE compared with non-transfected cells. Data are the mean ± SEM. * P

    Journal: Clinical and experimental pharmacology & physiology

    Article Title: Inhibition of glycogen synthase kinase 3? prevents peroxide-induced collapse of mitochondrial membrane potential in rat ventricular myocytes

    doi: 10.1111/j.1440-1681.2010.05372.x

    Figure Lengend Snippet: Rat isolated cardiomyocytes stained with tetramethylrhodamine ethyl ester (TMRE) and stressed for 40 min by exposure to 100 μmol/L H 2 O 2 (control) exhibited a significant reduction in mean fluorescence compared with untreated cells (no peroxide). Treatment of cells with SB216763 (SB) and the other GSK3β inhibitor SB415286 (SB41) significantly increased mean fluorescence, indicating that glycogen synthase kinase (GSK) 3β inhibition preserves mitochondrial membrane potential in the face of oxidative stress. A similar protective effect was seen after treatment of cells with the Δ-opioid receptor agonist [d-Ala 2 -d-Leu 5 ]-enkephalin (DADLE). Myocytes transfected with wild-type (wtGSK) GSK3β plasmid showed no changes in response to either H 2 O 2 , SB216763 or DADLE compared with non-transfected cells. Data are the mean ± SEM. * P

    Article Snippet: The wild-type cDNA of human GSK3β (NCBI reference: ) was obtained from Open Biosystems (Huntsville, AL, USA).

    Techniques: Isolation, Staining, Fluorescence, Inhibition, Transfection, Plasmid Preparation

    After adenoviral transfection of myocytes with a dominant-negative (dnGSK) glycogen synthase kinase (GSK) 3β plasmid, cells were protected against the H 2 O 2 load, confirming a role for GSK3β in a highly specific approach. Further treatment of cells with 1 μmol/L SB216763 (SB) or 1 μmol/L [d-Ala 2 -d-Leu 5 ]-enkephalin (DADLE) had no additive effect. Myocytes transfected with the constitutively active (caGSK) plasmid could no longer be protected by DADLE, indicating a role for GSK3β between the surface receptor and the mitochondria. Data are the mean ± SEM. * P

    Journal: Clinical and experimental pharmacology & physiology

    Article Title: Inhibition of glycogen synthase kinase 3? prevents peroxide-induced collapse of mitochondrial membrane potential in rat ventricular myocytes

    doi: 10.1111/j.1440-1681.2010.05372.x

    Figure Lengend Snippet: After adenoviral transfection of myocytes with a dominant-negative (dnGSK) glycogen synthase kinase (GSK) 3β plasmid, cells were protected against the H 2 O 2 load, confirming a role for GSK3β in a highly specific approach. Further treatment of cells with 1 μmol/L SB216763 (SB) or 1 μmol/L [d-Ala 2 -d-Leu 5 ]-enkephalin (DADLE) had no additive effect. Myocytes transfected with the constitutively active (caGSK) plasmid could no longer be protected by DADLE, indicating a role for GSK3β between the surface receptor and the mitochondria. Data are the mean ± SEM. * P

    Article Snippet: The wild-type cDNA of human GSK3β (NCBI reference: ) was obtained from Open Biosystems (Huntsville, AL, USA).

    Techniques: Transfection, Dominant Negative Mutation, Plasmid Preparation

    12B2 and 15C2 are specific for nonphospho-S GSK3 in brain lysates of human, mouse, and rat, and both effectively immunoprecipitate GSK3 from cell lysates. (A) Protein sequence alignments for GSK3β (amino acids 1–25) and GSK3α (amino acids 13–37) from human, mouse and rat (Uniprot IDs in parentheses). (B,C) Blots of lysates from human, mouse and rat cortical tissue and GAPDH was used as a loading control (40 μg/lane total protein loaded; experiment repeated three times). (B) 12B2 (red) specifically labeled GSK3β, not GSK3α, in lysates and total GSK3α/β (green) was used to identify both isoforms. (C) 15C2 (red) labeled both GSK3β and GSK3α in lysates and total GSK3α/β (green) was used to identify both isoforms. (D–F) The 12B2 (D) , 15C2 (E) , or control mouse IgG ( F , Ms IgG) were used to immunoprecipitate GSK3 enzymes from HEK293T cell lysates. The starting lysate (Input) was incubated with magnetic beads coated with 12B2 (D) , 15C2 (E) , or Ms IgG control (F) antibodies. 12B2 pulled down only GSK3β (12B2-IP), 15C2 pulled down both GSK3α and β (15C2-IP) and Ms IgG did not pull down GSK3α or β (MsIgG-IP). The post-IP lysates were also run for comparisons to the input samples. These experiments were performed three independent times.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: 12B2 and 15C2 are specific for nonphospho-S GSK3 in brain lysates of human, mouse, and rat, and both effectively immunoprecipitate GSK3 from cell lysates. (A) Protein sequence alignments for GSK3β (amino acids 1–25) and GSK3α (amino acids 13–37) from human, mouse and rat (Uniprot IDs in parentheses). (B,C) Blots of lysates from human, mouse and rat cortical tissue and GAPDH was used as a loading control (40 μg/lane total protein loaded; experiment repeated three times). (B) 12B2 (red) specifically labeled GSK3β, not GSK3α, in lysates and total GSK3α/β (green) was used to identify both isoforms. (C) 15C2 (red) labeled both GSK3β and GSK3α in lysates and total GSK3α/β (green) was used to identify both isoforms. (D–F) The 12B2 (D) , 15C2 (E) , or control mouse IgG ( F , Ms IgG) were used to immunoprecipitate GSK3 enzymes from HEK293T cell lysates. The starting lysate (Input) was incubated with magnetic beads coated with 12B2 (D) , 15C2 (E) , or Ms IgG control (F) antibodies. 12B2 pulled down only GSK3β (12B2-IP), 15C2 pulled down both GSK3α and β (15C2-IP) and Ms IgG did not pull down GSK3α or β (MsIgG-IP). The post-IP lysates were also run for comparisons to the input samples. These experiments were performed three independent times.

    Article Snippet: HEK293T cells were transfected with either the human GSK3β Stealth primer set (1299001-VHS40279, Thermo), the human GSK3α Stealth primer set (1299001-HSS104518, Thermo), the human GAPDH Stealth control (12935140, Thermo), or the medium GC siRNA negative control (12935300, Thermo) constructs according to manufacturer’s protocol.

    Techniques: Sequencing, Labeling, Mass Spectrometry, Incubation, Magnetic Beads

    Treating cells with protein phosphatase inhibitor decreases npS9 GSK3β in cells. (A) A standard curve of dephosphorylated GSK3β protein captured with 12B2 antibody was used for quantitative sandwich ELISAs ( r 2 = 0.999). (B) Untreated HEK293T lysates assayed in 12B2 sandwich ELISAs at 120, 60, 30, 15, and 7.5 μg total protein/well produces a linear dose response curve ( r 2 = 0.988). Interpolation using the standard curve in (A) indicates that the lysate samples contain 7.4, 5.2, 3.5, 2.0, and 0.9 ng of npS9 GSK3β, respectively. (C) HEK293T cells were either untreated (-) or treated with 10 nM calyculin A for 30 min (+) to reduce npS9 GSK3β levels ( n = 4 independent experiments). The lysates were used in 12B2 sandwich ELISAs. A significant reduction in npS9 GSK3β levels was detected in calyculin A (10 nM) treated cells compared to untreated cells ( ∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: Treating cells with protein phosphatase inhibitor decreases npS9 GSK3β in cells. (A) A standard curve of dephosphorylated GSK3β protein captured with 12B2 antibody was used for quantitative sandwich ELISAs ( r 2 = 0.999). (B) Untreated HEK293T lysates assayed in 12B2 sandwich ELISAs at 120, 60, 30, 15, and 7.5 μg total protein/well produces a linear dose response curve ( r 2 = 0.988). Interpolation using the standard curve in (A) indicates that the lysate samples contain 7.4, 5.2, 3.5, 2.0, and 0.9 ng of npS9 GSK3β, respectively. (C) HEK293T cells were either untreated (-) or treated with 10 nM calyculin A for 30 min (+) to reduce npS9 GSK3β levels ( n = 4 independent experiments). The lysates were used in 12B2 sandwich ELISAs. A significant reduction in npS9 GSK3β levels was detected in calyculin A (10 nM) treated cells compared to untreated cells ( ∗ p

    Article Snippet: HEK293T cells were transfected with either the human GSK3β Stealth primer set (1299001-VHS40279, Thermo), the human GSK3α Stealth primer set (1299001-HSS104518, Thermo), the human GAPDH Stealth control (12935140, Thermo), or the medium GC siRNA negative control (12935300, Thermo) constructs according to manufacturer’s protocol.

    Techniques:

    Protein phosphatases regulate GSK3β phosphorylation independent of Akt signaling. HEK293T cells were treated with an Akt inhibitor (AZD-5363, 1 μM), a protein phosphatase inhibitor (calyculin A, 10 nM) or the Akt inhibitor followed by the phosphatase inhibitor. Four independent experiments were run. (A) Western blots of samples were probed with 12B2 (npS9-GSK3β specific), total GSK3α/β, pS9-GSK3β and GAPDH (loading control). (B) Quantitation of the blots shows that inhibition of Akt (AZD) significantly increased npS9 GSK3β, while inhibition of protein phosphatases (Caly) significantly reduced npS9 GSK3β. When Akt signaling was blocked first and then the phosphatase inhibitor was applied (AZD + Caly) a significant reduction in the level of npS9 GSK3β occurred when compared to Akt inhibitor alone. (C) Quantitation of the pS9 GSK3β blots shows an opposite pattern where inhibition of Akt significantly decreased pS9 GSK3β, while inhibition of phosphatases significantly increased pS9 GSK3β. When Akt signaling was blocked and then the phosphatase inhibitor was applied a significant increase in the level of pS9 GSK3β occurred when compared to Akt treatment alone. (D) Western blots of samples were probed with 15C2 (npS9-GSK3α/β specific), total GSK3α/β, pS9-GSK3β and GAPDH (loading control). (E,F) Quantitation of the blots shows that inhibition of Akt significantly increased npS9 GSK3α and β, while inhibition of protein phosphatases significantly reduced npS9 GSK3α and β. When Akt signaling was blocked and then the phosphatase inhibitor was applied a significant reduction in the level of npS9 GSK3β and npS21 GSK3α occurred when compared to Akt inhibitor alone. Collectively, these results suggest protein phosphatases dephosphorylate Ser9/21 independent of Akt signaling. All bands are normalized to GAPDH. All groups were statistically significant from the others in ( B,C,E,F) , but only ∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: Protein phosphatases regulate GSK3β phosphorylation independent of Akt signaling. HEK293T cells were treated with an Akt inhibitor (AZD-5363, 1 μM), a protein phosphatase inhibitor (calyculin A, 10 nM) or the Akt inhibitor followed by the phosphatase inhibitor. Four independent experiments were run. (A) Western blots of samples were probed with 12B2 (npS9-GSK3β specific), total GSK3α/β, pS9-GSK3β and GAPDH (loading control). (B) Quantitation of the blots shows that inhibition of Akt (AZD) significantly increased npS9 GSK3β, while inhibition of protein phosphatases (Caly) significantly reduced npS9 GSK3β. When Akt signaling was blocked first and then the phosphatase inhibitor was applied (AZD + Caly) a significant reduction in the level of npS9 GSK3β occurred when compared to Akt inhibitor alone. (C) Quantitation of the pS9 GSK3β blots shows an opposite pattern where inhibition of Akt significantly decreased pS9 GSK3β, while inhibition of phosphatases significantly increased pS9 GSK3β. When Akt signaling was blocked and then the phosphatase inhibitor was applied a significant increase in the level of pS9 GSK3β occurred when compared to Akt treatment alone. (D) Western blots of samples were probed with 15C2 (npS9-GSK3α/β specific), total GSK3α/β, pS9-GSK3β and GAPDH (loading control). (E,F) Quantitation of the blots shows that inhibition of Akt significantly increased npS9 GSK3α and β, while inhibition of protein phosphatases significantly reduced npS9 GSK3α and β. When Akt signaling was blocked and then the phosphatase inhibitor was applied a significant reduction in the level of npS9 GSK3β and npS21 GSK3α occurred when compared to Akt inhibitor alone. Collectively, these results suggest protein phosphatases dephosphorylate Ser9/21 independent of Akt signaling. All bands are normalized to GAPDH. All groups were statistically significant from the others in ( B,C,E,F) , but only ∗ p

    Article Snippet: HEK293T cells were transfected with either the human GSK3β Stealth primer set (1299001-VHS40279, Thermo), the human GSK3α Stealth primer set (1299001-HSS104518, Thermo), the human GAPDH Stealth control (12935140, Thermo), or the medium GC siRNA negative control (12935300, Thermo) constructs according to manufacturer’s protocol.

    Techniques: Western Blot, Quantitation Assay, Inhibition

    Protein phosphatase inhibition significantly reduces GSK3β kinase activity in cells. (A) A standard curve of active GSK3β enzyme (300 – 9.4 ng) confirmed the signal in the experimental samples was within the linear range of detection in this assay ( r 2 = 0.97). Experiment was repeated three times. (B) Calyculin A treated cells showed a significant reduction in GSK3β kinase activity compared to control cells (the –TCS sample sets; all samples were used at 60 μg total protein/well). Interpolation from the recombinant GSK3β enzyme activity curve with known amounts of active GSK3β indicated that the control samples contained 29 ng of active GSK3β and calyculin A treated cells contained 15 ng. Addition of TCS-2002 (0.1 mM; +TCS), a potent GSK3β inhibitor, completely blocked kinase activity in control and calyculin A treated cells ( ∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: Protein phosphatase inhibition significantly reduces GSK3β kinase activity in cells. (A) A standard curve of active GSK3β enzyme (300 – 9.4 ng) confirmed the signal in the experimental samples was within the linear range of detection in this assay ( r 2 = 0.97). Experiment was repeated three times. (B) Calyculin A treated cells showed a significant reduction in GSK3β kinase activity compared to control cells (the –TCS sample sets; all samples were used at 60 μg total protein/well). Interpolation from the recombinant GSK3β enzyme activity curve with known amounts of active GSK3β indicated that the control samples contained 29 ng of active GSK3β and calyculin A treated cells contained 15 ng. Addition of TCS-2002 (0.1 mM; +TCS), a potent GSK3β inhibitor, completely blocked kinase activity in control and calyculin A treated cells ( ∗ p

    Article Snippet: HEK293T cells were transfected with either the human GSK3β Stealth primer set (1299001-VHS40279, Thermo), the human GSK3α Stealth primer set (1299001-HSS104518, Thermo), the human GAPDH Stealth control (12935140, Thermo), or the medium GC siRNA negative control (12935300, Thermo) constructs according to manufacturer’s protocol.

    Techniques: Inhibition, Activity Assay, Recombinant

    12B2 is specific for nonphospho-S9 recombinant GSK3β, and 15C2 is specific for nonphospho-S9/21 recombinant GSK3β/α. (A–C) Western blots of recombinant GSK3β and α alone (input, In), phosphorylated S9 GSK3β and S21 GSK3α proteins (Ph; i.e., Akt1 treated) and dephosphorylated S9 GSK3β and S21 GSK3α proteins (NP; i.e., alkaline phosphatase treated). The GSK3α is GST-tagged and GSK3β is his-tagged, and in each blot the 12B2 (A) , 15C2 (B) or pS9 GSK3β (C) antibodies are red, while total GSK3α/β antibody is green. (D) Quantitation of the 12B2 blots shows strong reactivity with npS9 GSK3β, and none with npS21 GSK3α, pS9 GSK3β or pS21 GSK3α. (E) Quantitation of the 15C2 blots shows strong reactivity with npS9 GSK3β and npS21 GSK3α, but none with pS9 GSK3β or pS21 GSK3α. (F) Quantitation of the pS9 GSK3 blots confirm that the Akt1 treatment produced robust phosphorylation of S9 in GSK3β (and S21 in α) and that the alkaline phosphatase treatment removed phosphorylation of S9 in GSK3β (and S21 in α). Each experiment was repeated three-four independent times and all samples were loaded at 50 ng GSK3/lane. The data are normalized to total GSK3 signal.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: 12B2 is specific for nonphospho-S9 recombinant GSK3β, and 15C2 is specific for nonphospho-S9/21 recombinant GSK3β/α. (A–C) Western blots of recombinant GSK3β and α alone (input, In), phosphorylated S9 GSK3β and S21 GSK3α proteins (Ph; i.e., Akt1 treated) and dephosphorylated S9 GSK3β and S21 GSK3α proteins (NP; i.e., alkaline phosphatase treated). The GSK3α is GST-tagged and GSK3β is his-tagged, and in each blot the 12B2 (A) , 15C2 (B) or pS9 GSK3β (C) antibodies are red, while total GSK3α/β antibody is green. (D) Quantitation of the 12B2 blots shows strong reactivity with npS9 GSK3β, and none with npS21 GSK3α, pS9 GSK3β or pS21 GSK3α. (E) Quantitation of the 15C2 blots shows strong reactivity with npS9 GSK3β and npS21 GSK3α, but none with pS9 GSK3β or pS21 GSK3α. (F) Quantitation of the pS9 GSK3 blots confirm that the Akt1 treatment produced robust phosphorylation of S9 in GSK3β (and S21 in α) and that the alkaline phosphatase treatment removed phosphorylation of S9 in GSK3β (and S21 in α). Each experiment was repeated three-four independent times and all samples were loaded at 50 ng GSK3/lane. The data are normalized to total GSK3 signal.

    Article Snippet: HEK293T cells were transfected with either the human GSK3β Stealth primer set (1299001-VHS40279, Thermo), the human GSK3α Stealth primer set (1299001-HSS104518, Thermo), the human GAPDH Stealth control (12935140, Thermo), or the medium GC siRNA negative control (12935300, Thermo) constructs according to manufacturer’s protocol.

    Techniques: Recombinant, Western Blot, Quantitation Assay, Produced

    siRNA knockdown of GSK3α and GSK3β demonstrate specificity of the 12B2 antibody. (A) HEK293T cells were treated with control, GSK3α, GSK3β or GAPDH siRNAs and probed with 12B2 (red) and total GSK3β/α (green) antibodies. (B) Quantitation of 12B2 signal shows that GSK3β siRNA caused a reduction of 50% for GSK3β when compared to control cells, while GSK3α siRNA caused an increase in GSK3β (+35%). (C) Quantitation of total GSK3α/β antibody signal shows that GSK3α siRNA caused a loss of 66% for GSK3α and an increase in GSK3β (+29%) when compared to controls. Quantitation of total GSK3α/β antibody signal shows that GSK3β siRNA caused a loss of 41% for GSK3β and an increase in the GSK3α (+17%) when compared to control cells. All immunoblotting data are normalized to GAPDH signal and expressed as percent of the control group to illustrate the siRNA-mediated changes in signal. (D) Immunocytofluorescence of HEK293T cells confirms the reduction in 12B2 detection of npS9 GSK3β, which produces a punctate staining pattern, in GSK3β siRNA treated cells compared to control and GSK3α siRNA treated cells. Scale bars = 20 μm. Four independent experiments were performed. GAPDH siRNA quantitation is provided in Supplementary Figure S4 .

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: siRNA knockdown of GSK3α and GSK3β demonstrate specificity of the 12B2 antibody. (A) HEK293T cells were treated with control, GSK3α, GSK3β or GAPDH siRNAs and probed with 12B2 (red) and total GSK3β/α (green) antibodies. (B) Quantitation of 12B2 signal shows that GSK3β siRNA caused a reduction of 50% for GSK3β when compared to control cells, while GSK3α siRNA caused an increase in GSK3β (+35%). (C) Quantitation of total GSK3α/β antibody signal shows that GSK3α siRNA caused a loss of 66% for GSK3α and an increase in GSK3β (+29%) when compared to controls. Quantitation of total GSK3α/β antibody signal shows that GSK3β siRNA caused a loss of 41% for GSK3β and an increase in the GSK3α (+17%) when compared to control cells. All immunoblotting data are normalized to GAPDH signal and expressed as percent of the control group to illustrate the siRNA-mediated changes in signal. (D) Immunocytofluorescence of HEK293T cells confirms the reduction in 12B2 detection of npS9 GSK3β, which produces a punctate staining pattern, in GSK3β siRNA treated cells compared to control and GSK3α siRNA treated cells. Scale bars = 20 μm. Four independent experiments were performed. GAPDH siRNA quantitation is provided in Supplementary Figure S4 .

    Article Snippet: HEK293T cells were transfected with either the human GSK3β Stealth primer set (1299001-VHS40279, Thermo), the human GSK3α Stealth primer set (1299001-HSS104518, Thermo), the human GAPDH Stealth control (12935140, Thermo), or the medium GC siRNA negative control (12935300, Thermo) constructs according to manufacturer’s protocol.

    Techniques: Quantitation Assay, Staining

    GSK3β antibody immunostaining in cultured cells and tissue sections. (A,B) HEK293T cells stained with 12B2 ( A , green) and 15C2 ( B , green). ( C,D ) Undifferentiated SH-SY5Y cells stained with 12B2 ( C , green) and 15C2 ( D , green). (E,F) Rat primary cortical neurons (E18) stained with 12B2 ( E , green) and 15C2 ( F , green). In A-F , all cells were also stained with total GSK3α/β (red) and DAPI (blue in merged image). The pattern of staining with 12B2 and 15C2 was punctate staining throughout the cells, and 12B2 produced stronger signal than 15C2 in each cell type. Scale bars = 25 μm. (G,H) Brain sections in rat (left, retrosplenial cortex displayed) and human (right, temporal cortex) stained with 12B2 (G) or 15C2 (H) . In general, both antibodies produced clear somatodendritic and parenchymal staining in human and rat brain sections. Scale bars = 50 μm.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: GSK3β antibody immunostaining in cultured cells and tissue sections. (A,B) HEK293T cells stained with 12B2 ( A , green) and 15C2 ( B , green). ( C,D ) Undifferentiated SH-SY5Y cells stained with 12B2 ( C , green) and 15C2 ( D , green). (E,F) Rat primary cortical neurons (E18) stained with 12B2 ( E , green) and 15C2 ( F , green). In A-F , all cells were also stained with total GSK3α/β (red) and DAPI (blue in merged image). The pattern of staining with 12B2 and 15C2 was punctate staining throughout the cells, and 12B2 produced stronger signal than 15C2 in each cell type. Scale bars = 25 μm. (G,H) Brain sections in rat (left, retrosplenial cortex displayed) and human (right, temporal cortex) stained with 12B2 (G) or 15C2 (H) . In general, both antibodies produced clear somatodendritic and parenchymal staining in human and rat brain sections. Scale bars = 50 μm.

    Article Snippet: HEK293T cells were transfected with either the human GSK3β Stealth primer set (1299001-VHS40279, Thermo), the human GSK3α Stealth primer set (1299001-HSS104518, Thermo), the human GAPDH Stealth control (12935140, Thermo), or the medium GC siRNA negative control (12935300, Thermo) constructs according to manufacturer’s protocol.

    Techniques: Immunostaining, Cell Culture, Staining, Produced

    siRNA knockdown of GSK3α and GSK3β demonstrate specificity of the 15C2 antibody. (A) HEK293T cells were treated with control, GSK3α, GSK3β or GAPDH siRNAs and probed with 15C2 (red) and total GSK3β/α (green) antibodies. (B) Quantitation of 15C2 signal shows that GSK3α siRNA caused a loss of 84% for GSK3α and an increase in GSK3β (+22%) when compared to control cells. Quantitation of 15C2 shows that GSK3β siRNA caused a loss of 49% for GSK3β and an increase in GSK3α (+18%) when compared to control. (C) Quantitation of total GSK3α/β antibody signal shows that GSK3α siRNA caused a loss of 66% in GSK3α and an increase in GSK3β (+24%) when compared to controls. Quantitation of total GSK3α/β antibody signal shows that GSK3β siRNA caused a loss of 40% for the GSK3β and an increase in GSK3α (+9%) when compared to control cells. All immunoblotting data are normalized to GAPDH signal and expressed as percent of the control group to illustrate the siRNA-mediated changes in signal. (D) Immunocytofluorescence of HEK293T cells confirms the reduction in 15C2 detection of npS21 GSK3α or npS9 GSK3β when treated with GSK3α siRNA or GSK3β siRNA, respectively. Scale bars = 20 μm. Four independent experiments were performed. GAPDH siRNA quantitation is provided in Supplementary Figure S4 .

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: siRNA knockdown of GSK3α and GSK3β demonstrate specificity of the 15C2 antibody. (A) HEK293T cells were treated with control, GSK3α, GSK3β or GAPDH siRNAs and probed with 15C2 (red) and total GSK3β/α (green) antibodies. (B) Quantitation of 15C2 signal shows that GSK3α siRNA caused a loss of 84% for GSK3α and an increase in GSK3β (+22%) when compared to control cells. Quantitation of 15C2 shows that GSK3β siRNA caused a loss of 49% for GSK3β and an increase in GSK3α (+18%) when compared to control. (C) Quantitation of total GSK3α/β antibody signal shows that GSK3α siRNA caused a loss of 66% in GSK3α and an increase in GSK3β (+24%) when compared to controls. Quantitation of total GSK3α/β antibody signal shows that GSK3β siRNA caused a loss of 40% for the GSK3β and an increase in GSK3α (+9%) when compared to control cells. All immunoblotting data are normalized to GAPDH signal and expressed as percent of the control group to illustrate the siRNA-mediated changes in signal. (D) Immunocytofluorescence of HEK293T cells confirms the reduction in 15C2 detection of npS21 GSK3α or npS9 GSK3β when treated with GSK3α siRNA or GSK3β siRNA, respectively. Scale bars = 20 μm. Four independent experiments were performed. GAPDH siRNA quantitation is provided in Supplementary Figure S4 .

    Article Snippet: HEK293T cells were transfected with either the human GSK3β Stealth primer set (1299001-VHS40279, Thermo), the human GSK3α Stealth primer set (1299001-HSS104518, Thermo), the human GAPDH Stealth control (12935140, Thermo), or the medium GC siRNA negative control (12935300, Thermo) constructs according to manufacturer’s protocol.

    Techniques: Quantitation Assay

    12B2 and 15C2 are specific for nonphospho-Ser GSK3β/α peptides. Each antibody was screened in indirect ELISA titers against npS9 GSK3β, pS9 GSK3β, npS21 GSK3α and pS21 GSK3α peptides ( n = 3 independent experiments). (A) 12B2 showed strong reactivity for npS9 GSK3β compared to npS21 GSK3α peptides and did not react with pS9 or pS21 GSK3 peptides (EC 50 values: npS9 = 2.1 nM; pS9 = indeterminate (id); npS21 = 6.4 nM; pS21 = id). (B) To further confirm the specificity of 12B2, ELISAs were performed by coating wells with a wide range of peptide amounts (0 – 6.4 μg peptide/well). 12B2 showed strong reactivity with the npS GSK3 peptides (β > α), but did not react with pS GSK3 peptides. (C) 15C2 showed stronger reactivity for npS21 GSK3α compared to npS9 GSK3β and did not react with pS9 or pS21 GSK3 peptides (EC 50 values: npS9 = 2.4 nM; pS9 = id; npS21 = 277 pM; pS21 = id). (D) To further confirm the specificity of 15C2, ELISAs were performed by coating wells with a wide range of peptide amounts (0 – 6.4 μg peptide/well). 15C2 showed strong reactivity with the npS GSK3 peptides, but did not react with pS GSK peptides. It is noteworthy that synthetic peptides provide a homogeneous source of modified peptides, and thus, are ideal for challenging the specificity of the antibodies against nonphospho-Ser and phospho-Ser residues in GSK3.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: 12B2 and 15C2 are specific for nonphospho-Ser GSK3β/α peptides. Each antibody was screened in indirect ELISA titers against npS9 GSK3β, pS9 GSK3β, npS21 GSK3α and pS21 GSK3α peptides ( n = 3 independent experiments). (A) 12B2 showed strong reactivity for npS9 GSK3β compared to npS21 GSK3α peptides and did not react with pS9 or pS21 GSK3 peptides (EC 50 values: npS9 = 2.1 nM; pS9 = indeterminate (id); npS21 = 6.4 nM; pS21 = id). (B) To further confirm the specificity of 12B2, ELISAs were performed by coating wells with a wide range of peptide amounts (0 – 6.4 μg peptide/well). 12B2 showed strong reactivity with the npS GSK3 peptides (β > α), but did not react with pS GSK3 peptides. (C) 15C2 showed stronger reactivity for npS21 GSK3α compared to npS9 GSK3β and did not react with pS9 or pS21 GSK3 peptides (EC 50 values: npS9 = 2.4 nM; pS9 = id; npS21 = 277 pM; pS21 = id). (D) To further confirm the specificity of 15C2, ELISAs were performed by coating wells with a wide range of peptide amounts (0 – 6.4 μg peptide/well). 15C2 showed strong reactivity with the npS GSK3 peptides, but did not react with pS GSK peptides. It is noteworthy that synthetic peptides provide a homogeneous source of modified peptides, and thus, are ideal for challenging the specificity of the antibodies against nonphospho-Ser and phospho-Ser residues in GSK3.

    Article Snippet: HEK293T cells were transfected with either the human GSK3β Stealth primer set (1299001-VHS40279, Thermo), the human GSK3α Stealth primer set (1299001-HSS104518, Thermo), the human GAPDH Stealth control (12935140, Thermo), or the medium GC siRNA negative control (12935300, Thermo) constructs according to manufacturer’s protocol.

    Techniques: Indirect ELISA, Modification

    The Akt-protein phosphatase signaling pathway involved in regulating GSK3β phosphorylation. Active Akt (i.e., phosphorylated) inactivates GSK3β by phosphorylation at S9. Protein phosphatases can modulate GSK3β phosphorylation at S9 via two routes. (1) Protein phosphatases inactivate Akt by dephosphorylation, and (2) protein phosphatases activate GSK3β by directly dephosphorylating S9. Inhibition of Akt (with inhibitors such as AZD-5363) increases non-phosphorylated GSK3β by suppressing Akt-mediated phosphorylation of GSK3β. Inhibition of protein phosphatases (with inhibitors such as calyculin A) causes a decrease in non-phosphorylated GSK3β through the Akt pathway by increasing active Akt (the grayed portion of the Akt cycle). Protein phosphatase inhibition also leads to decreased non-phosphorylated GSK3β independent of Akt by directly dephosphorylating S9 in GSK3β. If an Akt inhibitor is applied followed by a protein phosphatase inhibitor the Akt-independent pathway can be evaluated.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: The Akt-protein phosphatase signaling pathway involved in regulating GSK3β phosphorylation. Active Akt (i.e., phosphorylated) inactivates GSK3β by phosphorylation at S9. Protein phosphatases can modulate GSK3β phosphorylation at S9 via two routes. (1) Protein phosphatases inactivate Akt by dephosphorylation, and (2) protein phosphatases activate GSK3β by directly dephosphorylating S9. Inhibition of Akt (with inhibitors such as AZD-5363) increases non-phosphorylated GSK3β by suppressing Akt-mediated phosphorylation of GSK3β. Inhibition of protein phosphatases (with inhibitors such as calyculin A) causes a decrease in non-phosphorylated GSK3β through the Akt pathway by increasing active Akt (the grayed portion of the Akt cycle). Protein phosphatase inhibition also leads to decreased non-phosphorylated GSK3β independent of Akt by directly dephosphorylating S9 in GSK3β. If an Akt inhibitor is applied followed by a protein phosphatase inhibitor the Akt-independent pathway can be evaluated.

    Article Snippet: HEK293T cells were transfected with either the human GSK3β Stealth primer set (1299001-VHS40279, Thermo), the human GSK3α Stealth primer set (1299001-HSS104518, Thermo), the human GAPDH Stealth control (12935140, Thermo), or the medium GC siRNA negative control (12935300, Thermo) constructs according to manufacturer’s protocol.

    Techniques: De-Phosphorylation Assay, Inhibition

    Detection of recombinant npS9 GSK3β with 12B2 and 15C2 antibodies is linear and correlates with kinase activity. (A) The level of GSK3β kinase activity with 30, 60, 120, 180, 240, and 300 ng of npS9 GSK3β (“active”) was measured using an in vitro GSK3β kinase activity assay and there was a linear increase in kinase activity with increasing amounts of GSK3β ( r 2 = 0.93). Three independent experiments were performed. (B) For western blotting, recombinant GSK3β was incubated with alkaline phosphatase to generate nonphosphoS9 GSK3β or incubated with Akt1 to generate phosphoS9 GSK3β, and then 0, 30, 60, 120, 180, 240, or 300 ng of npS9 GSK3β was mixed with 300, 240, 180, 120, 60, or 0 ng of pS9 GSK3β to bring the total protein content to 300 ng/lane. The blot was probed with 12B2 (red) and total GSK3α/β antibodies (green). (C) Quantitation of signal from 12B2 shows a linear increase in reactivity with increasing npS9 GSK3β amount ( r 2 = 0.92). (D) The same samples were probed with 15C2 (red) and total GSK3α/β antibodies (green). (E) Quantitation of signal from 15C2 shows a linear increase in reactivity with increasing npS9 GSK3β amount ( r 2 = 0.90). It is notable that both 12B2 and 15C2 signals also showed a direct correlation with GSK3β activity levels (12B2: r = 0.99, p = 0.0002; 15C2: r = 0.99, p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: Detection of recombinant npS9 GSK3β with 12B2 and 15C2 antibodies is linear and correlates with kinase activity. (A) The level of GSK3β kinase activity with 30, 60, 120, 180, 240, and 300 ng of npS9 GSK3β (“active”) was measured using an in vitro GSK3β kinase activity assay and there was a linear increase in kinase activity with increasing amounts of GSK3β ( r 2 = 0.93). Three independent experiments were performed. (B) For western blotting, recombinant GSK3β was incubated with alkaline phosphatase to generate nonphosphoS9 GSK3β or incubated with Akt1 to generate phosphoS9 GSK3β, and then 0, 30, 60, 120, 180, 240, or 300 ng of npS9 GSK3β was mixed with 300, 240, 180, 120, 60, or 0 ng of pS9 GSK3β to bring the total protein content to 300 ng/lane. The blot was probed with 12B2 (red) and total GSK3α/β antibodies (green). (C) Quantitation of signal from 12B2 shows a linear increase in reactivity with increasing npS9 GSK3β amount ( r 2 = 0.92). (D) The same samples were probed with 15C2 (red) and total GSK3α/β antibodies (green). (E) Quantitation of signal from 15C2 shows a linear increase in reactivity with increasing npS9 GSK3β amount ( r 2 = 0.90). It is notable that both 12B2 and 15C2 signals also showed a direct correlation with GSK3β activity levels (12B2: r = 0.99, p = 0.0002; 15C2: r = 0.99, p

    Article Snippet: HEK293T cells were transfected with either the human GSK3β Stealth primer set (1299001-VHS40279, Thermo), the human GSK3α Stealth primer set (1299001-HSS104518, Thermo), the human GAPDH Stealth control (12935140, Thermo), or the medium GC siRNA negative control (12935300, Thermo) constructs according to manufacturer’s protocol.

    Techniques: Recombinant, Activity Assay, In Vitro, Kinase Assay, Western Blot, Incubation, Quantitation Assay

    Induction of gene expression in Gsk3β +/+ (wild-type) and Gsk3β −/− (knockout) mouse embryonic fibroblasts (MEFs) by IL-17 family cytokines ( A – D ) MEFs were treated with 20 ng/ml mouse recombinant IL-17A (A), IL-17F (B), IL-17C (C), and IL-17E (D) for 2 h. Gene expression was determined using qRT-PCR analysis. The levels of the control group (treated with phosphate-buffered saline) were taken as the basal levels. Data represent the mean ± standard deviation of three independent experiments ( n = 3); * P

    Journal: Oncotarget

    Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

    doi: 10.18632/oncotarget.7296

    Figure Lengend Snippet: Induction of gene expression in Gsk3β +/+ (wild-type) and Gsk3β −/− (knockout) mouse embryonic fibroblasts (MEFs) by IL-17 family cytokines ( A – D ) MEFs were treated with 20 ng/ml mouse recombinant IL-17A (A), IL-17F (B), IL-17C (C), and IL-17E (D) for 2 h. Gene expression was determined using qRT-PCR analysis. The levels of the control group (treated with phosphate-buffered saline) were taken as the basal levels. Data represent the mean ± standard deviation of three independent experiments ( n = 3); * P

    Article Snippet: In vitro kinase assay For human peptides, 5 μg non-phosphorylated peptides were incubated with or without 1 μl (0.35 μg) of recombinant human GSK3α (Invitrogen) or 1 μl (500 units) recombinant human or rabbit GSK3β (R & D Systems or New England Biolab) in 9 μl GSK3 kinase buffer (New England Biolab) with or without 200 μM ATP (New England Biolab) at 30°C for 1 h. One μl sample was printed onto nitrocellulose membrane, air dried, and probed with anti-phospho-IL-17RA antibodies.

    Techniques: Expressing, Knock-Out, Recombinant, Quantitative RT-PCR, Standard Deviation

    GSK3β binds to and phosphorylates IL-17RA ( A – D ) 293 cells were co-transfected with Flag-IL-17RA, V5-IL-17RC, and HA-Act1, labeled with 32 P orthophosphate, and treated with 20 ng/ml IL-17A for 20 min; IP with anti-HA; autoradiography (A) followed by IB (B-D). ( E ) Anti-Flag IP followed by IB. ( F – G ) Co-IP of Flag-IL-17RA and HA-GSK3β. ( H ) 293 cells were co-transfected with Flag-IL-17RA, WT HA-GSK3β or kinase-dead mutant HA-GSK3βK85A, labeled with 32 P orthophosphate, or treated with 20 mM LiCl for 2 h; autoradiography ( 32 P) followed by IB. WE, whole cell extract.

    Journal: Oncotarget

    Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

    doi: 10.18632/oncotarget.7296

    Figure Lengend Snippet: GSK3β binds to and phosphorylates IL-17RA ( A – D ) 293 cells were co-transfected with Flag-IL-17RA, V5-IL-17RC, and HA-Act1, labeled with 32 P orthophosphate, and treated with 20 ng/ml IL-17A for 20 min; IP with anti-HA; autoradiography (A) followed by IB (B-D). ( E ) Anti-Flag IP followed by IB. ( F – G ) Co-IP of Flag-IL-17RA and HA-GSK3β. ( H ) 293 cells were co-transfected with Flag-IL-17RA, WT HA-GSK3β or kinase-dead mutant HA-GSK3βK85A, labeled with 32 P orthophosphate, or treated with 20 mM LiCl for 2 h; autoradiography ( 32 P) followed by IB. WE, whole cell extract.

    Article Snippet: In vitro kinase assay For human peptides, 5 μg non-phosphorylated peptides were incubated with or without 1 μl (0.35 μg) of recombinant human GSK3α (Invitrogen) or 1 μl (500 units) recombinant human or rabbit GSK3β (R & D Systems or New England Biolab) in 9 μl GSK3 kinase buffer (New England Biolab) with or without 200 μM ATP (New England Biolab) at 30°C for 1 h. One μl sample was printed onto nitrocellulose membrane, air dried, and probed with anti-phospho-IL-17RA antibodies.

    Techniques: Transfection, Labeling, Autoradiography, Co-Immunoprecipitation Assay, Mutagenesis

    GSK3β phosphorylates IL-17RA at T780 ( A – B ) 293 cells were transfected with Flag-IL-17RA and its mutants and labeled with 32 P orthophosphate; autoradiography ( 32 P) followed by IB. ( C ) IL-17RA peptide phosphorylated at T780 (p-Peptide) was treated with CIP, while IL-17RA peptide with wild-type T780 (Peptide) or with mutant T780A (mut-Peptide) were treated with recombinant GSK3, followed with dot blot analysis using B4 antibodies. ( D – E ) Flag-IL-17RA, Flag-IL-17RAT780A mutant, or empty vector was transfected into 293 cells; HeLa cells were not transfected; IP with anti-Flag, B4 (+), or control IgG (−), followed by IB; *indicates endogenous P-IL-17RA.

    Journal: Oncotarget

    Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

    doi: 10.18632/oncotarget.7296

    Figure Lengend Snippet: GSK3β phosphorylates IL-17RA at T780 ( A – B ) 293 cells were transfected with Flag-IL-17RA and its mutants and labeled with 32 P orthophosphate; autoradiography ( 32 P) followed by IB. ( C ) IL-17RA peptide phosphorylated at T780 (p-Peptide) was treated with CIP, while IL-17RA peptide with wild-type T780 (Peptide) or with mutant T780A (mut-Peptide) were treated with recombinant GSK3, followed with dot blot analysis using B4 antibodies. ( D – E ) Flag-IL-17RA, Flag-IL-17RAT780A mutant, or empty vector was transfected into 293 cells; HeLa cells were not transfected; IP with anti-Flag, B4 (+), or control IgG (−), followed by IB; *indicates endogenous P-IL-17RA.

    Article Snippet: In vitro kinase assay For human peptides, 5 μg non-phosphorylated peptides were incubated with or without 1 μl (0.35 μg) of recombinant human GSK3α (Invitrogen) or 1 μl (500 units) recombinant human or rabbit GSK3β (R & D Systems or New England Biolab) in 9 μl GSK3 kinase buffer (New England Biolab) with or without 200 μM ATP (New England Biolab) at 30°C for 1 h. One μl sample was printed onto nitrocellulose membrane, air dried, and probed with anti-phospho-IL-17RA antibodies.

    Techniques: Transfection, Labeling, Autoradiography, Mutagenesis, Recombinant, Dot Blot, Plasmid Preparation

    T 613 is phosphorylated by GSK3. ( A–D ) Expression and localization of Pf GSK3 and its PKA dependent phosphorylation of AMA1. ( A ) Schematic drawing of the GSK3-GFP 3′replacement approach in 3D7 parasites and diagnostic PCR revealing plasmid integration. The gsk3 gene has a six exon structure and an open reading frame of 2472 base pairs. Approximately 1 kb of the 3′ end was fused with the coding sequence of GFP (black) and cloned into a pARL derivate (pARL-gsk3-3’repl-gfp). The human dihydrofolate reductase (hDHFR, grey box) of the plasmid allowed selection of transgenic parasites. Position of oligonucleotides used for diagnostic PCR are shown with blue and red arrows. Sizes are indicated in kilo bases (kb). ( B ) Expression of Pf GSK3-GFP in late stage parasites was analyzed by Western blot analysis using anti-GFP specific antibodies. Anti-Aldolase specific antibodies were used as a loading control. ( C , D ) Epifluorescence ( C ) and confocal ( D ) localization of Pf GSK3-GFP in late trophozoites (LT) schizonts (S) and merozoites (M) revealed perinuclear and cytosolic distribution. Nuclei are stained with DAPI (blue). Scale bars, 2 μm. E. SDS-PAGE and autoradiograph of in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) of AMA1 WT and AMA1 PM incubated with human GSK3β (hGSK3β). ( F ) Differential in vitro phosphorylation of AMA1 variants with single phosphorylation sites (AMA1 S588 , AMA1 S601 , AMA1 S610 , AMA1 T612 , AMA1 T613 ) by hGSK3β. SDS-PAGE and autoradiograph of the in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) are shown.

    Journal: Scientific Reports

    Article Title: Hierarchical phosphorylation of apical membrane antigen 1 is required for efficient red blood cell invasion by malaria parasites

    doi: 10.1038/srep34479

    Figure Lengend Snippet: T 613 is phosphorylated by GSK3. ( A–D ) Expression and localization of Pf GSK3 and its PKA dependent phosphorylation of AMA1. ( A ) Schematic drawing of the GSK3-GFP 3′replacement approach in 3D7 parasites and diagnostic PCR revealing plasmid integration. The gsk3 gene has a six exon structure and an open reading frame of 2472 base pairs. Approximately 1 kb of the 3′ end was fused with the coding sequence of GFP (black) and cloned into a pARL derivate (pARL-gsk3-3’repl-gfp). The human dihydrofolate reductase (hDHFR, grey box) of the plasmid allowed selection of transgenic parasites. Position of oligonucleotides used for diagnostic PCR are shown with blue and red arrows. Sizes are indicated in kilo bases (kb). ( B ) Expression of Pf GSK3-GFP in late stage parasites was analyzed by Western blot analysis using anti-GFP specific antibodies. Anti-Aldolase specific antibodies were used as a loading control. ( C , D ) Epifluorescence ( C ) and confocal ( D ) localization of Pf GSK3-GFP in late trophozoites (LT) schizonts (S) and merozoites (M) revealed perinuclear and cytosolic distribution. Nuclei are stained with DAPI (blue). Scale bars, 2 μm. E. SDS-PAGE and autoradiograph of in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) of AMA1 WT and AMA1 PM incubated with human GSK3β (hGSK3β). ( F ) Differential in vitro phosphorylation of AMA1 variants with single phosphorylation sites (AMA1 S588 , AMA1 S601 , AMA1 S610 , AMA1 T612 , AMA1 T613 ) by hGSK3β. SDS-PAGE and autoradiograph of the in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) are shown.

    Article Snippet: Reactions were initiated by addition of 3 μg/100 μL total parasite lysate, 1% bovine PKA, 4 μg/mL human GSK3β (Biaffin) or 25 μg/mL recombinant Pf GSK3.

    Techniques: Expressing, Diagnostic Assay, Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Clone Assay, Selection, Transgenic Assay, Western Blot, Staining, SDS Page, Autoradiography, In Vitro, Incubation

    S 610 phosphorylation is a prerequisite for efficient T 613 is phosphorylation. ( A ) Autoradiograph of in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) are shown. AMA1 variants displaying either all (AMA1 WT ), none (AMA1 PM ) or only single phosphorylation sites (AMA1 S610 , AMA1 T612 , AMA1 T613 ) were incubated with schizont material in the presence of cAMP and 32 P-γ-ATP. ( B) Phosphorylation of PKA pre-phosphorylated AMA1 WT and mutants AMA1 PM and AMA1 S610/T613 . Recombinant proteins were first incubated with PKA in the presence of non-labeled ATP. Subsequently, these pre-treated AMA1 were incubated with either PKA or a schizont extract in the presence of 32 P-γ-ATP. AMA1 PM was used as a control. SDS-PAGE and radiograph of in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) are shown. ( C) In vitro phosphorylation of AMA1-GST fusion variants (AMA1 WT , AMA1 S610 , AMA1 S610/T613 ) either as unphosphorylated (-) or PKA-pre-phosphorylated (+) substrates in the presence of recombinant Pf GSK3β and 32 P-γ-ATP or without the addition of Pf GSK3β (c). ( D) Signal intensities were quantified. Error bars correspond to standard deviation of two independent experiments done in triplicates. ( E) Sandwich ELISA demonstrating 5v-induced inhibition of native AMA1 threonine phosphorylation. Parasites were treated with 5v and a rabbit anti- Pf AMA1 antibody was used to capture Pf AMA1 from culture lysates. Threonine phosphorylation was detected in an ELISA format using a mouse anti-phosphothreonine antibody. Histograms were generated after normalizing against uninfected (0%, background) and untreated (100%) culture signals. Phosphatase-treatment was used to denote zero phosphorylation and chloroquine treatment was used to exclude parasite growth arrest as a cause for reduced phosphorylation. ( F) The Pf PKA inhibitor H89, weakly inhibited egress and strongly inhibited invasion in P. falciparum reporter parasites transfected with secreted Nanoluciferase. Luciferase activity in relative light units was measured and dose-response curves were plotted for egress (dashed line) and invasion (solid line) after normalizing against uninfected (0%) and untreated (100%) culture sample signals. Two biological replicates were performed in triplicate; error bars denote one standard deviation. ( G) The Pf GSK inhibitor 5v strongly inhibits invasion. The experiment was performed as per F.

    Journal: Scientific Reports

    Article Title: Hierarchical phosphorylation of apical membrane antigen 1 is required for efficient red blood cell invasion by malaria parasites

    doi: 10.1038/srep34479

    Figure Lengend Snippet: S 610 phosphorylation is a prerequisite for efficient T 613 is phosphorylation. ( A ) Autoradiograph of in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) are shown. AMA1 variants displaying either all (AMA1 WT ), none (AMA1 PM ) or only single phosphorylation sites (AMA1 S610 , AMA1 T612 , AMA1 T613 ) were incubated with schizont material in the presence of cAMP and 32 P-γ-ATP. ( B) Phosphorylation of PKA pre-phosphorylated AMA1 WT and mutants AMA1 PM and AMA1 S610/T613 . Recombinant proteins were first incubated with PKA in the presence of non-labeled ATP. Subsequently, these pre-treated AMA1 were incubated with either PKA or a schizont extract in the presence of 32 P-γ-ATP. AMA1 PM was used as a control. SDS-PAGE and radiograph of in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) are shown. ( C) In vitro phosphorylation of AMA1-GST fusion variants (AMA1 WT , AMA1 S610 , AMA1 S610/T613 ) either as unphosphorylated (-) or PKA-pre-phosphorylated (+) substrates in the presence of recombinant Pf GSK3β and 32 P-γ-ATP or without the addition of Pf GSK3β (c). ( D) Signal intensities were quantified. Error bars correspond to standard deviation of two independent experiments done in triplicates. ( E) Sandwich ELISA demonstrating 5v-induced inhibition of native AMA1 threonine phosphorylation. Parasites were treated with 5v and a rabbit anti- Pf AMA1 antibody was used to capture Pf AMA1 from culture lysates. Threonine phosphorylation was detected in an ELISA format using a mouse anti-phosphothreonine antibody. Histograms were generated after normalizing against uninfected (0%, background) and untreated (100%) culture signals. Phosphatase-treatment was used to denote zero phosphorylation and chloroquine treatment was used to exclude parasite growth arrest as a cause for reduced phosphorylation. ( F) The Pf PKA inhibitor H89, weakly inhibited egress and strongly inhibited invasion in P. falciparum reporter parasites transfected with secreted Nanoluciferase. Luciferase activity in relative light units was measured and dose-response curves were plotted for egress (dashed line) and invasion (solid line) after normalizing against uninfected (0%) and untreated (100%) culture sample signals. Two biological replicates were performed in triplicate; error bars denote one standard deviation. ( G) The Pf GSK inhibitor 5v strongly inhibits invasion. The experiment was performed as per F.

    Article Snippet: Reactions were initiated by addition of 3 μg/100 μL total parasite lysate, 1% bovine PKA, 4 μg/mL human GSK3β (Biaffin) or 25 μg/mL recombinant Pf GSK3.

    Techniques: Autoradiography, In Vitro, Staining, Incubation, Recombinant, Labeling, SDS Page, Standard Deviation, Sandwich ELISA, Inhibition, Enzyme-linked Immunosorbent Assay, Generated, Transfection, Luciferase, Activity Assay

    BSSM activates AKT/GSK3β pathway, promotes tau hyperphosphorylation and PHF accumulation and increases the expression of active asparagine endopeptidase (AEP) and tau cleavage. (A,B) Western blotting showed higher expressions of p-AKT and the downstream p-GSK in hippocampus in BSSM treated group. (C,D) Phosphorylation of tau at S396 was higher in BSSM group than in sham and USSM group. PHF accumulation also showed significant differences between sham and the two shear stress modifier groups. (E–G) Expression of active AEP and tau N368 truncation were higher in BSSM group. (H,I) Immunofluorescent staining further reconfirmed the finding that BSSM group had more tau N368 fragments in hippocampus than the other two groups. L, left hippocampus; R, right hippocampus. * p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Bilateral Implantation of Shear Stress Modifier in ApoE Knockout Mouse Induces Cognitive Impairment and Tau Abnormalities

    doi: 10.3389/fnagi.2018.00303

    Figure Lengend Snippet: BSSM activates AKT/GSK3β pathway, promotes tau hyperphosphorylation and PHF accumulation and increases the expression of active asparagine endopeptidase (AEP) and tau cleavage. (A,B) Western blotting showed higher expressions of p-AKT and the downstream p-GSK in hippocampus in BSSM treated group. (C,D) Phosphorylation of tau at S396 was higher in BSSM group than in sham and USSM group. PHF accumulation also showed significant differences between sham and the two shear stress modifier groups. (E–G) Expression of active AEP and tau N368 truncation were higher in BSSM group. (H,I) Immunofluorescent staining further reconfirmed the finding that BSSM group had more tau N368 fragments in hippocampus than the other two groups. L, left hippocampus; R, right hippocampus. * p

    Article Snippet: Anti-GSK3β rat (cat#MAB2506), Legumain/asparaginyl endopeptidase sheep (Cat#AF2058) antibodies were purchased from R & D System (Minneapolis, MN, USA).

    Techniques: Expressing, Western Blot, Staining