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  • 86
    Name:
    GSK 3β D5C5Z XP Rabbit mAb
    Description:
    Glycogen synthase kinase 3 GSK 3 was initially identified as an enzyme that regulates glycogen synthesis in response to insulin 1 GSK 3 is a ubiquitously expressed serine threonine protein kinase that phosphorylates and inactivates glycogen synthase GSK 3 is a critical downstream element of the PI3K Akt cell survival pathway whose activity can be inhibited by Akt mediated phosphorylation at Ser21 of GSK 3α and Ser9 of GSK 3β 2 3 GSK 3 has been implicated in the regulation of cell fate in Dictyostelium and is a component of the Wnt signaling pathway required for Drosophila Xenopus and mammalian development 4 GSK 3 has been shown to regulate cyclin D1 proteolysis and subcellular localization 5
    Catalog Number:
    12456
    Price:
    None
    Category:
    Primary Antibodies
    Source:
    Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human GSK-3β protein.
    Reactivity:
    Human Mouse Rat Monkey
    Applications:
    Western Blot, Immunoprecipitation, Immunohistochemistry, Immunofluorescence, Flow Cytometry
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc gsk3β
    Phosphorylation and expression level of proteins of the PI3K-AKT pathway in brain. Representative western blot showing immunoreactivity for phosphorylated and total forms of AKT, p70S6K and <t>GSK3β</t> (above), phosphorylation level (middle), and expression level (below) in control or HU14 groups, in sensorimotor cortex (A), striatum (B), cerebellum (C) and visual cortex (D). *and ** p
    Glycogen synthase kinase 3 GSK 3 was initially identified as an enzyme that regulates glycogen synthesis in response to insulin 1 GSK 3 is a ubiquitously expressed serine threonine protein kinase that phosphorylates and inactivates glycogen synthase GSK 3 is a critical downstream element of the PI3K Akt cell survival pathway whose activity can be inhibited by Akt mediated phosphorylation at Ser21 of GSK 3α and Ser9 of GSK 3β 2 3 GSK 3 has been implicated in the regulation of cell fate in Dictyostelium and is a component of the Wnt signaling pathway required for Drosophila Xenopus and mammalian development 4 GSK 3 has been shown to regulate cyclin D1 proteolysis and subcellular localization 5
    https://www.bioz.com/result/gsk3β/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gsk3β - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "Hypoactivity Affects IGF-1 Level and PI3K/AKT Signaling Pathway in Cerebral Structures Implied in Motor Control"

    Article Title: Hypoactivity Affects IGF-1 Level and PI3K/AKT Signaling Pathway in Cerebral Structures Implied in Motor Control

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107631

    Phosphorylation and expression level of proteins of the PI3K-AKT pathway in brain. Representative western blot showing immunoreactivity for phosphorylated and total forms of AKT, p70S6K and GSK3β (above), phosphorylation level (middle), and expression level (below) in control or HU14 groups, in sensorimotor cortex (A), striatum (B), cerebellum (C) and visual cortex (D). *and ** p
    Figure Legend Snippet: Phosphorylation and expression level of proteins of the PI3K-AKT pathway in brain. Representative western blot showing immunoreactivity for phosphorylated and total forms of AKT, p70S6K and GSK3β (above), phosphorylation level (middle), and expression level (below) in control or HU14 groups, in sensorimotor cortex (A), striatum (B), cerebellum (C) and visual cortex (D). *and ** p

    Techniques Used: Expressing, Western Blot

    2) Product Images from "Palmitate- and C6 ceramide-induced Tnnt3 pre-mRNA alternative splicing occurs in a PP2A dependent manner"

    Article Title: Palmitate- and C6 ceramide-induced Tnnt3 pre-mRNA alternative splicing occurs in a PP2A dependent manner

    Journal: Nutrition & Metabolism

    doi: 10.1186/s12986-018-0326-3

    Palmitate- and C6 ceramide-induced changes in Tnnt3 pre-mRNA alternative splicing are blocked by inhibition of PP2A. a-c L6 myotubes were pretreated for two hours with either 1 or 15 nM okadaic acid (OKA) or an equal volume of MeOH or DMSO for 24-h prior to Western blot analysis. The ratio of ( a ) GSK3β at S9 to total GSK3β, ( b ) ERK1/2 at T202/Y204 to total ERK1/2, and ( c ) phosphorylated Akt at S473 to total Akt was assessed. L6 myotubes were pretreated for two hours with okadaic acid or an equal volume of DMSO (Vehicle) prior to a 24-h treatment with ( d ) 150 μM PA conjugated to BSA (PA) or an equal volume of BSA alone or ( e ) 20 μM C6 ceramide (C6) or an equal volume of methanol (MeOH). The fold change in the relative abundance of the 737 base pair Tnnt3 splice form was assessed by capillary electrophoresis ( d and e ). Data are presented as means ± SEM from three independent experiments using three replicates per treatment. Statistical significance was assessed by Student’s t-test ( a - c ) or Two-way ANOVA with Fishers LSD post-hoc test for multiple comparisons ( d and e ). Statistically different means are denoted with an asterisk (*) or different letters above the bars ( p ≤ 0.05). # p = 0.08 vs. BSA/Vehicle
    Figure Legend Snippet: Palmitate- and C6 ceramide-induced changes in Tnnt3 pre-mRNA alternative splicing are blocked by inhibition of PP2A. a-c L6 myotubes were pretreated for two hours with either 1 or 15 nM okadaic acid (OKA) or an equal volume of MeOH or DMSO for 24-h prior to Western blot analysis. The ratio of ( a ) GSK3β at S9 to total GSK3β, ( b ) ERK1/2 at T202/Y204 to total ERK1/2, and ( c ) phosphorylated Akt at S473 to total Akt was assessed. L6 myotubes were pretreated for two hours with okadaic acid or an equal volume of DMSO (Vehicle) prior to a 24-h treatment with ( d ) 150 μM PA conjugated to BSA (PA) or an equal volume of BSA alone or ( e ) 20 μM C6 ceramide (C6) or an equal volume of methanol (MeOH). The fold change in the relative abundance of the 737 base pair Tnnt3 splice form was assessed by capillary electrophoresis ( d and e ). Data are presented as means ± SEM from three independent experiments using three replicates per treatment. Statistical significance was assessed by Student’s t-test ( a - c ) or Two-way ANOVA with Fishers LSD post-hoc test for multiple comparisons ( d and e ). Statistically different means are denoted with an asterisk (*) or different letters above the bars ( p ≤ 0.05). # p = 0.08 vs. BSA/Vehicle

    Techniques Used: Inhibition, Western Blot, Electrophoresis

    3) Product Images from "Alpha-enolase regulates the malignant phenotype of pulmonary artery smooth muscle cells via the AMPK-Akt pathway"

    Article Title: Alpha-enolase regulates the malignant phenotype of pulmonary artery smooth muscle cells via the AMPK-Akt pathway

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06376-x

    ENO1 activates Akt-GSK3β Pathway via the phosphorylation of AMPKα1. a We treated shENO1 and shCTRL PASMC with hypoxia (1% O 2 ) for 8 h, and the levels of p-AMPKα, AMPKα, p-ACC, and ACC were measured by western blotting. b Normalized quantification of p-AMPKα and AMPKα in shCTRL and shENO1–PASMC under normoxic and hypoxic conditions ( n = 4, * P
    Figure Legend Snippet: ENO1 activates Akt-GSK3β Pathway via the phosphorylation of AMPKα1. a We treated shENO1 and shCTRL PASMC with hypoxia (1% O 2 ) for 8 h, and the levels of p-AMPKα, AMPKα, p-ACC, and ACC were measured by western blotting. b Normalized quantification of p-AMPKα and AMPKα in shCTRL and shENO1–PASMC under normoxic and hypoxic conditions ( n = 4, * P

    Techniques Used: Western Blot

    ENO1 promotes PASMC proliferation via the activation of the Akt-GSK3β pathway. a We treated shENO1 and shCTRL PASMC with hypoxia (1% O 2 ) for 8 h, and measured the levels of p-Akt, pan-Akt, p-PRAS40, PRAS40, p-GSK3β, and GSK3β by western blotting. b Normal PASMCs were treated with 10 μM ENOblock and hypoxia for 8 h, and the levels of p-Akt, pan-Akt, p-PRAS40, PRAS40, p-GSK3β, and GSK3β were measured by western blotting. c PASMCs were transfected with pCMV3-ENO1-GFP, and afterwards we measured the level of p-Akt, pan-Akt, p-PRAS40, PRAS40, p-GSK3β, and GSK3β using western blotting . d PASMCs were transfected with pCMV3-ENO1-GFP and immunostained with antibody against p-GSK3β. The green fluorescence indicates the GFP-tagged ENO1 and the red fluorescence indicates p-GSK3β (Scale bars, 50 μm). e PASMC were transfected with pCMV3-ENO1-GFP and treated with 1 μM GSK690693 or MK2206 for 12 h. The level of p-Akt, pan-Akt, and PCNA were measured by western blotting in the cell lysate. We also measured the f cell proliferation (using BrdU assay) and g cell viability in ENO1-overexpressing PASMC after the treatment of two Akt inhibitors: GSK690693 or MK2206 ( n = 5 per group, * P
    Figure Legend Snippet: ENO1 promotes PASMC proliferation via the activation of the Akt-GSK3β pathway. a We treated shENO1 and shCTRL PASMC with hypoxia (1% O 2 ) for 8 h, and measured the levels of p-Akt, pan-Akt, p-PRAS40, PRAS40, p-GSK3β, and GSK3β by western blotting. b Normal PASMCs were treated with 10 μM ENOblock and hypoxia for 8 h, and the levels of p-Akt, pan-Akt, p-PRAS40, PRAS40, p-GSK3β, and GSK3β were measured by western blotting. c PASMCs were transfected with pCMV3-ENO1-GFP, and afterwards we measured the level of p-Akt, pan-Akt, p-PRAS40, PRAS40, p-GSK3β, and GSK3β using western blotting . d PASMCs were transfected with pCMV3-ENO1-GFP and immunostained with antibody against p-GSK3β. The green fluorescence indicates the GFP-tagged ENO1 and the red fluorescence indicates p-GSK3β (Scale bars, 50 μm). e PASMC were transfected with pCMV3-ENO1-GFP and treated with 1 μM GSK690693 or MK2206 for 12 h. The level of p-Akt, pan-Akt, and PCNA were measured by western blotting in the cell lysate. We also measured the f cell proliferation (using BrdU assay) and g cell viability in ENO1-overexpressing PASMC after the treatment of two Akt inhibitors: GSK690693 or MK2206 ( n = 5 per group, * P

    Techniques Used: Activation Assay, Western Blot, Transfection, Fluorescence, BrdU Staining

    4) Product Images from "Shifting eating to the circadian rest phase misaligns the peripheral clocks with the master SCN clock and leads to a metabolic syndrome"

    Article Title: Shifting eating to the circadian rest phase misaligns the peripheral clocks with the master SCN clock and leads to a metabolic syndrome

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

    doi: 10.1073/pnas.1519807112

    RF-induced increase in GSK3β activity plays a critical role in maintaining the peripheral CCs shift. ( A and B ) RNA transcript levels of genes, as indicated, in liver ( A ) and IECs ( B ) of control, RF15, and reversal of RF (RRF; RRF2 and RRF4) mice. ( C and D ) RNA transcript levels of CC components genes in IECs ( C ) and pancreas ( D ) of control, RF15, RRF2, and RRF4 mice. ( E ) ChIP-qPCR analysis of RevErbα recruitment to RORE DBSs present in genes, as indicated, in liver of RF mice, with or without i.p. administration of the GSK3β inhibitor ARA. ( F ) ChIP-qPCR analysis of Bmal1 recruitment to E-box DBS present in genes, as indicated, in liver of RF mice, with or without i.p. administration of the GSK3β inhibitor ARA. ( G ) ChIP-qPCR analysis of RevErbα recruitment to RORE DBSs present in genes, as indicated, in IECs of RF mice, with or without i.p. administration of the GSK3β inhibitor ARA. ( H ) ChIP-qPCR analysis of Bmal1 recruitment to E-box DBS present in genes, as indicated, in IECs of RF mice, with or without i.p. administration of the GSK3β inhibitor ARA. ( I and J ) RNA transcript levels of CC components in liver ( I ) and IECs ( J ) of RF mice with or without i.p. administration of LiCl. ( K ) Immunoblot analyses, at ZT0, of control and RF30 livers, with indicated antibodies. ( L ) Immunoblot analyses, at ZT0, of RF30 livers treated with or without i.p. administration of glucose, with indicated antibodies. ( M ) RNA transcript levels of CC components in the liver of control, RF30, and RF30+Glucose mice. All values are mean ± SEM.
    Figure Legend Snippet: RF-induced increase in GSK3β activity plays a critical role in maintaining the peripheral CCs shift. ( A and B ) RNA transcript levels of genes, as indicated, in liver ( A ) and IECs ( B ) of control, RF15, and reversal of RF (RRF; RRF2 and RRF4) mice. ( C and D ) RNA transcript levels of CC components genes in IECs ( C ) and pancreas ( D ) of control, RF15, RRF2, and RRF4 mice. ( E ) ChIP-qPCR analysis of RevErbα recruitment to RORE DBSs present in genes, as indicated, in liver of RF mice, with or without i.p. administration of the GSK3β inhibitor ARA. ( F ) ChIP-qPCR analysis of Bmal1 recruitment to E-box DBS present in genes, as indicated, in liver of RF mice, with or without i.p. administration of the GSK3β inhibitor ARA. ( G ) ChIP-qPCR analysis of RevErbα recruitment to RORE DBSs present in genes, as indicated, in IECs of RF mice, with or without i.p. administration of the GSK3β inhibitor ARA. ( H ) ChIP-qPCR analysis of Bmal1 recruitment to E-box DBS present in genes, as indicated, in IECs of RF mice, with or without i.p. administration of the GSK3β inhibitor ARA. ( I and J ) RNA transcript levels of CC components in liver ( I ) and IECs ( J ) of RF mice with or without i.p. administration of LiCl. ( K ) Immunoblot analyses, at ZT0, of control and RF30 livers, with indicated antibodies. ( L ) Immunoblot analyses, at ZT0, of RF30 livers treated with or without i.p. administration of glucose, with indicated antibodies. ( M ) RNA transcript levels of CC components in the liver of control, RF30, and RF30+Glucose mice. All values are mean ± SEM.

    Techniques Used: Activity Assay, Mouse Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Acetylene Reduction Assay

    GSK3β-dependent RevErbα phosphorylation is critical for maintaining the RF-induced CC shift. ( A ) Levels of blood components in control, RF15 mice, and in mice after 2 and 4 d of reversal of RF (RRF2 and RRF4). ( B ) ChIP-qPCR assays in liver to analyze the RevErbα and the Bmal1 recruitment to their respective DBSs in the genes as indicated. ( C ) RNA transcript levels of CC components in liver of control, RF15, RRF2, and RRF4 mice. ( D ) RNA transcript levels of genes, as indicated, in the liver of control, RF15, RRF2, and RRF4 mice. ( E ) Immunoblot analyses, at ZT0, of control, RF15, RRF2, and RRF4 livers with indicated antibodies. ( F ) A schematic representation of how RF-induced PCCs shift is maintained. ( G ) A schematic representation of how restoration of insulin signaling leads to the reversal of RF-induced PCCs shift. ( H ) RNA transcript levels of CC components in liver of control, RF15, and RF15+ARA mice. ( I ) RNA transcript levels of CC components in IEC of control, RF15, and RF15+ARA mice. All values are mean ± SEM. * P
    Figure Legend Snippet: GSK3β-dependent RevErbα phosphorylation is critical for maintaining the RF-induced CC shift. ( A ) Levels of blood components in control, RF15 mice, and in mice after 2 and 4 d of reversal of RF (RRF2 and RRF4). ( B ) ChIP-qPCR assays in liver to analyze the RevErbα and the Bmal1 recruitment to their respective DBSs in the genes as indicated. ( C ) RNA transcript levels of CC components in liver of control, RF15, RRF2, and RRF4 mice. ( D ) RNA transcript levels of genes, as indicated, in the liver of control, RF15, RRF2, and RRF4 mice. ( E ) Immunoblot analyses, at ZT0, of control, RF15, RRF2, and RRF4 livers with indicated antibodies. ( F ) A schematic representation of how RF-induced PCCs shift is maintained. ( G ) A schematic representation of how restoration of insulin signaling leads to the reversal of RF-induced PCCs shift. ( H ) RNA transcript levels of CC components in liver of control, RF15, and RF15+ARA mice. ( I ) RNA transcript levels of CC components in IEC of control, RF15, and RF15+ARA mice. All values are mean ± SEM. * P

    Techniques Used: Mouse Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Acetylene Reduction Assay

    5) Product Images from "Hirsutine induces mPTP-dependent apoptosis through ROCK1/PTEN/PI3K/GSK3β pathway in human lung cancer cells"

    Article Title: Hirsutine induces mPTP-dependent apoptosis through ROCK1/PTEN/PI3K/GSK3β pathway in human lung cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0641-7

    Inhibition of PI3K activity plays an important functional role in hirsutine-induced GSK3β dephosphorylation, mPTP opening, and apoptosis. a A549 cells were treated with various concentrations of hirsutine for 24 h or 80 μM hirsutine for different time intervals; whole-cell lysates were prepared and subjected to western blot analysis. b A549 cells were pretreated with 20 µM LY294002 (a specific PI3K inhibitor) for 2 h, followed by treatment with 60 μM hirsutine for 24 h. Whole-cell lysates were prepared and subjected to western blot analysis. c Cells were treated as indicated in b , the interaction of p-GSK3β, CypD, and ANT1 was determined by immunoprecipitation. d Cells were treated as indicated in b , the calcein fluorescence in the mitochondria was analyzed by microplate reader. e Cells were treated as indicated in b , ATP concentrations were measured by using ATP Determination Kit. f Cells were treated as indicated in b , C-PARP and C-Caspase 3 in whole-cell lysates were determined by immunoblotting. g Cells were treated as indicated in b , the percentage of apoptotic cells was determined by flow cytometry using Annexin V-FITC/PI staining. Data are expressed as the mean ± SD ( n = 3), ** P
    Figure Legend Snippet: Inhibition of PI3K activity plays an important functional role in hirsutine-induced GSK3β dephosphorylation, mPTP opening, and apoptosis. a A549 cells were treated with various concentrations of hirsutine for 24 h or 80 μM hirsutine for different time intervals; whole-cell lysates were prepared and subjected to western blot analysis. b A549 cells were pretreated with 20 µM LY294002 (a specific PI3K inhibitor) for 2 h, followed by treatment with 60 μM hirsutine for 24 h. Whole-cell lysates were prepared and subjected to western blot analysis. c Cells were treated as indicated in b , the interaction of p-GSK3β, CypD, and ANT1 was determined by immunoprecipitation. d Cells were treated as indicated in b , the calcein fluorescence in the mitochondria was analyzed by microplate reader. e Cells were treated as indicated in b , ATP concentrations were measured by using ATP Determination Kit. f Cells were treated as indicated in b , C-PARP and C-Caspase 3 in whole-cell lysates were determined by immunoblotting. g Cells were treated as indicated in b , the percentage of apoptotic cells was determined by flow cytometry using Annexin V-FITC/PI staining. Data are expressed as the mean ± SD ( n = 3), ** P

    Techniques Used: Inhibition, Activity Assay, Functional Assay, De-Phosphorylation Assay, Western Blot, Immunoprecipitation, Fluorescence, Flow Cytometry, Cytometry, Staining

    Model for hirsutine-induced apoptosis in human lung cancer cells. Hirsutine induces ROCK1 cleavage/activation, PTEN activation (phosphorylation), and PI3K/Akt inactivation (dephosphorylation), resulting in GSK3β dephosphorylation, leading to decreases in the binding of GSK3β and ANT1 and increases in the interaction of ANT1 to CypD, culminating in mPTP opening, caspase activation, and apoptosis
    Figure Legend Snippet: Model for hirsutine-induced apoptosis in human lung cancer cells. Hirsutine induces ROCK1 cleavage/activation, PTEN activation (phosphorylation), and PI3K/Akt inactivation (dephosphorylation), resulting in GSK3β dephosphorylation, leading to decreases in the binding of GSK3β and ANT1 and increases in the interaction of ANT1 to CypD, culminating in mPTP opening, caspase activation, and apoptosis

    Techniques Used: Activation Assay, De-Phosphorylation Assay, Binding Assay

    ROCK1/PTEN/PI3K pathway regulates hirsutine-mediated GSK3β dephosphorylation, mPTP opening, and apoptosis. a A549 cells were treated with various concentrations of hirsutine for 24 h or 80 μM hirsutine for different time intervals; whole-cell lysates were prepared and subjected to western blot analysis. CF: cleavage fragment. b , c A549 cells were pretreated with 20 µM Y-27632 (a ROCK1 activation inhibitor) for 2 h, followed by treatment with 80 μM hirsutine for 24 h. Whole-cell lysates were prepared and subjected to western blot analysis. d Cells were treated as indicated in b , the interaction of p-GSK3β, CypD, and ANT1 was determined by immunoprecipitation. e Cells were treated as indicated in b , the calcein fluorescence in the mitochondria was analyzed by microplate reader. f Cells were treated as indicated in b , ATP concentrations were measured by using ATP Determination Kit. g Cells were treated as indicated in b , C-PARP and C-Caspase 3 in whole-cell lysates were determined by immunoblotting. h Cells were treated as indicated in b , the percentage of apoptotic cells was determined by flow cytometry using Annexin V-FITC/PI staining. Data are expressed as the mean ± SD ( n = 3), ** P
    Figure Legend Snippet: ROCK1/PTEN/PI3K pathway regulates hirsutine-mediated GSK3β dephosphorylation, mPTP opening, and apoptosis. a A549 cells were treated with various concentrations of hirsutine for 24 h or 80 μM hirsutine for different time intervals; whole-cell lysates were prepared and subjected to western blot analysis. CF: cleavage fragment. b , c A549 cells were pretreated with 20 µM Y-27632 (a ROCK1 activation inhibitor) for 2 h, followed by treatment with 80 μM hirsutine for 24 h. Whole-cell lysates were prepared and subjected to western blot analysis. d Cells were treated as indicated in b , the interaction of p-GSK3β, CypD, and ANT1 was determined by immunoprecipitation. e Cells were treated as indicated in b , the calcein fluorescence in the mitochondria was analyzed by microplate reader. f Cells were treated as indicated in b , ATP concentrations were measured by using ATP Determination Kit. g Cells were treated as indicated in b , C-PARP and C-Caspase 3 in whole-cell lysates were determined by immunoblotting. h Cells were treated as indicated in b , the percentage of apoptotic cells was determined by flow cytometry using Annexin V-FITC/PI staining. Data are expressed as the mean ± SD ( n = 3), ** P

    Techniques Used: De-Phosphorylation Assay, Western Blot, Activation Assay, Immunoprecipitation, Fluorescence, Flow Cytometry, Cytometry, Staining

    GSK3β inhibitor CHIR potentiates hirsutine-induced mPTP opening and apoptosis. a A549 cells were treated with various concentrations of hirsutine for 24 h or 80 μM hirsutine for different time intervals; the expressions of GSK3β and p-GSK3β were determined by immunoblotting. b A549 cells were pretreated with 5 µM CHIR99021 (CHIR, a GSK3 inhibitor) for 2 h, followed by treatment with 60 μM hirsutine for 24 h. Whole-cell lysates were prepared and subjected to immunoprecipitation to determine the interaction of p-GSK3β, CypD, and ANT1. c After treatment as indicated in b , the cells were stained with p-GSK3β (Alexa Fluor 647, red), ANT1 (Alexa Fluor 488, green), and DAPI; images were captured by confocal microscope. Scale bar represents 20 μm. d Cells were treated as indicated in b , the calcein fluorescence in the mitochondria was analyzed by microplate reader. e Cells were treated as indicated in b , ATP concentrations were measured by using ATP Determination Kit. f Cells were treated as indicated in b , the percentage of apoptotic cells was determined by flow cytometry using Annexin V-FITC/PI staining. Data are expressed as the mean ± SD ( n = 3), ** P
    Figure Legend Snippet: GSK3β inhibitor CHIR potentiates hirsutine-induced mPTP opening and apoptosis. a A549 cells were treated with various concentrations of hirsutine for 24 h or 80 μM hirsutine for different time intervals; the expressions of GSK3β and p-GSK3β were determined by immunoblotting. b A549 cells were pretreated with 5 µM CHIR99021 (CHIR, a GSK3 inhibitor) for 2 h, followed by treatment with 60 μM hirsutine for 24 h. Whole-cell lysates were prepared and subjected to immunoprecipitation to determine the interaction of p-GSK3β, CypD, and ANT1. c After treatment as indicated in b , the cells were stained with p-GSK3β (Alexa Fluor 647, red), ANT1 (Alexa Fluor 488, green), and DAPI; images were captured by confocal microscope. Scale bar represents 20 μm. d Cells were treated as indicated in b , the calcein fluorescence in the mitochondria was analyzed by microplate reader. e Cells were treated as indicated in b , ATP concentrations were measured by using ATP Determination Kit. f Cells were treated as indicated in b , the percentage of apoptotic cells was determined by flow cytometry using Annexin V-FITC/PI staining. Data are expressed as the mean ± SD ( n = 3), ** P

    Techniques Used: Immunoprecipitation, Staining, Microscopy, Fluorescence, Flow Cytometry, Cytometry

    6) Product Images from "An inflammatory-CCRK circuitry drives mTORC1-dependent metabolic and immunosuppressive reprogramming in obesity-associated hepatocellular carcinoma"

    Article Title: An inflammatory-CCRK circuitry drives mTORC1-dependent metabolic and immunosuppressive reprogramming in obesity-associated hepatocellular carcinoma

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07402-8

    CCRK over-expression correlates with mTOR signaling activation in patients with NASH-associated HCCs. a Western blots showed the induced expressions of CCRK/mTOR signaling proteins in HCC tissues relative to matched non-tumor tissues (23 pairs), and representative results from three patients are shown. b The Western blot results of all patients from a were quantified and shown in dot plots, wherein the fold enrichments of target proteins relative to their corresponding controls are shown. c The positive correlations of STAT3/AR/CCRK/GSK3β/mTORC1 signaling components were confirmed in the patient specimens. d Schematic diagram showing the IL-6-triggered self-reinforcing circuitry of STAT3/AR/CCRK (highlighted in the yellow panel), which activates mTORC1 signaling (highlighted in the green panel) through GSK3β/TSC2 to promote NASH and HCC development. Data are presented as mean ± SD. * p
    Figure Legend Snippet: CCRK over-expression correlates with mTOR signaling activation in patients with NASH-associated HCCs. a Western blots showed the induced expressions of CCRK/mTOR signaling proteins in HCC tissues relative to matched non-tumor tissues (23 pairs), and representative results from three patients are shown. b The Western blot results of all patients from a were quantified and shown in dot plots, wherein the fold enrichments of target proteins relative to their corresponding controls are shown. c The positive correlations of STAT3/AR/CCRK/GSK3β/mTORC1 signaling components were confirmed in the patient specimens. d Schematic diagram showing the IL-6-triggered self-reinforcing circuitry of STAT3/AR/CCRK (highlighted in the yellow panel), which activates mTORC1 signaling (highlighted in the green panel) through GSK3β/TSC2 to promote NASH and HCC development. Data are presented as mean ± SD. * p

    Techniques Used: Over Expression, Activation Assay, Western Blot

    CCRK activates mTORC1 signaling through GSK3β/TSC2 cascade. a Ectopic CCRK expression in LO2 and CCRK KO Huh7 cells activates mTORC1 signaling, which was abrogated by suppression of GSK3β phosphorylation at Ser9 (p-GSK3β Ser9 ) via over-expression of the constitutively-active S9A-GSK3β mutant. Western blot analysis was used to detect the protein expression of mTORC1 downstream molecules. b Knockdown of CCRK in HepG2 and Huh7 cells impaired the activation of mTORC1 signaling, which was rescued by the silencing of TSC2. c , d Dietary obesity-induced CCRK expression is responsible for the activation of mTORC1 signaling cascades in both c NASH and d NASH-HCC models, and such CCRK-dependent effects were abolished following knockdown of Ccrk . The establishment of NASH and NASH-HCC models are as described in Figs. 1a and 2a
    Figure Legend Snippet: CCRK activates mTORC1 signaling through GSK3β/TSC2 cascade. a Ectopic CCRK expression in LO2 and CCRK KO Huh7 cells activates mTORC1 signaling, which was abrogated by suppression of GSK3β phosphorylation at Ser9 (p-GSK3β Ser9 ) via over-expression of the constitutively-active S9A-GSK3β mutant. Western blot analysis was used to detect the protein expression of mTORC1 downstream molecules. b Knockdown of CCRK in HepG2 and Huh7 cells impaired the activation of mTORC1 signaling, which was rescued by the silencing of TSC2. c , d Dietary obesity-induced CCRK expression is responsible for the activation of mTORC1 signaling cascades in both c NASH and d NASH-HCC models, and such CCRK-dependent effects were abolished following knockdown of Ccrk . The establishment of NASH and NASH-HCC models are as described in Figs. 1a and 2a

    Techniques Used: Expressing, Over Expression, Mutagenesis, Western Blot, Activation Assay

    7) Product Images from "Phosphorylation and Inactivation of Glycogen Synthase Kinase 3β (GSK3β) by Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) *"

    Article Title: Phosphorylation and Inactivation of Glycogen Synthase Kinase 3β (GSK3β) by Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.594952

    Dyrk1A interacts with GSK3β. A and B , co-IP assays in HEK293T cells. HEK293T cell lysates that were transfected with plasmids encoding GSK3β and Dyrk1A were immunoprecipitated with control IgG, anti-GSK3β ( A ), or anti-Dyrk1A antibodies ( B ) and then subjected to immunoblot analysis with the indicated antibodies. C , co-IP in brain lysates. Mouse brain lysates were immunoprecipitated with control IgG or anti-Dyrk1A antibodies and then subjected to immunoblot analysis with the indicated antibodies. D , GST pulldown assay to determine the direct interaction between GSK3β and Dyrk1A. Purified GST or GST-GSK3β fusion protein that was immobilized on beads was incubated with recombinant Dyrk1A protein and subjected to immunoblot analyses.
    Figure Legend Snippet: Dyrk1A interacts with GSK3β. A and B , co-IP assays in HEK293T cells. HEK293T cell lysates that were transfected with plasmids encoding GSK3β and Dyrk1A were immunoprecipitated with control IgG, anti-GSK3β ( A ), or anti-Dyrk1A antibodies ( B ) and then subjected to immunoblot analysis with the indicated antibodies. C , co-IP in brain lysates. Mouse brain lysates were immunoprecipitated with control IgG or anti-Dyrk1A antibodies and then subjected to immunoblot analysis with the indicated antibodies. D , GST pulldown assay to determine the direct interaction between GSK3β and Dyrk1A. Purified GST or GST-GSK3β fusion protein that was immobilized on beads was incubated with recombinant Dyrk1A protein and subjected to immunoblot analyses.

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, GST Pulldown Assay, Purification, Incubation, Recombinant

    Dyrk1A-mediated phosphorylation of GSK3β at Thr 356 inhibits its activity in cells. A , GSK3β, immunoprecipitated using an anti-Myc antibody from lysates of HEK293T cells that were transfected with plasmids that encode Myc-tagged GSK3β WT or mutants were subjected to kinase assays. The immunoblot shown below depicts immunoprecipitated GSK3β WT and mutants. **, p
    Figure Legend Snippet: Dyrk1A-mediated phosphorylation of GSK3β at Thr 356 inhibits its activity in cells. A , GSK3β, immunoprecipitated using an anti-Myc antibody from lysates of HEK293T cells that were transfected with plasmids that encode Myc-tagged GSK3β WT or mutants were subjected to kinase assays. The immunoblot shown below depicts immunoprecipitated GSK3β WT and mutants. **, p

    Techniques Used: Activity Assay, Immunoprecipitation, Transfection

    GSK3β is phosphorylated by Dyrk1A at the Thr 356 residue in vitro . A , autoradiograph of SDS-polyacrylamide gels that contained the products of in vitro kinase assays with an inactive form of GSK3β (GSK3β K85R) substrate and Dyrk1A WT or the Y321F kinase-inactive mutant. B , top row , autoradiograph of SDS-polyacrylamide gels that contained the products of the in vitro kinase assays that were performed using GSK3β K85R substrates (WT and mutants) and Dyrk1A. Bottom row , Coomassie staining of SDS-polyacrylamide gels that contained purified recombinant WT and mutant GSK3β proteins. C , left panel , autoradiography of SDS-polyacrylamide gels that contained the products of in vitro kinase assays that used GSK3β deletion mutant proteins and Dyrk1A. Right panel , Coomassie staining of SDS-polyacrylamide gels that contained purified GSK3β deletion mutant proteins. D , autoradiography ( top panel ) of SDS-polyacrylamide gels that contained the products of in vitro kinase assays that employed purified GSK3β (343–420) or GSK3β (343–420) (T356A) mutants in the presence or absence of Dyrk1A. Bottom panel , Coomassie staining of SDS-polyacrylamide gels that contained purified GSK3β mutant proteins.
    Figure Legend Snippet: GSK3β is phosphorylated by Dyrk1A at the Thr 356 residue in vitro . A , autoradiograph of SDS-polyacrylamide gels that contained the products of in vitro kinase assays with an inactive form of GSK3β (GSK3β K85R) substrate and Dyrk1A WT or the Y321F kinase-inactive mutant. B , top row , autoradiograph of SDS-polyacrylamide gels that contained the products of the in vitro kinase assays that were performed using GSK3β K85R substrates (WT and mutants) and Dyrk1A. Bottom row , Coomassie staining of SDS-polyacrylamide gels that contained purified recombinant WT and mutant GSK3β proteins. C , left panel , autoradiography of SDS-polyacrylamide gels that contained the products of in vitro kinase assays that used GSK3β deletion mutant proteins and Dyrk1A. Right panel , Coomassie staining of SDS-polyacrylamide gels that contained purified GSK3β deletion mutant proteins. D , autoradiography ( top panel ) of SDS-polyacrylamide gels that contained the products of in vitro kinase assays that employed purified GSK3β (343–420) or GSK3β (343–420) (T356A) mutants in the presence or absence of Dyrk1A. Bottom panel , Coomassie staining of SDS-polyacrylamide gels that contained purified GSK3β mutant proteins.

    Techniques Used: In Vitro, Autoradiography, Mutagenesis, Staining, Purification, Recombinant

    Thr(P) 356 -GSK3β is increased in the WAT of Dyrk1A TG mice that were fed a normal diet. Shown are representative immunoblots ( A ) and densitometric analysis ( B ) of WAT lysates of Dyrk1A TG mice and control ( Con ) mice fed a normal chow ( n = 4∼6). WAT lysates of mice at 5–9 months of age were used for analysis. The phospho-GSK3β signals in the immunoblots were normalized by GAPDH and GSK3β signals. The GSK3β, GS, and β-catenin signals were normalized by GAPDH. *, p
    Figure Legend Snippet: Thr(P) 356 -GSK3β is increased in the WAT of Dyrk1A TG mice that were fed a normal diet. Shown are representative immunoblots ( A ) and densitometric analysis ( B ) of WAT lysates of Dyrk1A TG mice and control ( Con ) mice fed a normal chow ( n = 4∼6). WAT lysates of mice at 5–9 months of age were used for analysis. The phospho-GSK3β signals in the immunoblots were normalized by GAPDH and GSK3β signals. The GSK3β, GS, and β-catenin signals were normalized by GAPDH. *, p

    Techniques Used: Mouse Assay, Western Blot

    Thr(P) 356 -GSK3β is increased in the WAT of Dyrk1A TG mice that were fed a HFD. A and B , representative immunoblots ( A ) and densitometric analysis ( B ) of WAT lysates of 5–9 month-old Dyrk1A TG and control ( Con ) mice fed a HFD ( n = 6∼10). The amounts of phospho-GSK3β, GS, and β-catenin of Dyrk1A TG are plotted as a percentage compared with control mice. *, p
    Figure Legend Snippet: Thr(P) 356 -GSK3β is increased in the WAT of Dyrk1A TG mice that were fed a HFD. A and B , representative immunoblots ( A ) and densitometric analysis ( B ) of WAT lysates of 5–9 month-old Dyrk1A TG and control ( Con ) mice fed a HFD ( n = 6∼10). The amounts of phospho-GSK3β, GS, and β-catenin of Dyrk1A TG are plotted as a percentage compared with control mice. *, p

    Techniques Used: Mouse Assay, Western Blot

    Specificity of Thr(P) 356 -GSK3β antibody and the expression of Thr(P) 356 -GSK3β in WAT and 3T3-L1 cells. A , purified GSK3β proteins were subjected to kinase assays in the presence or absence of Dyrk1A (WT or inactive Y321F mutants). The reaction mixtures were subjected to SDS-PAGE and immunoblotting with phospho-GSK3β antibody. B , HEK293T cells that were transfected with GSK3β WT or GSK3β T356A expression plasmids in the presence or absence of plasmids encoding Dyrk1A were analyzed by immunoblots with the indicated antibodies. C , the lysates of GSK3β WT or GSK3β −/− mouse embryonic fibroblast cells were analyzed by immunoblot with the indicated antibodies. D , the lysates of WAT were treated with (+) or without (−) λ-protein phosphatase and subsequently analyzed by immunoblot with the Thr(P) 356 -GSK3β or GSK3β antibodies. E. Peptide competition assay for the pT356-GSK3β antibody. Mouse WAT lysates were analyzed by immunoblot with the Thr(P) 356 -GSK3β antibody that was preincubated in the absence ( N ) or presence of GSK3β-non-phosphopeptide ( NP ) or GSK3β-phosphopeptide ( P ). F , 3T3-L1 cells transfected with Dyrk1A-specific or control siRNA were analyzed by immunoblot with the indicated antibodies. G , 2-day post-confluent 3T3-L1 preadipocytes ( day 0 ) were induced to differentiate as described under “Experimental Procedures.” Differentiated 3T3-L1 cell lysates were analyzed by immunoblot with the indicated antibodies.
    Figure Legend Snippet: Specificity of Thr(P) 356 -GSK3β antibody and the expression of Thr(P) 356 -GSK3β in WAT and 3T3-L1 cells. A , purified GSK3β proteins were subjected to kinase assays in the presence or absence of Dyrk1A (WT or inactive Y321F mutants). The reaction mixtures were subjected to SDS-PAGE and immunoblotting with phospho-GSK3β antibody. B , HEK293T cells that were transfected with GSK3β WT or GSK3β T356A expression plasmids in the presence or absence of plasmids encoding Dyrk1A were analyzed by immunoblots with the indicated antibodies. C , the lysates of GSK3β WT or GSK3β −/− mouse embryonic fibroblast cells were analyzed by immunoblot with the indicated antibodies. D , the lysates of WAT were treated with (+) or without (−) λ-protein phosphatase and subsequently analyzed by immunoblot with the Thr(P) 356 -GSK3β or GSK3β antibodies. E. Peptide competition assay for the pT356-GSK3β antibody. Mouse WAT lysates were analyzed by immunoblot with the Thr(P) 356 -GSK3β antibody that was preincubated in the absence ( N ) or presence of GSK3β-non-phosphopeptide ( NP ) or GSK3β-phosphopeptide ( P ). F , 3T3-L1 cells transfected with Dyrk1A-specific or control siRNA were analyzed by immunoblot with the indicated antibodies. G , 2-day post-confluent 3T3-L1 preadipocytes ( day 0 ) were induced to differentiate as described under “Experimental Procedures.” Differentiated 3T3-L1 cell lysates were analyzed by immunoblot with the indicated antibodies.

    Techniques Used: Expressing, Purification, SDS Page, Transfection, Western Blot, Competitive Binding Assay

    Dyrk1A-mediated phosphorylation of GSK3β at Thr 356 inhibits its activity in vitro . A , purified GST-GSK3β protein that was immobilized on glutathione-Sepharose beads was incubated with Dyrk1A in the presence or absence of ATP. The bead-bound GST fusion proteins were washed and subjected to kinase assays with a GSK3 substrate peptide, GSM. ***, p
    Figure Legend Snippet: Dyrk1A-mediated phosphorylation of GSK3β at Thr 356 inhibits its activity in vitro . A , purified GST-GSK3β protein that was immobilized on glutathione-Sepharose beads was incubated with Dyrk1A in the presence or absence of ATP. The bead-bound GST fusion proteins were washed and subjected to kinase assays with a GSK3 substrate peptide, GSM. ***, p

    Techniques Used: Activity Assay, In Vitro, Purification, Incubation

    8) Product Images from "GSK3? inhibition blocks melanoma cell/host interactions by downregulating N-cadherin expression and decreasing FAK phosphorylation"

    Article Title: GSK3? inhibition blocks melanoma cell/host interactions by downregulating N-cadherin expression and decreasing FAK phosphorylation

    Journal: The Journal of investigative dermatology

    doi: 10.1038/jid.2012.237

    GSK3β regulates focal adhesions in melanoma cells Inhibition of GSK3β and siRNA knockdown of GSK3β increases the size of focal adhesions in WM793 and 1205Lu melanoma cells. Doxycycline-inducible EGFP-FAK expressing WM793 and parental 1205Lu cells were treated with vehicle (control), scrambled siRNA control (Scr), NP309 (0.3 μM) or an siRNA against GSK3β. WM793 were imaged directly, and 1205Lu cells were fixed and stained for FAK expression. Scale bar: 25 μm.
    Figure Legend Snippet: GSK3β regulates focal adhesions in melanoma cells Inhibition of GSK3β and siRNA knockdown of GSK3β increases the size of focal adhesions in WM793 and 1205Lu melanoma cells. Doxycycline-inducible EGFP-FAK expressing WM793 and parental 1205Lu cells were treated with vehicle (control), scrambled siRNA control (Scr), NP309 (0.3 μM) or an siRNA against GSK3β. WM793 were imaged directly, and 1205Lu cells were fixed and stained for FAK expression. Scale bar: 25 μm.

    Techniques Used: Inhibition, Expressing, Staining

    GSK3β inhibition prevents the migration and invasion of melanoma cell lines A: NP309 (0.3 μM) and LiCl (50 mM) prevents the movement of melanoma cells into a scratch wound. B: siRNA knockdown of GSK3β prevents the movement of 1205Lu melanoma cells into the scratch. Western blot shows knockdown of GSK3β (Mock, no siRNA; NT: scrambled control and GSK3β siRNA). C: NP309 prevents the invasion of melanoma cells in a modified Boyden Chamber model. D: NP309 (0.3 and 1 μM, 48 hr) prevents the invasion melanoma cells in a 3D collagen implanted spheroid model. Scale bar: 100μm. Images were analyzed using ImageJ. Statistically significant differences from controls are indicated where *P
    Figure Legend Snippet: GSK3β inhibition prevents the migration and invasion of melanoma cell lines A: NP309 (0.3 μM) and LiCl (50 mM) prevents the movement of melanoma cells into a scratch wound. B: siRNA knockdown of GSK3β prevents the movement of 1205Lu melanoma cells into the scratch. Western blot shows knockdown of GSK3β (Mock, no siRNA; NT: scrambled control and GSK3β siRNA). C: NP309 prevents the invasion of melanoma cells in a modified Boyden Chamber model. D: NP309 (0.3 and 1 μM, 48 hr) prevents the invasion melanoma cells in a 3D collagen implanted spheroid model. Scale bar: 100μm. Images were analyzed using ImageJ. Statistically significant differences from controls are indicated where *P

    Techniques Used: Inhibition, Migration, Western Blot, Modification

    Inhibition of GSK3β leads to a reduction in N-cadherin expression A: Western blot showing NP309 increases β-catenin expression and decreases N-cadherin expression in melanoma cells. B: Immunofluorescence pictures demonstrating the ability of NP309 to increase membrane and nuclear β-catenin expression in WM793 cells. Scale bar: 50μm. C: siRNA knockdown of GSK3β reduces N-cadherin expression in WM793 cells. D: NP309 (0.3 μM, 24 hrs) decreases N-cadherin expression at the mRNA level. E: NP309 decreases expression of Slug. F: (top panel) Western blot showing NP309 (0–0.1μM, 24 hrs) decreases expression of fibronectin (bottom panel) Immunofluorescence staining showing decreased fibronectin expression following NP309 (0.3 μM, 24 hrs) treatment. G : NP309 (0.3 μM, 24 hrs) decreases fibronectin expression at the mRNA level.
    Figure Legend Snippet: Inhibition of GSK3β leads to a reduction in N-cadherin expression A: Western blot showing NP309 increases β-catenin expression and decreases N-cadherin expression in melanoma cells. B: Immunofluorescence pictures demonstrating the ability of NP309 to increase membrane and nuclear β-catenin expression in WM793 cells. Scale bar: 50μm. C: siRNA knockdown of GSK3β reduces N-cadherin expression in WM793 cells. D: NP309 (0.3 μM, 24 hrs) decreases N-cadherin expression at the mRNA level. E: NP309 decreases expression of Slug. F: (top panel) Western blot showing NP309 (0–0.1μM, 24 hrs) decreases expression of fibronectin (bottom panel) Immunofluorescence staining showing decreased fibronectin expression following NP309 (0.3 μM, 24 hrs) treatment. G : NP309 (0.3 μM, 24 hrs) decreases fibronectin expression at the mRNA level.

    Techniques Used: Inhibition, Expressing, Western Blot, Immunofluorescence, Staining

    GSK3β inhibition prevents the interaction of melanoma cells with fibroblasts and endothelial cells A: 24 hr NP309 (0.3 μM) and LiCl (50 mM) pre-treatment reduced the adhesion of melanoma cells to a fibroblast monolayer. B: Overexpression of N-cadherin reverses the effects of NP309 upon the adhesion of 1205Lu melanoma cells to a fibroblast monolayer C: NP309 prevents the transendothelial migration of melanoma cells. Scale bar: 100μm. Data shows quantification of cells migrating through the HUVEC layer. D: NP309, LiCl and siRNA knockdown of GSK3β prevents the transendothelial cell migration of 1205Lu melanoma cells. E: Overexpression of N-cadherin reverses the anti-transendothelial cell migratory effects of NP309 on 1205Lu cells. Statistically significant differences from controls are indicated where *P
    Figure Legend Snippet: GSK3β inhibition prevents the interaction of melanoma cells with fibroblasts and endothelial cells A: 24 hr NP309 (0.3 μM) and LiCl (50 mM) pre-treatment reduced the adhesion of melanoma cells to a fibroblast monolayer. B: Overexpression of N-cadherin reverses the effects of NP309 upon the adhesion of 1205Lu melanoma cells to a fibroblast monolayer C: NP309 prevents the transendothelial migration of melanoma cells. Scale bar: 100μm. Data shows quantification of cells migrating through the HUVEC layer. D: NP309, LiCl and siRNA knockdown of GSK3β prevents the transendothelial cell migration of 1205Lu melanoma cells. E: Overexpression of N-cadherin reverses the anti-transendothelial cell migratory effects of NP309 on 1205Lu cells. Statistically significant differences from controls are indicated where *P

    Techniques Used: Inhibition, Over Expression, Migration

    GSK3β is focally expressed in melanoma specimens A: Representative immunohistochemical staining of an invasive primary melanoma and a melanoma brain metastases for expression of total GSK3β and phospho-GSK3β. Scale bar: 250 μm. Inset: arrows indicate focal expression of GSK3β. Scale bar: 100μm. B: Number of primary and metastatic melanoma specimens with high levels (+2/3) of focal staining for GSK3β. C : High power images of two melanoma metastases, showing increased levels of total GSK3β staining at the invasive front.
    Figure Legend Snippet: GSK3β is focally expressed in melanoma specimens A: Representative immunohistochemical staining of an invasive primary melanoma and a melanoma brain metastases for expression of total GSK3β and phospho-GSK3β. Scale bar: 250 μm. Inset: arrows indicate focal expression of GSK3β. Scale bar: 100μm. B: Number of primary and metastatic melanoma specimens with high levels (+2/3) of focal staining for GSK3β. C : High power images of two melanoma metastases, showing increased levels of total GSK3β staining at the invasive front.

    Techniques Used: Immunohistochemistry, Staining, Expressing

    9) Product Images from "Neuroprotective Effects of the Multitarget Agent AVCRI104P3 in Brain of Middle-Aged Mice"

    Article Title: Neuroprotective Effects of the Multitarget Agent AVCRI104P3 in Brain of Middle-Aged Mice

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092615

    Effect of AVCRI104P3 on pGSK3β (Ser9) and GSK3β in the hippocampus and cortex of 12-month-old 129/Sv × C57BL/6 male mice. ( A ) Representative Western blot images of pGSK3β (Ser9) and GSK3β. β-actin were used as internal control. ( B ) Photodensitometric quantification of WB experiments was used to evaluate changes in pGSK3β and GSK3β expression. The results are the mean ± SEM of 3–4 experiments (5 mice/treatment group). The statistical analysis used was one-way ANOVA followed by Dunnett’s test, * p
    Figure Legend Snippet: Effect of AVCRI104P3 on pGSK3β (Ser9) and GSK3β in the hippocampus and cortex of 12-month-old 129/Sv × C57BL/6 male mice. ( A ) Representative Western blot images of pGSK3β (Ser9) and GSK3β. β-actin were used as internal control. ( B ) Photodensitometric quantification of WB experiments was used to evaluate changes in pGSK3β and GSK3β expression. The results are the mean ± SEM of 3–4 experiments (5 mice/treatment group). The statistical analysis used was one-way ANOVA followed by Dunnett’s test, * p

    Techniques Used: Mouse Assay, Western Blot, Expressing

    10) Product Images from "PERK inhibition prevents tau-mediated neurodegeneration in a mouse model of frontotemporal dementia"

    Article Title: PERK inhibition prevents tau-mediated neurodegeneration in a mouse model of frontotemporal dementia

    Journal: Acta Neuropathologica

    doi: 10.1007/s00401-015-1487-z

    PERK inhibitor treatment decreases tau phosphorylation and prevents neurodegeneration and clinical disease in mutant tau-expressing rTg4510 mice. a GSK2606414 treatment prevented clinical signs in 8-month-old tau P301L + mice, which showed normal grooming, posture and movement compared to vehicle-treated animals and were indistinguishable from transgene-negative animals of the same age (representative images, i–iii). Histologically, PERK inhibitor resulted in marked neuroprotection with preservation of hippocampal volume and CA1-3 neuronal ribbon (iv–vi, hematoxylin and eosin-stained sections), and immunostaining using AT8 showed a significant reduction in pSer 202 /Thr 205 -tau staining in the hippocampus after GSK2606414 treatment (vii–ix, representative images of hippocampal sections, scale bar 50 μm). b Average count of the number of CA1 pyramidal neurons in five consecutive slices from tau P301L + treated with vehicle ( grey bars ) or GSK2606414 ( blue bars ) mice relative to control mice (tau P301L − , white bars ) shows reduced loss of pyramidal neurons in PERK inhibitor-treated mice (i.e. prevention of neurodegeneration) to numbers similar to 6-month-old mutant tau-expressing mice ( black bar ) ( n = 3–4 mice) ( c ) GSK2606414 partially prevented brain atrophy in comparison to vehicle-treated mice ( n = 3–8 mice). d Total tau levels were not significantly different in PERK inhibitor or vehicle-treated animals ( n = 3 mice). e AlphaScreen analysis with AT8 and PHF-1 shows a reduction in soluble phospho-tau at pSer 202 /Thr 205 and pSer 396/404 epitopes after PERK inhibitor treatment compared to vehicle-treated animals ( n = 4 mice). f Levels of the active form, pTyr 216 -GSK3β, and total GSK3β levels increased compared to control mice. PERK inhibitor treatment significantly reduced pTyr 216 -GSK3β and total GSK3β levels in tau P301L + mice to levels seen in control mice. Representative immunoblots of hippocampal lysates and bar charts quantitating protein levels (in three independent samples). All bar charts show mean ± SEM, * p
    Figure Legend Snippet: PERK inhibitor treatment decreases tau phosphorylation and prevents neurodegeneration and clinical disease in mutant tau-expressing rTg4510 mice. a GSK2606414 treatment prevented clinical signs in 8-month-old tau P301L + mice, which showed normal grooming, posture and movement compared to vehicle-treated animals and were indistinguishable from transgene-negative animals of the same age (representative images, i–iii). Histologically, PERK inhibitor resulted in marked neuroprotection with preservation of hippocampal volume and CA1-3 neuronal ribbon (iv–vi, hematoxylin and eosin-stained sections), and immunostaining using AT8 showed a significant reduction in pSer 202 /Thr 205 -tau staining in the hippocampus after GSK2606414 treatment (vii–ix, representative images of hippocampal sections, scale bar 50 μm). b Average count of the number of CA1 pyramidal neurons in five consecutive slices from tau P301L + treated with vehicle ( grey bars ) or GSK2606414 ( blue bars ) mice relative to control mice (tau P301L − , white bars ) shows reduced loss of pyramidal neurons in PERK inhibitor-treated mice (i.e. prevention of neurodegeneration) to numbers similar to 6-month-old mutant tau-expressing mice ( black bar ) ( n = 3–4 mice) ( c ) GSK2606414 partially prevented brain atrophy in comparison to vehicle-treated mice ( n = 3–8 mice). d Total tau levels were not significantly different in PERK inhibitor or vehicle-treated animals ( n = 3 mice). e AlphaScreen analysis with AT8 and PHF-1 shows a reduction in soluble phospho-tau at pSer 202 /Thr 205 and pSer 396/404 epitopes after PERK inhibitor treatment compared to vehicle-treated animals ( n = 4 mice). f Levels of the active form, pTyr 216 -GSK3β, and total GSK3β levels increased compared to control mice. PERK inhibitor treatment significantly reduced pTyr 216 -GSK3β and total GSK3β levels in tau P301L + mice to levels seen in control mice. Representative immunoblots of hippocampal lysates and bar charts quantitating protein levels (in three independent samples). All bar charts show mean ± SEM, * p

    Techniques Used: Mutagenesis, Expressing, Mouse Assay, Preserving, Staining, Immunostaining, Amplified Luminescent Proximity Homogenous Assay, Western Blot

    11) Product Images from "GSK3? is a negative regulator of platelet function and thrombosis"

    Article Title: GSK3? is a negative regulator of platelet function and thrombosis

    Journal: Blood

    doi: 10.1182/blood-2007-09-111518

    MAP-tau thr212 is dephosphorylated after agonist treatment in GSK3β +/− platelets and in human platelets treated with GSK3 inhibitors . (A) Washed human platelets (4 × 10 7 ) were incubated for the indicated times with no agonist (Untx)
    Figure Legend Snippet: MAP-tau thr212 is dephosphorylated after agonist treatment in GSK3β +/− platelets and in human platelets treated with GSK3 inhibitors . (A) Washed human platelets (4 × 10 7 ) were incubated for the indicated times with no agonist (Untx)

    Techniques Used: Incubation

    Platelets from GSK3β +/− mice are hypersensitive to agonist-stimulated aggregation, secretion of dense granule contents, and fibrinogen binding . (A) PRP from WT and GSK3β +/− mice (2 × 10 8 /mL) were stimulated with
    Figure Legend Snippet: Platelets from GSK3β +/− mice are hypersensitive to agonist-stimulated aggregation, secretion of dense granule contents, and fibrinogen binding . (A) PRP from WT and GSK3β +/− mice (2 × 10 8 /mL) were stimulated with

    Techniques Used: Mouse Assay, Binding Assay

    GSK3β antagonists enhance aggregation to PAR1 agonists . (A) Washed human platelets (2.5 × 10 8 /mL) were incubated for 2 hours with LiCl (20 mM), SB216763 (10 μM), or Tyrode buffer, then stimulated with human TRAP (PAR1 agonist)
    Figure Legend Snippet: GSK3β antagonists enhance aggregation to PAR1 agonists . (A) Washed human platelets (2.5 × 10 8 /mL) were incubated for 2 hours with LiCl (20 mM), SB216763 (10 μM), or Tyrode buffer, then stimulated with human TRAP (PAR1 agonist)

    Techniques Used: Incubation

    Disseminated thrombosis in WT versus GSK3β +/− mice . A combination of collagen (170 μg/kg) and epinephrine (350 μM/kg) was injected into the tail vein of sedated mice (7 WT, 6 GSK3β +/− ), and the time until
    Figure Legend Snippet: Disseminated thrombosis in WT versus GSK3β +/− mice . A combination of collagen (170 μg/kg) and epinephrine (350 μM/kg) was injected into the tail vein of sedated mice (7 WT, 6 GSK3β +/− ), and the time until

    Techniques Used: Mouse Assay, Injection

    GSK3β is present and phosphorylated after agonist treatment in platelets . (A) Lysates of washed human or WT mouse platelets (10 8 ) and mouse liver (20 μg/mL) were immunoblotted with an antibody to GSK3β. The second immunoblot shows
    Figure Legend Snippet: GSK3β is present and phosphorylated after agonist treatment in platelets . (A) Lysates of washed human or WT mouse platelets (10 8 ) and mouse liver (20 μg/mL) were immunoblotted with an antibody to GSK3β. The second immunoblot shows

    Techniques Used:

    12) Product Images from "UBE2T promotes nasopharyngeal carcinoma cell proliferation, invasion, and metastasis by activating the AKT/GSK3β/β-catenin pathway"

    Article Title: UBE2T promotes nasopharyngeal carcinoma cell proliferation, invasion, and metastasis by activating the AKT/GSK3β/β-catenin pathway

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7805

    UBE2T promotes NPC cell proliferation and metastasis probably by activating the AKT/GSK3β/β-catenin pathway A. Western blot detected the effects of UBE2T on β-catenin, its downstream proliferation/metastasis-related target proteins (Cyclin D1, C-MYC, C-JUN, MMP2, and MMP9), and its upstream pathway proteins (p-AKT, p-GSK3β). B. Immunofluorescence determined the effects of UBE2T overexpression on nuclear translocation of β-catenin. Scales indicate 40 μm. (up; ×1000 field), and separate nuclear and cytoplasmic protein western blot verified the effects of UBE2T on nuclear translocation of β-catenin (down). C. and D. Transwell and matrix-coated transwell analysis detected the effects of AKT inhibitor (MK-2206 2HCl) on the pro-migration and invasion abilities. Representative images of the transwell (C) and matrix-coated transwell (D) assay from indicated groups at 6h (migration) and 24h (invasion). The bar chart represents mean ±SEM number of migration and invasive cells from 5 random 20X objective fields (analysis of variance [ANOVA] of factorial design, *** P
    Figure Legend Snippet: UBE2T promotes NPC cell proliferation and metastasis probably by activating the AKT/GSK3β/β-catenin pathway A. Western blot detected the effects of UBE2T on β-catenin, its downstream proliferation/metastasis-related target proteins (Cyclin D1, C-MYC, C-JUN, MMP2, and MMP9), and its upstream pathway proteins (p-AKT, p-GSK3β). B. Immunofluorescence determined the effects of UBE2T overexpression on nuclear translocation of β-catenin. Scales indicate 40 μm. (up; ×1000 field), and separate nuclear and cytoplasmic protein western blot verified the effects of UBE2T on nuclear translocation of β-catenin (down). C. and D. Transwell and matrix-coated transwell analysis detected the effects of AKT inhibitor (MK-2206 2HCl) on the pro-migration and invasion abilities. Representative images of the transwell (C) and matrix-coated transwell (D) assay from indicated groups at 6h (migration) and 24h (invasion). The bar chart represents mean ±SEM number of migration and invasive cells from 5 random 20X objective fields (analysis of variance [ANOVA] of factorial design, *** P

    Techniques Used: Western Blot, Immunofluorescence, Over Expression, Translocation Assay, Migration

    13) Product Images from "Cytosolic THUMPD1 promotes breast cancer cells invasion and metastasis via the AKT-GSK3-Snail pathway"

    Article Title: Cytosolic THUMPD1 promotes breast cancer cells invasion and metastasis via the AKT-GSK3-Snail pathway

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14528

    THUMPD1 promoted cancer cell invasion and migration via the AKT-GSK3β-Snail pathway THUMPD1 overexpression in MCF-7 and MDA-MB-468 cells upregulated Snail and downregulated E-cadherin, whereas THUMPD1 knockdown downregulated Snail and upregulated E-cadherin ( A ) p-AKT and its downstream target, p-GSK3β, were increased in THUMPD1-overexpressing MCF-7 and MDA-MD-468 cells, but decreased in THUMPD1-deficient MCF-7 and BT-549 cells ( B ) AKT inhibitor, LY294002 (10 μM), reversed the effects of THUMPD1 on p-AKT and p-GSK3β expression, decreasing Snail and increasing E-cadherin ( C ) and reducing THUMPD1-overexpressing MCF-7 cell invasion ( D ).
    Figure Legend Snippet: THUMPD1 promoted cancer cell invasion and migration via the AKT-GSK3β-Snail pathway THUMPD1 overexpression in MCF-7 and MDA-MB-468 cells upregulated Snail and downregulated E-cadherin, whereas THUMPD1 knockdown downregulated Snail and upregulated E-cadherin ( A ) p-AKT and its downstream target, p-GSK3β, were increased in THUMPD1-overexpressing MCF-7 and MDA-MD-468 cells, but decreased in THUMPD1-deficient MCF-7 and BT-549 cells ( B ) AKT inhibitor, LY294002 (10 μM), reversed the effects of THUMPD1 on p-AKT and p-GSK3β expression, decreasing Snail and increasing E-cadherin ( C ) and reducing THUMPD1-overexpressing MCF-7 cell invasion ( D ).

    Techniques Used: Migration, Over Expression, Multiple Displacement Amplification, Expressing

    14) Product Images from "Chlorogenic Acid Targeting of the AKT PH Domain Activates AKT/GSK3β/FOXO1 Signaling and Improves Glucose Metabolism"

    Article Title: Chlorogenic Acid Targeting of the AKT PH Domain Activates AKT/GSK3β/FOXO1 Signaling and Improves Glucose Metabolism

    Journal: Nutrients

    doi: 10.3390/nu10101366

    The effects of CGA on AKT phosphorylation and its downstream factors. ( A ) The time-dependent effect of CGA on the phosphorylation of AKT (T308 and S473). ( B ) The time-dependent effect of CGA on the phosphorylation of forkhead box O1 (FOXO1) and glycogen synthase kinase 3β (GSK3β). The histograms present the relative intensities of the detected protein bands. ( C ) The effect of CGA on the enzymatic activity of GSK3β. SC79 was used as the positive control. The histogram presents the activity of GSK3β at 600 s after the respective treatments. Each bar represents the mean ± SEM. * p
    Figure Legend Snippet: The effects of CGA on AKT phosphorylation and its downstream factors. ( A ) The time-dependent effect of CGA on the phosphorylation of AKT (T308 and S473). ( B ) The time-dependent effect of CGA on the phosphorylation of forkhead box O1 (FOXO1) and glycogen synthase kinase 3β (GSK3β). The histograms present the relative intensities of the detected protein bands. ( C ) The effect of CGA on the enzymatic activity of GSK3β. SC79 was used as the positive control. The histogram presents the activity of GSK3β at 600 s after the respective treatments. Each bar represents the mean ± SEM. * p

    Techniques Used: Activity Assay, Positive Control

    15) Product Images from "Sirt1 Inhibits Akt2-Mediated Porcine Adipogenesis Potentially by Direct Protein-Protein Interaction"

    Article Title: Sirt1 Inhibits Akt2-Mediated Porcine Adipogenesis Potentially by Direct Protein-Protein Interaction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071576

    Sirt1 interacts with upstream and downstream genes of PI3K/Akt signaling pathway. At day 8 after induction, porcine fat cells were collected and protein was isolated for immunoprecipitated analysis. A. Sirt1 interacts with PI3K which is an upstream gene in PI3K/Akt signaling pathway. Endogenous PI3K in fat cell lysate was immunoprecipitated with anti–PI3K antibody, and coprecipitation of Sirt1 was detected by Western blot assay. B. Sirt1 interacts with GSK3β which is a downstream gene in PI3K/Akt signaling pathway. Endogenous GSK3β in fat cell lysate was immunoprecipitated with anti–GSK3β antibody, and coprecipitation of Sirt1 was detected by Western blot assay.
    Figure Legend Snippet: Sirt1 interacts with upstream and downstream genes of PI3K/Akt signaling pathway. At day 8 after induction, porcine fat cells were collected and protein was isolated for immunoprecipitated analysis. A. Sirt1 interacts with PI3K which is an upstream gene in PI3K/Akt signaling pathway. Endogenous PI3K in fat cell lysate was immunoprecipitated with anti–PI3K antibody, and coprecipitation of Sirt1 was detected by Western blot assay. B. Sirt1 interacts with GSK3β which is a downstream gene in PI3K/Akt signaling pathway. Endogenous GSK3β in fat cell lysate was immunoprecipitated with anti–GSK3β antibody, and coprecipitation of Sirt1 was detected by Western blot assay.

    Techniques Used: Isolation, Immunoprecipitation, Western Blot

    16) Product Images from "Inhibition of Serine Protease Activity Protects Against High Fat Diet-Induced Inflammation and Insulin Resistance"

    Article Title: Inhibition of Serine Protease Activity Protects Against High Fat Diet-Induced Inflammation and Insulin Resistance

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-58361-4

    4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) recovered high-fat diet (HFD)-impaired insulin signaling and inflammation in visceral adipose (VAD) tissue of LDLR −/− mice. ( A ) The peripheral-tissue insulin signaling pathway [insulin receptor, pyruvate dehydrogenase kinase isozyme 1 (PDK1), Akt, glycogen synthase kinase 3β (GSK3β)] was analyzed by western immunoblots. ( B ) Quantification of insulin receptor-α and -β expression, measured by imageQuant. Amounts of phosphorylated and total PDK1 ( C ), Akt ( D ), and GSK3β ( E ). ( F ) VAD tissue inflammatory cytokines and macrophage markers [interleukin (IL)-6, IL-10, CD11b, F4/80] were analyzed by western immunoblots, with protein levels quantified by imageQuant. ( G ) Tumor necrosis factor-α (TNF-α) expression in VAD tissue was analyzed by ELISA. ( H ) Serine protease activity in VAD tissue of mice receiving the chow, HFD, and HFD + AEBSF treatments ( n = 6/group). * P
    Figure Legend Snippet: 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) recovered high-fat diet (HFD)-impaired insulin signaling and inflammation in visceral adipose (VAD) tissue of LDLR −/− mice. ( A ) The peripheral-tissue insulin signaling pathway [insulin receptor, pyruvate dehydrogenase kinase isozyme 1 (PDK1), Akt, glycogen synthase kinase 3β (GSK3β)] was analyzed by western immunoblots. ( B ) Quantification of insulin receptor-α and -β expression, measured by imageQuant. Amounts of phosphorylated and total PDK1 ( C ), Akt ( D ), and GSK3β ( E ). ( F ) VAD tissue inflammatory cytokines and macrophage markers [interleukin (IL)-6, IL-10, CD11b, F4/80] were analyzed by western immunoblots, with protein levels quantified by imageQuant. ( G ) Tumor necrosis factor-α (TNF-α) expression in VAD tissue was analyzed by ELISA. ( H ) Serine protease activity in VAD tissue of mice receiving the chow, HFD, and HFD + AEBSF treatments ( n = 6/group). * P

    Techniques Used: Mouse Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay

    17) Product Images from "Valproic Acid Induces Telomerase Reverse Transcriptase Expression during Cortical Development"

    Article Title: Valproic Acid Induces Telomerase Reverse Transcriptase Expression during Cortical Development

    Journal: Experimental Neurobiology

    doi: 10.5607/en.2017.26.5.252

    The HDAC inhibitor activity of VPA increases TERT expression in NPCs. The bands and graphs show the effects of VPA and other HDACi or GSK3βi drugs in the expression of TERT, AcH3, pGSK3β and GSK3β proteins. β-actin was used as the loading control. Results are mean±S.E.M. (n=3). NPCs were cultured with embryos from three different pregnant rats. * p
    Figure Legend Snippet: The HDAC inhibitor activity of VPA increases TERT expression in NPCs. The bands and graphs show the effects of VPA and other HDACi or GSK3βi drugs in the expression of TERT, AcH3, pGSK3β and GSK3β proteins. β-actin was used as the loading control. Results are mean±S.E.M. (n=3). NPCs were cultured with embryos from three different pregnant rats. * p

    Techniques Used: Activity Assay, Expressing, Cell Culture

    18) Product Images from "Caspase-mediated Cleavage of ?-Catenin Precedes Drug-induced Apoptosis in Resistant Cancer Cells *Caspase-mediated Cleavage of ?-Catenin Precedes Drug-induced Apoptosis in Resistant Cancer Cells * S⃞"

    Article Title: Caspase-mediated Cleavage of ?-Catenin Precedes Drug-induced Apoptosis in Resistant Cancer Cells *Caspase-mediated Cleavage of ?-Catenin Precedes Drug-induced Apoptosis in Resistant Cancer Cells * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M900248200

    TRAIL-TZD regulatesβ-catenin expression via a post-translational mechanism independent of GSK3β. A , Huh-7 cells were treated with DMSO or the TRAIL-TZD combination in the presence of 20 μg/ml cycloheximide ( CHX, lanes 3–12 ). Cells were harvested at 0, 2, 4, 6, and 8 h after treatment followed by Western blot analysis with the indicated antibodies. The Huh-7 cells treated with either DMSO or the TRAIL-TZD combination in the absence of cycloheximide were included in lanes 1 and 2 , respectively, as positive controls to show TRAIL-TZD effects on β-catenin. B , Huh-7 cells were treated with the TRAIL-TZD combination for 24 h in the presence (+) or absence (-) of a pretreatment with the GSK3β inhibitor AR-A014418. At the end of the incubation total cell extracts were prepared and Western blot analysis was performed with the indicated antibodies. C , wild type ( WT +/+ ) or GSK3β knock-out ( KO -/- ) MEFs were incubated with either DMSO or the TRAIL-TZD combination. Total cell extracts were prepared at the indicated time points followed by Western blot analysis with the antibodies shown. D , Huh-7 cells were treated as in B followed by apoptosis assay utilizing the cell death detection ELISA PLUS kit. The data represent the mean ± S.D. of two independent experiments. E , phase-contrast microscopic pictures showing apoptotic morphology of Huh-7 cells following treatment with either DMSO or the TRAIL-TZD combination in the presence (+) or absence (-) of AR-A014418.
    Figure Legend Snippet: TRAIL-TZD regulatesβ-catenin expression via a post-translational mechanism independent of GSK3β. A , Huh-7 cells were treated with DMSO or the TRAIL-TZD combination in the presence of 20 μg/ml cycloheximide ( CHX, lanes 3–12 ). Cells were harvested at 0, 2, 4, 6, and 8 h after treatment followed by Western blot analysis with the indicated antibodies. The Huh-7 cells treated with either DMSO or the TRAIL-TZD combination in the absence of cycloheximide were included in lanes 1 and 2 , respectively, as positive controls to show TRAIL-TZD effects on β-catenin. B , Huh-7 cells were treated with the TRAIL-TZD combination for 24 h in the presence (+) or absence (-) of a pretreatment with the GSK3β inhibitor AR-A014418. At the end of the incubation total cell extracts were prepared and Western blot analysis was performed with the indicated antibodies. C , wild type ( WT +/+ ) or GSK3β knock-out ( KO -/- ) MEFs were incubated with either DMSO or the TRAIL-TZD combination. Total cell extracts were prepared at the indicated time points followed by Western blot analysis with the antibodies shown. D , Huh-7 cells were treated as in B followed by apoptosis assay utilizing the cell death detection ELISA PLUS kit. The data represent the mean ± S.D. of two independent experiments. E , phase-contrast microscopic pictures showing apoptotic morphology of Huh-7 cells following treatment with either DMSO or the TRAIL-TZD combination in the presence (+) or absence (-) of AR-A014418.

    Techniques Used: Expressing, Western Blot, Incubation, Knock-Out, Apoptosis Assay, Enzyme-linked Immunosorbent Assay

    Related Articles

    Incubation:

    Article Title: LMO3 promotes gastric cancer cell invasion and proliferation through Akt-mTOR and Akt-GSK3β signaling
    Article Snippet: A total of 20 μ g protein was loaded per lane and proteins were separated by 6-12% SDS-PAGE under reducing conditions and transferred onto a nitrocellulose membrane (Thermo Fisher Scientific, Inc.). .. The membrane was blocked in PBS/Tween-20 containing 5% bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China), followed by incubation with the antibodies for LMO3 (1:1,000, cat. no. ab69573; Abcam, Cambridge, MA, USA), phospho-Akt (1:2,000, cat. no. 4060), total-Akt (1:2,000, cat. no. 4691), phospho-mTOR (1:2,000, cat. no. 5536), total-mTOR (1:2,000, cat. no. 2983), phospho-GSK3β (1:2,000, cat. no. 5558), total-GSK3β (1:2,000, cat. no. 12456; all from Cell Signaling Technology, Inc., Danvers, MA, USA) and GAPDH (1:1,000, cat. no. 9001-50-7; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 4°C overnight. .. Subsequently, the membrane was washed and incubated with IRDye® 680LT Goat anti-Rabbit IgG (H+L, 1:10,000, cat. no. P/N 925-68021) or IRDye® 800CW Goat anti-Mouse IgG (H+L, 1:10,000, cat. no. P/N 925-32210; LI-COR Biosciences, Lincoln, NE, USA) at room temperature for 1 h. The fluorescently-labeled secondary antibodies were directly detected with the Odyssey imaging system (LI-COR Biosciences).

    Western Blot:

    Article Title: Presenilin 1 deficiency suppresses autophagy in human neural stem cells through reducing γ-secretase-independent ERK/CREB signaling
    Article Snippet: .. Materials For western blotting, PS1-CTF (Cat#3622), actin (Cat#4967), GADPH (Cat#5174), LC3B (Cat#12741), Bcl2 (Cat#1507), Atg7 (Cat#8558), TFEB (Cat#4240), LAMP1 (Cat#9091), P-p70S6K (Cat#9205), Histone H3 (Cat#9715), P-Akt (Cat#4060), Akt (Cat#9272), P-ERK (Cat#9101), ERK (Cat#9102), P-GSK3β (Cat#9336), GSK3β (Cat#12456), Grb2 (Cat#3972), N-cadherin (Cat#14215), P-PI3Kp85 (Cat#4228), and PI3Kp85 (Cat#4257) antibodies were purchased from Cell Signaling Technology. .. PS1-NTF antibody (Cat#MAB1563) was from Millipore.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Presenilin 1 deficiency suppresses autophagy in human neural stem cells through reducing γ-secretase-independent ERK/CREB signaling
    Article Snippet: .. Materials For western blotting, PS1-CTF (Cat#3622), actin (Cat#4967), GADPH (Cat#5174), LC3B (Cat#12741), Bcl2 (Cat#1507), Atg7 (Cat#8558), TFEB (Cat#4240), LAMP1 (Cat#9091), P-p70S6K (Cat#9205), Histone H3 (Cat#9715), P-Akt (Cat#4060), Akt (Cat#9272), P-ERK (Cat#9101), ERK (Cat#9102), P-GSK3β (Cat#9336), GSK3β (Cat#12456), Grb2 (Cat#3972), N-cadherin (Cat#14215), P-PI3Kp85 (Cat#4228), and PI3Kp85 (Cat#4257) antibodies were purchased from Cell Signaling Technology. .. PS1-NTF antibody (Cat#MAB1563) was from Millipore.

    Chromatin Immunoprecipitation:

    Article Title: CHIP functions as an oncogene by promoting colorectal cancer metastasis via activation of MAPK and AKT signaling and suppression of E-cadherin
    Article Snippet: Abs against RelA (sc-372X), RelB (sc-226X), p105/p50 (sc-7178X), p100/p52 (sc-298) and c-Rel (sc-70) were purchased from Santa Cruz Biotechnology. .. Abs against CHIP (#2080) ERK1/2 (#4695), p-ERK1/2 (#4370S), AKT (#4691), p-AKT308 (#2965), p-AKT473 (#4060), cyclinD1 (#2978), GSK-3β (#12456), p-GSK-3β (#5558), E-cadherin (#3195), EpCAM (#14452), CK8/18 (#4546) and Slug (#9585) were obtained from Cell Signaling Technology. .. Actin (A01215a) was obtained from Abgent.

    ALP Assay:

    Article Title: Human umbilical cord blood mesenchymal stem cells engineered to overexpress growth factors accelerate outcomes in hair growth
    Article Snippet: Western blot analysis Western blotting was performed as previously described using total protein obtained from hDPCs. .. Antibodies for phosphorylated-AKT (1:1000, cat. no. #9271, Cell Signaling Technology, Inc., Danvers, MA, USA), AKT (1:1000, cat. no. #4685, Cell Signaling Technology, Inc.), phopholyrated-GSK3β (1:1000, cat. no. #9323, Cell Signaling Technology, Inc), GSK3β (1:1000, cat. no. #12456, Cell Signaling Technology, Inc.), β-catenin (1:1000, cat. no. 610153, BD Biosciences, Franklin Lakes, NJ, USA), proliferating cell nuclear antigen (1:1000, cat. no. #13110, Cell Signaling Technology, Inc.), β-actin (1:1000, cat. no. sc-4778, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ALP (1:100, cat. no. SC-15065, Santa Cruz), CD 133 (cat. no. NB120-16518, Novus Biologicals, CO, USA) were used as the primary antibodies, and goat anti-rabbit (cat. no. BA-1000, VECTOR LABORATORIES, INC.) and goat anti-mouse (HRP) (cat. no. BA-9200, VECTOR LABORATORIES, INC.) were used as the secondary antibodies (1:10000). .. The blots were analyzed using densitometry with ImageJ 1.44 software (National Institutes of Health).

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    Cell Signaling Technology Inc rabbit polyclonal anti phospho gsk 3β
    SNU-C5/5-FU cells have a markedly different phenotype compared to the SNU-C5/WT parental cells ( A ) Immunoblot analysis of P-AKT, AKT, P-mTOR, mTOR, E-cadherin, <t>P-GSK-3β,</t> and GSK-3β in SNU-C5/WT and SNU-C5/5-FU cells. ( B ) The localization of P-AKT, P-mTOR, E-cadherin, and P-GSK-3β was confirmed by confocal microscopy. Scale bars, 20 μm. ( C ) Immunoblot analysis of β-catenin levels in SNU-C5/WT and SNU-C5/5-FU cells. ( D ) The nuclear localization of β-catenin was confirmed by confocal microscopy. Scale bars, 20 μm.
    Rabbit Polyclonal Anti Phospho Gsk 3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gsk3β
    Overexpression of FKBP12.6 significantly protects cardiomyocytes from AngII‐induced hypertrophy through inhibiting the Ca 2+ /CaMKII, <t>AKT/GSK3β</t> and AKT/mTOR signalling pathways in H9c2 cells. FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with PBS or with 200 nmol/L AngII for 20 min. The expressions of the total and phosphorylated CaMKII (A), AKT, GSK3β and mTOR (D) were detected by Western blot. The quantitative expressions of total and phosphoryated CaMKII, AKT and GSK3β protein were performed by density analysis, and the protein expression of p‐CaMKII (B), p‐AKT (C), p‐GSK3β (E) and p‐mTOR (F) was normalized to total CaMKII, AKT, GSK3β and mTOR. GAPDH was used as the loading controls. The data represent the mean ± SEM from three independent experiments, * P
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    Cell Signaling Technology Inc p gsk3β
    Western blotting and reverse transcription-quantitative polymerase chain reaction analysis of the expression of p-PI3k, p-Akt and <t>p-GSK3β</t> in brain tissues from an MCAO rat model. (A) Levels of p-PI3k, p-Akt and p-GSK3β protein were analyzed using western blotting and GAPDH was used as a loading control. (B) Quantitative analysis of western blotting. Expression of p-PI3k, p-Akt and p-GSK3β were normalized to total PI3k, Akt and GSK3β. (C) Quantitative analysis of the relative levels of PI3k, Akt and GSK3β mRNA. Data are presented as the mean ± standard deviation. All experiments were performed in triplicate. *P
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    SNU-C5/5-FU cells have a markedly different phenotype compared to the SNU-C5/WT parental cells ( A ) Immunoblot analysis of P-AKT, AKT, P-mTOR, mTOR, E-cadherin, P-GSK-3β, and GSK-3β in SNU-C5/WT and SNU-C5/5-FU cells. ( B ) The localization of P-AKT, P-mTOR, E-cadherin, and P-GSK-3β was confirmed by confocal microscopy. Scale bars, 20 μm. ( C ) Immunoblot analysis of β-catenin levels in SNU-C5/WT and SNU-C5/5-FU cells. ( D ) The nuclear localization of β-catenin was confirmed by confocal microscopy. Scale bars, 20 μm.

    Journal: Oncotarget

    Article Title: Over-activation of AKT signaling leading to 5-Fluorouracil resistance in SNU-C5/5-FU cells

    doi: 10.18632/oncotarget.24952

    Figure Lengend Snippet: SNU-C5/5-FU cells have a markedly different phenotype compared to the SNU-C5/WT parental cells ( A ) Immunoblot analysis of P-AKT, AKT, P-mTOR, mTOR, E-cadherin, P-GSK-3β, and GSK-3β in SNU-C5/WT and SNU-C5/5-FU cells. ( B ) The localization of P-AKT, P-mTOR, E-cadherin, and P-GSK-3β was confirmed by confocal microscopy. Scale bars, 20 μm. ( C ) Immunoblot analysis of β-catenin levels in SNU-C5/WT and SNU-C5/5-FU cells. ( D ) The nuclear localization of β-catenin was confirmed by confocal microscopy. Scale bars, 20 μm.

    Article Snippet: Mouse monoclonal anti-E-cadherin, anti-PTEN, anti-α tubulin, and anti-ubiquitin antibodies, rabbit polyclonal anti-caspase-3, anti-β-catenin, and anti-IκB-α antibodies, and goat polyclonal anti-COX-2 and anti-survivin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz Biotech, CA, USA); rabbit monoclonal anti-p-NF-κB, anti-GSK-3β, and anti-cleaved caspase-3 antibodies, rabbit polyclonal anti-phospho-GSK-3β, anti-AKT, anti-phospho-AKT, anti-cleaved caspase-9, anti-phospho-mTOR, anti-mTOR, anti-N-cadherin, and anti-PARP antibodies were purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA); the mouse monoclonal anti-cyclin D1 antibody was purchased form BD Biosciences (BD Biosciences, San Jose, CA, USA); the mouse monoclonal anti-β-actin antibody was purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA).

    Techniques: Confocal Microscopy

    Overexpression of FKBP12.6 significantly protects cardiomyocytes from AngII‐induced hypertrophy through inhibiting the Ca 2+ /CaMKII, AKT/GSK3β and AKT/mTOR signalling pathways in H9c2 cells. FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with PBS or with 200 nmol/L AngII for 20 min. The expressions of the total and phosphorylated CaMKII (A), AKT, GSK3β and mTOR (D) were detected by Western blot. The quantitative expressions of total and phosphoryated CaMKII, AKT and GSK3β protein were performed by density analysis, and the protein expression of p‐CaMKII (B), p‐AKT (C), p‐GSK3β (E) and p‐mTOR (F) was normalized to total CaMKII, AKT, GSK3β and mTOR. GAPDH was used as the loading controls. The data represent the mean ± SEM from three independent experiments, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro, et al. FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro

    doi: 10.1111/jcmm.13645

    Figure Lengend Snippet: Overexpression of FKBP12.6 significantly protects cardiomyocytes from AngII‐induced hypertrophy through inhibiting the Ca 2+ /CaMKII, AKT/GSK3β and AKT/mTOR signalling pathways in H9c2 cells. FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with PBS or with 200 nmol/L AngII for 20 min. The expressions of the total and phosphorylated CaMKII (A), AKT, GSK3β and mTOR (D) were detected by Western blot. The quantitative expressions of total and phosphoryated CaMKII, AKT and GSK3β protein were performed by density analysis, and the protein expression of p‐CaMKII (B), p‐AKT (C), p‐GSK3β (E) and p‐mTOR (F) was normalized to total CaMKII, AKT, GSK3β and mTOR. GAPDH was used as the loading controls. The data represent the mean ± SEM from three independent experiments, * P

    Article Snippet: The antibodies for Calcineurin (Millipore 07‐068, 1:1000), p‐CaMKII (Thr286/287, millipore 06‐881, 1:1000), total CaMKII (Abcam, ab52476, 1:2000), TBP (Abcam, ab818, 1:1000), NFATc4 (Abcam, ab62613, 1:1000), MCIP1 (sc377507, 1:200), α‐tubulin (sc5286, 1:1000), TGFβ1 (sc‐146, 1:200), BNP (sc‐67455, 1:200) (Santa cruz), Bcl2 (Gene Tex, GTX100064, 1:1000), FKBP12.6 (Thermo pierce antibodies, PA1‐026A, 1:1000), p‐AKT (Ser473, #4060), p‐mTOR (Ser2448, #5536), p‐GSK3β (Ser9, #9336), p‐Smad3 (Ser423/425, #9520), p‐ERK1/2 (Thr202/Tyr204, #9101), total AKT (#9272), mTOR (#2983), GSK3β (#9315), Smad3 (#9523), ERK1/2 (#9102), Bax (#2772, Cell Signaling Technology, 1:1000), GAPDH (BBI, Shanghai, China, D190636, 1:5000) were used in this study.

    Techniques: Over Expression, Western Blot, Expressing

    Western blotting and reverse transcription-quantitative polymerase chain reaction analysis of the expression of p-PI3k, p-Akt and p-GSK3β in brain tissues from an MCAO rat model. (A) Levels of p-PI3k, p-Akt and p-GSK3β protein were analyzed using western blotting and GAPDH was used as a loading control. (B) Quantitative analysis of western blotting. Expression of p-PI3k, p-Akt and p-GSK3β were normalized to total PI3k, Akt and GSK3β. (C) Quantitative analysis of the relative levels of PI3k, Akt and GSK3β mRNA. Data are presented as the mean ± standard deviation. All experiments were performed in triplicate. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Protective effects of primary neural stem cell treatment in ischemic stroke models

    doi: 10.3892/etm.2018.6466

    Figure Lengend Snippet: Western blotting and reverse transcription-quantitative polymerase chain reaction analysis of the expression of p-PI3k, p-Akt and p-GSK3β in brain tissues from an MCAO rat model. (A) Levels of p-PI3k, p-Akt and p-GSK3β protein were analyzed using western blotting and GAPDH was used as a loading control. (B) Quantitative analysis of western blotting. Expression of p-PI3k, p-Akt and p-GSK3β were normalized to total PI3k, Akt and GSK3β. (C) Quantitative analysis of the relative levels of PI3k, Akt and GSK3β mRNA. Data are presented as the mean ± standard deviation. All experiments were performed in triplicate. *P

    Article Snippet: Membranes were then incubated with primary antibodies against phosphorylated (p)-PI3k (1:1,000; cat no. 4228), p-Akt (1:1,000; cat no. 4060) and p-GSK3β (1:1,000; cat no. 9323; all Cell Signaling Technology, Inc., Danvers, MA, USA) in 5% BSA blocking buffer overnight at 4°C.

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

    MPT0G211 significantly decreased the phosphorylation of tau by GSK3β inactivation. a , b SH-SY5Y and Neuro-2a cells were transfected for 24 h with pCAX APP 695 and pRK5-EGFP-Tau P301L and incubated with MPT0G211 or ACY-1215 (0.1 μM) for another 24 h, after which the cell lysates were subjected to immunoblotting. c SH-SY5Y cells were incubated with or without Aβ 1–40 (10 μM) for 24 h and then with MPT0G211 (0.1 μM) for a further 24 h. Cell lysates were prepared for western blot analysis of the indicated proteins. Results are presented as the mean ± SEM. * p

    Journal: Cell Death & Disease

    Article Title: The novel histone de acetylase 6 inhibitor, MPT0G211, ameliorates tau phosphorylation and cognitive deficits in an Alzheimer’s disease model

    doi: 10.1038/s41419-018-0688-5

    Figure Lengend Snippet: MPT0G211 significantly decreased the phosphorylation of tau by GSK3β inactivation. a , b SH-SY5Y and Neuro-2a cells were transfected for 24 h with pCAX APP 695 and pRK5-EGFP-Tau P301L and incubated with MPT0G211 or ACY-1215 (0.1 μM) for another 24 h, after which the cell lysates were subjected to immunoblotting. c SH-SY5Y cells were incubated with or without Aβ 1–40 (10 μM) for 24 h and then with MPT0G211 (0.1 μM) for a further 24 h. Cell lysates were prepared for western blot analysis of the indicated proteins. Results are presented as the mean ± SEM. * p

    Article Snippet: Primary antibodies against APP, acetyl-histone 3, histone 3, α-tubulin, acetyl-α-tubulin, Hsp90, HDAC6, acetyl-lysine, p-Akt (Ser473), p-GSK3β (Ser9), and p-GSK3β (Tye216) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Transfection, Incubation, Western Blot

    Summary of the proposed mechanism of the neuroprotective effect of MPT0G211. Tau phosphorylation can be facilitated by GSK3β, lead to p-tau aggregation, and cause neuron death. MPT0G211 treatment not only diminished tau phosphorylation by inhibition GSK3β activity but also enhanced the acetylation of Hsp90, which caused the downregulation of HDAC6/Hsp90 binding and facilitated proteasomal degradation of polyubiquitinated p-tau

    Journal: Cell Death & Disease

    Article Title: The novel histone de acetylase 6 inhibitor, MPT0G211, ameliorates tau phosphorylation and cognitive deficits in an Alzheimer’s disease model

    doi: 10.1038/s41419-018-0688-5

    Figure Lengend Snippet: Summary of the proposed mechanism of the neuroprotective effect of MPT0G211. Tau phosphorylation can be facilitated by GSK3β, lead to p-tau aggregation, and cause neuron death. MPT0G211 treatment not only diminished tau phosphorylation by inhibition GSK3β activity but also enhanced the acetylation of Hsp90, which caused the downregulation of HDAC6/Hsp90 binding and facilitated proteasomal degradation of polyubiquitinated p-tau

    Article Snippet: Primary antibodies against APP, acetyl-histone 3, histone 3, α-tubulin, acetyl-α-tubulin, Hsp90, HDAC6, acetyl-lysine, p-Akt (Ser473), p-GSK3β (Ser9), and p-GSK3β (Tye216) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Inhibition, Activity Assay, Binding Assay