anti-akt Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Cell Signaling Technology Inc akt
    Inhibition of <t>FOXO1</t> transcriptional activity through the <t>melatonin-PI3K-AKT</t> axis protects GCs from H 2 O 2 -induced autophagic PCD. (A) GCs transfected with Foxo1 siRNA or scrambled control siRNA for 24 h were cultured in media containing various concentrations of melatonin (0, 5, 10, 20 μM). 24 h later, cells were rinsed with PBS, and exposed to H 2 O 2 (200 μM) incubation for another 2 h. The expression of phosphorylated FOXO1 (p-FOXO1) was determined by western blotting. (B) The phosphorylation level of FOXO1 was quantified by densitometric analysis. TUBA1A served as the control for loading. Data represent mean ± S.E; n = 3. ** Represents P
    Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 52454 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 52454 article reviews
    Price from $9.99 to $1999.99
    akt - by Bioz Stars, 2021-02
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc phospho akt
    Inhibition of <t>FOXO1</t> transcriptional activity through the <t>melatonin-PI3K-AKT</t> axis protects GCs from H 2 O 2 -induced autophagic PCD. (A) GCs transfected with Foxo1 siRNA or scrambled control siRNA for 24 h were cultured in media containing various concentrations of melatonin (0, 5, 10, 20 μM). 24 h later, cells were rinsed with PBS, and exposed to H 2 O 2 (200 μM) incubation for another 2 h. The expression of phosphorylated FOXO1 (p-FOXO1) was determined by western blotting. (B) The phosphorylation level of FOXO1 was quantified by densitometric analysis. TUBA1A served as the control for loading. Data represent mean ± S.E; n = 3. ** Represents P
    Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 16290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho akt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 16290 article reviews
    Price from $9.99 to $1999.99
    phospho akt - by Bioz Stars, 2021-02
    99/100 stars
      Buy from Supplier

    99
    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 7796 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho akt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 7796 article reviews
    Price from $9.99 to $1999.99
    anti phospho akt - by Bioz Stars, 2021-02
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc p akt ser473
    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 .
    P Akt Ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4241 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p akt ser473/product/Cell Signaling Technology Inc
    Average 99 stars, based on 4241 article reviews
    Price from $9.99 to $1999.99
    p akt ser473 - by Bioz Stars, 2021-02
    99/100 stars
      Buy from Supplier

    97
    Cell Signaling Technology Inc anti pakt
    GPR84 signaling activates <t>AKT,</t> <t>ERK,</t> and NFκβ in WT, but not in GPR84 −/− macrophages. (A) Bone marrow-derived macrophages (BMDMs) were treated with 0.1 µg/ml LPS for 2 h before stimulated with either vehicle (0.3% DMSO) or 1 µM 6-OAU for 1, 5, 10, 30, and 60 min. Cell lysates were prepared and western blotting conducted for either phosphorylated Akt (P-AKT) or ERK 1/2 (P-ERK), followed by stripping and re-staining for β-actin as a loading control. Representative images from n = 3 independent experiments are shown. (B) BMDMs were treated with LPS (0.1 µg/ml) for 2 h before stimulation with vehicle (0.3% DMSO) or 1 µM 6-OAU for 30 min followed by p65 staining. Confocal microscopy images are illustrative of two separate experiments. (C) BMDMs were treated with LPS (0.1 µg/ml) for 2 h before stimulation with either vehicle (0.3% DMSO) or 1 µM 6-OAU for 5, 10, 30, and 60 min. Western blotting for p65 was performed using samples from cytoplasmic and nuclear fractions of cell lysates, followed by stripping and re-staining for histone-3 in the nuclear fraction and α-tubulin in the cytoplasmic fraction as a loading control. Representative images from n = 3 independent experiments are shown.
    Anti Pakt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 2819 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti pakt/product/Cell Signaling Technology Inc
    Average 97 stars, based on 2819 article reviews
    Price from $9.99 to $1999.99
    anti pakt - by Bioz Stars, 2021-02
    97/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc anti p akt
    GPR84 signaling activates <t>AKT,</t> <t>ERK,</t> and NFκβ in WT, but not in GPR84 −/− macrophages. (A) Bone marrow-derived macrophages (BMDMs) were treated with 0.1 µg/ml LPS for 2 h before stimulated with either vehicle (0.3% DMSO) or 1 µM 6-OAU for 1, 5, 10, 30, and 60 min. Cell lysates were prepared and western blotting conducted for either phosphorylated Akt (P-AKT) or ERK 1/2 (P-ERK), followed by stripping and re-staining for β-actin as a loading control. Representative images from n = 3 independent experiments are shown. (B) BMDMs were treated with LPS (0.1 µg/ml) for 2 h before stimulation with vehicle (0.3% DMSO) or 1 µM 6-OAU for 30 min followed by p65 staining. Confocal microscopy images are illustrative of two separate experiments. (C) BMDMs were treated with LPS (0.1 µg/ml) for 2 h before stimulation with either vehicle (0.3% DMSO) or 1 µM 6-OAU for 5, 10, 30, and 60 min. Western blotting for p65 was performed using samples from cytoplasmic and nuclear fractions of cell lysates, followed by stripping and re-staining for histone-3 in the nuclear fraction and α-tubulin in the cytoplasmic fraction as a loading control. Representative images from n = 3 independent experiments are shown.
    Anti P Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p akt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 3648 article reviews
    Price from $9.99 to $1999.99
    anti p akt - by Bioz Stars, 2021-02
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc pakt
    Effects of ACA, AEA, and AMCA on the migration pathway. Notes: Expression levels of integrin β1, FAK, pFAK, Akt, and <t>pAkt</t> proteins from MDA-MB-231 cells were analyzed via Western blot analysis ( A ). <t>GAPDH</t> was used as a loading control. The quantitative data ( B – D ) are presented as mean ± SEM from three independent experiments (* p ≤0.05 by two-tailed unpaired t -test). Abbreviations: ACA, 1′ S -1′-acetoxychavicol acetate; AEA, 1′-acetoxyeugenol acetate; Akt, protein kinase B; AMCA, 1′-acetoxy-3,5-dimethoxychavicol acetate; DMSO, dimethyl sulfoxide; FAK, focal adhesion kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; integrin β1, integrin subunit beta 1; pAkt, phosphorylated protein kinase B; pFAK, phosphorylated focal adhesion kinase; SEM, standard error of the mean.
    Pakt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6873 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pakt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 6873 article reviews
    Price from $9.99 to $1999.99
    pakt - by Bioz Stars, 2021-02
    99/100 stars
      Buy from Supplier

    96
    Cell Signaling Technology Inc phospho akt thr308
    Effects of ACA, AEA, and AMCA on the migration pathway. Notes: Expression levels of integrin β1, FAK, pFAK, Akt, and <t>pAkt</t> proteins from MDA-MB-231 cells were analyzed via Western blot analysis ( A ). <t>GAPDH</t> was used as a loading control. The quantitative data ( B – D ) are presented as mean ± SEM from three independent experiments (* p ≤0.05 by two-tailed unpaired t -test). Abbreviations: ACA, 1′ S -1′-acetoxychavicol acetate; AEA, 1′-acetoxyeugenol acetate; Akt, protein kinase B; AMCA, 1′-acetoxy-3,5-dimethoxychavicol acetate; DMSO, dimethyl sulfoxide; FAK, focal adhesion kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; integrin β1, integrin subunit beta 1; pAkt, phosphorylated protein kinase B; pFAK, phosphorylated focal adhesion kinase; SEM, standard error of the mean.
    Phospho Akt Thr308, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1440 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho akt thr308/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1440 article reviews
    Price from $9.99 to $1999.99
    phospho akt thr308 - by Bioz Stars, 2021-02
    96/100 stars
      Buy from Supplier

    99
    Abcam anti akt
    APS reduced LPS-induced inflammation injury by down-regulating miR-127 and inhibiting NF-κB and JNK and promoting <t>PI3K/AKT</t> signaling pathways in H9c2 cells. (a) H9c2 cells were treated with LPS or co-treated with APS and LPS, and then expression levels of miR-127 in H9c2 cells were measured by qRT-PCR. (b)–(d) Protein expression levels of p65/p-p65, lκBα/p-lκBα JNK/p-JNK, c-Jun/p-c-Jun, PI3K/p-PI3K, and AKT/p-AKT were detected by western blot. Different letters above the bars (a, b, c) indicate that the means of different groups were significantly different ( P
    Anti Akt, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1086 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti akt/product/Abcam
    Average 99 stars, based on 1086 article reviews
    Price from $9.99 to $1999.99
    anti akt - by Bioz Stars, 2021-02
    99/100 stars
      Buy from Supplier

    akt  (Abcam)
    99
    Abcam akt
    <t>BACE1</t> gene silencing activates the <t>PI3K/Akt</t> signaling pathway. (A) The mRNA expression of caspase-3 was reduced, and that of PI3K and Akt was increased following reverse transcription-quantitative polymerase chain reaction. (B) The protein expression of caspase-3 was reduced, and that of PI3K and Akt was increased by silencing BACE1, (C) as determined by western blot analysis. *P
    Akt, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2501 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt/product/Abcam
    Average 99 stars, based on 2501 article reviews
    Price from $9.99 to $1999.99
    akt - by Bioz Stars, 2021-02
    99/100 stars
      Buy from Supplier

    97
    Santa Cruz Biotechnology anti akt
    Effects of PSP on the expression of Klotho, <t>Akt,</t> <t>FOXO3a,</t> FGF23, p-Akt and p-FOXO3a proteins. ( A ) PSP increases the expression of Klotho protein (p
    Anti Akt, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 2421 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti akt/product/Santa Cruz Biotechnology
    Average 97 stars, based on 2421 article reviews
    Price from $9.99 to $1999.99
    anti akt - by Bioz Stars, 2021-02
    97/100 stars
      Buy from Supplier

    N/A
    The serine threonine protein kinase AKT1 is catalytically inactive in serum starved primary and immortalized fibroblasts AKT1 and the related AKT2 are activated by platelet derived growth factor The activation
      Buy from Supplier


    N/A
    The serine threonine protein kinase AKT1 is catalytically inactive in serum starved primary and immortalized fibroblasts Survival factors can suppress apoptosis in a transcription independent manner by activating the serine
      Buy from Supplier

    N/A
    Rabbit polyclonal AKT1 3 antibody Tyr437 434
      Buy from Supplier

    Image Search Results


    Inhibition of FOXO1 transcriptional activity through the melatonin-PI3K-AKT axis protects GCs from H 2 O 2 -induced autophagic PCD. (A) GCs transfected with Foxo1 siRNA or scrambled control siRNA for 24 h were cultured in media containing various concentrations of melatonin (0, 5, 10, 20 μM). 24 h later, cells were rinsed with PBS, and exposed to H 2 O 2 (200 μM) incubation for another 2 h. The expression of phosphorylated FOXO1 (p-FOXO1) was determined by western blotting. (B) The phosphorylation level of FOXO1 was quantified by densitometric analysis. TUBA1A served as the control for loading. Data represent mean ± S.E; n = 3. ** Represents P

    Journal: Redox Biology

    Article Title: Melatonin protects mouse granulosa cells against oxidative damage by inhibiting FOXO1-mediated autophagy: Implication of an antioxidation-independent mechanism

    doi: 10.1016/j.redox.2018.07.004

    Figure Lengend Snippet: Inhibition of FOXO1 transcriptional activity through the melatonin-PI3K-AKT axis protects GCs from H 2 O 2 -induced autophagic PCD. (A) GCs transfected with Foxo1 siRNA or scrambled control siRNA for 24 h were cultured in media containing various concentrations of melatonin (0, 5, 10, 20 μM). 24 h later, cells were rinsed with PBS, and exposed to H 2 O 2 (200 μM) incubation for another 2 h. The expression of phosphorylated FOXO1 (p-FOXO1) was determined by western blotting. (B) The phosphorylation level of FOXO1 was quantified by densitometric analysis. TUBA1A served as the control for loading. Data represent mean ± S.E; n = 3. ** Represents P

    Article Snippet: Antibodies against AKT (9272), phospho-AKT (4060), FOXO1 (2880), phospho-FOXO1 (9461), FLAG (2908), BECN1 (3495), MTOR (2983), ATG3 (3415), ATG5 (8540), ATG7 (2631), and ATG12 (4180) were obtained from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Inhibition, Activity Assay, Transfection, Cell Culture, Incubation, Expressing, Western Blot

    Oligomeric Aβ 1–42 specifically binds to TREM2 and activates TREM2 signaling pathway . a Schematic representation of human TREM2 extracellular domain (sTREM2, amino acid residues 1–171) tagged with human IgG1 Fc. SP: signal peptide. b The cDNA encoding sTREM2-Fc, sTREM1-Fc or Fc alone was transfected into HEK 293 T cells. Each protein was purified from the conditioned medium and analyzed by silver stained SDS-PAGE. c The prepared oAβ 1–42 peptides were analyzed by Western blotting using 4–12% Bis-Tris NuPAGE gel. d Solid phase binding assay showing the saturation binding curve and equilibrium dissociation constant (K D ) of oAβ 1–42 binding to sTREM2-Fc. Fc and sTREM1-Fc served as negative controls ( n = 3). e The Fc, sTREM2-Fc or sTREM1-Fc control was pre-bound to protein A agarose beads and used as baits for immunoprecipitation of oAβ 1–42 . The precipitated products were separated on 4–12% Bis-Tris NuPAGE gel and further subjected to Western blotting. ( f and g ) The binding profiles of oAβ 1–42 to different concentrations of sTREM2-Fc f or Fc g were generated by SPR assay. h The prepared monomeric Aβ 1–42 (mAβ 1–42 ) peptides were analyzed by Western blotting using 4–12% Bis-Tris NuPAGE gel. i The binding profiles of mAβ 1–42 to different concentrations of sTREM2-Fc were generated by SPR assay. j The binding profiles of scrambled Aβ 42 (scAβ 42 ) to different concentrations of sTREM2-Fc were generated by SPR assay. k Wild-type (WT) or Trem2 -knockout (KO) microglia were stimulated with 1.0 μM oAβ 1–42 for 1 h. Cell lysates were analyzed by Western blotting using antibodies specific for either total (T-Syk) or phosphorylated form (p-Syk) of Syk. l Quantification of Western blots as ratios of p-Syk/T-Syk. β-Actin was used as an internal control ( n = 3, two-way ANOVA). m WT or Trem2 -KO microglia were stimulated with 1.0 μM oAβ 1–42 for the indicated time. Cell lysates at each time point were analyzed by Western blotting using antibodies specific for either total (T-Akt) or phosphorylated form (p-Akt) of Akt. n Quantification of Western blots as ratios of p-Akt/T-Akt. β-Actin was used as an internal control (n = 3, two-way ANOVA). Data information: Data represent mean ± SD. **, p

    Journal: Molecular Neurodegeneration

    Article Title: Amyloid-beta modulates microglial responses by binding to the triggering receptor expressed on myeloid cells 2 (TREM2)

    doi: 10.1186/s13024-018-0247-7

    Figure Lengend Snippet: Oligomeric Aβ 1–42 specifically binds to TREM2 and activates TREM2 signaling pathway . a Schematic representation of human TREM2 extracellular domain (sTREM2, amino acid residues 1–171) tagged with human IgG1 Fc. SP: signal peptide. b The cDNA encoding sTREM2-Fc, sTREM1-Fc or Fc alone was transfected into HEK 293 T cells. Each protein was purified from the conditioned medium and analyzed by silver stained SDS-PAGE. c The prepared oAβ 1–42 peptides were analyzed by Western blotting using 4–12% Bis-Tris NuPAGE gel. d Solid phase binding assay showing the saturation binding curve and equilibrium dissociation constant (K D ) of oAβ 1–42 binding to sTREM2-Fc. Fc and sTREM1-Fc served as negative controls ( n = 3). e The Fc, sTREM2-Fc or sTREM1-Fc control was pre-bound to protein A agarose beads and used as baits for immunoprecipitation of oAβ 1–42 . The precipitated products were separated on 4–12% Bis-Tris NuPAGE gel and further subjected to Western blotting. ( f and g ) The binding profiles of oAβ 1–42 to different concentrations of sTREM2-Fc f or Fc g were generated by SPR assay. h The prepared monomeric Aβ 1–42 (mAβ 1–42 ) peptides were analyzed by Western blotting using 4–12% Bis-Tris NuPAGE gel. i The binding profiles of mAβ 1–42 to different concentrations of sTREM2-Fc were generated by SPR assay. j The binding profiles of scrambled Aβ 42 (scAβ 42 ) to different concentrations of sTREM2-Fc were generated by SPR assay. k Wild-type (WT) or Trem2 -knockout (KO) microglia were stimulated with 1.0 μM oAβ 1–42 for 1 h. Cell lysates were analyzed by Western blotting using antibodies specific for either total (T-Syk) or phosphorylated form (p-Syk) of Syk. l Quantification of Western blots as ratios of p-Syk/T-Syk. β-Actin was used as an internal control ( n = 3, two-way ANOVA). m WT or Trem2 -KO microglia were stimulated with 1.0 μM oAβ 1–42 for the indicated time. Cell lysates at each time point were analyzed by Western blotting using antibodies specific for either total (T-Akt) or phosphorylated form (p-Akt) of Akt. n Quantification of Western blots as ratios of p-Akt/T-Akt. β-Actin was used as an internal control (n = 3, two-way ANOVA). Data information: Data represent mean ± SD. **, p

    Article Snippet: Anti-Phospho-Syk (Tyr525/526) (2711 s), anti-total-Syk (13,198 s), anti-Phospho-Akt (Ser473) (4060 s), anti-total-Akt (4685 s) and anti-β-actin antibody (4970 s) were purchased from Cell Signaling Technology.

    Techniques: Transfection, Purification, Staining, SDS Page, Western Blot, Binding Assay, Immunoprecipitation, Generated, SPR Assay, Knock-Out

    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

    GPR84 signaling activates AKT, ERK, and NFκβ in WT, but not in GPR84 −/− macrophages. (A) Bone marrow-derived macrophages (BMDMs) were treated with 0.1 µg/ml LPS for 2 h before stimulated with either vehicle (0.3% DMSO) or 1 µM 6-OAU for 1, 5, 10, 30, and 60 min. Cell lysates were prepared and western blotting conducted for either phosphorylated Akt (P-AKT) or ERK 1/2 (P-ERK), followed by stripping and re-staining for β-actin as a loading control. Representative images from n = 3 independent experiments are shown. (B) BMDMs were treated with LPS (0.1 µg/ml) for 2 h before stimulation with vehicle (0.3% DMSO) or 1 µM 6-OAU for 30 min followed by p65 staining. Confocal microscopy images are illustrative of two separate experiments. (C) BMDMs were treated with LPS (0.1 µg/ml) for 2 h before stimulation with either vehicle (0.3% DMSO) or 1 µM 6-OAU for 5, 10, 30, and 60 min. Western blotting for p65 was performed using samples from cytoplasmic and nuclear fractions of cell lysates, followed by stripping and re-staining for histone-3 in the nuclear fraction and α-tubulin in the cytoplasmic fraction as a loading control. Representative images from n = 3 independent experiments are shown.

    Journal: Frontiers in Immunology

    Article Title: Activation of the Immune-Metabolic Receptor GPR84 Enhances Inflammation and Phagocytosis in Macrophages

    doi: 10.3389/fimmu.2018.01419

    Figure Lengend Snippet: GPR84 signaling activates AKT, ERK, and NFκβ in WT, but not in GPR84 −/− macrophages. (A) Bone marrow-derived macrophages (BMDMs) were treated with 0.1 µg/ml LPS for 2 h before stimulated with either vehicle (0.3% DMSO) or 1 µM 6-OAU for 1, 5, 10, 30, and 60 min. Cell lysates were prepared and western blotting conducted for either phosphorylated Akt (P-AKT) or ERK 1/2 (P-ERK), followed by stripping and re-staining for β-actin as a loading control. Representative images from n = 3 independent experiments are shown. (B) BMDMs were treated with LPS (0.1 µg/ml) for 2 h before stimulation with vehicle (0.3% DMSO) or 1 µM 6-OAU for 30 min followed by p65 staining. Confocal microscopy images are illustrative of two separate experiments. (C) BMDMs were treated with LPS (0.1 µg/ml) for 2 h before stimulation with either vehicle (0.3% DMSO) or 1 µM 6-OAU for 5, 10, 30, and 60 min. Western blotting for p65 was performed using samples from cytoplasmic and nuclear fractions of cell lysates, followed by stripping and re-staining for histone-3 in the nuclear fraction and α-tubulin in the cytoplasmic fraction as a loading control. Representative images from n = 3 independent experiments are shown.

    Article Snippet: Rabbit anti-phospho-ERK (D13.14.4E), rabbit anti-total-ERK, rabbit anti-phospho-Akt (D9E), rabbit anti-total-Akt, β-actin, were purchased from Cell Signaling Technologies (Danvers, MA, USA).

    Techniques: Derivative Assay, Western Blot, Stripping Membranes, Staining, Confocal Microscopy

    GPR84 antagonist abrogates the enhanced inflammatory response mediated by 6-OAU. (A) Intracellular cyclic AMP levels were measured in CHO-GPR84 cells pre-treated with the antagonist followed by forskolin and 6-OAU stimulation. Data are represented as the mean ± SEM of the percentage of the response to forskolin in the absence of agonists n = 3 independent experiments. (B–H) Bone marrow-derived macrophages were treated with LPS (0.1 µg/ml) for 2 h before pre-treatment with vehicle (0.3% DMSO), 10 µM antagonist for 30 min, or 200 ng/ml Pertussis toxin for 90 min. Afterward cells were stimulated with either vehicle (0.3% DMSO) or 1 µM 6-OAU for either 10′ for protein isolation or 1 h for RNA extraction. (B) Cell lysates were prepared and western blotting conducted for either phosphorylated Akt (P-AKT) or ERK 1/2 (P-ERK), followed by stripping and re-staining for β-actin as a loading control. Representative images from n = 3 independent experiments. (C–H) mRNA expression of Tnf α (C) , Il-6 (D) , Il-12 (E) , Ccl5 (F) , Ccl2 (G) , and Cxcl1 (H) was analyzed by q-PCR. Data are presented as mean ± SEM of n = 4–7 separate experiments. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparison post hoc test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 versus vehicle; # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001 versus 6-OAU.

    Journal: Frontiers in Immunology

    Article Title: Activation of the Immune-Metabolic Receptor GPR84 Enhances Inflammation and Phagocytosis in Macrophages

    doi: 10.3389/fimmu.2018.01419

    Figure Lengend Snippet: GPR84 antagonist abrogates the enhanced inflammatory response mediated by 6-OAU. (A) Intracellular cyclic AMP levels were measured in CHO-GPR84 cells pre-treated with the antagonist followed by forskolin and 6-OAU stimulation. Data are represented as the mean ± SEM of the percentage of the response to forskolin in the absence of agonists n = 3 independent experiments. (B–H) Bone marrow-derived macrophages were treated with LPS (0.1 µg/ml) for 2 h before pre-treatment with vehicle (0.3% DMSO), 10 µM antagonist for 30 min, or 200 ng/ml Pertussis toxin for 90 min. Afterward cells were stimulated with either vehicle (0.3% DMSO) or 1 µM 6-OAU for either 10′ for protein isolation or 1 h for RNA extraction. (B) Cell lysates were prepared and western blotting conducted for either phosphorylated Akt (P-AKT) or ERK 1/2 (P-ERK), followed by stripping and re-staining for β-actin as a loading control. Representative images from n = 3 independent experiments. (C–H) mRNA expression of Tnf α (C) , Il-6 (D) , Il-12 (E) , Ccl5 (F) , Ccl2 (G) , and Cxcl1 (H) was analyzed by q-PCR. Data are presented as mean ± SEM of n = 4–7 separate experiments. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparison post hoc test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 versus vehicle; # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001 versus 6-OAU.

    Article Snippet: Rabbit anti-phospho-ERK (D13.14.4E), rabbit anti-total-ERK, rabbit anti-phospho-Akt (D9E), rabbit anti-total-Akt, β-actin, were purchased from Cell Signaling Technologies (Danvers, MA, USA).

    Techniques: Derivative Assay, Isolation, RNA Extraction, Western Blot, Stripping Membranes, Staining, Expressing, Polymerase Chain Reaction

    Effects of ACA, AEA, and AMCA on the migration pathway. Notes: Expression levels of integrin β1, FAK, pFAK, Akt, and pAkt proteins from MDA-MB-231 cells were analyzed via Western blot analysis ( A ). GAPDH was used as a loading control. The quantitative data ( B – D ) are presented as mean ± SEM from three independent experiments (* p ≤0.05 by two-tailed unpaired t -test). Abbreviations: ACA, 1′ S -1′-acetoxychavicol acetate; AEA, 1′-acetoxyeugenol acetate; Akt, protein kinase B; AMCA, 1′-acetoxy-3,5-dimethoxychavicol acetate; DMSO, dimethyl sulfoxide; FAK, focal adhesion kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; integrin β1, integrin subunit beta 1; pAkt, phosphorylated protein kinase B; pFAK, phosphorylated focal adhesion kinase; SEM, standard error of the mean.

    Journal: Drug Design, Development and Therapy

    Article Title: Anti-proliferative, apoptotic induction, and anti-migration effects of hemi-synthetic 1′S-1′-acetoxychavicol acetate analogs on MDA-MB-231 breast cancer cells

    doi: 10.2147/DDDT.S130349

    Figure Lengend Snippet: Effects of ACA, AEA, and AMCA on the migration pathway. Notes: Expression levels of integrin β1, FAK, pFAK, Akt, and pAkt proteins from MDA-MB-231 cells were analyzed via Western blot analysis ( A ). GAPDH was used as a loading control. The quantitative data ( B – D ) are presented as mean ± SEM from three independent experiments (* p ≤0.05 by two-tailed unpaired t -test). Abbreviations: ACA, 1′ S -1′-acetoxychavicol acetate; AEA, 1′-acetoxyeugenol acetate; Akt, protein kinase B; AMCA, 1′-acetoxy-3,5-dimethoxychavicol acetate; DMSO, dimethyl sulfoxide; FAK, focal adhesion kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; integrin β1, integrin subunit beta 1; pAkt, phosphorylated protein kinase B; pFAK, phosphorylated focal adhesion kinase; SEM, standard error of the mean.

    Article Snippet: Membranes were blocked with 5% (w/v) bovine serum albumin and incubated with primary antibodies against GAPDH, Bcl-2, Bcl-xL, Bax, p53, PARP, β1 integrin, FAK, pFAK, Akt, and pAkt (1:1,000 dilutions; Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Migration, Expressing, Multiple Displacement Amplification, Western Blot, Two Tailed Test

    APS reduced LPS-induced inflammation injury by down-regulating miR-127 and inhibiting NF-κB and JNK and promoting PI3K/AKT signaling pathways in H9c2 cells. (a) H9c2 cells were treated with LPS or co-treated with APS and LPS, and then expression levels of miR-127 in H9c2 cells were measured by qRT-PCR. (b)–(d) Protein expression levels of p65/p-p65, lκBα/p-lκBα JNK/p-JNK, c-Jun/p-c-Jun, PI3K/p-PI3K, and AKT/p-AKT were detected by western blot. Different letters above the bars (a, b, c) indicate that the means of different groups were significantly different ( P

    Journal: International Journal of Immunopathology and Pharmacology

    Article Title: Astragalus polysaccharide alleviates LPS-induced inflammation injury by regulating miR-127 in H9c2 cardiomyoblasts

    doi: 10.1177/2058738418759180

    Figure Lengend Snippet: APS reduced LPS-induced inflammation injury by down-regulating miR-127 and inhibiting NF-κB and JNK and promoting PI3K/AKT signaling pathways in H9c2 cells. (a) H9c2 cells were treated with LPS or co-treated with APS and LPS, and then expression levels of miR-127 in H9c2 cells were measured by qRT-PCR. (b)–(d) Protein expression levels of p65/p-p65, lκBα/p-lκBα JNK/p-JNK, c-Jun/p-c-Jun, PI3K/p-PI3K, and AKT/p-AKT were detected by western blot. Different letters above the bars (a, b, c) indicate that the means of different groups were significantly different ( P

    Article Snippet: The primary antibodies used in this study included anti-Bax (ab32503, 1:1000), anti-pro-Caspase3 (ab44976, 1:500), anti-cleaved-Caspase3 (ab13847, 1:500), anti-Caspase9 (ab25758, 1:200), anti-IL-6 (ab9770, 1:5000), anti-IL-8 (ab7747, 1:10), anti-TNF-α (ab6671, 1:1000), anti-p65 (ab16502, 1:2000), anti-toll-like receptor 4 (anti-TLR4, ab13556, 1:500), anti-p-p65 (ab86299, 1:2000), anti-IκBα (ab7217, 1:2000), anti-JNK (ab179461, 1:1000), anti-p-JNK (ab124956, 1:5000), anti-c-Jun (ab32137, 1:1000) and anti-p-c-Jun (ab32385, 1:1000), anti-PI3K (ab191606, 1:1000), anti-p-PI3K (ab182651, 1:1000), anti-AKT (ab32505, 1:2000), anti-p-AKT (ab131443, 1:1000), all purchased from Abcam (Cambridge, UK).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    BACE1 gene silencing activates the PI3K/Akt signaling pathway. (A) The mRNA expression of caspase-3 was reduced, and that of PI3K and Akt was increased following reverse transcription-quantitative polymerase chain reaction. (B) The protein expression of caspase-3 was reduced, and that of PI3K and Akt was increased by silencing BACE1, (C) as determined by western blot analysis. *P

    Journal: Molecular Medicine Reports

    Article Title: BACE1 gene silencing alleviates isoflurane anesthesia-induced postoperative cognitive dysfunction in immature rats by activating the PI3K/Akt signaling pathway

    doi: 10.3892/mmr.2018.9453

    Figure Lengend Snippet: BACE1 gene silencing activates the PI3K/Akt signaling pathway. (A) The mRNA expression of caspase-3 was reduced, and that of PI3K and Akt was increased following reverse transcription-quantitative polymerase chain reaction. (B) The protein expression of caspase-3 was reduced, and that of PI3K and Akt was increased by silencing BACE1, (C) as determined by western blot analysis. *P

    Article Snippet: Five percent skim milk was used to block the membrane for 1 h. After gently shaking at 37°C for 2 h, GAPDH (ab9485, 1:5,000) and the primary antibodies BACE1 (ab108394, 1:1,000), PI3K (ab86714, 1:1,000), phosphorylated (p)-PI3K (ab182651, 1:1,000), Akt (ab8805, 1:500), p-Akt (ab8933, 1:1,000), caspase-3 (ab13847, 1:500) and cleaved-caspase-3 (ab49822, 1:500; all purchased from Abcam, Cambridge, MA, USA) were added for incubation at 4°C overnight.

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

    Silencing BACE1 promotes the activation of the PI3K/Akt signaling pathway, thereby inhibiting the apoptosis of the hippocampal CA1 region by TUNEL staining. (A) The positive rate of TUNEL staining in the rat hippocampus CA1 of the six groups; black arrows indicate apoptotic cells (magnification, ×200; scale bars, 50 µm). (B) The rate of apoptosis in the hippocampal CA1 region of the six groups. *P

    Journal: Molecular Medicine Reports

    Article Title: BACE1 gene silencing alleviates isoflurane anesthesia-induced postoperative cognitive dysfunction in immature rats by activating the PI3K/Akt signaling pathway

    doi: 10.3892/mmr.2018.9453

    Figure Lengend Snippet: Silencing BACE1 promotes the activation of the PI3K/Akt signaling pathway, thereby inhibiting the apoptosis of the hippocampal CA1 region by TUNEL staining. (A) The positive rate of TUNEL staining in the rat hippocampus CA1 of the six groups; black arrows indicate apoptotic cells (magnification, ×200; scale bars, 50 µm). (B) The rate of apoptosis in the hippocampal CA1 region of the six groups. *P

    Article Snippet: Five percent skim milk was used to block the membrane for 1 h. After gently shaking at 37°C for 2 h, GAPDH (ab9485, 1:5,000) and the primary antibodies BACE1 (ab108394, 1:1,000), PI3K (ab86714, 1:1,000), phosphorylated (p)-PI3K (ab182651, 1:1,000), Akt (ab8805, 1:500), p-Akt (ab8933, 1:1,000), caspase-3 (ab13847, 1:500) and cleaved-caspase-3 (ab49822, 1:500; all purchased from Abcam, Cambridge, MA, USA) were added for incubation at 4°C overnight.

    Techniques: Activation Assay, TUNEL Assay, Staining

    Effects of PSP on the expression of Klotho, Akt, FOXO3a, FGF23, p-Akt and p-FOXO3a proteins. ( A ) PSP increases the expression of Klotho protein (p

    Journal: Scientific Reports

    Article Title: Protective effect of Polygonatum sibiricum Polysaccharide on D-galactose-induced aging rats model

    doi: 10.1038/s41598-020-59055-7

    Figure Lengend Snippet: Effects of PSP on the expression of Klotho, Akt, FOXO3a, FGF23, p-Akt and p-FOXO3a proteins. ( A ) PSP increases the expression of Klotho protein (p

    Article Snippet: The first anti-Akt, phosphorylated Akt (p-Akt), FOXO3a, phosphorylated FOXO3a (p-FOXO3a), Beta-Actin (β-Actin) and the second anti-Akt were purchased from Santa Cruz Biotechnology Company, USA.

    Techniques: Expressing

    Western blot was used to detect the expression of Klotho, Akt, p-Akt, FOXO3a, p-FOXO3a and FGF23 protein.

    Journal: Scientific Reports

    Article Title: Protective effect of Polygonatum sibiricum Polysaccharide on D-galactose-induced aging rats model

    doi: 10.1038/s41598-020-59055-7

    Figure Lengend Snippet: Western blot was used to detect the expression of Klotho, Akt, p-Akt, FOXO3a, p-FOXO3a and FGF23 protein.

    Article Snippet: The first anti-Akt, phosphorylated Akt (p-Akt), FOXO3a, phosphorylated FOXO3a (p-FOXO3a), Beta-Actin (β-Actin) and the second anti-Akt were purchased from Santa Cruz Biotechnology Company, USA.

    Techniques: Western Blot, Expressing