phospho-akt ser473 Search Results


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    Cell Signaling Technology Inc phospho akt
    PKN1 rs34309238 variant influences pancreatic cancer risk by altering the level of phosphorylated PKN1 and thus affecting the <t>FAK/PI3K/AKT</t> signalling pathway. a Protein modification sites of PKN1. Annotations were obtained from the PhosphoSitePlus database. b Result of the iTRAQ-based comparative proteomics screen. PANC-1 cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector. The raw intensity values of cells transfection with PKN1[A] or PKN1[C] were divided by the intensity values of cells transfection with control vector to obtain the relative intensity values. The y axis shows the relative intensity values of cells' transfection with PKN1[A] minus the relative intensity values of cells' transfection with PKN1[C]. The x axis shows the molecular weight of detected peptides. The proteomics screen experiment was repeated independently for two times with similar results. c Levels of phosphorylated FAK and AKT were affected by the PKN1 rs34309238 variant. Cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector (left) and PKN1-targeting siRNAs or control siRNA (right). d Levels of phosphorylated FAK and AKT were reduced by the PKN1 inhibitors. Cells were seeded in six-well plates after transfection with PKN1 inhibitors Lestaurtinib and Ro318220 or DMSO as control. For c , d , the western blot experiment was repeated independently for three times with similar results
    Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 16418 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho akt
    GSK-3 phosphorylates RacE at Ser192 in response to the chemoattractant. a , WT and RacE-KO cells were stimulated with the chemoattractant cAMP (1 μM). Total amounts of RacE and its phosphorylation at Ser192 were analyzed by <t>immunoblotting</t> with antibodies to RacE and phospho-RacE(Ser192). b , WT cells expressing GFP fused to WT RacE, phospho-defective RacE S192A or phospho-mimetic RacE S192D were stimulated with cAMP. Whole cell lysates prepared at the indicated time points were analyzed by immunoblotting with antibodies to RacE and phospho-RacE(S192). c , The amino acid sequence in the vicinity of the phosphorylation site (Ser192, red) of RacE. A consensus motif for GSK-3 phosphorylation — a cluster of (S/TXXXS/T) — is underlined. A phosphopeptide used to raise anti-phospho-RacE (S192P) is highlighted. d , The location of serine 192 in a modeled RacE 3-D structure. (e–h) WT cells were treated with inhibitors to GSK-3 (250 nM LY2090314 in e and 10 mM lithium in f ), PI3K (250 μM LY294002 in g ), mTORC2 (0.5 μM PP242 in g ), or <t>AKT</t> (5 μM afuresertib in h ) for 10 min. Cells were then stimulated by the chemoattractant cAMP for the indicated amounts of time. Whole cell lysates were analyzed by immunoblotting with antibodies to RacE and phospho-RacE(Ser192). i , WT and cells lacking AKT (PkbA-KO or PkbR1-KO) were stimulated with cAMP for 30 s. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. j , Purified human GSK-3β was incubated with purified WT, GDP-bound RacE T25N or phospho-defective RacE S192A for 15 min. Ser192 phosphorylation of RacE was tested by immunoblotting. k , WT or GDP-bound RacE T25N was mixed with GSK-3β in the presence or absence of the GSK-3 inhibitor LY2090314 (10 nM) and examined for Ser192 phosphorylation using immunoblotting. l , Summary of the data. Experiments were repeated independently three times with similar results in a, b and e - k .
    Anti Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 15236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho akt s473
    Mechanical unloading of the failing myocardium increased cardiac <t>PI3K/Akt/Foxo</t> signaling. ( A ) Increased myocardial activation of Akt and Foxo following LVAD placement. ( B ) Quantitative analysis of Akt and Foxo activation. (empty bars – pre-LVAD;
    Phospho Akt S473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2493 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho akt s473
    Role of NF-κB, <t>PI3K</t> and <t>AKT</t> pathways in ASC-mediated latency-reactivation of U1 cells. ( A ) Effect of NF-κB inhibitor, bardoxolone-methyl (BM; a.k.a. CDDO-Me) on HIV-1 p24 production by U1 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). Exposure to BM (50 nM) decreased HIV-1 p24 production in both PMA-induced and ASC-CM-stimulated U1 cells. ( B,C ) effect of PI3K inhibitors, ( B ) LY294002 (10 μM) or ( C ) PX866 (500 nM) on HIV-1 p24 production in ASC-CM (50%) stimulated U1 cells. PI3K inhibitors suppressed latency-reactivation by ASC-CM. ( D,E ) effects of AKT inhibition by 124005 (5 μM) and AKT activation by SC-79 (2.5 μM) on ASC-CM (50%) induced HIV-1 p24 production. Latency-reactivation by ASC-CM is increased following AKT inhibition and decreased following AKT induction. ( F ) Effect of heat-inactivation (HI) of ASC-CM or BMSC-CM on HIV-1 LTR-directed GFP expression (MFI) by the U-494 cells. Heat labile factors in CMs are responsible for LTR activation. ( G ) Effect of PI3K and NF-κB inhibitors on HIV-1 LTR function in U-494 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). GFP expression (MFI) was monitored after 48 h and fold change (compared to control media) are shown. Both LY294002 (PI3K inhibitor) and BM (NFκB inhibitor) suppressed HIV-1 LTR function, but 124005 (AKT inhibitor) showed an inductive effect. Error bars show ± SEM and significant changes are represented as P-values (*p
    Anti Phospho Akt S473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3862 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti phospho akt ser473
    Role of NF-κB, <t>PI3K</t> and <t>AKT</t> pathways in ASC-mediated latency-reactivation of U1 cells. ( A ) Effect of NF-κB inhibitor, bardoxolone-methyl (BM; a.k.a. CDDO-Me) on HIV-1 p24 production by U1 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). Exposure to BM (50 nM) decreased HIV-1 p24 production in both PMA-induced and ASC-CM-stimulated U1 cells. ( B,C ) effect of PI3K inhibitors, ( B ) LY294002 (10 μM) or ( C ) PX866 (500 nM) on HIV-1 p24 production in ASC-CM (50%) stimulated U1 cells. PI3K inhibitors suppressed latency-reactivation by ASC-CM. ( D,E ) effects of AKT inhibition by 124005 (5 μM) and AKT activation by SC-79 (2.5 μM) on ASC-CM (50%) induced HIV-1 p24 production. Latency-reactivation by ASC-CM is increased following AKT inhibition and decreased following AKT induction. ( F ) Effect of heat-inactivation (HI) of ASC-CM or BMSC-CM on HIV-1 LTR-directed GFP expression (MFI) by the U-494 cells. Heat labile factors in CMs are responsible for LTR activation. ( G ) Effect of PI3K and NF-κB inhibitors on HIV-1 LTR function in U-494 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). GFP expression (MFI) was monitored after 48 h and fold change (compared to control media) are shown. Both LY294002 (PI3K inhibitor) and BM (NFκB inhibitor) suppressed HIV-1 LTR function, but 124005 (AKT inhibitor) showed an inductive effect. Error bars show ± SEM and significant changes are represented as P-values (*p
    Rabbit Anti Phospho Akt Ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 738 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho akt ser 473
    Role of NF-κB, <t>PI3K</t> and <t>AKT</t> pathways in ASC-mediated latency-reactivation of U1 cells. ( A ) Effect of NF-κB inhibitor, bardoxolone-methyl (BM; a.k.a. CDDO-Me) on HIV-1 p24 production by U1 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). Exposure to BM (50 nM) decreased HIV-1 p24 production in both PMA-induced and ASC-CM-stimulated U1 cells. ( B,C ) effect of PI3K inhibitors, ( B ) LY294002 (10 μM) or ( C ) PX866 (500 nM) on HIV-1 p24 production in ASC-CM (50%) stimulated U1 cells. PI3K inhibitors suppressed latency-reactivation by ASC-CM. ( D,E ) effects of AKT inhibition by 124005 (5 μM) and AKT activation by SC-79 (2.5 μM) on ASC-CM (50%) induced HIV-1 p24 production. Latency-reactivation by ASC-CM is increased following AKT inhibition and decreased following AKT induction. ( F ) Effect of heat-inactivation (HI) of ASC-CM or BMSC-CM on HIV-1 LTR-directed GFP expression (MFI) by the U-494 cells. Heat labile factors in CMs are responsible for LTR activation. ( G ) Effect of PI3K and NF-κB inhibitors on HIV-1 LTR function in U-494 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). GFP expression (MFI) was monitored after 48 h and fold change (compared to control media) are shown. Both LY294002 (PI3K inhibitor) and BM (NFκB inhibitor) suppressed HIV-1 LTR function, but 124005 (AKT inhibitor) showed an inductive effect. Error bars show ± SEM and significant changes are represented as P-values (*p
    Phospho Akt Ser 473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 552 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phosphorylated akt p akt
    Role of NF-κB, <t>PI3K</t> and <t>AKT</t> pathways in ASC-mediated latency-reactivation of U1 cells. ( A ) Effect of NF-κB inhibitor, bardoxolone-methyl (BM; a.k.a. CDDO-Me) on HIV-1 p24 production by U1 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). Exposure to BM (50 nM) decreased HIV-1 p24 production in both PMA-induced and ASC-CM-stimulated U1 cells. ( B,C ) effect of PI3K inhibitors, ( B ) LY294002 (10 μM) or ( C ) PX866 (500 nM) on HIV-1 p24 production in ASC-CM (50%) stimulated U1 cells. PI3K inhibitors suppressed latency-reactivation by ASC-CM. ( D,E ) effects of AKT inhibition by 124005 (5 μM) and AKT activation by SC-79 (2.5 μM) on ASC-CM (50%) induced HIV-1 p24 production. Latency-reactivation by ASC-CM is increased following AKT inhibition and decreased following AKT induction. ( F ) Effect of heat-inactivation (HI) of ASC-CM or BMSC-CM on HIV-1 LTR-directed GFP expression (MFI) by the U-494 cells. Heat labile factors in CMs are responsible for LTR activation. ( G ) Effect of PI3K and NF-κB inhibitors on HIV-1 LTR function in U-494 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). GFP expression (MFI) was monitored after 48 h and fold change (compared to control media) are shown. Both LY294002 (PI3K inhibitor) and BM (NFκB inhibitor) suppressed HIV-1 LTR function, but 124005 (AKT inhibitor) showed an inductive effect. Error bars show ± SEM and significant changes are represented as P-values (*p
    Phosphorylated Akt P Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 609 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho akt ser473 d9e rabbit mab
    Role of NF-κB, <t>PI3K</t> and <t>AKT</t> pathways in ASC-mediated latency-reactivation of U1 cells. ( A ) Effect of NF-κB inhibitor, bardoxolone-methyl (BM; a.k.a. CDDO-Me) on HIV-1 p24 production by U1 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). Exposure to BM (50 nM) decreased HIV-1 p24 production in both PMA-induced and ASC-CM-stimulated U1 cells. ( B,C ) effect of PI3K inhibitors, ( B ) LY294002 (10 μM) or ( C ) PX866 (500 nM) on HIV-1 p24 production in ASC-CM (50%) stimulated U1 cells. PI3K inhibitors suppressed latency-reactivation by ASC-CM. ( D,E ) effects of AKT inhibition by 124005 (5 μM) and AKT activation by SC-79 (2.5 μM) on ASC-CM (50%) induced HIV-1 p24 production. Latency-reactivation by ASC-CM is increased following AKT inhibition and decreased following AKT induction. ( F ) Effect of heat-inactivation (HI) of ASC-CM or BMSC-CM on HIV-1 LTR-directed GFP expression (MFI) by the U-494 cells. Heat labile factors in CMs are responsible for LTR activation. ( G ) Effect of PI3K and NF-κB inhibitors on HIV-1 LTR function in U-494 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). GFP expression (MFI) was monitored after 48 h and fold change (compared to control media) are shown. Both LY294002 (PI3K inhibitor) and BM (NFκB inhibitor) suppressed HIV-1 LTR function, but 124005 (AKT inhibitor) showed an inductive effect. Error bars show ± SEM and significant changes are represented as P-values (*p
    Phospho Akt Ser473 D9e Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho akt ser473 d9e rabbit mab/product/Cell Signaling Technology Inc
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    N/A
    Akt also known as PKB Protein kinase B is a family of serine threonine kinases that plays an important role in signal transduction There are three known isoforms of Akt
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    Rabbit polyclonal antibody to AKT1 Phospho Ser473 Isotype Note IgG Host Note Rabbit Conjugation Note Unconjugated Reactivity Note Human Mouse Rat Application Note WB
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    Rabbit polyclonal Phospho Akt antibody
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    Image Search Results


    PKN1 rs34309238 variant influences pancreatic cancer risk by altering the level of phosphorylated PKN1 and thus affecting the FAK/PI3K/AKT signalling pathway. a Protein modification sites of PKN1. Annotations were obtained from the PhosphoSitePlus database. b Result of the iTRAQ-based comparative proteomics screen. PANC-1 cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector. The raw intensity values of cells transfection with PKN1[A] or PKN1[C] were divided by the intensity values of cells transfection with control vector to obtain the relative intensity values. The y axis shows the relative intensity values of cells' transfection with PKN1[A] minus the relative intensity values of cells' transfection with PKN1[C]. The x axis shows the molecular weight of detected peptides. The proteomics screen experiment was repeated independently for two times with similar results. c Levels of phosphorylated FAK and AKT were affected by the PKN1 rs34309238 variant. Cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector (left) and PKN1-targeting siRNAs or control siRNA (right). d Levels of phosphorylated FAK and AKT were reduced by the PKN1 inhibitors. Cells were seeded in six-well plates after transfection with PKN1 inhibitors Lestaurtinib and Ro318220 or DMSO as control. For c , d , the western blot experiment was repeated independently for three times with similar results

    Journal: Nature Communications

    Article Title: Exome-wide analysis identifies three low-frequency missense variants associated with pancreatic cancer risk in Chinese populations

    doi: 10.1038/s41467-018-06136-x

    Figure Lengend Snippet: PKN1 rs34309238 variant influences pancreatic cancer risk by altering the level of phosphorylated PKN1 and thus affecting the FAK/PI3K/AKT signalling pathway. a Protein modification sites of PKN1. Annotations were obtained from the PhosphoSitePlus database. b Result of the iTRAQ-based comparative proteomics screen. PANC-1 cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector. The raw intensity values of cells transfection with PKN1[A] or PKN1[C] were divided by the intensity values of cells transfection with control vector to obtain the relative intensity values. The y axis shows the relative intensity values of cells' transfection with PKN1[A] minus the relative intensity values of cells' transfection with PKN1[C]. The x axis shows the molecular weight of detected peptides. The proteomics screen experiment was repeated independently for two times with similar results. c Levels of phosphorylated FAK and AKT were affected by the PKN1 rs34309238 variant. Cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector (left) and PKN1-targeting siRNAs or control siRNA (right). d Levels of phosphorylated FAK and AKT were reduced by the PKN1 inhibitors. Cells were seeded in six-well plates after transfection with PKN1 inhibitors Lestaurtinib and Ro318220 or DMSO as control. For c , d , the western blot experiment was repeated independently for three times with similar results

    Article Snippet: FAK (#3285), phospho-FAK (Tyr397, #8556), Akt (#4685) and phospho-Akt (Ser473, #4060) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Variant Assay, Modification, Transfection, Plasmid Preparation, Molecular Weight, Western Blot

    GSK-3 phosphorylates RacE at Ser192 in response to the chemoattractant. a , WT and RacE-KO cells were stimulated with the chemoattractant cAMP (1 μM). Total amounts of RacE and its phosphorylation at Ser192 were analyzed by immunoblotting with antibodies to RacE and phospho-RacE(Ser192). b , WT cells expressing GFP fused to WT RacE, phospho-defective RacE S192A or phospho-mimetic RacE S192D were stimulated with cAMP. Whole cell lysates prepared at the indicated time points were analyzed by immunoblotting with antibodies to RacE and phospho-RacE(S192). c , The amino acid sequence in the vicinity of the phosphorylation site (Ser192, red) of RacE. A consensus motif for GSK-3 phosphorylation — a cluster of (S/TXXXS/T) — is underlined. A phosphopeptide used to raise anti-phospho-RacE (S192P) is highlighted. d , The location of serine 192 in a modeled RacE 3-D structure. (e–h) WT cells were treated with inhibitors to GSK-3 (250 nM LY2090314 in e and 10 mM lithium in f ), PI3K (250 μM LY294002 in g ), mTORC2 (0.5 μM PP242 in g ), or AKT (5 μM afuresertib in h ) for 10 min. Cells were then stimulated by the chemoattractant cAMP for the indicated amounts of time. Whole cell lysates were analyzed by immunoblotting with antibodies to RacE and phospho-RacE(Ser192). i , WT and cells lacking AKT (PkbA-KO or PkbR1-KO) were stimulated with cAMP for 30 s. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. j , Purified human GSK-3β was incubated with purified WT, GDP-bound RacE T25N or phospho-defective RacE S192A for 15 min. Ser192 phosphorylation of RacE was tested by immunoblotting. k , WT or GDP-bound RacE T25N was mixed with GSK-3β in the presence or absence of the GSK-3 inhibitor LY2090314 (10 nM) and examined for Ser192 phosphorylation using immunoblotting. l , Summary of the data. Experiments were repeated independently three times with similar results in a, b and e - k .

    Journal: Nature cell biology

    Article Title: Phosphorylated Rho-GDP Directly Activates mTORC2 Kinase Toward AKT Through Dimerization with Ras-GTP to Regulate Cell Migration

    doi: 10.1038/s41556-019-0348-8

    Figure Lengend Snippet: GSK-3 phosphorylates RacE at Ser192 in response to the chemoattractant. a , WT and RacE-KO cells were stimulated with the chemoattractant cAMP (1 μM). Total amounts of RacE and its phosphorylation at Ser192 were analyzed by immunoblotting with antibodies to RacE and phospho-RacE(Ser192). b , WT cells expressing GFP fused to WT RacE, phospho-defective RacE S192A or phospho-mimetic RacE S192D were stimulated with cAMP. Whole cell lysates prepared at the indicated time points were analyzed by immunoblotting with antibodies to RacE and phospho-RacE(S192). c , The amino acid sequence in the vicinity of the phosphorylation site (Ser192, red) of RacE. A consensus motif for GSK-3 phosphorylation — a cluster of (S/TXXXS/T) — is underlined. A phosphopeptide used to raise anti-phospho-RacE (S192P) is highlighted. d , The location of serine 192 in a modeled RacE 3-D structure. (e–h) WT cells were treated with inhibitors to GSK-3 (250 nM LY2090314 in e and 10 mM lithium in f ), PI3K (250 μM LY294002 in g ), mTORC2 (0.5 μM PP242 in g ), or AKT (5 μM afuresertib in h ) for 10 min. Cells were then stimulated by the chemoattractant cAMP for the indicated amounts of time. Whole cell lysates were analyzed by immunoblotting with antibodies to RacE and phospho-RacE(Ser192). i , WT and cells lacking AKT (PkbA-KO or PkbR1-KO) were stimulated with cAMP for 30 s. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. j , Purified human GSK-3β was incubated with purified WT, GDP-bound RacE T25N or phospho-defective RacE S192A for 15 min. Ser192 phosphorylation of RacE was tested by immunoblotting. k , WT or GDP-bound RacE T25N was mixed with GSK-3β in the presence or absence of the GSK-3 inhibitor LY2090314 (10 nM) and examined for Ser192 phosphorylation using immunoblotting. l , Summary of the data. Experiments were repeated independently three times with similar results in a, b and e - k .

    Article Snippet: AKT phosphorylation was detected by immunoblotting with anti-phospho-AKT (serine 473) antibodies (Cell Signaling, 9271).

    Techniques: Expressing, Sequencing, Purification, Incubation

    RacE-GDP promotes chemoattractant-induced, mTORC2-mediated AKT phosphorylation in cells. The indicated Dictyostelium cell lines were stimulated with the chemoattractant cAMP (1 μM). a - f , WT cells and RacE-KO cells expressing different GFP-RacE constructs were analyzed. g and h , WT cells and RacE-KO cells expressing GFP-RacE were pretreated with 0.5 μM of the mTORC2 inhibitor PP242 for 10 min and then stimulated with cAMP. i and j , WT and RacE-KO cells expressing FLAG-tagged RasC or GTP-bound RasC Q62L were analyzed. a - j , Total amounts of two AKT homologs (PKBR1 and PKBA) and their phosphorylation (Red: hydrophobic motif, Green: activation loop) were analyzed by immunoblotting. PVDF membranes were stained with CBB as loading controls in a, c, e, g, and i . The band intensity of phosphorylated AKTs was quantified in b, d, f, h, and j : WT cells at 30 s (b, d, f and h) and WT cells expressing RasC at 30 s (j) were set at 100%. Values are average ± SD (n = 3 independent experiments).

    Journal: Nature cell biology

    Article Title: Phosphorylated Rho-GDP Directly Activates mTORC2 Kinase Toward AKT Through Dimerization with Ras-GTP to Regulate Cell Migration

    doi: 10.1038/s41556-019-0348-8

    Figure Lengend Snippet: RacE-GDP promotes chemoattractant-induced, mTORC2-mediated AKT phosphorylation in cells. The indicated Dictyostelium cell lines were stimulated with the chemoattractant cAMP (1 μM). a - f , WT cells and RacE-KO cells expressing different GFP-RacE constructs were analyzed. g and h , WT cells and RacE-KO cells expressing GFP-RacE were pretreated with 0.5 μM of the mTORC2 inhibitor PP242 for 10 min and then stimulated with cAMP. i and j , WT and RacE-KO cells expressing FLAG-tagged RasC or GTP-bound RasC Q62L were analyzed. a - j , Total amounts of two AKT homologs (PKBR1 and PKBA) and their phosphorylation (Red: hydrophobic motif, Green: activation loop) were analyzed by immunoblotting. PVDF membranes were stained with CBB as loading controls in a, c, e, g, and i . The band intensity of phosphorylated AKTs was quantified in b, d, f, h, and j : WT cells at 30 s (b, d, f and h) and WT cells expressing RasC at 30 s (j) were set at 100%. Values are average ± SD (n = 3 independent experiments).

    Article Snippet: AKT phosphorylation was detected by immunoblotting with anti-phospho-AKT (serine 473) antibodies (Cell Signaling, 9271).

    Techniques: Expressing, Construct, Activation Assay, Staining

    RacE-GDP specifically interacts with mTORC2. a , Dictyostelium cell lysates carrying GFP fused to the indicated forms of RacE were incubated with cell lysates carrying FLAG-Tor and subjected to immunoprecipitation with GFP-Trap. Quantification of interaction is shown. The band intensity of FLAG-Tor in immunoprecipitates of cells expressing WT RacE was set 100% (n=6, 6, 6 and 3 independent experiments for RacE, RacE T25N , RacE G20V and RacE T43A , respectively). Values are average ± SD. Significance was calculated using ANOVA with post-hoc Tukey. p values are shown for comparison between RacE and others. b , Dictyostelium cell lysates carrying the indicated GFP-RacE were subject to immunoprecipitation with GFP-Trap to analyze its association with endogenous PiaA. The band intensity of PiaA in immunoprecipitates of cells expressing WT RacE was set 100% (n = 3 independent experiments). Values are average ± SD. Significance was calculated using ANOVA with post-hoc Tukey. p values are shown for comparison betwen RacE with others. c , Dictyostelium cell lysates carrying GFP fused to the indicated forms of Rac1A and RacE were incubated with cell lysates carrying FLAG-Tor and subjected to immunoprecipitation with GFP-Trap. Experiment was repeated independently three times with similar results. d , HEK293T cells were transfected with YFP fused to the indicated constructs of human Rac1 and RhoA and subjected to immunoprecipitation using GFP-Trap. The band intensities of Tor and rictor in immunoprecipitates of cells expressing WT RhoA was set 100% (n = 3 and n = 4 independent experiments for Tor and Rictor, respectively). Values are average ± SD. Significance was calculated using ANOVA with post-hoc Tukey. p values are shown for comparison between YFP-RhoA and others. e , Summary of the data. f , AKTs are phosphorylated in the hydrophobic motif by mTORC2 and in the activation loop by PDK. g and h , WT, RacE-KO, and PiaA-KO Dictyostelium cells were stimulated with the chemoattractant cAMP (1 μM). (n = 3 independent experiments). The total amounts of two AKT homologs (PkbR1 and PkbA) and their phosphorylation (Red: hydrophobic motif, Green: activation loop) were analyzed by immunoblotting. PVDF membranes were stained with CBB as loading controls. The band intensity of phosphorylated AKTs was quantified in h : WT cells at 30 s were set at 100%. Values are average ± SD.

    Journal: Nature cell biology

    Article Title: Phosphorylated Rho-GDP Directly Activates mTORC2 Kinase Toward AKT Through Dimerization with Ras-GTP to Regulate Cell Migration

    doi: 10.1038/s41556-019-0348-8

    Figure Lengend Snippet: RacE-GDP specifically interacts with mTORC2. a , Dictyostelium cell lysates carrying GFP fused to the indicated forms of RacE were incubated with cell lysates carrying FLAG-Tor and subjected to immunoprecipitation with GFP-Trap. Quantification of interaction is shown. The band intensity of FLAG-Tor in immunoprecipitates of cells expressing WT RacE was set 100% (n=6, 6, 6 and 3 independent experiments for RacE, RacE T25N , RacE G20V and RacE T43A , respectively). Values are average ± SD. Significance was calculated using ANOVA with post-hoc Tukey. p values are shown for comparison between RacE and others. b , Dictyostelium cell lysates carrying the indicated GFP-RacE were subject to immunoprecipitation with GFP-Trap to analyze its association with endogenous PiaA. The band intensity of PiaA in immunoprecipitates of cells expressing WT RacE was set 100% (n = 3 independent experiments). Values are average ± SD. Significance was calculated using ANOVA with post-hoc Tukey. p values are shown for comparison betwen RacE with others. c , Dictyostelium cell lysates carrying GFP fused to the indicated forms of Rac1A and RacE were incubated with cell lysates carrying FLAG-Tor and subjected to immunoprecipitation with GFP-Trap. Experiment was repeated independently three times with similar results. d , HEK293T cells were transfected with YFP fused to the indicated constructs of human Rac1 and RhoA and subjected to immunoprecipitation using GFP-Trap. The band intensities of Tor and rictor in immunoprecipitates of cells expressing WT RhoA was set 100% (n = 3 and n = 4 independent experiments for Tor and Rictor, respectively). Values are average ± SD. Significance was calculated using ANOVA with post-hoc Tukey. p values are shown for comparison between YFP-RhoA and others. e , Summary of the data. f , AKTs are phosphorylated in the hydrophobic motif by mTORC2 and in the activation loop by PDK. g and h , WT, RacE-KO, and PiaA-KO Dictyostelium cells were stimulated with the chemoattractant cAMP (1 μM). (n = 3 independent experiments). The total amounts of two AKT homologs (PkbR1 and PkbA) and their phosphorylation (Red: hydrophobic motif, Green: activation loop) were analyzed by immunoblotting. PVDF membranes were stained with CBB as loading controls. The band intensity of phosphorylated AKTs was quantified in h : WT cells at 30 s were set at 100%. Values are average ± SD.

    Article Snippet: AKT phosphorylation was detected by immunoblotting with anti-phospho-AKT (serine 473) antibodies (Cell Signaling, 9271).

    Techniques: Incubation, Immunoprecipitation, Expressing, Transfection, Construct, Activation Assay, Staining

    Ser192 phosphorylated RacE-GDP forms a supercomplex with Tor and Ras-GTP. a and b , The indicated GFP-RacE proteins were purified from Dictyostelium cells with or without 1 μM cAMP stimulation for 30 s. FLAG-RasC proteins were purified without cAMP stimulation. GFP-RacE was incubated with FLAG-RasC and pulled down using GFP-Trap. The pellet fraction was analyzed by immunoblotting using antibodies to GFP and FLAG. c , GFP-RacE, GFP-RasC or GFP-RasG was incubated with FLAG-Tor that was purified in a high-salt condition and pulled down with GFP-Trap. The pellet fraction was analyzed by immunoblotting using antibodies to GFP and FLAG. d , GFP fused to GDP-bound RacE T25N or GTP-bound RacE G20V were purified from Dictyostelium cells under a high salt condition after stimulation with the chemoattractant cAMP. These GFP fusion proteins were incubated with high-salt washed FLAG-Tor and/or FLAG-RasC proteins. GFP-RacE was pulled down with GFP-Trap, and the pellet fractions were analyzed by immunoblotting. e , RacE forms a complex with Tor and RasC. The indicated proteins were purified under high-salt conditions and mixed for 15 min at room temperature. GFP-RasC proteins were pulled down with GFP-Trap, and the pellet fraction was analyzed by immunoblotting. f and g , Different GFP-RacE proteins were purified from Dictyostelium cells with or without 1 μM cAMP stimulation for 30 s in the presence or absence of the GSK-3 inhibitor LY2090314 (250 nM). GFP-RacE was incubated with FLAG-RasC in f or FLAG-Tor in g and pulled down using GFP-Trap. The pellet fraction was analyzed by immunoblotting using antibodies to GFP and FLAG. h , Model for GPCR-mediated mTORC2-AKT signaling. In response to GPCR activation by chemoattractant, Rho-GDP becomes phosphorylated by GSK-3 and assembles the super signaling complex with Ras-GTP and mTORC2 to promote AKT phosphorylation. Experiments were repeated independently three times with similar results in a - g .

    Journal: Nature cell biology

    Article Title: Phosphorylated Rho-GDP Directly Activates mTORC2 Kinase Toward AKT Through Dimerization with Ras-GTP to Regulate Cell Migration

    doi: 10.1038/s41556-019-0348-8

    Figure Lengend Snippet: Ser192 phosphorylated RacE-GDP forms a supercomplex with Tor and Ras-GTP. a and b , The indicated GFP-RacE proteins were purified from Dictyostelium cells with or without 1 μM cAMP stimulation for 30 s. FLAG-RasC proteins were purified without cAMP stimulation. GFP-RacE was incubated with FLAG-RasC and pulled down using GFP-Trap. The pellet fraction was analyzed by immunoblotting using antibodies to GFP and FLAG. c , GFP-RacE, GFP-RasC or GFP-RasG was incubated with FLAG-Tor that was purified in a high-salt condition and pulled down with GFP-Trap. The pellet fraction was analyzed by immunoblotting using antibodies to GFP and FLAG. d , GFP fused to GDP-bound RacE T25N or GTP-bound RacE G20V were purified from Dictyostelium cells under a high salt condition after stimulation with the chemoattractant cAMP. These GFP fusion proteins were incubated with high-salt washed FLAG-Tor and/or FLAG-RasC proteins. GFP-RacE was pulled down with GFP-Trap, and the pellet fractions were analyzed by immunoblotting. e , RacE forms a complex with Tor and RasC. The indicated proteins were purified under high-salt conditions and mixed for 15 min at room temperature. GFP-RasC proteins were pulled down with GFP-Trap, and the pellet fraction was analyzed by immunoblotting. f and g , Different GFP-RacE proteins were purified from Dictyostelium cells with or without 1 μM cAMP stimulation for 30 s in the presence or absence of the GSK-3 inhibitor LY2090314 (250 nM). GFP-RacE was incubated with FLAG-RasC in f or FLAG-Tor in g and pulled down using GFP-Trap. The pellet fraction was analyzed by immunoblotting using antibodies to GFP and FLAG. h , Model for GPCR-mediated mTORC2-AKT signaling. In response to GPCR activation by chemoattractant, Rho-GDP becomes phosphorylated by GSK-3 and assembles the super signaling complex with Ras-GTP and mTORC2 to promote AKT phosphorylation. Experiments were repeated independently three times with similar results in a - g .

    Article Snippet: AKT phosphorylation was detected by immunoblotting with anti-phospho-AKT (serine 473) antibodies (Cell Signaling, 9271).

    Techniques: Purification, Incubation, Activation Assay

    Phosphorylated RacE-GDP activates mTORC2 in vitro . mTORC2-mediated AKT phosphorylation was reconstituted using purified proteins. mTORC2 (FLAG-Tor, -PiaA or -Lst8), FLAG-RacE, and FLAG-RasC/G were purified from Dictyostelium cells. +cAMP indicates that cells were stimulated by the chemoattractant for 30 s before purification of FLAG-RacE. Purified proteins were mixed in the presence or absence of ATP and human unactive AKT for 5 min at room temperature. AKT phosphorylation was analyzed by immunoblotting using anti-phospho AKT (serine 473) antibodies. a , mTORC2 phosphorylates AKT in the presence of RacE and RasC. b and c , mTORC2 activation requires PiaA and Lst8, but not the mSIN1 homolog Rip3. FLAG-PiaA or FLAG-Lst8 was added to FLAG-Tor purified from the indicated KO cell lines in b . FLAG-PiaA and/or FLAG-Lst8 were incubated with high-salt washed FLAG-Tor in c . d , mTORC2 activation requires RacE-GDP. Purified RacE was incubated with EDTA (25 mM), GTPγS (0.5 mM) or GTPγS then GDP (2.5 mM) (GTPγS → GDP) before reconstitution. e , WT RacE or GDP-bound RacE T25N , but not GTP-bound RacE G20V , activates mTORC2 after the chemoattractant stimulation. f , mTORC2 activation needs RasC-GTP but not RasG-GTP. g , RacE phosphorylation controls mTORC2 activation. Phospho-mimetic mutation S192D in GDP-bound RacE T25N activates mTORC2 without chemoattractant stimulation, while the phospho-defective S192A mutation blocks it. h , mTORC2 activation requires RasC-GTP. Purified RacC was incubated with EDTA followed by either GTPγS or GDP before reconstitution. i , Summary of the data. j , RacE G23V -GDP activates mTORC2. Purified RacE T25N and RacE G23V were incubated with EDTA followed by GTPγS or GDP prior to reconstitution. Experiments were repeated independently three times with similar results in a - h and j .

    Journal: Nature cell biology

    Article Title: Phosphorylated Rho-GDP Directly Activates mTORC2 Kinase Toward AKT Through Dimerization with Ras-GTP to Regulate Cell Migration

    doi: 10.1038/s41556-019-0348-8

    Figure Lengend Snippet: Phosphorylated RacE-GDP activates mTORC2 in vitro . mTORC2-mediated AKT phosphorylation was reconstituted using purified proteins. mTORC2 (FLAG-Tor, -PiaA or -Lst8), FLAG-RacE, and FLAG-RasC/G were purified from Dictyostelium cells. +cAMP indicates that cells were stimulated by the chemoattractant for 30 s before purification of FLAG-RacE. Purified proteins were mixed in the presence or absence of ATP and human unactive AKT for 5 min at room temperature. AKT phosphorylation was analyzed by immunoblotting using anti-phospho AKT (serine 473) antibodies. a , mTORC2 phosphorylates AKT in the presence of RacE and RasC. b and c , mTORC2 activation requires PiaA and Lst8, but not the mSIN1 homolog Rip3. FLAG-PiaA or FLAG-Lst8 was added to FLAG-Tor purified from the indicated KO cell lines in b . FLAG-PiaA and/or FLAG-Lst8 were incubated with high-salt washed FLAG-Tor in c . d , mTORC2 activation requires RacE-GDP. Purified RacE was incubated with EDTA (25 mM), GTPγS (0.5 mM) or GTPγS then GDP (2.5 mM) (GTPγS → GDP) before reconstitution. e , WT RacE or GDP-bound RacE T25N , but not GTP-bound RacE G20V , activates mTORC2 after the chemoattractant stimulation. f , mTORC2 activation needs RasC-GTP but not RasG-GTP. g , RacE phosphorylation controls mTORC2 activation. Phospho-mimetic mutation S192D in GDP-bound RacE T25N activates mTORC2 without chemoattractant stimulation, while the phospho-defective S192A mutation blocks it. h , mTORC2 activation requires RasC-GTP. Purified RacC was incubated with EDTA followed by either GTPγS or GDP before reconstitution. i , Summary of the data. j , RacE G23V -GDP activates mTORC2. Purified RacE T25N and RacE G23V were incubated with EDTA followed by GTPγS or GDP prior to reconstitution. Experiments were repeated independently three times with similar results in a - h and j .

    Article Snippet: AKT phosphorylation was detected by immunoblotting with anti-phospho-AKT (serine 473) antibodies (Cell Signaling, 9271).

    Techniques: In Vitro, Purification, Activation Assay, Incubation, Mutagenesis

    The effects of estrogen stimulation on the expression of miR-182, miR-223, and miR-142-3p and their targets in infant female quadriceps femoris -derived myoblasts. (A) Quantitative PCR analyses of miRNA transcripts normalized with RNU44 in human myoblasts treated for 72 h with 100 n m estradiol or mock. Note: The expression of miR-182 in these myoblasts was too low to be accurately measured. (B) qPCR analyses of target mRNAs (IGF-1, FOXO3A, FOXO1A) in human myoblasts treated with 10 n m and 100 n m estradiol or mock for 72 h. (C) Representative Western blots of target proteins, IGF-1R, FOXO3A and FOXO1A, and GAPDH, in human myoblasts treated with 10 n m and 100 n m estradiol or mock for 72 h. (D) Densitometry data of Western blots normalized with GAPDH. (E) Representative Western blots showing phosphorylation of AKT and mTOR proteins in myoblasts treated with 10 n m and 100 n m estradiol or mock for 72 h. (F) Densitometry data of Western blots normalized with GAPDH. Data are presented as percentage of control (mock) and reported as means ± SD of three independent experiments. OD indicates optical density. t -test, *** P

    Journal: Aging Cell

    Article Title: Hormone replacement therapy enhances IGF-1 signaling in skeletal muscle by diminishing miR-182 and miR-223 expressions: a study on postmenopausal monozygotic twin pairs

    doi: 10.1111/acel.12245

    Figure Lengend Snippet: The effects of estrogen stimulation on the expression of miR-182, miR-223, and miR-142-3p and their targets in infant female quadriceps femoris -derived myoblasts. (A) Quantitative PCR analyses of miRNA transcripts normalized with RNU44 in human myoblasts treated for 72 h with 100 n m estradiol or mock. Note: The expression of miR-182 in these myoblasts was too low to be accurately measured. (B) qPCR analyses of target mRNAs (IGF-1, FOXO3A, FOXO1A) in human myoblasts treated with 10 n m and 100 n m estradiol or mock for 72 h. (C) Representative Western blots of target proteins, IGF-1R, FOXO3A and FOXO1A, and GAPDH, in human myoblasts treated with 10 n m and 100 n m estradiol or mock for 72 h. (D) Densitometry data of Western blots normalized with GAPDH. (E) Representative Western blots showing phosphorylation of AKT and mTOR proteins in myoblasts treated with 10 n m and 100 n m estradiol or mock for 72 h. (F) Densitometry data of Western blots normalized with GAPDH. Data are presented as percentage of control (mock) and reported as means ± SD of three independent experiments. OD indicates optical density. t -test, *** P

    Article Snippet: Experiments for phosphorylation of AKT and mTOR Myoblasts were treated for 72 h with 10 nm E2 , 100 nm E2 , or solvent alone and analyzed in Western blots to detect phosphorylation of AKT (SER 473, Cell Signalling #9271) and mTOR (SER 2448, Cell Signalling #2971).

    Techniques: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot

    Mechanical unloading of the failing myocardium increased cardiac PI3K/Akt/Foxo signaling. ( A ) Increased myocardial activation of Akt and Foxo following LVAD placement. ( B ) Quantitative analysis of Akt and Foxo activation. (empty bars – pre-LVAD;

    Journal: Circulation

    Article Title: Ventricular Assist Device Implantation Corrects Myocardial Lipotoxicity, Reverses Insulin Resistance and Normalizes Cardiac Metabolism in Patients with Advanced Heart Failure

    doi: 10.1161/CIRCULATIONAHA.111.060889

    Figure Lengend Snippet: Mechanical unloading of the failing myocardium increased cardiac PI3K/Akt/Foxo signaling. ( A ) Increased myocardial activation of Akt and Foxo following LVAD placement. ( B ) Quantitative analysis of Akt and Foxo activation. (empty bars – pre-LVAD;

    Article Snippet: Protein lysates from cells were resolved on 4–15 % SDS PAGE reducing gels (Bio-Rad), transferred to PVDF membranes (Bio-Rad), blocked in 5% milk/TRIS-buffered saline, and probed with antibodies for detection of phosphoAkt, total Akt, phosphoFoxo, total Foxo and GAPDH (all from Cell Signaling Technologies Inc, MA, USA).

    Techniques: Activation Assay

    Impairment of PI3Kinase/Akt signaling in human advanced HF. ( A ) Reduced activation indicated by decreased phosphorylation status of Akt and FOXO in samples from patients with advanced HF compared to control subjects (three representative samples per group).

    Journal: Circulation

    Article Title: Ventricular Assist Device Implantation Corrects Myocardial Lipotoxicity, Reverses Insulin Resistance and Normalizes Cardiac Metabolism in Patients with Advanced Heart Failure

    doi: 10.1161/CIRCULATIONAHA.111.060889

    Figure Lengend Snippet: Impairment of PI3Kinase/Akt signaling in human advanced HF. ( A ) Reduced activation indicated by decreased phosphorylation status of Akt and FOXO in samples from patients with advanced HF compared to control subjects (three representative samples per group).

    Article Snippet: Protein lysates from cells were resolved on 4–15 % SDS PAGE reducing gels (Bio-Rad), transferred to PVDF membranes (Bio-Rad), blocked in 5% milk/TRIS-buffered saline, and probed with antibodies for detection of phosphoAkt, total Akt, phosphoFoxo, total Foxo and GAPDH (all from Cell Signaling Technologies Inc, MA, USA).

    Techniques: Activation Assay

    Role of NF-κB, PI3K and AKT pathways in ASC-mediated latency-reactivation of U1 cells. ( A ) Effect of NF-κB inhibitor, bardoxolone-methyl (BM; a.k.a. CDDO-Me) on HIV-1 p24 production by U1 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). Exposure to BM (50 nM) decreased HIV-1 p24 production in both PMA-induced and ASC-CM-stimulated U1 cells. ( B,C ) effect of PI3K inhibitors, ( B ) LY294002 (10 μM) or ( C ) PX866 (500 nM) on HIV-1 p24 production in ASC-CM (50%) stimulated U1 cells. PI3K inhibitors suppressed latency-reactivation by ASC-CM. ( D,E ) effects of AKT inhibition by 124005 (5 μM) and AKT activation by SC-79 (2.5 μM) on ASC-CM (50%) induced HIV-1 p24 production. Latency-reactivation by ASC-CM is increased following AKT inhibition and decreased following AKT induction. ( F ) Effect of heat-inactivation (HI) of ASC-CM or BMSC-CM on HIV-1 LTR-directed GFP expression (MFI) by the U-494 cells. Heat labile factors in CMs are responsible for LTR activation. ( G ) Effect of PI3K and NF-κB inhibitors on HIV-1 LTR function in U-494 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). GFP expression (MFI) was monitored after 48 h and fold change (compared to control media) are shown. Both LY294002 (PI3K inhibitor) and BM (NFκB inhibitor) suppressed HIV-1 LTR function, but 124005 (AKT inhibitor) showed an inductive effect. Error bars show ± SEM and significant changes are represented as P-values (*p

    Journal: Scientific Reports

    Article Title: Mesenchymal stem cells are attracted to latent HIV-1-infected cells and enable virus reactivation via a non-canonical PI3K-NFκB signaling pathway

    doi: 10.1038/s41598-018-32657-y

    Figure Lengend Snippet: Role of NF-κB, PI3K and AKT pathways in ASC-mediated latency-reactivation of U1 cells. ( A ) Effect of NF-κB inhibitor, bardoxolone-methyl (BM; a.k.a. CDDO-Me) on HIV-1 p24 production by U1 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). Exposure to BM (50 nM) decreased HIV-1 p24 production in both PMA-induced and ASC-CM-stimulated U1 cells. ( B,C ) effect of PI3K inhibitors, ( B ) LY294002 (10 μM) or ( C ) PX866 (500 nM) on HIV-1 p24 production in ASC-CM (50%) stimulated U1 cells. PI3K inhibitors suppressed latency-reactivation by ASC-CM. ( D,E ) effects of AKT inhibition by 124005 (5 μM) and AKT activation by SC-79 (2.5 μM) on ASC-CM (50%) induced HIV-1 p24 production. Latency-reactivation by ASC-CM is increased following AKT inhibition and decreased following AKT induction. ( F ) Effect of heat-inactivation (HI) of ASC-CM or BMSC-CM on HIV-1 LTR-directed GFP expression (MFI) by the U-494 cells. Heat labile factors in CMs are responsible for LTR activation. ( G ) Effect of PI3K and NF-κB inhibitors on HIV-1 LTR function in U-494 cells, stimulated with either ASC-CM (50%) or PMA (10 ng/mL). GFP expression (MFI) was monitored after 48 h and fold change (compared to control media) are shown. Both LY294002 (PI3K inhibitor) and BM (NFκB inhibitor) suppressed HIV-1 LTR function, but 124005 (AKT inhibitor) showed an inductive effect. Error bars show ± SEM and significant changes are represented as P-values (*p

    Article Snippet: Antibodies against AKT, phospho-AKT and PI3K p110β were purchased from Cell signaling (Danvers, MA).

    Techniques: Inhibition, Activation Assay, Expressing

    Putative molecular effects of MSC-secretome on latency-reactivation from tissue HIV-1 reservoirs. ( A ) Schematics of possible interactions between MSCs latently-infected cells within tissue HIV-1 reservoirs. The following steps may be involved in persistence, propagation and transmission of HIV-1 from reservoirs microenvironments: (1) Factors secreted from latently-infected cells (MACs THLs) may recruit tissue-resident MSCs; (2) these reservoir-recruited MSCs may closely interact with latently-infected cells, where reciprocal signaling (cell-to-cell and/or secretome-mediated) may facilitate virus reactivation; (3) the reservoir-recruited MSCs may also be activated to secrete factors that constantly reactivate HIV-1 from latency, generating an inflammatory microenvironment that enable further recruitment of infectable cells; and (4) these productively infected reservoir cells may then migrate into the blood and enable rapid viral rebound. ( B ) Signaling pathways involved in MSC-secretome (ASC-CM) mediated HIV-1 reactivation from latently-infected MACs (U1 cells). The potent suppressive effects of LY294002 PX-866 implicate a direct role for the PI3K signaling pathway. The potent effect of Bardoxolone-Methyl (CDDO-Me) also suggests that NF-κB activation is a mechanism of this latency-reactivation. Interestingly however, increased reactivation by the AKT inhibitor (124005) and decreased reactivation by the AKT activator (SC-79) also suggest the involvement of a non-canonical PI3K-NFκB dependent pathway, where the downstream AKT pathway has a negative regulatory effect on MSC-mediated latency-reactivation from tissue resident MACs.

    Journal: Scientific Reports

    Article Title: Mesenchymal stem cells are attracted to latent HIV-1-infected cells and enable virus reactivation via a non-canonical PI3K-NFκB signaling pathway

    doi: 10.1038/s41598-018-32657-y

    Figure Lengend Snippet: Putative molecular effects of MSC-secretome on latency-reactivation from tissue HIV-1 reservoirs. ( A ) Schematics of possible interactions between MSCs latently-infected cells within tissue HIV-1 reservoirs. The following steps may be involved in persistence, propagation and transmission of HIV-1 from reservoirs microenvironments: (1) Factors secreted from latently-infected cells (MACs THLs) may recruit tissue-resident MSCs; (2) these reservoir-recruited MSCs may closely interact with latently-infected cells, where reciprocal signaling (cell-to-cell and/or secretome-mediated) may facilitate virus reactivation; (3) the reservoir-recruited MSCs may also be activated to secrete factors that constantly reactivate HIV-1 from latency, generating an inflammatory microenvironment that enable further recruitment of infectable cells; and (4) these productively infected reservoir cells may then migrate into the blood and enable rapid viral rebound. ( B ) Signaling pathways involved in MSC-secretome (ASC-CM) mediated HIV-1 reactivation from latently-infected MACs (U1 cells). The potent suppressive effects of LY294002 PX-866 implicate a direct role for the PI3K signaling pathway. The potent effect of Bardoxolone-Methyl (CDDO-Me) also suggests that NF-κB activation is a mechanism of this latency-reactivation. Interestingly however, increased reactivation by the AKT inhibitor (124005) and decreased reactivation by the AKT activator (SC-79) also suggest the involvement of a non-canonical PI3K-NFκB dependent pathway, where the downstream AKT pathway has a negative regulatory effect on MSC-mediated latency-reactivation from tissue resident MACs.

    Article Snippet: Antibodies against AKT, phospho-AKT and PI3K p110β were purchased from Cell signaling (Danvers, MA).

    Techniques: Infection, Transmission Assay, Magnetic Cell Separation, Activation Assay