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  • 99
    Name:
    Akt Antibody
    Description:
    Akt also referred to as PKB or Rac plays a critical role in controlling survival and apoptosis 1 3 This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin sensitive pathway involving PI3 kinase 2 3 Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 4 and by phosphorylation within the carboxy terminus at Ser473 The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin mTOR in a rapamycin insensitive complex with rictor and Sin1 5 6 Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets including Bad 7 forkhead transcription factors 8 c Raf 9 and caspase 9 PTEN phosphatase is a major negative regulator of the PI3 kinase Akt signaling pathway 10 LY294002 is a specific PI3 kinase inhibitor 11 Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK 3α and β 12 13 Akt may also play a role in insulin stimulation of glucose transport 12 In addition to its role in survival and glycogen synthesis Akt is involved in cell cycle regulation by preventing GSK 3β mediated phosphorylation and degradation of cyclin D1 14 and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 15 and p21 Waf1 Cip1 16 Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin sensitive complex containing raptor 17 More importantly Akt phosphorylates and inactivates tuberin TSC2 an inhibitor of mTOR within the mTOR raptor complex 18 19
    Catalog Number:
    9272
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation, Immunofluorescence, Flow Cytometry
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the carboxy-terminal sequence of mouse Akt. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Hamster Monkey Chicken D melanogaster Bovine Dog Pig Guinea Pig
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc anti akt
    S-nitrosylation in T cells is inversely correlated with plasma Hcy levels in human CAD patients. Peripheral blood mononuclear cells (PBMCs) from CAD patients with HHcy (above 10 μmol/L) were isolated, collected and stained with anti-CD3, anti-SNO-Cys and anti-IFN-γ antibodies (n = 18). The level of Gsnor in PBMCs, the SNO-Cys mean fluorescence intensity (MFI) and IFN-γ + subsets of CD3 + -gated T cells were assessed by RT-PCR and flow cytometry. ( A-C ), Correlations between the plasma Hcy concentration and Gsnor gene expression in PBMCs ( A ), SNO-Cys MFI in T cells ( B ), and IFN-γ + T cell percentage ( C ). ( D ), Schematic representation of the proposed mechanism of <t>GSNOR-Akt-dependent</t> T-cell activation in HHcy-induced atherosclerosis. Akt was abundantly S-nitrosylated at Cys224 during inactivation of T cells. On HHcy stimulation, elevated GSNOR denitrosylated Akt and led to Akt phosphorylation at <t>Ser473</t> with activated Akt signaling pathways, which promoted T-cell proliferation and secretion of proinflammatory cytokines, including IL-2 and IFN-γ. These events were associated with aggravating vascular immune inflammation and early atherosclerotic development, which suggests an essential switch role for the GSNOR-Akt axis dependent denitrosylation in T cell-driven atherosclerosis.
    Akt also referred to as PKB or Rac plays a critical role in controlling survival and apoptosis 1 3 This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin sensitive pathway involving PI3 kinase 2 3 Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 4 and by phosphorylation within the carboxy terminus at Ser473 The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin mTOR in a rapamycin insensitive complex with rictor and Sin1 5 6 Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets including Bad 7 forkhead transcription factors 8 c Raf 9 and caspase 9 PTEN phosphatase is a major negative regulator of the PI3 kinase Akt signaling pathway 10 LY294002 is a specific PI3 kinase inhibitor 11 Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK 3α and β 12 13 Akt may also play a role in insulin stimulation of glucose transport 12 In addition to its role in survival and glycogen synthesis Akt is involved in cell cycle regulation by preventing GSK 3β mediated phosphorylation and degradation of cyclin D1 14 and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 15 and p21 Waf1 Cip1 16 Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin sensitive complex containing raptor 17 More importantly Akt phosphorylates and inactivates tuberin TSC2 an inhibitor of mTOR within the mTOR raptor complex 18 19
    https://www.bioz.com/result/anti akt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 127 article reviews
    Price from $9.99 to $1999.99
    anti akt - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "GSNOR modulates hyperhomocysteinemia-induced T cell activation and atherosclerosis by switching Akt S-nitrosylation to phosphorylation"

    Article Title: GSNOR modulates hyperhomocysteinemia-induced T cell activation and atherosclerosis by switching Akt S-nitrosylation to phosphorylation

    Journal: Redox Biology

    doi: 10.1016/j.redox.2018.04.021

    S-nitrosylation in T cells is inversely correlated with plasma Hcy levels in human CAD patients. Peripheral blood mononuclear cells (PBMCs) from CAD patients with HHcy (above 10 μmol/L) were isolated, collected and stained with anti-CD3, anti-SNO-Cys and anti-IFN-γ antibodies (n = 18). The level of Gsnor in PBMCs, the SNO-Cys mean fluorescence intensity (MFI) and IFN-γ + subsets of CD3 + -gated T cells were assessed by RT-PCR and flow cytometry. ( A-C ), Correlations between the plasma Hcy concentration and Gsnor gene expression in PBMCs ( A ), SNO-Cys MFI in T cells ( B ), and IFN-γ + T cell percentage ( C ). ( D ), Schematic representation of the proposed mechanism of GSNOR-Akt-dependent T-cell activation in HHcy-induced atherosclerosis. Akt was abundantly S-nitrosylated at Cys224 during inactivation of T cells. On HHcy stimulation, elevated GSNOR denitrosylated Akt and led to Akt phosphorylation at Ser473 with activated Akt signaling pathways, which promoted T-cell proliferation and secretion of proinflammatory cytokines, including IL-2 and IFN-γ. These events were associated with aggravating vascular immune inflammation and early atherosclerotic development, which suggests an essential switch role for the GSNOR-Akt axis dependent denitrosylation in T cell-driven atherosclerosis.
    Figure Legend Snippet: S-nitrosylation in T cells is inversely correlated with plasma Hcy levels in human CAD patients. Peripheral blood mononuclear cells (PBMCs) from CAD patients with HHcy (above 10 μmol/L) were isolated, collected and stained with anti-CD3, anti-SNO-Cys and anti-IFN-γ antibodies (n = 18). The level of Gsnor in PBMCs, the SNO-Cys mean fluorescence intensity (MFI) and IFN-γ + subsets of CD3 + -gated T cells were assessed by RT-PCR and flow cytometry. ( A-C ), Correlations between the plasma Hcy concentration and Gsnor gene expression in PBMCs ( A ), SNO-Cys MFI in T cells ( B ), and IFN-γ + T cell percentage ( C ). ( D ), Schematic representation of the proposed mechanism of GSNOR-Akt-dependent T-cell activation in HHcy-induced atherosclerosis. Akt was abundantly S-nitrosylated at Cys224 during inactivation of T cells. On HHcy stimulation, elevated GSNOR denitrosylated Akt and led to Akt phosphorylation at Ser473 with activated Akt signaling pathways, which promoted T-cell proliferation and secretion of proinflammatory cytokines, including IL-2 and IFN-γ. These events were associated with aggravating vascular immune inflammation and early atherosclerotic development, which suggests an essential switch role for the GSNOR-Akt axis dependent denitrosylation in T cell-driven atherosclerosis.

    Techniques Used: Isolation, Staining, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry, Concentration Assay, Expressing, Activation Assay

    HHcy activation of T cells depends on GSNOR-Akt axis. GSNOR -/- mice or GSNOR +/+ littermates were fed a normal chow diet and given drinking water supplemented with or without 1.8 g/L Hcy for 4 weeks. ( A ), Total cell numbers of splenic T cells. ( B ), Purified T cells were labeled with CFSE before culture with plate-bound anti-CD3 antibody, then cell proliferation was assessed by flow cytometry after 48 h. ( C-D ), Purified T cells were cultured for an additional 24 h with plate-bound anti-CD3 antibody. ELISA of interleukin 2 (IL-2) ( C ) and interferon-γ (IFN-γ) ( D ) levels in supernatants of cultured T cells. ( E-I ), In freshly isolated splenic T cells, gene expression of Il-2 ( E ) and Ifn-γ ( F ) measured by quantitative PCR. Phosphorylated proteins in Akt signaling networks ( G ) were simultaneously detected by using the Akt signaling antibody array kit and presented as a heat map. Akt kinase enzyme activity ( H ) was analyzed, and phosphorylation of Akt at Ser473 and Thr308 ( I ) was detected by western blot analysis. GAPDH was an internal control. Data are mean ± SEM of at least three independent experiments (n = 3–6 mice in each group). * P ﹤.05 compared with the control. # P ﹤.05 compared with HHcy.
    Figure Legend Snippet: HHcy activation of T cells depends on GSNOR-Akt axis. GSNOR -/- mice or GSNOR +/+ littermates were fed a normal chow diet and given drinking water supplemented with or without 1.8 g/L Hcy for 4 weeks. ( A ), Total cell numbers of splenic T cells. ( B ), Purified T cells were labeled with CFSE before culture with plate-bound anti-CD3 antibody, then cell proliferation was assessed by flow cytometry after 48 h. ( C-D ), Purified T cells were cultured for an additional 24 h with plate-bound anti-CD3 antibody. ELISA of interleukin 2 (IL-2) ( C ) and interferon-γ (IFN-γ) ( D ) levels in supernatants of cultured T cells. ( E-I ), In freshly isolated splenic T cells, gene expression of Il-2 ( E ) and Ifn-γ ( F ) measured by quantitative PCR. Phosphorylated proteins in Akt signaling networks ( G ) were simultaneously detected by using the Akt signaling antibody array kit and presented as a heat map. Akt kinase enzyme activity ( H ) was analyzed, and phosphorylation of Akt at Ser473 and Thr308 ( I ) was detected by western blot analysis. GAPDH was an internal control. Data are mean ± SEM of at least three independent experiments (n = 3–6 mice in each group). * P ﹤.05 compared with the control. # P ﹤.05 compared with HHcy.

    Techniques Used: Activation Assay, Mouse Assay, Purification, Labeling, Flow Cytometry, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Real-time Polymerase Chain Reaction, Ab Array, Activity Assay, Western Blot

    2) Product Images from "Physiological electric field works via the VEGF receptor to stimulate neovessel formation of vascular endothelial cells in a 3D environment"

    Article Title: Physiological electric field works via the VEGF receptor to stimulate neovessel formation of vascular endothelial cells in a 3D environment

    Journal: Biology Open

    doi: 10.1242/bio.035204

    Activation of VEGFR2, Akt, Erk1/2 and JNK following EF treatment. Tube-like structures of endothelial cells cultured in 3D were treated with an EF (150 mV/mm). After 15 min, they were fixed and stained with antibodies directed against the active (phosphorylated) form of the proteins. Protein expression was quantified by confocal laser scanning microscopy. The images show representative immunolabeled tube-like structures (A–D). The histogram depicts the relative immunofluorescence of the phosphorylated proteins (E–H). The error bars represent the S.E. ** P
    Figure Legend Snippet: Activation of VEGFR2, Akt, Erk1/2 and JNK following EF treatment. Tube-like structures of endothelial cells cultured in 3D were treated with an EF (150 mV/mm). After 15 min, they were fixed and stained with antibodies directed against the active (phosphorylated) form of the proteins. Protein expression was quantified by confocal laser scanning microscopy. The images show representative immunolabeled tube-like structures (A–D). The histogram depicts the relative immunofluorescence of the phosphorylated proteins (E–H). The error bars represent the S.E. ** P

    Techniques Used: Activation Assay, Cell Culture, Staining, Expressing, Confocal Laser Scanning Microscopy, Immunolabeling, Immunofluorescence

    Effects of various drugs on EF -induced tube formation of endothelial cells. Inhibition of Akt (Akt-i), Erk1/2 (Erk1/2 -i) and JNK (JNK-i) significantly decreased tube length, whereas inhibition of VEGFR2 (VEGFR2-i) potently abolished the EF-mediated enhancement of tube length. The tube lengths were expressed as a percentage relative to that obtained in the untreated control in EF culture. VEGFR-i, VEGFR inhibitor SU1498 (50 μM); Akt-i, Akt inhibitor MK-2206 2HCl (10 μM); Erk 1/2-i, Erk 1/2 inhibitor U0126 (20 μM); JNK-i, JNK inhibitor Sp600125 (10 μM). Endothelial cells cultured in 3D were subjected to EFs of 150 mV/mm for 6 h. Each treatment was performed in duplicate in at least three independent experiments. The error bars represent the S.E. ***, P
    Figure Legend Snippet: Effects of various drugs on EF -induced tube formation of endothelial cells. Inhibition of Akt (Akt-i), Erk1/2 (Erk1/2 -i) and JNK (JNK-i) significantly decreased tube length, whereas inhibition of VEGFR2 (VEGFR2-i) potently abolished the EF-mediated enhancement of tube length. The tube lengths were expressed as a percentage relative to that obtained in the untreated control in EF culture. VEGFR-i, VEGFR inhibitor SU1498 (50 μM); Akt-i, Akt inhibitor MK-2206 2HCl (10 μM); Erk 1/2-i, Erk 1/2 inhibitor U0126 (20 μM); JNK-i, JNK inhibitor Sp600125 (10 μM). Endothelial cells cultured in 3D were subjected to EFs of 150 mV/mm for 6 h. Each treatment was performed in duplicate in at least three independent experiments. The error bars represent the S.E. ***, P

    Techniques Used: Inhibition, Cell Culture

    3) Product Images from "A loss-of-function genetic screening reveals synergistic targeting of AKT/mTOR and WTN/β-catenin pathways for treatment of AML with high PRL-3 phosphatase"

    Article Title: A loss-of-function genetic screening reveals synergistic targeting of AKT/mTOR and WTN/β-catenin pathways for treatment of AML with high PRL-3 phosphatase

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-018-0581-9

    Evaluations of apoptotic response induced by simultaneous inhibition of AKT/mTOR and WNT/β-catenin pathways. a ” section. Upper representative images show the original FACS plots and lower bar figures represent the percentage of annexin V-positive cells, including both early and late apoptotic cells. b Luminescent assays for caspase 3 and caspase 7 activities in MOLM-14 cells and primary AML cells from unique patient number 4 (Pt#4, PRL-3 high) ( c ) incubated with either VS-5584, ICG-001 alone, or combination ( n = 3, mean ± SD). Five-point constant ratio of combination-based single drug IC 50 were used here. V + I: combination of VS-5584 and ICG-001. d Caspase 3 and caspase 7 activities in primary AML cells from unique patient number 9 (Pt#9, PRL-3 low) incubated with either VS-5584, ICG-001 alone, or combination ( n = 3, mean ± SD). Five-point constant ratio of combination-based single drug IC 50 were used here. V + I: combination of VS-5584 and ICG-001. e The cell lysates extracted from MOLM-14 cells treated with DMSO control, VS-5584 (800 nM), ICG-001 (14 μM) single agent or in combination for 48 h were subjected to Western blot analysis for AKT, p-AKT, Survivin, full length (FL) or cleaved (CL) caspase 3, 7 and PARP. Beta-actin was used as the loading control. Protein levels were determined by densitometric analysis. The experiments were duplicated, and representative images were shown
    Figure Legend Snippet: Evaluations of apoptotic response induced by simultaneous inhibition of AKT/mTOR and WNT/β-catenin pathways. a ” section. Upper representative images show the original FACS plots and lower bar figures represent the percentage of annexin V-positive cells, including both early and late apoptotic cells. b Luminescent assays for caspase 3 and caspase 7 activities in MOLM-14 cells and primary AML cells from unique patient number 4 (Pt#4, PRL-3 high) ( c ) incubated with either VS-5584, ICG-001 alone, or combination ( n = 3, mean ± SD). Five-point constant ratio of combination-based single drug IC 50 were used here. V + I: combination of VS-5584 and ICG-001. d Caspase 3 and caspase 7 activities in primary AML cells from unique patient number 9 (Pt#9, PRL-3 low) incubated with either VS-5584, ICG-001 alone, or combination ( n = 3, mean ± SD). Five-point constant ratio of combination-based single drug IC 50 were used here. V + I: combination of VS-5584 and ICG-001. e The cell lysates extracted from MOLM-14 cells treated with DMSO control, VS-5584 (800 nM), ICG-001 (14 μM) single agent or in combination for 48 h were subjected to Western blot analysis for AKT, p-AKT, Survivin, full length (FL) or cleaved (CL) caspase 3, 7 and PARP. Beta-actin was used as the loading control. Protein levels were determined by densitometric analysis. The experiments were duplicated, and representative images were shown

    Techniques Used: Inhibition, FACS, Incubation, Western Blot

    4) Product Images from "LncRNA TP73-AS1 promoted the progression of lung adenocarcinoma via PI3K/AKT pathway"

    Article Title: LncRNA TP73-AS1 promoted the progression of lung adenocarcinoma via PI3K/AKT pathway

    Journal: Bioscience Reports

    doi: 10.1042/BSR20180999

    TP73-AS1 aggravated the progression of LAD via PI3K/Akt signaling pathway ( A ) The expression of proteins involved in PI3K/AKT and MRK/ERK pathway was determined by Western blotting in shCtrl or shTP73-AS1 transfected A549 and HCC827 cells after treated with or without 740Y-P, the PI3K activator. ( B ) Relative protein level of total AKT and p-AKT in LAD tissues was evaluated by Western blot assay and quantitated using ImageJ software. ( C , D ) MTT assay and colony formation assay were applied to detect the changes of cell proliferation ability of HCC827 and A549 cells after the transfection with shCtrl or shTP73-AS1 and the treatment with or without 740Y-P. ( E , F ) Cell cycle distribution and apoptosis rate altered in shCtrl or shTP73-AS1 transfected HCC827 and A549 cells after treating with or without 740Y-P was estimated by flow cytometry analysis. ( G ) Transwell assay was utilized to test the alterations of migration and invasion capacities in TP73-AS1 silenced LAD cells with or without the treatment of 740Y-P. The experiments were performed in triplicate. * P
    Figure Legend Snippet: TP73-AS1 aggravated the progression of LAD via PI3K/Akt signaling pathway ( A ) The expression of proteins involved in PI3K/AKT and MRK/ERK pathway was determined by Western blotting in shCtrl or shTP73-AS1 transfected A549 and HCC827 cells after treated with or without 740Y-P, the PI3K activator. ( B ) Relative protein level of total AKT and p-AKT in LAD tissues was evaluated by Western blot assay and quantitated using ImageJ software. ( C , D ) MTT assay and colony formation assay were applied to detect the changes of cell proliferation ability of HCC827 and A549 cells after the transfection with shCtrl or shTP73-AS1 and the treatment with or without 740Y-P. ( E , F ) Cell cycle distribution and apoptosis rate altered in shCtrl or shTP73-AS1 transfected HCC827 and A549 cells after treating with or without 740Y-P was estimated by flow cytometry analysis. ( G ) Transwell assay was utilized to test the alterations of migration and invasion capacities in TP73-AS1 silenced LAD cells with or without the treatment of 740Y-P. The experiments were performed in triplicate. * P

    Techniques Used: Expressing, Western Blot, Transfection, Software, MTT Assay, Colony Assay, Flow Cytometry, Cytometry, Transwell Assay, Migration

    5) Product Images from "Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M"

    Article Title: Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01828

    Impaired IKKα and IRF7 activation in plasmacytoid dendritic cells (pDCs) with reduced Blimp-1 expression. (A) Immunoblot analysis of nuclear extracts showing the levels of IRF7 in 4-OHT treated Ctrl-ER and CKO-ER FLpDCs following stimulation with 1 µM CpG-A at indicated time points. (B) Immunoblot analysis of total cell lysates showing the levels of IRF7 phosphorylation at Ser471/472 in 1 µM CpG-A stimulated Ctrl-ER and CKO-ER FLpDCs. (C) Immunoblot analysis showing the levels of phospho-IKKα at Ser176/180, OPN and phospho-AKT at Ser473, in 4-OHT treated and CpG-A stimulated Ctrl-ER and CKO-ER FLpDCs. The quantification of pIKKα was presented by the ratios of pIKKα band intensity vs. IKKα band intensity at each time point. (D) Immunoblot analysis using nuclear extracts of 4-OHT treated Ctrl-ER and CKO-ER FLpDCs showing the levels of p50 and p65 translocation after 1 µM CpG-A treatment at indicated time points. (E) Immunoblot analysis showing the levels of phosphorylated STAT1 at Tyr701 at the indicated time points in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A at the indicated time points. (F) Immunoblot analysis showing levels of phosphorylated STAT1 at Tyr701 in FLpDCs in the presence of 500 U/ml mIFN-α.
    Figure Legend Snippet: Impaired IKKα and IRF7 activation in plasmacytoid dendritic cells (pDCs) with reduced Blimp-1 expression. (A) Immunoblot analysis of nuclear extracts showing the levels of IRF7 in 4-OHT treated Ctrl-ER and CKO-ER FLpDCs following stimulation with 1 µM CpG-A at indicated time points. (B) Immunoblot analysis of total cell lysates showing the levels of IRF7 phosphorylation at Ser471/472 in 1 µM CpG-A stimulated Ctrl-ER and CKO-ER FLpDCs. (C) Immunoblot analysis showing the levels of phospho-IKKα at Ser176/180, OPN and phospho-AKT at Ser473, in 4-OHT treated and CpG-A stimulated Ctrl-ER and CKO-ER FLpDCs. The quantification of pIKKα was presented by the ratios of pIKKα band intensity vs. IKKα band intensity at each time point. (D) Immunoblot analysis using nuclear extracts of 4-OHT treated Ctrl-ER and CKO-ER FLpDCs showing the levels of p50 and p65 translocation after 1 µM CpG-A treatment at indicated time points. (E) Immunoblot analysis showing the levels of phosphorylated STAT1 at Tyr701 at the indicated time points in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A at the indicated time points. (F) Immunoblot analysis showing levels of phosphorylated STAT1 at Tyr701 in FLpDCs in the presence of 500 U/ml mIFN-α.

    Techniques Used: Activation Assay, Expressing, Translocation Assay

    6) Product Images from "Loss of KIBRA function activates EGFR signaling by inducing AREG"

    Article Title: Loss of KIBRA function activates EGFR signaling by inducing AREG

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25724

    KIBRA knockdown induces the secretion of the EGFR ligand AREG (A) Conditioned media collected from shControl or shKIBRA 3D cell cultures was used to treat parental MCF10A cells. (B) Human growth factor/cytokine antibody array analyses were performed using conditioned media from shControl- or shKIBRA-transduced cells grown in the absence of EGF. Four positive and four negative controls are shown in the upper left corner. (C) Immunoblot detection of AREG, phosphor-YAP1 (S127) and YAP1 expression in the presence or absence of EGF in shControl or shKIBRA cells. GAPDH was used as the loading control. (D) AKT and ERK activation was detected in the presence or absence of EGF in shControl or shKIBRA cells by immunoblot. GAPDH was used as the loading control.
    Figure Legend Snippet: KIBRA knockdown induces the secretion of the EGFR ligand AREG (A) Conditioned media collected from shControl or shKIBRA 3D cell cultures was used to treat parental MCF10A cells. (B) Human growth factor/cytokine antibody array analyses were performed using conditioned media from shControl- or shKIBRA-transduced cells grown in the absence of EGF. Four positive and four negative controls are shown in the upper left corner. (C) Immunoblot detection of AREG, phosphor-YAP1 (S127) and YAP1 expression in the presence or absence of EGF in shControl or shKIBRA cells. GAPDH was used as the loading control. (D) AKT and ERK activation was detected in the presence or absence of EGF in shControl or shKIBRA cells by immunoblot. GAPDH was used as the loading control.

    Techniques Used: Ab Array, Expressing, Activation Assay

    7) Product Images from "Tissue inhibitor of metalloproteinases 1 enhances rod survival in the rd1 mouse retina"

    Article Title: Tissue inhibitor of metalloproteinases 1 enhances rod survival in the rd1 mouse retina

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197322

    Induction of pERK1/2 by TIMP1 in rd1 mouse retina. Immunoblot analysis of SDS-PAGE transferred samples of phosphorylated ERK1/2 (44/42 kDa) and AKT (60 kDa) were examined from saline- (-) and TIMP1-treated (+) (A-C) and SB-3CT-treated (D-F) rd1 retinas. Retinas were collected at 5 min, 1 hour, and 6 hours after injection at P15 and processed. Activation of ERK1/2 was detected at 1 hour after TIMP1 injection (A, B). No pAKT increase was detectable in TIMP1 treated retinas (A, C). In addition, no detectable increase in either pERK1/2 or pAKT was noted in SB-3CT-treated retinas (D, E, F). Densitometry analysis of immunoblots was generated in the histogram. Immunoreactive β-actin served as the loading control to gain relative pERK1/2 and pAKT activation value. Data represent mean ± SEM, *** P
    Figure Legend Snippet: Induction of pERK1/2 by TIMP1 in rd1 mouse retina. Immunoblot analysis of SDS-PAGE transferred samples of phosphorylated ERK1/2 (44/42 kDa) and AKT (60 kDa) were examined from saline- (-) and TIMP1-treated (+) (A-C) and SB-3CT-treated (D-F) rd1 retinas. Retinas were collected at 5 min, 1 hour, and 6 hours after injection at P15 and processed. Activation of ERK1/2 was detected at 1 hour after TIMP1 injection (A, B). No pAKT increase was detectable in TIMP1 treated retinas (A, C). In addition, no detectable increase in either pERK1/2 or pAKT was noted in SB-3CT-treated retinas (D, E, F). Densitometry analysis of immunoblots was generated in the histogram. Immunoreactive β-actin served as the loading control to gain relative pERK1/2 and pAKT activation value. Data represent mean ± SEM, *** P

    Techniques Used: SDS Page, Injection, Activation Assay, Western Blot, Generated

    8) Product Images from "Bufalin suppresses the proliferation and metastasis of renal cell carcinoma by inhibiting the PI3K/Akt/mTOR signaling pathway"

    Article Title: Bufalin suppresses the proliferation and metastasis of renal cell carcinoma by inhibiting the PI3K/Akt/mTOR signaling pathway

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.9111

    Bufalin suppressed the expression of p-Akt Ser473 in ACHN cells. (A) Two-color immunofluorescence analysis showing the expression of E-cadherin (green) and DAPI (blue) in ACHN cells treated with bufalin (concentrations of 0 or 20 nM) (magnification, ×200). (B) PI3K/Akt signaling pathway-related protein expression was detected by Western blot analysis after treatment with bufalin (concentrations of 0, 10, and 20 nM) for 24 h. GAPDH was considered as an internal standard. (C) Bufalin treatment downregulated the expression of p-Akt, PI3K, and p-mTOR in both the 10- and 20-nM treatment groups compared with the DMSO control. The test was repeated three times, and the data are presented as mean ± SD. *P
    Figure Legend Snippet: Bufalin suppressed the expression of p-Akt Ser473 in ACHN cells. (A) Two-color immunofluorescence analysis showing the expression of E-cadherin (green) and DAPI (blue) in ACHN cells treated with bufalin (concentrations of 0 or 20 nM) (magnification, ×200). (B) PI3K/Akt signaling pathway-related protein expression was detected by Western blot analysis after treatment with bufalin (concentrations of 0, 10, and 20 nM) for 24 h. GAPDH was considered as an internal standard. (C) Bufalin treatment downregulated the expression of p-Akt, PI3K, and p-mTOR in both the 10- and 20-nM treatment groups compared with the DMSO control. The test was repeated three times, and the data are presented as mean ± SD. *P

    Techniques Used: Expressing, Immunofluorescence, Western Blot

    9) Product Images from "Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment"

    Article Title: Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05707-2

    Abd-7 results on RAS-dependent signalling pathways and cell viability. a , b DLD-1 ( a ) or H358 ( b ) cells were serum-starved for 24 h, incubated in mono-layer with Abd-7 in a range from 2, 5, 10 and 20 μM for 3 h and stimulated with EGF 10 min. Proteins were extracted and separated by SDS-PAGE and transferred to membranes for Western analysis with anti-pAKT, anti-pERK, anti-pan AKT and anti-pan ERK. Anti-cyclophilin B antibody was the loading control. Signal was developed using standard ECL. c – e Effects of compounds Abd-2, Abd-4, Abd-5, Abd-6, and Abd-7 on the viability of human cancer cells lines was assessed for a 2-D culture of DLD-1 (mutant KRAS G13D ) cells and HT1080 cells (mutant NRAS Q61K ). The cells were treated with a dose range from 0 μM to 20 μ and incubated for 72 h when cell viability was assessed using CellTitreGlo. In each case, the data are normalized to cells treated with DMSO only. c is DLD-1 and d is HT1080. e shows the colour coding for the different compounds. Each experiment was repeated at least three times ( a , b ) and four times ( c, d ). Where error bars are presented, they correspond to mean values ± SD of biological repeats
    Figure Legend Snippet: Abd-7 results on RAS-dependent signalling pathways and cell viability. a , b DLD-1 ( a ) or H358 ( b ) cells were serum-starved for 24 h, incubated in mono-layer with Abd-7 in a range from 2, 5, 10 and 20 μM for 3 h and stimulated with EGF 10 min. Proteins were extracted and separated by SDS-PAGE and transferred to membranes for Western analysis with anti-pAKT, anti-pERK, anti-pan AKT and anti-pan ERK. Anti-cyclophilin B antibody was the loading control. Signal was developed using standard ECL. c – e Effects of compounds Abd-2, Abd-4, Abd-5, Abd-6, and Abd-7 on the viability of human cancer cells lines was assessed for a 2-D culture of DLD-1 (mutant KRAS G13D ) cells and HT1080 cells (mutant NRAS Q61K ). The cells were treated with a dose range from 0 μM to 20 μ and incubated for 72 h when cell viability was assessed using CellTitreGlo. In each case, the data are normalized to cells treated with DMSO only. c is DLD-1 and d is HT1080. e shows the colour coding for the different compounds. Each experiment was repeated at least three times ( a , b ) and four times ( c, d ). Where error bars are presented, they correspond to mean values ± SD of biological repeats

    Techniques Used: Incubation, SDS Page, Western Blot, Mutagenesis

    10) Product Images from "Autophagy of macrophages is regulated by PI3k/Akt/mTOR signalling in the development of diabetic encephalopathy"

    Article Title: Autophagy of macrophages is regulated by PI3k/Akt/mTOR signalling in the development of diabetic encephalopathy

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101586

    Enhanced PI3k/AKT/mTOR signalling is detected in brain macrophages from STZ-treated rats. ( A-D ) Western blot analysis of activation of PI3k (A), AKT (B), mTOR (C) and S6K1 (D) in brain macrophages. *p
    Figure Legend Snippet: Enhanced PI3k/AKT/mTOR signalling is detected in brain macrophages from STZ-treated rats. ( A-D ) Western blot analysis of activation of PI3k (A), AKT (B), mTOR (C) and S6K1 (D) in brain macrophages. *p

    Techniques Used: Western Blot, Activation Assay

    PI3k/AKT/mTOR signalling in RAP/CQ-treated and STZ-treated rat brain macrophages. ( A-D ) Western blot analysis of activation of PI3k (A), AKT (B), mTOR (C) and S6K1 (D) in brain macrophages from RAP or CQ treated rats. *p
    Figure Legend Snippet: PI3k/AKT/mTOR signalling in RAP/CQ-treated and STZ-treated rat brain macrophages. ( A-D ) Western blot analysis of activation of PI3k (A), AKT (B), mTOR (C) and S6K1 (D) in brain macrophages from RAP or CQ treated rats. *p

    Techniques Used: Western Blot, Activation Assay

    Schematic. Diabetes induces increases in brain inflammatory macrophages, through increased PI3k/AKT/mTOR/S6K1 signalling and suppression of autophagy. mTOR and autophagy inhibit each other. RAP inhibits mTOR. CQ inhibits autophagy.
    Figure Legend Snippet: Schematic. Diabetes induces increases in brain inflammatory macrophages, through increased PI3k/AKT/mTOR/S6K1 signalling and suppression of autophagy. mTOR and autophagy inhibit each other. RAP inhibits mTOR. CQ inhibits autophagy.

    Techniques Used:

    11) Product Images from "Evaluation of the Inhibitory Effects of Genipin on the Fluoxetine-Induced Invasive and Metastatic Model in Human HepG2 Cells"

    Article Title: Evaluation of the Inhibitory Effects of Genipin on the Fluoxetine-Induced Invasive and Metastatic Model in Human HepG2 Cells

    Journal: Molecules

    doi: 10.3390/molecules23123327

    Western blot showing the expressions of p38 and Akt ( A ), transcription factors NF-κB and c-Jun/c-Fos ( B ) in HepG2 cells induced by FXT and the attenuating effect of GNP. HepG2 cells were seeded onto a 6-well plate and incubated overnight, then treated with FXT and GNP at dose as indicated and further incubated for 72 h. DMSO (0.1%) was used as vehicle control for 72 h. The intensity was subsequently quantified by the densitometer.
    Figure Legend Snippet: Western blot showing the expressions of p38 and Akt ( A ), transcription factors NF-κB and c-Jun/c-Fos ( B ) in HepG2 cells induced by FXT and the attenuating effect of GNP. HepG2 cells were seeded onto a 6-well plate and incubated overnight, then treated with FXT and GNP at dose as indicated and further incubated for 72 h. DMSO (0.1%) was used as vehicle control for 72 h. The intensity was subsequently quantified by the densitometer.

    Techniques Used: Western Blot, Incubation

    12) Product Images from "Leukemia inhibitory factor regulates marmoset induced pluripotent stem cell proliferation via a PI3K/Akt-dependent Tbx-3 activation pathway"

    Article Title: Leukemia inhibitory factor regulates marmoset induced pluripotent stem cell proliferation via a PI3K/Akt-dependent Tbx-3 activation pathway

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3610

    Involvement of the PI3K/Akt signaling pathway in LIF-induced activation of Tbx-3 in marmoset induced pluripotent stem cells. The protein expression levels of STAT3, Tbx3, Nanog, p-Akt and PI3K(85α) in the presence or absence of LIF were analyzed by western blot analysis. Actin is shown as a loading control. *** P
    Figure Legend Snippet: Involvement of the PI3K/Akt signaling pathway in LIF-induced activation of Tbx-3 in marmoset induced pluripotent stem cells. The protein expression levels of STAT3, Tbx3, Nanog, p-Akt and PI3K(85α) in the presence or absence of LIF were analyzed by western blot analysis. Actin is shown as a loading control. *** P

    Techniques Used: Activation Assay, Expressing, Western Blot

    Model of LIF-mediated expression levels of pluripotency-associated gene by activation of the PI3K/Akt signaling pathway. LIF, leukemia inhibitory factor; PI3K, phosphoinositide 3-kinase; Tbx-3, T-box 3.
    Figure Legend Snippet: Model of LIF-mediated expression levels of pluripotency-associated gene by activation of the PI3K/Akt signaling pathway. LIF, leukemia inhibitory factor; PI3K, phosphoinositide 3-kinase; Tbx-3, T-box 3.

    Techniques Used: Expressing, Activation Assay

    13) Product Images from "Chemotherapy induced PRL3 expression promotes cancer growth via plasma membrane remodeling and specific alterations of caveolae-associated signaling"

    Article Title: Chemotherapy induced PRL3 expression promotes cancer growth via plasma membrane remodeling and specific alterations of caveolae-associated signaling

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0264-8

    PRL3 expression affects cellular growth-associated signaling ( a ) Rac1-GTP loading increases upon PRL3 expression. b AKT phosphorylation increases at Ser473 upon PRL3 expression. c GSK phosphorylation increases at Ser9 upon PRL3 expression. d Cyclin D1 levels are upregulated upon PRL3 expression. e Representative confocal images of the localization of cyclin D1 in the indicated cells. Cyclin D1 localizes to the nucleus. f Rac1 inhibition decreases the elevated cyclin D1 levels. The indicated cells were treated with 10 μM NSC23766 for 24 h in order to inhibit Rac1-dependent elevation of cyclin D1 levels
    Figure Legend Snippet: PRL3 expression affects cellular growth-associated signaling ( a ) Rac1-GTP loading increases upon PRL3 expression. b AKT phosphorylation increases at Ser473 upon PRL3 expression. c GSK phosphorylation increases at Ser9 upon PRL3 expression. d Cyclin D1 levels are upregulated upon PRL3 expression. e Representative confocal images of the localization of cyclin D1 in the indicated cells. Cyclin D1 localizes to the nucleus. f Rac1 inhibition decreases the elevated cyclin D1 levels. The indicated cells were treated with 10 μM NSC23766 for 24 h in order to inhibit Rac1-dependent elevation of cyclin D1 levels

    Techniques Used: Expressing, Inhibition

    14) Product Images from "Green Tea Catechin Is an Alternative Immune Checkpoint Inhibitor that Inhibits PD-L1 Expression and Lung Tumor Growth"

    Article Title: Green Tea Catechin Is an Alternative Immune Checkpoint Inhibitor that Inhibits PD-L1 Expression and Lung Tumor Growth

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules23082071

    Downregulation of IFN-γ–induced PD-L1 protein and inhibition of STAT1- and Akt-phosphorylation in A549 cells treated with (−)-epigallocatechin gallate (EGCG). ( A ) PD-L1 mRNA expression, ( B ) PD-L1 protein, ( C ) phosphorylation of STAT1 and Akt, and ( D ) cell-surface PD-L1. “−“ and “+” indicate in the absence or presence of IFN-γ (10 ng/mL). Numbers indicate average percentage compared with IFN-γ–treated cells. * p
    Figure Legend Snippet: Downregulation of IFN-γ–induced PD-L1 protein and inhibition of STAT1- and Akt-phosphorylation in A549 cells treated with (−)-epigallocatechin gallate (EGCG). ( A ) PD-L1 mRNA expression, ( B ) PD-L1 protein, ( C ) phosphorylation of STAT1 and Akt, and ( D ) cell-surface PD-L1. “−“ and “+” indicate in the absence or presence of IFN-γ (10 ng/mL). Numbers indicate average percentage compared with IFN-γ–treated cells. * p

    Techniques Used: Inhibition, Expressing

    Downregulation of EGF-induced PD-L1 protein and inhibition of Akt phosphorylation in Lu99 cells treated with EGCG. ( A ) PD-L1 mRNA expression, ( B ) PD-L1 protein, ( C ) phosphorylation of STAT1 and Akt, and ( D ) cell-surface PD-L1. “−“ and “+” indicate in the absence or presence of EGF (10 ng/mL). Numbers indicate average percentage compared with EGF-treated cells. * p
    Figure Legend Snippet: Downregulation of EGF-induced PD-L1 protein and inhibition of Akt phosphorylation in Lu99 cells treated with EGCG. ( A ) PD-L1 mRNA expression, ( B ) PD-L1 protein, ( C ) phosphorylation of STAT1 and Akt, and ( D ) cell-surface PD-L1. “−“ and “+” indicate in the absence or presence of EGF (10 ng/mL). Numbers indicate average percentage compared with EGF-treated cells. * p

    Techniques Used: Inhibition, Expressing

    15) Product Images from "Formin-like 3 regulates RhoC/FAK pathway and actin assembly to promote cell invasion in colorectal carcinoma"

    Article Title: Formin-like 3 regulates RhoC/FAK pathway and actin assembly to promote cell invasion in colorectal carcinoma

    Journal: World Journal of Gastroenterology

    doi: 10.3748/wjg.v24.i34.3884

    Formin-like 3 regulates the RhoC/FAK signaling pathway to promote colorectal carcinoma invasion. A and B: Analysis of VEGF, MMP-2 and MMP-9 expression in FMNL3-overexpressing or -depleted colorectal carcinoma cells by western blot and gelatin zymography experiments, respectively. C and D: Analysis of the effects of FMNL3 overexpression or depletion on the expression of RhoC, (p-)Pyk2, (p-)FAK, (p-)MAPK and (p-)AKT by western blot. E: Effects of Pyk2 silencing on the expression of FMNL3, (p-)FAK, (p-)MAPK and (p-)AKT in FMNL3-expressing cells by western blot. FMNL3: Formin-like 3; VEGF: vascular endothelial growth factor; MMP: matrix metalloprotein; Pyk2: Proline rich tyrosine kinase 2; FAK: Focal adhesion kinase; MAPK: Mitogen activated protein kinases; AKT: Protein kinase B.
    Figure Legend Snippet: Formin-like 3 regulates the RhoC/FAK signaling pathway to promote colorectal carcinoma invasion. A and B: Analysis of VEGF, MMP-2 and MMP-9 expression in FMNL3-overexpressing or -depleted colorectal carcinoma cells by western blot and gelatin zymography experiments, respectively. C and D: Analysis of the effects of FMNL3 overexpression or depletion on the expression of RhoC, (p-)Pyk2, (p-)FAK, (p-)MAPK and (p-)AKT by western blot. E: Effects of Pyk2 silencing on the expression of FMNL3, (p-)FAK, (p-)MAPK and (p-)AKT in FMNL3-expressing cells by western blot. FMNL3: Formin-like 3; VEGF: vascular endothelial growth factor; MMP: matrix metalloprotein; Pyk2: Proline rich tyrosine kinase 2; FAK: Focal adhesion kinase; MAPK: Mitogen activated protein kinases; AKT: Protein kinase B.

    Techniques Used: Expressing, Western Blot, Zymography, Over Expression

    16) Product Images from "Cytoprotective and pro-angiogenic functions of thrombomodulin are preserved in the C loop of the fifth epidermal growth factor-like domain"

    Article Title: Cytoprotective and pro-angiogenic functions of thrombomodulin are preserved in the C loop of the fifth epidermal growth factor-like domain

    Journal: Haematologica

    doi: 10.3324/haematol.2017.184481

    TME5C increases the levels of p-ERK, p-AKT, p-p38, and Mcl-1 in endothelial cells. (A and C). HUVECs or HHSECs were exposed to control diluents (PBS as control) or TM mutants (500 nM). After 48 h, proteins were extracted and subjected to western blot analyses. The membrane was sequentially probed with the indicated antibodies. (B and D). Relative quantifications of p-ERK, p-AKT, p-p38, and Mcl-1. ImageJ software was used to measure the band intensities after western blotting. All experiments were performed three times. Results represent the mean ± SD. * P
    Figure Legend Snippet: TME5C increases the levels of p-ERK, p-AKT, p-p38, and Mcl-1 in endothelial cells. (A and C). HUVECs or HHSECs were exposed to control diluents (PBS as control) or TM mutants (500 nM). After 48 h, proteins were extracted and subjected to western blot analyses. The membrane was sequentially probed with the indicated antibodies. (B and D). Relative quantifications of p-ERK, p-AKT, p-p38, and Mcl-1. ImageJ software was used to measure the band intensities after western blotting. All experiments were performed three times. Results represent the mean ± SD. * P

    Techniques Used: Western Blot, Software

    17) Product Images from "The effect of noise exposure on insulin sensitivity in mice may be mediated by the JNK/IRS1 pathway"

    Article Title: The effect of noise exposure on insulin sensitivity in mice may be mediated by the JNK/IRS1 pathway

    Journal: Environmental Health and Preventive Medicine

    doi: 10.1186/s12199-018-0694-3

    Effect of noise exposure on insulin signaling pathways in the gastrocnemius muscle. a Levels of total and Thr 183 /Tyr 185 phosphorylated JNK, total and Ser 307 phosphorylated IRS1, and total Akt and phosphorylated Akt at Ser 473 and Thr 308 were detected via immunoblotting, and representative Western blots are presented. b – e Graphs showing the relative intensities of phospho-protein bands are normalized to the corresponding total protein levels in each case. The data are expressed relative to values for age-matched controls. The average of each age-matched control group was set to 1. Values are presented as the mean ± SEM of 8 mice per group. * p
    Figure Legend Snippet: Effect of noise exposure on insulin signaling pathways in the gastrocnemius muscle. a Levels of total and Thr 183 /Tyr 185 phosphorylated JNK, total and Ser 307 phosphorylated IRS1, and total Akt and phosphorylated Akt at Ser 473 and Thr 308 were detected via immunoblotting, and representative Western blots are presented. b – e Graphs showing the relative intensities of phospho-protein bands are normalized to the corresponding total protein levels in each case. The data are expressed relative to values for age-matched controls. The average of each age-matched control group was set to 1. Values are presented as the mean ± SEM of 8 mice per group. * p

    Techniques Used: Western Blot, Mouse Assay

    18) Product Images from "Hypoxia negates hyperglycaemia-induced chemo-resistance in breast cancer cells: the role of insulin-like growth factor binding protein 2"

    Article Title: Hypoxia negates hyperglycaemia-induced chemo-resistance in breast cancer cells: the role of insulin-like growth factor binding protein 2

    Journal: Oncotarget

    doi: 10.18632/oncotarget.20287

    The involvement of the FASN/ERα/Akt pathway in hypoxia regulation of IGFBP-2
    Figure Legend Snippet: The involvement of the FASN/ERα/Akt pathway in hypoxia regulation of IGFBP-2

    Techniques Used:

    19) Product Images from "Lysine methyltransferase SMYD2 promotes cyst growth in autosomal dominant polycystic kidney disease"

    Article Title: Lysine methyltransferase SMYD2 promotes cyst growth in autosomal dominant polycystic kidney disease

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI90921

    Working model of SMYD2 in regulation of cyst growth in ADPKD. A schematic diagram depicting SMYD2-mediated pathways and processes in Pkd1 mutant renal epithelial cells and kidneys. Pkd1 knockout or mutation results in the upregulation of SMYD2, which may be stabilized via Hsp90, induced by cyst fluid TNF-α or through other unknown mechanisms. Upregulated SMYD2 in Pkd1 mutant renal epithelial cells (a) methylates STAT3, leading to its activation and cystic renal epithelial cell proliferation; (b) methylates the p65 subunit of NF-κB, leading to its activation, which represses cystic renal epithelial cell apoptosis; (c) methylates p53, leading to the repression of p53 and cystic renal epithelial cell apoptosis; and (d) methylates histones to regulate the transcription of the SMYD2 target gene Ptpn13 , a protein of the PTP family, which may regulate the phosphorylation and activation of ERK, mTOR, Akt, and Rb signaling. Targeting SMYD2 with its specific inhibitor AZ505 delays cyst growth in Pkd1 -knockout mouse kidneys. In addition, two positive feedback loops can be observed: SMYD2/IL-6/STAT3/SMYD2 and SMYD2/TNF-α/NF-κB/SMYD2, which may further increase the levels of SMYD2 in cystic renal epithelial cells.
    Figure Legend Snippet: Working model of SMYD2 in regulation of cyst growth in ADPKD. A schematic diagram depicting SMYD2-mediated pathways and processes in Pkd1 mutant renal epithelial cells and kidneys. Pkd1 knockout or mutation results in the upregulation of SMYD2, which may be stabilized via Hsp90, induced by cyst fluid TNF-α or through other unknown mechanisms. Upregulated SMYD2 in Pkd1 mutant renal epithelial cells (a) methylates STAT3, leading to its activation and cystic renal epithelial cell proliferation; (b) methylates the p65 subunit of NF-κB, leading to its activation, which represses cystic renal epithelial cell apoptosis; (c) methylates p53, leading to the repression of p53 and cystic renal epithelial cell apoptosis; and (d) methylates histones to regulate the transcription of the SMYD2 target gene Ptpn13 , a protein of the PTP family, which may regulate the phosphorylation and activation of ERK, mTOR, Akt, and Rb signaling. Targeting SMYD2 with its specific inhibitor AZ505 delays cyst growth in Pkd1 -knockout mouse kidneys. In addition, two positive feedback loops can be observed: SMYD2/IL-6/STAT3/SMYD2 and SMYD2/TNF-α/NF-κB/SMYD2, which may further increase the levels of SMYD2 in cystic renal epithelial cells.

    Techniques Used: Mutagenesis, Knock-Out, Activation Assay

    PKD-associated signaling pathways could be affected by SMYD2 in Pkd1 mutant renal epithelial cells and cystic tissues. ( A and B ) Western blot analysis of the phosphorylation of ERK, S6, AKT, and Rb as well as the total protein levels of these proteins in Pkd1- null MEK cells with or without knockdown of Smyd2 with siRNA for 24 hours ( A ) and with or without AZ505 treatment for 2 hours ( B ). Representative data are shown from 3 independent experiments. ( C ) Western blot analysis of the phosphorylation of ERK, S6, AKT, and Rb in kidneys from Pkd1 conditional knockout mice. Data are representative of 2 independent experiments. ( D ) Western blot analysis of the phosphorylation of ERK, S6, AKT, and Rb, as well as the expression of SMYD2 and PTPN13 in mIMCD3 cells transfected with or without Smyd2 siRNA and/or Ptpn13 siRNA. Representative data from 3 independent experiments are shown.
    Figure Legend Snippet: PKD-associated signaling pathways could be affected by SMYD2 in Pkd1 mutant renal epithelial cells and cystic tissues. ( A and B ) Western blot analysis of the phosphorylation of ERK, S6, AKT, and Rb as well as the total protein levels of these proteins in Pkd1- null MEK cells with or without knockdown of Smyd2 with siRNA for 24 hours ( A ) and with or without AZ505 treatment for 2 hours ( B ). Representative data are shown from 3 independent experiments. ( C ) Western blot analysis of the phosphorylation of ERK, S6, AKT, and Rb in kidneys from Pkd1 conditional knockout mice. Data are representative of 2 independent experiments. ( D ) Western blot analysis of the phosphorylation of ERK, S6, AKT, and Rb, as well as the expression of SMYD2 and PTPN13 in mIMCD3 cells transfected with or without Smyd2 siRNA and/or Ptpn13 siRNA. Representative data from 3 independent experiments are shown.

    Techniques Used: Mutagenesis, Western Blot, Knock-Out, Mouse Assay, Expressing, Transfection

    20) Product Images from "Cigarette smoke extracts and cadmium induce COX-2 expression through γ-secretase-mediated p38 MAPK activation in C6 astroglia cells"

    Article Title: Cigarette smoke extracts and cadmium induce COX-2 expression through γ-secretase-mediated p38 MAPK activation in C6 astroglia cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0212749

    Phosphorylation of CREB by Cd is mediated by γ-secretase activation. (A) C6 cells were treated for 6 h with 0, 1, 10, and 25 μM Cd and lysed in RIPA buffer. (B) C6 cells were pretreated for 1 h with γ-secretase inhibitors (2.5 μM DAPT and 1 μM L-685,486) and then exposed to 25 μM Cd for 6 h. (C) Cells were transfected and incubated with siRNA against nicastrin (siRNA NCT) and non-targeting control (siRNA NON) for 48 h followed by treatment with 25 μM Cd for 6 h. (D) C6 cells were preincubated for 1 h in the presence of BAPTA-AM (10 μM) or NAC (10 mM), and then treated with 25 μM Cd. Total cell extracts were analyzed by western blotting using antibodies against p-CREB (Ser133), CREB, p-AKT (Thr308), and AKT. β-actin indicated equal loading of lysates.
    Figure Legend Snippet: Phosphorylation of CREB by Cd is mediated by γ-secretase activation. (A) C6 cells were treated for 6 h with 0, 1, 10, and 25 μM Cd and lysed in RIPA buffer. (B) C6 cells were pretreated for 1 h with γ-secretase inhibitors (2.5 μM DAPT and 1 μM L-685,486) and then exposed to 25 μM Cd for 6 h. (C) Cells were transfected and incubated with siRNA against nicastrin (siRNA NCT) and non-targeting control (siRNA NON) for 48 h followed by treatment with 25 μM Cd for 6 h. (D) C6 cells were preincubated for 1 h in the presence of BAPTA-AM (10 μM) or NAC (10 mM), and then treated with 25 μM Cd. Total cell extracts were analyzed by western blotting using antibodies against p-CREB (Ser133), CREB, p-AKT (Thr308), and AKT. β-actin indicated equal loading of lysates.

    Techniques Used: Activation Assay, Transfection, Incubation, Western Blot

    21) Product Images from "YAP1 overexpression is associated with poor prognosis of breast cancer patients and induces breast cancer cell growth by inhibiting PTEN"

    Article Title: YAP1 overexpression is associated with poor prognosis of breast cancer patients and induces breast cancer cell growth by inhibiting PTEN

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12597

    YAP 1 affects cell apoptosis and proliferation through regulation of PTEN – AKT signaling. (A) Western blot analysis of PTEN , phosphorylated AKT (p‐AKT), and total AKT protein in the indicated BC cell lines. (B) Flow cytometric assays revealed the role of PTEN ‐specific inhibitor bpV( HO pic) in the apoptosis of YAP 1‐ RNA i1‐transduced cells. (C) CCK 8 assays revealed the role of bpV( HO pic) in the proliferation of YAP 1‐ RNA i1‐transduced cells. (D) Western blot analysis of p‐ PTEN , PTEN , p‐AKT, and total AKT protein in bpV ( HO pic)‐treated YAP 1 silenced cells. Data are presented as mean ± SD of three biological replicates and were analyzed by two‐tailed Student's t test; * P
    Figure Legend Snippet: YAP 1 affects cell apoptosis and proliferation through regulation of PTEN – AKT signaling. (A) Western blot analysis of PTEN , phosphorylated AKT (p‐AKT), and total AKT protein in the indicated BC cell lines. (B) Flow cytometric assays revealed the role of PTEN ‐specific inhibitor bpV( HO pic) in the apoptosis of YAP 1‐ RNA i1‐transduced cells. (C) CCK 8 assays revealed the role of bpV( HO pic) in the proliferation of YAP 1‐ RNA i1‐transduced cells. (D) Western blot analysis of p‐ PTEN , PTEN , p‐AKT, and total AKT protein in bpV ( HO pic)‐treated YAP 1 silenced cells. Data are presented as mean ± SD of three biological replicates and were analyzed by two‐tailed Student's t test; * P

    Techniques Used: Western Blot, Flow Cytometry, CCK-8 Assay, Two Tailed Test

    22) Product Images from "VEGF receptor 2 and the adherens junction as a mechanical transducer in vascular endothelial cells"

    Article Title: VEGF receptor 2 and the adherens junction as a mechanical transducer in vascular endothelial cells

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

    doi: 10.1073/pnas.142224299

    Shear stress-mediated activation (phosphorylation) of p38 does not occur in endothelial cells lacking the VE-cadherin gene. ( A ) Endothelial cells null for VE-cadherin (VE-cad −/−) and the same cells stably retransfected with VE-cadherin (VE-cad +/+) were grown under static conditions or exposed to LSS (10 dynes/cm 2 ) for various time intervals. Whole-cell extracts were tested for P38 expression or phosphorylation (pP38). ( B ) Endothelial cells null for VE-cadherin (VE-cad −/−) and the same cells stably retransfected with VE-cadherin (VE-cad +/+) were grown under static conditions or exposed to LSS (10 dynes/cm 2 ) for various time intervals. The expression and activation of Akt were tested and are presented as the ratio of Akt to phosphorylated Akt (mean ± SD, n = 4).
    Figure Legend Snippet: Shear stress-mediated activation (phosphorylation) of p38 does not occur in endothelial cells lacking the VE-cadherin gene. ( A ) Endothelial cells null for VE-cadherin (VE-cad −/−) and the same cells stably retransfected with VE-cadherin (VE-cad +/+) were grown under static conditions or exposed to LSS (10 dynes/cm 2 ) for various time intervals. Whole-cell extracts were tested for P38 expression or phosphorylation (pP38). ( B ) Endothelial cells null for VE-cadherin (VE-cad −/−) and the same cells stably retransfected with VE-cadherin (VE-cad +/+) were grown under static conditions or exposed to LSS (10 dynes/cm 2 ) for various time intervals. The expression and activation of Akt were tested and are presented as the ratio of Akt to phosphorylated Akt (mean ± SD, n = 4).

    Techniques Used: Activation Assay, Stable Transfection, Expressing

    23) Product Images from "PECAM-1 Affects GSK-3?-Mediated ?-Catenin Phosphorylation and Degradation"

    Article Title: PECAM-1 Affects GSK-3?-Mediated ?-Catenin Phosphorylation and Degradation

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2006.051112

    Levels of pP85 (pPI3K), pAkt, and pS-GSK-3β correlate directly with PECAM-1 expression levels. A: Representative Western blots of HUVECs treated with either a scrambled PECAM-1 oligonucleotide (scrambled CD31) and/or antisense PECAM-1 oligonucleotide (antisense CD31), revealing a significant knockdown of PECAM-1 expression in the antisense CD31-treated cultures, which correlated with significant knockdowns of tyrosine-phosphorylated (active) PI3K and Akt. n = 3. B: Quantitation of three independent experiments consisting of HUVECs treated with either a scrambled PECAM-1 oligonucleotide (scrambled CD31) and/or antisense PECAM-1 oligonucleotide (antisense CD31), illustrating significant knockdown of phospho-Akt and phospho-PI3K. C: Representative Western blots of three independent experiments consisting of lung-derived RC and CD31 KO and brain-derived WT and CD31 KO endothelial cell cultures, illustrating decreased phospho-PI3K and phospho-Akt levels in the CD31 KO cultures compared with the WT and RC cultures.
    Figure Legend Snippet: Levels of pP85 (pPI3K), pAkt, and pS-GSK-3β correlate directly with PECAM-1 expression levels. A: Representative Western blots of HUVECs treated with either a scrambled PECAM-1 oligonucleotide (scrambled CD31) and/or antisense PECAM-1 oligonucleotide (antisense CD31), revealing a significant knockdown of PECAM-1 expression in the antisense CD31-treated cultures, which correlated with significant knockdowns of tyrosine-phosphorylated (active) PI3K and Akt. n = 3. B: Quantitation of three independent experiments consisting of HUVECs treated with either a scrambled PECAM-1 oligonucleotide (scrambled CD31) and/or antisense PECAM-1 oligonucleotide (antisense CD31), illustrating significant knockdown of phospho-Akt and phospho-PI3K. C: Representative Western blots of three independent experiments consisting of lung-derived RC and CD31 KO and brain-derived WT and CD31 KO endothelial cell cultures, illustrating decreased phospho-PI3K and phospho-Akt levels in the CD31 KO cultures compared with the WT and RC cultures.

    Techniques Used: Expressing, Western Blot, Quantitation Assay, Derivative Assay

    Working model illustrating the effects of presence or absence of PECAM-1 on GSK-3β-mediated β-catenin phosphorylation and degradation. A: In PECAM-1-expressing endothelial cells, tyrosine phosphorylated β-catenin is efficiently complexed with SHP-2 in a tripartite complex with immunoreceptor tyrosine-based activation motif tyrosine phosphorylated PECAM-1, dephosphorylated, and made available to participate in reformation of adherens junctional complexes and translocation to the nucleus where it modulates gene expression on complexing with Lef/Tcf. PECAM-1 expression is also associated with increased activity (phosphorylation) of PI3K and Akt, which increase the serine phosphorylation of GSK-3β, inactivating it, thus reducing the fraction of β-catenin that is serine/threonine phosphorylated and thus targeted for proteosomal degradation. B: In the absence/reduction of PECAM-1 expression, tyrosine-phosphorylated β-catenin is inefficiently complexed with and dephosphorylated by SHP-2, reducing its ability to participate in the reformation of adherens junctions ( dashed lines ). In addition, the absence/reduction of PECAM-1 expression is also associated with a reduction in PI3K and Akt activity (phosphorylation), leading to a persistent high activity of GSK-3β ( dashed line ), resulting in an increased serine/threonine phosphorylation of β-catenin and its targeting for proteosomal degradation ( heavy solid lines ) and reduced nuclear translocation ( light solid lines ).
    Figure Legend Snippet: Working model illustrating the effects of presence or absence of PECAM-1 on GSK-3β-mediated β-catenin phosphorylation and degradation. A: In PECAM-1-expressing endothelial cells, tyrosine phosphorylated β-catenin is efficiently complexed with SHP-2 in a tripartite complex with immunoreceptor tyrosine-based activation motif tyrosine phosphorylated PECAM-1, dephosphorylated, and made available to participate in reformation of adherens junctional complexes and translocation to the nucleus where it modulates gene expression on complexing with Lef/Tcf. PECAM-1 expression is also associated with increased activity (phosphorylation) of PI3K and Akt, which increase the serine phosphorylation of GSK-3β, inactivating it, thus reducing the fraction of β-catenin that is serine/threonine phosphorylated and thus targeted for proteosomal degradation. B: In the absence/reduction of PECAM-1 expression, tyrosine-phosphorylated β-catenin is inefficiently complexed with and dephosphorylated by SHP-2, reducing its ability to participate in the reformation of adherens junctions ( dashed lines ). In addition, the absence/reduction of PECAM-1 expression is also associated with a reduction in PI3K and Akt activity (phosphorylation), leading to a persistent high activity of GSK-3β ( dashed line ), resulting in an increased serine/threonine phosphorylation of β-catenin and its targeting for proteosomal degradation ( heavy solid lines ) and reduced nuclear translocation ( light solid lines ).

    Techniques Used: Expressing, Activation Assay, Translocation Assay, Activity Assay

    24) Product Images from "Neurofibromatosis type 1 alternative splicing is a key regulator of Ras/ERK signaling and learning behaviors in mice"

    Article Title: Neurofibromatosis type 1 alternative splicing is a key regulator of Ras/ERK signaling and learning behaviors in mice

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddx264

    Western blot analysis showing the expression of downstream targets of Ras in lysates from whole mouse brains. ( A ) Phospho-ERK1/2 (∼42/44 kDa) and total ERK1/2 (∼42/44 kDa). Error bars represent standard errors. N = 3. * P = 1 × 10 −2 , ** P = 5.85 × 10 −6 . ( B ) Phospho-Akt (∼60 kDa) and total Akt (∼60 kDa). Error bars represent standard errors. N = 3. ( C ) Phospho-S6 (∼32 kDa), total S6 (∼32 kDa) and γ-tubulin (∼48 kDa, a loading control). Error bars represent standard errors. N = 3.
    Figure Legend Snippet: Western blot analysis showing the expression of downstream targets of Ras in lysates from whole mouse brains. ( A ) Phospho-ERK1/2 (∼42/44 kDa) and total ERK1/2 (∼42/44 kDa). Error bars represent standard errors. N = 3. * P = 1 × 10 −2 , ** P = 5.85 × 10 −6 . ( B ) Phospho-Akt (∼60 kDa) and total Akt (∼60 kDa). Error bars represent standard errors. N = 3. ( C ) Phospho-S6 (∼32 kDa), total S6 (∼32 kDa) and γ-tubulin (∼48 kDa, a loading control). Error bars represent standard errors. N = 3.

    Techniques Used: Western Blot, Expressing

    25) Product Images from "Differentiation capacities of skeletal muscle satellite cells in Lantang and Landrace piglets"

    Article Title: Differentiation capacities of skeletal muscle satellite cells in Lantang and Landrace piglets

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17860

    Protein levels of mammalian target of rapamycin (mTOR) pathway regulators in Lantang and Landrace piglet satellite cells (SCs) Western blot analysis of satellite cells for protein kinase B (Akt), p-Akt (Ser437), mTOR, p-mTOR (Ser2448, Ser2481), p70 ribosomal S6 protein kinase 1 (S6K1), p-S6K1(Thr389), ribosomal protein S6 (S6), p-S6 (Ser235/236), eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), and p-4EBP1(Thr70) at 72 hours during differentiation. The results show phosphorylation levels relative to total levels. Values are mean ± SEM. Asterisk (*) indicates a significant difference ( P
    Figure Legend Snippet: Protein levels of mammalian target of rapamycin (mTOR) pathway regulators in Lantang and Landrace piglet satellite cells (SCs) Western blot analysis of satellite cells for protein kinase B (Akt), p-Akt (Ser437), mTOR, p-mTOR (Ser2448, Ser2481), p70 ribosomal S6 protein kinase 1 (S6K1), p-S6K1(Thr389), ribosomal protein S6 (S6), p-S6 (Ser235/236), eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), and p-4EBP1(Thr70) at 72 hours during differentiation. The results show phosphorylation levels relative to total levels. Values are mean ± SEM. Asterisk (*) indicates a significant difference ( P

    Techniques Used: Western Blot, Binding Assay

    26) Product Images from "Syndecan-4-/- mice have smaller muscle fibers, increased Akt/mTOR/S6K1 and Notch/HES-1 pathways, and alterations in extracellular matrix components"

    Article Title: Syndecan-4-/- mice have smaller muscle fibers, increased Akt/mTOR/S6K1 and Notch/HES-1 pathways, and alterations in extracellular matrix components

    Journal: bioRxiv

    doi: 10.1101/2020.06.10.143982

    Illustration of alterations in the syndecan-4 -/- mice. ( A ) The syndecan-4 -/- mice had reduced body weight, muscle weight, muscle fibers and expression of myogenic regulatory transcription factors. ( B ) The syndecan-4 -/- muscle had increased mRNA levels of syndecan-2 and the ECM components decorin, fibromodulin, biglycan, collagen 1, collagen 3 and the collagen cross-linking enzyme lysyl oxidase (LOX). However, fibromodulin, biglycan and LOX were reduced at the protein level (denoted with star), suggesting that these ECM components are more susceptible to degradation or less efficiently translated when syndecan-4 is absent. The syndecan-4 -/- muscle had also increased activation of the ( C ) Akt/mTOR/S6K1 and ( D ) Cleaved Notch1-HES-1 pathways.
    Figure Legend Snippet: Illustration of alterations in the syndecan-4 -/- mice. ( A ) The syndecan-4 -/- mice had reduced body weight, muscle weight, muscle fibers and expression of myogenic regulatory transcription factors. ( B ) The syndecan-4 -/- muscle had increased mRNA levels of syndecan-2 and the ECM components decorin, fibromodulin, biglycan, collagen 1, collagen 3 and the collagen cross-linking enzyme lysyl oxidase (LOX). However, fibromodulin, biglycan and LOX were reduced at the protein level (denoted with star), suggesting that these ECM components are more susceptible to degradation or less efficiently translated when syndecan-4 is absent. The syndecan-4 -/- muscle had also increased activation of the ( C ) Akt/mTOR/S6K1 and ( D ) Cleaved Notch1-HES-1 pathways.

    Techniques Used: Mouse Assay, Expressing, Activation Assay

    27) Product Images from "Menin Upregulates FOXO1 Protein Stability by Repressing Skp2-Mediated Degradation in β Cells"

    Article Title: Menin Upregulates FOXO1 Protein Stability by Repressing Skp2-Mediated Degradation in β Cells

    Journal: Pancreas

    doi: 10.1097/MPA.0000000000001239

    Menin represses FOXO1 ubiquitination and AKT phosphorylation. A, C, INS-1 cells expressing vector or ectopic menin were lysed for Western blot with indicated antibodies. B and D, Quantitation of p-AKT or p-FOXO1 protein level from panel A or C. Band density analysis was executed with ImageJ, and p-AKT or p-FOXO1 protein levels were quantified and normalized to total AKT or total FOXO1. E and G, INS-1 cells expressing vector or sShMen1 were lysed for Western blot with indicated antibodies. F and H, Quantitation of p-AKT or p-FOXO1 protein level from panel E or G. Band density analysis was executed with ImageJ, and p-AKT or p-FOXO1 protein levels were quantified and normalized to total AKT or total FOXO1. I, Equal amounts of menin and/or HA-ubiquitin constructs were transfected into 293T cells in the presence or absence of EGF (100 ng/mL) for 24 hours, followed by MG132 (20 μmol) treatment for 4 hours and lysed for IP and Western blot with the indicated antibodies. * P
    Figure Legend Snippet: Menin represses FOXO1 ubiquitination and AKT phosphorylation. A, C, INS-1 cells expressing vector or ectopic menin were lysed for Western blot with indicated antibodies. B and D, Quantitation of p-AKT or p-FOXO1 protein level from panel A or C. Band density analysis was executed with ImageJ, and p-AKT or p-FOXO1 protein levels were quantified and normalized to total AKT or total FOXO1. E and G, INS-1 cells expressing vector or sShMen1 were lysed for Western blot with indicated antibodies. F and H, Quantitation of p-AKT or p-FOXO1 protein level from panel E or G. Band density analysis was executed with ImageJ, and p-AKT or p-FOXO1 protein levels were quantified and normalized to total AKT or total FOXO1. I, Equal amounts of menin and/or HA-ubiquitin constructs were transfected into 293T cells in the presence or absence of EGF (100 ng/mL) for 24 hours, followed by MG132 (20 μmol) treatment for 4 hours and lysed for IP and Western blot with the indicated antibodies. * P

    Techniques Used: Expressing, Plasmid Preparation, Western Blot, Quantitation Assay, Construct, Transfection

    28) Product Images from "LncRNA TP73-AS1 promoted the progression of lung adenocarcinoma via PI3K/AKT pathway"

    Article Title: LncRNA TP73-AS1 promoted the progression of lung adenocarcinoma via PI3K/AKT pathway

    Journal: Bioscience Reports

    doi: 10.1042/BSR20180999

    TP73-AS1 aggravated the progression of LAD via PI3K/Akt signaling pathway ( A ) The expression of proteins involved in PI3K/AKT and MRK/ERK pathway was determined by Western blotting in shCtrl or shTP73-AS1 transfected A549 and HCC827 cells after treated with or without 740Y-P, the PI3K activator. ( B ) Relative protein level of total AKT and p-AKT in LAD tissues was evaluated by Western blot assay and quantitated using ImageJ software. ( C , D ) MTT assay and colony formation assay were applied to detect the changes of cell proliferation ability of HCC827 and A549 cells after the transfection with shCtrl or shTP73-AS1 and the treatment with or without 740Y-P. ( E , F ) Cell cycle distribution and apoptosis rate altered in shCtrl or shTP73-AS1 transfected HCC827 and A549 cells after treating with or without 740Y-P was estimated by flow cytometry analysis. ( G ) Transwell assay was utilized to test the alterations of migration and invasion capacities in TP73-AS1 silenced LAD cells with or without the treatment of 740Y-P. The experiments were performed in triplicate. * P
    Figure Legend Snippet: TP73-AS1 aggravated the progression of LAD via PI3K/Akt signaling pathway ( A ) The expression of proteins involved in PI3K/AKT and MRK/ERK pathway was determined by Western blotting in shCtrl or shTP73-AS1 transfected A549 and HCC827 cells after treated with or without 740Y-P, the PI3K activator. ( B ) Relative protein level of total AKT and p-AKT in LAD tissues was evaluated by Western blot assay and quantitated using ImageJ software. ( C , D ) MTT assay and colony formation assay were applied to detect the changes of cell proliferation ability of HCC827 and A549 cells after the transfection with shCtrl or shTP73-AS1 and the treatment with or without 740Y-P. ( E , F ) Cell cycle distribution and apoptosis rate altered in shCtrl or shTP73-AS1 transfected HCC827 and A549 cells after treating with or without 740Y-P was estimated by flow cytometry analysis. ( G ) Transwell assay was utilized to test the alterations of migration and invasion capacities in TP73-AS1 silenced LAD cells with or without the treatment of 740Y-P. The experiments were performed in triplicate. * P

    Techniques Used: Expressing, Western Blot, Transfection, Software, MTT Assay, Colony Assay, Flow Cytometry, Cytometry, Transwell Assay, Migration

    29) Product Images from "Hydroxytyrosol contributes to cell proliferation and inhibits apoptosis in pulsed electromagnetic fields treated human umbilical vein endothelial cells in vitro"

    Article Title: Hydroxytyrosol contributes to cell proliferation and inhibits apoptosis in pulsed electromagnetic fields treated human umbilical vein endothelial cells in vitro

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.7701

    Effect of HTY and PEMF on protein expression levels of Akt, mTOR, TGF-β and p53 at 24 h. Representative western blot images and quantification of (A) Akt, (B) mTOR, (C) TGF-β and (D) p53 protein expression levels in human umbilical vein endothelial cells. Data are expressed as the mean ± standard error of three independent experiments. **P
    Figure Legend Snippet: Effect of HTY and PEMF on protein expression levels of Akt, mTOR, TGF-β and p53 at 24 h. Representative western blot images and quantification of (A) Akt, (B) mTOR, (C) TGF-β and (D) p53 protein expression levels in human umbilical vein endothelial cells. Data are expressed as the mean ± standard error of three independent experiments. **P

    Techniques Used: Expressing, Western Blot

    Effect of HTY and PEMF on mRNA expression levels of Akt, mTOR, TGF-β and p53 at 24 h. (A) Akt, (B) mTOR, (C) TGF-β and (D) p53 mRNA expression levels in human umbilical vein endothelial cells. Data are expressed as the mean ± standard error of six independent experiments. *P
    Figure Legend Snippet: Effect of HTY and PEMF on mRNA expression levels of Akt, mTOR, TGF-β and p53 at 24 h. (A) Akt, (B) mTOR, (C) TGF-β and (D) p53 mRNA expression levels in human umbilical vein endothelial cells. Data are expressed as the mean ± standard error of six independent experiments. *P

    Techniques Used: Expressing

    30) Product Images from "miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer"

    Article Title: miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101656

    miR-150-5p inhibited VEGFA/VEGFR2/Akt/mTOR signaling pathway in CRC. Western blot was used to measure the expression of VEGFA, VEGFR2, p-VEGFR2, Akt, p-Akt, mTOR, p-mTOR in transfected HCT116 and HCT8 cells. GAPDH was used as a loading control. Data are shown as the mean±SD of three independent experiments. * p
    Figure Legend Snippet: miR-150-5p inhibited VEGFA/VEGFR2/Akt/mTOR signaling pathway in CRC. Western blot was used to measure the expression of VEGFA, VEGFR2, p-VEGFR2, Akt, p-Akt, mTOR, p-mTOR in transfected HCT116 and HCT8 cells. GAPDH was used as a loading control. Data are shown as the mean±SD of three independent experiments. * p

    Techniques Used: Western Blot, Expressing, Transfection

    31) Product Images from "Interferon regulatory factor 4 (IRF4) is overexpressed in human non-small cell lung cancer (NSCLC) and activates the Notch signaling pathway"

    Article Title: Interferon regulatory factor 4 (IRF4) is overexpressed in human non-small cell lung cancer (NSCLC) and activates the Notch signaling pathway

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.7319

    IRF4 activates Notch-Akt signaling in NSCLC cells (A) IRF4 knockdown inhibits Notch1 and Notch2 expression in A549 cells. A549 cells were infected with lentivirus expressing sh-Ctrl or sh-IRF4 vectors for 48 h. qRT-PCR was performed to analyzed mRNA level. **P
    Figure Legend Snippet: IRF4 activates Notch-Akt signaling in NSCLC cells (A) IRF4 knockdown inhibits Notch1 and Notch2 expression in A549 cells. A549 cells were infected with lentivirus expressing sh-Ctrl or sh-IRF4 vectors for 48 h. qRT-PCR was performed to analyzed mRNA level. **P

    Techniques Used: Expressing, Infection, Quantitative RT-PCR

    32) Product Images from "Leukemia inhibitory factor regulates marmoset induced pluripotent stem cell proliferation via a PI3K/Akt-dependent Tbx-3 activation pathway"

    Article Title: Leukemia inhibitory factor regulates marmoset induced pluripotent stem cell proliferation via a PI3K/Akt-dependent Tbx-3 activation pathway

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3610

    Involvement of the PI3K/Akt signaling pathway in LIF-induced activation of Tbx-3 in marmoset induced pluripotent stem cells. The protein expression levels of STAT3, Tbx3, Nanog, p-Akt and PI3K(85α) in the presence or absence of LIF were analyzed by western blot analysis. Actin is shown as a loading control. *** P
    Figure Legend Snippet: Involvement of the PI3K/Akt signaling pathway in LIF-induced activation of Tbx-3 in marmoset induced pluripotent stem cells. The protein expression levels of STAT3, Tbx3, Nanog, p-Akt and PI3K(85α) in the presence or absence of LIF were analyzed by western blot analysis. Actin is shown as a loading control. *** P

    Techniques Used: Activation Assay, Expressing, Western Blot

    Model of LIF-mediated expression levels of pluripotency-associated gene by activation of the PI3K/Akt signaling pathway. LIF, leukemia inhibitory factor; PI3K, phosphoinositide 3-kinase; Tbx-3, T-box 3.
    Figure Legend Snippet: Model of LIF-mediated expression levels of pluripotency-associated gene by activation of the PI3K/Akt signaling pathway. LIF, leukemia inhibitory factor; PI3K, phosphoinositide 3-kinase; Tbx-3, T-box 3.

    Techniques Used: Expressing, Activation Assay

    33) Product Images from "Sanhuang Xiexin Tang Ameliorates Type 2 Diabetic Rats via Modulation of the Metabolic Profiles and NF-κB/PI-3K/Akt Signaling Pathways"

    Article Title: Sanhuang Xiexin Tang Ameliorates Type 2 Diabetic Rats via Modulation of the Metabolic Profiles and NF-κB/PI-3K/Akt Signaling Pathways

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.00955

    (A) mRNA expression level of PI-3K, (B) mRNA expression level of Akt, (C) mRNA expression level of GLUT4, (D) protein expression level of phosphorylated PI-3K, (E) protein expression level of phosphorylated Akt and (F) protein expression level of GLUT4 in skeletal muscle of the normal group (N), model group (M), and groups gavaged with metformin (Met), 5 g/kg SXT (SL), 10 g/kg SXT (SM), and 15 g/kg SXT (SH). The values were shown as mean ± SD for seven animals. ** P
    Figure Legend Snippet: (A) mRNA expression level of PI-3K, (B) mRNA expression level of Akt, (C) mRNA expression level of GLUT4, (D) protein expression level of phosphorylated PI-3K, (E) protein expression level of phosphorylated Akt and (F) protein expression level of GLUT4 in skeletal muscle of the normal group (N), model group (M), and groups gavaged with metformin (Met), 5 g/kg SXT (SL), 10 g/kg SXT (SM), and 15 g/kg SXT (SH). The values were shown as mean ± SD for seven animals. ** P

    Techniques Used: Expressing

    34) Product Images from "c-RET Molecule in Malignant Melanoma from Oncogenic RET-Carrying Transgenic Mice and Human Cell Lines"

    Article Title: c-RET Molecule in Malignant Melanoma from Oncogenic RET-Carrying Transgenic Mice and Human Cell Lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010279

    Signal transduction molecules potentially sited downstream of c-RET in HM3KO cells. Expression and phosphorylation levels of ERK and AKT in HM3KO cells before (0 min) and at 15 and 60 min after stimulation with GDNF (100 ng/ml) were examined by immunoblotting. Equality of protein amounts in each lane was confirmed by immunoblotting with anti-β-actin antibody.
    Figure Legend Snippet: Signal transduction molecules potentially sited downstream of c-RET in HM3KO cells. Expression and phosphorylation levels of ERK and AKT in HM3KO cells before (0 min) and at 15 and 60 min after stimulation with GDNF (100 ng/ml) were examined by immunoblotting. Equality of protein amounts in each lane was confirmed by immunoblotting with anti-β-actin antibody.

    Techniques Used: Transduction, Expressing

    35) Product Images from "Targeting phosphatidylinositol 3 kinase-β and -δ for Bruton tyrosine kinase resistance in diffuse large B-cell lymphoma"

    Article Title: Targeting phosphatidylinositol 3 kinase-β and -δ for Bruton tyrosine kinase resistance in diffuse large B-cell lymphoma

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2020001685

    PI3K-β/δ dual inhibitor sensitized ibrutinib-resistant cells to cytotoxic effects of chemotherapeutic agents. (A) Colony formation assays were performed using ibrutinib-resistant DLBCL cells treated with KA2237. (B) OCI-LY10-IbR tumor cells suspended in a 1:1 Matrigel mixture were implanted subcutaneously into nude mice. Intraperitoneal administration of either KA2237 (100 mg/kg) or saline control was initiated every other day after tumors reached ∼300 mm 3 . Tumor volumes were reported for all mice for 15 days. (C) Western blot analysis for PI3K/AKT/mTOR signaling pathway from tumor lysates treated with KA2237. (D) Drug dose matrix data of respective ibrutinib-resistant cells. The numbers in each matrix indicate the percent of growth inhibition in cells treated with KA2237 plus 1 of 3 chemotherapeutic agents compared with cells treated with vehicle alone. (E) Graphical image representing the PI3K-β/δ–dependent activation of survival PI3K/AKT signaling in acquired ibrutinib resistance DLBCL cells. This activation of survival-PI3K signaling is blocked by the dual PI3K-β/δ selective inhibitor KA2237. Note: font sizes of the molecules in panel E represent their experimentally observed expression levels. Error bars represent standard error. ** P
    Figure Legend Snippet: PI3K-β/δ dual inhibitor sensitized ibrutinib-resistant cells to cytotoxic effects of chemotherapeutic agents. (A) Colony formation assays were performed using ibrutinib-resistant DLBCL cells treated with KA2237. (B) OCI-LY10-IbR tumor cells suspended in a 1:1 Matrigel mixture were implanted subcutaneously into nude mice. Intraperitoneal administration of either KA2237 (100 mg/kg) or saline control was initiated every other day after tumors reached ∼300 mm 3 . Tumor volumes were reported for all mice for 15 days. (C) Western blot analysis for PI3K/AKT/mTOR signaling pathway from tumor lysates treated with KA2237. (D) Drug dose matrix data of respective ibrutinib-resistant cells. The numbers in each matrix indicate the percent of growth inhibition in cells treated with KA2237 plus 1 of 3 chemotherapeutic agents compared with cells treated with vehicle alone. (E) Graphical image representing the PI3K-β/δ–dependent activation of survival PI3K/AKT signaling in acquired ibrutinib resistance DLBCL cells. This activation of survival-PI3K signaling is blocked by the dual PI3K-β/δ selective inhibitor KA2237. Note: font sizes of the molecules in panel E represent their experimentally observed expression levels. Error bars represent standard error. ** P

    Techniques Used: Mouse Assay, Western Blot, Inhibition, Activation Assay, Expressing

    IR DLBCL cells show enhanced PI3K/AKT/mTOR signaling. (A) Western blot analyses of BTK expression in IR/PT DLBCL cells. (B) Heat maps derived from reverse-phase protein array analyses of IR/PT DLBCL pairs represent the differential expression of proteins identified by Wilcoxon rank-sum test (red indicates above median; green indicates below median). (C) Western blot analyses for AKT and mTOR and its substrates show activity in IR/PT DLBCL cell pairs. (D) Western blot analysis for AKT, mTOR, and mTOR substrates in OCI-LY10-IbR cells 48 hours after electroporation with WT-BTK or empty vector. The upregulation of BTK in resistant clones reverses AKT and mTOR activation.
    Figure Legend Snippet: IR DLBCL cells show enhanced PI3K/AKT/mTOR signaling. (A) Western blot analyses of BTK expression in IR/PT DLBCL cells. (B) Heat maps derived from reverse-phase protein array analyses of IR/PT DLBCL pairs represent the differential expression of proteins identified by Wilcoxon rank-sum test (red indicates above median; green indicates below median). (C) Western blot analyses for AKT and mTOR and its substrates show activity in IR/PT DLBCL cell pairs. (D) Western blot analysis for AKT, mTOR, and mTOR substrates in OCI-LY10-IbR cells 48 hours after electroporation with WT-BTK or empty vector. The upregulation of BTK in resistant clones reverses AKT and mTOR activation.

    Techniques Used: Western Blot, Expressing, Derivative Assay, Protein Array, Activity Assay, Electroporation, Plasmid Preparation, Clone Assay, Activation Assay

    36) Product Images from "Extraocular muscle regeneration in zebrafish requires late signals from Insulin-like growth factors"

    Article Title: Extraocular muscle regeneration in zebrafish requires late signals from Insulin-like growth factors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0192214

    Role of Akt in the regenerating muscle. The activation of Akt in injured muscles (non-BMS754807 treated fish) was assessed by western blot in a time course experiment (A). Immunoblotting was performed with anti-phosphorylated Akt antibody. Total amounts of Akt were monitored by reprobing membranes with anti-Akt antibody. Note that phosphorylated Akt (pAkt) was rapidly and persistently reduced in the injured muscle. Tubulin was used as a loading control. The densitometric quantification of the Akt bands is shown (B, C). The intensity of pAkt (B) and tAkt bands (C) was normalized to the tubulin content. The ratio between pAkt and tAkt was used to represent the fraction of active Akt (D). For comparative purposes, the pAkt/tAkt ratio of the injured muscle was divided by the pAkt/tAkt ratio of the uninjured muscle at each time point (E). U, uninjured muscle; I, injured muscle; R.U., relative units.
    Figure Legend Snippet: Role of Akt in the regenerating muscle. The activation of Akt in injured muscles (non-BMS754807 treated fish) was assessed by western blot in a time course experiment (A). Immunoblotting was performed with anti-phosphorylated Akt antibody. Total amounts of Akt were monitored by reprobing membranes with anti-Akt antibody. Note that phosphorylated Akt (pAkt) was rapidly and persistently reduced in the injured muscle. Tubulin was used as a loading control. The densitometric quantification of the Akt bands is shown (B, C). The intensity of pAkt (B) and tAkt bands (C) was normalized to the tubulin content. The ratio between pAkt and tAkt was used to represent the fraction of active Akt (D). For comparative purposes, the pAkt/tAkt ratio of the injured muscle was divided by the pAkt/tAkt ratio of the uninjured muscle at each time point (E). U, uninjured muscle; I, injured muscle; R.U., relative units.

    Techniques Used: Activation Assay, Fluorescence In Situ Hybridization, Western Blot

    37) Product Images from "Inhibition of fatty acid desaturation is detrimental to cancer cell survival in metabolically compromised environments"

    Article Title: Inhibition of fatty acid desaturation is detrimental to cancer cell survival in metabolically compromised environments

    Journal: Cancer & Metabolism

    doi: 10.1186/s40170-016-0146-8

    SCD inhibition alters cellular cardiolipin composition leading to cytochrome C release and sensitisation towards apoptosis. a DU145 cells were treated with different concentrations of SCD inhibitor (SCDi II) and proliferation was determined by BrdU labelling. b Cells treated as in a were used to determine apoptosis using Annexin V staining. Data represent mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t test (* p ≤ 0.05). c Quantitative lipid profiling of cardiolipin (CL) content of DU145 cells grown for 48 h in medium containing full (FS) or low (LS) serum using LC-MS/MS. Concentrations of mono-unsaturated (≤4 double bonds) and poly-unsaturated ( > 4 double bonds) CL species are displayed. Data represent the mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t test (*** p ≤ 0.001). d DU145 cells were treated with SCD inhibitors (100 nM) or solvent (DMSO) for 48 h in medium containing low serum or medium supplemented with BSA or BSA-coupled oleic acid (BSA-Oleate). Quantitative lipid profiling of CL content was determined using LC-MS/MS. Concentrations of mono- and poly-unsaturated CL species are displayed. Data represent the mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t tests (* p ≤ 0.05). e DU145 cells were treated with SCD inhibitor (SCDi I) or solvent (DMSO) for 48 h in medium containing low serum or medium supplemented with BSA or BSA-oleate. Cells were lysed by digitonin, and the presence of cytoplasmic cytochrome C was determined. UV treatment was used as positive control. Vinculin is shown as loading control. Levels of phosphorylated Akt (S473) and total Akt were detected in total lysates. f DU145 cells were treated as in e but 3 μM of Akt inhibitor was added prior to addition of BSA-oleate. Cytoplasmic cytochrome C was detected in digitonin lysates. g DU145 cells were treated with SCD inhibitor (SCDi I) or solvent (DMSO) in medium containing full (FS) or low (LS) serum or medium supplemented with BSA or BSA-oleate. The presence of full-length and cleaved (cl.) PARP was determined. Actin is shown as loading control. h DU145 cells were transfected with siRNA targeting SCD (siSCD) or non-targeting controls (siCtrl) and cultured as in e . The presence of full-length and cleaved (cl.) PARP was determined. Actin is shown as loading control. i DU145 or MDA-MB-468 cells were treated with SCD inhibitor (100 nM) for 48 h, and oxygen consumption rate (OCR) before and after addition of oligomycin, FCCP and rotenone was determined using a Seahorse Bioanalyzer. j – m DU145 cells were treated with the indicated doses of SCD inhibitor (SCDi II) either alone or in combination with different doses of metformin (Metf, j ), rotenone (RN, k ), paclitaxel (PTX, l ) or staurosporin (STP, m ) for 72 h in medium containing low serum. Cell viability was determined by crystal violet staining. Data represent the mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t tests (* p ≤ 0.05, ** p ≤ 0.01 compared to SCDi alone; # p ≤ 0.05, ## p ≤ 0.01 compared to no SCDi)
    Figure Legend Snippet: SCD inhibition alters cellular cardiolipin composition leading to cytochrome C release and sensitisation towards apoptosis. a DU145 cells were treated with different concentrations of SCD inhibitor (SCDi II) and proliferation was determined by BrdU labelling. b Cells treated as in a were used to determine apoptosis using Annexin V staining. Data represent mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t test (* p ≤ 0.05). c Quantitative lipid profiling of cardiolipin (CL) content of DU145 cells grown for 48 h in medium containing full (FS) or low (LS) serum using LC-MS/MS. Concentrations of mono-unsaturated (≤4 double bonds) and poly-unsaturated ( > 4 double bonds) CL species are displayed. Data represent the mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t test (*** p ≤ 0.001). d DU145 cells were treated with SCD inhibitors (100 nM) or solvent (DMSO) for 48 h in medium containing low serum or medium supplemented with BSA or BSA-coupled oleic acid (BSA-Oleate). Quantitative lipid profiling of CL content was determined using LC-MS/MS. Concentrations of mono- and poly-unsaturated CL species are displayed. Data represent the mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t tests (* p ≤ 0.05). e DU145 cells were treated with SCD inhibitor (SCDi I) or solvent (DMSO) for 48 h in medium containing low serum or medium supplemented with BSA or BSA-oleate. Cells were lysed by digitonin, and the presence of cytoplasmic cytochrome C was determined. UV treatment was used as positive control. Vinculin is shown as loading control. Levels of phosphorylated Akt (S473) and total Akt were detected in total lysates. f DU145 cells were treated as in e but 3 μM of Akt inhibitor was added prior to addition of BSA-oleate. Cytoplasmic cytochrome C was detected in digitonin lysates. g DU145 cells were treated with SCD inhibitor (SCDi I) or solvent (DMSO) in medium containing full (FS) or low (LS) serum or medium supplemented with BSA or BSA-oleate. The presence of full-length and cleaved (cl.) PARP was determined. Actin is shown as loading control. h DU145 cells were transfected with siRNA targeting SCD (siSCD) or non-targeting controls (siCtrl) and cultured as in e . The presence of full-length and cleaved (cl.) PARP was determined. Actin is shown as loading control. i DU145 or MDA-MB-468 cells were treated with SCD inhibitor (100 nM) for 48 h, and oxygen consumption rate (OCR) before and after addition of oligomycin, FCCP and rotenone was determined using a Seahorse Bioanalyzer. j – m DU145 cells were treated with the indicated doses of SCD inhibitor (SCDi II) either alone or in combination with different doses of metformin (Metf, j ), rotenone (RN, k ), paclitaxel (PTX, l ) or staurosporin (STP, m ) for 72 h in medium containing low serum. Cell viability was determined by crystal violet staining. Data represent the mean ± SEM of three independent biological replicates. Statistical comparisons were performed using Student t tests (* p ≤ 0.05, ** p ≤ 0.01 compared to SCDi alone; # p ≤ 0.05, ## p ≤ 0.01 compared to no SCDi)

    Techniques Used: Inhibition, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Positive Control, Transfection, Cell Culture, Multiple Displacement Amplification

    38) Product Images from "Endothelial S1pr1 regulates pressure overload‐induced cardiac remodelling through AKT‐eNOS pathway, et al. Endothelial S1pr1 regulates pressure overload‐induced cardiac remodelling through AKT‐eNOS pathway"

    Article Title: Endothelial S1pr1 regulates pressure overload‐induced cardiac remodelling through AKT‐eNOS pathway, et al. Endothelial S1pr1 regulates pressure overload‐induced cardiac remodelling through AKT‐eNOS pathway

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14900

    Endothelial S1pr1 inhibits fibroblast proliferation, migration and its conversion towards myofibroblasts via AKT/eNOS pathway. A, Condition medium of lentiviral S1PR1‐overexpressing ECs decreases fibroblast cell proliferation, while blockade of AKT or eNOS in ECs and inhibition of sGC in fibroblasts increase cell proliferation, as shown by MTT assay. n = 5. B, Relative expression levels of collagen 3 in fibroblasts treated with HUVEC‐conditioned medium in the indicated groups. n = 5. C, D, Condition medium of lentiviral S1PR1‐overexpressing ECs reduced MCF cell migration, while blockade of AKT or eNOS in ECs and inhibition of sGC in fibroblasts enhance cell migration, as shown by transwell assay. HPF, high‐power field. n = 3. E, Relative expression levels of α‐SMA in fibroblasts treated with HUVEC‐conditioned medium in the indicated groups. n = 5. F, Western blotting of AKT activation status in HUVECs treated with or without S1P in the indicated groups with quantification. n = 5. G, Western blotting of eNOS activation status in HUVECs treated with or without S1P in the indicated groups with quantification. n = 5. H, Nitric oxide (NO) production in HUVEC‐conditioned medium in the indicated groups. n = 5. I, cGMP levels in fibroblasts of the indicated groups. n = 5. NC, scramble shRNA lentivirus. S1PR1, S1PR1‐overexpressing lentivirus. shRNA, S1PR1 shRNA lentivirus. L‐NAME, eNOS antagonist. LY294002 (LY), AKT inhibitor. NS‐2028, sGC inhibitor. Scale bar, 100 μm. Data are mean ± SEM. * P
    Figure Legend Snippet: Endothelial S1pr1 inhibits fibroblast proliferation, migration and its conversion towards myofibroblasts via AKT/eNOS pathway. A, Condition medium of lentiviral S1PR1‐overexpressing ECs decreases fibroblast cell proliferation, while blockade of AKT or eNOS in ECs and inhibition of sGC in fibroblasts increase cell proliferation, as shown by MTT assay. n = 5. B, Relative expression levels of collagen 3 in fibroblasts treated with HUVEC‐conditioned medium in the indicated groups. n = 5. C, D, Condition medium of lentiviral S1PR1‐overexpressing ECs reduced MCF cell migration, while blockade of AKT or eNOS in ECs and inhibition of sGC in fibroblasts enhance cell migration, as shown by transwell assay. HPF, high‐power field. n = 3. E, Relative expression levels of α‐SMA in fibroblasts treated with HUVEC‐conditioned medium in the indicated groups. n = 5. F, Western blotting of AKT activation status in HUVECs treated with or without S1P in the indicated groups with quantification. n = 5. G, Western blotting of eNOS activation status in HUVECs treated with or without S1P in the indicated groups with quantification. n = 5. H, Nitric oxide (NO) production in HUVEC‐conditioned medium in the indicated groups. n = 5. I, cGMP levels in fibroblasts of the indicated groups. n = 5. NC, scramble shRNA lentivirus. S1PR1, S1PR1‐overexpressing lentivirus. shRNA, S1PR1 shRNA lentivirus. L‐NAME, eNOS antagonist. LY294002 (LY), AKT inhibitor. NS‐2028, sGC inhibitor. Scale bar, 100 μm. Data are mean ± SEM. * P

    Techniques Used: Migration, Inhibition, MTT Assay, Expressing, Transwell Assay, Western Blot, Activation Assay, shRNA

    Endothelial S1pr1 reduced cardiomyocyte hypertrophy via AKT/eNOS pathway. A, Condition medium of lentiviral S1PR1‐overexpressing ECs decreases CM area, while S1PR1 shRNA knockdown increases CM area, as shown by immunostaining of filament actin in rat neonatal cardiomyocytes (left), with quantification of cardiomyocyte size (right). Ang II, angiotensin II. n = 5. B‐D, Condition medium of lentiviral S1PR1‐overexpressing ECs decreases MYH7 (B), BNP (C) and ANP (D) expression, while S1PR1 shRNA knockdown increases MYH7 (B), BNP (C) and ANP (D) expression, as shown by RT‐PCR. n = 5. NC, scramble shRNA lentivirus. S1PR1, S1PR1‐overexpressing lentivirus. shRNA, S1PR1 shRNA lentivirus. L‐NAME, eNOS antagonist. LY294002 (LY), AKT inhibitor. Data are mean ± SEM. Scale Bars: A, 50 µm
    Figure Legend Snippet: Endothelial S1pr1 reduced cardiomyocyte hypertrophy via AKT/eNOS pathway. A, Condition medium of lentiviral S1PR1‐overexpressing ECs decreases CM area, while S1PR1 shRNA knockdown increases CM area, as shown by immunostaining of filament actin in rat neonatal cardiomyocytes (left), with quantification of cardiomyocyte size (right). Ang II, angiotensin II. n = 5. B‐D, Condition medium of lentiviral S1PR1‐overexpressing ECs decreases MYH7 (B), BNP (C) and ANP (D) expression, while S1PR1 shRNA knockdown increases MYH7 (B), BNP (C) and ANP (D) expression, as shown by RT‐PCR. n = 5. NC, scramble shRNA lentivirus. S1PR1, S1PR1‐overexpressing lentivirus. shRNA, S1PR1 shRNA lentivirus. L‐NAME, eNOS antagonist. LY294002 (LY), AKT inhibitor. Data are mean ± SEM. Scale Bars: A, 50 µm

    Techniques Used: shRNA, Immunostaining, Aqueous Normal-phase Chromatography, Expressing, Reverse Transcription Polymerase Chain Reaction

    39) Product Images from "FoxM1 drives ADAM17/EGFR activation loop to promote mesenchymal transition in glioblastoma"

    Article Title: FoxM1 drives ADAM17/EGFR activation loop to promote mesenchymal transition in glioblastoma

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0482-4

    FoxM1/ADAM17 axis drives EGFR/AKT/GSK3β signaling and maintains FoxM1 stability in glioma cells. a Heatmap revealed that the expression of FoxM1 and ADAM17 were correlated with that of EGFR in glioblastoma samples from TCGA database. b , c The effects of FoxM1 on EGFR/AKT/GSK3β signaling pathway in glioma cells were investigated by western blot. d The key components of EGFR/AKT/GSK3β signaling were detected after knocking down FoxM1 and overexpressing ADAM17 by western blot in U251MG and U87MG cells. e The effects of FoxM1 upregulation and ADAM17 knockdown on EGFR/AKT/GSK3β signaling pathway were confirmed by western blot in SW1783 and LN229 cells. f U87MG cells were treated with TAPI-2(ADAM17 inhibitor), and then indicated proteins were measured by western blot. g After transfected with sh-ADAM17, U87MG cells were treated with CHX (0, 2, 4, 6 h) and then western blot was used to determine the expression of FoxM1. h FoxM1 levels were measured in MEF GSK3β+/+ and GSK3β−/− cells with CHX (0, 2, 4, 6 h) . i The effects of ADAM17 downregulation on FoxM1 expression in MEF GSK3β+/+ and GSK3β−/− cells. j The levels of indicated proteins were detected by western blot in U87MG-sh-ADAM17 cells treated with CHX and LiCl (0, 2, 4, 6 h)
    Figure Legend Snippet: FoxM1/ADAM17 axis drives EGFR/AKT/GSK3β signaling and maintains FoxM1 stability in glioma cells. a Heatmap revealed that the expression of FoxM1 and ADAM17 were correlated with that of EGFR in glioblastoma samples from TCGA database. b , c The effects of FoxM1 on EGFR/AKT/GSK3β signaling pathway in glioma cells were investigated by western blot. d The key components of EGFR/AKT/GSK3β signaling were detected after knocking down FoxM1 and overexpressing ADAM17 by western blot in U251MG and U87MG cells. e The effects of FoxM1 upregulation and ADAM17 knockdown on EGFR/AKT/GSK3β signaling pathway were confirmed by western blot in SW1783 and LN229 cells. f U87MG cells were treated with TAPI-2(ADAM17 inhibitor), and then indicated proteins were measured by western blot. g After transfected with sh-ADAM17, U87MG cells were treated with CHX (0, 2, 4, 6 h) and then western blot was used to determine the expression of FoxM1. h FoxM1 levels were measured in MEF GSK3β+/+ and GSK3β−/− cells with CHX (0, 2, 4, 6 h) . i The effects of ADAM17 downregulation on FoxM1 expression in MEF GSK3β+/+ and GSK3β−/− cells. j The levels of indicated proteins were detected by western blot in U87MG-sh-ADAM17 cells treated with CHX and LiCl (0, 2, 4, 6 h)

    Techniques Used: Expressing, Western Blot, Transfection

    40) Product Images from "Menin Upregulates FOXO1 Protein Stability by Repressing Skp2-Mediated Degradation in β Cells"

    Article Title: Menin Upregulates FOXO1 Protein Stability by Repressing Skp2-Mediated Degradation in β Cells

    Journal: Pancreas

    doi: 10.1097/MPA.0000000000001239

    Menin represses FOXO1 ubiquitination and AKT phosphorylation. A, C, INS-1 cells expressing vector or ectopic menin were lysed for Western blot with indicated antibodies. B and D, Quantitation of p-AKT or p-FOXO1 protein level from panel A or C. Band density analysis was executed with ImageJ, and p-AKT or p-FOXO1 protein levels were quantified and normalized to total AKT or total FOXO1. E and G, INS-1 cells expressing vector or sShMen1 were lysed for Western blot with indicated antibodies. F and H, Quantitation of p-AKT or p-FOXO1 protein level from panel E or G. Band density analysis was executed with ImageJ, and p-AKT or p-FOXO1 protein levels were quantified and normalized to total AKT or total FOXO1. I, Equal amounts of menin and/or HA-ubiquitin constructs were transfected into 293T cells in the presence or absence of EGF (100 ng/mL) for 24 hours, followed by MG132 (20 μmol) treatment for 4 hours and lysed for IP and Western blot with the indicated antibodies. * P
    Figure Legend Snippet: Menin represses FOXO1 ubiquitination and AKT phosphorylation. A, C, INS-1 cells expressing vector or ectopic menin were lysed for Western blot with indicated antibodies. B and D, Quantitation of p-AKT or p-FOXO1 protein level from panel A or C. Band density analysis was executed with ImageJ, and p-AKT or p-FOXO1 protein levels were quantified and normalized to total AKT or total FOXO1. E and G, INS-1 cells expressing vector or sShMen1 were lysed for Western blot with indicated antibodies. F and H, Quantitation of p-AKT or p-FOXO1 protein level from panel E or G. Band density analysis was executed with ImageJ, and p-AKT or p-FOXO1 protein levels were quantified and normalized to total AKT or total FOXO1. I, Equal amounts of menin and/or HA-ubiquitin constructs were transfected into 293T cells in the presence or absence of EGF (100 ng/mL) for 24 hours, followed by MG132 (20 μmol) treatment for 4 hours and lysed for IP and Western blot with the indicated antibodies. * P

    Techniques Used: Expressing, Plasmid Preparation, Western Blot, Quantitation Assay, Construct, Transfection

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    Article Snippet: .. The antibodies used for Western blot analysis included (a) anti-SMYD2 (sc-79084), anti-STAT3 (sc-482), anti-p65 (sc372 and sc-8008), anti-p53 (sc-6423), and anti-PTPN13 (sc-15356), which were purchased from Santa Cruz Biotechnology Inc.; (b) anti-SMYD2 (no. 9734), anti-ERK (no. 4696), anti-S6 (no. 2217), anti-Rb (no. 9309), anti-GFP (no. 2956), anti-AKT (no. 9272), and anti–lamin A/C (no. 2032), purchased from Cell Signaling Technology; and (c) the phosphorylated antibodies for STAT3-Y705 (no. 9131), p65-S536 (no. 3031), ERK-T202/Y204 (no. 9101), S6-S235/236 (no. 2211), AKT-S473 (no. 9271), and Rb-S780 (no. 9307), also purchased from Cell Signaling Technology. .. All of the anti–methylated histone antibodies, including H3K4-mono (ab8895), H3K4-di (ab7766), H3K4-tri (ab8580), H3K36-mono (ab9048), H3K36-di (ab9049), and H3K36-tri (ab9050), anti-Ki67 antibody (ab15580), anti-T7 antibody (ab9138), and anti–pan–methyl lysine antibody (ab23366) were purchased from Abcam.

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    Cell Signaling Technology Inc anti phosphot akt ser473
    Immunohistochemistry of Qdot-GRP78 antibody in MDA-MB-231/GFP breast cells and LNCaP prostate cancer cells. ( A ) and ( B ) Fluorescence images of cells staining with Qdot625-GRP78 antibody. ( A ) control cells staining with unlabeled nanobeads; ( B ) MDA-MB-231/GFP cells staining with Qdot-GRP78 antibody; ( C ) Scanned confocal microscopy imaging in LNCaP prostate cancer cell, stained with anti-GRP78 mouse monoclonal antibody with secondary Goat anti-mouse IgG labeled with Alexa488(green)and Qdot-GRP78(red dots) and overlapped with two probes (yellow); ( D ) and ( E ) Internalization of Qdot-GRP78 antibody by MDA-MB-231/GFP cells. Cells were incubated with unlabelled nanobeads. ( D ) or Qdot-GRP78 antibody; ( E ) at 37 °C for 16 h. Cells were then washed with PBS and analyzed by fluorescence microscopy; ( F ) Scanned confocal microscopy imaging in MDA-MB-231/GFP cell (green), treated with control unlabelled beads; ( G ) Qdot-GRP78(red dots) was detected inside MDA-MB-231/GFP cell; ( H ) 3D reconstruction of confocal Z stack with 0.8-μM, GFP cell showing in green channel, while Qdot-GRP78 showing in red channel. Scale bar represents 20 μm; ( I ) Western blot analysis of GRP78 protein in Qdot-GRP78 antibody treated cells. Cells either treated with control (unlabelled Qdot) or Qdot-labeled antibody. Phosphorylated <t>Akt-ser473</t> protein was detected by an anti-anti-Akt-se473 antibody and Pan-Akt antibody was used as a loading control. The western blot represents three independent experiments. ( J ) MDA-MB-231/GFP cells were incubated with various concentrations of Qot-GRP78 antibody for 24h. Apoptotic nuclei or nuclear DNA strand breaks were visualized using Hoechst DNA dye H33342 (10). A minimum of 200 cells were counted in each sample and condensed or fragmented nuclei were expressed as a percentage of the total number of nuclei. Values are presented as mean ± S.D. of three determinations. * indicates significant difference ( p
    Anti Phosphot Akt Ser473, supplied by Cell Signaling Technology Inc, 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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    Cell Signaling Technology Inc rabbit anti akt
    Loss of FlnB induces Cdk1 activity changes through β1 <t>integrin-Pi3k/Akt</t> pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p
    Rabbit Anti Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 587 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti phosphorylated ser505 akt
    Dgk regulates dILP secretion and insulin signaling pathway activity. ( A ) Knockdown of Dgk using dIlp2-Gal4 shows elevated TAG, glucose, and glycogen phenotypes. ( B ) dIlp2-Gal4 -driven overexpression of kinase-dead Dgk but not wild-type Dgk also decreases glucose and glycogen levels. These phenotypes in ( A–B ) are similar to those seen using fru-Gal4 . ( C ) fru > Dgk RNAi increases dILP2 and dILP5 levels in the hemolymph. ( D ) Overexpression of either Dgk.V5 or Dgk G509D .V5 using fru-Gal4 does not affect hemolymph dILP2 or dILP5 levels. ( A–D ) Data is represented as percent or fold-change relative to a Gal4/+ control ± SD, n = 5. Assays were performed three times but results from only one representative assay are shown. ( E–F ) Knockdown or overexpression of Dgk with fru-Gal4 doesn't affect dIlp2 or dIlp5 transcript levels. Overexpression of Dgk G509D .V5 increases dIlp3 levels. ( G ) fru-Gal4 -driven Dgk RNAi or overexpression of Dgk G509D .V5 result in lowered insulin pathway activity as measured by <t>p-Ser505</t> <t>Akt/total</t> Akt levels. fru > Dgk . V5 did not affect pAkt/Akt levels. Quantification is from 3 biological replicates and is normalized to a fru-Gal4/+ control ±SD. Asterisks denote p-values based on student's t-test: *p
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    Immunohistochemistry of Qdot-GRP78 antibody in MDA-MB-231/GFP breast cells and LNCaP prostate cancer cells. ( A ) and ( B ) Fluorescence images of cells staining with Qdot625-GRP78 antibody. ( A ) control cells staining with unlabeled nanobeads; ( B ) MDA-MB-231/GFP cells staining with Qdot-GRP78 antibody; ( C ) Scanned confocal microscopy imaging in LNCaP prostate cancer cell, stained with anti-GRP78 mouse monoclonal antibody with secondary Goat anti-mouse IgG labeled with Alexa488(green)and Qdot-GRP78(red dots) and overlapped with two probes (yellow); ( D ) and ( E ) Internalization of Qdot-GRP78 antibody by MDA-MB-231/GFP cells. Cells were incubated with unlabelled nanobeads. ( D ) or Qdot-GRP78 antibody; ( E ) at 37 °C for 16 h. Cells were then washed with PBS and analyzed by fluorescence microscopy; ( F ) Scanned confocal microscopy imaging in MDA-MB-231/GFP cell (green), treated with control unlabelled beads; ( G ) Qdot-GRP78(red dots) was detected inside MDA-MB-231/GFP cell; ( H ) 3D reconstruction of confocal Z stack with 0.8-μM, GFP cell showing in green channel, while Qdot-GRP78 showing in red channel. Scale bar represents 20 μm; ( I ) Western blot analysis of GRP78 protein in Qdot-GRP78 antibody treated cells. Cells either treated with control (unlabelled Qdot) or Qdot-labeled antibody. Phosphorylated Akt-ser473 protein was detected by an anti-anti-Akt-se473 antibody and Pan-Akt antibody was used as a loading control. The western blot represents three independent experiments. ( J ) MDA-MB-231/GFP cells were incubated with various concentrations of Qot-GRP78 antibody for 24h. Apoptotic nuclei or nuclear DNA strand breaks were visualized using Hoechst DNA dye H33342 (10). A minimum of 200 cells were counted in each sample and condensed or fragmented nuclei were expressed as a percentage of the total number of nuclei. Values are presented as mean ± S.D. of three determinations. * indicates significant difference ( p

    Journal: Molecules

    Article Title: Quantum Dot- Conjugated Anti-GRP78 scFv Inhibits Cancer Growth in Mice

    doi: 10.3390/molecules17010796

    Figure Lengend Snippet: Immunohistochemistry of Qdot-GRP78 antibody in MDA-MB-231/GFP breast cells and LNCaP prostate cancer cells. ( A ) and ( B ) Fluorescence images of cells staining with Qdot625-GRP78 antibody. ( A ) control cells staining with unlabeled nanobeads; ( B ) MDA-MB-231/GFP cells staining with Qdot-GRP78 antibody; ( C ) Scanned confocal microscopy imaging in LNCaP prostate cancer cell, stained with anti-GRP78 mouse monoclonal antibody with secondary Goat anti-mouse IgG labeled with Alexa488(green)and Qdot-GRP78(red dots) and overlapped with two probes (yellow); ( D ) and ( E ) Internalization of Qdot-GRP78 antibody by MDA-MB-231/GFP cells. Cells were incubated with unlabelled nanobeads. ( D ) or Qdot-GRP78 antibody; ( E ) at 37 °C for 16 h. Cells were then washed with PBS and analyzed by fluorescence microscopy; ( F ) Scanned confocal microscopy imaging in MDA-MB-231/GFP cell (green), treated with control unlabelled beads; ( G ) Qdot-GRP78(red dots) was detected inside MDA-MB-231/GFP cell; ( H ) 3D reconstruction of confocal Z stack with 0.8-μM, GFP cell showing in green channel, while Qdot-GRP78 showing in red channel. Scale bar represents 20 μm; ( I ) Western blot analysis of GRP78 protein in Qdot-GRP78 antibody treated cells. Cells either treated with control (unlabelled Qdot) or Qdot-labeled antibody. Phosphorylated Akt-ser473 protein was detected by an anti-anti-Akt-se473 antibody and Pan-Akt antibody was used as a loading control. The western blot represents three independent experiments. ( J ) MDA-MB-231/GFP cells were incubated with various concentrations of Qot-GRP78 antibody for 24h. Apoptotic nuclei or nuclear DNA strand breaks were visualized using Hoechst DNA dye H33342 (10). A minimum of 200 cells were counted in each sample and condensed or fragmented nuclei were expressed as a percentage of the total number of nuclei. Values are presented as mean ± S.D. of three determinations. * indicates significant difference ( p

    Article Snippet: Anti-phosphot-Akt (ser473) and anti-Akt (pan) were from the Cell Signaling Technology, Inc (Danvers, USA).

    Techniques: Immunohistochemistry, Multiple Displacement Amplification, Fluorescence, Staining, Confocal Microscopy, Imaging, Labeling, Incubation, Microscopy, Western Blot

    Loss of FlnB induces Cdk1 activity changes through β1 integrin-Pi3k/Akt pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p

    Journal: PLoS ONE

    Article Title: Filamin B Regulates Chondrocyte Proliferation and Differentiation through Cdk1 Signaling

    doi: 10.1371/journal.pone.0089352

    Figure Lengend Snippet: Loss of FlnB induces Cdk1 activity changes through β1 integrin-Pi3k/Akt pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p

    Article Snippet: The primary antibodies (for immunostaining and some also for western blotting) were: rabbit anti-FlnA monoclonal antibody (1∶300, Cat.# 2242, Epitomics, Burlingame, CA, USA); rabbit anti-FlnB polyclonal antibody (Gifted by Dr. Kao, CWRU); mouse anti-Col2a1 (Cat.# Ab3092, ABCAM, USA); rabbit anti-Col10a1 (kindly gifted by Dr. Horton and Dr. Lunstrum, Shriners Hospital for Children, Portland, OR, USA; ); rabbit anti-Pthr1 (Cat.# Ab75150, ABCAM, USA);rabbit anti-Ihh (Cat.# sc-13088, Santa Cruz); rabbit anti-Runx2 (Cat.# sc-10758, Santa Cruz); rabbit anti-Sox9 pab (1∶300, AB5535, Millipore); rabbit anti-Sox9 pab (O9-1, gift of Professor Dr. Michael Wegner, Institute of Biochemistry, Friedrich-Alexander-University, Erlangen-Nurnberg, Germany); rat anti-BrdU (1∶150, Cat.# MCA2060, AbD Serotec, Raleigh, NC, USA); rabbit anti-Ki-67 mab (1∶200, Cat.# 4203, Epitomics); rabbit anti-PH3 pab (1∶250, Cat:# 06-570, Millipore, Billerica, MA, USA); mouse and rabbit anti-Wee1 (1∶50, Cat.# sc-5285 and sc-325, Santa Cruz); rabbit anti-Pkmyt1 (1∶100, Cat.# 3303, Epitomics); anti-pan 14-3-3 (Santa Cruz, sc-629); mouse and rabbit anti-cyclin B1 (1∶100, Cat.# sc-245 and sc-752, Santa Cruz); mouse anti-Cdc20 (1∶100, Cat.# sc-13162, Santa Cruz); mouse and rabbit anti-cdc25c (1∶100, Cat.# sc-55513 and sc-327, Santa Cruz); rabbit-anti-Cdk1 (Cat.# PC25,Calbiochem, San Diego, CA, USA); mouse anti-Cdk1(pY15) (Cat.# BD612306, BD, Franklin Lakes, NJ USA); rabbit anti-Pi3k (p85 subunit alpha, Cat#: 1675, Epitomics); rabbit anti-Akt (phospho-S473, Cat# 4060, Cell Signaling); rabbit anti-Erk1/2 (phospho-T202/Y204, Cat.# 4370, Cell Signaling); rat anti-β1 integrin (Cat.# mab1997, Millipore); rabbit anti- β1 integrin(pS785) (Cat.# OPA1-03177, Affinity BioReagents).

    Techniques: Activity Assay, Immunostaining, Knock-Out, Western Blot, Activation Assay, Incubation, Laser Capture Microdissection

    Reduction in phosphorylation/activation of Akt by GT1b . (A) Phosphorylation of Akt was detected by immunoblotting with phospho-Akt (Ser473) and phospho-Akt (Thr308) antibody in cultures treated with 20 μg/ml GT1b at indicated time points. (B) The histogram shows quantification of phospho-Akt levels. (C-F) The activity of Akt was measured using immunoprecipitated Akt, and then it was mixed with the GSK-3α/β fusion protein (1 μg/assay). Phosphorylated GSK-3α/β was detected by immunoblotting with phospho-GSK-3α/β antibody. (D,F) The histograms show quantification of phospho-GSK-3α/β levels of C and E, respectively. The values represent the mean ± SEM of four to five separate experiments; *P

    Journal: BMC Neuroscience

    Article Title: GT1b-induced neurotoxicity is mediated by the Akt/GSK-3/tau signaling pathway but not caspase-3 in mesencephalic dopaminergic neurons

    doi: 10.1186/1471-2202-11-74

    Figure Lengend Snippet: Reduction in phosphorylation/activation of Akt by GT1b . (A) Phosphorylation of Akt was detected by immunoblotting with phospho-Akt (Ser473) and phospho-Akt (Thr308) antibody in cultures treated with 20 μg/ml GT1b at indicated time points. (B) The histogram shows quantification of phospho-Akt levels. (C-F) The activity of Akt was measured using immunoprecipitated Akt, and then it was mixed with the GSK-3α/β fusion protein (1 μg/assay). Phosphorylated GSK-3α/β was detected by immunoblotting with phospho-GSK-3α/β antibody. (D,F) The histograms show quantification of phospho-GSK-3α/β levels of C and E, respectively. The values represent the mean ± SEM of four to five separate experiments; *P

    Article Snippet: Equivalent amounts of Akt were immunoprecipitated by rabbit-Akt antibody prebound to protein A-agarose beads, and kinase assays were carried out according to the manufacturer's instruction manual of the Akt kinase Assay Kit (Cell Signaling Technology).

    Techniques: Activation Assay, Activity Assay, Immunoprecipitation

    Dgk regulates dILP secretion and insulin signaling pathway activity. ( A ) Knockdown of Dgk using dIlp2-Gal4 shows elevated TAG, glucose, and glycogen phenotypes. ( B ) dIlp2-Gal4 -driven overexpression of kinase-dead Dgk but not wild-type Dgk also decreases glucose and glycogen levels. These phenotypes in ( A–B ) are similar to those seen using fru-Gal4 . ( C ) fru > Dgk RNAi increases dILP2 and dILP5 levels in the hemolymph. ( D ) Overexpression of either Dgk.V5 or Dgk G509D .V5 using fru-Gal4 does not affect hemolymph dILP2 or dILP5 levels. ( A–D ) Data is represented as percent or fold-change relative to a Gal4/+ control ± SD, n = 5. Assays were performed three times but results from only one representative assay are shown. ( E–F ) Knockdown or overexpression of Dgk with fru-Gal4 doesn't affect dIlp2 or dIlp5 transcript levels. Overexpression of Dgk G509D .V5 increases dIlp3 levels. ( G ) fru-Gal4 -driven Dgk RNAi or overexpression of Dgk G509D .V5 result in lowered insulin pathway activity as measured by p-Ser505 Akt/total Akt levels. fru > Dgk . V5 did not affect pAkt/Akt levels. Quantification is from 3 biological replicates and is normalized to a fru-Gal4/+ control ±SD. Asterisks denote p-values based on student's t-test: *p

    Journal: Molecular Metabolism

    Article Title: An in vivo screen for neuronal genes involved in obesity identifies Diacylglycerol kinase as a regulator of insulin secretion

    doi: 10.1016/j.molmet.2018.10.006

    Figure Lengend Snippet: Dgk regulates dILP secretion and insulin signaling pathway activity. ( A ) Knockdown of Dgk using dIlp2-Gal4 shows elevated TAG, glucose, and glycogen phenotypes. ( B ) dIlp2-Gal4 -driven overexpression of kinase-dead Dgk but not wild-type Dgk also decreases glucose and glycogen levels. These phenotypes in ( A–B ) are similar to those seen using fru-Gal4 . ( C ) fru > Dgk RNAi increases dILP2 and dILP5 levels in the hemolymph. ( D ) Overexpression of either Dgk.V5 or Dgk G509D .V5 using fru-Gal4 does not affect hemolymph dILP2 or dILP5 levels. ( A–D ) Data is represented as percent or fold-change relative to a Gal4/+ control ± SD, n = 5. Assays were performed three times but results from only one representative assay are shown. ( E–F ) Knockdown or overexpression of Dgk with fru-Gal4 doesn't affect dIlp2 or dIlp5 transcript levels. Overexpression of Dgk G509D .V5 increases dIlp3 levels. ( G ) fru-Gal4 -driven Dgk RNAi or overexpression of Dgk G509D .V5 result in lowered insulin pathway activity as measured by p-Ser505 Akt/total Akt levels. fru > Dgk . V5 did not affect pAkt/Akt levels. Quantification is from 3 biological replicates and is normalized to a fru-Gal4/+ control ±SD. Asterisks denote p-values based on student's t-test: *p

    Article Snippet: Primary antibodies used: mouse anti-V5 (1:2000, Invitrogen), rabbit anti-Akt (1:500, Cell Signalling Technology), rabbit anti-phosphorylated Ser505 Akt (1:1000, Cell Signalling Technology) and mouse anti-actin (1:4000, Abcam).

    Techniques: Activity Assay, Over Expression