gsk3α Search Results


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  • 97
    Cell Signaling Technology Inc anti phospho glycogen synthase kinase 3α
    <t>GSK-3</t> expression, body weight, plasma insulin concentration, glucose tolerance and insulin sensitivity in <t>GSK-3α</t> liver KO animals. Tissue extracts were prepared from eight wk old male mice of the genotypes indicated and immunoblotted with antibodies against (A) <t>GSK-3α/β.</t> Even loading was determined using antibodies to GAPDH. Blots are representative from three separate experiments. (B) Body weight of male Alb Cre − (open squares) and Alb Cre + (filled squares) littermate control mice was monitored weekly from age 4 to 22 weeks. Values are the mean ± SEM from nine separate animals. (C) Plasma insulin concentration was determined in eight week old male Alb Cre − (open bars) and Alb Cre + (filled bars) littermate control animals under fasted and fed conditions. Values are the mean ± SEM from at least seven separate animals. Blood glucose concentration in eight week old male Alb Cre − (open squares) and Alb Cre + (filled squares) littermate control mice was measured at the indicated times following administration of (D) 2 mg/g glucose or (E) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from fourteen separate animals for Alb Cre – and nine separate animals for Alb Cre +. Genetic background was C57BL/6/129.
    Anti Phospho Glycogen Synthase Kinase 3α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho glycogen synthase kinase 3α/product/Cell Signaling Technology Inc
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    85
    Cell Signaling Technology Inc glycogen synthase kinase 3α gsk3α
    CAPE inhibits Akt signaling-related proteins in PC-3 cells. Protein expression of Akt, Akt1, Akt2, Akt3, total Akt, phospho-Akt S473, phospho-Akt T308, mTOR, phospho-mTOR Ser2448 and Ser2481, <t>GSK3α,</t> GSK3β, phopho-GSK3α S21, phospho-GSK3β S9, PDK1, phospho-PDK1 Ser241, Bcl-2, KLF6, β-tubulin, and β-actin in PC-3 cells treated with 20 µM CAPE for 24, 48, and 5, 10, 20 µM CAPE for 72 h were assayed by Western blotting.
    Glycogen Synthase Kinase 3α Gsk3α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glycogen synthase kinase 3α gsk3α/product/Cell Signaling Technology Inc
    Average 85 stars, based on 46 article reviews
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    88
    Upstate Biotechnology Inc anti gsk3α
    CAPE inhibits Akt signaling-related proteins in PC-3 cells. Protein expression of Akt, Akt1, Akt2, Akt3, total Akt, phospho-Akt S473, phospho-Akt T308, mTOR, phospho-mTOR Ser2448 and Ser2481, <t>GSK3α,</t> GSK3β, phopho-GSK3α S21, phospho-GSK3β S9, PDK1, phospho-PDK1 Ser241, Bcl-2, KLF6, β-tubulin, and β-actin in PC-3 cells treated with 20 µM CAPE for 24, 48, and 5, 10, 20 µM CAPE for 72 h were assayed by Western blotting.
    Anti Gsk3α, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Cell Signaling Technology Inc p gsk3α β
    Western blot analyses of steady-state levels of AKT, P -AKT, <t>GSK3α/β,</t> p-GSK3α/β, mTOR and p-mTOR following exposure to MK2206 and GSK690693 in 2D culture (CM+Y). (A) Representative western blots of AKT, p-AKT(S473), GSK3α/β, p-GSK3α/β, mTOR, p-mTOR and β-actin from GUMC220 and GUMC221 exposed to MK2206 (0.6 μM and 1.2 µM) compared with DMSO vehicle control. (B) Steady-state expression levels of pAKT(S473), GSK3α/β, p-GSK3α/β and p-mTOR in GUMC220 and GUMC221 cells after 1, 2 and 3 days’ exposure to the two different concentrations of MK2206 and DMSO. Data are mean±s.e.m., n =3/timepoint/concentration. (C) Representative western blots of AKT, p-AKT(S473), p-AKT(T308), GSK3α/β, p-GSK3α/β, mTOR, p-mTOR and β-actin from GUMC220 and GUMC221 in 2D CM+Y culture exposed to GSK690693 (15 μM and 30 µM) compared with DMSO vehicle control. (D) Steady-state expression levels of pAKT(S473), p-AKT(T308), GSK3α/β, p-GSK3α/β and p-mTOR in GUMC220 and GUMC221 cells after 1, 2 and 3 days’ exposure to the two different concentrations of GSK690693 compared with DMSO vehicle control. Phosphoprotein levels were normalized to total proteins, which were normalized to actin. *P ≤0.05, **P ≤0.01, ***P ≤0.001, one tailed, unpaired t -test. Protein lysates were collected after 1, 2 and 3 days of drug exposure. Data are mean±s.e.m., n =3/timepoint/concentration. Molecular weights (kDa) are indicated for each protein.
    P Gsk3α β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Santa Cruz Biotechnology p gsk3α
    The inactivation of GSK3 proteins at various stages of OTSCC progression. Representative immunostaining showing the differential expression of pS 21 <t>GSK3α</t> and pS 9 GSK3β in the consecutive sections of (a, b) normal tongue tissue, (c, d) mild hyperplasia of tumor adjacent tongue, and (e, f) OTSCC tissue samples (original magnification 100X).
    P Gsk3α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p gsk3α/product/Santa Cruz Biotechnology
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    Image Search Results


    GSK-3 expression, body weight, plasma insulin concentration, glucose tolerance and insulin sensitivity in GSK-3α liver KO animals. Tissue extracts were prepared from eight wk old male mice of the genotypes indicated and immunoblotted with antibodies against (A) GSK-3α/β. Even loading was determined using antibodies to GAPDH. Blots are representative from three separate experiments. (B) Body weight of male Alb Cre − (open squares) and Alb Cre + (filled squares) littermate control mice was monitored weekly from age 4 to 22 weeks. Values are the mean ± SEM from nine separate animals. (C) Plasma insulin concentration was determined in eight week old male Alb Cre − (open bars) and Alb Cre + (filled bars) littermate control animals under fasted and fed conditions. Values are the mean ± SEM from at least seven separate animals. Blood glucose concentration in eight week old male Alb Cre − (open squares) and Alb Cre + (filled squares) littermate control mice was measured at the indicated times following administration of (D) 2 mg/g glucose or (E) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from fourteen separate animals for Alb Cre – and nine separate animals for Alb Cre +. Genetic background was C57BL/6/129.

    Journal: PLoS ONE

    Article Title: Tissue-Specific Analysis of Glycogen Synthase Kinase-3? (GSK-3?) in Glucose Metabolism: Effect of Strain Variation

    doi: 10.1371/journal.pone.0015845

    Figure Lengend Snippet: GSK-3 expression, body weight, plasma insulin concentration, glucose tolerance and insulin sensitivity in GSK-3α liver KO animals. Tissue extracts were prepared from eight wk old male mice of the genotypes indicated and immunoblotted with antibodies against (A) GSK-3α/β. Even loading was determined using antibodies to GAPDH. Blots are representative from three separate experiments. (B) Body weight of male Alb Cre − (open squares) and Alb Cre + (filled squares) littermate control mice was monitored weekly from age 4 to 22 weeks. Values are the mean ± SEM from nine separate animals. (C) Plasma insulin concentration was determined in eight week old male Alb Cre − (open bars) and Alb Cre + (filled bars) littermate control animals under fasted and fed conditions. Values are the mean ± SEM from at least seven separate animals. Blood glucose concentration in eight week old male Alb Cre − (open squares) and Alb Cre + (filled squares) littermate control mice was measured at the indicated times following administration of (D) 2 mg/g glucose or (E) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from fourteen separate animals for Alb Cre – and nine separate animals for Alb Cre +. Genetic background was C57BL/6/129.

    Article Snippet: Membranes were blocked for 1 h at room temperature in 5% non-fat milk/Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 0.1% Tween-20 prior to probing with antibodies directed against the following antigens: GSK-3α/β (1∶10,000, Biosource), phospho-GSK-3β (1∶1000, Cell Signaling), β-catenin (1∶1000, BD Transduction Laboratories), PKB (1∶1000, Cell Signaling), phospho-PKB Ser473 (1∶1000, Cell Signaling Technologies), GS (GYS1, 1∶1000, Chemicon), GS (GYS2, 1∶1000, gift from J. Guinovart, University of Barcelona), phospho-GS (1∶1000, Cell Signaling Technologies), β-actin (1∶20,000, Abcam), GAPDH (1∶100,000, Abcam), overnight at 4°C.

    Techniques: Expressing, Concentration Assay, Mouse Assay, Injection

    Effect of GSK-3α liver KO on insulin signaling. (A) liver, (B) muscle from eight week old Alb Cre − (−) and Alb Cre + (+) littermate control animals were extracted following either an overnight (16–18 h) fast alone or an overnight fast followed by i.p. administration of 150 mU/g insulin for 15 min. Twenty µg of the protein lysates was resolved by SDS-PAGE. Proteins were detected by immunoblotting with antibodies against phospho-PKB, PKB, phospho-GSK-3β, GSK-3β, phospho-GS and GS. Even loading was determined by blotting for either β-actin or GAPDH. Blots are representative from 4 separate experiments. (C) GSK-3 kinase activity was determined from liver tissue extracted from male Alb Cre − control (open bars) and Alb Cre + (filled bars) mice as described in experimental procedures. GSK-3 kinase activity was determined using a quantitative peptide phosphorylation assay. GSK-3 kinase activity is expressed relative to the Cre− control (which is set at 100%) and is the mean ± SEM of four different liver samples with each assayed in triplicate. Genetic background was C57BL/6/129.

    Journal: PLoS ONE

    Article Title: Tissue-Specific Analysis of Glycogen Synthase Kinase-3? (GSK-3?) in Glucose Metabolism: Effect of Strain Variation

    doi: 10.1371/journal.pone.0015845

    Figure Lengend Snippet: Effect of GSK-3α liver KO on insulin signaling. (A) liver, (B) muscle from eight week old Alb Cre − (−) and Alb Cre + (+) littermate control animals were extracted following either an overnight (16–18 h) fast alone or an overnight fast followed by i.p. administration of 150 mU/g insulin for 15 min. Twenty µg of the protein lysates was resolved by SDS-PAGE. Proteins were detected by immunoblotting with antibodies against phospho-PKB, PKB, phospho-GSK-3β, GSK-3β, phospho-GS and GS. Even loading was determined by blotting for either β-actin or GAPDH. Blots are representative from 4 separate experiments. (C) GSK-3 kinase activity was determined from liver tissue extracted from male Alb Cre − control (open bars) and Alb Cre + (filled bars) mice as described in experimental procedures. GSK-3 kinase activity was determined using a quantitative peptide phosphorylation assay. GSK-3 kinase activity is expressed relative to the Cre− control (which is set at 100%) and is the mean ± SEM of four different liver samples with each assayed in triplicate. Genetic background was C57BL/6/129.

    Article Snippet: Membranes were blocked for 1 h at room temperature in 5% non-fat milk/Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 0.1% Tween-20 prior to probing with antibodies directed against the following antigens: GSK-3α/β (1∶10,000, Biosource), phospho-GSK-3β (1∶1000, Cell Signaling), β-catenin (1∶1000, BD Transduction Laboratories), PKB (1∶1000, Cell Signaling), phospho-PKB Ser473 (1∶1000, Cell Signaling Technologies), GS (GYS1, 1∶1000, Chemicon), GS (GYS2, 1∶1000, gift from J. Guinovart, University of Barcelona), phospho-GS (1∶1000, Cell Signaling Technologies), β-actin (1∶20,000, Abcam), GAPDH (1∶100,000, Abcam), overnight at 4°C.

    Techniques: SDS Page, Activity Assay, Mouse Assay, Phosphorylation Assay

    GSK-3 expression, body weight, plasma insulin concentration, glucose tolerance and insulin sensitivity in C57BL/6/129 GSK-3α muscle KO animals. (A) Brain, heart, liver, testes and quad, gastroc, soleus and EDL muscles and (B) brain, heart, testes, liver and gastroc from eight week old MLC Cre − (−) and MLC Cre + (+) littermate control animals were extracted and lysed as described in Experimental Procedures and proteins resolved by SDS-PAGE. Proteins were detected by immunoblotting with antibodies against GSK-3α/β. Even loading was determined using antibodies to GAPDH. Blots are representative from three separate experiments. (B) Body weight of male MLC Cre − (open squares) and MLC Cre + (filled squares) littermate control mice was monitored weekly from age 4 to 22 weeks. Values are the mean ± SEM from eight separate animals. (C) Plasma insulin concentration was determined in eight week old male MLC Cre − (open bars) and MLC Cre + (filled bars) littermate control animals under fasted and fed conditions. Values are the mean ± SEM from at least seven separate animals. Blood glucose concentration in eight week old male MLC Cre − (open squares) and MLC Cre + (filled squares) littermate control mice was measured at the indicated times following administration of (D) 2 mg/g glucose or (E) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from at least seven separate animals.

    Journal: PLoS ONE

    Article Title: Tissue-Specific Analysis of Glycogen Synthase Kinase-3? (GSK-3?) in Glucose Metabolism: Effect of Strain Variation

    doi: 10.1371/journal.pone.0015845

    Figure Lengend Snippet: GSK-3 expression, body weight, plasma insulin concentration, glucose tolerance and insulin sensitivity in C57BL/6/129 GSK-3α muscle KO animals. (A) Brain, heart, liver, testes and quad, gastroc, soleus and EDL muscles and (B) brain, heart, testes, liver and gastroc from eight week old MLC Cre − (−) and MLC Cre + (+) littermate control animals were extracted and lysed as described in Experimental Procedures and proteins resolved by SDS-PAGE. Proteins were detected by immunoblotting with antibodies against GSK-3α/β. Even loading was determined using antibodies to GAPDH. Blots are representative from three separate experiments. (B) Body weight of male MLC Cre − (open squares) and MLC Cre + (filled squares) littermate control mice was monitored weekly from age 4 to 22 weeks. Values are the mean ± SEM from eight separate animals. (C) Plasma insulin concentration was determined in eight week old male MLC Cre − (open bars) and MLC Cre + (filled bars) littermate control animals under fasted and fed conditions. Values are the mean ± SEM from at least seven separate animals. Blood glucose concentration in eight week old male MLC Cre − (open squares) and MLC Cre + (filled squares) littermate control mice was measured at the indicated times following administration of (D) 2 mg/g glucose or (E) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from at least seven separate animals.

    Article Snippet: Membranes were blocked for 1 h at room temperature in 5% non-fat milk/Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 0.1% Tween-20 prior to probing with antibodies directed against the following antigens: GSK-3α/β (1∶10,000, Biosource), phospho-GSK-3β (1∶1000, Cell Signaling), β-catenin (1∶1000, BD Transduction Laboratories), PKB (1∶1000, Cell Signaling), phospho-PKB Ser473 (1∶1000, Cell Signaling Technologies), GS (GYS1, 1∶1000, Chemicon), GS (GYS2, 1∶1000, gift from J. Guinovart, University of Barcelona), phospho-GS (1∶1000, Cell Signaling Technologies), β-actin (1∶20,000, Abcam), GAPDH (1∶100,000, Abcam), overnight at 4°C.

    Techniques: Expressing, Concentration Assay, SDS Page, Mouse Assay, Injection

    Effect of GSK-3α muscle KO on insulin signaling. (A) Quad, (B) gastroc and (C) liver from eight week old MLC Cre − (−) and MLC Cre + (+) littermate control animals was extracted following either an over night (16–18 h) fast alone or an overnight fast followed by i.p. administration of 150 mU/g insulin for 15 min. Twenty µg of the protein lysate was resolved by SDS-PAGE. Proteins were detected by immunoblotting with antibodies against phospho-PKB (pSer473), PKB, phospho-GSK-3β (pSer9), GSK-3β, phospho-GS (pSer641) and GS (lower left inset in C). Loading was determined using antibodies to either GAPDH or β-actin. Blots are representative from five separate experiments. (D) GSK-3 kinase activity was determined from muscle tissue extracted from male MLC Cre − control (open bars) and MLC Cre + (filled bars) mice as described in experimental procedures. GSK-3 kinase activity was determined using a quantitative peptide phosphorylation assay. GSK-3 kinase activity is expressed relative to the MLC Cre- control (which is set at 100%) and is the mean ± SEM of four different muscle samples with each assayed in triplicate. Genetic background was C57BL/6/129.

    Journal: PLoS ONE

    Article Title: Tissue-Specific Analysis of Glycogen Synthase Kinase-3? (GSK-3?) in Glucose Metabolism: Effect of Strain Variation

    doi: 10.1371/journal.pone.0015845

    Figure Lengend Snippet: Effect of GSK-3α muscle KO on insulin signaling. (A) Quad, (B) gastroc and (C) liver from eight week old MLC Cre − (−) and MLC Cre + (+) littermate control animals was extracted following either an over night (16–18 h) fast alone or an overnight fast followed by i.p. administration of 150 mU/g insulin for 15 min. Twenty µg of the protein lysate was resolved by SDS-PAGE. Proteins were detected by immunoblotting with antibodies against phospho-PKB (pSer473), PKB, phospho-GSK-3β (pSer9), GSK-3β, phospho-GS (pSer641) and GS (lower left inset in C). Loading was determined using antibodies to either GAPDH or β-actin. Blots are representative from five separate experiments. (D) GSK-3 kinase activity was determined from muscle tissue extracted from male MLC Cre − control (open bars) and MLC Cre + (filled bars) mice as described in experimental procedures. GSK-3 kinase activity was determined using a quantitative peptide phosphorylation assay. GSK-3 kinase activity is expressed relative to the MLC Cre- control (which is set at 100%) and is the mean ± SEM of four different muscle samples with each assayed in triplicate. Genetic background was C57BL/6/129.

    Article Snippet: Membranes were blocked for 1 h at room temperature in 5% non-fat milk/Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 0.1% Tween-20 prior to probing with antibodies directed against the following antigens: GSK-3α/β (1∶10,000, Biosource), phospho-GSK-3β (1∶1000, Cell Signaling), β-catenin (1∶1000, BD Transduction Laboratories), PKB (1∶1000, Cell Signaling), phospho-PKB Ser473 (1∶1000, Cell Signaling Technologies), GS (GYS1, 1∶1000, Chemicon), GS (GYS2, 1∶1000, gift from J. Guinovart, University of Barcelona), phospho-GS (1∶1000, Cell Signaling Technologies), β-actin (1∶20,000, Abcam), GAPDH (1∶100,000, Abcam), overnight at 4°C.

    Techniques: SDS Page, Activity Assay, Mouse Assay, Phosphorylation Assay

    Glycogen synthase activity in quad, gastroc and liver of GSK-3α muscle KO animals. Glycogen synthase activity was determined in (A) quad, (B) gastroc and (C) liver of eight weeks old MLC Cre – (open bars) and MLC Cre + (filled bars) tissues by assaying incorporation of glucose from uridine diphospho-{6- 3 H)-D-glucose into glycogen and expressed as a ratio of activity in the absence divided by that in the presence of glucose-6-phosphate. Values are the mean ± SEM for at least five experiments carried out in duplicate. Glycogen content was measured in (D) quad, (E) gastroc and (F) liver from eight week old MLC Cre − (open bars) and MLC Cre + (filled bars) following either an overnight fast or random feeding. Tissues were extracted, acid-hydrolyzed and glycosyl units assayed using a glucose reagent hexokinase method (Amresco, Ohio) as described in Experimental Procedures. Glycogen content is expressed as µmol glucose/g tissue. Values are mean ± SEM from at least five separate animals with each assayed in triplicate. Genetic background of the animals was C57BL/6/129.

    Journal: PLoS ONE

    Article Title: Tissue-Specific Analysis of Glycogen Synthase Kinase-3? (GSK-3?) in Glucose Metabolism: Effect of Strain Variation

    doi: 10.1371/journal.pone.0015845

    Figure Lengend Snippet: Glycogen synthase activity in quad, gastroc and liver of GSK-3α muscle KO animals. Glycogen synthase activity was determined in (A) quad, (B) gastroc and (C) liver of eight weeks old MLC Cre – (open bars) and MLC Cre + (filled bars) tissues by assaying incorporation of glucose from uridine diphospho-{6- 3 H)-D-glucose into glycogen and expressed as a ratio of activity in the absence divided by that in the presence of glucose-6-phosphate. Values are the mean ± SEM for at least five experiments carried out in duplicate. Glycogen content was measured in (D) quad, (E) gastroc and (F) liver from eight week old MLC Cre − (open bars) and MLC Cre + (filled bars) following either an overnight fast or random feeding. Tissues were extracted, acid-hydrolyzed and glycosyl units assayed using a glucose reagent hexokinase method (Amresco, Ohio) as described in Experimental Procedures. Glycogen content is expressed as µmol glucose/g tissue. Values are mean ± SEM from at least five separate animals with each assayed in triplicate. Genetic background of the animals was C57BL/6/129.

    Article Snippet: Membranes were blocked for 1 h at room temperature in 5% non-fat milk/Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 0.1% Tween-20 prior to probing with antibodies directed against the following antigens: GSK-3α/β (1∶10,000, Biosource), phospho-GSK-3β (1∶1000, Cell Signaling), β-catenin (1∶1000, BD Transduction Laboratories), PKB (1∶1000, Cell Signaling), phospho-PKB Ser473 (1∶1000, Cell Signaling Technologies), GS (GYS1, 1∶1000, Chemicon), GS (GYS2, 1∶1000, gift from J. Guinovart, University of Barcelona), phospho-GS (1∶1000, Cell Signaling Technologies), β-actin (1∶20,000, Abcam), GAPDH (1∶100,000, Abcam), overnight at 4°C.

    Techniques: Activity Assay

    Glucose tolerance and insulin sensitivity in C57BL/6-GSK-3α global and ICR-GSK-3α liver KO animals. Blood glucose concentration in eight week old male WT - (open squares) and C57BL/6-GSK-3α KO (filled squares) mice was measured at the indicated times following administration of (A) 2 mg/g glucose or (B) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from at least seven separate animals. Blood glucose concentration in eight week old male ICR-GSK-3α Alb Cre − (open squares) and ICR-GSK-3α Alb Cre + (filled squares) littermate control mice was measured at the indicated times following administration of (C) 2 mg/g glucose or (D) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from nine separate animals for Alb Cre − and ten separate animals for Alb Cre +. Glycogen content was measured in liver (E) from ICR-GSK-3α Alb Cre − (open squares) and ICR-GSK-3α Alb Cre + (filled squares) following either an overnight fast or random feeding. Values are mean ± SEM from at least five separate animals with each assayed in triplicate.

    Journal: PLoS ONE

    Article Title: Tissue-Specific Analysis of Glycogen Synthase Kinase-3? (GSK-3?) in Glucose Metabolism: Effect of Strain Variation

    doi: 10.1371/journal.pone.0015845

    Figure Lengend Snippet: Glucose tolerance and insulin sensitivity in C57BL/6-GSK-3α global and ICR-GSK-3α liver KO animals. Blood glucose concentration in eight week old male WT - (open squares) and C57BL/6-GSK-3α KO (filled squares) mice was measured at the indicated times following administration of (A) 2 mg/g glucose or (B) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from at least seven separate animals. Blood glucose concentration in eight week old male ICR-GSK-3α Alb Cre − (open squares) and ICR-GSK-3α Alb Cre + (filled squares) littermate control mice was measured at the indicated times following administration of (C) 2 mg/g glucose or (D) 1 mU/g insulin by i.p. injection as described in Experimental Procedures. Values are the mean ± SEM from nine separate animals for Alb Cre − and ten separate animals for Alb Cre +. Glycogen content was measured in liver (E) from ICR-GSK-3α Alb Cre − (open squares) and ICR-GSK-3α Alb Cre + (filled squares) following either an overnight fast or random feeding. Values are mean ± SEM from at least five separate animals with each assayed in triplicate.

    Article Snippet: Membranes were blocked for 1 h at room temperature in 5% non-fat milk/Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 0.1% Tween-20 prior to probing with antibodies directed against the following antigens: GSK-3α/β (1∶10,000, Biosource), phospho-GSK-3β (1∶1000, Cell Signaling), β-catenin (1∶1000, BD Transduction Laboratories), PKB (1∶1000, Cell Signaling), phospho-PKB Ser473 (1∶1000, Cell Signaling Technologies), GS (GYS1, 1∶1000, Chemicon), GS (GYS2, 1∶1000, gift from J. Guinovart, University of Barcelona), phospho-GS (1∶1000, Cell Signaling Technologies), β-actin (1∶20,000, Abcam), GAPDH (1∶100,000, Abcam), overnight at 4°C.

    Techniques: Concentration Assay, Mouse Assay, Injection

    GSK3α/β KI/KI phenotype. A: Immunoblots show no phosphorylation of serine 21 of GSK3α or serine 9 of GSK3β in the GSK3α/β KI/KI animals. Protein lysates derived from the small intestine of control wild-type or mutant GSK3α/β KI/KI mice were analysed with an antibody for phosphorylated GSK3α/β. Immunoblotting for GSK3α/β and ERK2 was used to determine equal protein loading. B: Kaplan-Meier survival analysis of GSK3α/β mutant mice. All mice were kept on study until they became moribund. None of these animals demonstrated specific symptoms prior to this. Genotypes of mice are as follows: WT/WT = GSK3α +/+ ;GSK3β +/+ (n = 6), KI/KI = GSK3α S21A/S21A ;GSK3β S9A/S9A (n = 7), HET/HET = GSK3α +/S21A ;GSK3β +/S9A (n = 7), WT/KI = GSK3α +/+ ;GSK3β S9A/S9A (n = 6), KI/WT = GSK3α S21A/S21A ;GSK3β +/+ (n = 5), HET/KI = GSK3α +/S21A ;GSK3β S9A/S9A (n = 5), KI/HET = GSK3α S21A/S21A ;GSK3β +/S9A (n = 6). Black tick marks show censored data. Median survival was as follows: WT/WT = 637d, KI/KI = 686d, HET/HET = 778d, WT/KI = undefined, KI/WT = undefined, HET/KI = 568d and KI/HET = 519d. C: Weight analysis. Age matched males and females were weighed at 10 weeks of age. Genotypes of mice were: WT/WT = GSK3α +/+ ;GSK3β +/+ (n = 3), KI/KI = GSK3α S21A/S21A ;GSK3β S9A/S9A (n = 3) for each sex. Mean + SEM are shown, demonstrating no statistically significant difference in the mean weights for any group using the Student’s t-test. D: Intestine length. The small intestine length was measured and mean length + SEM is shown. Genotypes of mice were: WT/WT = GSK3α +/+ ;GSK3β +/+ (n = 6), KI/KI = GSK3α S21A/S21A ;GSK3β S9A/S9A (n = 7). No statistically significant difference was observed using the Student’s t-test. E: Villus length. The length of villi was measured in age matched males and females at 10 weeks of age. Genotypes of mice were: WT/WT = GSK3α +/+ ;GSK3β +/+ (n = 5), KI/KI = GSK3α S21A/S21A ;GSK3β S9A/S9A (n = 7). 50 villi were measured per animal. No statistically significant difference was observed using the Student’s t-test. F: H E stained small intestinal sections of GSK3α/β WT/WT or mutant GSK3α/β KI/KI mice are shown. Scale bars, 50μm.

    Journal: PLoS ONE

    Article Title: Phosphorylations of Serines 21/9 in Glycogen Synthase Kinase 3α/β Are Not Required for Cell Lineage Commitment or WNT Signaling in the Normal Mouse Intestine

    doi: 10.1371/journal.pone.0156877

    Figure Lengend Snippet: GSK3α/β KI/KI phenotype. A: Immunoblots show no phosphorylation of serine 21 of GSK3α or serine 9 of GSK3β in the GSK3α/β KI/KI animals. Protein lysates derived from the small intestine of control wild-type or mutant GSK3α/β KI/KI mice were analysed with an antibody for phosphorylated GSK3α/β. Immunoblotting for GSK3α/β and ERK2 was used to determine equal protein loading. B: Kaplan-Meier survival analysis of GSK3α/β mutant mice. All mice were kept on study until they became moribund. None of these animals demonstrated specific symptoms prior to this. Genotypes of mice are as follows: WT/WT = GSK3α +/+ ;GSK3β +/+ (n = 6), KI/KI = GSK3α S21A/S21A ;GSK3β S9A/S9A (n = 7), HET/HET = GSK3α +/S21A ;GSK3β +/S9A (n = 7), WT/KI = GSK3α +/+ ;GSK3β S9A/S9A (n = 6), KI/WT = GSK3α S21A/S21A ;GSK3β +/+ (n = 5), HET/KI = GSK3α +/S21A ;GSK3β S9A/S9A (n = 5), KI/HET = GSK3α S21A/S21A ;GSK3β +/S9A (n = 6). Black tick marks show censored data. Median survival was as follows: WT/WT = 637d, KI/KI = 686d, HET/HET = 778d, WT/KI = undefined, KI/WT = undefined, HET/KI = 568d and KI/HET = 519d. C: Weight analysis. Age matched males and females were weighed at 10 weeks of age. Genotypes of mice were: WT/WT = GSK3α +/+ ;GSK3β +/+ (n = 3), KI/KI = GSK3α S21A/S21A ;GSK3β S9A/S9A (n = 3) for each sex. Mean + SEM are shown, demonstrating no statistically significant difference in the mean weights for any group using the Student’s t-test. D: Intestine length. The small intestine length was measured and mean length + SEM is shown. Genotypes of mice were: WT/WT = GSK3α +/+ ;GSK3β +/+ (n = 6), KI/KI = GSK3α S21A/S21A ;GSK3β S9A/S9A (n = 7). No statistically significant difference was observed using the Student’s t-test. E: Villus length. The length of villi was measured in age matched males and females at 10 weeks of age. Genotypes of mice were: WT/WT = GSK3α +/+ ;GSK3β +/+ (n = 5), KI/KI = GSK3α S21A/S21A ;GSK3β S9A/S9A (n = 7). 50 villi were measured per animal. No statistically significant difference was observed using the Student’s t-test. F: H E stained small intestinal sections of GSK3α/β WT/WT or mutant GSK3α/β KI/KI mice are shown. Scale bars, 50μm.

    Article Snippet: Primary antibodies used were: β-Catenin (BD Transduction 610153), phospho-GSK3α/β (Ser21/9) (Cell Signalling 9331), GSK3α/β (Cell Signalling 5676), ERK2 (Santa Cruz sc-1647) GAPDH (Millipore, MAB374), cleaved Caspase 3 (Cell Signalling 7661) and cleaved PARP (Enzo BML-SA249).

    Techniques: Western Blot, Derivative Assay, Mutagenesis, Mouse Assay, Staining

    Proliferation and apoptosis analysis of the small intestine. A: Quantification of the total number of cells per crypt. A minimum of 50 crypts were quantitated per animal of each genotype and mean values are shown + SEM. Genotypes were: GSK3α +/+ ;GSK3β +/+ (n = 3), KI/KI = GSK3α S21A/S21A ;GSK3β S9A/S9A (n = 3). No statistically significant difference was observed using the Student’s t-test. B: Immunohistochemical staining of WT/WT and KI/KI small intestinal crypts using antibodies for BrdU or phospho-Histone H3. Scale bars, 20μm. C: Quantification of BrdU-positive cells per crypt within the WT/WT and KI/KI small intestine. Staining in 50 crypts of three mice of each genotype was quantified. No statistically significant difference was observed using the Student’s t-test. D: The position of BrdU-positive cells within the WT/WT and KI/KI crypts was quantified. Position 0 represents the base of the crypt. A minimum of 50 crypts were quantified in three mice of each genotype. No statistically significant difference was observed using the Student’s t-test. E: Immunohistochemical staining of WT/WT and KI/KI villi using an antibody for cleaved caspase 3. Scale bars, 50μm. F: Western blot analysis of small intestinal protein samples from WT/WT or KI/KI mice using antibodies for phosphorylated GSK3α/β, cleaved PARP or cleaved caspase-3. Immunoblotting for ERK2 and GAPDH was used to determine equal protein loading.

    Journal: PLoS ONE

    Article Title: Phosphorylations of Serines 21/9 in Glycogen Synthase Kinase 3α/β Are Not Required for Cell Lineage Commitment or WNT Signaling in the Normal Mouse Intestine

    doi: 10.1371/journal.pone.0156877

    Figure Lengend Snippet: Proliferation and apoptosis analysis of the small intestine. A: Quantification of the total number of cells per crypt. A minimum of 50 crypts were quantitated per animal of each genotype and mean values are shown + SEM. Genotypes were: GSK3α +/+ ;GSK3β +/+ (n = 3), KI/KI = GSK3α S21A/S21A ;GSK3β S9A/S9A (n = 3). No statistically significant difference was observed using the Student’s t-test. B: Immunohistochemical staining of WT/WT and KI/KI small intestinal crypts using antibodies for BrdU or phospho-Histone H3. Scale bars, 20μm. C: Quantification of BrdU-positive cells per crypt within the WT/WT and KI/KI small intestine. Staining in 50 crypts of three mice of each genotype was quantified. No statistically significant difference was observed using the Student’s t-test. D: The position of BrdU-positive cells within the WT/WT and KI/KI crypts was quantified. Position 0 represents the base of the crypt. A minimum of 50 crypts were quantified in three mice of each genotype. No statistically significant difference was observed using the Student’s t-test. E: Immunohistochemical staining of WT/WT and KI/KI villi using an antibody for cleaved caspase 3. Scale bars, 50μm. F: Western blot analysis of small intestinal protein samples from WT/WT or KI/KI mice using antibodies for phosphorylated GSK3α/β, cleaved PARP or cleaved caspase-3. Immunoblotting for ERK2 and GAPDH was used to determine equal protein loading.

    Article Snippet: Primary antibodies used were: β-Catenin (BD Transduction 610153), phospho-GSK3α/β (Ser21/9) (Cell Signalling 9331), GSK3α/β (Cell Signalling 5676), ERK2 (Santa Cruz sc-1647) GAPDH (Millipore, MAB374), cleaved Caspase 3 (Cell Signalling 7661) and cleaved PARP (Enzo BML-SA249).

    Techniques: Immunohistochemistry, Staining, Mouse Assay, Western Blot

    Orai1, STIM1, STIM2 and calbindin-D28k mRNA and protein abundance in DCs from gsk3 KI and gsk3 WT mice. A. Original western blot showing the protein abundance of phosphorylated (p, Ser21/9) GSK3α,β, total GSK3α,β and respective GAPDH, STIM1, Orai1 and respective GAPDH, STIM2 and respective GAPDH in DCs derived from bone marrow of gsk3 KI and gsk3 WT mice. Blots were stripped and reprobed with a GAPDH antibody to determine equal protein loading. Also the blot of p-GSK3 was stripped and reprobed with GSK3 antibody. B. Arithmetic means ± SEM (n = 4 independent experiments) of the relative (to GAPDH) protein abundance of Orai1, STIM1 and STIM2 in gsk3 WT DCs (white bars) and gsk3 KI DCs (black bars). *(p

    Journal: PLoS ONE

    Article Title: Decreased Store Operated Ca2+ Entry in Dendritic Cells Isolated from Mice Expressing PKB/SGK-Resistant GSK3

    doi: 10.1371/journal.pone.0088637

    Figure Lengend Snippet: Orai1, STIM1, STIM2 and calbindin-D28k mRNA and protein abundance in DCs from gsk3 KI and gsk3 WT mice. A. Original western blot showing the protein abundance of phosphorylated (p, Ser21/9) GSK3α,β, total GSK3α,β and respective GAPDH, STIM1, Orai1 and respective GAPDH, STIM2 and respective GAPDH in DCs derived from bone marrow of gsk3 KI and gsk3 WT mice. Blots were stripped and reprobed with a GAPDH antibody to determine equal protein loading. Also the blot of p-GSK3 was stripped and reprobed with GSK3 antibody. B. Arithmetic means ± SEM (n = 4 independent experiments) of the relative (to GAPDH) protein abundance of Orai1, STIM1 and STIM2 in gsk3 WT DCs (white bars) and gsk3 KI DCs (black bars). *(p

    Article Snippet: For immunoblotting the membranes were incubated overnight at 4 °C with antibodies directed against GSK3α/β (D75D3, XP™ antibody, 1∶1000, Cell Signaling Technology, Inc., New England Biolabs, 46, 51 kDa), phospho-GSK3α/β (Ser21/9, 1∶1000, Cell Signaling Technology, Inc., New England Biolabs, 46, 51 kDa), Orai1 (1∶500, Proteintech, Manchester), STIM1 (1∶300, Cell Signaling Technology, Inc., New England Biolabs), STIM2 (1∶300 Cell Signaling Technology, Inc., New England Biolabs) or calbindin-D28k (1∶200, SWANT, Switzerland).

    Techniques: Mouse Assay, Western Blot, Derivative Assay

    Activation of Wnt/β-Catenin signaling requires GSK3β Y216 phosphorylation. ( A ) IB analysis using whole cell lysates from indicated MEFs treated with control-CM or Wnt3a-CM for 6 hr. Anti-GSK3 was used to detect endogenous GSK3 and HA-GSK3β. This antibody recognized HA-GSK3β Y216F but not HA-GSK3β Y216E or GSK3β Y216E/S4 mutant proteins. ( B ) The GSK3β KO/GSK3α KD MEFs were transfected with vectors expressing indicated proteins, treated with Wnt3a-CM and proteasome inhibitor MG132 (10 µM) for 4 hr as indicated, and then harvested for IP and IB analyses using indicated antibodies. ( C ) Left panel, SW480 cells and GSK3α/β KD SW480 cell lines expressing vector control, shRNA-resistant WT HA-GSK3β or its mutants were used for soft agar assays. Pictures were taken 16 days after plating at 40×. Right panel, quantification of soft agar assays. Data are presented as mean ±SD. Expressions of GSK3α/β (endogenous or mutants) and β-catenin were evaluated by IB at the time of plating. ( D ) Upper left panel, SW480 cell lines used in ( C ) were injected into both flanks of nude mice (5 mice in each cell group). Tumor sizes were measured at indicated time points. Tumor volumes were calculated and plotted. * denotes a statistically significant difference (p

    Journal: eLife

    Article Title: FAK/PYK2 promotes the Wnt/β-catenin pathway and intestinal tumorigenesis by phosphorylating GSK3β

    doi: 10.7554/eLife.10072

    Figure Lengend Snippet: Activation of Wnt/β-Catenin signaling requires GSK3β Y216 phosphorylation. ( A ) IB analysis using whole cell lysates from indicated MEFs treated with control-CM or Wnt3a-CM for 6 hr. Anti-GSK3 was used to detect endogenous GSK3 and HA-GSK3β. This antibody recognized HA-GSK3β Y216F but not HA-GSK3β Y216E or GSK3β Y216E/S4 mutant proteins. ( B ) The GSK3β KO/GSK3α KD MEFs were transfected with vectors expressing indicated proteins, treated with Wnt3a-CM and proteasome inhibitor MG132 (10 µM) for 4 hr as indicated, and then harvested for IP and IB analyses using indicated antibodies. ( C ) Left panel, SW480 cells and GSK3α/β KD SW480 cell lines expressing vector control, shRNA-resistant WT HA-GSK3β or its mutants were used for soft agar assays. Pictures were taken 16 days after plating at 40×. Right panel, quantification of soft agar assays. Data are presented as mean ±SD. Expressions of GSK3α/β (endogenous or mutants) and β-catenin were evaluated by IB at the time of plating. ( D ) Upper left panel, SW480 cell lines used in ( C ) were injected into both flanks of nude mice (5 mice in each cell group). Tumor sizes were measured at indicated time points. Tumor volumes were calculated and plotted. * denotes a statistically significant difference (p

    Article Snippet: The following antibodies were used in this study: Anti-HA, anti-FLAG, anti-phospho-β-catenin (Ser33/37/Thr41), anti-β-catenin, anti-GSK3α, anti-β-TrCP, anti-α-Tubulin, anti-GSK3β, anti-FAK, anti-phospho-FAK (Tyr397), anti-PYK2 and anti-phospho-PYK2 (Tyr402) (Cell Signaling); anti-FLAG M2 (Sigma); anti-ubiquitin and anti-β-catenin (BD Bioscience, San Jose, CA); anti-GSK3, anti-c-Myc, anti-PYK2, anti-β-TrCP, anti-phospho-GSK3β (Tyr216) and anti-β-Actin (Santa Cruz Biotechnology, Dallas, TX); anti-phospho-GSK3 (Tyr279/Tyr216) (Millipore).

    Techniques: Activation Assay, Mutagenesis, Transfection, Expressing, Plasmid Preparation, shRNA, Injection, Mouse Assay

    β-TrCP-mediated GSK3β ubiquitination requires GSK3β Y216 phosphorylation. ( A ) The whole cells lysates from HEK293T cells transfected with empty vector or constructs expressing HA-tagged GSK3β or its phosphorylation mutants were used for immunoprecipitation (IP) and immunoblotting (IB) analysis with indicated antibodies. ( B ) Ubiquitination of HA-GSK3β was evaluated by IP with anti-FLAG using whole cell lysates derived from HEK293T cells transfected with indicated constructs followed by IB analysis with anti-HA. ( C ) In vitro synthesized wild type (WT) and mutant HA-GSK3β proteins were immunoprecipitated with anti-HA affinity gel. Left panel: in vitro ubiquitination assay was performed by incubating immunoprecipitated HA-GSK3β with E1, E2, Ub and SCF β-TrCP1 in the presence of ATP at 37°C for 2 hr. The reaction mixtures were analyzed by IB. Right panel: the immunoprecipitated HA-GSK3β was incubated with ATP at 37°C for 2 hr before subjecting to IB using antibody recognizing phosphorylated GSK3β Y216 . ( D ) HEK293T cells were treated with Wnt3a-conditioned medium (CM) for the indicated time before harvesting for IB analysis. ( E ) HEK293T cells were transfected with indicated plasmids and treated with control-CM or Wnt3a-CM for 6 hr. Ubiquitination of HA-GSK3β WT and Y216F mutant protein were evaluated by IP with anti-FLAG followed by IB with anti-HA. ( F ) HEK293T cells were transfected with constructs expressing HA-GSK3β or its mutant. 24 hr post-transfection, the cells were harvested for IP with anti-HA followed by IB with anti-Axin1. ( G ) Upper panel: HA-tagged GSK3β or its mutants were ectopically expressed in HEK293T cells and immunoprecipitated with anti-HA. The precipitated proteins were incubated with purified CK1 and β-catenin in the presence of ATP at 37°C for 1 hr. Kinase activity was evaluated by IB with antibody recognizing phosphorylation of β-catenin S33/S37/T41 catalyzed by GSK3β. Lower panel: the whole cell lysates from WT MEFs, GSK3β knockout (KO) and GSK3α knockdown (KD) MEFs reconstituted with empty vector or indicated HA-GSK3β mutants were used for IB. Antibody recognizing both GSK3α and GSK3β was used for detecting endogenous GSK3 and HA-tagged GSK3β. This antibody recognized HA-GSK3β Y216F but not HA-GSK3β Y216E mutant protein. DOI: http://dx.doi.org/10.7554/eLife.10072.003

    Journal: eLife

    Article Title: FAK/PYK2 promotes the Wnt/β-catenin pathway and intestinal tumorigenesis by phosphorylating GSK3β

    doi: 10.7554/eLife.10072

    Figure Lengend Snippet: β-TrCP-mediated GSK3β ubiquitination requires GSK3β Y216 phosphorylation. ( A ) The whole cells lysates from HEK293T cells transfected with empty vector or constructs expressing HA-tagged GSK3β or its phosphorylation mutants were used for immunoprecipitation (IP) and immunoblotting (IB) analysis with indicated antibodies. ( B ) Ubiquitination of HA-GSK3β was evaluated by IP with anti-FLAG using whole cell lysates derived from HEK293T cells transfected with indicated constructs followed by IB analysis with anti-HA. ( C ) In vitro synthesized wild type (WT) and mutant HA-GSK3β proteins were immunoprecipitated with anti-HA affinity gel. Left panel: in vitro ubiquitination assay was performed by incubating immunoprecipitated HA-GSK3β with E1, E2, Ub and SCF β-TrCP1 in the presence of ATP at 37°C for 2 hr. The reaction mixtures were analyzed by IB. Right panel: the immunoprecipitated HA-GSK3β was incubated with ATP at 37°C for 2 hr before subjecting to IB using antibody recognizing phosphorylated GSK3β Y216 . ( D ) HEK293T cells were treated with Wnt3a-conditioned medium (CM) for the indicated time before harvesting for IB analysis. ( E ) HEK293T cells were transfected with indicated plasmids and treated with control-CM or Wnt3a-CM for 6 hr. Ubiquitination of HA-GSK3β WT and Y216F mutant protein were evaluated by IP with anti-FLAG followed by IB with anti-HA. ( F ) HEK293T cells were transfected with constructs expressing HA-GSK3β or its mutant. 24 hr post-transfection, the cells were harvested for IP with anti-HA followed by IB with anti-Axin1. ( G ) Upper panel: HA-tagged GSK3β or its mutants were ectopically expressed in HEK293T cells and immunoprecipitated with anti-HA. The precipitated proteins were incubated with purified CK1 and β-catenin in the presence of ATP at 37°C for 1 hr. Kinase activity was evaluated by IB with antibody recognizing phosphorylation of β-catenin S33/S37/T41 catalyzed by GSK3β. Lower panel: the whole cell lysates from WT MEFs, GSK3β knockout (KO) and GSK3α knockdown (KD) MEFs reconstituted with empty vector or indicated HA-GSK3β mutants were used for IB. Antibody recognizing both GSK3α and GSK3β was used for detecting endogenous GSK3 and HA-tagged GSK3β. This antibody recognized HA-GSK3β Y216F but not HA-GSK3β Y216E mutant protein. DOI: http://dx.doi.org/10.7554/eLife.10072.003

    Article Snippet: The following antibodies were used in this study: Anti-HA, anti-FLAG, anti-phospho-β-catenin (Ser33/37/Thr41), anti-β-catenin, anti-GSK3α, anti-β-TrCP, anti-α-Tubulin, anti-GSK3β, anti-FAK, anti-phospho-FAK (Tyr397), anti-PYK2 and anti-phospho-PYK2 (Tyr402) (Cell Signaling); anti-FLAG M2 (Sigma); anti-ubiquitin and anti-β-catenin (BD Bioscience, San Jose, CA); anti-GSK3, anti-c-Myc, anti-PYK2, anti-β-TrCP, anti-phospho-GSK3β (Tyr216) and anti-β-Actin (Santa Cruz Biotechnology, Dallas, TX); anti-phospho-GSK3 (Tyr279/Tyr216) (Millipore).

    Techniques: Transfection, Plasmid Preparation, Construct, Expressing, Immunoprecipitation, Derivative Assay, In Vitro, Synthesized, Mutagenesis, Ubiquitin Assay, Incubation, Purification, Activity Assay, Knock-Out

    LBP, l-arabinose and β-carotene prevented cellular steatosis and insulin/glucose metabolism dysfunction in a palmitate acid-induced cell model. (a) Representative image of sodium palmitate-induced fat accumulation in rat hepatocyte cell line BRL-3A after 24-hr incubation. Fat droplets were stained by Oil Red O. Magnification: ×100. Bar: 10 μm. (b) Cellular viability measured by MTT conversion in BRL-3A cells with different treatment for 24 hrs. (c) Quantitative Oil Red O (normalized by MTT results) in BRL-3A cells with different treatment for 24 hrs. In addition, (d) Glucose production and (e) change of the resistin, IRS-1/GSK3α pathways were measured in BRL-3A cells with different treatment for 24 hrs. Data from each group were expressed as means ± SEMs (n = 5). Statistical comparisons between groups were done using the Kruskal–Wallis test followed by Dunn's post hoc test to detect differences in all groups. Different letters (e.g. a and b) mean a statistical significant change ( p

    Journal: Scientific Reports

    Article Title: Lycium barbarum polysaccharides therapeutically improve hepatic functions in non-alcoholic steatohepatitis rats and cellular steatosis model

    doi: 10.1038/srep05587

    Figure Lengend Snippet: LBP, l-arabinose and β-carotene prevented cellular steatosis and insulin/glucose metabolism dysfunction in a palmitate acid-induced cell model. (a) Representative image of sodium palmitate-induced fat accumulation in rat hepatocyte cell line BRL-3A after 24-hr incubation. Fat droplets were stained by Oil Red O. Magnification: ×100. Bar: 10 μm. (b) Cellular viability measured by MTT conversion in BRL-3A cells with different treatment for 24 hrs. (c) Quantitative Oil Red O (normalized by MTT results) in BRL-3A cells with different treatment for 24 hrs. In addition, (d) Glucose production and (e) change of the resistin, IRS-1/GSK3α pathways were measured in BRL-3A cells with different treatment for 24 hrs. Data from each group were expressed as means ± SEMs (n = 5). Statistical comparisons between groups were done using the Kruskal–Wallis test followed by Dunn's post hoc test to detect differences in all groups. Different letters (e.g. a and b) mean a statistical significant change ( p

    Article Snippet: Antibodies of nitrotyrosine, phosphorylated IRS-1 at Ser307, total IRS-1, phosphorylated GSK3α at Ser21, total GSK3α, phosphorylated p38 MAPK at Thr180/Tyr182, total p38 MAPK, phosphorylated JNK at Thr183/Tyr185, total JNK, phosphorylated ERK1/2 at Thr202/Tyr204, total ERK1/2, IκBα, cleaved caspase-3, cytochrome c, p62, beclin-1, Atg5, phosphorylated mTOR at Ser2448, and total mTOR were purchased from Cell Signaling (Beverly, MA).

    Techniques: Incubation, Staining, MTT Assay

    LBP therapeutically improved NASH-induced obesity, insulin resistance and glucose metabolism dysfunction. (a) The body weight of each rat was recorded every week throughout the 12-week high-fat diet induction of NASH. (b) After the induction, the wet liver weight was weighed for each rat. (c) The food intake of each rat (in ml/d) was recorded everyday throughout the 12-week high-fat diet induction of NASH. Each dot represents the average food intake of a week. (d) Representative Western blot results of resistin, phosphorylated IRS-1, total IRS-1, phosphorylated GSK3α, and total GSK3α in all group of rats from 3 repeated Western blot experiments. (e) Blood glucose level was assessed after an intraperitoneal (i.p.) injection with recombinant insulin (a fixed bolus of 0.17 IU) and the serum glucose level was recorded 0, 20, 40, 60, 80, and 100 min later after the 12-week induction of NASH. (f) Area under the curve for glucose (AUCglucose) was calculated using the trapezoidal rule. (g) Blood glucose level of each rat was recorded 0, 20, 40, 60, 80, and 100 min after an i.p. injection of D-glucose (a fixed bolus of 350 mg) after the 12-week induction of NASH. Data were normalized with basal glucose levels. (h) AUCglucose was calculated using the trapezoidal rule. Data from each group were expressed as means ± SEMs (n = 6). Statistical comparisons between groups were done using the Kruskal–Wallis test followed by Dunn's post hoc test to detect differences in all groups. A p

    Journal: Scientific Reports

    Article Title: Lycium barbarum polysaccharides therapeutically improve hepatic functions in non-alcoholic steatohepatitis rats and cellular steatosis model

    doi: 10.1038/srep05587

    Figure Lengend Snippet: LBP therapeutically improved NASH-induced obesity, insulin resistance and glucose metabolism dysfunction. (a) The body weight of each rat was recorded every week throughout the 12-week high-fat diet induction of NASH. (b) After the induction, the wet liver weight was weighed for each rat. (c) The food intake of each rat (in ml/d) was recorded everyday throughout the 12-week high-fat diet induction of NASH. Each dot represents the average food intake of a week. (d) Representative Western blot results of resistin, phosphorylated IRS-1, total IRS-1, phosphorylated GSK3α, and total GSK3α in all group of rats from 3 repeated Western blot experiments. (e) Blood glucose level was assessed after an intraperitoneal (i.p.) injection with recombinant insulin (a fixed bolus of 0.17 IU) and the serum glucose level was recorded 0, 20, 40, 60, 80, and 100 min later after the 12-week induction of NASH. (f) Area under the curve for glucose (AUCglucose) was calculated using the trapezoidal rule. (g) Blood glucose level of each rat was recorded 0, 20, 40, 60, 80, and 100 min after an i.p. injection of D-glucose (a fixed bolus of 350 mg) after the 12-week induction of NASH. Data were normalized with basal glucose levels. (h) AUCglucose was calculated using the trapezoidal rule. Data from each group were expressed as means ± SEMs (n = 6). Statistical comparisons between groups were done using the Kruskal–Wallis test followed by Dunn's post hoc test to detect differences in all groups. A p

    Article Snippet: Antibodies of nitrotyrosine, phosphorylated IRS-1 at Ser307, total IRS-1, phosphorylated GSK3α at Ser21, total GSK3α, phosphorylated p38 MAPK at Thr180/Tyr182, total p38 MAPK, phosphorylated JNK at Thr183/Tyr185, total JNK, phosphorylated ERK1/2 at Thr202/Tyr204, total ERK1/2, IκBα, cleaved caspase-3, cytochrome c, p62, beclin-1, Atg5, phosphorylated mTOR at Ser2448, and total mTOR were purchased from Cell Signaling (Beverly, MA).

    Techniques: Western Blot, Injection, Recombinant

    GSK3 represses HSF1 activity in HeLa cells independent of S303 phosphorylation. ( A ) HeLa cells were treated with DMSO solvent or the GSK3 inhibitor SB-216763 (25 µM) for 15 h. Total protein was analyzed for pS303, HSF1, and β-catenin by immunoblotting. GAPDH serves as a loading control. ( B ) HeLa cells were treated with siRNA specific for GSK3α and GSK3β either individually or together or a scrambled siRNA for 72 h. Total protein was analyzed for pS303, total HSF1, β-catenin, GSK3α/β and GAPDH by immunoblotting.

    Journal: PLoS ONE

    Article Title: Deciphering Human Heat Shock Transcription Factor 1 Regulation via Post-Translational Modification in Yeast

    doi: 10.1371/journal.pone.0015976

    Figure Lengend Snippet: GSK3 represses HSF1 activity in HeLa cells independent of S303 phosphorylation. ( A ) HeLa cells were treated with DMSO solvent or the GSK3 inhibitor SB-216763 (25 µM) for 15 h. Total protein was analyzed for pS303, HSF1, and β-catenin by immunoblotting. GAPDH serves as a loading control. ( B ) HeLa cells were treated with siRNA specific for GSK3α and GSK3β either individually or together or a scrambled siRNA for 72 h. Total protein was analyzed for pS303, total HSF1, β-catenin, GSK3α/β and GAPDH by immunoblotting.

    Article Snippet: Antibodies used in this study were anti-phospho-S303(pS303) (ab47369, Abcam), anti-HSF1 , anti-Pgk1, anti-FLAG (M2, Sigma), anti-Hsp70 (C92, Stressmarq), anti-β-catenin (6B3, Cell Signaling), anti-GAPDH (6C5, Ambion) and anti-GSK3α/β (D75D3, Cell Signaling).

    Techniques: Activity Assay

    Proteomic analysis of tivantinib’s target profile. ( a ) Chemical structures of (−)-tivantinib and couplable c-(−)-tivantinib ( b ) Kinases enriched from drug affinity chromatography in HL60 cells passing SaintScore > 0.95, CRAPomePCT ≥ 95%, and -ln(NSAF) ≥ −7 cutoffs. Bubble size represents the sum of total unique spectra. Bubble color represents probability of a specific interaction based on the CRAPome ( c ) Total unique spectra of GSK3α and GSK3β for tivantinib and ampicillin control pulldowns. ( d ) Western blot of GSK3α and GKS3β following drug affinity chromatography experiments with c-(−)-tivantinib and c-(+)-tivantinib in HL60 and U937 cells. Competition experiments were performed with 20 μM BIO. TCL = total cell lysate, BB = blocked beads

    Journal: Scientific Reports

    Article Title: Off-target based drug repurposing opportunities for tivantinib in acute myeloid leukemia

    doi: 10.1038/s41598-018-37174-6

    Figure Lengend Snippet: Proteomic analysis of tivantinib’s target profile. ( a ) Chemical structures of (−)-tivantinib and couplable c-(−)-tivantinib ( b ) Kinases enriched from drug affinity chromatography in HL60 cells passing SaintScore > 0.95, CRAPomePCT ≥ 95%, and -ln(NSAF) ≥ −7 cutoffs. Bubble size represents the sum of total unique spectra. Bubble color represents probability of a specific interaction based on the CRAPome ( c ) Total unique spectra of GSK3α and GSK3β for tivantinib and ampicillin control pulldowns. ( d ) Western blot of GSK3α and GKS3β following drug affinity chromatography experiments with c-(−)-tivantinib and c-(+)-tivantinib in HL60 and U937 cells. Competition experiments were performed with 20 μM BIO. TCL = total cell lysate, BB = blocked beads

    Article Snippet: Antibodies against GSK3α (#4337), GSK3β (#9315), pSer10 Histone H3 (#3377), Cleaved Caspase 3 (#9661), PARP-1 (#9542), and BCL-XL (#2764) were from Cell Signaling.

    Techniques: Affinity Chromatography, Western Blot

    Staphylococcus aureus inactivates glycogen synthase kinase 3 (GSK3α) and GSK3β by phosphorylation at Ser21 and Ser9. Bovine endothelial cells were left uninfected (−) or infected at a multiplicity of infection of 20 CFU/cell of S. aureus for 20–120 min (A,B) or 20–60 min (C) . The relative abundance of phosphorylated GSK3α at Ser21 (A) , GSK3β at Ser9 (B) , and glycogen synthase at Ser641 (C) was evaluated by Western blotting. The detection of total GSK3α, GSK3β, and β-actin was done to ensure equal protein loading. Error bars in graphs A–C are presented as the mean ± SEM of three independent experiments (SEM; n = 3 per experiment). * P

    Journal: Frontiers in Immunology

    Article Title: Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus

    doi: 10.3389/fimmu.2018.00092

    Figure Lengend Snippet: Staphylococcus aureus inactivates glycogen synthase kinase 3 (GSK3α) and GSK3β by phosphorylation at Ser21 and Ser9. Bovine endothelial cells were left uninfected (−) or infected at a multiplicity of infection of 20 CFU/cell of S. aureus for 20–120 min (A,B) or 20–60 min (C) . The relative abundance of phosphorylated GSK3α at Ser21 (A) , GSK3β at Ser9 (B) , and glycogen synthase at Ser641 (C) was evaluated by Western blotting. The detection of total GSK3α, GSK3β, and β-actin was done to ensure equal protein loading. Error bars in graphs A–C are presented as the mean ± SEM of three independent experiments (SEM; n = 3 per experiment). * P

    Article Snippet: Antibodies against phospho-glycogen synthase (Ser641), phospho-GSK3α (Ser21), phospho-GSK3α (Ser9), phospho-NF-κB p65 (Ser536), phospho-CREB (Ser133), GSK3β, GSK3α, and NF-κB p65 were purchased from Cell Signaling Technology (Boston, MA, USA).

    Techniques: Infection, Western Blot

    CRMP2 phosphorylation in retinae of GSK3 S/A or conditional GSK3 KOs correlates with axon regeneration. ( A ) Representative pictures of flat-mounted retinae isolated from WT, GSK3α S/A (α S/A ), GSK3β S/A (β S/A ), and GSK3(α/β) S/A [(α/β) S/A ] mice that were either left untreated (con) or subjected to optic nerve crush (ONC). Five days after surgery, retinae were fixed and stained for T514-phosphorylated CRMP2 (pCRMP2; red) and βIII-tubulin (tubulin; green in lower row to visualize RGCs). In WT animals, pCRMP2 was reduced after ONC in RGC somas and axons (arrows). No difference was detectable upon ONC compared with con in cell bodies of α S/A , β S/A , and (α/β) S/A RGCs, while axonal staining appeared slightly reduced after ONC but still much stronger than in the ONC-treated WT. (Scale bar: 50 μm.) ( B ) Representative pictures of flat-mounted retinae isolated from WT or conditional GSK3α −/− (α −/− ), GSK3β −/− (β −/− ) and GSK3α −/− /β −/− (α/β) −/− mice treated as in A and stained for pCRMP2 (red) and βIII-tubulin (green). Phospho-CRMP2 levels were reduced in β −/− and (α/β) −/− , but not in α −/− RGCs compared with retinae from respectively treated WT mice. ONC reduced pCRMP2 in RGC somas and particularly in axons (arrows) in all four genotypes. (Scale bar: 50 μm.) ( C ) Higher magnification of retinal flat-mount from an untreated (α/β) −/− animal as indicated in B by the dashed white box. Phospho-CRMP2 was not detected in RGCs, but only in other retinal cells with weak βIII-tubulin staining and distinct morphology (arrows). (Scale bar: 10 μm.) ( D and E ) Photographs of retinal flat-mounts from WT, or (α/β) S/A mice ( D ), or respective WT and conditional (α/β) −/− animals ( E ) treated as described in A and stained for total CRMP2 protein (CRMP2; red). RGCs were identified using a βIII-tubulin staining (green). Total CRMP2 levels remained unchanged in all treatment groups. (Scale bar: 50 μm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Boosting CNS axon regeneration by harnessing antagonistic effects of GSK3 activity

    doi: 10.1073/pnas.1621225114

    Figure Lengend Snippet: CRMP2 phosphorylation in retinae of GSK3 S/A or conditional GSK3 KOs correlates with axon regeneration. ( A ) Representative pictures of flat-mounted retinae isolated from WT, GSK3α S/A (α S/A ), GSK3β S/A (β S/A ), and GSK3(α/β) S/A [(α/β) S/A ] mice that were either left untreated (con) or subjected to optic nerve crush (ONC). Five days after surgery, retinae were fixed and stained for T514-phosphorylated CRMP2 (pCRMP2; red) and βIII-tubulin (tubulin; green in lower row to visualize RGCs). In WT animals, pCRMP2 was reduced after ONC in RGC somas and axons (arrows). No difference was detectable upon ONC compared with con in cell bodies of α S/A , β S/A , and (α/β) S/A RGCs, while axonal staining appeared slightly reduced after ONC but still much stronger than in the ONC-treated WT. (Scale bar: 50 μm.) ( B ) Representative pictures of flat-mounted retinae isolated from WT or conditional GSK3α −/− (α −/− ), GSK3β −/− (β −/− ) and GSK3α −/− /β −/− (α/β) −/− mice treated as in A and stained for pCRMP2 (red) and βIII-tubulin (green). Phospho-CRMP2 levels were reduced in β −/− and (α/β) −/− , but not in α −/− RGCs compared with retinae from respectively treated WT mice. ONC reduced pCRMP2 in RGC somas and particularly in axons (arrows) in all four genotypes. (Scale bar: 50 μm.) ( C ) Higher magnification of retinal flat-mount from an untreated (α/β) −/− animal as indicated in B by the dashed white box. Phospho-CRMP2 was not detected in RGCs, but only in other retinal cells with weak βIII-tubulin staining and distinct morphology (arrows). (Scale bar: 10 μm.) ( D and E ) Photographs of retinal flat-mounts from WT, or (α/β) S/A mice ( D ), or respective WT and conditional (α/β) −/− animals ( E ) treated as described in A and stained for total CRMP2 protein (CRMP2; red). RGCs were identified using a βIII-tubulin staining (green). Total CRMP2 levels remained unchanged in all treatment groups. (Scale bar: 50 μm.)

    Article Snippet: Blots were blocked in 5% dried milk in Tris- or phosphate-buffered saline solution with 0.05% Tween-20 (TBS-T and PBS-T, respectively; Sigma) and incubated with antibodies against βIII-tubulin (1:2,000; BioLegend; RRID:AB_2313773), S9-phosphorylated GSK3β (1:1,000; RRID:AB_2115196), S21-phosphorylated GSK3α (1:2,000; RRID:AB_2114897), total GSK3α/β (1:500; RRID:AB_10547140; all from Cell Signaling Technologies), T514-phosphorylated CRMP2 (1:2,000; Abcam; RRID:AB_942229), total CRMP2 (1:500; Cell Signaling Technologies; RRID:AB_2094339), or T308-phosphorylated AKT (1:1,000; Cell Signaling Technologies, RRID:AB_2629447) at 4 °C overnight.

    Techniques: Isolation, Mouse Assay, Staining

    ONC elicits inhibitory GSK3α and GSK3β phosphorylation in RGCs. ( A ) Retinal flat-mounts and cross-sections of WT mice isolated 5 d after ONC and ONC+IS, respectively. Compared with untreated mice (con), S21-phosphorylation of GSK3α (pGSK3α; red) and S9-phosphorylation of GSK3β (pGSK3β; red) were similarly increased in βIII-tubulin–positive RGCs (green) after ONC and ONC+IS. Retinal cross-sections reveal that GSK3α/β phosphorylation occurred in RGCs in the ganglion cell layer (GCL). Total GSK3α/β (red) remained unchanged. Phospho-GSK3 staining was absent in retinae of GSK3(α/β) S/A knock-in [(α/β) S/A ] mice, indicating antibody specificity. Nuclei were labeled by DAPI (blue). INL, inner nuclear layer; ONL, outer nuclear layer. (Scale bars: 50 µm.) ( B ) Western blots of retinal lysates from WT mice untreated (con), 5 d after ONC or ONC+IS. pGSK3α and pGSK3β signals increased after ONC compared with untreated controls, but additional IS did not further enhance GSK3 phosphorylation. No pGSK3 signals were detected in retinal lysates from (α/β) S/A mice, verifying antibody specificities. Levels of T308-phosphorylated AKT (pAKT) were elevated after injury in WT and (α/β) S/A mice. Total GSK3α and GSK3β levels were comparable in all experimental groups. βIII-tubulin served as a loading control. ( C – G ) Densitometric quantification of pGSK3α ( C ), pGSK3β ( D ), pAKT ( E ), total GSK3α ( F ), and total GSK3β ( G ) relative to βIII-tubulin and normalized to WT control on different Western blots as depicted in B . Significances of intergroup differences were evaluated by one-way ANOVA with Tukey post hoc test. Treatment effects compared with WT control: ** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Boosting CNS axon regeneration by harnessing antagonistic effects of GSK3 activity

    doi: 10.1073/pnas.1621225114

    Figure Lengend Snippet: ONC elicits inhibitory GSK3α and GSK3β phosphorylation in RGCs. ( A ) Retinal flat-mounts and cross-sections of WT mice isolated 5 d after ONC and ONC+IS, respectively. Compared with untreated mice (con), S21-phosphorylation of GSK3α (pGSK3α; red) and S9-phosphorylation of GSK3β (pGSK3β; red) were similarly increased in βIII-tubulin–positive RGCs (green) after ONC and ONC+IS. Retinal cross-sections reveal that GSK3α/β phosphorylation occurred in RGCs in the ganglion cell layer (GCL). Total GSK3α/β (red) remained unchanged. Phospho-GSK3 staining was absent in retinae of GSK3(α/β) S/A knock-in [(α/β) S/A ] mice, indicating antibody specificity. Nuclei were labeled by DAPI (blue). INL, inner nuclear layer; ONL, outer nuclear layer. (Scale bars: 50 µm.) ( B ) Western blots of retinal lysates from WT mice untreated (con), 5 d after ONC or ONC+IS. pGSK3α and pGSK3β signals increased after ONC compared with untreated controls, but additional IS did not further enhance GSK3 phosphorylation. No pGSK3 signals were detected in retinal lysates from (α/β) S/A mice, verifying antibody specificities. Levels of T308-phosphorylated AKT (pAKT) were elevated after injury in WT and (α/β) S/A mice. Total GSK3α and GSK3β levels were comparable in all experimental groups. βIII-tubulin served as a loading control. ( C – G ) Densitometric quantification of pGSK3α ( C ), pGSK3β ( D ), pAKT ( E ), total GSK3α ( F ), and total GSK3β ( G ) relative to βIII-tubulin and normalized to WT control on different Western blots as depicted in B . Significances of intergroup differences were evaluated by one-way ANOVA with Tukey post hoc test. Treatment effects compared with WT control: ** P

    Article Snippet: Blots were blocked in 5% dried milk in Tris- or phosphate-buffered saline solution with 0.05% Tween-20 (TBS-T and PBS-T, respectively; Sigma) and incubated with antibodies against βIII-tubulin (1:2,000; BioLegend; RRID:AB_2313773), S9-phosphorylated GSK3β (1:1,000; RRID:AB_2115196), S21-phosphorylated GSK3α (1:2,000; RRID:AB_2114897), total GSK3α/β (1:500; RRID:AB_10547140; all from Cell Signaling Technologies), T514-phosphorylated CRMP2 (1:2,000; Abcam; RRID:AB_942229), total CRMP2 (1:500; Cell Signaling Technologies; RRID:AB_2094339), or T308-phosphorylated AKT (1:1,000; Cell Signaling Technologies, RRID:AB_2629447) at 4 °C overnight.

    Techniques: Mouse Assay, Isolation, Staining, Knock-In, Labeling, Western Blot

    β-Adrenergic GSK3 inactivation is PKA dependent. ( a ) The PKA consensus sites around Ser21 and Ser9 in GSK3α and GSK3β, respectively, are conserved in various species. ( b ) Immunoblot analysis of total and phosphorylated GSK3α and GSK3β in immortalized brown adipocytes treated with 40 μM H89 for 1 h before stimulation with 0.1 μM ISO for additional 15 min. Some of the cells were stimulated with 100 μM 6-MB-cAMP (6-MB) for 15 min. ( c ) Immunoblot analysis of total and phosphorylated GSK3α and GSK3β as well as phosphorylated HSL (Ser660) in iBAT from AdipoQ -caPKA and wild-type mice housed at room temperature. ( d ) Medium glycerol of immortalized brown adipocytes pre-treated with 10 μM SB216763 for 1 h before stimulation with 0.1 μM for 24 h. ( e ) Immunoblot analysis of phosphorylated and total HSL (Ser660), CREB (Ser133) and phosphorylated PKA substrates in immortalized brown adipocytes pre-treated with 10 μM SB216763 for 1 h before stimulation with 0.1 μM ISO for 1 h. Vinculin (tissues) or TFIIB (cells) serves as loading control. Data presented as mean of means +SEM (n = 4). Statistical significance was determined by two-way ANOVA with repeated measures and Tukey’s post hoc test for multiple comparisons. *p

    Journal: Scientific Reports

    Article Title: GSK3 is a negative regulator of the thermogenic program in brown adipocytes

    doi: 10.1038/s41598-018-21795-y

    Figure Lengend Snippet: β-Adrenergic GSK3 inactivation is PKA dependent. ( a ) The PKA consensus sites around Ser21 and Ser9 in GSK3α and GSK3β, respectively, are conserved in various species. ( b ) Immunoblot analysis of total and phosphorylated GSK3α and GSK3β in immortalized brown adipocytes treated with 40 μM H89 for 1 h before stimulation with 0.1 μM ISO for additional 15 min. Some of the cells were stimulated with 100 μM 6-MB-cAMP (6-MB) for 15 min. ( c ) Immunoblot analysis of total and phosphorylated GSK3α and GSK3β as well as phosphorylated HSL (Ser660) in iBAT from AdipoQ -caPKA and wild-type mice housed at room temperature. ( d ) Medium glycerol of immortalized brown adipocytes pre-treated with 10 μM SB216763 for 1 h before stimulation with 0.1 μM for 24 h. ( e ) Immunoblot analysis of phosphorylated and total HSL (Ser660), CREB (Ser133) and phosphorylated PKA substrates in immortalized brown adipocytes pre-treated with 10 μM SB216763 for 1 h before stimulation with 0.1 μM ISO for 1 h. Vinculin (tissues) or TFIIB (cells) serves as loading control. Data presented as mean of means +SEM (n = 4). Statistical significance was determined by two-way ANOVA with repeated measures and Tukey’s post hoc test for multiple comparisons. *p

    Article Snippet: Primary antibodies used were: CREB (#9192), p-CREB (Ser133) (#9198), GSK3α (#4337), p-GSK3α (Ser21) (#9316), GSK3β (#12456), p-GSK3β (Ser9) (#5558), p38 MAPK (#9212), p-p38 MAPK (Thr180/Tyr182) (#9211), MKK3 (#8535), MKK6 (#8550), p-MKK3/6 (Ser189/Ser207) (#12280), ATF2 (#9226), p-ATF2 (Thr71) (#5112), HSL (#4107), p-HSL (Ser660) (#4126), Phospho-(Ser/Thr) PKA substrate (#9621) (all from Cell Signaling Technology), TFIIB (#sc-225) (Santa Cruz Biotechnology), Vinculin (#V9264) (Sigma-Aldrich), UCP1 (#10983) (Abcam) and HA (#11583816001) (Roche).

    Techniques: Mouse Assay

    Ca 2+ influx via voltage-gated calcium channels is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM flunarizine (Flun) for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 5 for both P-Akt and P-GSK3 with 80 Hz, n = 6 for P-Akt IONO and n = 7 for P-GSK3 IONO (students t test, ns non-significant, * p

    Journal: Neurochemical Research

    Article Title: Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals

    doi: 10.1007/s11064-015-1663-5

    Figure Lengend Snippet: Ca 2+ influx via voltage-gated calcium channels is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM flunarizine (Flun) for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 5 for both P-Akt and P-GSK3 with 80 Hz, n = 6 for P-Akt IONO and n = 7 for P-GSK3 IONO (students t test, ns non-significant, * p

    Article Snippet: The primary antibodies phospho-Akt Ser473 and GSK3α/β Ser21/9 were from Cell Signalling.

    Techniques: Incubation

    PI3K activity is essential for activity-dependent Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with either 10 μM LY294002 or 200 nM wortmannin for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after action potential stimulation in the presence of either LY294002 ( a ) or wortmannin ( d ). b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in the presence of either LY294002 ( b ) or wortmannin ( e ). c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in the presence of either LY294002 ( c ) or wortmannin ( f ). In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; LY294002—n = 6 for P-Akt, n = 16 for P-GSK3; wortmannin—n = 4 for P-Akt, n = 6 for P-GSK3 (students t test, ns non-significant, * p

    Journal: Neurochemical Research

    Article Title: Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals

    doi: 10.1007/s11064-015-1663-5

    Figure Lengend Snippet: PI3K activity is essential for activity-dependent Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with either 10 μM LY294002 or 200 nM wortmannin for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after action potential stimulation in the presence of either LY294002 ( a ) or wortmannin ( d ). b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in the presence of either LY294002 ( b ) or wortmannin ( e ). c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in the presence of either LY294002 ( c ) or wortmannin ( f ). In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; LY294002—n = 6 for P-Akt, n = 16 for P-GSK3; wortmannin—n = 4 for P-Akt, n = 6 for P-GSK3 (students t test, ns non-significant, * p

    Article Snippet: The primary antibodies phospho-Akt Ser473 and GSK3α/β Ser21/9 were from Cell Signalling.

    Techniques: Activity Assay, Incubation

    Localised Ca 2+ influx is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with either 100 μM BAPTA-AM or EGTA-AM for 30 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 4 for P-Akt 80 Hz, n = 5 for P-GSK3 80 Hz, n = 7 for P-Akt IONO and n = 6 for P-GSK3 IONO (students t test, ns non-significant * p

    Journal: Neurochemical Research

    Article Title: Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals

    doi: 10.1007/s11064-015-1663-5

    Figure Lengend Snippet: Localised Ca 2+ influx is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with either 100 μM BAPTA-AM or EGTA-AM for 30 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 4 for P-Akt 80 Hz, n = 5 for P-GSK3 80 Hz, n = 7 for P-Akt IONO and n = 6 for P-GSK3 IONO (students t test, ns non-significant * p

    Article Snippet: The primary antibodies phospho-Akt Ser473 and GSK3α/β Ser21/9 were from Cell Signalling.

    Techniques: Incubation

    [Ca 2+ ] i increases are essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then either rested (Basal) or stimulated with ionomycin (IONO, 5 µM) for 1 min in incubation buffer containing either 1.3 mM (+Ca 2+ ) or low (−Ca 2+ ) calcium. a Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ). b , c The fold increase in phosphorylation of either Akt Ser473 ( b , open bars ) or GSK3α/β Ser21/9 ( c , closed bars ) is displayed after correction for protein levels using β-Actin and normalisation to the low calcium control. All error bars represent ±SEM; n = 5 for P-Akt and n = 4 for P-GSK3 (students t test, ns non-significant, * p

    Journal: Neurochemical Research

    Article Title: Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals

    doi: 10.1007/s11064-015-1663-5

    Figure Lengend Snippet: [Ca 2+ ] i increases are essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then either rested (Basal) or stimulated with ionomycin (IONO, 5 µM) for 1 min in incubation buffer containing either 1.3 mM (+Ca 2+ ) or low (−Ca 2+ ) calcium. a Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ). b , c The fold increase in phosphorylation of either Akt Ser473 ( b , open bars ) or GSK3α/β Ser21/9 ( c , closed bars ) is displayed after correction for protein levels using β-Actin and normalisation to the low calcium control. All error bars represent ±SEM; n = 5 for P-Akt and n = 4 for P-GSK3 (students t test, ns non-significant, * p

    Article Snippet: The primary antibodies phospho-Akt Ser473 and GSK3α/β Ser21/9 were from Cell Signalling.

    Techniques: Incubation

    Calmodulin is not the calcium sensor for activity-dependent Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM calmidazolium for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 6 for P-Akt 80 Hz, n = 9 for P-GSK3 80 Hz, n = 8 for P-Akt IONO and n = 9 for P-GSK3 IONO (students t test, ns non-significant, * p

    Journal: Neurochemical Research

    Article Title: Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals

    doi: 10.1007/s11064-015-1663-5

    Figure Lengend Snippet: Calmodulin is not the calcium sensor for activity-dependent Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM calmidazolium for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 6 for P-Akt 80 Hz, n = 9 for P-GSK3 80 Hz, n = 8 for P-Akt IONO and n = 9 for P-GSK3 IONO (students t test, ns non-significant, * p

    Article Snippet: The primary antibodies phospho-Akt Ser473 and GSK3α/β Ser21/9 were from Cell Signalling.

    Techniques: Activity Assay, Incubation

    Effect of HHcy on GSK3 levels in Tg2576 mice. A ) Representative Western blots of brain homogenates probed with specific antibodies against total GSK3α/β, and phosphorylated GSK3α/β on Ser-21/9, respectively. B ) Densitometric

    Journal: The FASEB Journal

    Article Title: Normalization of hyperhomocysteinemia improves cognitive deficits and ameliorates brain amyloidosis of a transgenic mouse model of Alzheimer's disease

    doi: 10.1096/fj.10-161828

    Figure Lengend Snippet: Effect of HHcy on GSK3 levels in Tg2576 mice. A ) Representative Western blots of brain homogenates probed with specific antibodies against total GSK3α/β, and phosphorylated GSK3α/β on Ser-21/9, respectively. B ) Densitometric

    Article Snippet: Antibodies used in the current study were as follows: anti-APP (22C11; Chemicon International, Temecula, CA, USA), anti-secreted APPβ (sAPPβ) (6A1, IBL America, Minneapolis, MN, USA), anti-β-site APP cleaving enzyme 1 (BACE-1) (IBL America), anti-ADAM10 (Chemicon International), anti-APP C-terminal for C-terminal fragments (CTFs) (EMD Biosciences Inc, La Jolla, CA, USA), anti-presenilin 1 (PS1) (Sigma-Aldrich, St. Louis, MO, USA), anti-presenilin enhancer 2 (Pen-2) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti-Nicastrin (Cell Signaling Technology, Danvers, MA, USA), anti-anterior pharynx-defective 1 (APH1) (Sigma-Aldrich), anti-apolipoprotein E (apoE) (Santa Cruz Biotechnologies), anti-insulin-degrading enzyme (IDE) N-terminal (EMD Biosciences Inc.), anti-neprilysin (NEP) (Santa Cruz Biotechnologies), anti-glial fibrillary acidic protein (GFAP) (Cell Signaling Technology), anti-glycogen synthase kinase 3 α/β (GSK3α/β) (Santa Cruz Biotechnologies), anti-phospho- GSK3α/β (Ser-21/9) (Cell Signaling Technology), and anti-β-actin (1:4000; Santa Cruz Biotechnologies).

    Techniques: Mouse Assay, Western Blot

    CAPE inhibits Akt signaling-related proteins in PC-3 cells. Protein expression of Akt, Akt1, Akt2, Akt3, total Akt, phospho-Akt S473, phospho-Akt T308, mTOR, phospho-mTOR Ser2448 and Ser2481, GSK3α, GSK3β, phopho-GSK3α S21, phospho-GSK3β S9, PDK1, phospho-PDK1 Ser241, Bcl-2, KLF6, β-tubulin, and β-actin in PC-3 cells treated with 20 µM CAPE for 24, 48, and 5, 10, 20 µM CAPE for 72 h were assayed by Western blotting.

    Journal: PLoS ONE

    Article Title: Caffeic Acid Phenethyl Ester Causes p21Cip1 Induction, Akt Signaling Reduction, and Growth Inhibition in PC-3 Human Prostate Cancer Cells

    doi: 10.1371/journal.pone.0031286

    Figure Lengend Snippet: CAPE inhibits Akt signaling-related proteins in PC-3 cells. Protein expression of Akt, Akt1, Akt2, Akt3, total Akt, phospho-Akt S473, phospho-Akt T308, mTOR, phospho-mTOR Ser2448 and Ser2481, GSK3α, GSK3β, phopho-GSK3α S21, phospho-GSK3β S9, PDK1, phospho-PDK1 Ser241, Bcl-2, KLF6, β-tubulin, and β-actin in PC-3 cells treated with 20 µM CAPE for 24, 48, and 5, 10, 20 µM CAPE for 72 h were assayed by Western blotting.

    Article Snippet: Cyclin D1, Cyclin E, p-Akt (Ser 473), p-Akt (Thr 308), p-ERK1/2, GSK3α, GSK3β, p-GSK3α, p-GSK3β, mTOR, p-mTOR(Ser2481), p-PDK1(Ser241), Rb, and p-Rb(Ser807/811) were from Cell Signaling (Danvers, MA, U.S.A.). c-Myc was purchased from Epitomics (Burlingame, CA, U.S.A.). p21Cip1 , p27Kip1 and SKP2 were purchased from Santa Cruz (Santa Cruz, CA, U.S.A.).

    Techniques: Expressing, Western Blot

    Western blot analyses of steady-state levels of AKT, P -AKT, GSK3α/β, p-GSK3α/β, mTOR and p-mTOR following exposure to MK2206 and GSK690693 in 2D culture (CM+Y). (A) Representative western blots of AKT, p-AKT(S473), GSK3α/β, p-GSK3α/β, mTOR, p-mTOR and β-actin from GUMC220 and GUMC221 exposed to MK2206 (0.6 μM and 1.2 µM) compared with DMSO vehicle control. (B) Steady-state expression levels of pAKT(S473), GSK3α/β, p-GSK3α/β and p-mTOR in GUMC220 and GUMC221 cells after 1, 2 and 3 days’ exposure to the two different concentrations of MK2206 and DMSO. Data are mean±s.e.m., n =3/timepoint/concentration. (C) Representative western blots of AKT, p-AKT(S473), p-AKT(T308), GSK3α/β, p-GSK3α/β, mTOR, p-mTOR and β-actin from GUMC220 and GUMC221 in 2D CM+Y culture exposed to GSK690693 (15 μM and 30 µM) compared with DMSO vehicle control. (D) Steady-state expression levels of pAKT(S473), p-AKT(T308), GSK3α/β, p-GSK3α/β and p-mTOR in GUMC220 and GUMC221 cells after 1, 2 and 3 days’ exposure to the two different concentrations of GSK690693 compared with DMSO vehicle control. Phosphoprotein levels were normalized to total proteins, which were normalized to actin. *P ≤0.05, **P ≤0.01, ***P ≤0.001, one tailed, unpaired t -test. Protein lysates were collected after 1, 2 and 3 days of drug exposure. Data are mean±s.e.m., n =3/timepoint/concentration. Molecular weights (kDa) are indicated for each protein.

    Journal: Disease Models & Mechanisms

    Article Title: Expanding primary cells from mucoepidermoid and other salivary gland neoplasms for genetic and chemosensitivity testing

    doi: 10.1242/dmm.031716

    Figure Lengend Snippet: Western blot analyses of steady-state levels of AKT, P -AKT, GSK3α/β, p-GSK3α/β, mTOR and p-mTOR following exposure to MK2206 and GSK690693 in 2D culture (CM+Y). (A) Representative western blots of AKT, p-AKT(S473), GSK3α/β, p-GSK3α/β, mTOR, p-mTOR and β-actin from GUMC220 and GUMC221 exposed to MK2206 (0.6 μM and 1.2 µM) compared with DMSO vehicle control. (B) Steady-state expression levels of pAKT(S473), GSK3α/β, p-GSK3α/β and p-mTOR in GUMC220 and GUMC221 cells after 1, 2 and 3 days’ exposure to the two different concentrations of MK2206 and DMSO. Data are mean±s.e.m., n =3/timepoint/concentration. (C) Representative western blots of AKT, p-AKT(S473), p-AKT(T308), GSK3α/β, p-GSK3α/β, mTOR, p-mTOR and β-actin from GUMC220 and GUMC221 in 2D CM+Y culture exposed to GSK690693 (15 μM and 30 µM) compared with DMSO vehicle control. (D) Steady-state expression levels of pAKT(S473), p-AKT(T308), GSK3α/β, p-GSK3α/β and p-mTOR in GUMC220 and GUMC221 cells after 1, 2 and 3 days’ exposure to the two different concentrations of GSK690693 compared with DMSO vehicle control. Phosphoprotein levels were normalized to total proteins, which were normalized to actin. *P ≤0.05, **P ≤0.01, ***P ≤0.001, one tailed, unpaired t -test. Protein lysates were collected after 1, 2 and 3 days of drug exposure. Data are mean±s.e.m., n =3/timepoint/concentration. Molecular weights (kDa) are indicated for each protein.

    Article Snippet: GSK3α/β (5676, CST); WB (1:1000); Antibodypedia. p-GSK3α/β (9331, CST); WB (1:1000); Antibodypedia. β-Actin (3700, CST); WB (1:1000); Antibodypedia.

    Techniques: Western Blot, Expressing, Concentration Assay, One-tailed Test

    The inactivation of GSK3 proteins at various stages of OTSCC progression. Representative immunostaining showing the differential expression of pS 21 GSK3α and pS 9 GSK3β in the consecutive sections of (a, b) normal tongue tissue, (c, d) mild hyperplasia of tumor adjacent tongue, and (e, f) OTSCC tissue samples (original magnification 100X).

    Journal: Molecular Cancer

    Article Title: Expression and inactivation of glycogen synthase kinase 3 alpha/ beta and their association with the expression of cyclin D1 and p53 in oral squamous cell carcinoma progression

    doi: 10.1186/s12943-015-0300-x

    Figure Lengend Snippet: The inactivation of GSK3 proteins at various stages of OTSCC progression. Representative immunostaining showing the differential expression of pS 21 GSK3α and pS 9 GSK3β in the consecutive sections of (a, b) normal tongue tissue, (c, d) mild hyperplasia of tumor adjacent tongue, and (e, f) OTSCC tissue samples (original magnification 100X).

    Article Snippet: Primary antibodies (Santa Cruz Biotechnology) p/GSK3α (dilution 1: 15) and p/GSK3β (dilution 1: 40) were incubated at 4°C overnight.

    Techniques: Immunostaining, Expressing

    The correlation of pSer 21 GSK3α /pSer 9 GSK3β expression with cyclin D1 transcription in various OSCC samples. (A) RT-PCR showing cyclin D1 mRNA expression (201 bp PCR product) in different normals (lane 1–3), PMLs (lane 4–6) and oral cancer samples of various stages (T1-T2 samples lane 7–10; T3-T4 samples lane 11–15). GAPDH expression (410 bp PCR product) was used as a control in this experiment. (B) A histogram showing the level of expression of cyclin D1 mRNA in various groups (N-Normal, PML, T-Tumor) of samples as indicated. No significant correlation (C) of cyclin D1 mRNA expression and pSer 21 GSK3α protein expression was observed, and a positive correlation (D) between cyclin D1 mRNA expression and the expression of pSer 9 GSK3β was observed.

    Journal: Molecular Cancer

    Article Title: Expression and inactivation of glycogen synthase kinase 3 alpha/ beta and their association with the expression of cyclin D1 and p53 in oral squamous cell carcinoma progression

    doi: 10.1186/s12943-015-0300-x

    Figure Lengend Snippet: The correlation of pSer 21 GSK3α /pSer 9 GSK3β expression with cyclin D1 transcription in various OSCC samples. (A) RT-PCR showing cyclin D1 mRNA expression (201 bp PCR product) in different normals (lane 1–3), PMLs (lane 4–6) and oral cancer samples of various stages (T1-T2 samples lane 7–10; T3-T4 samples lane 11–15). GAPDH expression (410 bp PCR product) was used as a control in this experiment. (B) A histogram showing the level of expression of cyclin D1 mRNA in various groups (N-Normal, PML, T-Tumor) of samples as indicated. No significant correlation (C) of cyclin D1 mRNA expression and pSer 21 GSK3α protein expression was observed, and a positive correlation (D) between cyclin D1 mRNA expression and the expression of pSer 9 GSK3β was observed.

    Article Snippet: Primary antibodies (Santa Cruz Biotechnology) p/GSK3α (dilution 1: 15) and p/GSK3β (dilution 1: 40) were incubated at 4°C overnight.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    The expression of GSK3α /β proteins at various stages of OSCC progression. (A) Representative immunostaining showing the differential expression of GSK3α and GSK3β from consecutive sections of lip SCC tissue progression, including (a, b) normal lip, (c, d) hyperplasia of the lip and (e, f) SCC of the lip. (B) Representative immunostaining showing the differential expression of GSK3α and GSK3β from consecutive sections of OTSCC tissue progression, including (a, b) normal tongue; (c, d) hyperplasia of the tongue and (e, f) SCC of the tongue. (C) Photomicrographs showing distant metastasis of SCC cells at the lymph nodes from various OSCC showing immunoreactivity to GSK3α and GSK3β antibodies to different extents in the consecutive sections (a to h). The metastatic OTSCC showed maximum expression of GSK3β (original magnification 100X).

    Journal: Molecular Cancer

    Article Title: Expression and inactivation of glycogen synthase kinase 3 alpha/ beta and their association with the expression of cyclin D1 and p53 in oral squamous cell carcinoma progression

    doi: 10.1186/s12943-015-0300-x

    Figure Lengend Snippet: The expression of GSK3α /β proteins at various stages of OSCC progression. (A) Representative immunostaining showing the differential expression of GSK3α and GSK3β from consecutive sections of lip SCC tissue progression, including (a, b) normal lip, (c, d) hyperplasia of the lip and (e, f) SCC of the lip. (B) Representative immunostaining showing the differential expression of GSK3α and GSK3β from consecutive sections of OTSCC tissue progression, including (a, b) normal tongue; (c, d) hyperplasia of the tongue and (e, f) SCC of the tongue. (C) Photomicrographs showing distant metastasis of SCC cells at the lymph nodes from various OSCC showing immunoreactivity to GSK3α and GSK3β antibodies to different extents in the consecutive sections (a to h). The metastatic OTSCC showed maximum expression of GSK3β (original magnification 100X).

    Article Snippet: Primary antibodies (Santa Cruz Biotechnology) p/GSK3α (dilution 1: 15) and p/GSK3β (dilution 1: 40) were incubated at 4°C overnight.

    Techniques: Expressing, Immunostaining

    WB analysis to show the expression of in/active GSK3, cyclin D1 and p53 proteins at various stages of OSCC. (A) Representative blot showing the expression of GSK3α, GSK3β, pSer 21 GSK3α, pSer 9 GSK3β, cyclin D1 and p53 in various normal, PMLs and OSCC samples. β-Actin was used as a loading control in this experiment. (B) The mean and SD of each protein band has been plotted for the normal, PML and OSCC samples. Statistical analysis was performed using Student’s t -test. Statistical significance was observed among various groups: **p = 0.001, ***p

    Journal: Molecular Cancer

    Article Title: Expression and inactivation of glycogen synthase kinase 3 alpha/ beta and their association with the expression of cyclin D1 and p53 in oral squamous cell carcinoma progression

    doi: 10.1186/s12943-015-0300-x

    Figure Lengend Snippet: WB analysis to show the expression of in/active GSK3, cyclin D1 and p53 proteins at various stages of OSCC. (A) Representative blot showing the expression of GSK3α, GSK3β, pSer 21 GSK3α, pSer 9 GSK3β, cyclin D1 and p53 in various normal, PMLs and OSCC samples. β-Actin was used as a loading control in this experiment. (B) The mean and SD of each protein band has been plotted for the normal, PML and OSCC samples. Statistical analysis was performed using Student’s t -test. Statistical significance was observed among various groups: **p = 0.001, ***p

    Article Snippet: Primary antibodies (Santa Cruz Biotechnology) p/GSK3α (dilution 1: 15) and p/GSK3β (dilution 1: 40) were incubated at 4°C overnight.

    Techniques: Western Blot, Expressing

    The expression of GSK3 α and GSK3β proteins in the tumor/ normal tissues of various anatomical sites of the mouth. (A) Representative immunostaining showing the differential expression of GSK3α and GSK3β from consecutive sections in various types of oral tumor tissue samples as indicated in the figure. (a, b) SCC (cheek); (c, d) Mucoepidermoid carcinoma (palate); (e, f) Adamantinoma (mandible); (g, h) SCC (gingiva); (i, j) SCC (lower mandible); (k, l) Adenoid cystic carcinoma (palate); (m, n) Basal cell carcinoma (lip); (o, p) Acinic cell carcinoma (parotid gland); (q, r) Mucoepidermoid carcinoma (root of the tongue). (s, t) Normal salivary gland, (u, v) Mucoepidermoid carcinoma (parotid gland) (w, x) and SCC (lip) showed differential expression of GSK3α and GSK3β. The maximum intense immunoreactivity of GSK3β was observed in SCC compared to other types of oral tumors. Original magnification 100X. (B) Overexpression of GSK3β is significantly higher in SCC than in the other types of (non-SCC) oral cancers (p

    Journal: Molecular Cancer

    Article Title: Expression and inactivation of glycogen synthase kinase 3 alpha/ beta and their association with the expression of cyclin D1 and p53 in oral squamous cell carcinoma progression

    doi: 10.1186/s12943-015-0300-x

    Figure Lengend Snippet: The expression of GSK3 α and GSK3β proteins in the tumor/ normal tissues of various anatomical sites of the mouth. (A) Representative immunostaining showing the differential expression of GSK3α and GSK3β from consecutive sections in various types of oral tumor tissue samples as indicated in the figure. (a, b) SCC (cheek); (c, d) Mucoepidermoid carcinoma (palate); (e, f) Adamantinoma (mandible); (g, h) SCC (gingiva); (i, j) SCC (lower mandible); (k, l) Adenoid cystic carcinoma (palate); (m, n) Basal cell carcinoma (lip); (o, p) Acinic cell carcinoma (parotid gland); (q, r) Mucoepidermoid carcinoma (root of the tongue). (s, t) Normal salivary gland, (u, v) Mucoepidermoid carcinoma (parotid gland) (w, x) and SCC (lip) showed differential expression of GSK3α and GSK3β. The maximum intense immunoreactivity of GSK3β was observed in SCC compared to other types of oral tumors. Original magnification 100X. (B) Overexpression of GSK3β is significantly higher in SCC than in the other types of (non-SCC) oral cancers (p

    Article Snippet: Primary antibodies (Santa Cruz Biotechnology) p/GSK3α (dilution 1: 15) and p/GSK3β (dilution 1: 40) were incubated at 4°C overnight.

    Techniques: Expressing, Immunostaining, Over Expression