phospho-gsk3β Search Results


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  • 95
    Cell Signaling Technology Inc phospho gsk3β
    Effect of EVs isolated from primary NPCE cells and NPCE cell line on β‐catenin and <t>GSK3β</t> protein expression in primary TM cells. Primary TM cells were treated with NPCE primary or cell line‐derived EVs and the effect of EVs on protein level of β‐catenin and pGSK3β in TM cells was detected by Western blotting at the indicated time‐points. A, Representative Western blots showing the levels of β‐catenin or (C) pGSK3β protein, phosphorylated at Ser9 in primary TM cells following EV treatment. B, The graph shows the densitometry quantification for the expression of β‐catenin or (D) the expression of pGSK3β in primary TM cells. Data presented as mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences from untreated control (* P
    Phospho Gsk3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 961 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti phospho gsk3β
    PPAR β/δ knockdown or lithium treatment attenuate the PPAR β/δ-effects on the <t>AKT/GSK3β/β-catenin</t> pathway and apoptosis ( a ) Immunoblot of phosphorylated AKT, GSKβ, and β-catenin in control cells or cells transfected with PPAR β/δ siRNA or a non-targeting siRNA (N-con) and treated with GW0742 (10 μM, 24h). ( b ) Immunoblot of PARP and phosphorylated AKT, GSK3β, and β-catenin after pretreatment of cancer cells with lithium and subsequent 24 hour GW0742 (10 μM, 24h) treatment. (U) indicates uncleaved PARP and (C) indicates cleaved PARP. Hybridization signals were normalized to β-actin and are presented as the fold change as compared to the control. Blots are representative of n=3 replicates.
    Anti Phospho Gsk3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p gsk3β s9
    Akt activation is necessary for the enhanced transformation caused by LCMT-1 knockdown. (A) Lysates from VC and L3 cells stably expressing an empty control plasmid (VC-control and L3-control, respectively) and lysates from L3 cells stably expressing dominant-negative Akt-AA (L3-Akt-AA) were analyzed by Western blot analysis for the expression of Akt-AA, LCMT-1, and GAPDH. (B) knockdownknockdownAnchorage-independent growth of VC-control, L3-control, and L3-Akt-AA cells in soft agar. Photographs show small, single, representative fields within the agars. Average colony numbers (C) and average colony volumes (D) were determined, and data are shown in graphs as fold change relative to VC-control. Error bars represent SD of three independent experiments performed in triplicate. (E) Lysates from VC-control, L3-control, and L3-Akt-AA suspension colonies were analyzed by Western blot analysis for changes in activation of endogenous Akt. GAPDH was used as a loading control. (F) Graph depicts the average fold change in the levels of phospho-Akt T308, phospho-Akt S473, and <t>phospho-GSK3β</t> S9 in three immunoblot experiments relative to VC-control. (G) Probing of VC-control, L3-control, and L3-Akt-AA lysates with phospho-Akt substrate antibody shows that dominant-negative Akt expression prevents the increased phosphorylation of many proteins caused by LCMT-1 knockdown (arrows show examples). The bracket with asterisk indicates phospho-rpS6, which ran at the bottom of this 7.5% SDS-PAGE gel. (H and I) Lysates were probed for changes in p70 and p85 S6K activation and rpS6 phosphorylation. Total p70 and p85 S6K and total rpS6 were used as controls. (J) Graph depicts the average fold change in the levels of phospho-p85, phospho-p70, and phospho-rpS6 in three immunoblot experiments relative to VC-control. Error bars represent SD of three independent experiments. For all graphs: * P ≤ .05. ** P ≤ .01.
    P Gsk3β S9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    WuXi AppTec phospho gsk3β s
    Akt activation is necessary for the enhanced transformation caused by LCMT-1 knockdown. (A) Lysates from VC and L3 cells stably expressing an empty control plasmid (VC-control and L3-control, respectively) and lysates from L3 cells stably expressing dominant-negative Akt-AA (L3-Akt-AA) were analyzed by Western blot analysis for the expression of Akt-AA, LCMT-1, and GAPDH. (B) knockdownknockdownAnchorage-independent growth of VC-control, L3-control, and L3-Akt-AA cells in soft agar. Photographs show small, single, representative fields within the agars. Average colony numbers (C) and average colony volumes (D) were determined, and data are shown in graphs as fold change relative to VC-control. Error bars represent SD of three independent experiments performed in triplicate. (E) Lysates from VC-control, L3-control, and L3-Akt-AA suspension colonies were analyzed by Western blot analysis for changes in activation of endogenous Akt. GAPDH was used as a loading control. (F) Graph depicts the average fold change in the levels of phospho-Akt T308, phospho-Akt S473, and <t>phospho-GSK3β</t> S9 in three immunoblot experiments relative to VC-control. (G) Probing of VC-control, L3-control, and L3-Akt-AA lysates with phospho-Akt substrate antibody shows that dominant-negative Akt expression prevents the increased phosphorylation of many proteins caused by LCMT-1 knockdown (arrows show examples). The bracket with asterisk indicates phospho-rpS6, which ran at the bottom of this 7.5% SDS-PAGE gel. (H and I) Lysates were probed for changes in p70 and p85 S6K activation and rpS6 phosphorylation. Total p70 and p85 S6K and total rpS6 were used as controls. (J) Graph depicts the average fold change in the levels of phospho-p85, phospho-p70, and phospho-rpS6 in three immunoblot experiments relative to VC-control. Error bars represent SD of three independent experiments. For all graphs: * P ≤ .05. ** P ≤ .01.
    Phospho Gsk3β S, supplied by WuXi AppTec, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho gsk3β s/product/WuXi AppTec
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    85
    Abcam phospho gsk3β serine9
    Akt activation is necessary for the enhanced transformation caused by LCMT-1 knockdown. (A) Lysates from VC and L3 cells stably expressing an empty control plasmid (VC-control and L3-control, respectively) and lysates from L3 cells stably expressing dominant-negative Akt-AA (L3-Akt-AA) were analyzed by Western blot analysis for the expression of Akt-AA, LCMT-1, and GAPDH. (B) knockdownknockdownAnchorage-independent growth of VC-control, L3-control, and L3-Akt-AA cells in soft agar. Photographs show small, single, representative fields within the agars. Average colony numbers (C) and average colony volumes (D) were determined, and data are shown in graphs as fold change relative to VC-control. Error bars represent SD of three independent experiments performed in triplicate. (E) Lysates from VC-control, L3-control, and L3-Akt-AA suspension colonies were analyzed by Western blot analysis for changes in activation of endogenous Akt. GAPDH was used as a loading control. (F) Graph depicts the average fold change in the levels of phospho-Akt T308, phospho-Akt S473, and <t>phospho-GSK3β</t> S9 in three immunoblot experiments relative to VC-control. (G) Probing of VC-control, L3-control, and L3-Akt-AA lysates with phospho-Akt substrate antibody shows that dominant-negative Akt expression prevents the increased phosphorylation of many proteins caused by LCMT-1 knockdown (arrows show examples). The bracket with asterisk indicates phospho-rpS6, which ran at the bottom of this 7.5% SDS-PAGE gel. (H and I) Lysates were probed for changes in p70 and p85 S6K activation and rpS6 phosphorylation. Total p70 and p85 S6K and total rpS6 were used as controls. (J) Graph depicts the average fold change in the levels of phospho-p85, phospho-p70, and phospho-rpS6 in three immunoblot experiments relative to VC-control. Error bars represent SD of three independent experiments. For all graphs: * P ≤ .05. ** P ≤ .01.
    Phospho Gsk3β Serine9, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology phospho gsk3β
    TLR4 decreases Wnt signaling at the level of the LRP6 receptor activation in Muller glia MIO-M1 cells. (A) Representative Western blots showing changes in phosphorylation status with Wnt signaling and LPS. Wnt3a conditioned media (W) increases the levels of phospho-Ser1490 LRP6 (p-LRP6) compared with control conditioned media (C). The addition of LPS reduces p-LRPS in Wnt3a treated cultures. In contrast, the amount of phospho-Ser9 in <t>GSK3β</t> is not affected by LPS. (B–C) Quantification of phospho-LRP6 and phospho-GSK3β. Twenty micrograms of total protein were loaded in each lane. The phosphorylated proteins were detected by phospho-specific antibodies and normalized to β-actin as a labeled control, and total LRP6 and GSK3β proteins were detected by antibodies that recognize both their respective phospho- and unphosphorylated forms, and normalized to β-actin. #, Wnt3a compared with control p
    Phospho Gsk3β, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher anti phospho gsk3β
    TLR4 decreases Wnt signaling at the level of the LRP6 receptor activation in Muller glia MIO-M1 cells. (A) Representative Western blots showing changes in phosphorylation status with Wnt signaling and LPS. Wnt3a conditioned media (W) increases the levels of phospho-Ser1490 LRP6 (p-LRP6) compared with control conditioned media (C). The addition of LPS reduces p-LRPS in Wnt3a treated cultures. In contrast, the amount of phospho-Ser9 in <t>GSK3β</t> is not affected by LPS. (B–C) Quantification of phospho-LRP6 and phospho-GSK3β. Twenty micrograms of total protein were loaded in each lane. The phosphorylated proteins were detected by phospho-specific antibodies and normalized to β-actin as a labeled control, and total LRP6 and GSK3β proteins were detected by antibodies that recognize both their respective phospho- and unphosphorylated forms, and normalized to β-actin. #, Wnt3a compared with control p
    Anti Phospho Gsk3β, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho gsk3β α
    TLR4 decreases Wnt signaling at the level of the LRP6 receptor activation in Muller glia MIO-M1 cells. (A) Representative Western blots showing changes in phosphorylation status with Wnt signaling and LPS. Wnt3a conditioned media (W) increases the levels of phospho-Ser1490 LRP6 (p-LRP6) compared with control conditioned media (C). The addition of LPS reduces p-LRPS in Wnt3a treated cultures. In contrast, the amount of phospho-Ser9 in <t>GSK3β</t> is not affected by LPS. (B–C) Quantification of phospho-LRP6 and phospho-GSK3β. Twenty micrograms of total protein were loaded in each lane. The phosphorylated proteins were detected by phospho-specific antibodies and normalized to β-actin as a labeled control, and total LRP6 and GSK3β proteins were detected by antibodies that recognize both their respective phospho- and unphosphorylated forms, and normalized to β-actin. #, Wnt3a compared with control p
    Phospho Gsk3β α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of EVs isolated from primary NPCE cells and NPCE cell line on β‐catenin and GSK3β protein expression in primary TM cells. Primary TM cells were treated with NPCE primary or cell line‐derived EVs and the effect of EVs on protein level of β‐catenin and pGSK3β in TM cells was detected by Western blotting at the indicated time‐points. A, Representative Western blots showing the levels of β‐catenin or (C) pGSK3β protein, phosphorylated at Ser9 in primary TM cells following EV treatment. B, The graph shows the densitometry quantification for the expression of β‐catenin or (D) the expression of pGSK3β in primary TM cells. Data presented as mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences from untreated control (* P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Extracellular vesicle‐mediated crosstalk between NPCE cells and TM cells result in modulation of Wnt signalling pathway and ECM remodelling. Extracellular vesicle‐mediated crosstalk between NPCE cells and TM cells result in modulation of Wnt signalling pathway and ECM remodelling

    doi: 10.1111/jcmm.15129

    Figure Lengend Snippet: Effect of EVs isolated from primary NPCE cells and NPCE cell line on β‐catenin and GSK3β protein expression in primary TM cells. Primary TM cells were treated with NPCE primary or cell line‐derived EVs and the effect of EVs on protein level of β‐catenin and pGSK3β in TM cells was detected by Western blotting at the indicated time‐points. A, Representative Western blots showing the levels of β‐catenin or (C) pGSK3β protein, phosphorylated at Ser9 in primary TM cells following EV treatment. B, The graph shows the densitometry quantification for the expression of β‐catenin or (D) the expression of pGSK3β in primary TM cells. Data presented as mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences from untreated control (* P

    Article Snippet: After blocking (5% BSA, 1 hour at room temperature), membranes were incubated overnight at 4°C with phospho‐GSK3β (1:1000, Cell Signaling, 9323) and β‐catenin (1:1000, Cell Signaling, 8480).

    Techniques: Isolation, Expressing, Derivative Assay, Western Blot

    HDAC inhibitors promote GSK3β-mediated Mcl-1 phosphorylation and apoptosis. (A) Western blotting of indicated proteins in HCT116 cells treated with SAHA or MS-275 at indicated time points. p-Mcl-1: S159/T163; p-GSK3β: S9; p-ERK: T202/Y204; p-AKT: S473. (B) HCT116 cells were treated with SAHA with or without the GSK3 inhibitor SB216763 (5 µM) for 24 hr. Left , western blotting of indicated proteins; right , apoptosis was analyzed by counting condensed and fragmented nuclei after nuclear staining with Hoechst 33258. (C) Western blotting of Mcl-1 in HCT116 cells treated with indicated agents for 24 hr. ABT-263: 5 µM; ABT-737: 5 µM; SAHA: 4 µM; MS-275: 5 µM; regorafenib: 40 µM; sorafenib: 20 µM; UCN-01: 1 µM; sunitinib: 15 µM. (D) Western blotting of indicated proteins in HCT116 cells transfected with V5-tagged WT or 4A mutant Mcl-1 (S121A/E125A/S159A/T163A) and treated with SAHA or MS-275 for 24 hr. (E) Apoptosis in HCT116 cells transfected and treated as in (D) was analyzed as in (B). (F) Western blotting of indicated proteins in WT and Mcl-1 knock-in ( Mcl-1- KI) HCT116 cells treated with SAHA or MS-275 for 24 hr. In (A)-(F), SAHA: 4 µM; MS-275: 5 µM. In (B) and (E), results were expressed as means ± s.d. of three independent experiments. * , P

    Journal: Cancer research

    Article Title: Mcl-1 phosphorylation without degradation mediates sensitivity to HDAC inhibitors by liberating BH3-only proteins

    doi: 10.1158/0008-5472.CAN-18-0399

    Figure Lengend Snippet: HDAC inhibitors promote GSK3β-mediated Mcl-1 phosphorylation and apoptosis. (A) Western blotting of indicated proteins in HCT116 cells treated with SAHA or MS-275 at indicated time points. p-Mcl-1: S159/T163; p-GSK3β: S9; p-ERK: T202/Y204; p-AKT: S473. (B) HCT116 cells were treated with SAHA with or without the GSK3 inhibitor SB216763 (5 µM) for 24 hr. Left , western blotting of indicated proteins; right , apoptosis was analyzed by counting condensed and fragmented nuclei after nuclear staining with Hoechst 33258. (C) Western blotting of Mcl-1 in HCT116 cells treated with indicated agents for 24 hr. ABT-263: 5 µM; ABT-737: 5 µM; SAHA: 4 µM; MS-275: 5 µM; regorafenib: 40 µM; sorafenib: 20 µM; UCN-01: 1 µM; sunitinib: 15 µM. (D) Western blotting of indicated proteins in HCT116 cells transfected with V5-tagged WT or 4A mutant Mcl-1 (S121A/E125A/S159A/T163A) and treated with SAHA or MS-275 for 24 hr. (E) Apoptosis in HCT116 cells transfected and treated as in (D) was analyzed as in (B). (F) Western blotting of indicated proteins in WT and Mcl-1 knock-in ( Mcl-1- KI) HCT116 cells treated with SAHA or MS-275 for 24 hr. In (A)-(F), SAHA: 4 µM; MS-275: 5 µM. In (B) and (E), results were expressed as means ± s.d. of three independent experiments. * , P

    Article Snippet: Antibodies include those for PUMA, phospho-Mcl-1 (S159/T163), cleaved caspases 3, 8, and 9, ERK, phospho-ERK (T202/Y204), AKT, phospho-AKT (S473), GSK3β, phospho-GSK3β (S9), Bim, Noxa, Bad (Cell Signaling), V5, cytochrome oxidase subunit IV (Invitrogen), Bax, HA (Santa Cruz), Bcl-XL , Mcl-1 (BD Biosciences), Bid (EMD Biosciences), cytochrome c , β-actin (Sigma), Bak (Millipore), and Bcl-2 (Dako), and FBW7 (Bethyl).

    Techniques: Western Blot, Mass Spectrometry, Staining, Transfection, Mutagenesis, Knock-In

    Skeletal muscle GSK3α, Ser 21 phospho-GSK3α, GSK3β, Ser 9 phospho-GSK3β, GS and Ser 641 phospho-GS protein abundance in four month old lambs of normal weight ewes (CC), normal weight ewes put on a dietary restriction regime for one month before and one week after conception (CR), overnourished, obese ewes (HH) and obese ewes put on a dietary restriction regime for one month before and one week after conception (HR). Different superscripts denote mean values that are significantly different. n =6 lambs, CC, 2 males, 3 females; CR, HH HR, 3 males, 3 females. All data are mean ± s.e.m.

    Journal: PLoS ONE

    Article Title: Differential Effects of Exposure to Maternal Obesity or Maternal Weight Loss during the Periconceptional Period in the Sheep on Insulin Signalling Molecules in Skeletal Muscle of the Offspring at 4 Months of Age

    doi: 10.1371/journal.pone.0084594

    Figure Lengend Snippet: Skeletal muscle GSK3α, Ser 21 phospho-GSK3α, GSK3β, Ser 9 phospho-GSK3β, GS and Ser 641 phospho-GS protein abundance in four month old lambs of normal weight ewes (CC), normal weight ewes put on a dietary restriction regime for one month before and one week after conception (CR), overnourished, obese ewes (HH) and obese ewes put on a dietary restriction regime for one month before and one week after conception (HR). Different superscripts denote mean values that are significantly different. n =6 lambs, CC, 2 males, 3 females; CR, HH HR, 3 males, 3 females. All data are mean ± s.e.m.

    Article Snippet: The proteins were transferred to polyvinylidene diflouride membrane (Millipore, MA, USA), blocked overnight and then incubated with primary antisera raised against: IRβ subunit (IRB ) and GLUT-4 (Abcam, Cambridge, UK), IRS1 and PI3K p85α subunit (Upstate Biotechnology, Millipore, Billerica, USA), Tyr 1162/1163 phospho-IR, PI3K p110β subunit and aPKCζ (Santa Cruz Biotechnology, Santa Cruz, USA) and PDK1, Ser 241 phospho-PDK1, PTEN, Akt1, Akt2, Ser 473 phospho-Akt, Thr 308 phospho-Akt, AS160, Thr 642 phospho-AS160, GSK3α, Ser 21 phospho-GSK3α, GSK3β, Ser 9 phospho-GSK3β, glycogen synthase (GS) and Ser 641 phospho-GS (Cell Signalling Technology, Danvers, USA) [ ].

    Techniques:

    PPAR β/δ knockdown or lithium treatment attenuate the PPAR β/δ-effects on the AKT/GSK3β/β-catenin pathway and apoptosis ( a ) Immunoblot of phosphorylated AKT, GSKβ, and β-catenin in control cells or cells transfected with PPAR β/δ siRNA or a non-targeting siRNA (N-con) and treated with GW0742 (10 μM, 24h). ( b ) Immunoblot of PARP and phosphorylated AKT, GSK3β, and β-catenin after pretreatment of cancer cells with lithium and subsequent 24 hour GW0742 (10 μM, 24h) treatment. (U) indicates uncleaved PARP and (C) indicates cleaved PARP. Hybridization signals were normalized to β-actin and are presented as the fold change as compared to the control. Blots are representative of n=3 replicates.

    Journal: Hormones & cancer

    Article Title: Ligand-Activated Peroxisome Proliferator-activated Receptor β/δ Modulates Human Endometrial Cancer Cell Survival

    doi: 10.1007/s12672-013-0157-7

    Figure Lengend Snippet: PPAR β/δ knockdown or lithium treatment attenuate the PPAR β/δ-effects on the AKT/GSK3β/β-catenin pathway and apoptosis ( a ) Immunoblot of phosphorylated AKT, GSKβ, and β-catenin in control cells or cells transfected with PPAR β/δ siRNA or a non-targeting siRNA (N-con) and treated with GW0742 (10 μM, 24h). ( b ) Immunoblot of PARP and phosphorylated AKT, GSK3β, and β-catenin after pretreatment of cancer cells with lithium and subsequent 24 hour GW0742 (10 μM, 24h) treatment. (U) indicates uncleaved PARP and (C) indicates cleaved PARP. Hybridization signals were normalized to β-actin and are presented as the fold change as compared to the control. Blots are representative of n=3 replicates.

    Article Snippet: The following antibodies were used: PARP, caspase9, cleaved-caspase9, anti-Akt, anti-phospho-Akt, anti-β-catenin, anti-phospho-β-catenin, anti-GSK3β and anti-phospho-GSK3β (all from Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (Rockland, Gilbertsville, PA, USA).

    Techniques: Transfection, Hybridization

    Effects of hepaCAM alone and co-treatment with Wnt inhibitor DKK1 or GSK3β inhibitor LiCL on protein expression. ( A ) T24 cells cultured for 12 hours were pretreated with Ad-GFP and Ad-GFP-hepaCAM, followed by treatment with 10uM LiCL for

    Journal: Cancer Biology & Therapy

    Article Title: Expression of hepaCAM inhibits bladder cancer cell proliferation via a Wnt/β-catenin-dependent pathway in vitro and in vivo

    doi: 10.1080/15384047.2015.1071732

    Figure Lengend Snippet: Effects of hepaCAM alone and co-treatment with Wnt inhibitor DKK1 or GSK3β inhibitor LiCL on protein expression. ( A ) T24 cells cultured for 12 hours were pretreated with Ad-GFP and Ad-GFP-hepaCAM, followed by treatment with 10uM LiCL for

    Article Snippet: Membranes were first incubated overnight at 4°C with the primary antibodies against phospho-GSK3β (ser9,try216), total GSK3β, phosphor-AKT (ser473), total AKT, phosphor-β-catenin, total β-catenin(Cell Signaling Technology, Boston, MA USA), c-Myc, cyclinD1 and GAPDH(Santa Cruz Biotechnology, Santa Cruz, CA USA).

    Techniques: Expressing, Cell Culture

    Effects of overexpression of hepaCAM on p-β-catenin, p-GSK3β, p-AKT, total AKT, total GSK3β in T24 cells. ( A ) Expression levels of p-β-catenin, p-GSK3β (tyr216/ser9), total GSK3β, p-AKT (ser473) and total

    Journal: Cancer Biology & Therapy

    Article Title: Expression of hepaCAM inhibits bladder cancer cell proliferation via a Wnt/β-catenin-dependent pathway in vitro and in vivo

    doi: 10.1080/15384047.2015.1071732

    Figure Lengend Snippet: Effects of overexpression of hepaCAM on p-β-catenin, p-GSK3β, p-AKT, total AKT, total GSK3β in T24 cells. ( A ) Expression levels of p-β-catenin, p-GSK3β (tyr216/ser9), total GSK3β, p-AKT (ser473) and total

    Article Snippet: Membranes were first incubated overnight at 4°C with the primary antibodies against phospho-GSK3β (ser9,try216), total GSK3β, phosphor-AKT (ser473), total AKT, phosphor-β-catenin, total β-catenin(Cell Signaling Technology, Boston, MA USA), c-Myc, cyclinD1 and GAPDH(Santa Cruz Biotechnology, Santa Cruz, CA USA).

    Techniques: Over Expression, Expressing

    Proliferation effects of hepaCAM alone or co-treatment with Wnt inhibitor DKK1 or GSK3β inhibitor LiCL by using Colony-formation assay and CCK-8 assay in T24 cells. ( A ) Representative colony-formation assay of hepaCAM alone or co-treatment with

    Journal: Cancer Biology & Therapy

    Article Title: Expression of hepaCAM inhibits bladder cancer cell proliferation via a Wnt/β-catenin-dependent pathway in vitro and in vivo

    doi: 10.1080/15384047.2015.1071732

    Figure Lengend Snippet: Proliferation effects of hepaCAM alone or co-treatment with Wnt inhibitor DKK1 or GSK3β inhibitor LiCL by using Colony-formation assay and CCK-8 assay in T24 cells. ( A ) Representative colony-formation assay of hepaCAM alone or co-treatment with

    Article Snippet: Membranes were first incubated overnight at 4°C with the primary antibodies against phospho-GSK3β (ser9,try216), total GSK3β, phosphor-AKT (ser473), total AKT, phosphor-β-catenin, total β-catenin(Cell Signaling Technology, Boston, MA USA), c-Myc, cyclinD1 and GAPDH(Santa Cruz Biotechnology, Santa Cruz, CA USA).

    Techniques: Colony Assay, CCK-8 Assay

    Inhibition of Aurora A kinase by B6 promotes inactivation of GSK3β. ( a ) Both mock-infected and 4F-infected (day 3) MEFs were treated with 1 μM B6 for 2 days and then collected for western blotting of AurkA. Actin served as a loading control. ( b ) MEFs were infected with 4F for 3 days and mock-treated or treated with DMSO or 1 μM B6 for 2 days before isolation of total RNA and RT–qPCR analysis. B6 treatment did not alter induction of AurkA by 4F. Error bar represents variation between two experiments with triplicate wells. ( c ) 4F-infected MEFs were treated with the indicated doses of B6 inhibitor starting on day 3 post-infection and 2 days later were collected for western blotting analysis. ( d ) Inhibition of AurkA by MLN8237 also increases AurkA protein level and dose-dependently promotes GSK3β phosphorylation. The experiment was performed the same as for c . Actin served as the loading control. ( e ) Expression of a dominant-negative form of AurkA promotes GSK3β (Ser9) phosphorylation. MEFs were infected with 4F plus expression vectors for red fluorescent protein (RFP), wild-type (wt) murine AurkA or the D274A kinase-dead mutant of human AurkA. Expression of wt AurkA inhibited GSK3β phosphorylation on Ser9, whereas overexpression of the mutant AurkA D274A enhanced GSK3β phosphorylation. Exposure time was almost doubled for 4F-infected samples. ( f ) MEFs were transfected with 50 nM AurkA and control siRNAs for 2 days. Cells were then collected for western blotting of AurkA, total and phosphorylated GSK3β (Ser9), and actin as a loading control. ( g ) MEFs were infected with pMX-RFP (control) or pMX-GSK3β virus for 4 days and then collected for RT–qPCR analysis. Error bar represents variation of duplicate wells. ( h ) Overexpression of GSK3β blocked the effect of B6 on reprogramming. pMX-Gsk3β was transduced into MEFs together with 4F viruses. B6 (1 μM) was added at day 3 post-transduction and GFP+ colonies were quantified on day 12 post-transduction. Data are mean±s.d. of three independent wells. * P

    Journal: Nature Communications

    Article Title: A kinase inhibitor screen identifies small-molecule enhancers of reprogramming and iPS cell generation

    doi: 10.1038/ncomms2059

    Figure Lengend Snippet: Inhibition of Aurora A kinase by B6 promotes inactivation of GSK3β. ( a ) Both mock-infected and 4F-infected (day 3) MEFs were treated with 1 μM B6 for 2 days and then collected for western blotting of AurkA. Actin served as a loading control. ( b ) MEFs were infected with 4F for 3 days and mock-treated or treated with DMSO or 1 μM B6 for 2 days before isolation of total RNA and RT–qPCR analysis. B6 treatment did not alter induction of AurkA by 4F. Error bar represents variation between two experiments with triplicate wells. ( c ) 4F-infected MEFs were treated with the indicated doses of B6 inhibitor starting on day 3 post-infection and 2 days later were collected for western blotting analysis. ( d ) Inhibition of AurkA by MLN8237 also increases AurkA protein level and dose-dependently promotes GSK3β phosphorylation. The experiment was performed the same as for c . Actin served as the loading control. ( e ) Expression of a dominant-negative form of AurkA promotes GSK3β (Ser9) phosphorylation. MEFs were infected with 4F plus expression vectors for red fluorescent protein (RFP), wild-type (wt) murine AurkA or the D274A kinase-dead mutant of human AurkA. Expression of wt AurkA inhibited GSK3β phosphorylation on Ser9, whereas overexpression of the mutant AurkA D274A enhanced GSK3β phosphorylation. Exposure time was almost doubled for 4F-infected samples. ( f ) MEFs were transfected with 50 nM AurkA and control siRNAs for 2 days. Cells were then collected for western blotting of AurkA, total and phosphorylated GSK3β (Ser9), and actin as a loading control. ( g ) MEFs were infected with pMX-RFP (control) or pMX-GSK3β virus for 4 days and then collected for RT–qPCR analysis. Error bar represents variation of duplicate wells. ( h ) Overexpression of GSK3β blocked the effect of B6 on reprogramming. pMX-Gsk3β was transduced into MEFs together with 4F viruses. B6 (1 μM) was added at day 3 post-transduction and GFP+ colonies were quantified on day 12 post-transduction. Data are mean±s.d. of three independent wells. * P

    Article Snippet: Blots were then incubated with the following antibodies: anti-mNanog (R & D Systems, AF2729, 1:400), anti-h/mSSEA1 (R & D Systems, MAB2156, 1:400), anti-actin (Thermo Scientific, MS1295P0, 1:5,000), anti-AFP (Abcam, ab7751, 1:400), anti-β III tubulin (R & D Systems, MAB1368, 1:400), anti-α actin (Sigma, A7811, 1:400), anti-mAurkA (Bethyl Labs, A300-072A, 1:3,000), anti-hAurkA (Bethyl Labs, A300-071A, 1:1,000), anti-total-GSK3β (Cell Signaling Technology, 9315S, 1:1,000), anti-phospho-GSK3β (Ser9) (Cell Signaling Technology, 9323S, 1:1,000), anti-total Akt (Cell Signaling Technology, 9272S, 1:1,000) and anti-phospho-Akt (Ser473) (Cell Signaling Technology, 9271S, 1:1,000).

    Techniques: Inhibition, Infection, Western Blot, Isolation, Quantitative RT-PCR, Expressing, Dominant Negative Mutation, Mutagenesis, Over Expression, Transfection, Transduction

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep22999

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

    Article Snippet: Cellular proteins associated with GST-tagged fusion proteins were separated by SDS-PAGE, and analyzed by standard Western blotting using specific rabbit monoclonal GSK3β antibody (27C10, Cell Signaling Technology, MA), phospho-GSK3β (Ser9) antibody (9323, Cell Signaling Technology, MA).

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep22999

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

    Article Snippet: Cellular proteins associated with GST-tagged fusion proteins were separated by SDS-PAGE, and analyzed by standard Western blotting using specific rabbit monoclonal GSK3β antibody (27C10, Cell Signaling Technology, MA), phospho-GSK3β (Ser9) antibody (9323, Cell Signaling Technology, MA).

    Techniques: Transfection, Western Blot

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

    Journal: Scientific Reports

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

    doi: 10.1038/srep22999

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

    Article Snippet: Cellular proteins associated with GST-tagged fusion proteins were separated by SDS-PAGE, and analyzed by standard Western blotting using specific rabbit monoclonal GSK3β antibody (27C10, Cell Signaling Technology, MA), phospho-GSK3β (Ser9) antibody (9323, Cell Signaling Technology, MA).

    Techniques: Expressing

    A, Western blots from MEF cells probed with GSK3β Ser(P)-9 ( Ser9-P ) or total GSK3β ( green ) and actin ( red ) antibodies show basal and insulin-mediated GSK3β Ser-9 phosphorylation increased in Oga KO MEF cells compared with WT cells

    Journal: The Journal of Biological Chemistry

    Article Title: Conditional Knock-out Reveals a Requirement for O-Linked N-Acetylglucosaminase (O-GlcNAcase) in Metabolic Homeostasis *

    doi: 10.1074/jbc.M114.617779

    Figure Lengend Snippet: A, Western blots from MEF cells probed with GSK3β Ser(P)-9 ( Ser9-P ) or total GSK3β ( green ) and actin ( red ) antibodies show basal and insulin-mediated GSK3β Ser-9 phosphorylation increased in Oga KO MEF cells compared with WT cells

    Article Snippet: Total GSK3β and phospho-GSK3β Ser-9 antibodies were from Cell Signaling Technology (GSK, catalog no. 9332 and GSKβ3 Ser-9 catalog no. 9336S).

    Techniques: Western Blot

    Activation of PI3K/Akt pathway and inactivation of GSK3 by E. chaffeensis TRPs. (A) THP-1 cells were treated with thioredoxin (control) or TRP120, TRP32, or TRP47 in suspension (1 μg/ml) for 24 h. Data represent results of immunoblot analysis of levels of phospho-PI3K, total PI3K, phospho-Akt, total Akt, phospho-GSK3β, and GSK3. Data are representative of results from n = 4 experiments. (B to D) Quantitative analysis of the Western blot data was performed using Image J software (****, P

    Journal: Infection and Immunity

    Article Title: Ehrlichia Activation of Wnt-PI3K-mTOR Signaling Inhibits Autolysosome Generation and Autophagic Destruction by the Mononuclear Phagocyte

    doi: 10.1128/IAI.00690-17

    Figure Lengend Snippet: Activation of PI3K/Akt pathway and inactivation of GSK3 by E. chaffeensis TRPs. (A) THP-1 cells were treated with thioredoxin (control) or TRP120, TRP32, or TRP47 in suspension (1 μg/ml) for 24 h. Data represent results of immunoblot analysis of levels of phospho-PI3K, total PI3K, phospho-Akt, total Akt, phospho-GSK3β, and GSK3. Data are representative of results from n = 4 experiments. (B to D) Quantitative analysis of the Western blot data was performed using Image J software (****, P

    Article Snippet: Anti-LC3A/B (D3U4C) antibody, anti-SQSTM1p62 (D5E2) antibody, anti-phospho-p70 S6 kinase (Thr389) (108D2) antibody, anti-phospho-Akt (Ser473; D9E) antibody, anti-Akt (pan, C67E7) antibody, anti-phospho-PI3K p85 (Tyr458)/p55 (Tyr 199) antibody, anti-PI3Kinase p85 (19H8) antibody, anti-Tuberin/TSC2 antibody, anti-PTEN antibody, anti-TFEB antibody, anti-Rheb antibody, anti-phospho-GSK3β antibody, anti-GSK3 (Cell Signaling Technology) antibody, anti-MAP1LC3B (catalog no. NB100-2220; NovusBio) antibody, and anti-GAPDH (clone 6C5; EMD Millipore) antibody were used for Western blotting.

    Techniques: Activation Assay, Western Blot, Software

    Activation of PI3K-Akt pathway and Wnt-dependent inhibition of GSK3 during E. chaffeensis infection. (A to C) Western blot analysis of control and E. chaffeensis- infected cells to determine phospho-PI3K (Tyr 458), total PI3K, phospho-Akt (Ser 473), and total Akt levels. E. chaffeensis infection was confirmed by detecting ehrlichial DSB protein. (D and E) PTEN levels were determined and quantified relative to GAPDH (1, 2, and 3 days pi). (F and G) THP-1 cells were infected with E. chaffeensis (24 h) and treated with either vehicle (DMSO) or Wnt inhibitor (3289-8625; 30 μM). Immunoblot analysis was performed to determine phospho-GSK3β and total GSK3 levels (48 and 60 h pi), and the results were quantified relative to GAPDH. (H and I) TSC2 levels were determined in E. chaffeensis- infected and uninfected controls by Western immunoblotting, and the results were quantified relative to GAPDH. Data are representative of results from n = 4 experiments. Quantitative analysis of the Western blot data was performed using Image J software (***, P

    Journal: Infection and Immunity

    Article Title: Ehrlichia Activation of Wnt-PI3K-mTOR Signaling Inhibits Autolysosome Generation and Autophagic Destruction by the Mononuclear Phagocyte

    doi: 10.1128/IAI.00690-17

    Figure Lengend Snippet: Activation of PI3K-Akt pathway and Wnt-dependent inhibition of GSK3 during E. chaffeensis infection. (A to C) Western blot analysis of control and E. chaffeensis- infected cells to determine phospho-PI3K (Tyr 458), total PI3K, phospho-Akt (Ser 473), and total Akt levels. E. chaffeensis infection was confirmed by detecting ehrlichial DSB protein. (D and E) PTEN levels were determined and quantified relative to GAPDH (1, 2, and 3 days pi). (F and G) THP-1 cells were infected with E. chaffeensis (24 h) and treated with either vehicle (DMSO) or Wnt inhibitor (3289-8625; 30 μM). Immunoblot analysis was performed to determine phospho-GSK3β and total GSK3 levels (48 and 60 h pi), and the results were quantified relative to GAPDH. (H and I) TSC2 levels were determined in E. chaffeensis- infected and uninfected controls by Western immunoblotting, and the results were quantified relative to GAPDH. Data are representative of results from n = 4 experiments. Quantitative analysis of the Western blot data was performed using Image J software (***, P

    Article Snippet: Anti-LC3A/B (D3U4C) antibody, anti-SQSTM1p62 (D5E2) antibody, anti-phospho-p70 S6 kinase (Thr389) (108D2) antibody, anti-phospho-Akt (Ser473; D9E) antibody, anti-Akt (pan, C67E7) antibody, anti-phospho-PI3K p85 (Tyr458)/p55 (Tyr 199) antibody, anti-PI3Kinase p85 (19H8) antibody, anti-Tuberin/TSC2 antibody, anti-PTEN antibody, anti-TFEB antibody, anti-Rheb antibody, anti-phospho-GSK3β antibody, anti-GSK3 (Cell Signaling Technology) antibody, anti-MAP1LC3B (catalog no. NB100-2220; NovusBio) antibody, and anti-GAPDH (clone 6C5; EMD Millipore) antibody were used for Western blotting.

    Techniques: Activation Assay, Inhibition, Infection, Western Blot, Software

    Akt activation is necessary for the enhanced transformation caused by LCMT-1 knockdown. (A) Lysates from VC and L3 cells stably expressing an empty control plasmid (VC-control and L3-control, respectively) and lysates from L3 cells stably expressing dominant-negative Akt-AA (L3-Akt-AA) were analyzed by Western blot analysis for the expression of Akt-AA, LCMT-1, and GAPDH. (B) knockdownknockdownAnchorage-independent growth of VC-control, L3-control, and L3-Akt-AA cells in soft agar. Photographs show small, single, representative fields within the agars. Average colony numbers (C) and average colony volumes (D) were determined, and data are shown in graphs as fold change relative to VC-control. Error bars represent SD of three independent experiments performed in triplicate. (E) Lysates from VC-control, L3-control, and L3-Akt-AA suspension colonies were analyzed by Western blot analysis for changes in activation of endogenous Akt. GAPDH was used as a loading control. (F) Graph depicts the average fold change in the levels of phospho-Akt T308, phospho-Akt S473, and phospho-GSK3β S9 in three immunoblot experiments relative to VC-control. (G) Probing of VC-control, L3-control, and L3-Akt-AA lysates with phospho-Akt substrate antibody shows that dominant-negative Akt expression prevents the increased phosphorylation of many proteins caused by LCMT-1 knockdown (arrows show examples). The bracket with asterisk indicates phospho-rpS6, which ran at the bottom of this 7.5% SDS-PAGE gel. (H and I) Lysates were probed for changes in p70 and p85 S6K activation and rpS6 phosphorylation. Total p70 and p85 S6K and total rpS6 were used as controls. (J) Graph depicts the average fold change in the levels of phospho-p85, phospho-p70, and phospho-rpS6 in three immunoblot experiments relative to VC-control. Error bars represent SD of three independent experiments. For all graphs: * P ≤ .05. ** P ≤ .01.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Circumventing Cellular Control of PP2A by Methylation Promotes Transformation in an Akt-Dependent Manner 1

    doi:

    Figure Lengend Snippet: Akt activation is necessary for the enhanced transformation caused by LCMT-1 knockdown. (A) Lysates from VC and L3 cells stably expressing an empty control plasmid (VC-control and L3-control, respectively) and lysates from L3 cells stably expressing dominant-negative Akt-AA (L3-Akt-AA) were analyzed by Western blot analysis for the expression of Akt-AA, LCMT-1, and GAPDH. (B) knockdownknockdownAnchorage-independent growth of VC-control, L3-control, and L3-Akt-AA cells in soft agar. Photographs show small, single, representative fields within the agars. Average colony numbers (C) and average colony volumes (D) were determined, and data are shown in graphs as fold change relative to VC-control. Error bars represent SD of three independent experiments performed in triplicate. (E) Lysates from VC-control, L3-control, and L3-Akt-AA suspension colonies were analyzed by Western blot analysis for changes in activation of endogenous Akt. GAPDH was used as a loading control. (F) Graph depicts the average fold change in the levels of phospho-Akt T308, phospho-Akt S473, and phospho-GSK3β S9 in three immunoblot experiments relative to VC-control. (G) Probing of VC-control, L3-control, and L3-Akt-AA lysates with phospho-Akt substrate antibody shows that dominant-negative Akt expression prevents the increased phosphorylation of many proteins caused by LCMT-1 knockdown (arrows show examples). The bracket with asterisk indicates phospho-rpS6, which ran at the bottom of this 7.5% SDS-PAGE gel. (H and I) Lysates were probed for changes in p70 and p85 S6K activation and rpS6 phosphorylation. Total p70 and p85 S6K and total rpS6 were used as controls. (J) Graph depicts the average fold change in the levels of phospho-p85, phospho-p70, and phospho-rpS6 in three immunoblot experiments relative to VC-control. Error bars represent SD of three independent experiments. For all graphs: * P ≤ .05. ** P ≤ .01.

    Article Snippet: Other antibodies used included sepharose bead-conjugated anti-HA-tag antibody used for immunoprecipitation (F-7 AC; Santa Cruz Biotechnologies, Santa Cruz, CA), anti-HA antibody used for Western blot analysis (16B12; Covance, Princeton, NJ), anti-SVST rabbit polyclonal antibody (gift from W. Hahn), anti-PyST rabbit polyclonal antibody [ ], PP2A Bα mouse monoclonal antibody (clone 2G9; Millipore, Bedford, MA), PP2Ac mouse monoclonal antibody (BD Transduction Laboratories, Franklin Lakes, NJ), unmethylated PP2A C subunit mouse monoclonal antibody (clone 1D6 [ ]; available from Millipore, Santa Cruz Biotechnology, or request to the corresponding author), p-Akt T308 and p-Akt S473 rabbit monoclonal antibodies (Epitomics, Burlingame, CA), GAPDH mouse monoclonal antibody (Abmart, Shanghai, China), Pme1 mouse monoclonal antibody (B12; Santa Cruz Biotechnology), mouse monoclonal antibodies to p-S6K (p70 T389 and p85 T412) and total rpS6, and rabbit polyclonal antibodies to p-GSK3β S9, p-rpS6 S235/236, total Akt, total p70/p85 S6K, and pAkt substrate ((R/K)X(R/K)XX(pT/pS)) obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Activation Assay, Transformation Assay, Stable Transfection, Expressing, Plasmid Preparation, Dominant Negative Mutation, Western Blot, SDS Page

    LCMT-1 knockdown activates Akt and S6K signaling in anchorage-independent conditions. (A) Equal numbers of VC and L3 cells were seeded on low-binding tissue culture dishes to analyze differences during anchorage-independent growth. After 1 week, suspension cells were weighed to assess growth. In the graph, data are presented as average fold change relative to VC for three independent experiments. (B) Lysates from VC and L3 suspension cells were analyzed by Western blot analysis for changes in activation of Akt. GAPDH and total Akt were used as loading controls. Western blot analysis for LCMT-1 confirmed the knockdown of LCMT-1 in suspension cultures. (C) Graph depicts the average levels of phospho-Akt T308, phospho-Akt S473, and phospho-GSK3β S9 in three independent immunoblot experiments as fold change relative to VC. (D) An anti-Akt phospho-substrate motif (R/K)X(R/K)XX(pT/pS) antibody was used to probe the VC and L3 suspension cell lysates. Arrows highlight some proteins with increased phosphorylation in the L3 cells relative to VC. Bracket with asterisk indicates phospho-rpS6, which is known to cross-react with this antibody. (E and F) Lysates were probed for changes in p70 and p85 S6K activation and rpS6 phosphorylation. Total p70 and p85 S6K and total rpS6 were used as loading controls. (G) Graph represents average fold change in the levels of phospho-p85, phospho-p70 and phospho-rpS6 in three independent immunoblot experiments relative to VC. Experiments were performed in triplicate and error bars in all panels represent SD. * P ≤ .05. ** P ≤ .01.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Circumventing Cellular Control of PP2A by Methylation Promotes Transformation in an Akt-Dependent Manner 1

    doi:

    Figure Lengend Snippet: LCMT-1 knockdown activates Akt and S6K signaling in anchorage-independent conditions. (A) Equal numbers of VC and L3 cells were seeded on low-binding tissue culture dishes to analyze differences during anchorage-independent growth. After 1 week, suspension cells were weighed to assess growth. In the graph, data are presented as average fold change relative to VC for three independent experiments. (B) Lysates from VC and L3 suspension cells were analyzed by Western blot analysis for changes in activation of Akt. GAPDH and total Akt were used as loading controls. Western blot analysis for LCMT-1 confirmed the knockdown of LCMT-1 in suspension cultures. (C) Graph depicts the average levels of phospho-Akt T308, phospho-Akt S473, and phospho-GSK3β S9 in three independent immunoblot experiments as fold change relative to VC. (D) An anti-Akt phospho-substrate motif (R/K)X(R/K)XX(pT/pS) antibody was used to probe the VC and L3 suspension cell lysates. Arrows highlight some proteins with increased phosphorylation in the L3 cells relative to VC. Bracket with asterisk indicates phospho-rpS6, which is known to cross-react with this antibody. (E and F) Lysates were probed for changes in p70 and p85 S6K activation and rpS6 phosphorylation. Total p70 and p85 S6K and total rpS6 were used as loading controls. (G) Graph represents average fold change in the levels of phospho-p85, phospho-p70 and phospho-rpS6 in three independent immunoblot experiments relative to VC. Experiments were performed in triplicate and error bars in all panels represent SD. * P ≤ .05. ** P ≤ .01.

    Article Snippet: Other antibodies used included sepharose bead-conjugated anti-HA-tag antibody used for immunoprecipitation (F-7 AC; Santa Cruz Biotechnologies, Santa Cruz, CA), anti-HA antibody used for Western blot analysis (16B12; Covance, Princeton, NJ), anti-SVST rabbit polyclonal antibody (gift from W. Hahn), anti-PyST rabbit polyclonal antibody [ ], PP2A Bα mouse monoclonal antibody (clone 2G9; Millipore, Bedford, MA), PP2Ac mouse monoclonal antibody (BD Transduction Laboratories, Franklin Lakes, NJ), unmethylated PP2A C subunit mouse monoclonal antibody (clone 1D6 [ ]; available from Millipore, Santa Cruz Biotechnology, or request to the corresponding author), p-Akt T308 and p-Akt S473 rabbit monoclonal antibodies (Epitomics, Burlingame, CA), GAPDH mouse monoclonal antibody (Abmart, Shanghai, China), Pme1 mouse monoclonal antibody (B12; Santa Cruz Biotechnology), mouse monoclonal antibodies to p-S6K (p70 T389 and p85 T412) and total rpS6, and rabbit polyclonal antibodies to p-GSK3β S9, p-rpS6 S235/236, total Akt, total p70/p85 S6K, and pAkt substrate ((R/K)X(R/K)XX(pT/pS)) obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Binding Assay, Western Blot, Activation Assay

    TLR4 decreases Wnt signaling at the level of the LRP6 receptor activation in Muller glia MIO-M1 cells. (A) Representative Western blots showing changes in phosphorylation status with Wnt signaling and LPS. Wnt3a conditioned media (W) increases the levels of phospho-Ser1490 LRP6 (p-LRP6) compared with control conditioned media (C). The addition of LPS reduces p-LRPS in Wnt3a treated cultures. In contrast, the amount of phospho-Ser9 in GSK3β is not affected by LPS. (B–C) Quantification of phospho-LRP6 and phospho-GSK3β. Twenty micrograms of total protein were loaded in each lane. The phosphorylated proteins were detected by phospho-specific antibodies and normalized to β-actin as a labeled control, and total LRP6 and GSK3β proteins were detected by antibodies that recognize both their respective phospho- and unphosphorylated forms, and normalized to β-actin. #, Wnt3a compared with control p

    Journal: PLoS ONE

    Article Title: Novel Role for the Innate Immune Receptor Toll-Like Receptor 4 (TLR4) in the Regulation of the Wnt Signaling Pathway and Photoreceptor Apoptosis

    doi: 10.1371/journal.pone.0036560

    Figure Lengend Snippet: TLR4 decreases Wnt signaling at the level of the LRP6 receptor activation in Muller glia MIO-M1 cells. (A) Representative Western blots showing changes in phosphorylation status with Wnt signaling and LPS. Wnt3a conditioned media (W) increases the levels of phospho-Ser1490 LRP6 (p-LRP6) compared with control conditioned media (C). The addition of LPS reduces p-LRPS in Wnt3a treated cultures. In contrast, the amount of phospho-Ser9 in GSK3β is not affected by LPS. (B–C) Quantification of phospho-LRP6 and phospho-GSK3β. Twenty micrograms of total protein were loaded in each lane. The phosphorylated proteins were detected by phospho-specific antibodies and normalized to β-actin as a labeled control, and total LRP6 and GSK3β proteins were detected by antibodies that recognize both their respective phospho- and unphosphorylated forms, and normalized to β-actin. #, Wnt3a compared with control p

    Article Snippet: Twenty micrograms of total protein were resolved in 10% SDS–PAGE gels using Tris-glycine buffer and the proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes and probed using the antibodies that detect total GSK3β, total LRP6, phospho-GSK3β at Ser9, phospho-LRP6 at Ser1490, and β-actin, followed by several washes, incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology Inc, Santa Cruz CA), and incubation with enhanced chemiluminescence reagent (ECL-plus) (GE Amersham, Pistacataway NJ).

    Techniques: Activation Assay, Western Blot, Labeling