polyclonal antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc polyclonal antibodies
    Polyclonal Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal antibodies/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    polyclonal antibodies - by Bioz Stars, 2023-01
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    polyclonal antibodies  (Cell Signaling Technology Inc)


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  • 94

    Structured Review

    Cell Signaling Technology Inc polyclonal antibodies
    Polyclonal Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal antibodies/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    anti phospho foxo1 ser319  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho foxo1 ser319
    Cells were transfected with either siCon or siGRP78 RNA duplexes for 24 hour before treatment with tunicamycin for 24 hour. Proteins were extracted for immunoblot analysis with GRP78, P-PDK1(Ser241), PDK1, P-AKT(Thr308), P-AKT(Ser473), AKT, P-AKT substrate (RXRXXS/T), P-mTOR(Ser2448), P-HDM2(Ser166), <t>P-FOXO1(Ser319)</t> and P-GSK-3α/β(Ser21/9). Densitometry of band intensity is expressed relative to siCon untreated control (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are mean±SEM for 3 independent experiments. ** indicates P≤ 0.01; n.s indicates non-significant change. A & B ) Down-regulation of GRP78 elevates Ser473 phosphorylation but not at Thr308. C ) Knock-down of GRP78 alters AKT downstream substrates recognition. Arrows indicate the substrates changed their phosphorylation pattern in tunicamycin-treated siGRP78 cells.
    Anti Phospho Foxo1 Ser319, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Regulation of AKT Phosphorylation at Ser473 and Thr308 by Endoplasmic Reticulum Stress Modulates Substrate Specificity in a Severity Dependent Manner"

    Article Title: Regulation of AKT Phosphorylation at Ser473 and Thr308 by Endoplasmic Reticulum Stress Modulates Substrate Specificity in a Severity Dependent Manner

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017894

    Cells were transfected with either siCon or siGRP78 RNA duplexes for 24 hour before treatment with tunicamycin for 24 hour. Proteins were extracted for immunoblot analysis with GRP78, P-PDK1(Ser241), PDK1, P-AKT(Thr308), P-AKT(Ser473), AKT, P-AKT substrate (RXRXXS/T), P-mTOR(Ser2448), P-HDM2(Ser166), P-FOXO1(Ser319) and P-GSK-3α/β(Ser21/9). Densitometry of band intensity is expressed relative to siCon untreated control (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are mean±SEM for 3 independent experiments. ** indicates P≤ 0.01; n.s indicates non-significant change. A & B ) Down-regulation of GRP78 elevates Ser473 phosphorylation but not at Thr308. C ) Knock-down of GRP78 alters AKT downstream substrates recognition. Arrows indicate the substrates changed their phosphorylation pattern in tunicamycin-treated siGRP78 cells.
    Figure Legend Snippet: Cells were transfected with either siCon or siGRP78 RNA duplexes for 24 hour before treatment with tunicamycin for 24 hour. Proteins were extracted for immunoblot analysis with GRP78, P-PDK1(Ser241), PDK1, P-AKT(Thr308), P-AKT(Ser473), AKT, P-AKT substrate (RXRXXS/T), P-mTOR(Ser2448), P-HDM2(Ser166), P-FOXO1(Ser319) and P-GSK-3α/β(Ser21/9). Densitometry of band intensity is expressed relative to siCon untreated control (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are mean±SEM for 3 independent experiments. ** indicates P≤ 0.01; n.s indicates non-significant change. A & B ) Down-regulation of GRP78 elevates Ser473 phosphorylation but not at Thr308. C ) Knock-down of GRP78 alters AKT downstream substrates recognition. Arrows indicate the substrates changed their phosphorylation pattern in tunicamycin-treated siGRP78 cells.

    Techniques Used: Transfection, Western Blot

    anti phospho foxo1 ser319  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho foxo1 ser319
    GLK-IKKβ signaling attenuates Foxp3 promoter activity through <t>FoxO1.</t> (A) Purified CD4 + T cells were cultured under the iTreg-differentiated condition with or without the treatment of the GLK inhibitor (1 μM or 5 μM), with a quantitative graph of CD4 + Foxp3 + T cells among CD4 + T cells. Mean ± SEM are shown. (B) Flow cytometry analysis of CD4 + Foxp3 + T cells among in vitro -differentiated iTreg cells from wild-type (WT) and GLK-deficient mice, with a quantitative graph of CD4 + Foxp3 + T cells among CD4 + T cells. Mean ± SEM are shown. (C) Flow cytometry analysis of CD4 + Foxp3 + T cells among in vitro -differentiated iTreg cells from WT, Lck-GLK Tg mice, and Lck-GLK Tg/ IKKβ cKO mice, with a quantitative graph of CD4 + Foxp3 + T cells among CD4 + T cells. Mean ± SEM are shown. (D) For immunoblotting analysis of phosphorylated (p)-FoxO1, FoxO1, p-FoxO3a, FoxO3a, and vinculin in protein lysates from WT or Lck-GLK Tg T cells, Phos-tag SDS-PAGE gel was used. (E) The schematic diagram of FoxO1 or FoxO3a binding site on the Foxp3 promoter. Arrows indicate FoxO-binding sites. (F) Chromatin immunoprecipitation (ChIP)-PCR analysis of immunoprecipitated FoxO1- or FoxO3a-binding DNA fragments of the Foxp3 promoter from T cells of WT and Lck-GLK Tg mice. (G) The Foxp3 promoter reporter, the FoxO1 plasmid, and the plasmid encoding IKKβ or IKKβ kinase-dead (K44M) mutant were co-transfected into Jurkat T cells; the luciferase reporter activity of the Foxp3 promoter was determined. Mean ± SEM are shown. (H) Immunoblotting analysis of p-FoxO1, FoxO1, GLK, IKKβ, and actin in protein lysates from WT, Lck-GLK Tg, and Lck-GLK Tg/IKKβ cKO T cells using Phos-tag SDS-PAGE gel. Data shown are representative of three independent experiments.
    Anti Phospho Foxo1 Ser319, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho foxo1 ser319/product/Cell Signaling Technology Inc
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    1) Product Images from "MAP4K3/GLK inhibits Treg differentiation by direct phosphorylating IKKβ and inducing IKKβ-mediated FoxO1 nuclear export and Foxp3 downregulation"

    Article Title: MAP4K3/GLK inhibits Treg differentiation by direct phosphorylating IKKβ and inducing IKKβ-mediated FoxO1 nuclear export and Foxp3 downregulation

    Journal: Theranostics

    doi: 10.7150/thno.72148

    GLK-IKKβ signaling attenuates Foxp3 promoter activity through FoxO1. (A) Purified CD4 + T cells were cultured under the iTreg-differentiated condition with or without the treatment of the GLK inhibitor (1 μM or 5 μM), with a quantitative graph of CD4 + Foxp3 + T cells among CD4 + T cells. Mean ± SEM are shown. (B) Flow cytometry analysis of CD4 + Foxp3 + T cells among in vitro -differentiated iTreg cells from wild-type (WT) and GLK-deficient mice, with a quantitative graph of CD4 + Foxp3 + T cells among CD4 + T cells. Mean ± SEM are shown. (C) Flow cytometry analysis of CD4 + Foxp3 + T cells among in vitro -differentiated iTreg cells from WT, Lck-GLK Tg mice, and Lck-GLK Tg/ IKKβ cKO mice, with a quantitative graph of CD4 + Foxp3 + T cells among CD4 + T cells. Mean ± SEM are shown. (D) For immunoblotting analysis of phosphorylated (p)-FoxO1, FoxO1, p-FoxO3a, FoxO3a, and vinculin in protein lysates from WT or Lck-GLK Tg T cells, Phos-tag SDS-PAGE gel was used. (E) The schematic diagram of FoxO1 or FoxO3a binding site on the Foxp3 promoter. Arrows indicate FoxO-binding sites. (F) Chromatin immunoprecipitation (ChIP)-PCR analysis of immunoprecipitated FoxO1- or FoxO3a-binding DNA fragments of the Foxp3 promoter from T cells of WT and Lck-GLK Tg mice. (G) The Foxp3 promoter reporter, the FoxO1 plasmid, and the plasmid encoding IKKβ or IKKβ kinase-dead (K44M) mutant were co-transfected into Jurkat T cells; the luciferase reporter activity of the Foxp3 promoter was determined. Mean ± SEM are shown. (H) Immunoblotting analysis of p-FoxO1, FoxO1, GLK, IKKβ, and actin in protein lysates from WT, Lck-GLK Tg, and Lck-GLK Tg/IKKβ cKO T cells using Phos-tag SDS-PAGE gel. Data shown are representative of three independent experiments.
    Figure Legend Snippet: GLK-IKKβ signaling attenuates Foxp3 promoter activity through FoxO1. (A) Purified CD4 + T cells were cultured under the iTreg-differentiated condition with or without the treatment of the GLK inhibitor (1 μM or 5 μM), with a quantitative graph of CD4 + Foxp3 + T cells among CD4 + T cells. Mean ± SEM are shown. (B) Flow cytometry analysis of CD4 + Foxp3 + T cells among in vitro -differentiated iTreg cells from wild-type (WT) and GLK-deficient mice, with a quantitative graph of CD4 + Foxp3 + T cells among CD4 + T cells. Mean ± SEM are shown. (C) Flow cytometry analysis of CD4 + Foxp3 + T cells among in vitro -differentiated iTreg cells from WT, Lck-GLK Tg mice, and Lck-GLK Tg/ IKKβ cKO mice, with a quantitative graph of CD4 + Foxp3 + T cells among CD4 + T cells. Mean ± SEM are shown. (D) For immunoblotting analysis of phosphorylated (p)-FoxO1, FoxO1, p-FoxO3a, FoxO3a, and vinculin in protein lysates from WT or Lck-GLK Tg T cells, Phos-tag SDS-PAGE gel was used. (E) The schematic diagram of FoxO1 or FoxO3a binding site on the Foxp3 promoter. Arrows indicate FoxO-binding sites. (F) Chromatin immunoprecipitation (ChIP)-PCR analysis of immunoprecipitated FoxO1- or FoxO3a-binding DNA fragments of the Foxp3 promoter from T cells of WT and Lck-GLK Tg mice. (G) The Foxp3 promoter reporter, the FoxO1 plasmid, and the plasmid encoding IKKβ or IKKβ kinase-dead (K44M) mutant were co-transfected into Jurkat T cells; the luciferase reporter activity of the Foxp3 promoter was determined. Mean ± SEM are shown. (H) Immunoblotting analysis of p-FoxO1, FoxO1, GLK, IKKβ, and actin in protein lysates from WT, Lck-GLK Tg, and Lck-GLK Tg/IKKβ cKO T cells using Phos-tag SDS-PAGE gel. Data shown are representative of three independent experiments.

    Techniques Used: Activity Assay, Purification, Cell Culture, Flow Cytometry, In Vitro, Western Blot, SDS Page, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Plasmid Preparation, Mutagenesis, Transfection, Luciferase

    IKKβ directly interacts with FoxO1. (A) Co-immunoprecipitation experiments of Flag-tagged FoxO1 and HA-tagged IKKβ using lysates of HEK293T cells. IB, immunoblotting. (B) Amplified luminescent proximity homogeneous assay (ALPHA) analysis of the interaction between Flag-FoxO1 and either HA-IKKβ or Flag-GLK in lysates of HEK293T transfectants. Mean ± SEM are shown. ** P < 0.01 and *** P < 0.001 (two-tailed student's test). (C) Purified Flag-tagged FoxO1 and GST-tagged IKKβ proteins were used for in vitro binding assay. The relative protein levels of the protein complexes determined by densitometry analysis were shown at the bottom of panels. (D) FRET analysis of the direct interaction between YFP-fused FoxO1 and CFP-fused IKKβ proteins in HEK293T transfectants. ** P < 0.01 and *** P < 0.001 (two-tailed student's test). (E) Confocal microscopy analysis of intracellular localization of IKKβ and FoxO1 proteins in iTreg cells differentiated in vitro from T cells of wild-type (WT) and Lck-GLK Tg mice (left). Original magnification, x630; scale bars, 10 μm. FoxO1 localizations in the cytoplasm or nucleus of iTreg cells were quantified (right). (F) Confocal microscopy analysis of proximity ligation assay (PLA) for the interaction between endogenous FoxO1 and IKKβ proteins in iTreg differentiated from T cells of WT and Lck-GLK Tg mice (upper panel). Red dots represent direct interactions. Original magnification, x630; scale bars, 5 μm. PLA signals in the cytoplasm or nucleus of iTreg cells were quantified (lower panel). Data shown are representative of three independent experiments.
    Figure Legend Snippet: IKKβ directly interacts with FoxO1. (A) Co-immunoprecipitation experiments of Flag-tagged FoxO1 and HA-tagged IKKβ using lysates of HEK293T cells. IB, immunoblotting. (B) Amplified luminescent proximity homogeneous assay (ALPHA) analysis of the interaction between Flag-FoxO1 and either HA-IKKβ or Flag-GLK in lysates of HEK293T transfectants. Mean ± SEM are shown. ** P < 0.01 and *** P < 0.001 (two-tailed student's test). (C) Purified Flag-tagged FoxO1 and GST-tagged IKKβ proteins were used for in vitro binding assay. The relative protein levels of the protein complexes determined by densitometry analysis were shown at the bottom of panels. (D) FRET analysis of the direct interaction between YFP-fused FoxO1 and CFP-fused IKKβ proteins in HEK293T transfectants. ** P < 0.01 and *** P < 0.001 (two-tailed student's test). (E) Confocal microscopy analysis of intracellular localization of IKKβ and FoxO1 proteins in iTreg cells differentiated in vitro from T cells of wild-type (WT) and Lck-GLK Tg mice (left). Original magnification, x630; scale bars, 10 μm. FoxO1 localizations in the cytoplasm or nucleus of iTreg cells were quantified (right). (F) Confocal microscopy analysis of proximity ligation assay (PLA) for the interaction between endogenous FoxO1 and IKKβ proteins in iTreg differentiated from T cells of WT and Lck-GLK Tg mice (upper panel). Red dots represent direct interactions. Original magnification, x630; scale bars, 5 μm. PLA signals in the cytoplasm or nucleus of iTreg cells were quantified (lower panel). Data shown are representative of three independent experiments.

    Techniques Used: Immunoprecipitation, Western Blot, Amplification, Two Tailed Test, Purification, In Vitro, Binding Assay, Confocal Microscopy, Proximity Ligation Assay

    IKKβ directly phosphorylates FoxO1 at Ser319, leading to FoxO1 nuclear export. (A) Tandem MS (MS/MS) fragmentation spectra of the trypsin-digested FoxO1 peptides contain the phosphorylation of Ser319 with observed b/y ions. m/z, mass/charge ratio. (B) In vitro kinase assays using purified GST-tagged FoxO1 plus either Flag-tagged IKKβ or IKKβ kinase-dead (K44M) mutant proteins. Three individual phosphorylated residues of FoxO1 proteins were determined by immunoblotting analyses. The relative phosphorylation levels (phosphorylated protein level/total protein level) determined by densitometry analysis were shown at the bottom of panels. (C) Immunoblotting analysis of phosphorylated (p-) FoxO1 (Ser319), FoxO1, p-FoxO3a (Ser256), FoxO3a, and actin in splenic wild-type (WT) or Lck-GLK Tg T cells stimulated with or without anti-CD3 antibodies. The relative phosphorylation levels (phosphorylated protein level/total protein level) determined by densitometry analysis were shown at the bottom of panels. (D) Immunoblotting analysis of p-FoxO1 (Ser319), FoxO1, and actin in the lysates of HEK293T cells co-transfected with Flag-FoxO1 plus individual Flag-GLK, Flag-PKCθ, Myc-IKKα, or Flag-IKKβ plasmids. The relative phospho-Ser319-FoxO1 levels determined by densitometry analysis were shown at the bottom of the panel. (E) The Foxp3 promoter reporter with either FoxO1 or FoxO1 (Ser319A) plasmid plus IKKβ plasmid were co-transfected into Jurkat T cells; the luciferase reporter activity of the Foxp3 promoter was determined. Mean ± SEM are shown. (F) Confocal microscopy analysis of intracellular localization of HA-tagged IKKβ and Flag-tagged-FoxO1 proteins in Jurkat (J-TAg) cells co-transfected with HA-IKKβ plus either Flag-FoxO1 or Flag-FoxO1 (S319A) plasmids. Original magnification, x630; scale bars, 10 μm. Localizations of FoxO1 and FoxO1 (S319A) localizations in the cytoplasm or nucleus of Jurkat (J-TAg) cells were quantified (right panel). Data shown (B-F) are representative of three independent experiments.
    Figure Legend Snippet: IKKβ directly phosphorylates FoxO1 at Ser319, leading to FoxO1 nuclear export. (A) Tandem MS (MS/MS) fragmentation spectra of the trypsin-digested FoxO1 peptides contain the phosphorylation of Ser319 with observed b/y ions. m/z, mass/charge ratio. (B) In vitro kinase assays using purified GST-tagged FoxO1 plus either Flag-tagged IKKβ or IKKβ kinase-dead (K44M) mutant proteins. Three individual phosphorylated residues of FoxO1 proteins were determined by immunoblotting analyses. The relative phosphorylation levels (phosphorylated protein level/total protein level) determined by densitometry analysis were shown at the bottom of panels. (C) Immunoblotting analysis of phosphorylated (p-) FoxO1 (Ser319), FoxO1, p-FoxO3a (Ser256), FoxO3a, and actin in splenic wild-type (WT) or Lck-GLK Tg T cells stimulated with or without anti-CD3 antibodies. The relative phosphorylation levels (phosphorylated protein level/total protein level) determined by densitometry analysis were shown at the bottom of panels. (D) Immunoblotting analysis of p-FoxO1 (Ser319), FoxO1, and actin in the lysates of HEK293T cells co-transfected with Flag-FoxO1 plus individual Flag-GLK, Flag-PKCθ, Myc-IKKα, or Flag-IKKβ plasmids. The relative phospho-Ser319-FoxO1 levels determined by densitometry analysis were shown at the bottom of the panel. (E) The Foxp3 promoter reporter with either FoxO1 or FoxO1 (Ser319A) plasmid plus IKKβ plasmid were co-transfected into Jurkat T cells; the luciferase reporter activity of the Foxp3 promoter was determined. Mean ± SEM are shown. (F) Confocal microscopy analysis of intracellular localization of HA-tagged IKKβ and Flag-tagged-FoxO1 proteins in Jurkat (J-TAg) cells co-transfected with HA-IKKβ plus either Flag-FoxO1 or Flag-FoxO1 (S319A) plasmids. Original magnification, x630; scale bars, 10 μm. Localizations of FoxO1 and FoxO1 (S319A) localizations in the cytoplasm or nucleus of Jurkat (J-TAg) cells were quantified (right panel). Data shown (B-F) are representative of three independent experiments.

    Techniques Used: Tandem Mass Spectroscopy, In Vitro, Purification, Mutagenesis, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Confocal Microscopy

    GLK directly phosphorylates IKKβ at Ser733, leading to IKKβ-induced FoxO1 Ser319 phosphorylation. (A) For immunoblotting analysis of phosphorylated (p-) IKKβ, IKKβ proteins in protein lysates from HEK293T co-transfected with Flag-GLK and Flag-IKKβ kinase-dead (K44M) plasmids, Phos-tag SDS-PAGE gel was used. (B) Co-immunoprecipitation experiments of Flag-tagged GLK and HA-tagged IKKβ using lysates of transfected HEK293T cells. IB, immunoblotting. (C) Tandem MS (MS/MS) fragmentation spectra of the trypsin-digested peptides of IKKβ contain the phosphorylation of Ser733 with observed b/y ions. m/z, mass/charge ratio. Flag-tagged-IKKβ kinase-dead (K44M) mutant proteins were immunoprecipitated from lysates of HEK293T cells co-transfected with Flag-IKKβ kinase-dead (K44M) mutant and Flag-GLK plasmids. (D) Immunoblotting analyses of p-IKKβ (S733), p-IKKα/β (S180/S181), IKKβ, GLK, and PKCθ in HEK293T cells. Cells were co-transfected with Flag-IKKβ kinase-dead (K44M) mutant plus individual Flag-GLK, Flag-GLK kinase-dead (K45E) mutant, Myc-PKCθ, or Myc-PKCθ kinase-dead (K409W) mutant plasmids. (E) In vitro kinase assays of Flag-tagged GLK with either Flag-tagged IKKβ kinase-dead (K44M) mutant or Flag-tagged IKKβ phospho-deficient (K44M/S733A) mutant proteins immunopurified from individual HEK293T transfectants. (F) Immunoblotting analyses of p-FoxO1 (Ser319) and FoxO1 proteins in HEK293T cells co-transfected with Flag-FoxO1 plus individual Flag-IKKβ, phosphomimetic Flag-IKKβ (S733E) mutant, or phosphomimetic Flag-IKKβ (S180E/S181E) mutant plasmids. (G) Immunoblotting analyses of the endogenous p-FoxO1 (Ser319), FoxO1, p-IKKβ (Ser733), IKKβ, GLK, and PKCθ proteins in primary splenic T cells from wild-type (WT), Lck-GLK Tg, and Lck-GLK Tg/PKCθ KO mice. (H) Confocal microscopy analysis of proximity ligation assay (PLA) for the interaction between endogenous GLK and IKKβ proteins in T cells from WT, Lck-GLK Tg, and Lck-GLK Tg/PKCθ KO mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg/ml). Red dots represent direct interactions. Original magnification, x630; scale bars, 10 μm. (I) Quantification of the PLA signals (H) in each cell is shown. (J) Confocal microscopy analysis of PLA for the interaction between endogenous GLK and PKCθ proteins in T cells from WT, Lck-GLK Tg, and Lck-GLK Tg/PKCθ KO mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg/ml). Red dots represent direct interactions. Original magnification, x630; scale bars, 10 μm. (K) Quantification of the PLA signals (J) in each cell is shown. Data shown (A, B, D-K) are representative of three independent experiments.
    Figure Legend Snippet: GLK directly phosphorylates IKKβ at Ser733, leading to IKKβ-induced FoxO1 Ser319 phosphorylation. (A) For immunoblotting analysis of phosphorylated (p-) IKKβ, IKKβ proteins in protein lysates from HEK293T co-transfected with Flag-GLK and Flag-IKKβ kinase-dead (K44M) plasmids, Phos-tag SDS-PAGE gel was used. (B) Co-immunoprecipitation experiments of Flag-tagged GLK and HA-tagged IKKβ using lysates of transfected HEK293T cells. IB, immunoblotting. (C) Tandem MS (MS/MS) fragmentation spectra of the trypsin-digested peptides of IKKβ contain the phosphorylation of Ser733 with observed b/y ions. m/z, mass/charge ratio. Flag-tagged-IKKβ kinase-dead (K44M) mutant proteins were immunoprecipitated from lysates of HEK293T cells co-transfected with Flag-IKKβ kinase-dead (K44M) mutant and Flag-GLK plasmids. (D) Immunoblotting analyses of p-IKKβ (S733), p-IKKα/β (S180/S181), IKKβ, GLK, and PKCθ in HEK293T cells. Cells were co-transfected with Flag-IKKβ kinase-dead (K44M) mutant plus individual Flag-GLK, Flag-GLK kinase-dead (K45E) mutant, Myc-PKCθ, or Myc-PKCθ kinase-dead (K409W) mutant plasmids. (E) In vitro kinase assays of Flag-tagged GLK with either Flag-tagged IKKβ kinase-dead (K44M) mutant or Flag-tagged IKKβ phospho-deficient (K44M/S733A) mutant proteins immunopurified from individual HEK293T transfectants. (F) Immunoblotting analyses of p-FoxO1 (Ser319) and FoxO1 proteins in HEK293T cells co-transfected with Flag-FoxO1 plus individual Flag-IKKβ, phosphomimetic Flag-IKKβ (S733E) mutant, or phosphomimetic Flag-IKKβ (S180E/S181E) mutant plasmids. (G) Immunoblotting analyses of the endogenous p-FoxO1 (Ser319), FoxO1, p-IKKβ (Ser733), IKKβ, GLK, and PKCθ proteins in primary splenic T cells from wild-type (WT), Lck-GLK Tg, and Lck-GLK Tg/PKCθ KO mice. (H) Confocal microscopy analysis of proximity ligation assay (PLA) for the interaction between endogenous GLK and IKKβ proteins in T cells from WT, Lck-GLK Tg, and Lck-GLK Tg/PKCθ KO mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg/ml). Red dots represent direct interactions. Original magnification, x630; scale bars, 10 μm. (I) Quantification of the PLA signals (H) in each cell is shown. (J) Confocal microscopy analysis of PLA for the interaction between endogenous GLK and PKCθ proteins in T cells from WT, Lck-GLK Tg, and Lck-GLK Tg/PKCθ KO mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg/ml). Red dots represent direct interactions. Original magnification, x630; scale bars, 10 μm. (K) Quantification of the PLA signals (J) in each cell is shown. Data shown (A, B, D-K) are representative of three independent experiments.

    Techniques Used: Western Blot, Transfection, SDS Page, Immunoprecipitation, Tandem Mass Spectroscopy, Mutagenesis, In Vitro, Confocal Microscopy, Proximity Ligation Assay

    Foxp3 mRNA levels are inversely correlated with FoxO1 mRNA levels in GLK transgenic T cells. (A) Two-dimensional tSNE plot of T cell single-cell transcriptomes with different areas identified by clustering. The T cell subset in each area is indicated. (B) tSNE plot showing GLK + T-cell subpopulation (6.86%) in total T cells. (C) Heatmap showing Foxp3 transcripts in GLK + T-cell subpopulation. (D) tSNE plot showing GLK + CD4 + T-cell subpopulation (4.96%) in total T cells. (E) Heatmap showing Foxp3 transcripts in GLK + CD4 + T-cell subpopulation. (F) tSNE plots showing 1.14% of Foxp3 + T cells in GLK + FoxO1 + T cells (upper panel) or 3.9% of Foxp3 + T cells in GLK - FoxO1 + T cells (lower panel). (G) tSNE plot showing FoxO1 + T-cell subpopulation (36.8%) in total T cells. (H) Heatmap showing Foxp3 downregulation in FoxO1 + T-cell subpopulation with concomitantly elevated GLK expression. (I) tSNE plot showing FoxO1 + T-cell subpopulation (36.8%) in CD4 + T cells. (J) Heatmap showing Foxp3 expression in FoxO1 + CD4 + T-cell subpopulation with concomitantly elevated GLK expression.
    Figure Legend Snippet: Foxp3 mRNA levels are inversely correlated with FoxO1 mRNA levels in GLK transgenic T cells. (A) Two-dimensional tSNE plot of T cell single-cell transcriptomes with different areas identified by clustering. The T cell subset in each area is indicated. (B) tSNE plot showing GLK + T-cell subpopulation (6.86%) in total T cells. (C) Heatmap showing Foxp3 transcripts in GLK + T-cell subpopulation. (D) tSNE plot showing GLK + CD4 + T-cell subpopulation (4.96%) in total T cells. (E) Heatmap showing Foxp3 transcripts in GLK + CD4 + T-cell subpopulation. (F) tSNE plots showing 1.14% of Foxp3 + T cells in GLK + FoxO1 + T cells (upper panel) or 3.9% of Foxp3 + T cells in GLK - FoxO1 + T cells (lower panel). (G) tSNE plot showing FoxO1 + T-cell subpopulation (36.8%) in total T cells. (H) Heatmap showing Foxp3 downregulation in FoxO1 + T-cell subpopulation with concomitantly elevated GLK expression. (I) tSNE plot showing FoxO1 + T-cell subpopulation (36.8%) in CD4 + T cells. (J) Heatmap showing Foxp3 expression in FoxO1 + CD4 + T-cell subpopulation with concomitantly elevated GLK expression.

    Techniques Used: Transgenic Assay, Expressing

    Schematic model of GLK overexpression-induced attenuation of Foxp3 transcription. GLK overexpression in Treg cells phosphorylates IKKβ at Ser733 residue in a PKCθ-independent manner. The transcription factor FoxO1 binds to the Foxp3 promoter and promotes Foxp3 transcription in normal Treg cells. The GLK-phosphorylated IKKβ interacts with and induces FoxO1 Ser319 phosphorylation, leading to FoxO1 nuclear export and Foxp3 downregulation. Thus, GLK-IKKβ-FoxO1 signaling attenuates differentiation and function of Treg cells.
    Figure Legend Snippet: Schematic model of GLK overexpression-induced attenuation of Foxp3 transcription. GLK overexpression in Treg cells phosphorylates IKKβ at Ser733 residue in a PKCθ-independent manner. The transcription factor FoxO1 binds to the Foxp3 promoter and promotes Foxp3 transcription in normal Treg cells. The GLK-phosphorylated IKKβ interacts with and induces FoxO1 Ser319 phosphorylation, leading to FoxO1 nuclear export and Foxp3 downregulation. Thus, GLK-IKKβ-FoxO1 signaling attenuates differentiation and function of Treg cells.

    Techniques Used: Over Expression

    p foxo1  (Cell Signaling Technology Inc)


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    P Foxo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho foxo1
    Activation of RUNX2 transcriptional activity by CSE in the periosteum-derived cells. A: Western analysis of expression of the indicated proteins in periosteum-derived cells treated with the indicated concentrations of CSE B: Luciferase activity showing RUNX2 transcriptional activity in cells co-nucleofected with wild-type <t>FOXO1</t> and the AKT phosphorylation-resistant mutant FOXO1-A3 (b) . FOXO1-WT, wild-type FOXO1; FOXO1-A3, constitutively active FOXO1. ** P < 0.01.
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    1) Product Images from "FOXO1 Is Involved in the Effects of Cigarette Smoke Extract on Osteoblastic Differentiation of Cultured Human Periosteum-derived Cells"

    Article Title: FOXO1 Is Involved in the Effects of Cigarette Smoke Extract on Osteoblastic Differentiation of Cultured Human Periosteum-derived Cells

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.13172

    Activation of RUNX2 transcriptional activity by CSE in the periosteum-derived cells. A: Western analysis of expression of the indicated proteins in periosteum-derived cells treated with the indicated concentrations of CSE B: Luciferase activity showing RUNX2 transcriptional activity in cells co-nucleofected with wild-type FOXO1 and the AKT phosphorylation-resistant mutant FOXO1-A3 (b) . FOXO1-WT, wild-type FOXO1; FOXO1-A3, constitutively active FOXO1. ** P < 0.01.
    Figure Legend Snippet: Activation of RUNX2 transcriptional activity by CSE in the periosteum-derived cells. A: Western analysis of expression of the indicated proteins in periosteum-derived cells treated with the indicated concentrations of CSE B: Luciferase activity showing RUNX2 transcriptional activity in cells co-nucleofected with wild-type FOXO1 and the AKT phosphorylation-resistant mutant FOXO1-A3 (b) . FOXO1-WT, wild-type FOXO1; FOXO1-A3, constitutively active FOXO1. ** P < 0.01.

    Techniques Used: Activation Assay, Activity Assay, Derivative Assay, Western Blot, Expressing, Luciferase, Mutagenesis

    p foxo1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p foxo1
    E 2 -stimulated activation of SGK1 attenuates TLR4-mediated apoptosis of DSCs. (A) Relative efficiencies of DSC growth were assessed with the MTT assay. (B) TLR-4-triggered apoptosis of E 2 -pretreated DSCs in the presence or absence of SGK1 inhibitor GSK650394 was determined by flow cytometry based on PI staining and annexin expression. Transcript levels the of anti-apoptotic genes BCL2 and XIAP (C), and the decidual marker gene PRL (D) in LPS-stimulated DSCs incubated with E 2 with or without GSK650394. Protein levels of BCL2 (E), XIAP (F) and PRL (G) were analyzed by immunoblotting in LPS-stimulated DSCs incubated with E 2 with or without GSK650394. (H) Western blot analysis of cell lysates of LPS-stimulated DSCs pretreated with E 2 with or without GSK650394. Blots were probed with antibodies to <t>total-FOXO1</t> (t-FOXO1), phosphorylated-FOXO1 (p-FOXO1) and β-actin as a loading control. (I) Densitometric quantification of phosphorylated- and total FOXO1 to β-actin (left), and mean (SEM) ratio of phosphorylated-to-total protein for FOXO1 (right). Control group, phenol red-free RPMI-1640 media; LPS group, 10 ng/mL LPS; LPS+E 2 group, 10 ng/mL LPS + 10 nM E 2 ; LPS+E 2 +GSK650394 group, 10 ng/mL LPS + 10 nM E 2 + 10 μM GSK650394. Data are represented as arithmetic means ± SEM for 3 individual DSCs from early normal pregnancy. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with control group; ∆ P < 0.05, ∆∆ P < 0.01, ∆∆∆ P < 0.001, compared with LPS group; # P < 0.05, ## P < 0.01, ### P < 0.001, compared with LPS+E 2 group.
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    1) Product Images from "Estradiol Suppresses TLR4-triggered Apoptosis of Decidual Stromal Cells and Drives an Anti-inflammatory T H 2 Shift by Activating SGK1"

    Article Title: Estradiol Suppresses TLR4-triggered Apoptosis of Decidual Stromal Cells and Drives an Anti-inflammatory T H 2 Shift by Activating SGK1

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.18278

    E 2 -stimulated activation of SGK1 attenuates TLR4-mediated apoptosis of DSCs. (A) Relative efficiencies of DSC growth were assessed with the MTT assay. (B) TLR-4-triggered apoptosis of E 2 -pretreated DSCs in the presence or absence of SGK1 inhibitor GSK650394 was determined by flow cytometry based on PI staining and annexin expression. Transcript levels the of anti-apoptotic genes BCL2 and XIAP (C), and the decidual marker gene PRL (D) in LPS-stimulated DSCs incubated with E 2 with or without GSK650394. Protein levels of BCL2 (E), XIAP (F) and PRL (G) were analyzed by immunoblotting in LPS-stimulated DSCs incubated with E 2 with or without GSK650394. (H) Western blot analysis of cell lysates of LPS-stimulated DSCs pretreated with E 2 with or without GSK650394. Blots were probed with antibodies to total-FOXO1 (t-FOXO1), phosphorylated-FOXO1 (p-FOXO1) and β-actin as a loading control. (I) Densitometric quantification of phosphorylated- and total FOXO1 to β-actin (left), and mean (SEM) ratio of phosphorylated-to-total protein for FOXO1 (right). Control group, phenol red-free RPMI-1640 media; LPS group, 10 ng/mL LPS; LPS+E 2 group, 10 ng/mL LPS + 10 nM E 2 ; LPS+E 2 +GSK650394 group, 10 ng/mL LPS + 10 nM E 2 + 10 μM GSK650394. Data are represented as arithmetic means ± SEM for 3 individual DSCs from early normal pregnancy. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with control group; ∆ P < 0.05, ∆∆ P < 0.01, ∆∆∆ P < 0.001, compared with LPS group; # P < 0.05, ## P < 0.01, ### P < 0.001, compared with LPS+E 2 group.
    Figure Legend Snippet: E 2 -stimulated activation of SGK1 attenuates TLR4-mediated apoptosis of DSCs. (A) Relative efficiencies of DSC growth were assessed with the MTT assay. (B) TLR-4-triggered apoptosis of E 2 -pretreated DSCs in the presence or absence of SGK1 inhibitor GSK650394 was determined by flow cytometry based on PI staining and annexin expression. Transcript levels the of anti-apoptotic genes BCL2 and XIAP (C), and the decidual marker gene PRL (D) in LPS-stimulated DSCs incubated with E 2 with or without GSK650394. Protein levels of BCL2 (E), XIAP (F) and PRL (G) were analyzed by immunoblotting in LPS-stimulated DSCs incubated with E 2 with or without GSK650394. (H) Western blot analysis of cell lysates of LPS-stimulated DSCs pretreated with E 2 with or without GSK650394. Blots were probed with antibodies to total-FOXO1 (t-FOXO1), phosphorylated-FOXO1 (p-FOXO1) and β-actin as a loading control. (I) Densitometric quantification of phosphorylated- and total FOXO1 to β-actin (left), and mean (SEM) ratio of phosphorylated-to-total protein for FOXO1 (right). Control group, phenol red-free RPMI-1640 media; LPS group, 10 ng/mL LPS; LPS+E 2 group, 10 ng/mL LPS + 10 nM E 2 ; LPS+E 2 +GSK650394 group, 10 ng/mL LPS + 10 nM E 2 + 10 μM GSK650394. Data are represented as arithmetic means ± SEM for 3 individual DSCs from early normal pregnancy. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with control group; ∆ P < 0.05, ∆∆ P < 0.01, ∆∆∆ P < 0.001, compared with LPS group; # P < 0.05, ## P < 0.01, ### P < 0.001, compared with LPS+E 2 group.

    Techniques Used: Activation Assay, MTT Assay, Flow Cytometry, Staining, Expressing, Marker, Incubation, Western Blot

    Working model of E 2 -activated SGK1 involvement in negative regulation of TLR-4-mediated apoptosis and pro-inflammatory T H 1 immune responses at the feto-maternal interface. E 2 -activated SGK1 via the PI3K signaling pathway promoted cell viability and suppressed LPS-induced apoptosis of DSCs by up-regulating the expressions of anti-apoptotic genes, and attenuating pro-apoptotic FOXO1 activation. E 2 -sensitive activation of SGK1 down-regulates TLR4-mediated NF-κB activation, leading to reduced production of pro-inflammatory cytokines and promoting T H 2 shift at the feto-maternal interface, which is beneficial to successful gestation.
    Figure Legend Snippet: Working model of E 2 -activated SGK1 involvement in negative regulation of TLR-4-mediated apoptosis and pro-inflammatory T H 1 immune responses at the feto-maternal interface. E 2 -activated SGK1 via the PI3K signaling pathway promoted cell viability and suppressed LPS-induced apoptosis of DSCs by up-regulating the expressions of anti-apoptotic genes, and attenuating pro-apoptotic FOXO1 activation. E 2 -sensitive activation of SGK1 down-regulates TLR4-mediated NF-κB activation, leading to reduced production of pro-inflammatory cytokines and promoting T H 2 shift at the feto-maternal interface, which is beneficial to successful gestation.

    Techniques Used: Activation Assay

    phospho foxo1 ser319  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho foxo1 ser319
    Phospho Foxo1 Ser319, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phospho foxo1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho foxo1
    PHLPP2/1 knock-down results in a significant improvement of insulin-stimulated phosphorylation of Akt, <t>FoxO1,</t> and mTor. (a) Representative western blot images of Akt phosphorylation on the Serine 473 residue (upper panel) and of total Akt levels (middle panel) in mock-infected INS-1 HG, INS-1 HG AV-LD, INS-1 HG AV-HD-stimulated (+I), or not (b) with 10 −7 M insulin. Graphs of the mean changes over insulin-stimulated mock-infected INS-1 HG values INS-1 NG values of the densitometric values of pAkt obtained in 3 independent experiments and normalized for total Akt levels; ∗ indicates significant ( p < 0.05) differences for AV-infected INS-1 HG vs mock-infected INS-1 HG. (b) Representative Western Blot images of FoxO1 phosphorylation (upper panel), total FoxO1 levels (upper-middle panel), mTor phosphorylation (lower-middle panel), total mTor levels (lower panel) in mock-infected INS-1 HG, INS-1 HG AV-LD, INS-1 HG AV-HD-stimulated (+I), or not (b) with 10 −7 M insulin. Graphs of the mean changes over insulin-stimulated mock-infected INS-1 HG values and INS-1 NG values of the densitometric values of pFoxO1 or pmTor obtained in 3–5 independent experiments and normalized for total levels of the unphosphorylated protein; ∗ indicates significant ( p < 0.05) differences for AV-infected INS-1 HG vs mock-infected INS-1 HG.
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    1) Product Images from "The Phosphatase PHLPP2 Plays a Key Role in the Regulation of Pancreatic Beta-Cell Survival"

    Article Title: The Phosphatase PHLPP2 Plays a Key Role in the Regulation of Pancreatic Beta-Cell Survival

    Journal: International Journal of Endocrinology

    doi: 10.1155/2020/1027386

    PHLPP2/1 knock-down results in a significant improvement of insulin-stimulated phosphorylation of Akt, FoxO1, and mTor. (a) Representative western blot images of Akt phosphorylation on the Serine 473 residue (upper panel) and of total Akt levels (middle panel) in mock-infected INS-1 HG, INS-1 HG AV-LD, INS-1 HG AV-HD-stimulated (+I), or not (b) with 10 −7 M insulin. Graphs of the mean changes over insulin-stimulated mock-infected INS-1 HG values INS-1 NG values of the densitometric values of pAkt obtained in 3 independent experiments and normalized for total Akt levels; ∗ indicates significant ( p < 0.05) differences for AV-infected INS-1 HG vs mock-infected INS-1 HG. (b) Representative Western Blot images of FoxO1 phosphorylation (upper panel), total FoxO1 levels (upper-middle panel), mTor phosphorylation (lower-middle panel), total mTor levels (lower panel) in mock-infected INS-1 HG, INS-1 HG AV-LD, INS-1 HG AV-HD-stimulated (+I), or not (b) with 10 −7 M insulin. Graphs of the mean changes over insulin-stimulated mock-infected INS-1 HG values and INS-1 NG values of the densitometric values of pFoxO1 or pmTor obtained in 3–5 independent experiments and normalized for total levels of the unphosphorylated protein; ∗ indicates significant ( p < 0.05) differences for AV-infected INS-1 HG vs mock-infected INS-1 HG.
    Figure Legend Snippet: PHLPP2/1 knock-down results in a significant improvement of insulin-stimulated phosphorylation of Akt, FoxO1, and mTor. (a) Representative western blot images of Akt phosphorylation on the Serine 473 residue (upper panel) and of total Akt levels (middle panel) in mock-infected INS-1 HG, INS-1 HG AV-LD, INS-1 HG AV-HD-stimulated (+I), or not (b) with 10 −7 M insulin. Graphs of the mean changes over insulin-stimulated mock-infected INS-1 HG values INS-1 NG values of the densitometric values of pAkt obtained in 3 independent experiments and normalized for total Akt levels; ∗ indicates significant ( p < 0.05) differences for AV-infected INS-1 HG vs mock-infected INS-1 HG. (b) Representative Western Blot images of FoxO1 phosphorylation (upper panel), total FoxO1 levels (upper-middle panel), mTor phosphorylation (lower-middle panel), total mTor levels (lower panel) in mock-infected INS-1 HG, INS-1 HG AV-LD, INS-1 HG AV-HD-stimulated (+I), or not (b) with 10 −7 M insulin. Graphs of the mean changes over insulin-stimulated mock-infected INS-1 HG values and INS-1 NG values of the densitometric values of pFoxO1 or pmTor obtained in 3–5 independent experiments and normalized for total levels of the unphosphorylated protein; ∗ indicates significant ( p < 0.05) differences for AV-infected INS-1 HG vs mock-infected INS-1 HG.

    Techniques Used: Western Blot, Infection

    p foxo1 ser319 no 2486 1 1000  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p foxo1 ser319 no 2486 1 1000
    RSK2 upregulates cyclin D1 via promoting <t>FOXO1</t> degradation. (A) Western blot was used to measure FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (B) RT-qPCR was used to measure FOXO1 mRNA expression in A375 cells transfected with RSK2 siRNA. (C) The influence of RSK2 on FOXO1 degradation was estimated in A375 cells under MG132 treatment. (D) The effect of RSK2 on FOXO1 protein stability was evaluated in A375 cells under CHX treatment for the indicated time. (E) HEK293T cells were co-transfected with Flag-FOXO1 and HA-RSK2 plasmid as indicated, immunoprecipitation with anti-Flag antibody was performed. (F) Western blot was used to measure p-FOXO1 (S319) and FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (G) Western blot was used to measure cyclin D1 expression in A375 cells co-transfected with Flag-FOXO1 and HA-RSK2 as indicated. (H) A schematic model of RSK2–FOXO1–cyclin D1 axis leading to melanoma proliferation and vemurafenib resistance.
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    1) Product Images from "RSK2 promotes melanoma cell proliferation and vemurafenib resistance via upregulating cyclin D1"

    Article Title: RSK2 promotes melanoma cell proliferation and vemurafenib resistance via upregulating cyclin D1

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2022.950571

    RSK2 upregulates cyclin D1 via promoting FOXO1 degradation. (A) Western blot was used to measure FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (B) RT-qPCR was used to measure FOXO1 mRNA expression in A375 cells transfected with RSK2 siRNA. (C) The influence of RSK2 on FOXO1 degradation was estimated in A375 cells under MG132 treatment. (D) The effect of RSK2 on FOXO1 protein stability was evaluated in A375 cells under CHX treatment for the indicated time. (E) HEK293T cells were co-transfected with Flag-FOXO1 and HA-RSK2 plasmid as indicated, immunoprecipitation with anti-Flag antibody was performed. (F) Western blot was used to measure p-FOXO1 (S319) and FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (G) Western blot was used to measure cyclin D1 expression in A375 cells co-transfected with Flag-FOXO1 and HA-RSK2 as indicated. (H) A schematic model of RSK2–FOXO1–cyclin D1 axis leading to melanoma proliferation and vemurafenib resistance.
    Figure Legend Snippet: RSK2 upregulates cyclin D1 via promoting FOXO1 degradation. (A) Western blot was used to measure FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (B) RT-qPCR was used to measure FOXO1 mRNA expression in A375 cells transfected with RSK2 siRNA. (C) The influence of RSK2 on FOXO1 degradation was estimated in A375 cells under MG132 treatment. (D) The effect of RSK2 on FOXO1 protein stability was evaluated in A375 cells under CHX treatment for the indicated time. (E) HEK293T cells were co-transfected with Flag-FOXO1 and HA-RSK2 plasmid as indicated, immunoprecipitation with anti-Flag antibody was performed. (F) Western blot was used to measure p-FOXO1 (S319) and FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (G) Western blot was used to measure cyclin D1 expression in A375 cells co-transfected with Flag-FOXO1 and HA-RSK2 as indicated. (H) A schematic model of RSK2–FOXO1–cyclin D1 axis leading to melanoma proliferation and vemurafenib resistance.

    Techniques Used: Western Blot, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunoprecipitation

    phospho foxo1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho foxo1
    ( A ) There is an increased expression of <t>FoxO1</t> mRNA in the EDL (fast twitch) when this is compared to the soleus (slow twitch) from the control animals. ( B ) A trend for the upregulation of FoxO1 mRNA in soleus of SU/Hx-induced PH is compared to that of the control animals. ( C ) A trend for the upregulation of FoxO1 mRNA in diaphragm of SU/Hx-induced PH is compared to that of the control animals. Expression is normalized to Nup 133 . Values are expressed as the mean ± SEM. Statistical analysis is conducted by a Student’s t -test, ** p < 0.01, ns: not significant. ( D ) There is an increased expression of myogenin and FoxO1 mRNA in the whole cell lysates from differentiated L6 cells (Day 7) when this is compared to the whole cell lysates from undifferentiated cells. Expression is normalized to GAPDH . Values are expressed as the mean ± SEM. Statistical analysis was conducted by a Student’s t -test, ** p < 0.01. ( E ) Immunoblots and quantitative analysis showing that there was a cellular fractionation of the L6 myoblasts and myotube lysates. There was predominantly nuclear FoxO1 localization in myoblasts. There was cytosolic p-FoxO1 localization in myoblasts and myotubes. GAPDH was used as a marker for cytosolic fraction, and H3 was used as a marker for nuclear fraction. Values are expressed as the mean ± SEM. Statistical analysis was conducted by a Student’s t -test, * p < 0.05. ( F ) L6 myotube structure formation (indicated with arrows) after FoxO1 inhibitor treatment (AS1842856, 100 nM, 24 h). Immunoblots showing that there were unchanged levels of type II marker MYH1/2/4/6 and increased levels of type I marker Hexokinase in L6 myotube lysates after FoxO1 inhibitor treatment.
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    1) Product Images from "Skeletal Muscle Dysfunction in Experimental Pulmonary Hypertension"

    Article Title: Skeletal Muscle Dysfunction in Experimental Pulmonary Hypertension

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms231810912

    ( A ) There is an increased expression of FoxO1 mRNA in the EDL (fast twitch) when this is compared to the soleus (slow twitch) from the control animals. ( B ) A trend for the upregulation of FoxO1 mRNA in soleus of SU/Hx-induced PH is compared to that of the control animals. ( C ) A trend for the upregulation of FoxO1 mRNA in diaphragm of SU/Hx-induced PH is compared to that of the control animals. Expression is normalized to Nup 133 . Values are expressed as the mean ± SEM. Statistical analysis is conducted by a Student’s t -test, ** p < 0.01, ns: not significant. ( D ) There is an increased expression of myogenin and FoxO1 mRNA in the whole cell lysates from differentiated L6 cells (Day 7) when this is compared to the whole cell lysates from undifferentiated cells. Expression is normalized to GAPDH . Values are expressed as the mean ± SEM. Statistical analysis was conducted by a Student’s t -test, ** p < 0.01. ( E ) Immunoblots and quantitative analysis showing that there was a cellular fractionation of the L6 myoblasts and myotube lysates. There was predominantly nuclear FoxO1 localization in myoblasts. There was cytosolic p-FoxO1 localization in myoblasts and myotubes. GAPDH was used as a marker for cytosolic fraction, and H3 was used as a marker for nuclear fraction. Values are expressed as the mean ± SEM. Statistical analysis was conducted by a Student’s t -test, * p < 0.05. ( F ) L6 myotube structure formation (indicated with arrows) after FoxO1 inhibitor treatment (AS1842856, 100 nM, 24 h). Immunoblots showing that there were unchanged levels of type II marker MYH1/2/4/6 and increased levels of type I marker Hexokinase in L6 myotube lysates after FoxO1 inhibitor treatment.
    Figure Legend Snippet: ( A ) There is an increased expression of FoxO1 mRNA in the EDL (fast twitch) when this is compared to the soleus (slow twitch) from the control animals. ( B ) A trend for the upregulation of FoxO1 mRNA in soleus of SU/Hx-induced PH is compared to that of the control animals. ( C ) A trend for the upregulation of FoxO1 mRNA in diaphragm of SU/Hx-induced PH is compared to that of the control animals. Expression is normalized to Nup 133 . Values are expressed as the mean ± SEM. Statistical analysis is conducted by a Student’s t -test, ** p < 0.01, ns: not significant. ( D ) There is an increased expression of myogenin and FoxO1 mRNA in the whole cell lysates from differentiated L6 cells (Day 7) when this is compared to the whole cell lysates from undifferentiated cells. Expression is normalized to GAPDH . Values are expressed as the mean ± SEM. Statistical analysis was conducted by a Student’s t -test, ** p < 0.01. ( E ) Immunoblots and quantitative analysis showing that there was a cellular fractionation of the L6 myoblasts and myotube lysates. There was predominantly nuclear FoxO1 localization in myoblasts. There was cytosolic p-FoxO1 localization in myoblasts and myotubes. GAPDH was used as a marker for cytosolic fraction, and H3 was used as a marker for nuclear fraction. Values are expressed as the mean ± SEM. Statistical analysis was conducted by a Student’s t -test, * p < 0.05. ( F ) L6 myotube structure formation (indicated with arrows) after FoxO1 inhibitor treatment (AS1842856, 100 nM, 24 h). Immunoblots showing that there were unchanged levels of type II marker MYH1/2/4/6 and increased levels of type I marker Hexokinase in L6 myotube lysates after FoxO1 inhibitor treatment.

    Techniques Used: Expressing, Western Blot, Cell Fractionation, Marker

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    Cell Signaling Technology Inc anti phospho foxo1 ser319
    Cells were transfected with either siCon or siGRP78 RNA duplexes for 24 hour before treatment with tunicamycin for 24 hour. Proteins were extracted for immunoblot analysis with GRP78, P-PDK1(Ser241), PDK1, P-AKT(Thr308), P-AKT(Ser473), AKT, P-AKT substrate (RXRXXS/T), P-mTOR(Ser2448), P-HDM2(Ser166), <t>P-FOXO1(Ser319)</t> and P-GSK-3α/β(Ser21/9). Densitometry of band intensity is expressed relative to siCon untreated control (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are mean±SEM for 3 independent experiments. ** indicates P≤ 0.01; n.s indicates non-significant change. A & B ) Down-regulation of GRP78 elevates Ser473 phosphorylation but not at Thr308. C ) Knock-down of GRP78 alters AKT downstream substrates recognition. Arrows indicate the substrates changed their phosphorylation pattern in tunicamycin-treated siGRP78 cells.
    Anti Phospho Foxo1 Ser319, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cells were transfected with either siCon or siGRP78 RNA duplexes for 24 hour before treatment with tunicamycin for 24 hour. Proteins were extracted for immunoblot analysis with GRP78, P-PDK1(Ser241), PDK1, P-AKT(Thr308), P-AKT(Ser473), AKT, P-AKT substrate (RXRXXS/T), P-mTOR(Ser2448), P-HDM2(Ser166), <t>P-FOXO1(Ser319)</t> and P-GSK-3α/β(Ser21/9). Densitometry of band intensity is expressed relative to siCon untreated control (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are mean±SEM for 3 independent experiments. ** indicates P≤ 0.01; n.s indicates non-significant change. A & B ) Down-regulation of GRP78 elevates Ser473 phosphorylation but not at Thr308. C ) Knock-down of GRP78 alters AKT downstream substrates recognition. Arrows indicate the substrates changed their phosphorylation pattern in tunicamycin-treated siGRP78 cells.
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    Activation of RUNX2 transcriptional activity by CSE in the periosteum-derived cells. A: Western analysis of expression of the indicated proteins in periosteum-derived cells treated with the indicated concentrations of CSE B: Luciferase activity showing RUNX2 transcriptional activity in cells co-nucleofected with wild-type <t>FOXO1</t> and the AKT phosphorylation-resistant mutant FOXO1-A3 (b) . FOXO1-WT, wild-type FOXO1; FOXO1-A3, constitutively active FOXO1. ** P < 0.01.
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    Activation of RUNX2 transcriptional activity by CSE in the periosteum-derived cells. A: Western analysis of expression of the indicated proteins in periosteum-derived cells treated with the indicated concentrations of CSE B: Luciferase activity showing RUNX2 transcriptional activity in cells co-nucleofected with wild-type <t>FOXO1</t> and the AKT phosphorylation-resistant mutant FOXO1-A3 (b) . FOXO1-WT, wild-type FOXO1; FOXO1-A3, constitutively active FOXO1. ** P < 0.01.
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    Cell Signaling Technology Inc p foxo1 ser319 no 2486 1 1000
    RSK2 upregulates cyclin D1 via promoting <t>FOXO1</t> degradation. (A) Western blot was used to measure FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (B) RT-qPCR was used to measure FOXO1 mRNA expression in A375 cells transfected with RSK2 siRNA. (C) The influence of RSK2 on FOXO1 degradation was estimated in A375 cells under MG132 treatment. (D) The effect of RSK2 on FOXO1 protein stability was evaluated in A375 cells under CHX treatment for the indicated time. (E) HEK293T cells were co-transfected with Flag-FOXO1 and HA-RSK2 plasmid as indicated, immunoprecipitation with anti-Flag antibody was performed. (F) Western blot was used to measure p-FOXO1 (S319) and FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (G) Western blot was used to measure cyclin D1 expression in A375 cells co-transfected with Flag-FOXO1 and HA-RSK2 as indicated. (H) A schematic model of RSK2–FOXO1–cyclin D1 axis leading to melanoma proliferation and vemurafenib resistance.
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    Image Search Results


    Cells were transfected with either siCon or siGRP78 RNA duplexes for 24 hour before treatment with tunicamycin for 24 hour. Proteins were extracted for immunoblot analysis with GRP78, P-PDK1(Ser241), PDK1, P-AKT(Thr308), P-AKT(Ser473), AKT, P-AKT substrate (RXRXXS/T), P-mTOR(Ser2448), P-HDM2(Ser166), P-FOXO1(Ser319) and P-GSK-3α/β(Ser21/9). Densitometry of band intensity is expressed relative to siCon untreated control (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are mean±SEM for 3 independent experiments. ** indicates P≤ 0.01; n.s indicates non-significant change. A & B ) Down-regulation of GRP78 elevates Ser473 phosphorylation but not at Thr308. C ) Knock-down of GRP78 alters AKT downstream substrates recognition. Arrows indicate the substrates changed their phosphorylation pattern in tunicamycin-treated siGRP78 cells.

    Journal: PLoS ONE

    Article Title: Regulation of AKT Phosphorylation at Ser473 and Thr308 by Endoplasmic Reticulum Stress Modulates Substrate Specificity in a Severity Dependent Manner

    doi: 10.1371/journal.pone.0017894

    Figure Lengend Snippet: Cells were transfected with either siCon or siGRP78 RNA duplexes for 24 hour before treatment with tunicamycin for 24 hour. Proteins were extracted for immunoblot analysis with GRP78, P-PDK1(Ser241), PDK1, P-AKT(Thr308), P-AKT(Ser473), AKT, P-AKT substrate (RXRXXS/T), P-mTOR(Ser2448), P-HDM2(Ser166), P-FOXO1(Ser319) and P-GSK-3α/β(Ser21/9). Densitometry of band intensity is expressed relative to siCon untreated control (100%). Phosphorylation status is presented as the ratio between phosphorylated and total protein. Data are mean±SEM for 3 independent experiments. ** indicates P≤ 0.01; n.s indicates non-significant change. A & B ) Down-regulation of GRP78 elevates Ser473 phosphorylation but not at Thr308. C ) Knock-down of GRP78 alters AKT downstream substrates recognition. Arrows indicate the substrates changed their phosphorylation pattern in tunicamycin-treated siGRP78 cells.

    Article Snippet: Anti-phospho-AKT (Ser473), anti-AKT, anti-AKT1, anti-phospho-mTOR (Ser2448), anti-phospho-HDM2 (Ser166), anti-phospho-FOXO1 (Ser319), anti-phospho-GSK3α/β (Ser21/9), immobilized AKT (1G1) antibodies (bead conjugated), immobilized phospho-AKT (Ser473) (D9E) antibodies (bead conjugated) and immobilized IgG mouse (bead conjugated) were from Cell Signaling Technology (New England Biolabs Ltd, UK).

    Techniques: Transfection, Western Blot

    Activation of RUNX2 transcriptional activity by CSE in the periosteum-derived cells. A: Western analysis of expression of the indicated proteins in periosteum-derived cells treated with the indicated concentrations of CSE B: Luciferase activity showing RUNX2 transcriptional activity in cells co-nucleofected with wild-type FOXO1 and the AKT phosphorylation-resistant mutant FOXO1-A3 (b) . FOXO1-WT, wild-type FOXO1; FOXO1-A3, constitutively active FOXO1. ** P < 0.01.

    Journal: International Journal of Medical Sciences

    Article Title: FOXO1 Is Involved in the Effects of Cigarette Smoke Extract on Osteoblastic Differentiation of Cultured Human Periosteum-derived Cells

    doi: 10.7150/ijms.13172

    Figure Lengend Snippet: Activation of RUNX2 transcriptional activity by CSE in the periosteum-derived cells. A: Western analysis of expression of the indicated proteins in periosteum-derived cells treated with the indicated concentrations of CSE B: Luciferase activity showing RUNX2 transcriptional activity in cells co-nucleofected with wild-type FOXO1 and the AKT phosphorylation-resistant mutant FOXO1-A3 (b) . FOXO1-WT, wild-type FOXO1; FOXO1-A3, constitutively active FOXO1. ** P < 0.01.

    Article Snippet: The membranes were blotted with antibodies against FOXO1, phospho-FOXO1, AKT, and phospho-AKT (Cell Signaling Technology, USA), and the proteins were identified using the Pierce ECL detection system (Thermo Scientific, USA).

    Techniques: Activation Assay, Activity Assay, Derivative Assay, Western Blot, Expressing, Luciferase, Mutagenesis

    RSK2 upregulates cyclin D1 via promoting FOXO1 degradation. (A) Western blot was used to measure FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (B) RT-qPCR was used to measure FOXO1 mRNA expression in A375 cells transfected with RSK2 siRNA. (C) The influence of RSK2 on FOXO1 degradation was estimated in A375 cells under MG132 treatment. (D) The effect of RSK2 on FOXO1 protein stability was evaluated in A375 cells under CHX treatment for the indicated time. (E) HEK293T cells were co-transfected with Flag-FOXO1 and HA-RSK2 plasmid as indicated, immunoprecipitation with anti-Flag antibody was performed. (F) Western blot was used to measure p-FOXO1 (S319) and FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (G) Western blot was used to measure cyclin D1 expression in A375 cells co-transfected with Flag-FOXO1 and HA-RSK2 as indicated. (H) A schematic model of RSK2–FOXO1–cyclin D1 axis leading to melanoma proliferation and vemurafenib resistance.

    Journal: Frontiers in Pharmacology

    Article Title: RSK2 promotes melanoma cell proliferation and vemurafenib resistance via upregulating cyclin D1

    doi: 10.3389/fphar.2022.950571

    Figure Lengend Snippet: RSK2 upregulates cyclin D1 via promoting FOXO1 degradation. (A) Western blot was used to measure FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (B) RT-qPCR was used to measure FOXO1 mRNA expression in A375 cells transfected with RSK2 siRNA. (C) The influence of RSK2 on FOXO1 degradation was estimated in A375 cells under MG132 treatment. (D) The effect of RSK2 on FOXO1 protein stability was evaluated in A375 cells under CHX treatment for the indicated time. (E) HEK293T cells were co-transfected with Flag-FOXO1 and HA-RSK2 plasmid as indicated, immunoprecipitation with anti-Flag antibody was performed. (F) Western blot was used to measure p-FOXO1 (S319) and FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (G) Western blot was used to measure cyclin D1 expression in A375 cells co-transfected with Flag-FOXO1 and HA-RSK2 as indicated. (H) A schematic model of RSK2–FOXO1–cyclin D1 axis leading to melanoma proliferation and vemurafenib resistance.

    Article Snippet: Antibodies used in immunoblotting were purchased from Cell Signaling Technologies: RSK2 (No.5528, 1:1000), FOXO1 (No.2880,1:1000), p-FOXO1/Ser319(No.2486,1:1000) and cyclin D1 (No.55506, 1:1000).

    Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunoprecipitation