anti-gsk3β Search Results


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  • 92
    Millipore anti gsk3β
    Strain inhibits <t>GSK3β</t> action through an LRP-independent process. A , Western blot of GSK3β phosphorylation (serine 9) showed an increase at 30 min of strain and a peak response at 1 h. Actin protein was blotted as a control for equal
    Anti Gsk3β, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti gsk3β
    Proposed model of SF-1 action in the centrosome. In the centrosome, SF-1 interacts with Ku and cyclin A to prevent the DNA-PK/Akt signaling cascade. In the absence of SF-1, DNA-PKcs and cyclin A are recruited to the centrosome, leading to the activation of DNA-PK and Akt. This signaling cascade leads to <t>GSK3β</t> inactivation and β-catenin accumulation in the centrosome, therefore inducing centriole splitting. On the other hand, accumulation of cyclin A leads to CDK2 activation, thus facilitating centriole amplification. Solid arrows: proven activation step. Dotted arrows: indirect activation, perpendicular lines: inhibitory step.
    Anti Gsk3β, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Proteintech gsk3β
    PAK7 inhibits the degradation of β-catenin via phosphorylating <t>GSK3β.</t> A, PAK7 knockdown suppressed p-GSK3β (S9) expression. pEGFP-β-catenin was transfected into HEK293T cells along with siR-PAK7 or negative control. Forty-eight hours after transfection, cells were harvested for western blotting analysis to detect the expression of PAK7, GFP-β-catenin, GSK3β, p-GSK3β. B, PAK7 knockdown inhibited β-catenin mediated transcriptional activity of TCF. HEK293T cells were cotransfected with siR-PAK7 or siR-NC, pEGFP-β-catenin, TOP flash (TOP) or FOP flash (FOP), and Renilla. 48 h after transfection, cells were harvested for luciferase activity assay. C, Inhibiting proteasomes degradation by MG132 reverses the effect of PAK7 knockdown on β-catenin degradation. HEK293T cells were cotransfected with pEGFP-β-catenin and siR-PAK7 or siR-NC. Forty-four hours after transfection, the cells were treated with 30μM MG132. Cells were harvested 4 hours later and subjected to western blotting analysis to detect the expression of GFP-β-catenin and PAK7. D, Inhibiting GSK3β activity by Licl reduced β-catenin expression inhibition by PAK7 knockdown. siR-PAK7 or siR-NC was transfected into MDA-MB-231 cells. 36 hours after transfection, the cells were treated with 30 mM Licl for 12 hours and then harvested for western blotting analysis to detect the expression of GFP-β-catenin, p-GSK3β (S9) and PAK7. E, siR-PAK7 or siR-NC was transfected into MDA-MB-231 cells. 36 hours after transfection, the cells were treated with 30 mM Licl for 12 hours and harvested for luciferase activity assay. F, MDA-MB-231 cells were transfected with flag-NC and flag-PAK7, treated with Licl for 48 hours and harvested for western blot assay.
    Gsk3β, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc anti gsk3β
    Regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression by <t>GSK3β</t> . A: The GSK3β inhibitor LiCl prolongs phosphorylated H2AX increase in response to 4 Gy irradiation. HeLa cells were pretreated with 40 μM LiCl for 2 h, irradiated with 4 Gy γ rays and harvested at 0, 0.5, 1, 4 and 10 h after irradiation. Protein expression was assayed by Western blotting. B: GSK3β inhibitor LiCl enhanced the phosphorylation of H2AX in G2/M phase cells. To release the cells from G1 block and inhibit GSK3β activity, synchronized HeLa cells were grown in DMEM medium supplemented with 40 μM LiCl. S-phase cells were harvested at 5 h and G2/M phase cells at 8, 9 and10 h after released. Protein expression was assayed by Western blotting. C: RNAi depletion of GSK3β. HeLa cells were transfected with 50 nM GSK3β siRNA or non-specific (ns) control siRNA molecules. GSK3β expression was determined by Western blotting. D: Effect of GSK3β depletion on the phosphorylation of H2AX induced by 4 Gy γ-irradiation. After 48 h incubation with 50 nM GSK3β-specific siRNA or control non-specific (ns), cells were irradiated with 4 Gy γ rays, and harvested at 0, 0.25, 1, 4 and 10 h post-irradiation. Protein expression was assayed by Western blotting. E: Effect of the PP2A inhibitor fostriecin on H2AX phosphorylation. HeLa cells were pretreated with 50 nM fostriecin for 2 h, and then irradiated with 4 Gy γ rays, and harvested at 0, 0.25, 1, 4 and 10 h post-irradiation. Protein expression was assayed by Western blotting.
    Anti Gsk3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 865 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology gsk3β
    <t>GSK3β</t> controls microtubule dynamics by regulating Tau and CRMP2. A, differentiated podocytes were transfected with vectors encoding the hemagglutinin (HA)-conjugated wild type GSK3β ( WT ), kinase-dead mutant of GSK3β ( KD ), or constitutively
    Gsk3β, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 559 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti gsk3β
    VACV-Expressed B14 Associates with the IKK Signaling Complex Cells were co-transfected with expression vectors for indicated proteins tagged with HA (A) or FLAG (B) epitopes and the indicated protein fused with luciferase. The indicated tagged bait protein was immunoprecipated by correspondent monoclonal Ab and then the immune complex was eluted to be analysed for luciferase activity. Data presented are from one of the three independent experiments. (C) HeLa cells were infected with vB14 and a cell extract was prepared and analysed by size exclusion chromatography on a Superose 6 column. An aliquot of each fraction was separated in 4%–12% NuPAGE and analysed by immunoblotting with the indicated Abs. The position of protein size markers is indicated in kDa at the top. (D) Extracts from HeLa cells infected with the vΔB14 (Δ) or vB14-HA (HA) were pre-cleared and immunoprecipitated with mAbs against HA, NEMO, and <t>GSK3β</t> (isotype control). The immunoprecipitated proteins were resolved in 4%–12% NuPAGE and then were analysed by immunoblotting with the Abs indicated on the right, respectively. Sizes of bands detected are indicated on the left in kDa.
    Anti Gsk3β, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioworld Antibodies gsk3β
    VACV-Expressed B14 Associates with the IKK Signaling Complex Cells were co-transfected with expression vectors for indicated proteins tagged with HA (A) or FLAG (B) epitopes and the indicated protein fused with luciferase. The indicated tagged bait protein was immunoprecipated by correspondent monoclonal Ab and then the immune complex was eluted to be analysed for luciferase activity. Data presented are from one of the three independent experiments. (C) HeLa cells were infected with vB14 and a cell extract was prepared and analysed by size exclusion chromatography on a Superose 6 column. An aliquot of each fraction was separated in 4%–12% NuPAGE and analysed by immunoblotting with the indicated Abs. The position of protein size markers is indicated in kDa at the top. (D) Extracts from HeLa cells infected with the vΔB14 (Δ) or vB14-HA (HA) were pre-cleared and immunoprecipitated with mAbs against HA, NEMO, and <t>GSK3β</t> (isotype control). The immunoprecipitated proteins were resolved in 4%–12% NuPAGE and then were analysed by immunoblotting with the Abs indicated on the right, respectively. Sizes of bands detected are indicated on the left in kDa.
    Gsk3β, supplied by Bioworld Antibodies, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Affinity Biosciences gsk3β
    VACV-Expressed B14 Associates with the IKK Signaling Complex Cells were co-transfected with expression vectors for indicated proteins tagged with HA (A) or FLAG (B) epitopes and the indicated protein fused with luciferase. The indicated tagged bait protein was immunoprecipated by correspondent monoclonal Ab and then the immune complex was eluted to be analysed for luciferase activity. Data presented are from one of the three independent experiments. (C) HeLa cells were infected with vB14 and a cell extract was prepared and analysed by size exclusion chromatography on a Superose 6 column. An aliquot of each fraction was separated in 4%–12% NuPAGE and analysed by immunoblotting with the indicated Abs. The position of protein size markers is indicated in kDa at the top. (D) Extracts from HeLa cells infected with the vΔB14 (Δ) or vB14-HA (HA) were pre-cleared and immunoprecipitated with mAbs against HA, NEMO, and <t>GSK3β</t> (isotype control). The immunoprecipitated proteins were resolved in 4%–12% NuPAGE and then were analysed by immunoblotting with the Abs indicated on the right, respectively. Sizes of bands detected are indicated on the left in kDa.
    Gsk3β, supplied by Affinity Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioss anti gsk3β
    VACV-Expressed B14 Associates with the IKK Signaling Complex Cells were co-transfected with expression vectors for indicated proteins tagged with HA (A) or FLAG (B) epitopes and the indicated protein fused with luciferase. The indicated tagged bait protein was immunoprecipated by correspondent monoclonal Ab and then the immune complex was eluted to be analysed for luciferase activity. Data presented are from one of the three independent experiments. (C) HeLa cells were infected with vB14 and a cell extract was prepared and analysed by size exclusion chromatography on a Superose 6 column. An aliquot of each fraction was separated in 4%–12% NuPAGE and analysed by immunoblotting with the indicated Abs. The position of protein size markers is indicated in kDa at the top. (D) Extracts from HeLa cells infected with the vΔB14 (Δ) or vB14-HA (HA) were pre-cleared and immunoprecipitated with mAbs against HA, NEMO, and <t>GSK3β</t> (isotype control). The immunoprecipitated proteins were resolved in 4%–12% NuPAGE and then were analysed by immunoblotting with the Abs indicated on the right, respectively. Sizes of bands detected are indicated on the left in kDa.
    Anti Gsk3β, supplied by Bioss, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Sangon Biotech anti gsk3β
    Inhibiting SLC26A4 ameliorates the expression levels of cardiomyocyte hypertrophy markers in PE-induced cardiac hypertrophy. RT-qPCR results showing the relative mRNA expression levels of (A) ANP; (B) BNP and (C) <t>GSK3β</t> in PE-induced H9C2 cells transfected by siRNA-SLC26A4. The relative expression level of ANP, BNP and GSK3β was determined using the 2 −ΔΔ Ct method. All experiments were performed at least three times. Data represent mean ± SD. * P -value
    Anti Gsk3β, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GeneTex anti gsk3β
    The <t>TRAX/DISC1/GSK3β</t> (TDG) complex is involved in the protective effect of an A 2A R agonist. a , b PC12 cells were treated with an inhibitor of protein kinase A (H89; 10 μM and KT5720; 10 μM) or vehicle for 30 min and then treated with CGS (10 μM) or vehicle for 1 h ( a ) and 8 h ( b ). Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-GSK3β Ser9, anti-GSK3β, anti-β-catenin and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. c – e In PC12 cells and human MSN neurons, cells were infected with lentivirus expressing GSK3β or a constitutively active GSK3β mutant (GSK3β-S9A) for 3 days. Cells were treated with an A 2A R agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-α-Tubulin and anti-β-Actin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin or β-Actin, the loading controls. These experiments were repeated three times ( c , d ). Apoptosis was assessed by co-staining with Annexin V-Cy5 and PI for 10 min followed by flow cytometric analysis ( e ). f Protein–protein interactions were monitored by the proximity ligation assay [ 31 ] using the corresponding antibodies as described in the Methods section [ 31 ]. Each red dot represents the detection of protein–protein interaction. Scale bar, 10 μm. g PC12 cells were treated with an A 2A R agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h. Cells were lysed, subjected to immunoprecipitation with anti-DISC1 and immunoblotted with anti-DISC1, anti-TRAX and anti-GSK3β antibodies. The amount of target protein was quantified and normalized to that of the input. These experiments were repeated three times. h PC12 cells were infected with the lentivirus expressing A 2A R 253–410 -Flag (M7) for 3 days. Cells were lysed, subjected to immunoprecipitation with an anti-A 2A R antibody or a control IgG, followed by Western blot analysis using the indicated antibody. The amount of TRAX that interacted with A 2A R was quantified and normalized to that of the input. These experiments were repeated three times. i PC12 cells were infected with lentivirus expressing A 2A R 253–410 -Flag for 3 days. Cells were treated with CGS (10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-Flag and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading controls. These experiments were repeated three times. j PC12 cells were treated with CGS (10 μM) during the indicated time period. Cells were subjected to immunostaining with an anti-TRAX antibody (Green). Scale bar, 10 μm. k PC12-shTRAX cells were transfected with TRAX-V5 and ΔNLS TRAX-V5 plasmids. Cells were treated with CGS (10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were subjected to immunostaining with anti-γH2AX (Green) and anti-V5 (Red) antibodies. Scale bar, 10 μm. Data are presented as the mean ± SEM from at least three independent experiments. * P
    Anti Gsk3β, supplied by GeneTex, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare anti gsk3β
    Fap1-inhibition with SLV peptide increases Fas and <t>Gsk3β</t> phosphorylation in CD133 + cells in a murine xenograft model SW620 cells were injected in the flanks of athymic Nude mice and tumor volume was determination biweekly. Mice were treated weekly with oxaliplatin (days 0, 7 and 14) and injected daily with Fap1 blocking SLV peptide or VLS control peptide, or treated with SLV or VLS peptide alone (n=12 per cohort). Tumors were simultaneously harvested from cohorts of mice when control tumors were > 2,000 mm3. (A) SLV peptide increases Fas phosphorylation in CD133 + xenograft tumors with or without oxaliplatin. Immunofluorescent detection of phospho-Fas or CD133 was performed with DAPI staining of nuclei. (B) SLV peptide increases Gsk3β phosphorylation in CD133 + xenograft tumors with or without oxaliplatin. Immunofluorescent detection of phospho-Gsk3β or CD133 was performed with DAPI staining of nuclei.
    Anti Gsk3β, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Epitomics gsk3β
    <t>GSK3β</t> 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.
    Gsk3β, supplied by Epitomics, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore gsk3β
    Localization of signaling components necessary for A–P guidance in rat E13 and mouse E11.5 spinal cord. A–A″ , Phosphorylated PKCζ in transverse ( A , A′ ) and postcrossing ( A″ ) sections. Arrows indicate immunoreactivity in postcrossing segments. B–B″ , Par6 staining in transverse ( B , B′ ) and postcrossing ( B″ ) sections. Arrows indicate immunoreactivity in postcrossing segments. C–C′ , Phosphorylated <t>GSK3β</t> (S9) staining in transverse sections. Arrows indicate immunoreactivity in postcrossing segments. D , D″ , L1 staining in transverse ( D ) and postcrossing ( D″ ) sections. Arrows indicate postcrossing staining. E , E″ , Tag-1 staining in transverse ( E ) and postcrossing ( E″ ) sections. Precrossing staining is present ( E , E″ , arrows), whereas postcrossing segments are negative ( E , arrowhead). Inset, Differential interference contrast image indicates presence of axons (black arrow). F , G , Diagrams indicating axon trajectories in transverse ( F ) and postcrossing ( G ) sections. The precrossing segment is labeled blue, the crossing segment is labeled red, and the postcrossing segment is labeled green. mE11.5, Mouse E11.5; rE13, rat E13.
    Gsk3β, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 504 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher anti gsk3β py216
    Localization of signaling components necessary for A–P guidance in rat E13 and mouse E11.5 spinal cord. A–A″ , Phosphorylated PKCζ in transverse ( A , A′ ) and postcrossing ( A″ ) sections. Arrows indicate immunoreactivity in postcrossing segments. B–B″ , Par6 staining in transverse ( B , B′ ) and postcrossing ( B″ ) sections. Arrows indicate immunoreactivity in postcrossing segments. C–C′ , Phosphorylated <t>GSK3β</t> (S9) staining in transverse sections. Arrows indicate immunoreactivity in postcrossing segments. D , D″ , L1 staining in transverse ( D ) and postcrossing ( D″ ) sections. Arrows indicate postcrossing staining. E , E″ , Tag-1 staining in transverse ( E ) and postcrossing ( E″ ) sections. Precrossing staining is present ( E , E″ , arrows), whereas postcrossing segments are negative ( E , arrowhead). Inset, Differential interference contrast image indicates presence of axons (black arrow). F , G , Diagrams indicating axon trajectories in transverse ( F ) and postcrossing ( G ) sections. The precrossing segment is labeled blue, the crossing segment is labeled red, and the postcrossing segment is labeled green. mE11.5, Mouse E11.5; rE13, rat E13.
    Anti Gsk3β Py216, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems gsk3β
    Localization of signaling components necessary for A–P guidance in rat E13 and mouse E11.5 spinal cord. A–A″ , Phosphorylated PKCζ in transverse ( A , A′ ) and postcrossing ( A″ ) sections. Arrows indicate immunoreactivity in postcrossing segments. B–B″ , Par6 staining in transverse ( B , B′ ) and postcrossing ( B″ ) sections. Arrows indicate immunoreactivity in postcrossing segments. C–C′ , Phosphorylated <t>GSK3β</t> (S9) staining in transverse sections. Arrows indicate immunoreactivity in postcrossing segments. D , D″ , L1 staining in transverse ( D ) and postcrossing ( D″ ) sections. Arrows indicate postcrossing staining. E , E″ , Tag-1 staining in transverse ( E ) and postcrossing ( E″ ) sections. Precrossing staining is present ( E , E″ , arrows), whereas postcrossing segments are negative ( E , arrowhead). Inset, Differential interference contrast image indicates presence of axons (black arrow). F , G , Diagrams indicating axon trajectories in transverse ( F ) and postcrossing ( G ) sections. The precrossing segment is labeled blue, the crossing segment is labeled red, and the postcrossing segment is labeled green. mE11.5, Mouse E11.5; rE13, rat E13.
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    Cell Signaling Technology Inc gsk3β ser9
    Phosphorylation of Akt and <t>GSK3β</t> at R30 in heart ( A – D ) and brain ( E – H ) was analyzed by Western blot. A and E : p-Akt Thr308, p-Akt Ser473, and p-GSK3β <t>Ser9</t> at R30. α-Tubulin and β-actin were used as loading controls for heart and brain, respectively. B and F : densitometric analysis of p-Akt Thr308 ( * P
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    Image Search Results


    Strain inhibits GSK3β action through an LRP-independent process. A , Western blot of GSK3β phosphorylation (serine 9) showed an increase at 30 min of strain and a peak response at 1 h. Actin protein was blotted as a control for equal

    Journal: The Journal of Biological Chemistry

    Article Title: ?-Catenin Levels Influence Rapid Mechanical Responses in Osteoblasts *

    doi: 10.1074/jbc.M801907200

    Figure Lengend Snippet: Strain inhibits GSK3β action through an LRP-independent process. A , Western blot of GSK3β phosphorylation (serine 9) showed an increase at 30 min of strain and a peak response at 1 h. Actin protein was blotted as a control for equal

    Article Snippet: The primary antibodies used include anti-active β-catenin (clone 8E7; Upstate Biotechnology, Inc., Lake Placid, NY), anti-total β-catenin (BD Biosciences), anti-phospho-GSK3β (serine 9, clone 2D3; Upstate Biotechnology), anti-GSK3β (Chemicon, Billerica, MA), anti-phospho-Akt (serine 473; Cell Signaling, Danvers, MA), anti-Akt (clone 11E7; Cell Signaling), and anti-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

    Techniques: Western Blot

    Proposed model of SF-1 action in the centrosome. In the centrosome, SF-1 interacts with Ku and cyclin A to prevent the DNA-PK/Akt signaling cascade. In the absence of SF-1, DNA-PKcs and cyclin A are recruited to the centrosome, leading to the activation of DNA-PK and Akt. This signaling cascade leads to GSK3β inactivation and β-catenin accumulation in the centrosome, therefore inducing centriole splitting. On the other hand, accumulation of cyclin A leads to CDK2 activation, thus facilitating centriole amplification. Solid arrows: proven activation step. Dotted arrows: indirect activation, perpendicular lines: inhibitory step.

    Journal: Cell Communication and Signaling : CCS

    Article Title: NR5A1 prevents centriole splitting by inhibiting centrosomal DNA-PK activation and β-catenin accumulation

    doi: 10.1186/s12964-014-0055-9

    Figure Lengend Snippet: Proposed model of SF-1 action in the centrosome. In the centrosome, SF-1 interacts with Ku and cyclin A to prevent the DNA-PK/Akt signaling cascade. In the absence of SF-1, DNA-PKcs and cyclin A are recruited to the centrosome, leading to the activation of DNA-PK and Akt. This signaling cascade leads to GSK3β inactivation and β-catenin accumulation in the centrosome, therefore inducing centriole splitting. On the other hand, accumulation of cyclin A leads to CDK2 activation, thus facilitating centriole amplification. Solid arrows: proven activation step. Dotted arrows: indirect activation, perpendicular lines: inhibitory step.

    Article Snippet: Antibodies The following antibodies were obtained commercially: anti-γ-tubulin, polyclonal anti-FLAG, anti-Cyclin A, monoclonal anti-FLAG M2, anti-α-tubulin and anti-acetylated-α-tubulin (all from Sigma, St. Louis, MO), anti-Cyclin E, anti-CDK2 phospho-Thr160, anti-Akt and anti-Akt phospho-Thr308 (Cell Signaling, Beverly, MA), anti-centrin 20H5 (Millipore, Billerica, MA), polyclonal anti-β-catenin and anti-GSK3β (Abcam, Cambridge, UK), anti-Ku70 (Genetex, Trvine, CA), anti-DNA-PKcs, and anti-DNA-PKcs phospho-Thr 2609 (Santa Cruz Biotech, Santa Cruz, CA).

    Techniques: Activation Assay, Amplification

    SF-1 depletion activates DNA-PK signaling and causes accumulation of β-catenin and GSK3β in the centrosome. (A, C) Centrosomal or (B) whole cell extracts (WCE) of shluc or shsf1#3 lentivirus infected Y1 cells in the presence or absence of DNA-PK inhibitor, vanillin, were analyzed by immunoblotting with antibodies against β-catenin (β-cat), phosphorylated GSK3β (pGSK3β), GSK3β, SF-1, γ-tubulin (γ-tub), phsophorylated Akt (pAkt), Actin and Hsc70.

    Journal: Cell Communication and Signaling : CCS

    Article Title: NR5A1 prevents centriole splitting by inhibiting centrosomal DNA-PK activation and β-catenin accumulation

    doi: 10.1186/s12964-014-0055-9

    Figure Lengend Snippet: SF-1 depletion activates DNA-PK signaling and causes accumulation of β-catenin and GSK3β in the centrosome. (A, C) Centrosomal or (B) whole cell extracts (WCE) of shluc or shsf1#3 lentivirus infected Y1 cells in the presence or absence of DNA-PK inhibitor, vanillin, were analyzed by immunoblotting with antibodies against β-catenin (β-cat), phosphorylated GSK3β (pGSK3β), GSK3β, SF-1, γ-tubulin (γ-tub), phsophorylated Akt (pAkt), Actin and Hsc70.

    Article Snippet: Antibodies The following antibodies were obtained commercially: anti-γ-tubulin, polyclonal anti-FLAG, anti-Cyclin A, monoclonal anti-FLAG M2, anti-α-tubulin and anti-acetylated-α-tubulin (all from Sigma, St. Louis, MO), anti-Cyclin E, anti-CDK2 phospho-Thr160, anti-Akt and anti-Akt phospho-Thr308 (Cell Signaling, Beverly, MA), anti-centrin 20H5 (Millipore, Billerica, MA), polyclonal anti-β-catenin and anti-GSK3β (Abcam, Cambridge, UK), anti-Ku70 (Genetex, Trvine, CA), anti-DNA-PKcs, and anti-DNA-PKcs phospho-Thr 2609 (Santa Cruz Biotech, Santa Cruz, CA).

    Techniques: Infection

    eMaSCs have inherently more active Wnt/β-catenin signaling compared with cMaSCs. A , whole cell lysates were immunoblotted as indicated. ABC and p-Dvl-2 were detected in both low and high passage eMaSCs, whereas these proteins were only detected in low passage cMaSCs. Western blot images of three independent experiments were quantified using the Bio-Rad ChemiDoc MP system ( panel ii ), and a representative Western blot image is shown ( panel i ; n = 3). B , panel i , representative fluorescent microscopy of nuclear β-catenin expression in eMaSCs, which is similar to expression in Rat2/Wnt1 cells, and cMaSCs, which is similar to expression in control Rat2/MV7 cells. Scale bar , 25 μm. Panel ii , the percentage of cells with nuclear β-catenin expression was quantified by analyzing random fields containing at least 100 cells. C , panel i , representative fluorescent microscopy images of GSK-3β expression in eMaSCs and cMaSCs on days 1 and 4 in culture. Scale bars , 20 μm. Panel ii , schematic representation of the particle size distribution analyses of GSK3β in immunostained cells. Left panel , part of an original immunofluorescent image. Right panel , image adjustment performed in ImageJ used to define the fluorescent particles software (detailed under “Experimental Procedures”). Inset/bottom panel , enlarged part of image, highlighting examples of two particles ( red ) and their size, as measured by ImageJ software. Panel iii , quantitation of particle size distribution in GSK3β-stained cells, comparing the percentage of small (particle size 1) versus large (particle size 2–99) fluorescent particles in cMaSCs and eMaSCs between days 1 and 4 in culture ( n = 3). ****, p

    Journal: The Journal of Biological Chemistry

    Article Title: Microvesicle-mediated Wnt/β-Catenin Signaling Promotes Interspecies Mammary Stem/Progenitor Cell Growth *

    doi: 10.1074/jbc.M116.726117

    Figure Lengend Snippet: eMaSCs have inherently more active Wnt/β-catenin signaling compared with cMaSCs. A , whole cell lysates were immunoblotted as indicated. ABC and p-Dvl-2 were detected in both low and high passage eMaSCs, whereas these proteins were only detected in low passage cMaSCs. Western blot images of three independent experiments were quantified using the Bio-Rad ChemiDoc MP system ( panel ii ), and a representative Western blot image is shown ( panel i ; n = 3). B , panel i , representative fluorescent microscopy of nuclear β-catenin expression in eMaSCs, which is similar to expression in Rat2/Wnt1 cells, and cMaSCs, which is similar to expression in control Rat2/MV7 cells. Scale bar , 25 μm. Panel ii , the percentage of cells with nuclear β-catenin expression was quantified by analyzing random fields containing at least 100 cells. C , panel i , representative fluorescent microscopy images of GSK-3β expression in eMaSCs and cMaSCs on days 1 and 4 in culture. Scale bars , 20 μm. Panel ii , schematic representation of the particle size distribution analyses of GSK3β in immunostained cells. Left panel , part of an original immunofluorescent image. Right panel , image adjustment performed in ImageJ used to define the fluorescent particles software (detailed under “Experimental Procedures”). Inset/bottom panel , enlarged part of image, highlighting examples of two particles ( red ) and their size, as measured by ImageJ software. Panel iii , quantitation of particle size distribution in GSK3β-stained cells, comparing the percentage of small (particle size 1) versus large (particle size 2–99) fluorescent particles in cMaSCs and eMaSCs between days 1 and 4 in culture ( n = 3). ****, p

    Article Snippet: The cells were incubated with β-catenin (BD Biosciences) or GSK3β (Abcam) antibodies, washed with 1% BSA in PBS, and incubated with a 488-conjugated secondary antibody (Jackson) and DAPI (Sigma) to label nuclei.

    Techniques: Western Blot, Microscopy, Expressing, Software, Quantitation Assay, Staining

    PAK7 inhibits the degradation of β-catenin via phosphorylating GSK3β. A, PAK7 knockdown suppressed p-GSK3β (S9) expression. pEGFP-β-catenin was transfected into HEK293T cells along with siR-PAK7 or negative control. Forty-eight hours after transfection, cells were harvested for western blotting analysis to detect the expression of PAK7, GFP-β-catenin, GSK3β, p-GSK3β. B, PAK7 knockdown inhibited β-catenin mediated transcriptional activity of TCF. HEK293T cells were cotransfected with siR-PAK7 or siR-NC, pEGFP-β-catenin, TOP flash (TOP) or FOP flash (FOP), and Renilla. 48 h after transfection, cells were harvested for luciferase activity assay. C, Inhibiting proteasomes degradation by MG132 reverses the effect of PAK7 knockdown on β-catenin degradation. HEK293T cells were cotransfected with pEGFP-β-catenin and siR-PAK7 or siR-NC. Forty-four hours after transfection, the cells were treated with 30μM MG132. Cells were harvested 4 hours later and subjected to western blotting analysis to detect the expression of GFP-β-catenin and PAK7. D, Inhibiting GSK3β activity by Licl reduced β-catenin expression inhibition by PAK7 knockdown. siR-PAK7 or siR-NC was transfected into MDA-MB-231 cells. 36 hours after transfection, the cells were treated with 30 mM Licl for 12 hours and then harvested for western blotting analysis to detect the expression of GFP-β-catenin, p-GSK3β (S9) and PAK7. E, siR-PAK7 or siR-NC was transfected into MDA-MB-231 cells. 36 hours after transfection, the cells were treated with 30 mM Licl for 12 hours and harvested for luciferase activity assay. F, MDA-MB-231 cells were transfected with flag-NC and flag-PAK7, treated with Licl for 48 hours and harvested for western blot assay.

    Journal: Journal of Cancer

    Article Title: P21-activated kinase 7 (PAK7) interacts with and activates Wnt/β-catenin signaling pathway in breast cancer

    doi: 10.7150/jca.24934

    Figure Lengend Snippet: PAK7 inhibits the degradation of β-catenin via phosphorylating GSK3β. A, PAK7 knockdown suppressed p-GSK3β (S9) expression. pEGFP-β-catenin was transfected into HEK293T cells along with siR-PAK7 or negative control. Forty-eight hours after transfection, cells were harvested for western blotting analysis to detect the expression of PAK7, GFP-β-catenin, GSK3β, p-GSK3β. B, PAK7 knockdown inhibited β-catenin mediated transcriptional activity of TCF. HEK293T cells were cotransfected with siR-PAK7 or siR-NC, pEGFP-β-catenin, TOP flash (TOP) or FOP flash (FOP), and Renilla. 48 h after transfection, cells were harvested for luciferase activity assay. C, Inhibiting proteasomes degradation by MG132 reverses the effect of PAK7 knockdown on β-catenin degradation. HEK293T cells were cotransfected with pEGFP-β-catenin and siR-PAK7 or siR-NC. Forty-four hours after transfection, the cells were treated with 30μM MG132. Cells were harvested 4 hours later and subjected to western blotting analysis to detect the expression of GFP-β-catenin and PAK7. D, Inhibiting GSK3β activity by Licl reduced β-catenin expression inhibition by PAK7 knockdown. siR-PAK7 or siR-NC was transfected into MDA-MB-231 cells. 36 hours after transfection, the cells were treated with 30 mM Licl for 12 hours and then harvested for western blotting analysis to detect the expression of GFP-β-catenin, p-GSK3β (S9) and PAK7. E, siR-PAK7 or siR-NC was transfected into MDA-MB-231 cells. 36 hours after transfection, the cells were treated with 30 mM Licl for 12 hours and harvested for luciferase activity assay. F, MDA-MB-231 cells were transfected with flag-NC and flag-PAK7, treated with Licl for 48 hours and harvested for western blot assay.

    Article Snippet: The following primary antibodies were used for western blotting and analyses: PAK7 (Proteintech, USA), flag (Proteintech, USA), GFP (Proteintech, USA), GSK3β (Proteintech, USA), p-GSK3β (Cell Signaling Technology, USA), β-catenin (Proteintech, USA), p-β-catenin (Cell Signaling Technology, USA), c-myc (Proteintech, USA), cyclin D1 (Proteintech, USA) and GAPDH (Proteintech, USA),and the goat anti-rabbit IgG (Proteintech, USA) and goat anti-mouse IgG (Proteintech, USA).

    Techniques: Expressing, Transfection, Negative Control, Western Blot, Activity Assay, Luciferase, Inhibition, Multiple Displacement Amplification

    PAK7 activates Wnt/β-catenin signaling pathway in breast cancer cells. A, The effect of transfecting with flag-PAK7 overexpression plasmid or flag-NC negative control vector on the protein levels of flag-PAK7, β-catenin, p-β-catenin (S33/S37/T41), GSK3β, p-GSK3β(S9), c-myc, cyclin D1 and β-catenin (nucleus), c-myc (nucleus) in MCF-7 cells. B, The effect of transfecting with small interfering RNA of PAK7 (siR-PAK7) or negative control (siR-NC) vector on the protein levels of flag-PAK7, β-catenin, p-β-catenin (S33/S37/T41), GSK3β, p-GSK3β(S9), c-myc, cyclin D1 and β-catenin (nucleus), c-myc (nucleus) in MDA-MB-231 cells. C, PAK7 overexpression increases the activity of Wnt/β-catenin signaling pathway which was detected by TOP/FOP flash assay in MCF-7 cells. D, PAK7 knockdown inhibits the activity of Wnt/β-catenin signaling pathway which was detected by TOP/FOP flash assay in MDA-MB-231 cells. E, The expression of PAK7 was positively correlated with β-catenin (CTNNB1), c-myc (MYC) expression which was analyzed in GEPIA database by bioinformatics methods. Values represent the mean ± SD from three independent measurements. *P

    Journal: Journal of Cancer

    Article Title: P21-activated kinase 7 (PAK7) interacts with and activates Wnt/β-catenin signaling pathway in breast cancer

    doi: 10.7150/jca.24934

    Figure Lengend Snippet: PAK7 activates Wnt/β-catenin signaling pathway in breast cancer cells. A, The effect of transfecting with flag-PAK7 overexpression plasmid or flag-NC negative control vector on the protein levels of flag-PAK7, β-catenin, p-β-catenin (S33/S37/T41), GSK3β, p-GSK3β(S9), c-myc, cyclin D1 and β-catenin (nucleus), c-myc (nucleus) in MCF-7 cells. B, The effect of transfecting with small interfering RNA of PAK7 (siR-PAK7) or negative control (siR-NC) vector on the protein levels of flag-PAK7, β-catenin, p-β-catenin (S33/S37/T41), GSK3β, p-GSK3β(S9), c-myc, cyclin D1 and β-catenin (nucleus), c-myc (nucleus) in MDA-MB-231 cells. C, PAK7 overexpression increases the activity of Wnt/β-catenin signaling pathway which was detected by TOP/FOP flash assay in MCF-7 cells. D, PAK7 knockdown inhibits the activity of Wnt/β-catenin signaling pathway which was detected by TOP/FOP flash assay in MDA-MB-231 cells. E, The expression of PAK7 was positively correlated with β-catenin (CTNNB1), c-myc (MYC) expression which was analyzed in GEPIA database by bioinformatics methods. Values represent the mean ± SD from three independent measurements. *P

    Article Snippet: The following primary antibodies were used for western blotting and analyses: PAK7 (Proteintech, USA), flag (Proteintech, USA), GFP (Proteintech, USA), GSK3β (Proteintech, USA), p-GSK3β (Cell Signaling Technology, USA), β-catenin (Proteintech, USA), p-β-catenin (Cell Signaling Technology, USA), c-myc (Proteintech, USA), cyclin D1 (Proteintech, USA) and GAPDH (Proteintech, USA),and the goat anti-rabbit IgG (Proteintech, USA) and goat anti-mouse IgG (Proteintech, USA).

    Techniques: Over Expression, Plasmid Preparation, Negative Control, Small Interfering RNA, Multiple Displacement Amplification, Activity Assay, Expressing

    PAK7 interacts with β-catenin and GSK3β. A, PAK7 was binding to β-catenin and GSK3β by immunoprecipitation. β-catenin (GFP tagged) was cotransfected with PAK7 (flag tagged) or empty vector into HEK293T cells. Immunoprecipitation was performed with flag antibody and GFP antibody, respectively. β-catenin, GSK3β and PAK7 were analyzed with a GFP antibody, GSK3β antibody, and flag antibody, respectively. B, PAK7 and β-catenin were co-localized in the cell cytoplasm and nucleus. PAK7 (GFP tagged) and pCherry-β-catenin (Cherry tagged) were cotransfected into HEK293T cells. Co-localization (yellow fluorescence) of PAK7 (green fluorescence) and β-catenin (red fluorescence) was detected in the cytoplasm and nucleus. C, Working model for the regulation of β-catenin degradation by PAK7 via phosphorylating GSK3β in Wnt/β-catenin signaling pathway.

    Journal: Journal of Cancer

    Article Title: P21-activated kinase 7 (PAK7) interacts with and activates Wnt/β-catenin signaling pathway in breast cancer

    doi: 10.7150/jca.24934

    Figure Lengend Snippet: PAK7 interacts with β-catenin and GSK3β. A, PAK7 was binding to β-catenin and GSK3β by immunoprecipitation. β-catenin (GFP tagged) was cotransfected with PAK7 (flag tagged) or empty vector into HEK293T cells. Immunoprecipitation was performed with flag antibody and GFP antibody, respectively. β-catenin, GSK3β and PAK7 were analyzed with a GFP antibody, GSK3β antibody, and flag antibody, respectively. B, PAK7 and β-catenin were co-localized in the cell cytoplasm and nucleus. PAK7 (GFP tagged) and pCherry-β-catenin (Cherry tagged) were cotransfected into HEK293T cells. Co-localization (yellow fluorescence) of PAK7 (green fluorescence) and β-catenin (red fluorescence) was detected in the cytoplasm and nucleus. C, Working model for the regulation of β-catenin degradation by PAK7 via phosphorylating GSK3β in Wnt/β-catenin signaling pathway.

    Article Snippet: The following primary antibodies were used for western blotting and analyses: PAK7 (Proteintech, USA), flag (Proteintech, USA), GFP (Proteintech, USA), GSK3β (Proteintech, USA), p-GSK3β (Cell Signaling Technology, USA), β-catenin (Proteintech, USA), p-β-catenin (Cell Signaling Technology, USA), c-myc (Proteintech, USA), cyclin D1 (Proteintech, USA) and GAPDH (Proteintech, USA),and the goat anti-rabbit IgG (Proteintech, USA) and goat anti-mouse IgG (Proteintech, USA).

    Techniques: Binding Assay, Immunoprecipitation, Plasmid Preparation, Fluorescence

    PALLD interacts with AKT1 to maintain AKT1-GSK3β activation and spindle orientation. ( a and b ) Results of co-immunoprecipitation between endogenous AKT1 and PALLD in mitotic HeLa cells. ( c ) After synchronization using double Thymidine, HeLa cells in the scramble and KD groups were collected, and proteins were extracted. Levels of proteins in the AKT1–GSK3β pathway were examined by Western blot. ( d and e ) Spindle angles of scramble and KD cells overexpressing AKT1 CA, AKT1 DN, GSK3β CA, or GSK3β DN were analyzed to determine the extent of rescue of spindle misorientation caused by PALLD knockdown (n = 50). ( f ) Molecular model illustrating the mechanism by which PALLD regulates spindle orientation and mitotic progress. ***P

    Journal: Scientific Reports

    Article Title: Palladin is a novel microtubule-associated protein responsible for spindle orientation

    doi: 10.1038/s41598-017-12051-w

    Figure Lengend Snippet: PALLD interacts with AKT1 to maintain AKT1-GSK3β activation and spindle orientation. ( a and b ) Results of co-immunoprecipitation between endogenous AKT1 and PALLD in mitotic HeLa cells. ( c ) After synchronization using double Thymidine, HeLa cells in the scramble and KD groups were collected, and proteins were extracted. Levels of proteins in the AKT1–GSK3β pathway were examined by Western blot. ( d and e ) Spindle angles of scramble and KD cells overexpressing AKT1 CA, AKT1 DN, GSK3β CA, or GSK3β DN were analyzed to determine the extent of rescue of spindle misorientation caused by PALLD knockdown (n = 50). ( f ) Molecular model illustrating the mechanism by which PALLD regulates spindle orientation and mitotic progress. ***P

    Article Snippet: The following antibodies were applied: anti-Palladin (Proteintech), anti-α-tubulin (Sigma, Proteintech), anti-Akt (Cell Signaling), anti-phospho-Thr308-Akt (Cell Signaling), anti-phospho-Ser473-Akt (Cell Signaling), anti-GSK3β (Proteintech), anti-phospho-Ser9-GSK3β (Cell Signaling), anti-β-actin (Sigma), anti-FLAG tag (Abmart, Cell Signaling), and anti-HA tag (Cell Signaling).

    Techniques: Activation Assay, Immunoprecipitation, Western Blot

    Regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression by GSK3β . A: The GSK3β inhibitor LiCl prolongs phosphorylated H2AX increase in response to 4 Gy irradiation. HeLa cells were pretreated with 40 μM LiCl for 2 h, irradiated with 4 Gy γ rays and harvested at 0, 0.5, 1, 4 and 10 h after irradiation. Protein expression was assayed by Western blotting. B: GSK3β inhibitor LiCl enhanced the phosphorylation of H2AX in G2/M phase cells. To release the cells from G1 block and inhibit GSK3β activity, synchronized HeLa cells were grown in DMEM medium supplemented with 40 μM LiCl. S-phase cells were harvested at 5 h and G2/M phase cells at 8, 9 and10 h after released. Protein expression was assayed by Western blotting. C: RNAi depletion of GSK3β. HeLa cells were transfected with 50 nM GSK3β siRNA or non-specific (ns) control siRNA molecules. GSK3β expression was determined by Western blotting. D: Effect of GSK3β depletion on the phosphorylation of H2AX induced by 4 Gy γ-irradiation. After 48 h incubation with 50 nM GSK3β-specific siRNA or control non-specific (ns), cells were irradiated with 4 Gy γ rays, and harvested at 0, 0.25, 1, 4 and 10 h post-irradiation. Protein expression was assayed by Western blotting. E: Effect of the PP2A inhibitor fostriecin on H2AX phosphorylation. HeLa cells were pretreated with 50 nM fostriecin for 2 h, and then irradiated with 4 Gy γ rays, and harvested at 0, 0.25, 1, 4 and 10 h post-irradiation. Protein expression was assayed by Western blotting.

    Journal: BMC Molecular Biology

    Article Title: DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression

    doi: 10.1186/1471-2199-11-18

    Figure Lengend Snippet: Regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression by GSK3β . A: The GSK3β inhibitor LiCl prolongs phosphorylated H2AX increase in response to 4 Gy irradiation. HeLa cells were pretreated with 40 μM LiCl for 2 h, irradiated with 4 Gy γ rays and harvested at 0, 0.5, 1, 4 and 10 h after irradiation. Protein expression was assayed by Western blotting. B: GSK3β inhibitor LiCl enhanced the phosphorylation of H2AX in G2/M phase cells. To release the cells from G1 block and inhibit GSK3β activity, synchronized HeLa cells were grown in DMEM medium supplemented with 40 μM LiCl. S-phase cells were harvested at 5 h and G2/M phase cells at 8, 9 and10 h after released. Protein expression was assayed by Western blotting. C: RNAi depletion of GSK3β. HeLa cells were transfected with 50 nM GSK3β siRNA or non-specific (ns) control siRNA molecules. GSK3β expression was determined by Western blotting. D: Effect of GSK3β depletion on the phosphorylation of H2AX induced by 4 Gy γ-irradiation. After 48 h incubation with 50 nM GSK3β-specific siRNA or control non-specific (ns), cells were irradiated with 4 Gy γ rays, and harvested at 0, 0.25, 1, 4 and 10 h post-irradiation. Protein expression was assayed by Western blotting. E: Effect of the PP2A inhibitor fostriecin on H2AX phosphorylation. HeLa cells were pretreated with 50 nM fostriecin for 2 h, and then irradiated with 4 Gy γ rays, and harvested at 0, 0.25, 1, 4 and 10 h post-irradiation. Protein expression was assayed by Western blotting.

    Article Snippet: All antibodies were purchased commercially: anti-β-actin (I-19-R, Santa Cruz, CA), anti-DNA-PKcs (H-163, Santa Cruz, CA), γH2AX (05-636, Upstate Biotechnology, Charlottesville, VA), anti-GSK3β (#9332, Cell Signaling, Danvers, MA), anti-phospho-GSK3β (Ser-9, #9336, Cell Signaling, Danvers, MA), anti-PDK1 (#3062, Cell signaling, Danvers, MA), anti-phospho-Akt (Ser-473, #9271, Cell Signaling, Danvers, MA), anti-phospho-ATM (Ser-1981, Cell Signaling, Danvers, MA), anti-rabbit IgG(H+L)/HRP (ZB-2301, Zhongshan, Beijing, China), and anti-mouse IgG(H+L)/HRP (ZB-2305, Zhongshan, Beijing, China).

    Techniques: Irradiation, Expressing, Western Blot, Blocking Assay, Activity Assay, Transfection, Incubation

    Regulation of phosphoinositide-dependent kinase (PDK) on the phosphorylation of H2AX . A: RNAi mediated depletion of PDK protein. HeLa cells were transfected with 50 nM PDK specific siRNA molecules or non-specific (ns) control siRNA. Western blotting shows PDK expression. B: Phosphorylation of Akt at Ser473 and GSK3β at Ser9 was decreased in the DNA-PKcs depleted HeLa-H1 cells compared to control HeLa-NC cells. C: PDK regulates the phosphorylation of H2AX in response to DNA damage induced by 4 Gy of γ-irradiation. After 48 h incubation with 50 nM PDK-specific siRNA or non-specific (ns) control siRNA, cells were irradiated with 4 Gy γ rays, then harvested 0, 0.5, 1, 4, 10 h post-irradiation and analyzed by Western blotting. D: PDK regulates the phosphorylation of H2AX associated with cell cycle progression. After 24 h incubation with 50 nM PDK-specific siRNA or non-specific (ns) control siRNA, cells were synchronized in G1 phase by TdR double-blocking, then released and harvested after 5 h, for S-phase, and at 8, 9, and 10 h, for G2/M phase. The culture medium was supplemented with 50 nM siRNA molecules during the period of synchronization and cell cycle progression. Protein expression was assayed by Western blotting.

    Journal: BMC Molecular Biology

    Article Title: DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression

    doi: 10.1186/1471-2199-11-18

    Figure Lengend Snippet: Regulation of phosphoinositide-dependent kinase (PDK) on the phosphorylation of H2AX . A: RNAi mediated depletion of PDK protein. HeLa cells were transfected with 50 nM PDK specific siRNA molecules or non-specific (ns) control siRNA. Western blotting shows PDK expression. B: Phosphorylation of Akt at Ser473 and GSK3β at Ser9 was decreased in the DNA-PKcs depleted HeLa-H1 cells compared to control HeLa-NC cells. C: PDK regulates the phosphorylation of H2AX in response to DNA damage induced by 4 Gy of γ-irradiation. After 48 h incubation with 50 nM PDK-specific siRNA or non-specific (ns) control siRNA, cells were irradiated with 4 Gy γ rays, then harvested 0, 0.5, 1, 4, 10 h post-irradiation and analyzed by Western blotting. D: PDK regulates the phosphorylation of H2AX associated with cell cycle progression. After 24 h incubation with 50 nM PDK-specific siRNA or non-specific (ns) control siRNA, cells were synchronized in G1 phase by TdR double-blocking, then released and harvested after 5 h, for S-phase, and at 8, 9, and 10 h, for G2/M phase. The culture medium was supplemented with 50 nM siRNA molecules during the period of synchronization and cell cycle progression. Protein expression was assayed by Western blotting.

    Article Snippet: All antibodies were purchased commercially: anti-β-actin (I-19-R, Santa Cruz, CA), anti-DNA-PKcs (H-163, Santa Cruz, CA), γH2AX (05-636, Upstate Biotechnology, Charlottesville, VA), anti-GSK3β (#9332, Cell Signaling, Danvers, MA), anti-phospho-GSK3β (Ser-9, #9336, Cell Signaling, Danvers, MA), anti-PDK1 (#3062, Cell signaling, Danvers, MA), anti-phospho-Akt (Ser-473, #9271, Cell Signaling, Danvers, MA), anti-phospho-ATM (Ser-1981, Cell Signaling, Danvers, MA), anti-rabbit IgG(H+L)/HRP (ZB-2301, Zhongshan, Beijing, China), and anti-mouse IgG(H+L)/HRP (ZB-2305, Zhongshan, Beijing, China).

    Techniques: Transfection, Western Blot, Expressing, Irradiation, Incubation, Blocking Assay

    RASAL2 modulated VEGFA expression via p-GSK3β/c-FOS pathway in RCC. a Western blot analysis of c-FOS expression in ACHN or 786O sublines. b Western blot analysis of c-FOS and VEGFA expression in ACHN sublines transfected with c-FOS siRNA. c Western blot analysis of p-GSK3β S9 and GSK3β in ACHN or 786O sublines. d , e Western blot analysis of c-FOS and VEGFA in ACHN sublines treated with XAV-939 or 786O sublines treated with CT99021. The densitometric analysis of VEGFA expression was shown. f Immunoprecipitation assay of RASAL2 and GSK3β in 786O sublines. GAPDH was used as loading control

    Journal: Cell Death & Disease

    Article Title: The expression and function of RASAL2 in renal cell carcinoma angiogenesis

    doi: 10.1038/s41419-018-0898-x

    Figure Lengend Snippet: RASAL2 modulated VEGFA expression via p-GSK3β/c-FOS pathway in RCC. a Western blot analysis of c-FOS expression in ACHN or 786O sublines. b Western blot analysis of c-FOS and VEGFA expression in ACHN sublines transfected with c-FOS siRNA. c Western blot analysis of p-GSK3β S9 and GSK3β in ACHN or 786O sublines. d , e Western blot analysis of c-FOS and VEGFA in ACHN sublines treated with XAV-939 or 786O sublines treated with CT99021. The densitometric analysis of VEGFA expression was shown. f Immunoprecipitation assay of RASAL2 and GSK3β in 786O sublines. GAPDH was used as loading control

    Article Snippet: The antibodies used were as follows: RASAL2 (Rabbit, GeneTex, Inc., Irvine, CA, USA), GAPDH (mouse, KangChen Bio-tech, Shanghai, China), VEGFA (Rabbit, abcam, Inc., Cambridge, Britain), c-FOS (Rabbit, Santa Cruz Biotechnology, Dallas, TX, USA), GSK3β (Rabbit, Cell Signaling Technology, Danvers, MA, USA), p-GSK3βSer9 (Rabbit, Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Expressing, Western Blot, Transfection, Immunoprecipitation

    Summary of the mechanism of Dicer regulation in osteogenesis. Model demonstrates that Runx2 directly regulates Dicer expression to improve osteogenesis by up-regulating miR21a-5p targeting PTEN through activating Akt/Gsk3β/β-catenin signaling

    Journal: American Journal of Translational Research

    Article Title: Dicer-dependent pathway contribute to the osteogenesis mediated by regulation of Runx2

    doi:

    Figure Lengend Snippet: Summary of the mechanism of Dicer regulation in osteogenesis. Model demonstrates that Runx2 directly regulates Dicer expression to improve osteogenesis by up-regulating miR21a-5p targeting PTEN through activating Akt/Gsk3β/β-catenin signaling

    Article Snippet: Anti-phospho-AKT antibody, anti-AKT antibody, anti-phospho-GSK3β (Ser9) antibody, anti-GSK3β antibody, anti-β-catenin antibody and anti-β-actin antibody were purchased from Cell Signaling Technology (CST, MA, USA).

    Techniques: Expressing

    GSK3β controls microtubule dynamics by regulating Tau and CRMP2. A, differentiated podocytes were transfected with vectors encoding the hemagglutinin (HA)-conjugated wild type GSK3β ( WT ), kinase-dead mutant of GSK3β ( KD ), or constitutively

    Journal: The Journal of Biological Chemistry

    Article Title: Glycogen Synthase Kinase 3β Orchestrates Microtubule Remodeling in Compensatory Glomerular Adaptation to Podocyte Depletion *

    doi: 10.1074/jbc.M114.593830

    Figure Lengend Snippet: GSK3β controls microtubule dynamics by regulating Tau and CRMP2. A, differentiated podocytes were transfected with vectors encoding the hemagglutinin (HA)-conjugated wild type GSK3β ( WT ), kinase-dead mutant of GSK3β ( KD ), or constitutively

    Article Snippet: Podocytes or kidney cryosections were fixed with 4% paraformaldehyde, permeabilized, and stained with primary antibodies against detyrosinated tubulin (Millipore), GSK3β (Santa Cruz Biotechnology, Dallas), Tau (Santa Cruz Biotechnology), CRMP2 (Abcam, Cambridge, MA), phosphorylated Tau (Ser-396) (Sigma), phosphorylated CRMP2 (Abcam), HA (Santa Cruz Biotechnology), synaptopodin (Santa Cruz Biotechnology), and WT-1 (Santa Cruz Biotechnology), and then with the Alexa Fluorophore-conjugated secondary antibody (Invitrogen).

    Techniques: Transfection, Mutagenesis

    Rescue treatment with a single low dose of lithium, an inhibitor of GSK3β and Food and Drug Administration-approved mood stabilizer, in experimental adriamycin nephropathy promotes major process elongation in remnant surviving podocytes to compensate

    Journal: The Journal of Biological Chemistry

    Article Title: Glycogen Synthase Kinase 3β Orchestrates Microtubule Remodeling in Compensatory Glomerular Adaptation to Podocyte Depletion *

    doi: 10.1074/jbc.M114.593830

    Figure Lengend Snippet: Rescue treatment with a single low dose of lithium, an inhibitor of GSK3β and Food and Drug Administration-approved mood stabilizer, in experimental adriamycin nephropathy promotes major process elongation in remnant surviving podocytes to compensate

    Article Snippet: Podocytes or kidney cryosections were fixed with 4% paraformaldehyde, permeabilized, and stained with primary antibodies against detyrosinated tubulin (Millipore), GSK3β (Santa Cruz Biotechnology, Dallas), Tau (Santa Cruz Biotechnology), CRMP2 (Abcam, Cambridge, MA), phosphorylated Tau (Ser-396) (Sigma), phosphorylated CRMP2 (Abcam), HA (Santa Cruz Biotechnology), synaptopodin (Santa Cruz Biotechnology), and WT-1 (Santa Cruz Biotechnology), and then with the Alexa Fluorophore-conjugated secondary antibody (Invitrogen).

    Techniques:

    Lithium counteracts GSK3β overactivity, diminishes Tau and CRMP2 phosphorylation, and reinstates microtubule integrity in adriamycin-injured glomeruli. A, immunoblot analysis of homogenates of isolated glomeruli for indicated molecules. B, arbitrary

    Journal: The Journal of Biological Chemistry

    Article Title: Glycogen Synthase Kinase 3β Orchestrates Microtubule Remodeling in Compensatory Glomerular Adaptation to Podocyte Depletion *

    doi: 10.1074/jbc.M114.593830

    Figure Lengend Snippet: Lithium counteracts GSK3β overactivity, diminishes Tau and CRMP2 phosphorylation, and reinstates microtubule integrity in adriamycin-injured glomeruli. A, immunoblot analysis of homogenates of isolated glomeruli for indicated molecules. B, arbitrary

    Article Snippet: Podocytes or kidney cryosections were fixed with 4% paraformaldehyde, permeabilized, and stained with primary antibodies against detyrosinated tubulin (Millipore), GSK3β (Santa Cruz Biotechnology, Dallas), Tau (Santa Cruz Biotechnology), CRMP2 (Abcam, Cambridge, MA), phosphorylated Tau (Ser-396) (Sigma), phosphorylated CRMP2 (Abcam), HA (Santa Cruz Biotechnology), synaptopodin (Santa Cruz Biotechnology), and WT-1 (Santa Cruz Biotechnology), and then with the Alexa Fluorophore-conjugated secondary antibody (Invitrogen).

    Techniques: Isolation

    Tau and CRMP2 colocalize and physically interact with GSK3β as its putative substrates in podocytes. A, representative micrographs of laser scanning confocal microscopy of dual color fluorescent immunocytochemistry staining of GSK3β and

    Journal: The Journal of Biological Chemistry

    Article Title: Glycogen Synthase Kinase 3β Orchestrates Microtubule Remodeling in Compensatory Glomerular Adaptation to Podocyte Depletion *

    doi: 10.1074/jbc.M114.593830

    Figure Lengend Snippet: Tau and CRMP2 colocalize and physically interact with GSK3β as its putative substrates in podocytes. A, representative micrographs of laser scanning confocal microscopy of dual color fluorescent immunocytochemistry staining of GSK3β and

    Article Snippet: Podocytes or kidney cryosections were fixed with 4% paraformaldehyde, permeabilized, and stained with primary antibodies against detyrosinated tubulin (Millipore), GSK3β (Santa Cruz Biotechnology, Dallas), Tau (Santa Cruz Biotechnology), CRMP2 (Abcam, Cambridge, MA), phosphorylated Tau (Ser-396) (Sigma), phosphorylated CRMP2 (Abcam), HA (Santa Cruz Biotechnology), synaptopodin (Santa Cruz Biotechnology), and WT-1 (Santa Cruz Biotechnology), and then with the Alexa Fluorophore-conjugated secondary antibody (Invitrogen).

    Techniques: Confocal Microscopy, Immunocytochemistry, Staining

    Delayed Inhibition of GSK3β Promotes Podocyte Process Elongation to Compensate for Podocyte Depletion in Adriamycin Nephropathy

    Journal: The Journal of Biological Chemistry

    Article Title: Glycogen Synthase Kinase 3β Orchestrates Microtubule Remodeling in Compensatory Glomerular Adaptation to Podocyte Depletion *

    doi: 10.1074/jbc.M114.593830

    Figure Lengend Snippet: Delayed Inhibition of GSK3β Promotes Podocyte Process Elongation to Compensate for Podocyte Depletion in Adriamycin Nephropathy

    Article Snippet: Podocytes or kidney cryosections were fixed with 4% paraformaldehyde, permeabilized, and stained with primary antibodies against detyrosinated tubulin (Millipore), GSK3β (Santa Cruz Biotechnology, Dallas), Tau (Santa Cruz Biotechnology), CRMP2 (Abcam, Cambridge, MA), phosphorylated Tau (Ser-396) (Sigma), phosphorylated CRMP2 (Abcam), HA (Santa Cruz Biotechnology), synaptopodin (Santa Cruz Biotechnology), and WT-1 (Santa Cruz Biotechnology), and then with the Alexa Fluorophore-conjugated secondary antibody (Invitrogen).

    Techniques: Inhibition

    miR-UL112-3p negatively regulates TUSC3 expression in GBM. ( A ) The predicted miR-UL112-3p target sequences in TUSC3 mRNA is shown (green bases indicate matching base pairs, and the red bases represent non-matching base pairs). ( B ) Luciferase assays of HEK293 and GBM cells co-transfected with wild-type/mutant pMIR-REPORT 3′UTR of TUSC3 and miR-UL112-3p mimics/inhibitor or the negative control, as indicated. ( C ) Immunohistochemistry analysis of TUSC3 expression in GBM specimens. ( D ) Spearman’s correlation analysis was used to determine the correlation between the expression levels of TUSC3 and miR-UL112-3p in human GBM samples; Spearman’s correlation, r = −0.7589 (n = 20). ( E ) The expression of TUSC3 was detected by Western blotting in GBM cells. ( F,G ) The protein levels of total and phosphorylated AKT, caspase 9, p27, p-GSK3β and GSK3β in GBM cells, as determined by Western blot analysis. Relative protein expression was calculated based on densitometric analysis of band intensities. Full-length blots are presented in Supplementary Figures S4–S6 . The data are expressed as the means ± SD. * P

    Journal: Scientific Reports

    Article Title: HCMV-encoded miR-UL112-3p promotes glioblastoma progression via tumour suppressor candidate 3

    doi: 10.1038/srep44705

    Figure Lengend Snippet: miR-UL112-3p negatively regulates TUSC3 expression in GBM. ( A ) The predicted miR-UL112-3p target sequences in TUSC3 mRNA is shown (green bases indicate matching base pairs, and the red bases represent non-matching base pairs). ( B ) Luciferase assays of HEK293 and GBM cells co-transfected with wild-type/mutant pMIR-REPORT 3′UTR of TUSC3 and miR-UL112-3p mimics/inhibitor or the negative control, as indicated. ( C ) Immunohistochemistry analysis of TUSC3 expression in GBM specimens. ( D ) Spearman’s correlation analysis was used to determine the correlation between the expression levels of TUSC3 and miR-UL112-3p in human GBM samples; Spearman’s correlation, r = −0.7589 (n = 20). ( E ) The expression of TUSC3 was detected by Western blotting in GBM cells. ( F,G ) The protein levels of total and phosphorylated AKT, caspase 9, p27, p-GSK3β and GSK3β in GBM cells, as determined by Western blot analysis. Relative protein expression was calculated based on densitometric analysis of band intensities. Full-length blots are presented in Supplementary Figures S4–S6 . The data are expressed as the means ± SD. * P

    Article Snippet: Primary antibodies anti-TUSC3, anti-phospho-AKT (Ser 473), anti-AKT, anti-caspase 9, anti-GSK3β, anti-p-GSK3β (Ser 9), anti-p27 and anti-GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Luciferase, Transfection, Mutagenesis, Negative Control, Immunohistochemistry, Western Blot

    High glucose-induced phosphorylation of GSK3β. Endothelial cells were grown in serum-free medium containing normal glucose (NG, 5.5 mM) or high glucose (HG, 25 mM) with or without VEGFR inhibitor (VEGFRI) for 1 to 5 days. Equal amounts of SDS extracted protein samples were immunoprecipitated (IP) by anti-GSK3β antibody and subjected to SDS-PAGE and Western blotting (A). Densitometric analysis of phospho-GSK3β bands, normalized for the corresponding GSK3β bands, showed that phosho-GSK3β levels were significantly increased by HG or VEGF treatment and that this effect was reduced in VEGFI treated samples compared to HG or VEGF treated endothelial cells (B). * = P

    Journal: PLoS ONE

    Article Title: Diabetes-Induced Superoxide Anion and Breakdown of the Blood-Retinal Barrier: Role of the VEGF/uPAR Pathway

    doi: 10.1371/journal.pone.0071868

    Figure Lengend Snippet: High glucose-induced phosphorylation of GSK3β. Endothelial cells were grown in serum-free medium containing normal glucose (NG, 5.5 mM) or high glucose (HG, 25 mM) with or without VEGFR inhibitor (VEGFRI) for 1 to 5 days. Equal amounts of SDS extracted protein samples were immunoprecipitated (IP) by anti-GSK3β antibody and subjected to SDS-PAGE and Western blotting (A). Densitometric analysis of phospho-GSK3β bands, normalized for the corresponding GSK3β bands, showed that phosho-GSK3β levels were significantly increased by HG or VEGF treatment and that this effect was reduced in VEGFI treated samples compared to HG or VEGF treated endothelial cells (B). * = P

    Article Snippet: Aliquots were taken for protein assay and the remaining material was used immediately or stored at -70° C. Anti-GSK3β antibody (Santa Cruz Biotechnology) and protein-A/G conjugated sepharose beads were added to the cell extracts and placed in a shaker at 4o C overnight.

    Techniques: Immunoprecipitation, SDS Page, Western Blot

    VACV-Expressed B14 Associates with the IKK Signaling Complex Cells were co-transfected with expression vectors for indicated proteins tagged with HA (A) or FLAG (B) epitopes and the indicated protein fused with luciferase. The indicated tagged bait protein was immunoprecipated by correspondent monoclonal Ab and then the immune complex was eluted to be analysed for luciferase activity. Data presented are from one of the three independent experiments. (C) HeLa cells were infected with vB14 and a cell extract was prepared and analysed by size exclusion chromatography on a Superose 6 column. An aliquot of each fraction was separated in 4%–12% NuPAGE and analysed by immunoblotting with the indicated Abs. The position of protein size markers is indicated in kDa at the top. (D) Extracts from HeLa cells infected with the vΔB14 (Δ) or vB14-HA (HA) were pre-cleared and immunoprecipitated with mAbs against HA, NEMO, and GSK3β (isotype control). The immunoprecipitated proteins were resolved in 4%–12% NuPAGE and then were analysed by immunoblotting with the Abs indicated on the right, respectively. Sizes of bands detected are indicated on the left in kDa.

    Journal: PLoS Pathogens

    Article Title: Inhibition of I?B Kinase by Vaccinia Virus Virulence Factor B14

    doi: 10.1371/journal.ppat.0040022

    Figure Lengend Snippet: VACV-Expressed B14 Associates with the IKK Signaling Complex Cells were co-transfected with expression vectors for indicated proteins tagged with HA (A) or FLAG (B) epitopes and the indicated protein fused with luciferase. The indicated tagged bait protein was immunoprecipated by correspondent monoclonal Ab and then the immune complex was eluted to be analysed for luciferase activity. Data presented are from one of the three independent experiments. (C) HeLa cells were infected with vB14 and a cell extract was prepared and analysed by size exclusion chromatography on a Superose 6 column. An aliquot of each fraction was separated in 4%–12% NuPAGE and analysed by immunoblotting with the indicated Abs. The position of protein size markers is indicated in kDa at the top. (D) Extracts from HeLa cells infected with the vΔB14 (Δ) or vB14-HA (HA) were pre-cleared and immunoprecipitated with mAbs against HA, NEMO, and GSK3β (isotype control). The immunoprecipitated proteins were resolved in 4%–12% NuPAGE and then were analysed by immunoblotting with the Abs indicated on the right, respectively. Sizes of bands detected are indicated on the left in kDa.

    Article Snippet: Anti-IKKγ (NEMO) (BD Biosciences), anti-HA (Cambridge Biosciences), anti-GSK3β (BD Biosciences) mAbs were used for immunoprecipitation or immunoblotting.

    Techniques: Transfection, Expressing, Luciferase, Activity Assay, Infection, Size-exclusion Chromatography, Immunoprecipitation

    Loss of ERK1/2 signaling does not affect Wnt signaling in the developing hippocampus. A , Western analysis of GSK3β Ser9 phosphorylation in E14.5 and P2 WT, CKO, and DKO mice (normalized to total GSK3β). Mean ± SEM: E14.5, WT: 1.000

    Journal: The Journal of Neuroscience

    Article Title: Dentate Gyrus Development Requires ERK Activity to Maintain Progenitor Population and MAPK Pathway Feedback Regulation

    doi: 10.1523/JNEUROSCI.4196-14.2015

    Figure Lengend Snippet: Loss of ERK1/2 signaling does not affect Wnt signaling in the developing hippocampus. A , Western analysis of GSK3β Ser9 phosphorylation in E14.5 and P2 WT, CKO, and DKO mice (normalized to total GSK3β). Mean ± SEM: E14.5, WT: 1.000

    Article Snippet: Primary antibodies used were as follows: phospho-ERK1/2 (Cell Signaling Technology, 1:5000), ERK1/2 (Cell Signaling Technology, 1:5000), phospho-MEK (Cell Signaling Technology, 1:1000), MEK1/2 (Cell Signaling Technology, 1:1000), phospho-C-Raf (S259) (Cell Signaling Technology, 1:2000), C-Raf (Cell Signaling Technology, 1:1000), Sprouty1 (Cell Signaling Technology, 1:1000), phospho-GSK3β (Ser9) (BD Biosciences, 1:100), GSK3β (BD Biosciences PharMingen, 1:1000), β-catenin (BD Biosciences, 1:10,000), Axin2 (Abcam, 1:1000), Reelin (Millipore, 1:1000), and tubulin (LI-COR, 1:1000).

    Techniques: Western Blot, Mouse Assay

    ILK overexpression enhanced PLC cell growth and motility. ( A ) FLAG-tagged ILK was transduced into PLC cells and stable clone of ILK was established. Western blot analysis confirmed stable FLAG-ILK expression in PLC cells but not in vector control clone. ( B ) PLC/vector and PLC/FLAG-ILK cells were counted in triplicates for 8 consecutive days. ( C ) PLC ILK overexpressing cells were subjected to migration assay. Cells were seeded in triplicates and allowed to migrate for 16 hours. Migrated cells were fixed and stained by crystal violet. ( D ) PLC and HEK293T cells overexpressing ILK were collected for western blot analysis to study the phosphorylation of Akt and GSK3β. Expression of β-actin was included as an internal loading control. * P

    Journal: PLoS ONE

    Article Title: Integrin-Linked Kinase Overexpression and Its Oncogenic Role in Promoting Tumorigenicity of Hepatocellular Carcinoma

    doi: 10.1371/journal.pone.0016984

    Figure Lengend Snippet: ILK overexpression enhanced PLC cell growth and motility. ( A ) FLAG-tagged ILK was transduced into PLC cells and stable clone of ILK was established. Western blot analysis confirmed stable FLAG-ILK expression in PLC cells but not in vector control clone. ( B ) PLC/vector and PLC/FLAG-ILK cells were counted in triplicates for 8 consecutive days. ( C ) PLC ILK overexpressing cells were subjected to migration assay. Cells were seeded in triplicates and allowed to migrate for 16 hours. Migrated cells were fixed and stained by crystal violet. ( D ) PLC and HEK293T cells overexpressing ILK were collected for western blot analysis to study the phosphorylation of Akt and GSK3β. Expression of β-actin was included as an internal loading control. * P

    Article Snippet: Anti-GSK3β antibody was purchased from BD Bioscience and anti-β-actin antibody was from Sigma-Aldrich.

    Techniques: Over Expression, Planar Chromatography, Stable Transfection, Western Blot, Expressing, Plasmid Preparation, Migration, Staining

    ILK knockdown suppressed phosphorylation of Akt and GSK3β. Insulin was added to cells to stimulate the PKB/Akt signaling pathway. Cell lysates were then collected for western blotting analysis. Expression levels of pAkt, total Akt, pGSK3β, total GSK3β and ILK were shown. Expression of β-actin was included as an internal loading control. The band intensities were determined by densitometry, and the amount of pAkt and pGSK3β was normalized with that of total Akt and GSK3β respectively.

    Journal: PLoS ONE

    Article Title: Integrin-Linked Kinase Overexpression and Its Oncogenic Role in Promoting Tumorigenicity of Hepatocellular Carcinoma

    doi: 10.1371/journal.pone.0016984

    Figure Lengend Snippet: ILK knockdown suppressed phosphorylation of Akt and GSK3β. Insulin was added to cells to stimulate the PKB/Akt signaling pathway. Cell lysates were then collected for western blotting analysis. Expression levels of pAkt, total Akt, pGSK3β, total GSK3β and ILK were shown. Expression of β-actin was included as an internal loading control. The band intensities were determined by densitometry, and the amount of pAkt and pGSK3β was normalized with that of total Akt and GSK3β respectively.

    Article Snippet: Anti-GSK3β antibody was purchased from BD Bioscience and anti-β-actin antibody was from Sigma-Aldrich.

    Techniques: Western Blot, Expressing

    Inhibiting SLC26A4 ameliorates the expression levels of cardiomyocyte hypertrophy markers in PE-induced cardiac hypertrophy. RT-qPCR results showing the relative mRNA expression levels of (A) ANP; (B) BNP and (C) GSK3β in PE-induced H9C2 cells transfected by siRNA-SLC26A4. The relative expression level of ANP, BNP and GSK3β was determined using the 2 −ΔΔ Ct method. All experiments were performed at least three times. Data represent mean ± SD. * P -value

    Journal: PeerJ

    Article Title: Inhibiting SLC26A4 reverses cardiac hypertrophy in H9C2 cells and in rats

    doi: 10.7717/peerj.8253

    Figure Lengend Snippet: Inhibiting SLC26A4 ameliorates the expression levels of cardiomyocyte hypertrophy markers in PE-induced cardiac hypertrophy. RT-qPCR results showing the relative mRNA expression levels of (A) ANP; (B) BNP and (C) GSK3β in PE-induced H9C2 cells transfected by siRNA-SLC26A4. The relative expression level of ANP, BNP and GSK3β was determined using the 2 −ΔΔ Ct method. All experiments were performed at least three times. Data represent mean ± SD. * P -value

    Article Snippet: The sections were incubated with primary antibodies including anti-ANP (Sangon Biotech Co., Ltd.), anti-BNP (Sangon Biotech Co., Ltd.) and anti-GSK3β (Sangon Biotech Co., Ltd.) overnight, followed by secondary antibody.

    Techniques: Expressing, Quantitative RT-PCR, Aqueous Normal-phase Chromatography, Transfection

    The TRAX/DISC1/GSK3β (TDG) complex is involved in the protective effect of an A 2A R agonist. a , b PC12 cells were treated with an inhibitor of protein kinase A (H89; 10 μM and KT5720; 10 μM) or vehicle for 30 min and then treated with CGS (10 μM) or vehicle for 1 h ( a ) and 8 h ( b ). Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-GSK3β Ser9, anti-GSK3β, anti-β-catenin and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. c – e In PC12 cells and human MSN neurons, cells were infected with lentivirus expressing GSK3β or a constitutively active GSK3β mutant (GSK3β-S9A) for 3 days. Cells were treated with an A 2A R agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-α-Tubulin and anti-β-Actin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin or β-Actin, the loading controls. These experiments were repeated three times ( c , d ). Apoptosis was assessed by co-staining with Annexin V-Cy5 and PI for 10 min followed by flow cytometric analysis ( e ). f Protein–protein interactions were monitored by the proximity ligation assay [ 31 ] using the corresponding antibodies as described in the Methods section [ 31 ]. Each red dot represents the detection of protein–protein interaction. Scale bar, 10 μm. g PC12 cells were treated with an A 2A R agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h. Cells were lysed, subjected to immunoprecipitation with anti-DISC1 and immunoblotted with anti-DISC1, anti-TRAX and anti-GSK3β antibodies. The amount of target protein was quantified and normalized to that of the input. These experiments were repeated three times. h PC12 cells were infected with the lentivirus expressing A 2A R 253–410 -Flag (M7) for 3 days. Cells were lysed, subjected to immunoprecipitation with an anti-A 2A R antibody or a control IgG, followed by Western blot analysis using the indicated antibody. The amount of TRAX that interacted with A 2A R was quantified and normalized to that of the input. These experiments were repeated three times. i PC12 cells were infected with lentivirus expressing A 2A R 253–410 -Flag for 3 days. Cells were treated with CGS (10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-Flag and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading controls. These experiments were repeated three times. j PC12 cells were treated with CGS (10 μM) during the indicated time period. Cells were subjected to immunostaining with an anti-TRAX antibody (Green). Scale bar, 10 μm. k PC12-shTRAX cells were transfected with TRAX-V5 and ΔNLS TRAX-V5 plasmids. Cells were treated with CGS (10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were subjected to immunostaining with anti-γH2AX (Green) and anti-V5 (Red) antibodies. Scale bar, 10 μm. Data are presented as the mean ± SEM from at least three independent experiments. * P

    Journal: Molecular Psychiatry

    Article Title: GSK3β negatively regulates TRAX, a scaffold protein implicated in mental disorders, for NHEJ-mediated DNA repair in neurons

    doi: 10.1038/s41380-017-0007-z

    Figure Lengend Snippet: The TRAX/DISC1/GSK3β (TDG) complex is involved in the protective effect of an A 2A R agonist. a , b PC12 cells were treated with an inhibitor of protein kinase A (H89; 10 μM and KT5720; 10 μM) or vehicle for 30 min and then treated with CGS (10 μM) or vehicle for 1 h ( a ) and 8 h ( b ). Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-GSK3β Ser9, anti-GSK3β, anti-β-catenin and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. c – e In PC12 cells and human MSN neurons, cells were infected with lentivirus expressing GSK3β or a constitutively active GSK3β mutant (GSK3β-S9A) for 3 days. Cells were treated with an A 2A R agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-α-Tubulin and anti-β-Actin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin or β-Actin, the loading controls. These experiments were repeated three times ( c , d ). Apoptosis was assessed by co-staining with Annexin V-Cy5 and PI for 10 min followed by flow cytometric analysis ( e ). f Protein–protein interactions were monitored by the proximity ligation assay [ 31 ] using the corresponding antibodies as described in the Methods section [ 31 ]. Each red dot represents the detection of protein–protein interaction. Scale bar, 10 μm. g PC12 cells were treated with an A 2A R agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h. Cells were lysed, subjected to immunoprecipitation with anti-DISC1 and immunoblotted with anti-DISC1, anti-TRAX and anti-GSK3β antibodies. The amount of target protein was quantified and normalized to that of the input. These experiments were repeated three times. h PC12 cells were infected with the lentivirus expressing A 2A R 253–410 -Flag (M7) for 3 days. Cells were lysed, subjected to immunoprecipitation with an anti-A 2A R antibody or a control IgG, followed by Western blot analysis using the indicated antibody. The amount of TRAX that interacted with A 2A R was quantified and normalized to that of the input. These experiments were repeated three times. i PC12 cells were infected with lentivirus expressing A 2A R 253–410 -Flag for 3 days. Cells were treated with CGS (10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-Flag and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading controls. These experiments were repeated three times. j PC12 cells were treated with CGS (10 μM) during the indicated time period. Cells were subjected to immunostaining with an anti-TRAX antibody (Green). Scale bar, 10 μm. k PC12-shTRAX cells were transfected with TRAX-V5 and ΔNLS TRAX-V5 plasmids. Cells were treated with CGS (10 μM) or vehicle for 1 h to activate the A 2A R and then treated with H 2 O 2 (100 μM) for 4 h. Cells were subjected to immunostaining with anti-γH2AX (Green) and anti-V5 (Red) antibodies. Scale bar, 10 μm. Data are presented as the mean ± SEM from at least three independent experiments. * P

    Article Snippet: The sources of the antibodies used in western blot analyses are listed below: anti-γH2AX (Millipore; 05–636), anti-α-tubulin (Genetex, Irvine, CA, USA; GTX76511), anti-TRAX [ ], anti-β-actin (Genetex; GTX11003), anti-DNA-PKCS Thr2609 (Abnova, Cambridge, United Kingdom; PAB10324), anti-DNA-PKCS (Abcam, Cambridge, United Kingdom; ab1832), anti-GSK3β Ser9 (Cell Signaling, Danvers, MA, USA; 9336), anti-GSK3β (Genetex; GTX83315), anti-β-catenin (Genetex; GTX61089), anti-DISC1 (Thermo; 710203), anti-A2A R (Santa cruz, Dallas, Texas, USA; SC-32261), and anti-PARP (Genetex; GTX112864).

    Techniques: SDS Page, Western Blot, Infection, Expressing, Mutagenesis, Staining, Flow Cytometry, Proximity Ligation Assay, Immunoprecipitation, Immunostaining, Transfection

    Inhibition of GSK3β enables TRAX-dependent DNA repair. a , b PC12-pSuper and PC12-shTRAX cells were treated with a GSK3β inhibitor (SB216763, 10 μM) or vehicle for 2 days. After the abovementioned treatment, cells were subjected to H 2 O 2 (100 μM) for 1–4 h as indicated. The levels of γH2AX, β-catenin, and α-Tubulin (a loading control) were evaluated by Western blot analysis using the indicated antibodies. The relative amounts of target proteins were quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. c Primary hippocampal neurons (DIV14) harvested from wild-type B6/C57 mice were treated with SB216763 (10 μM) or vehicle for 1 day and then treated with H 2 O 2 (100 μM) for 2 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX and anti-α-Tubulin antibodies, as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. d Human iPSCs-derived MSN neurons were treated with SB216763 (10 μM) or vehicle for 1 day and then treated with H 2 O 2 (100 μM) for 4 h. DNA damage was assessed by determining the intensity of the DNA damage marker γH2AX by immunofluorescence staining using the anti-γH2AX antibody (green) in neurons identified by a neuronal marker (TUJ1, red). Scale bar, 50 μm. e PC12 cells were treated with a GSK3β inhibitor (SB216763, 10 μM) or vehicle for 2 days and nuclear and cytosolic fractions were isolated. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-TRAX, anti-α-Tubulin and anti-PARP antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin or PARP, the loading controls for post-nucleus and nucleus fractions, respectively. These experiments were repeated three times. The data are presented as the mean ± SEM from at least three independent experiments0. * P

    Journal: Molecular Psychiatry

    Article Title: GSK3β negatively regulates TRAX, a scaffold protein implicated in mental disorders, for NHEJ-mediated DNA repair in neurons

    doi: 10.1038/s41380-017-0007-z

    Figure Lengend Snippet: Inhibition of GSK3β enables TRAX-dependent DNA repair. a , b PC12-pSuper and PC12-shTRAX cells were treated with a GSK3β inhibitor (SB216763, 10 μM) or vehicle for 2 days. After the abovementioned treatment, cells were subjected to H 2 O 2 (100 μM) for 1–4 h as indicated. The levels of γH2AX, β-catenin, and α-Tubulin (a loading control) were evaluated by Western blot analysis using the indicated antibodies. The relative amounts of target proteins were quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. c Primary hippocampal neurons (DIV14) harvested from wild-type B6/C57 mice were treated with SB216763 (10 μM) or vehicle for 1 day and then treated with H 2 O 2 (100 μM) for 2 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX and anti-α-Tubulin antibodies, as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. d Human iPSCs-derived MSN neurons were treated with SB216763 (10 μM) or vehicle for 1 day and then treated with H 2 O 2 (100 μM) for 4 h. DNA damage was assessed by determining the intensity of the DNA damage marker γH2AX by immunofluorescence staining using the anti-γH2AX antibody (green) in neurons identified by a neuronal marker (TUJ1, red). Scale bar, 50 μm. e PC12 cells were treated with a GSK3β inhibitor (SB216763, 10 μM) or vehicle for 2 days and nuclear and cytosolic fractions were isolated. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-TRAX, anti-α-Tubulin and anti-PARP antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin or PARP, the loading controls for post-nucleus and nucleus fractions, respectively. These experiments were repeated three times. The data are presented as the mean ± SEM from at least three independent experiments0. * P

    Article Snippet: The sources of the antibodies used in western blot analyses are listed below: anti-γH2AX (Millipore; 05–636), anti-α-tubulin (Genetex, Irvine, CA, USA; GTX76511), anti-TRAX [ ], anti-β-actin (Genetex; GTX11003), anti-DNA-PKCS Thr2609 (Abnova, Cambridge, United Kingdom; PAB10324), anti-DNA-PKCS (Abcam, Cambridge, United Kingdom; ab1832), anti-GSK3β Ser9 (Cell Signaling, Danvers, MA, USA; 9336), anti-GSK3β (Genetex; GTX83315), anti-β-catenin (Genetex; GTX61089), anti-DISC1 (Thermo; 710203), anti-A2A R (Santa cruz, Dallas, Texas, USA; SC-32261), and anti-PARP (Genetex; GTX112864).

    Techniques: Inhibition, Western Blot, Mouse Assay, SDS Page, Derivative Assay, Marker, Immunofluorescence, Staining, Isolation

    Fap1-inhibition with SLV peptide increases Fas and Gsk3β phosphorylation in CD133 + cells in a murine xenograft model SW620 cells were injected in the flanks of athymic Nude mice and tumor volume was determination biweekly. Mice were treated weekly with oxaliplatin (days 0, 7 and 14) and injected daily with Fap1 blocking SLV peptide or VLS control peptide, or treated with SLV or VLS peptide alone (n=12 per cohort). Tumors were simultaneously harvested from cohorts of mice when control tumors were > 2,000 mm3. (A) SLV peptide increases Fas phosphorylation in CD133 + xenograft tumors with or without oxaliplatin. Immunofluorescent detection of phospho-Fas or CD133 was performed with DAPI staining of nuclei. (B) SLV peptide increases Gsk3β phosphorylation in CD133 + xenograft tumors with or without oxaliplatin. Immunofluorescent detection of phospho-Gsk3β or CD133 was performed with DAPI staining of nuclei.

    Journal: Oncotarget

    Article Title: Inhibition of Fas associated phosphatase 1 (Fap1) facilitates apoptosis of colon cancer stem cells and enhances the effects of oxaliplatin

    doi: 10.18632/oncotarget.25401

    Figure Lengend Snippet: Fap1-inhibition with SLV peptide increases Fas and Gsk3β phosphorylation in CD133 + cells in a murine xenograft model SW620 cells were injected in the flanks of athymic Nude mice and tumor volume was determination biweekly. Mice were treated weekly with oxaliplatin (days 0, 7 and 14) and injected daily with Fap1 blocking SLV peptide or VLS control peptide, or treated with SLV or VLS peptide alone (n=12 per cohort). Tumors were simultaneously harvested from cohorts of mice when control tumors were > 2,000 mm3. (A) SLV peptide increases Fas phosphorylation in CD133 + xenograft tumors with or without oxaliplatin. Immunofluorescent detection of phospho-Fas or CD133 was performed with DAPI staining of nuclei. (B) SLV peptide increases Gsk3β phosphorylation in CD133 + xenograft tumors with or without oxaliplatin. Immunofluorescent detection of phospho-Gsk3β or CD133 was performed with DAPI staining of nuclei.

    Article Snippet: Antibodies include: anti-Fap1 or anti-Fas (Abcam, Cambridge, MA; ab198882, ab110021); anti-phospho Fas or anti-phospho Gsk3 beta (Thermo Fisher, Rockford, IL; PAS-38490, 702230); anti-Gsk3β (Novus, Littleton, CO; NBP1-47470); Cy3 AffiniPure Donkey Anti-Rabbit IgG, Cy2 AffiniPure Donkey Anti-Goat IgG, or Cy2 AffiniPure Donkey Anti-Mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA; 711-165-152, 705-225-147, 715-225-150).

    Techniques: Inhibition, Injection, Mouse Assay, Blocking Assay, Staining

    Fap1-inhibition with SLV peptide increases phosphorylation of Fap1-substrates Fas and Gsk3β in a murine xenograft model SW620 cells were injected in the flanks of athymic Nude mice and tumor volume was determination biweekly. Mice were treated weekly with oxaliplatin (days 0, 7 and 14) and injected daily with Fap1 blocking SLV peptide or VLS control peptide, or treated with SLV or VLS peptide alone (n=12 per cohort). Tumors were simultaneously harvested from cohorts of mice when control tumors were > 2,000 mm3. (A) SLV peptide increases gland formation in xenograft tumors with or without oxaliplatin. Histology was analyzed by hematoxylin/ eosin staining. Fap1 expression was determined by immunofluorescence. Relative fluorescent intensity (RFI) of Fap1 staining is indicated below relevant panels. (B) SLV peptide increases Fas-phosphorylation in xenograft tumors with or without by oxaliplatin. Immunofluorescent detection of total versus phospho-Fas was performed with DAPI staining of nuclei. Areas without gland formation were selected for this study. (C) SLV peptide increases Gsk3β-phosphorylation with or without oxaliplatin. Immunofluorescent detection of total versus phospho- Gsk3β was performed with DAPI staining of nuclei. Areas without gland formation were selected for this study.

    Journal: Oncotarget

    Article Title: Inhibition of Fas associated phosphatase 1 (Fap1) facilitates apoptosis of colon cancer stem cells and enhances the effects of oxaliplatin

    doi: 10.18632/oncotarget.25401

    Figure Lengend Snippet: Fap1-inhibition with SLV peptide increases phosphorylation of Fap1-substrates Fas and Gsk3β in a murine xenograft model SW620 cells were injected in the flanks of athymic Nude mice and tumor volume was determination biweekly. Mice were treated weekly with oxaliplatin (days 0, 7 and 14) and injected daily with Fap1 blocking SLV peptide or VLS control peptide, or treated with SLV or VLS peptide alone (n=12 per cohort). Tumors were simultaneously harvested from cohorts of mice when control tumors were > 2,000 mm3. (A) SLV peptide increases gland formation in xenograft tumors with or without oxaliplatin. Histology was analyzed by hematoxylin/ eosin staining. Fap1 expression was determined by immunofluorescence. Relative fluorescent intensity (RFI) of Fap1 staining is indicated below relevant panels. (B) SLV peptide increases Fas-phosphorylation in xenograft tumors with or without by oxaliplatin. Immunofluorescent detection of total versus phospho-Fas was performed with DAPI staining of nuclei. Areas without gland formation were selected for this study. (C) SLV peptide increases Gsk3β-phosphorylation with or without oxaliplatin. Immunofluorescent detection of total versus phospho- Gsk3β was performed with DAPI staining of nuclei. Areas without gland formation were selected for this study.

    Article Snippet: Antibodies include: anti-Fap1 or anti-Fas (Abcam, Cambridge, MA; ab198882, ab110021); anti-phospho Fas or anti-phospho Gsk3 beta (Thermo Fisher, Rockford, IL; PAS-38490, 702230); anti-Gsk3β (Novus, Littleton, CO; NBP1-47470); Cy3 AffiniPure Donkey Anti-Rabbit IgG, Cy2 AffiniPure Donkey Anti-Goat IgG, or Cy2 AffiniPure Donkey Anti-Mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA; 711-165-152, 705-225-147, 715-225-150).

    Techniques: Inhibition, Injection, Mouse Assay, Blocking Assay, Staining, Expressing, Immunofluorescence

    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.

    Journal: The Journal of investigative dermatology

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

    doi: 10.1038/jid.2012.237

    Figure Lengend 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.

    Article Snippet: The rabbit primary antibody for p-GSK3β was from Cell Signaling Technology and the antibody for total GSK3β was from Epitomics (Burlingame, CA).

    Techniques: 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

    Journal: The Journal of investigative dermatology

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

    doi: 10.1038/jid.2012.237

    Figure Lengend 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

    Article Snippet: The rabbit primary antibody for p-GSK3β was from Cell Signaling Technology and the antibody for total GSK3β was from Epitomics (Burlingame, CA).

    Techniques: 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.

    Journal: The Journal of investigative dermatology

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

    doi: 10.1038/jid.2012.237

    Figure Lengend 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.

    Article Snippet: The rabbit primary antibody for p-GSK3β was from Cell Signaling Technology and the antibody for total GSK3β was from Epitomics (Burlingame, CA).

    Techniques: 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

    Journal: The Journal of investigative dermatology

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

    doi: 10.1038/jid.2012.237

    Figure Lengend 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

    Article Snippet: The rabbit primary antibody for p-GSK3β was from Cell Signaling Technology and the antibody for total GSK3β was from Epitomics (Burlingame, CA).

    Techniques: 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.

    Journal: The Journal of investigative dermatology

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

    doi: 10.1038/jid.2012.237

    Figure Lengend 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.

    Article Snippet: The rabbit primary antibody for p-GSK3β was from Cell Signaling Technology and the antibody for total GSK3β was from Epitomics (Burlingame, CA).

    Techniques: Immunohistochemistry, Staining, Expressing

    Localization of signaling components necessary for A–P guidance in rat E13 and mouse E11.5 spinal cord. A–A″ , Phosphorylated PKCζ in transverse ( A , A′ ) and postcrossing ( A″ ) sections. Arrows indicate immunoreactivity in postcrossing segments. B–B″ , Par6 staining in transverse ( B , B′ ) and postcrossing ( B″ ) sections. Arrows indicate immunoreactivity in postcrossing segments. C–C′ , Phosphorylated GSK3β (S9) staining in transverse sections. Arrows indicate immunoreactivity in postcrossing segments. D , D″ , L1 staining in transverse ( D ) and postcrossing ( D″ ) sections. Arrows indicate postcrossing staining. E , E″ , Tag-1 staining in transverse ( E ) and postcrossing ( E″ ) sections. Precrossing staining is present ( E , E″ , arrows), whereas postcrossing segments are negative ( E , arrowhead). Inset, Differential interference contrast image indicates presence of axons (black arrow). F , G , Diagrams indicating axon trajectories in transverse ( F ) and postcrossing ( G ) sections. The precrossing segment is labeled blue, the crossing segment is labeled red, and the postcrossing segment is labeled green. mE11.5, Mouse E11.5; rE13, rat E13.

    Journal: The Journal of Neuroscience

    Article Title: Phosphatidylinositol-3-Kinase–Atypical Protein Kinase C Signaling Is Required for Wnt Attraction and Anterior–Posterior Axon Guidance

    doi: 10.1523/JNEUROSCI.0029-08.2008

    Figure Lengend Snippet: Localization of signaling components necessary for A–P guidance in rat E13 and mouse E11.5 spinal cord. A–A″ , Phosphorylated PKCζ in transverse ( A , A′ ) and postcrossing ( A″ ) sections. Arrows indicate immunoreactivity in postcrossing segments. B–B″ , Par6 staining in transverse ( B , B′ ) and postcrossing ( B″ ) sections. Arrows indicate immunoreactivity in postcrossing segments. C–C′ , Phosphorylated GSK3β (S9) staining in transverse sections. Arrows indicate immunoreactivity in postcrossing segments. D , D″ , L1 staining in transverse ( D ) and postcrossing ( D″ ) sections. Arrows indicate postcrossing staining. E , E″ , Tag-1 staining in transverse ( E ) and postcrossing ( E″ ) sections. Precrossing staining is present ( E , E″ , arrows), whereas postcrossing segments are negative ( E , arrowhead). Inset, Differential interference contrast image indicates presence of axons (black arrow). F , G , Diagrams indicating axon trajectories in transverse ( F ) and postcrossing ( G ) sections. The precrossing segment is labeled blue, the crossing segment is labeled red, and the postcrossing segment is labeled green. mE11.5, Mouse E11.5; rE13, rat E13.

    Article Snippet: The following antibodies were purchased from vendors: PKCζ (rabbit polyclonal; catalog #s.c.-216; Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated PKCζ (Thr 410; rabbit polyclonal; catalog #s.c.-12894; Santa Cruz Biotechnology), PAR6 (goat polyclonal; catalog #s.c.-14405; Santa Cruz Biotechnology), GSK3β (catalog #AB8687; Millipore, Billerica, MA), GSK3β pS9 (catalog #44–600G; Biosource, Carlsbad CA), enhanced green fluorescent protein (EGFP; rabbit polyclonal; catalog #A111122; Invitrogen, Carlsbad, CA) and β-tubulin E7 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA).

    Techniques: Staining, Labeling

    Phosphorylation of Akt and GSK3β at R30 in heart ( A – D ) and brain ( E – H ) was analyzed by Western blot. A and E : p-Akt Thr308, p-Akt Ser473, and p-GSK3β Ser9 at R30. α-Tubulin and β-actin were used as loading controls for heart and brain, respectively. B and F : densitometric analysis of p-Akt Thr308 ( * P

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: A novel pharmacological strategy by PTEN inhibition for improving metabolic resuscitation and survival after mouse cardiac arrest

    doi: 10.1152/ajpheart.00748.2014

    Figure Lengend Snippet: Phosphorylation of Akt and GSK3β at R30 in heart ( A – D ) and brain ( E – H ) was analyzed by Western blot. A and E : p-Akt Thr308, p-Akt Ser473, and p-GSK3β Ser9 at R30. α-Tubulin and β-actin were used as loading controls for heart and brain, respectively. B and F : densitometric analysis of p-Akt Thr308 ( * P

    Article Snippet: The protein phosphorylation and expression were detected with antibodies against phosphorylation of Akt Thr308, p-Akt Ser473, and GSK3β Ser9 (Cell Signaling Technology, Danvers, MA), pyruvate dehydrogenase (PDH) E1-α subunit (p Ser293; Novus Biologicals, Littleton, CO), phospholamban (p-PLB Thr17), sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA2α; Santa Cruz Biotechnology, Dallas, TX), α-tubulin (NeoMarkers, Fremont, CA), and β-actin (Sigma-Aldrich, St. Louis, MO).

    Techniques: Western Blot