akt Cell Signaling Technology Inc Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 81
    Cell Signaling Technology Inc p akt akt1
    ActA stimulates SMAD2 phosphorylation, <t>AKT1</t> activation and increases IκBα levels in mature osteoclasts. (A,B) Stroma-free BMM OCL precursors cultured in the presence of MCSF and RANKL for 2 or 5 days. Cells were serum starved for 6 hours
    P Akt Akt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 81/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p akt akt1/product/Cell Signaling Technology Inc
    Average 81 stars, based on 181 article reviews
    Price from $9.99 to $1999.99
    p akt akt1 - by Bioz Stars, 2020-02
    81/100 stars
      Buy from Supplier

    91
    Cell Signaling Technology Inc akt1 2
    Insulin stimulates phosphorylation of <t>Akt1/2</t> in a PI3K-dependent manner in 3T3-L1-GLUT4myc adipocytes. Cells were left untreated or treated with insulin (Ins) (100 nM) for 10 min in the presence and absence of wortmannin (+WM) (20 nM) (A) or BX912 (+BX) (100 nM) (B). Western blotting was carried out using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graphs, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.
    Akt1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt1 2/product/Cell Signaling Technology Inc
    Average 91 stars, based on 62 article reviews
    Price from $9.99 to $1999.99
    akt1 2 - by Bioz Stars, 2020-02
    91/100 stars
      Buy from Supplier

    86
    Cell Signaling Technology Inc gst akt1
    Insulin stimulates phosphorylation of <t>Akt1/2</t> in a PI3K-dependent manner in 3T3-L1-GLUT4myc adipocytes. Cells were left untreated or treated with insulin (Ins) (100 nM) for 10 min in the presence and absence of wortmannin (+WM) (20 nM) (A) or BX912 (+BX) (100 nM) (B). Western blotting was carried out using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graphs, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.
    Gst Akt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gst akt1/product/Cell Signaling Technology Inc
    Average 86 stars, based on 28 article reviews
    Price from $9.99 to $1999.99
    gst akt1 - by Bioz Stars, 2020-02
    86/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc total akt1
    Effects of Cr(VI) on pro-apoptotic and pro-survival signaling pathways in TM3 and TM4 cells. ( a , b ) Western blot analysis of mitochondria-dependent apoptotic pathways in TM3 cells after treatment with different concentrations of Cr(VI) for 24 h. ( c ) Western blot analysis of phospho and total <t>AKT1,</t> MAPK and P53 proteins in TM3 cells after treatment with different concentrations of Cr(VI) for 24 h. ( d – e ) Western blot analysis of mitochondria-dependent apoptotic pathways in TM4 cells after treatment with different concentrations of Cr(VI) for 24 h. ( f ) Western blot analysis of phospho and total AKT1, MAPK and P53 proteins in TM4 cells after treatment with different concentrations of Cr(VI) for 24 h. BAX, BCL2, CASP9, CASP3, PARP, AKT1, ERK 1/2, JNK 1/2, P38, and P53 proteins were analyzed in the whole cell protein lysate. CYCS was analyzed in the cytosolic fraction. For the BCL2/BAX ratio, values are expressed as mean ± S.E.M. ( n = 3 ). * P
    Total Akt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/total akt1/product/Cell Signaling Technology Inc
    Average 99 stars, based on 120 article reviews
    Price from $9.99 to $1999.99
    total akt1 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    78
    Cell Signaling Technology Inc akt 1 cell signaling technology
    CMP treatment of SH-SY5Y cells affects intermediary cell metabolic responses to stimulatory ligands. Representative western blots and associated histograms depict the changes in ERK1/2 ( A ), c-Src ( B ) and <t>Akt-1</t> ( C ) activation in response to β-methylcholine (MeCh, 10 nM) or brain-derived neurotrophic factor (BDNF, 10 ng/mL) stimulating ligands in both control (blue bars) or CMP-treated (red bars) SH-SY5Y cells. The time courses (0-60 minutes) for stimulation are denoted in the associated histograms depicting the mean ± SEM from at least three separate experiments. Statistical significance is indicated for changes in kinase activity in the CMP state relative to their time-matched control in vehicle-treated (control) cells. Statistical significance was measured using a Student's t-test (GraphPad Prism v.3): * - p
    Akt 1 Cell Signaling Technology, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt 1 cell signaling technology/product/Cell Signaling Technology Inc
    Average 78 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    akt 1 cell signaling technology - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    80
    Cell Signaling Technology Inc akt c67e7 cell signaling technology
    CMP treatment of SH-SY5Y cells affects intermediary cell metabolic responses to stimulatory ligands. Representative western blots and associated histograms depict the changes in ERK1/2 ( A ), c-Src ( B ) and <t>Akt-1</t> ( C ) activation in response to β-methylcholine (MeCh, 10 nM) or brain-derived neurotrophic factor (BDNF, 10 ng/mL) stimulating ligands in both control (blue bars) or CMP-treated (red bars) SH-SY5Y cells. The time courses (0-60 minutes) for stimulation are denoted in the associated histograms depicting the mean ± SEM from at least three separate experiments. Statistical significance is indicated for changes in kinase activity in the CMP state relative to their time-matched control in vehicle-treated (control) cells. Statistical significance was measured using a Student's t-test (GraphPad Prism v.3): * - p
    Akt C67e7 Cell Signaling Technology, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt c67e7 cell signaling technology/product/Cell Signaling Technology Inc
    Average 80 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    akt c67e7 cell signaling technology - by Bioz Stars, 2020-02
    80/100 stars
      Buy from Supplier

    90
    Cell Signaling Technology Inc anti akt
    NF-κB nuclear translocations and <t>Akt</t> phosphorylations were associated with O-GlcNAcylation. The activations of Akt, <t>β-catenin</t> and NF-kB were determined in 48 h treated siOGT; and siOGA cells. ( A ) Total Akt and phosphorylated-Akt were determined by western blot analysis. ( B ) Total cellular and nuclear β-catenin and ( C ) NF-κB were examined by western blotting. ( D ) Cellular localization of NF-κB (green) is demonstrated by immunocytofluorescent staining; nuclei (blue) are stained with Hoechst 33342. The quantitative analyses are compared in each graph by using the scramble control as 100%. The results (mean ± SEM) are the averages from three independent experiments; * P
    Anti Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 13710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti akt/product/Cell Signaling Technology Inc
    Average 90 stars, based on 13710 article reviews
    Price from $9.99 to $1999.99
    anti akt - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    81
    Cell Signaling Technology Inc akt1 small interfering rna akt1 sirna
    Hyperoxia-induced Nrf2-target gene expression in <t>AKT1-depleted</t> cells. (A) qRT-PCR analysis of hyperoxia-induced Nrf2-target gene expression in cells transfected with either scrambled <t>siRNA</t> (Scr-Si) or AKT1 siRNA. (A) Expression of AKT1 was calculated relative to Scr-Si-transfected room air-exposed samples. (B) Nrf2-target gene expression was calculated relative to Scr-Si-transfected room air-exposed samples. Values from the Scr-si transfected cells exposed to room air are considered as one unit. p ≤ 0.05, room air (RA) vs. hyperoxia (hyp); † p ≤ 0.05, Scr siRNA vs AKT1-siRNA. Data are expressed as mean ± SEM (n = 3–4).
    Akt1 Small Interfering Rna Akt1 Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 81/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt1 small interfering rna akt1 sirna/product/Cell Signaling Technology Inc
    Average 81 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    akt1 small interfering rna akt1 sirna - by Bioz Stars, 2020-02
    81/100 stars
      Buy from Supplier

    77
    Cell Signaling Technology Inc akt1 2 3ser473
    Hyperoxia-induced Nrf2-target gene expression in <t>AKT1-depleted</t> cells. (A) qRT-PCR analysis of hyperoxia-induced Nrf2-target gene expression in cells transfected with either scrambled <t>siRNA</t> (Scr-Si) or AKT1 siRNA. (A) Expression of AKT1 was calculated relative to Scr-Si-transfected room air-exposed samples. (B) Nrf2-target gene expression was calculated relative to Scr-Si-transfected room air-exposed samples. Values from the Scr-si transfected cells exposed to room air are considered as one unit. p ≤ 0.05, room air (RA) vs. hyperoxia (hyp); † p ≤ 0.05, Scr siRNA vs AKT1-siRNA. Data are expressed as mean ± SEM (n = 3–4).
    Akt1 2 3ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt1 2 3ser473/product/Cell Signaling Technology Inc
    Average 77 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    akt1 2 3ser473 - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    80
    Cell Signaling Technology Inc recombinant akt1
    Phosphorylation of palladin at Ser507 by <t>Akt1</t> inhibits cell migration A , MDA-MB-231 cells infected with palladin shRNA or empty lentiviral vectors were transfected with HA-Myr-Akt1 or control vector. 24 h after transfection cells were subjected to Transwell migration assays and lysates were immunoblotted with the indicated antibodies. B , MDA-MB-231 cells were infected with palladin shRNA lentiviral vector or empty vector. Forty-eight hours after infection cells were serum-starved overnight then stimulated with IGF-1 (100 ng ml −1 ) for 18 h, followed by Transwell migration assays. Total cell lysates were subjected to immunoblot analysis. C , MDA-MB-231 cells were infected with palladin shRNA and/or Akt1 shRNA lentiviral vectors, or empty vector. Forty-eight hours after infection, cells were subjected to Transwell migration assays and lysates were immunoblotted. D .
    Recombinant Akt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 80/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant akt1/product/Cell Signaling Technology Inc
    Average 80 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    recombinant akt1 - by Bioz Stars, 2020-02
    80/100 stars
      Buy from Supplier

    78
    Cell Signaling Technology Inc α pt450 akt1
    Phosphorylation of palladin at Ser507 by <t>Akt1</t> inhibits cell migration A , MDA-MB-231 cells infected with palladin shRNA or empty lentiviral vectors were transfected with HA-Myr-Akt1 or control vector. 24 h after transfection cells were subjected to Transwell migration assays and lysates were immunoblotted with the indicated antibodies. B , MDA-MB-231 cells were infected with palladin shRNA lentiviral vector or empty vector. Forty-eight hours after infection cells were serum-starved overnight then stimulated with IGF-1 (100 ng ml −1 ) for 18 h, followed by Transwell migration assays. Total cell lysates were subjected to immunoblot analysis. C , MDA-MB-231 cells were infected with palladin shRNA and/or Akt1 shRNA lentiviral vectors, or empty vector. Forty-eight hours after infection, cells were subjected to Transwell migration assays and lysates were immunoblotted. D .
    α Pt450 Akt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α pt450 akt1/product/Cell Signaling Technology Inc
    Average 78 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    α pt450 akt1 - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc phospho akt1 ser473
    Figure 6. Knockdown of LAMP2A triggers <t>AKT1</t> phosphorylation at <t>Ser473.</t> Proliferating T47D cells were either transfected with LAMP2A, or empty pcDNA3 vector for 48 h ( A, C and E ) and with LAMP2A siRNA or control siRNA for 72 h ( B, D and F ). ( A and B ) showing western blots analysis of total AKT1 and phospho AKT1 (Ser 473). ( C and D ) demonstrate AKT1 kinase activity using AKT1 substrate GSK3 fusion protein as a measure of AKT1 activation. ( E and F ) demonstrate the AKT1 substrate assay under oxidative stress condition rendered by 150 nM H 2 O 2 . Densitometric analyses from ( E and F ) are given under each panel. Data are mean ± SEMs, and significant changes are indicated by an asterisk (*p
    Phospho Akt1 Ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho akt1 ser473/product/Cell Signaling Technology Inc
    Average 99 stars, based on 56 article reviews
    Price from $9.99 to $1999.99
    phospho akt1 ser473 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    83
    Cell Signaling Technology Inc akt1 2 3
    MG132 treatment depresses PDGF-stimulated phosphorylation of ERK and also reduces the activation of upstream signaling components. a ) Immunoblot results, representative of 3 independent experiments, showing the phosphorylation kinetics of PDGF β-receptor Tyr 751 (pPDGFR), <t>Akt1/2/3</t> Ser 473 (pAkt), MEK1/2 Ser 217 /Ser 221 (pMEK), and ERK1/2 Thr 202 /Tyr 204 (pERK) in cells pretreated with either DMSO or 25 µM MG132 for 6 h and then stimulated with the indicated concentration of PDGF-BB. Stimulation times are 5, 15, 30, 60, and 120 minutes. Total ERK1/2 (tERK) serves as a loading control. For each antigen, the DMSO and MG132 bands are cropped from the same gel. At right it is shown that total Akt (tAkt) protein expression is not affected by MG132 treatment, whereas total MEK1/2 (tMEK) is only modestly increased in MG132-treated cells, relative to β-actin loading control. b-e ) Quantification of the phosphorylation kinetics represented in a . Each readout is normalized by total ERK and expressed as mean ± s.e.m. ( n = 3): b , pPDGFR; c , pAkt; d , pMEK; e , pERK. The indicated p value for each time course is from two-way ANOVA analysis comparing MG132-treated and control measurements.
    Akt1 2 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 83/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt1 2 3/product/Cell Signaling Technology Inc
    Average 83 stars, based on 28 article reviews
    Price from $9.99 to $1999.99
    akt1 2 3 - by Bioz Stars, 2020-02
    83/100 stars
      Buy from Supplier

    78
    Cell Signaling Technology Inc α y326 akt1
    MG132 treatment depresses PDGF-stimulated phosphorylation of ERK and also reduces the activation of upstream signaling components. a ) Immunoblot results, representative of 3 independent experiments, showing the phosphorylation kinetics of PDGF β-receptor Tyr 751 (pPDGFR), <t>Akt1/2/3</t> Ser 473 (pAkt), MEK1/2 Ser 217 /Ser 221 (pMEK), and ERK1/2 Thr 202 /Tyr 204 (pERK) in cells pretreated with either DMSO or 25 µM MG132 for 6 h and then stimulated with the indicated concentration of PDGF-BB. Stimulation times are 5, 15, 30, 60, and 120 minutes. Total ERK1/2 (tERK) serves as a loading control. For each antigen, the DMSO and MG132 bands are cropped from the same gel. At right it is shown that total Akt (tAkt) protein expression is not affected by MG132 treatment, whereas total MEK1/2 (tMEK) is only modestly increased in MG132-treated cells, relative to β-actin loading control. b-e ) Quantification of the phosphorylation kinetics represented in a . Each readout is normalized by total ERK and expressed as mean ± s.e.m. ( n = 3): b , pPDGFR; c , pAkt; d , pMEK; e , pERK. The indicated p value for each time course is from two-way ANOVA analysis comparing MG132-treated and control measurements.
    α Y326 Akt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α y326 akt1/product/Cell Signaling Technology Inc
    Average 78 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    α y326 akt1 - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    78
    Cell Signaling Technology Inc phospho specific akt1
    Effects of PPP3CA siRNA on VEGF-induced <t>AKT1</t> phosphorylation in OFPAE cells. Cells were transfected with the scrambled or PPP3CA siRNA for 48 h. After serum starvation, cells were treated with 10 ng/ml of VEGF. Proteins (15 μg/lane) were subjected
    Phospho Specific Akt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho specific akt1/product/Cell Signaling Technology Inc
    Average 78 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    phospho specific akt1 - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    80
    Cell Signaling Technology Inc akt1 phospho t308
    Effects of PPP3CA siRNA on VEGF-induced <t>AKT1</t> phosphorylation in OFPAE cells. Cells were transfected with the scrambled or PPP3CA siRNA for 48 h. After serum starvation, cells were treated with 10 ng/ml of VEGF. Proteins (15 μg/lane) were subjected
    Akt1 Phospho T308, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt1 phospho t308/product/Cell Signaling Technology Inc
    Average 80 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    akt1 phospho t308 - by Bioz Stars, 2020-02
    80/100 stars
      Buy from Supplier

    86
    Cell Signaling Technology Inc rabbit monoclonal akt 1 antibody
    Alteration of the insulin receptor signaling pathway in adipocytes cultured with amyloid‐β. ( A ) Insulin receptor substrate‐2 ( IRS2 ) gene expression in differentiated adipocytes incubated for 6 days with 100 or 1,000 pg/mL amyloid‐β40 ( n = 10–11 per condition). ( B ) IRS2 transcription in cultures treated with amyloid‐β42 at 10 or 100 pg/mL for 6 days ( n = 10–11 per condition). Representative immunoblot and quantification of ( C ) total <t>Akt‐1</t> and ( D ), phosphor‐serine 473 Akt‐1 and ( E ) ratio of phospho/total Akt‐1 in adipocyte cultures grown at 5.5 mM glucose with 10 nM insulin with 100 pg/mL or 1,000 pg/mL amyloid‐β40; n = 3 per condition, log‐transformed data. * P
    Rabbit Monoclonal Akt 1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit monoclonal akt 1 antibody/product/Cell Signaling Technology Inc
    Average 86 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    rabbit monoclonal akt 1 antibody - by Bioz Stars, 2020-02
    86/100 stars
      Buy from Supplier

    84
    Cell Signaling Technology Inc pathscan total akt1 sandwich elisa kit
    Alteration of the insulin receptor signaling pathway in adipocytes cultured with amyloid‐β. ( A ) Insulin receptor substrate‐2 ( IRS2 ) gene expression in differentiated adipocytes incubated for 6 days with 100 or 1,000 pg/mL amyloid‐β40 ( n = 10–11 per condition). ( B ) IRS2 transcription in cultures treated with amyloid‐β42 at 10 or 100 pg/mL for 6 days ( n = 10–11 per condition). Representative immunoblot and quantification of ( C ) total <t>Akt‐1</t> and ( D ), phosphor‐serine 473 Akt‐1 and ( E ) ratio of phospho/total Akt‐1 in adipocyte cultures grown at 5.5 mM glucose with 10 nM insulin with 100 pg/mL or 1,000 pg/mL amyloid‐β40; n = 3 per condition, log‐transformed data. * P
    Pathscan Total Akt1 Sandwich Elisa Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 84/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pathscan total akt1 sandwich elisa kit/product/Cell Signaling Technology Inc
    Average 84 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    pathscan total akt1 sandwich elisa kit - by Bioz Stars, 2020-02
    84/100 stars
      Buy from Supplier

    84
    Cell Signaling Technology Inc akt1 thr308
    The mitochondrial cycle of <t>Akt1</t> under redox stimuli. In (A) and (B), Akt1 requires to be phosphorylated in Ser 473 to enter mitochondria. Inactive Akt1-His tagged and mTORC2 were incubated in kinase buffer to allow phosphorylation. In absence of P-mTORC2 Akt1 remains outside mitochondria, In the presence of mTORC2, P-Akt Ser 473 translocates to mitochondria and becomes phosphorylated in Thr 308 ; approximately after 50 min biphosphorylated P-Akt Ser 473 /Thr 308 becomes detectable in the supernatant. These extracts were incubated with purified mitochondria in import buffer and the samples were centrifuged and prepared to run in SDS-PAGE. (C) Imaging of wb using a His-tag ab anti mouse conjugated to Cy3 and a <t>P-Thr308</t> ab anti rabbit conjugated to Cy2 shows colocalization (yellow) in the presence of P-mTORC2. Addition of PDK1 alone is unable to phosphorylate Akt1 in Thr 308 .
    Akt1 Thr308, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 84/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt1 thr308/product/Cell Signaling Technology Inc
    Average 84 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    akt1 thr308 - by Bioz Stars, 2020-02
    84/100 stars
      Buy from Supplier

    81
    Cell Signaling Technology Inc anti akt1 pser473
    The mitochondrial cycle of <t>Akt1</t> under redox stimuli. In (A) and (B), Akt1 requires to be phosphorylated in Ser 473 to enter mitochondria. Inactive Akt1-His tagged and mTORC2 were incubated in kinase buffer to allow phosphorylation. In absence of P-mTORC2 Akt1 remains outside mitochondria, In the presence of mTORC2, P-Akt Ser 473 translocates to mitochondria and becomes phosphorylated in Thr 308 ; approximately after 50 min biphosphorylated P-Akt Ser 473 /Thr 308 becomes detectable in the supernatant. These extracts were incubated with purified mitochondria in import buffer and the samples were centrifuged and prepared to run in SDS-PAGE. (C) Imaging of wb using a His-tag ab anti mouse conjugated to Cy3 and a <t>P-Thr308</t> ab anti rabbit conjugated to Cy2 shows colocalization (yellow) in the presence of P-mTORC2. Addition of PDK1 alone is unable to phosphorylate Akt1 in Thr 308 .
    Anti Akt1 Pser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 81/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti akt1 pser473/product/Cell Signaling Technology Inc
    Average 81 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    anti akt1 pser473 - by Bioz Stars, 2020-02
    81/100 stars
      Buy from Supplier

    77
    Cell Signaling Technology Inc protein akt1
    The mitochondrial cycle of <t>Akt1</t> under redox stimuli. In (A) and (B), Akt1 requires to be phosphorylated in Ser 473 to enter mitochondria. Inactive Akt1-His tagged and mTORC2 were incubated in kinase buffer to allow phosphorylation. In absence of P-mTORC2 Akt1 remains outside mitochondria, In the presence of mTORC2, P-Akt Ser 473 translocates to mitochondria and becomes phosphorylated in Thr 308 ; approximately after 50 min biphosphorylated P-Akt Ser 473 /Thr 308 becomes detectable in the supernatant. These extracts were incubated with purified mitochondria in import buffer and the samples were centrifuged and prepared to run in SDS-PAGE. (C) Imaging of wb using a His-tag ab anti mouse conjugated to Cy3 and a <t>P-Thr308</t> ab anti rabbit conjugated to Cy2 shows colocalization (yellow) in the presence of P-mTORC2. Addition of PDK1 alone is unable to phosphorylate Akt1 in Thr 308 .
    Protein Akt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein akt1/product/Cell Signaling Technology Inc
    Average 77 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    protein akt1 - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    76
    Cell Signaling Technology Inc akt1 2967 antibodies
    The mitochondrial cycle of <t>Akt1</t> under redox stimuli. In (A) and (B), Akt1 requires to be phosphorylated in Ser 473 to enter mitochondria. Inactive Akt1-His tagged and mTORC2 were incubated in kinase buffer to allow phosphorylation. In absence of P-mTORC2 Akt1 remains outside mitochondria, In the presence of mTORC2, P-Akt Ser 473 translocates to mitochondria and becomes phosphorylated in Thr 308 ; approximately after 50 min biphosphorylated P-Akt Ser 473 /Thr 308 becomes detectable in the supernatant. These extracts were incubated with purified mitochondria in import buffer and the samples were centrifuged and prepared to run in SDS-PAGE. (C) Imaging of wb using a His-tag ab anti mouse conjugated to Cy3 and a <t>P-Thr308</t> ab anti rabbit conjugated to Cy2 shows colocalization (yellow) in the presence of P-mTORC2. Addition of PDK1 alone is unable to phosphorylate Akt1 in Thr 308 .
    Akt1 2967 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt1 2967 antibodies/product/Cell Signaling Technology Inc
    Average 76 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    akt1 2967 antibodies - by Bioz Stars, 2020-02
    76/100 stars
      Buy from Supplier

    79
    Cell Signaling Technology Inc akt1 c73h10
    The mitochondrial cycle of <t>Akt1</t> under redox stimuli. In (A) and (B), Akt1 requires to be phosphorylated in Ser 473 to enter mitochondria. Inactive Akt1-His tagged and mTORC2 were incubated in kinase buffer to allow phosphorylation. In absence of P-mTORC2 Akt1 remains outside mitochondria, In the presence of mTORC2, P-Akt Ser 473 translocates to mitochondria and becomes phosphorylated in Thr 308 ; approximately after 50 min biphosphorylated P-Akt Ser 473 /Thr 308 becomes detectable in the supernatant. These extracts were incubated with purified mitochondria in import buffer and the samples were centrifuged and prepared to run in SDS-PAGE. (C) Imaging of wb using a His-tag ab anti mouse conjugated to Cy3 and a <t>P-Thr308</t> ab anti rabbit conjugated to Cy2 shows colocalization (yellow) in the presence of P-mTORC2. Addition of PDK1 alone is unable to phosphorylate Akt1 in Thr 308 .
    Akt1 C73h10, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 79/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt1 c73h10/product/Cell Signaling Technology Inc
    Average 79 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    akt1 c73h10 - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    78
    Cell Signaling Technology Inc akt1 t308
    The mitochondrial cycle of <t>Akt1</t> under redox stimuli. In (A) and (B), Akt1 requires to be phosphorylated in Ser 473 to enter mitochondria. Inactive Akt1-His tagged and mTORC2 were incubated in kinase buffer to allow phosphorylation. In absence of P-mTORC2 Akt1 remains outside mitochondria, In the presence of mTORC2, P-Akt Ser 473 translocates to mitochondria and becomes phosphorylated in Thr 308 ; approximately after 50 min biphosphorylated P-Akt Ser 473 /Thr 308 becomes detectable in the supernatant. These extracts were incubated with purified mitochondria in import buffer and the samples were centrifuged and prepared to run in SDS-PAGE. (C) Imaging of wb using a His-tag ab anti mouse conjugated to Cy3 and a <t>P-Thr308</t> ab anti rabbit conjugated to Cy2 shows colocalization (yellow) in the presence of P-mTORC2. Addition of PDK1 alone is unable to phosphorylate Akt1 in Thr 308 .
    Akt1 T308, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt1 t308/product/Cell Signaling Technology Inc
    Average 78 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    akt1 t308 - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    77
    Cell Signaling Technology Inc akt1 2938
    The mitochondrial cycle of <t>Akt1</t> under redox stimuli. In (A) and (B), Akt1 requires to be phosphorylated in Ser 473 to enter mitochondria. Inactive Akt1-His tagged and mTORC2 were incubated in kinase buffer to allow phosphorylation. In absence of P-mTORC2 Akt1 remains outside mitochondria, In the presence of mTORC2, P-Akt Ser 473 translocates to mitochondria and becomes phosphorylated in Thr 308 ; approximately after 50 min biphosphorylated P-Akt Ser 473 /Thr 308 becomes detectable in the supernatant. These extracts were incubated with purified mitochondria in import buffer and the samples were centrifuged and prepared to run in SDS-PAGE. (C) Imaging of wb using a His-tag ab anti mouse conjugated to Cy3 and a <t>P-Thr308</t> ab anti rabbit conjugated to Cy2 shows colocalization (yellow) in the presence of P-mTORC2. Addition of PDK1 alone is unable to phosphorylate Akt1 in Thr 308 .
    Akt1 2938, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt1 2938/product/Cell Signaling Technology Inc
    Average 77 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    akt1 2938 - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    76
    Cell Signaling Technology Inc p akt1 ser 473
    ZINC4085554 inhibits pressure-stimulated FAK-Tyr-397 yet not <t>Akt1-Ser-473</t> phosphorylation, and Akt-Thr-308 remains unaffected by pressure and/or ZINC4085554. SW620 cells were treated with vehicle (0.1% DMSO) or 50 µM ZINC4085554 and subjected to ambient or 15 mmHg increased extracellular pressure for 30 min. (A) Representative blots probed for FAK-Tyr-397 and total FAK. (B) Densitometric quantitation of the blots in A. (C) Representative blots probed for Akt1-Ser-473 and total Akt1. (D) Densitometric quantitation of the blots in C. (E) Representative blots probed for Akt-Thr-308 and total Akt1. (F) Densitometric quantitation of the blots in E. (n=5-8) *P
    P Akt1 Ser 473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 76/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p akt1 ser 473/product/Cell Signaling Technology Inc
    Average 76 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    p akt1 ser 473 - by Bioz Stars, 2020-02
    76/100 stars
      Buy from Supplier

    80
    Cell Signaling Technology Inc recombinant akt1 protein
    <t>Akt1</t> sequentially phosphorylates p27 at S10 and a neighboring serine . A , Akt1 does not phosphorylate hp27S10A. Upper panel: Recombinant Akt1 was incubated in a kinase reaction with the indicated forms of p27 as described. Samples were separated by SDS/PAGE and visualized by autoradiography. Bottom panel: His-tagged cyclin-E-CDK2 was purified from transfected HEK293 cells (see Methods), then incubated with indicated forms of p27 in the presence of [ 32 P]-γ-ATP. Samples were separated by SDS-PAGE and visualized by autoradiography. Commassie staining shows equal amounts of hwtp27 and p27S10A were present in the reactions. B , Akt1 phosphorylates hp27S10T. Kinase reaction was performed with recombinant Akt1 and indicated substrates. Upper panel: autoradiograph showing phosphorylated p27. Bottom panel: western blot showing similar levels of p27 in the reaction. C , Phospho-amino acid analysis comparing hwtp27 and hp27S10T. Kinase reaction described in B was repeated and radiolabeled p27 was subjected to PAA as in Figure 3C. Top panel: scheme representing migration of phospho-amino acid standards. Second panel: Phospho-amino acid standards separated by electrophoresis and visualized by 0.25% ninhydrin. Third panel: phospho-amino acid analysis of radiolabeled hwtp27. Bottom panel: phospho-amino acid analysis of hp27S10T. Radiolabeled peptides and amino acids were detected by phosphoimager.
    Recombinant Akt1 Protein, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant akt1 protein/product/Cell Signaling Technology Inc
    Average 80 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    recombinant akt1 protein - by Bioz Stars, 2020-02
    80/100 stars
      Buy from Supplier

    79
    Cell Signaling Technology Inc rabbit phospho akt1
    <t>AKT1</t> and AKT3 are phosphorylated in different types of spermatogonia. (A) Whole-mount wild-type or Tg(Gdnf) tubules co-immunostained for phosphorylated AKT1 Ser473 (P-AKT1) and GFRA1. GFRA + cells in wild-type testes (arrows) and large clusters of GFRA + cells in Tg(GDNF) testes (asterisks) are P-AKT1 negative. (B) Whole-mount wild-type or Tg(Gdnf) tubules co-immunostained for phosphorylated AKT3 Ser472 (P-AKT3) and GFRA1. Arrows indicate GFRA1 + P-AKT3 + A s and A pr spermatogonia. Arrowheads indicate GFRA1 + P-AKT3 + cluster in Tg(Gdnf) . Scale bars: 100 µm.
    Rabbit Phospho Akt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit phospho akt1/product/Cell Signaling Technology Inc
    Average 79 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    rabbit phospho akt1 - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    76
    Cell Signaling Technology Inc akt1 s473
    <t>AKT1</t> and AKT3 are phosphorylated in different types of spermatogonia. (A) Whole-mount wild-type or Tg(Gdnf) tubules co-immunostained for phosphorylated AKT1 Ser473 (P-AKT1) and GFRA1. GFRA + cells in wild-type testes (arrows) and large clusters of GFRA + cells in Tg(GDNF) testes (asterisks) are P-AKT1 negative. (B) Whole-mount wild-type or Tg(Gdnf) tubules co-immunostained for phosphorylated AKT3 Ser472 (P-AKT3) and GFRA1. Arrows indicate GFRA1 + P-AKT3 + A s and A pr spermatogonia. Arrowheads indicate GFRA1 + P-AKT3 + cluster in Tg(Gdnf) . Scale bars: 100 µm.
    Akt1 S473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt1 s473/product/Cell Signaling Technology Inc
    Average 76 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    akt1 s473 - by Bioz Stars, 2020-02
    76/100 stars
      Buy from Supplier

    88
    Cell Signaling Technology Inc pathscan phospho akt1 ser473 sandwich elisa kit
    <t>AKT1</t> and AKT3 are phosphorylated in different types of spermatogonia. (A) Whole-mount wild-type or Tg(Gdnf) tubules co-immunostained for phosphorylated AKT1 Ser473 (P-AKT1) and GFRA1. GFRA + cells in wild-type testes (arrows) and large clusters of GFRA + cells in Tg(GDNF) testes (asterisks) are P-AKT1 negative. (B) Whole-mount wild-type or Tg(Gdnf) tubules co-immunostained for phosphorylated AKT3 Ser472 (P-AKT3) and GFRA1. Arrows indicate GFRA1 + P-AKT3 + A s and A pr spermatogonia. Arrowheads indicate GFRA1 + P-AKT3 + cluster in Tg(Gdnf) . Scale bars: 100 µm.
    Pathscan Phospho Akt1 Ser473 Sandwich Elisa Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pathscan phospho akt1 ser473 sandwich elisa kit/product/Cell Signaling Technology Inc
    Average 88 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    pathscan phospho akt1 ser473 sandwich elisa kit - by Bioz Stars, 2020-02
    88/100 stars
      Buy from Supplier

    78
    Cell Signaling Technology Inc akt1 kinase assay akt1 kinase assay
    Deficit in BDNF-stimulated protein translation in cultured cortical primary neurons from APP/PS1 mice is rescued by myristoylated <t>Akt1</t> overexpression. Increased protein translation was observed in both soma and neurites of primary cortical neurons isolated from WT mice after BDNF stimulation. Stimulation of protein translation at soma and neurites after BDNF treatment was not observed in primary cortical neurons isolated from APP/PS1 mice. Overexpression of myristoylated Akt1 (myr-Akt1), which is constitutively active, rescues the compromised stimulated protein translation in APP/PS1 neurons. Values are mean ± SEM ( n = 31–36 neurons) and * denotes values significantly different from corresponding controls ( p
    Akt1 Kinase Assay Akt1 Kinase Assay, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/akt1 kinase assay akt1 kinase assay/product/Cell Signaling Technology Inc
    Average 78 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    akt1 kinase assay akt1 kinase assay - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    84
    Cell Signaling Technology Inc anti akt1 2 3
    Deficit in BDNF-stimulated protein translation in cultured cortical primary neurons from APP/PS1 mice is rescued by myristoylated <t>Akt1</t> overexpression. Increased protein translation was observed in both soma and neurites of primary cortical neurons isolated from WT mice after BDNF stimulation. Stimulation of protein translation at soma and neurites after BDNF treatment was not observed in primary cortical neurons isolated from APP/PS1 mice. Overexpression of myristoylated Akt1 (myr-Akt1), which is constitutively active, rescues the compromised stimulated protein translation in APP/PS1 neurons. Values are mean ± SEM ( n = 31–36 neurons) and * denotes values significantly different from corresponding controls ( p
    Anti Akt1 2 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 84/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti akt1 2 3/product/Cell Signaling Technology Inc
    Average 84 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    anti akt1 2 3 - by Bioz Stars, 2020-02
    84/100 stars
      Buy from Supplier

    Image Search Results


    ActA stimulates SMAD2 phosphorylation, AKT1 activation and increases IκBα levels in mature osteoclasts. (A,B) Stroma-free BMM OCL precursors cultured in the presence of MCSF and RANKL for 2 or 5 days. Cells were serum starved for 6 hours

    Journal: Journal of Cell Science

    Article Title: Activin A inhibits RANKL-mediated osteoclast formation, movement and function in murine bone marrow macrophage cultures

    doi: 10.1242/jcs.157834

    Figure Lengend Snippet: ActA stimulates SMAD2 phosphorylation, AKT1 activation and increases IκBα levels in mature osteoclasts. (A,B) Stroma-free BMM OCL precursors cultured in the presence of MCSF and RANKL for 2 or 5 days. Cells were serum starved for 6 hours

    Article Snippet: Antibodies were against CatK (Abcam, Cambridge, MA # ab19027), cleaved caspase-3, P-SMAD2, P-AKT1, total AKT1, total IκBα (Cell Signaling Technology, Danvers, MA, catalog # 9664, 3101, 9271, 9272, 4812, respectively) and β-tubulin (Thermo Scientific, Rockford, IL, clone #TBN06).

    Techniques: Activation Assay, Cell Culture

    DSS mice display lowered SUMOylated Akt1 and compromised kinase activity. ( a ) Immunoblots of total AKT, Akt1 and pAkt1 (S473) of proximal colon (PC), distal colon (DC) and colonic intestinal epithelium (IEC). ( b ) Various colonic tissue lysates were immunoprecipitated with anti-SUMO1 antibodies (upper panel) and immunoblotted for Akt1 showing reduced SUMO Akt1 in DSS-treated mice and IP with anti-Akt1 antibody and probed for SUMO-1 to detect SUMOylated-Akt1. Ten per cent of the input was loaded as shown in the blot (bottom panel). The ratios of SUMO-Akt1/Akt1 as well as pAkt1/Akt1 are shown based on densitometric values obtained from the IP immunoblots. ( c ) Immunoblots of total Akt1 and phospho-Akt1 (S473) from colonic lysates of mice. ( d ) Immunoblot of phospho-GSK3β (S9) and total GSK3β from mice samples. ( e ) Colonic epithelial lysates from control and DSS-treated animals were added to Akt-specific kinase activity mixture involving recombinant GSK3β (28 kDa), ATP and optimal buffer for 30 min at 30°C. Akt kinase activity was then determined by immunoblotting for pGSK3β. ( f ) Ubc9 and Akt1 interaction was tested using IP of colonic lysates with anti-Ubc9 antibody and probed with anti-phospho-Akt1 antibody, input lysates are represented at the bottom. β-Actin was used as a loading control. In all IP experiments and kinase assays, an equal amount of total protein (500 µg) was used in each category of samples. Mice samples DSS-7 1 (Ubc9

    Journal: Open Biology

    Article Title: SUMOylation pathway alteration coupled with downregulation of SUMO E2 enzyme at mucosal epithelium modulates inflammation in inflammatory bowel disease

    doi: 10.1098/rsob.170024

    Figure Lengend Snippet: DSS mice display lowered SUMOylated Akt1 and compromised kinase activity. ( a ) Immunoblots of total AKT, Akt1 and pAkt1 (S473) of proximal colon (PC), distal colon (DC) and colonic intestinal epithelium (IEC). ( b ) Various colonic tissue lysates were immunoprecipitated with anti-SUMO1 antibodies (upper panel) and immunoblotted for Akt1 showing reduced SUMO Akt1 in DSS-treated mice and IP with anti-Akt1 antibody and probed for SUMO-1 to detect SUMOylated-Akt1. Ten per cent of the input was loaded as shown in the blot (bottom panel). The ratios of SUMO-Akt1/Akt1 as well as pAkt1/Akt1 are shown based on densitometric values obtained from the IP immunoblots. ( c ) Immunoblots of total Akt1 and phospho-Akt1 (S473) from colonic lysates of mice. ( d ) Immunoblot of phospho-GSK3β (S9) and total GSK3β from mice samples. ( e ) Colonic epithelial lysates from control and DSS-treated animals were added to Akt-specific kinase activity mixture involving recombinant GSK3β (28 kDa), ATP and optimal buffer for 30 min at 30°C. Akt kinase activity was then determined by immunoblotting for pGSK3β. ( f ) Ubc9 and Akt1 interaction was tested using IP of colonic lysates with anti-Ubc9 antibody and probed with anti-phospho-Akt1 antibody, input lysates are represented at the bottom. β-Actin was used as a loading control. In all IP experiments and kinase assays, an equal amount of total protein (500 µg) was used in each category of samples. Mice samples DSS-7 1 (Ubc9

    Article Snippet: Blots were probed with antibodies against Ubc9 (Sigma), SUMO1 (Sigma), SUMO2/3 (Sigma), AKT (CST, USA), p-AKT (CST, USA), Akt1 (CST, USA), p-Akt1 (CST, USA), GSK3β (Abcam, USA) and p-GSK3β (CST, USA).

    Techniques: Mouse Assay, Activity Assay, Western Blot, Immunoprecipitation, Recombinant

    SUMOylation-deficient Akt1 exacerbates inflammation accompanied with impaired wound healing. ( a ) Values of IL-8 ELISA of supernatants from HCT-8 cells knocked-down for Ubc9 (UKD) or untreated cells. ( b ) ELISA of indicated cytokine with supernatants from HCT-8 cells transfected with WT-Akt1, DN-Akt1 (kinase dead) and SMUT-Akt1 (SUMO-deficient) plasmids. ( c ) Cell migration assessed by wound-healing assay in untreated (Ctl) or WT-Akt1, kinase dead Akt1 (DN) and SUMOylation-deficient Akt1 encoding plasmid transfected HCT-8 cells and Ubc9 knocked-down cells (UKD). The wound closure scores with statistics are plotted (lower panel), which show the compromised healing seen in SMUT and UKD samples. ( d ) qRT-PCR gene expression of wound-healing and inflammatory markers in WT-Akt1, DN-Akt-1 (kinase dead) and SMUT-Akt1 (SUMO-deficient) samples compared with control. ( e ) qRT-PCR gene expression of TNFAIP3, TNFAIP8 and PTGES in human IBD patient samples. UC 1 and CD 1 correspond to UC UBC9-Low and CD UBC9-Low , respectively. UC 2 and CD 2 correspond to UC UBC9-HyperLow and CD UBC9-HyperLow , respectively. For qRT, GAPDH and HPRT were taken for normalization.

    Journal: Open Biology

    Article Title: SUMOylation pathway alteration coupled with downregulation of SUMO E2 enzyme at mucosal epithelium modulates inflammation in inflammatory bowel disease

    doi: 10.1098/rsob.170024

    Figure Lengend Snippet: SUMOylation-deficient Akt1 exacerbates inflammation accompanied with impaired wound healing. ( a ) Values of IL-8 ELISA of supernatants from HCT-8 cells knocked-down for Ubc9 (UKD) or untreated cells. ( b ) ELISA of indicated cytokine with supernatants from HCT-8 cells transfected with WT-Akt1, DN-Akt1 (kinase dead) and SMUT-Akt1 (SUMO-deficient) plasmids. ( c ) Cell migration assessed by wound-healing assay in untreated (Ctl) or WT-Akt1, kinase dead Akt1 (DN) and SUMOylation-deficient Akt1 encoding plasmid transfected HCT-8 cells and Ubc9 knocked-down cells (UKD). The wound closure scores with statistics are plotted (lower panel), which show the compromised healing seen in SMUT and UKD samples. ( d ) qRT-PCR gene expression of wound-healing and inflammatory markers in WT-Akt1, DN-Akt-1 (kinase dead) and SMUT-Akt1 (SUMO-deficient) samples compared with control. ( e ) qRT-PCR gene expression of TNFAIP3, TNFAIP8 and PTGES in human IBD patient samples. UC 1 and CD 1 correspond to UC UBC9-Low and CD UBC9-Low , respectively. UC 2 and CD 2 correspond to UC UBC9-HyperLow and CD UBC9-HyperLow , respectively. For qRT, GAPDH and HPRT were taken for normalization.

    Article Snippet: Blots were probed with antibodies against Ubc9 (Sigma), SUMO1 (Sigma), SUMO2/3 (Sigma), AKT (CST, USA), p-AKT (CST, USA), Akt1 (CST, USA), p-Akt1 (CST, USA), GSK3β (Abcam, USA) and p-GSK3β (CST, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Migration, Wound Healing Assay, CTL Assay, Plasmid Preparation, Quantitative RT-PCR, Expressing

    The SUMO pathway plays a crucial role in IBD: model representation of importance of SUMOylation in intestinal health and disease. Steady-state events of the healthy epithelium are represented on the left. A fine balance between SUMOylated and non-SUMOylated proteins is maintained. During the onset of disease, due to downregulation of the sole E2 SUMO conjugating enzyme Ubc9, the balance of SUMOylated and non-SUMOylated proteins is tilted as shown in the see-saw. This activates the NFκB signalling pathway via master signalling regulator Akt1. These changes results in upregulation of transcriptional factors like cFOS, cJUN, RelA, STAT1, BIRC3 (upward arrows). Release of chemokine IL-8/CXCL8 for neutrophil recruitment and lower levels of anti-inflammatory cytokine IL-10 and TSLP further aggravate the condition (severely inflamed cell on the right side). These events leads to compromised expression of wound-healing markers TNFAIP3 and TNFAIP8 (downward arrows) leading to impaired wound healing.

    Journal: Open Biology

    Article Title: SUMOylation pathway alteration coupled with downregulation of SUMO E2 enzyme at mucosal epithelium modulates inflammation in inflammatory bowel disease

    doi: 10.1098/rsob.170024

    Figure Lengend Snippet: The SUMO pathway plays a crucial role in IBD: model representation of importance of SUMOylation in intestinal health and disease. Steady-state events of the healthy epithelium are represented on the left. A fine balance between SUMOylated and non-SUMOylated proteins is maintained. During the onset of disease, due to downregulation of the sole E2 SUMO conjugating enzyme Ubc9, the balance of SUMOylated and non-SUMOylated proteins is tilted as shown in the see-saw. This activates the NFκB signalling pathway via master signalling regulator Akt1. These changes results in upregulation of transcriptional factors like cFOS, cJUN, RelA, STAT1, BIRC3 (upward arrows). Release of chemokine IL-8/CXCL8 for neutrophil recruitment and lower levels of anti-inflammatory cytokine IL-10 and TSLP further aggravate the condition (severely inflamed cell on the right side). These events leads to compromised expression of wound-healing markers TNFAIP3 and TNFAIP8 (downward arrows) leading to impaired wound healing.

    Article Snippet: Blots were probed with antibodies against Ubc9 (Sigma), SUMO1 (Sigma), SUMO2/3 (Sigma), AKT (CST, USA), p-AKT (CST, USA), Akt1 (CST, USA), p-Akt1 (CST, USA), GSK3β (Abcam, USA) and p-GSK3β (CST, USA).

    Techniques: Expressing

    Experimental repression of Ubc9 in primary and cultured epithelial cells downmodulates active Akt1. ( a ) Immunoblots with indicated antibodies from lysates of HCT-8 cells with either Akt1 knockdown using Akt1-siRNA (AKD), Ubc9 knock-down using Ubc9-siRNA (UKD), Ubc9 over-expression using pUbc9 (UOE), along with corresponding control lysates (Ctl). Corresponding densitometric values of total Akt1 ( b ), ratios of pAkt1/Akt1 ( c ) and Ubc9 ( d ) were also represented as bar graphs. ( e ) Immunoblots of Ubc9 from HCT-8 cells treated specifically with Akt1 kinase inhibitor (AKTinh), siRNA-for Akt1 (AKD), UKD or left untreated (Ctl). ( f ) Schematic overview of steps involved in ex vivo culturing of PIECs from mice colon (actual picture of epithelial culture represented here). ( g ) Immunoblots of Ubc9, p-Akt1 and total Akt1 from lysates of PIEC treated with the indicated inhibitors (AKTinh, Akt1 inhibitor; LLO, listeriolysin; SAM, S -adenosyl methionine). Right panel: densitometric analysis of immunoblots. ( h , i ) ELISA for IFN-γ and IL-10 of supernatant of PIECs treated as indicated. Supernatant of DSS-7 mice primary epithelial cells were used as positive controls for inflammation.

    Journal: Open Biology

    Article Title: SUMOylation pathway alteration coupled with downregulation of SUMO E2 enzyme at mucosal epithelium modulates inflammation in inflammatory bowel disease

    doi: 10.1098/rsob.170024

    Figure Lengend Snippet: Experimental repression of Ubc9 in primary and cultured epithelial cells downmodulates active Akt1. ( a ) Immunoblots with indicated antibodies from lysates of HCT-8 cells with either Akt1 knockdown using Akt1-siRNA (AKD), Ubc9 knock-down using Ubc9-siRNA (UKD), Ubc9 over-expression using pUbc9 (UOE), along with corresponding control lysates (Ctl). Corresponding densitometric values of total Akt1 ( b ), ratios of pAkt1/Akt1 ( c ) and Ubc9 ( d ) were also represented as bar graphs. ( e ) Immunoblots of Ubc9 from HCT-8 cells treated specifically with Akt1 kinase inhibitor (AKTinh), siRNA-for Akt1 (AKD), UKD or left untreated (Ctl). ( f ) Schematic overview of steps involved in ex vivo culturing of PIECs from mice colon (actual picture of epithelial culture represented here). ( g ) Immunoblots of Ubc9, p-Akt1 and total Akt1 from lysates of PIEC treated with the indicated inhibitors (AKTinh, Akt1 inhibitor; LLO, listeriolysin; SAM, S -adenosyl methionine). Right panel: densitometric analysis of immunoblots. ( h , i ) ELISA for IFN-γ and IL-10 of supernatant of PIECs treated as indicated. Supernatant of DSS-7 mice primary epithelial cells were used as positive controls for inflammation.

    Article Snippet: Blots were probed with antibodies against Ubc9 (Sigma), SUMO1 (Sigma), SUMO2/3 (Sigma), AKT (CST, USA), p-AKT (CST, USA), Akt1 (CST, USA), p-Akt1 (CST, USA), GSK3β (Abcam, USA) and p-GSK3β (CST, USA).

    Techniques: Cell Culture, Western Blot, Over Expression, CTL Assay, Ex Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Human CD and UC patient samples display severe downregulation of Ubc9 and Akt1 function. ( a ) Colonoscopy images showing the colon of healthy, UC and CD patients. ( b , c ) qPCR analysis of SUMOylation-pathway genes in colonic biopsy samples of human UC ( b ) and CD ( c ) patients normalized to averaged control values ( n = 22) are plotted as relative fold expression. Each dot represents data from one individual, black dots represent CD UBC9-Low and UC UBC9-Low and red dots represent CD UBC9-HyperLow and UC UBC9-HyperLow . ( d ) Immunoblots of SUMO-1 and Ubc9 of patient samples showing overall decrease in SUMO proteome. ( e ) ELISA for IFN-γ, IL-6, TNF-α, TGF-β, IL-10 and TSLP from pooled patient tissue lysates ( n = 5 in each group) were performed and the specific values as indicated were plotted. UC 1 and CD 1 correspond to UC UBC9Low and CD UBC9Low , respectively. UC 2 and CD 2 correspond to UC UBC9HyperLow and CD UBC9HyperLow , respectively. ( f ) qPCR-based fold change expression of Akt1 in human CD and UC patients relative to averaged control values ( n = 22) are plotted (red dots showing more repression represent CD UBC9-HyperLow and UC UBC9-HyperLow and black dots represent CD UBC9-Low and UC UBC9-Low ). ( g ) Pooled lysates of control, CD and UC samples ( n = 5 in each group) were used for IP with anti-SUMO-1 antibody followed by immunoblotting with anti-phospho-Akt1 antibody. ( h ) Akt1-specific kinase activity was assayed by immunoblotting for pGSK3β from pooled lysates ( n = 5). The densitometry values representing lowered pGSK3β in CD and UC are also represented in the graph (lower panel). For human samples, statistical testing was performed between control and each diseased group using the Mann–Whitney U -test ( p -values as indicated). For qRT, GAPDH and HPRT ertr taken for normalization.

    Journal: Open Biology

    Article Title: SUMOylation pathway alteration coupled with downregulation of SUMO E2 enzyme at mucosal epithelium modulates inflammation in inflammatory bowel disease

    doi: 10.1098/rsob.170024

    Figure Lengend Snippet: Human CD and UC patient samples display severe downregulation of Ubc9 and Akt1 function. ( a ) Colonoscopy images showing the colon of healthy, UC and CD patients. ( b , c ) qPCR analysis of SUMOylation-pathway genes in colonic biopsy samples of human UC ( b ) and CD ( c ) patients normalized to averaged control values ( n = 22) are plotted as relative fold expression. Each dot represents data from one individual, black dots represent CD UBC9-Low and UC UBC9-Low and red dots represent CD UBC9-HyperLow and UC UBC9-HyperLow . ( d ) Immunoblots of SUMO-1 and Ubc9 of patient samples showing overall decrease in SUMO proteome. ( e ) ELISA for IFN-γ, IL-6, TNF-α, TGF-β, IL-10 and TSLP from pooled patient tissue lysates ( n = 5 in each group) were performed and the specific values as indicated were plotted. UC 1 and CD 1 correspond to UC UBC9Low and CD UBC9Low , respectively. UC 2 and CD 2 correspond to UC UBC9HyperLow and CD UBC9HyperLow , respectively. ( f ) qPCR-based fold change expression of Akt1 in human CD and UC patients relative to averaged control values ( n = 22) are plotted (red dots showing more repression represent CD UBC9-HyperLow and UC UBC9-HyperLow and black dots represent CD UBC9-Low and UC UBC9-Low ). ( g ) Pooled lysates of control, CD and UC samples ( n = 5 in each group) were used for IP with anti-SUMO-1 antibody followed by immunoblotting with anti-phospho-Akt1 antibody. ( h ) Akt1-specific kinase activity was assayed by immunoblotting for pGSK3β from pooled lysates ( n = 5). The densitometry values representing lowered pGSK3β in CD and UC are also represented in the graph (lower panel). For human samples, statistical testing was performed between control and each diseased group using the Mann–Whitney U -test ( p -values as indicated). For qRT, GAPDH and HPRT ertr taken for normalization.

    Article Snippet: Blots were probed with antibodies against Ubc9 (Sigma), SUMO1 (Sigma), SUMO2/3 (Sigma), AKT (CST, USA), p-AKT (CST, USA), Akt1 (CST, USA), p-Akt1 (CST, USA), GSK3β (Abcam, USA) and p-GSK3β (CST, USA).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, MANN-WHITNEY

    Cell proliferation assay in human iPSCs after RA exposure a GSEA analysis showing enrichment of Akt1/mTOR signature in RA 8 treated cells. hiPSCs-F RA 8 ( p

    Journal: Cell Death & Disease

    Article Title: Short-term retinoic acid treatment sustains pluripotency and suppresses differentiation of human induced pluripotent stem cells

    doi: 10.1038/s41419-017-0028-1

    Figure Lengend Snippet: Cell proliferation assay in human iPSCs after RA exposure a GSEA analysis showing enrichment of Akt1/mTOR signature in RA 8 treated cells. hiPSCs-F RA 8 ( p

    Article Snippet: After blocking in TBS-T (0.05% Tween 20) containing 5% nonfat dry milk for 1 h at room temperature, the membrane was incubated overnight at 4 °C with primary antibodies against Nanog (PA1-097, Thermo Fisher Scientific), Oct4 (60093, STEMCELL Technologies), β-catenin (sc-7963, Santacruz), phospho-Akt Ser473 (9271, Cell Signalling), phospho-Akt Thr308 (4056, Cell Signalling), Akt1 (2967, Cell Signalling), mTOR (2983, Cell Signalling), phospho-mTOR Ser2448 (5536, Cell Signalling), S6K1 (2708, Cell Signalling), phospho-S6K1 Thr389 (9206, Cell Signalling), and Actin (sc-1616, Santa Cruz).

    Techniques: Proliferation Assay

    Butein selectively inhibits Akt1 and Akt1 is necessary for effects of butein in adipocytes. a C3H10T1/2 adipocytes were treated with 10, 20, 40 μM of Pan Akt inhibitor (Akt1/2 i) for 6 h and expression levels of Ucp1 and Prdm4 mRNA were determined by real-time PCR. b C3H10T1/2 adipocytes were treated with Akt1/2 i (10, 20, or 40 μM) for 6 h and expression levels of Prdm4 and Ucp1 protein were measured by western blotting. c C3H10T1/2 adipocytes were treated with 20 μM of Pan Akt inhibitor (Akt1/2 i) for 6 h and consumption rates (OCR) was measured in approximately 8 min intervals using XF24 Extracellular Flux Analyzer. Data represent means ± s.d. ( n = 3). d C3H10T1/2 adipocytes were treated with butein, and Akt1 and Akt2 phosphorylation levels were determined by western blot analysis. e Akt1 and Akt2 phosphorylation levels in epididymal adipose tissue (eWAT) of HFD-fed C57BL/6J mice treated with butein or control for 3 weeks ( n = 4 per group) were determined by western blot analysis. f Mouse embryonic fibroblast isolated from wild-type (WT) mice or Akt1 knockout (KO) mice were treated with DMSO (control) or butein for 12 h and expression of Prdm4 and thermogenic genes were determined. g Expression of Akt1 in WT and KO MEF was verified by western blot analysis. h Model of adipocyte browning by butein. Butein inhibit the PI3Kα–Akt1 pathway in adipocytes, leading to upregulation of Prdm4 followed by expression of thermogenic genes. Data represent mean ± s.e.m. and statistically significant differences were determined by Student’s t -test. * P

    Journal: Cell Death & Disease

    Article Title: PI3Ka-Akt1-mediated Prdm4 induction in adipose tissue increases energy expenditure, inhibits weight gain, and improves insulin resistance in diet-induced obese mice

    doi: 10.1038/s41419-018-0904-3

    Figure Lengend Snippet: Butein selectively inhibits Akt1 and Akt1 is necessary for effects of butein in adipocytes. a C3H10T1/2 adipocytes were treated with 10, 20, 40 μM of Pan Akt inhibitor (Akt1/2 i) for 6 h and expression levels of Ucp1 and Prdm4 mRNA were determined by real-time PCR. b C3H10T1/2 adipocytes were treated with Akt1/2 i (10, 20, or 40 μM) for 6 h and expression levels of Prdm4 and Ucp1 protein were measured by western blotting. c C3H10T1/2 adipocytes were treated with 20 μM of Pan Akt inhibitor (Akt1/2 i) for 6 h and consumption rates (OCR) was measured in approximately 8 min intervals using XF24 Extracellular Flux Analyzer. Data represent means ± s.d. ( n = 3). d C3H10T1/2 adipocytes were treated with butein, and Akt1 and Akt2 phosphorylation levels were determined by western blot analysis. e Akt1 and Akt2 phosphorylation levels in epididymal adipose tissue (eWAT) of HFD-fed C57BL/6J mice treated with butein or control for 3 weeks ( n = 4 per group) were determined by western blot analysis. f Mouse embryonic fibroblast isolated from wild-type (WT) mice or Akt1 knockout (KO) mice were treated with DMSO (control) or butein for 12 h and expression of Prdm4 and thermogenic genes were determined. g Expression of Akt1 in WT and KO MEF was verified by western blot analysis. h Model of adipocyte browning by butein. Butein inhibit the PI3Kα–Akt1 pathway in adipocytes, leading to upregulation of Prdm4 followed by expression of thermogenic genes. Data represent mean ± s.e.m. and statistically significant differences were determined by Student’s t -test. * P

    Article Snippet: These membranes were probed with primary antibodies against Prdm4 (ab156867, Abcam, Cambridge, MA, USA), Ucp1 (ab10983, Abcam), Akt (4685s, Cell Signaling, Danvers, MA, USA), p-Akt (4060s, Cell Signaling), Akt1 (75692s, Cell Signaling) p-Akt1 (9018s, Cell Signaling), Akt2 (5239s, Cell Signaling), p-Akt2 (8599s, Cell Signaling), or actin (sc-47778 horseradish peroxidase (HRP), Santa Cruz Biotech, Santa Cruz, CA, USA) followed by incubation with HRP-conjugated secondary antibodies (AbFrontier).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Mouse Assay, Isolation, Knock-Out

    Figure 5. DMSO-induced autophagy is controlled through ATF4-mediated downregulation of phosphorylated AKT1. Expression of ATF4 and LC3 were assessed by western blotting and RT-PCR in the presence of siRNA against ATF4. (A and B) ATF4 siRNA were

    Journal: Autophagy

    Article Title: Dimethyl sulfoxide reduces hepatocellular lipid accumulation through autophagy induction

    doi: 10.4161/auto.20260

    Figure Lengend Snippet: Figure 5. DMSO-induced autophagy is controlled through ATF4-mediated downregulation of phosphorylated AKT1. Expression of ATF4 and LC3 were assessed by western blotting and RT-PCR in the presence of siRNA against ATF4. (A and B) ATF4 siRNA were

    Article Snippet: Proteins were detected using the following primary antibodies: LC3B (Cell Signaling Technology, 2275), ATF4 (Santa Cruz Biotechnology, C-20), HSPA5 (Abcam, ab21685), p-EIF2AK3 (Santa Cruz Biotechnology, SC-32577), p-EIF2S1 (Cell Signaling Technology, 3597), ATF6 (Abcam, ab11909), p-ERN1 (Santa Cruz Biotechnology, SC-20790), sXbp1 (BioLegend, 619502), DDIT3 (Santa Cruz Biotechnology, SC-575), AKT1 (Cell Signaling Technology, 4691), phospho-AKT1 (Cell Signaling Technology, 4060), phospho-MTOR (Cell Signaling Technology, 2971), phopho-RPS6KB2 (Cell Signaling Technology, 9208), GFP (Cell Signaling Technology, 2956), SQSTM1 (Cell Signaling Technology, 5114) and actin (Santa Cruz Biotechnology, SC-47778) were used as primary antibodies.

    Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Akt1 silencing disrupts the promotion of O -GlcNAcylation on invasion of thyroid anaplastic cancer cells. Notes: ( A ) Akt1-silenced or -non-silenced 8305C cells were treated or untreated with 5 μM Thiamet-G (Thia-G) for 24 hours. Invasion assay in vitro was carried out. Error bars represent the mean ± standard error of the mean of three independent experiments, * P

    Journal: OncoTargets and therapy

    Article Title: O-GlcNAcylation enhances the invasion of thyroid anaplastic cancer cells partially by PI3K/Akt1 pathway

    doi: 10.2147/OTT.S82845

    Figure Lengend Snippet: Akt1 silencing disrupts the promotion of O -GlcNAcylation on invasion of thyroid anaplastic cancer cells. Notes: ( A ) Akt1-silenced or -non-silenced 8305C cells were treated or untreated with 5 μM Thiamet-G (Thia-G) for 24 hours. Invasion assay in vitro was carried out. Error bars represent the mean ± standard error of the mean of three independent experiments, * P

    Article Snippet: Antibodies to O -GlcNAc (RL2, Affinity Bio-Reagents, Golden, CO, USA; CTD110.6, Abcam plc, Cambridge, UK), OGT (F-12; Santa Cruz, CA, USA), Akt1 (Cell Signaling Technology, Beverly, MA, USA), phospho-Akt1 (Ser473, Cell Signaling Technology), and GAPDH (Santa Cruz, CA, USA) were used for detection by ECL detection reagent (GE Healthcare UK Ltd, Little Chalfont, UK).

    Techniques: Invasion Assay, In Vitro

    The inhibition of LY294002 on the regulation of O -GlcNAcylation on invasion of thyroid anaplastic cancer cells. Notes: For 24 hours, 5 μM Thiamet-G (Thia-G) treated or untreated and OGT overexpression or underexpressed 8305C cells were treated with 2 μM LY294002. ( A ) The inhibition of LY294002, a specific inhibitor of PI3K, on Akt1 phosphorylation (Ser473) was assessed by Western blot. ( B ) Transwell assay analyzed the impact of LY294002 on invasion of Thiamet-G treated (on the left) or OGT overexpression (on the right) in 8305C cells. Western blot analysis showed the corresponding phosphorylation (Ser473) of Akt1, demonstrating the changes of Akt1 activity in respectively treated cells with Thiamet-G, OGT overexpression or LY294002. Anti-p-Akt1 (Ser473), anti-Akt1, and anti-GAPDH antibodies were used in the experiments. Error bars represent the mean ± standard error of the mean of three independent experiments, * P

    Journal: OncoTargets and therapy

    Article Title: O-GlcNAcylation enhances the invasion of thyroid anaplastic cancer cells partially by PI3K/Akt1 pathway

    doi: 10.2147/OTT.S82845

    Figure Lengend Snippet: The inhibition of LY294002 on the regulation of O -GlcNAcylation on invasion of thyroid anaplastic cancer cells. Notes: For 24 hours, 5 μM Thiamet-G (Thia-G) treated or untreated and OGT overexpression or underexpressed 8305C cells were treated with 2 μM LY294002. ( A ) The inhibition of LY294002, a specific inhibitor of PI3K, on Akt1 phosphorylation (Ser473) was assessed by Western blot. ( B ) Transwell assay analyzed the impact of LY294002 on invasion of Thiamet-G treated (on the left) or OGT overexpression (on the right) in 8305C cells. Western blot analysis showed the corresponding phosphorylation (Ser473) of Akt1, demonstrating the changes of Akt1 activity in respectively treated cells with Thiamet-G, OGT overexpression or LY294002. Anti-p-Akt1 (Ser473), anti-Akt1, and anti-GAPDH antibodies were used in the experiments. Error bars represent the mean ± standard error of the mean of three independent experiments, * P

    Article Snippet: Antibodies to O -GlcNAc (RL2, Affinity Bio-Reagents, Golden, CO, USA; CTD110.6, Abcam plc, Cambridge, UK), OGT (F-12; Santa Cruz, CA, USA), Akt1 (Cell Signaling Technology, Beverly, MA, USA), phospho-Akt1 (Ser473, Cell Signaling Technology), and GAPDH (Santa Cruz, CA, USA) were used for detection by ECL detection reagent (GE Healthcare UK Ltd, Little Chalfont, UK).

    Techniques: Inhibition, Over Expression, Western Blot, Transwell Assay, Activity Assay

    RNAi-mediated down-regulation of Akt1. Notes: The 8305C cells were treated with two different small interfering RNA duplexes (siAkt1-1 and siAkt1-2) or control (Ctrl) scrambled siRNA. ( A ) Relative amount of Akt1 mRNA 48 hours after cells were transfected with 30 nM siRNAs (siAkt1-1, siAkt1-2) as assessed by real-time RT-PCR. ( B ) Western blot analysis of changes in Akt1 protein expression level in lysates of 8305C cells treated with 30 nM siRNAs for 48 hours. Western blot analysis was performed with Akt1 and GAPDH antibodies. Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; mRNA, messenger RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Ctrl, control.

    Journal: OncoTargets and therapy

    Article Title: O-GlcNAcylation enhances the invasion of thyroid anaplastic cancer cells partially by PI3K/Akt1 pathway

    doi: 10.2147/OTT.S82845

    Figure Lengend Snippet: RNAi-mediated down-regulation of Akt1. Notes: The 8305C cells were treated with two different small interfering RNA duplexes (siAkt1-1 and siAkt1-2) or control (Ctrl) scrambled siRNA. ( A ) Relative amount of Akt1 mRNA 48 hours after cells were transfected with 30 nM siRNAs (siAkt1-1, siAkt1-2) as assessed by real-time RT-PCR. ( B ) Western blot analysis of changes in Akt1 protein expression level in lysates of 8305C cells treated with 30 nM siRNAs for 48 hours. Western blot analysis was performed with Akt1 and GAPDH antibodies. Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; mRNA, messenger RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Ctrl, control.

    Article Snippet: Antibodies to O -GlcNAc (RL2, Affinity Bio-Reagents, Golden, CO, USA; CTD110.6, Abcam plc, Cambridge, UK), OGT (F-12; Santa Cruz, CA, USA), Akt1 (Cell Signaling Technology, Beverly, MA, USA), phospho-Akt1 (Ser473, Cell Signaling Technology), and GAPDH (Santa Cruz, CA, USA) were used for detection by ECL detection reagent (GE Healthcare UK Ltd, Little Chalfont, UK).

    Techniques: Small Interfering RNA, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

    Up-regulation of O -GlcNAcylation increases Akt1 phosphorylation. Notes: The 8305C cells were treated with ( A ) 5 μM Thiamet-G (Thia-G) or ( B ) OGT overexpression for 24 hours. Cell lysates were prepared and used for Western blots for p-Akt1 (Ser473), Akt1, and GAPDH (loading control) with anti-p-Akt1 (Ser473), anti-Akt1, and anti-GAPDH antibodies. The blots of p-Akt1 were quantified by densitometry, and the results were expressed as a ratio relative to the values obtained in untreated control cells. Error bars represent the mean ± standard error of the mean of three independent experiments, ** P

    Journal: OncoTargets and therapy

    Article Title: O-GlcNAcylation enhances the invasion of thyroid anaplastic cancer cells partially by PI3K/Akt1 pathway

    doi: 10.2147/OTT.S82845

    Figure Lengend Snippet: Up-regulation of O -GlcNAcylation increases Akt1 phosphorylation. Notes: The 8305C cells were treated with ( A ) 5 μM Thiamet-G (Thia-G) or ( B ) OGT overexpression for 24 hours. Cell lysates were prepared and used for Western blots for p-Akt1 (Ser473), Akt1, and GAPDH (loading control) with anti-p-Akt1 (Ser473), anti-Akt1, and anti-GAPDH antibodies. The blots of p-Akt1 were quantified by densitometry, and the results were expressed as a ratio relative to the values obtained in untreated control cells. Error bars represent the mean ± standard error of the mean of three independent experiments, ** P

    Article Snippet: Antibodies to O -GlcNAc (RL2, Affinity Bio-Reagents, Golden, CO, USA; CTD110.6, Abcam plc, Cambridge, UK), OGT (F-12; Santa Cruz, CA, USA), Akt1 (Cell Signaling Technology, Beverly, MA, USA), phospho-Akt1 (Ser473, Cell Signaling Technology), and GAPDH (Santa Cruz, CA, USA) were used for detection by ECL detection reagent (GE Healthcare UK Ltd, Little Chalfont, UK).

    Techniques: Over Expression, Western Blot

    Changes in muscle hypertrophy and atrophy signaling pathways in myocilin expressing transgenic mouse. A , total lysates from C2C12 myotubes differentiated on the plates with or without laminin were probed with antibodies against phospho-AKT1 ( P-AKT1 ; 1:1000

    Journal: The Journal of Biological Chemistry

    Article Title: Myocilin Interacts with Syntrophins and Is Member of Dystrophin-associated Protein Complex

    doi: 10.1074/jbc.M111.224063

    Figure Lengend Snippet: Changes in muscle hypertrophy and atrophy signaling pathways in myocilin expressing transgenic mouse. A , total lysates from C2C12 myotubes differentiated on the plates with or without laminin were probed with antibodies against phospho-AKT1 ( P-AKT1 ; 1:1000

    Article Snippet: Other antibodies were purchased from the following sources: anti-α1-Syn, anti-pan-syntrophin (1351), and anti-laminin from Abcam (Cambridge, MA); anti-FoxO3a from Sigma; anti-heat shock cognate 70 (HSC70) and anti-β-dystroglycan (β-DG) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-phospho-FoxO3a and anti-α-dystroglycan (α-DG, IIH6) from Millipore (Bedford, MA); anti-α-dystrobrevin, anti-neuronal nitric-oxide synthase (nNOS), and anti-protein-disulfide isomerase from BD Transduction Laboratories (San Diego, CA); anti-AKT1, anti-phospho-AKT1, anti-mTOR, anti-phospho-mTOR, and anti-phospho-p70 S6 kinase from Cell Signaling (Beverly, MA); anti-dystrophin from Vector Laboratories (Burlingame, CA); anti-myosin heavy chain from R & D Systems (Minneapolis, MN); and anti-pigment epithelium-derived factor from BioProducts MD (Middletown, MD).

    Techniques: Expressing, Transgenic Assay

    Therapeutic efficiency of GT–SPE as an aerosol gene delivery carrier in lung cancer model K -ras LA1 mice. Aerosol delivery of GT–SPE/Akt1 shRNA significantly inhibited lung tumor numbers and tumorigenesis. ( A ) Lung tumor lesions. ( B ) Total tumor numbers (n = 4, ** P

    Journal: International Journal of Nanomedicine

    Article Title: Suppression of lung cancer progression by biocompatible glycerol triacrylate-spermine-mediated delivery of shAkt1

    doi: 10.2147/IJN.S29152

    Figure Lengend Snippet: Therapeutic efficiency of GT–SPE as an aerosol gene delivery carrier in lung cancer model K -ras LA1 mice. Aerosol delivery of GT–SPE/Akt1 shRNA significantly inhibited lung tumor numbers and tumorigenesis. ( A ) Lung tumor lesions. ( B ) Total tumor numbers (n = 4, ** P

    Article Snippet: Akt1 antibodies were purchased from Cell Signaling (Danvers, MA).

    Techniques: Mouse Assay, shRNA

    shAkt1 aerosol delivery significantly decreased Akt1 expression level and induced apoptosis. ( A ) Western blot bands. Statistical analyses of Western blot ( B ) Akt1, ( C ) Bax, and ( D ) Bcl-xL. Bands were analyzed by densitometer (n = 4, * P

    Journal: International Journal of Nanomedicine

    Article Title: Suppression of lung cancer progression by biocompatible glycerol triacrylate-spermine-mediated delivery of shAkt1

    doi: 10.2147/IJN.S29152

    Figure Lengend Snippet: shAkt1 aerosol delivery significantly decreased Akt1 expression level and induced apoptosis. ( A ) Western blot bands. Statistical analyses of Western blot ( B ) Akt1, ( C ) Bax, and ( D ) Bcl-xL. Bands were analyzed by densitometer (n = 4, * P

    Article Snippet: Akt1 antibodies were purchased from Cell Signaling (Danvers, MA).

    Techniques: Expressing, Western Blot

    Specific knockout Akt1 in BAT increases Ucp1 expression. ( a ) Body mass of wild-type mice (WT) and Akt1 conditional knockout mice (Akt1 cKO) fed on a chow diet for 24 weeks(WT, n =17; Akt1 cKO, n =16). ( b ) MRI analysis body lean and fat mass from 25-week-old Akt1 BAT-specific knockout (Akt1 cKO) and WT littermates mice (WT, n =9; Akt1 cKO, n =9). ( c ) HE and Ucp1 staining of sWAT and BAT from 4-week-old mice of the Akt1 cKO and WT mice. ( d ) Lysates of BAT from 4-week-old WT and Akt1 cKO mice were subjected to immunoblotting with indicated antibody. ( e ) Model depicting the role of the DJ-1-Mib2-PTEN-Akt1- FoxO1 signaling cascade in energy balance and glucose homeostasis of BAT.

    Journal: Cell Discovery

    Article Title: DJ-1 maintains energy and glucose homeostasis by regulating the function of brown adipose tissue

    doi: 10.1038/celldisc.2016.54

    Figure Lengend Snippet: Specific knockout Akt1 in BAT increases Ucp1 expression. ( a ) Body mass of wild-type mice (WT) and Akt1 conditional knockout mice (Akt1 cKO) fed on a chow diet for 24 weeks(WT, n =17; Akt1 cKO, n =16). ( b ) MRI analysis body lean and fat mass from 25-week-old Akt1 BAT-specific knockout (Akt1 cKO) and WT littermates mice (WT, n =9; Akt1 cKO, n =9). ( c ) HE and Ucp1 staining of sWAT and BAT from 4-week-old mice of the Akt1 cKO and WT mice. ( d ) Lysates of BAT from 4-week-old WT and Akt1 cKO mice were subjected to immunoblotting with indicated antibody. ( e ) Model depicting the role of the DJ-1-Mib2-PTEN-Akt1- FoxO1 signaling cascade in energy balance and glucose homeostasis of BAT.

    Article Snippet: The antibodies used were anti-phosphor-Akt (Ser473; #4060, Cell Signaling, Cambridge, MA, USA), anti-Akt (#4691, Cell Signaling), anti-Akt1 (#2938, Cell Signaling), anti-FoxO1 (sc-11350, Santa Cruz, Dallas, TX, USA), anti-Akt2 (#3063, Cell Signaling), anti-phosphor-FoxO (Thr24-FoxO1/ Thr32-FoxO3a; #9464, Cell Signaling), anti-phosphor-HSL (Ser660; #4126, Cell Signaling), anti-HSL (#4107, Cell Signaling), anti-PTEN (#9188, Cell Signaling), anti-DJ-1 (#5933, Cell Signaling), anti-Ucp1 (ab10983), anti-Ubiquitin (#3936, Cell Signaling), anti-Mib2 (Ls-C295379, LifeSpan BioSciences, Seattle, WA, USA), anti-FLAG (Sigma), anti-GFP (Invitrogen), anti-HA (Santa Cruz), anti-β-tubulin (CW0098A, CWBiotech, Beijing, China), anti-β-actin (60008-1-Ig, Proteintech Group, Campbell Park, Chicago, IL, USA) and anti-GAPDH (CW0266A, CWBiotech).

    Techniques: Knock-Out, Expressing, Mouse Assay, Magnetic Resonance Imaging, Staining

    Ablation of Akt1 mitigates the obesity and BAT dysfunction induced by DJ-1 transgene. ( a , b ) The effect of BYL-719 on gene and protein expression in BAT. ( c ) HE or anti-Ucp1 antibody staining in BAT was performed after BYL-719 treatment. ( d ) Body mass of wild-type mice (WT), DJ-1 transgenic mice (Tg, Line 17 C57BL/6J background), Akt1 KO mice (Akt1 −/− ), DJ-1 transgenic and Akt1 knockout mice (Akt1 −/− ; Tg) fed on a chow diet for 24 weeks (WT, n =15; Akt1 −/− , n =9; Tg, n =14; Akt1 −/− ; Tg, n =17). ( e ) Body lean and fat mass of 12-month-old indicated genotype mice measured by MRI (WT, n =7; Akt1 −/− , n =11; Tg, n =8; Akt1 −/− ; Tg, n =8). ( f ) Statistical analysis by General Linear Model (GLM) and ANCOVA analysis of the indicated genotype mice energy expenditure (EE) (WT, n =9; Akt1 −/− , n =8; Akt1 −/− ; Tg, n =9). ( g ) Lysates of BAT from mice of the genotypes indicated immunoblotted with Ucp1, Akt1, DJ-1 or β-tubulin antibodies. ( h ) HE and Ucp1 staining of BAT from 4-week-old mice of the indicated genotypes. ( i ) GTT results for 9-month-old indicated genotype mice fed on a chow diet (WT, n =8; Tg, n =7; Akt1 −/− , n =7; Akt1 −/− ; Tg, n =8).

    Journal: Cell Discovery

    Article Title: DJ-1 maintains energy and glucose homeostasis by regulating the function of brown adipose tissue

    doi: 10.1038/celldisc.2016.54

    Figure Lengend Snippet: Ablation of Akt1 mitigates the obesity and BAT dysfunction induced by DJ-1 transgene. ( a , b ) The effect of BYL-719 on gene and protein expression in BAT. ( c ) HE or anti-Ucp1 antibody staining in BAT was performed after BYL-719 treatment. ( d ) Body mass of wild-type mice (WT), DJ-1 transgenic mice (Tg, Line 17 C57BL/6J background), Akt1 KO mice (Akt1 −/− ), DJ-1 transgenic and Akt1 knockout mice (Akt1 −/− ; Tg) fed on a chow diet for 24 weeks (WT, n =15; Akt1 −/− , n =9; Tg, n =14; Akt1 −/− ; Tg, n =17). ( e ) Body lean and fat mass of 12-month-old indicated genotype mice measured by MRI (WT, n =7; Akt1 −/− , n =11; Tg, n =8; Akt1 −/− ; Tg, n =8). ( f ) Statistical analysis by General Linear Model (GLM) and ANCOVA analysis of the indicated genotype mice energy expenditure (EE) (WT, n =9; Akt1 −/− , n =8; Akt1 −/− ; Tg, n =9). ( g ) Lysates of BAT from mice of the genotypes indicated immunoblotted with Ucp1, Akt1, DJ-1 or β-tubulin antibodies. ( h ) HE and Ucp1 staining of BAT from 4-week-old mice of the indicated genotypes. ( i ) GTT results for 9-month-old indicated genotype mice fed on a chow diet (WT, n =8; Tg, n =7; Akt1 −/− , n =7; Akt1 −/− ; Tg, n =8).

    Article Snippet: The antibodies used were anti-phosphor-Akt (Ser473; #4060, Cell Signaling, Cambridge, MA, USA), anti-Akt (#4691, Cell Signaling), anti-Akt1 (#2938, Cell Signaling), anti-FoxO1 (sc-11350, Santa Cruz, Dallas, TX, USA), anti-Akt2 (#3063, Cell Signaling), anti-phosphor-FoxO (Thr24-FoxO1/ Thr32-FoxO3a; #9464, Cell Signaling), anti-phosphor-HSL (Ser660; #4126, Cell Signaling), anti-HSL (#4107, Cell Signaling), anti-PTEN (#9188, Cell Signaling), anti-DJ-1 (#5933, Cell Signaling), anti-Ucp1 (ab10983), anti-Ubiquitin (#3936, Cell Signaling), anti-Mib2 (Ls-C295379, LifeSpan BioSciences, Seattle, WA, USA), anti-FLAG (Sigma), anti-GFP (Invitrogen), anti-HA (Santa Cruz), anti-β-tubulin (CW0098A, CWBiotech, Beijing, China), anti-β-actin (60008-1-Ig, Proteintech Group, Campbell Park, Chicago, IL, USA) and anti-GAPDH (CW0266A, CWBiotech).

    Techniques: Expressing, Staining, Mouse Assay, Transgenic Assay, Knock-Out, Magnetic Resonance Imaging

    Event-Related Spectral Perturbation (ERSP) in WT, Akt1 +/− and Akt1 −/− mice. Figure A Top shows response following saline while Figure A Bottom shows response following 50 mg/kg ketamine. B. Quantification and analysis of ERSP response

    Journal: Psychopharmacology

    Article Title: Electrophysiological and behavioral responses to ketamine in mice with reduced Akt1 expression

    doi: 10.1007/s00213-013-2997-9

    Figure Lengend Snippet: Event-Related Spectral Perturbation (ERSP) in WT, Akt1 +/− and Akt1 −/− mice. Figure A Top shows response following saline while Figure A Bottom shows response following 50 mg/kg ketamine. B. Quantification and analysis of ERSP response

    Article Snippet: The membrane was incubated with the Akt1 antibody (rabbit monoclonal, #2938, cell signaling, 1/2000) followed by the secondary antibody (HRP conjugated Donkey anti Rabbit IgG (H+L), Jackson Immunoresearch, 1/10000).

    Techniques: Mouse Assay

    Intertrial Coherence (ITC) in WT, Akt1 +/− and Akt1 −/− mice. Figure A Top shows ITC heat map following saline while Figure A bottom shows ITC heat map following 50 mg/kg ketamine. B. ITC for the theta frequency band was significantly

    Journal: Psychopharmacology

    Article Title: Electrophysiological and behavioral responses to ketamine in mice with reduced Akt1 expression

    doi: 10.1007/s00213-013-2997-9

    Figure Lengend Snippet: Intertrial Coherence (ITC) in WT, Akt1 +/− and Akt1 −/− mice. Figure A Top shows ITC heat map following saline while Figure A bottom shows ITC heat map following 50 mg/kg ketamine. B. ITC for the theta frequency band was significantly

    Article Snippet: The membrane was incubated with the Akt1 antibody (rabbit monoclonal, #2938, cell signaling, 1/2000) followed by the secondary antibody (HRP conjugated Donkey anti Rabbit IgG (H+L), Jackson Immunoresearch, 1/10000).

    Techniques: Mouse Assay

    Effect of ketamine on P20 response in WT, Akt1 +/− and Akt1 −/− mice matched for P20 amplitude. A. The effect of ketamine on P20 amplitude was significantly greater in both Akt1 +/− and Akt1 −/− mice compared

    Journal: Psychopharmacology

    Article Title: Electrophysiological and behavioral responses to ketamine in mice with reduced Akt1 expression

    doi: 10.1007/s00213-013-2997-9

    Figure Lengend Snippet: Effect of ketamine on P20 response in WT, Akt1 +/− and Akt1 −/− mice matched for P20 amplitude. A. The effect of ketamine on P20 amplitude was significantly greater in both Akt1 +/− and Akt1 −/− mice compared

    Article Snippet: The membrane was incubated with the Akt1 antibody (rabbit monoclonal, #2938, cell signaling, 1/2000) followed by the secondary antibody (HRP conjugated Donkey anti Rabbit IgG (H+L), Jackson Immunoresearch, 1/10000).

    Techniques: Mouse Assay

    Western blot analysis for Akt1 in WT and Akt1 +/− mice. A significant decrease in Akt1 expression was observed in Akt1 +/− mice relative to WT controls (p

    Journal: Psychopharmacology

    Article Title: Electrophysiological and behavioral responses to ketamine in mice with reduced Akt1 expression

    doi: 10.1007/s00213-013-2997-9

    Figure Lengend Snippet: Western blot analysis for Akt1 in WT and Akt1 +/− mice. A significant decrease in Akt1 expression was observed in Akt1 +/− mice relative to WT controls (p

    Article Snippet: The membrane was incubated with the Akt1 antibody (rabbit monoclonal, #2938, cell signaling, 1/2000) followed by the secondary antibody (HRP conjugated Donkey anti Rabbit IgG (H+L), Jackson Immunoresearch, 1/10000).

    Techniques: Western Blot, Mouse Assay, Expressing

    Event-Related Potentials (ERP) in WT, Akt1 +/− and Akt1 −/− mice. A. Grand Average Waveform for the 200 msec period following presentation of the first white noise click of a paired-click stimulus (S1). WT, Akt1 +/− and Akt1

    Journal: Psychopharmacology

    Article Title: Electrophysiological and behavioral responses to ketamine in mice with reduced Akt1 expression

    doi: 10.1007/s00213-013-2997-9

    Figure Lengend Snippet: Event-Related Potentials (ERP) in WT, Akt1 +/− and Akt1 −/− mice. A. Grand Average Waveform for the 200 msec period following presentation of the first white noise click of a paired-click stimulus (S1). WT, Akt1 +/− and Akt1

    Article Snippet: The membrane was incubated with the Akt1 antibody (rabbit monoclonal, #2938, cell signaling, 1/2000) followed by the secondary antibody (HRP conjugated Donkey anti Rabbit IgG (H+L), Jackson Immunoresearch, 1/10000).

    Techniques: Mouse Assay

    Pre-pulse inhibition (PPI) in WT and Akt1 +/− mice following either saline or ketamine. A significant effect was observed for gene, with Akt1 +/− animals displaying reduced PPI relative to WT mice. Additionally, 50 mg/kg ketamine produced

    Journal: Psychopharmacology

    Article Title: Electrophysiological and behavioral responses to ketamine in mice with reduced Akt1 expression

    doi: 10.1007/s00213-013-2997-9

    Figure Lengend Snippet: Pre-pulse inhibition (PPI) in WT and Akt1 +/− mice following either saline or ketamine. A significant effect was observed for gene, with Akt1 +/− animals displaying reduced PPI relative to WT mice. Additionally, 50 mg/kg ketamine produced

    Article Snippet: The membrane was incubated with the Akt1 antibody (rabbit monoclonal, #2938, cell signaling, 1/2000) followed by the secondary antibody (HRP conjugated Donkey anti Rabbit IgG (H+L), Jackson Immunoresearch, 1/10000).

    Techniques: Inhibition, Mouse Assay, Produced

    Active Akt enhances phosphorylation of SGK1 Ser 397 . A , FLAG-SGK1 was coexpressed with vector or HA-Myr-Akt1 in HeLa cells. Immunoprecipitated SGK1 was subjected to mass spectrometry. Mass spectroscopic data of the phosphopeptide phospho-Ser 401 in the

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of a Third Conserved Phosphorylation Site in SGK1

    doi: 10.1074/jbc.M807502200

    Figure Lengend Snippet: Active Akt enhances phosphorylation of SGK1 Ser 397 . A , FLAG-SGK1 was coexpressed with vector or HA-Myr-Akt1 in HeLa cells. Immunoprecipitated SGK1 was subjected to mass spectrometry. Mass spectroscopic data of the phosphopeptide phospho-Ser 401 in the

    Article Snippet: The anti-Akt1 antibody was from Cell Signaling Technology.

    Techniques: Plasmid Preparation, Immunoprecipitation, Mass Spectrometry

    Overexpressed WNK1-(1–491) co-immunoprecipitates with overexpressed Akt1. HA-Akt1, Myc-WNK1-(1–491), Myc-WNK1-(1–491)(T58A), Myc-WNK1-(1–220), or Myc-WNK1-(1–220)(T58A) was coexpressed in HeLa cells in the indicated

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of a Third Conserved Phosphorylation Site in SGK1

    doi: 10.1074/jbc.M807502200

    Figure Lengend Snippet: Overexpressed WNK1-(1–491) co-immunoprecipitates with overexpressed Akt1. HA-Akt1, Myc-WNK1-(1–491), Myc-WNK1-(1–491)(T58A), Myc-WNK1-(1–220), or Myc-WNK1-(1–220)(T58A) was coexpressed in HeLa cells in the indicated

    Article Snippet: The anti-Akt1 antibody was from Cell Signaling Technology.

    Techniques:

    WNK1 and Akt1 are involved in SGK1 activation by H 2 O 2 and IGF1. HeLa cells were cotransfected with plasmid encoding FLAG-ΔSGK1 and WNK1 ( Wi ), Akt1 ( Ai ), or scrambled ( Ci ) small interfering RNA ( siRNA ) oligonucleotides. Cells were untreated

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of a Third Conserved Phosphorylation Site in SGK1

    doi: 10.1074/jbc.M807502200

    Figure Lengend Snippet: WNK1 and Akt1 are involved in SGK1 activation by H 2 O 2 and IGF1. HeLa cells were cotransfected with plasmid encoding FLAG-ΔSGK1 and WNK1 ( Wi ), Akt1 ( Ai ), or scrambled ( Ci ) small interfering RNA ( siRNA ) oligonucleotides. Cells were untreated

    Article Snippet: The anti-Akt1 antibody was from Cell Signaling Technology.

    Techniques: Activation Assay, Plasmid Preparation, Small Interfering RNA

    SGK1 is activated by active Akt1. Wild-type ( WT ) or kinase-dead ( KD ) ΔSGK1 was coexpressed with Myc-PDK1, HA-Akt1, or HA-Myr-Akt1 in HeLa cells. Protein expression was probed with anti-Myc, anti-FLAG, and anti-HA antibodies ( upper two panels

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of a Third Conserved Phosphorylation Site in SGK1

    doi: 10.1074/jbc.M807502200

    Figure Lengend Snippet: SGK1 is activated by active Akt1. Wild-type ( WT ) or kinase-dead ( KD ) ΔSGK1 was coexpressed with Myc-PDK1, HA-Akt1, or HA-Myr-Akt1 in HeLa cells. Protein expression was probed with anti-Myc, anti-FLAG, and anti-HA antibodies ( upper two panels

    Article Snippet: The anti-Akt1 antibody was from Cell Signaling Technology.

    Techniques: Expressing

    WNK1 and Akt1 cooperate to activate SGK1. A , FLAG-ΔSGK1 was coexpressed with Myc-WNK1-(1–491), Myc-WNK1-(1–491)(T58A), HA-Akt1, HA-Myr-Akt1, or HA-Myr-Akt1KM (kinase-dead) in different combinations in HeLa cells. Protein expression

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of a Third Conserved Phosphorylation Site in SGK1

    doi: 10.1074/jbc.M807502200

    Figure Lengend Snippet: WNK1 and Akt1 cooperate to activate SGK1. A , FLAG-ΔSGK1 was coexpressed with Myc-WNK1-(1–491), Myc-WNK1-(1–491)(T58A), HA-Akt1, HA-Myr-Akt1, or HA-Myr-Akt1KM (kinase-dead) in different combinations in HeLa cells. Protein expression

    Article Snippet: The anti-Akt1 antibody was from Cell Signaling Technology.

    Techniques: Expressing

    Predicted target sites of miR-302 in 3’ UTRs of AKT1 and RAD52 genes ( a ) The putative targeted sites in the 3’ UTRs of AKT1 and RAD52 . ( b ) and ( c ) Luciferase reporter assay results. MDA-MB-231 cells were transfected with the firefly luciferase reporter plasmid containing partial 3’-UTR of AKT1 ( b ) or RAD52 ( c ) with (3’UTR) or without (Mut 3’UTR) the putative miR-302 binding site. Blank vector was used as mock control (Mock). Luciferase activity was measured at 48 h post the co-transfection of luciferase reporter vector with miR-302a mimics. * P

    Journal: Pharmaceutical research

    Article Title: MicroRNA-302 Replacement Therapy Sensitizes Breast Cancer Cells to Ionizing Radiation

    doi: 10.1007/s11095-012-0936-9

    Figure Lengend Snippet: Predicted target sites of miR-302 in 3’ UTRs of AKT1 and RAD52 genes ( a ) The putative targeted sites in the 3’ UTRs of AKT1 and RAD52 . ( b ) and ( c ) Luciferase reporter assay results. MDA-MB-231 cells were transfected with the firefly luciferase reporter plasmid containing partial 3’-UTR of AKT1 ( b ) or RAD52 ( c ) with (3’UTR) or without (Mut 3’UTR) the putative miR-302 binding site. Blank vector was used as mock control (Mock). Luciferase activity was measured at 48 h post the co-transfection of luciferase reporter vector with miR-302a mimics. * P

    Article Snippet: The membranes were blocked for 0.5 hour in a blocking solution (5% milk in TBS-T [Tris-buffered saline containing Tween-20]) and incubated overnight at 4°C with polyclonal antibodies against AKT1, phosphorylated AKT1, and RAD52 (Cellsignaling, Danvers, MA), and monoclonal antibody against β-actin (Sigma–Aldrich, St. Louis, MO).

    Techniques: Luciferase, Reporter Assay, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Binding Assay, Activity Assay, Cotransfection

    Overexpression of miR-302 reduced the expression of AKT1 and RAD52 ( a ) Pre-miRNA double-strand oligo sequence inserted into a miRNA expression plasmid, Block-iT Pol II miR RNAi Expression Vector (Invitrogen). ( b ) Representative image (GFP) shows the transfection efficiency of the vectors after selection. ( c ) Levels of miR-302a were increased in miR-302a plasmid-transfected MDA-MB-231RR cells compared to mock MDA-MB-231RR cells. Levels of miR-302a were not significantly increased in control vector-trasfected MDA-MB-231 RR cells compared to mock MDA-MB-231RR cells ( d ) Levels of phosphorylated AKT1, total AKT1 and RAD52 proteins were reduced in miR-302a plasmid-transfected MDA-MB-231RR cells compared to control vector-transfected or mock MDA-MB-231RR cells. β-actin was used as a loading control.

    Journal: Pharmaceutical research

    Article Title: MicroRNA-302 Replacement Therapy Sensitizes Breast Cancer Cells to Ionizing Radiation

    doi: 10.1007/s11095-012-0936-9

    Figure Lengend Snippet: Overexpression of miR-302 reduced the expression of AKT1 and RAD52 ( a ) Pre-miRNA double-strand oligo sequence inserted into a miRNA expression plasmid, Block-iT Pol II miR RNAi Expression Vector (Invitrogen). ( b ) Representative image (GFP) shows the transfection efficiency of the vectors after selection. ( c ) Levels of miR-302a were increased in miR-302a plasmid-transfected MDA-MB-231RR cells compared to mock MDA-MB-231RR cells. Levels of miR-302a were not significantly increased in control vector-trasfected MDA-MB-231 RR cells compared to mock MDA-MB-231RR cells ( d ) Levels of phosphorylated AKT1, total AKT1 and RAD52 proteins were reduced in miR-302a plasmid-transfected MDA-MB-231RR cells compared to control vector-transfected or mock MDA-MB-231RR cells. β-actin was used as a loading control.

    Article Snippet: The membranes were blocked for 0.5 hour in a blocking solution (5% milk in TBS-T [Tris-buffered saline containing Tween-20]) and incubated overnight at 4°C with polyclonal antibodies against AKT1, phosphorylated AKT1, and RAD52 (Cellsignaling, Danvers, MA), and monoclonal antibody against β-actin (Sigma–Aldrich, St. Louis, MO).

    Techniques: Over Expression, Expressing, Sequencing, Plasmid Preparation, Blocking Assay, Transfection, Selection, Multiple Displacement Amplification

    Long-term effect of Ang-1 and VEGF on endothelial-barrier protection is reliant on Akt1-mediated TJ stabilization

    Journal: Cellular and molecular life sciences : CMLS

    Article Title: Akt1 promotes stimuli-induced endothelial-barrier protection through FoxO-mediated tight-junction protein turnover

    doi: 10.1007/s00018-016-2232-z

    Figure Lengend Snippet: Long-term effect of Ang-1 and VEGF on endothelial-barrier protection is reliant on Akt1-mediated TJ stabilization

    Article Snippet: For tissue immunofluorescent staining, sections were blocked with 10% goat serum followed by incubation with primary antibodies against Akt1 (1:100, rabbit anti-mouse, Cell Signaling, Danvers, MA) and CD31 (1:100, mouse anti-mouse, Abcam, Cambridge, MA) at 4 °C overnight.

    Techniques:

    Endothelial specific Akt1 depletion lead to vascular leakage in vivo

    Journal: Cellular and molecular life sciences : CMLS

    Article Title: Akt1 promotes stimuli-induced endothelial-barrier protection through FoxO-mediated tight-junction protein turnover

    doi: 10.1007/s00018-016-2232-z

    Figure Lengend Snippet: Endothelial specific Akt1 depletion lead to vascular leakage in vivo

    Article Snippet: For tissue immunofluorescent staining, sections were blocked with 10% goat serum followed by incubation with primary antibodies against Akt1 (1:100, rabbit anti-mouse, Cell Signaling, Danvers, MA) and CD31 (1:100, mouse anti-mouse, Abcam, Cambridge, MA) at 4 °C overnight.

    Techniques: In Vivo

    Akt1 augments VEGF-induced vascular permeability and Ang-1-induced vascular-barrier protection in vivo

    Journal: Cellular and molecular life sciences : CMLS

    Article Title: Akt1 promotes stimuli-induced endothelial-barrier protection through FoxO-mediated tight-junction protein turnover

    doi: 10.1007/s00018-016-2232-z

    Figure Lengend Snippet: Akt1 augments VEGF-induced vascular permeability and Ang-1-induced vascular-barrier protection in vivo

    Article Snippet: For tissue immunofluorescent staining, sections were blocked with 10% goat serum followed by incubation with primary antibodies against Akt1 (1:100, rabbit anti-mouse, Cell Signaling, Danvers, MA) and CD31 (1:100, mouse anti-mouse, Abcam, Cambridge, MA) at 4 °C overnight.

    Techniques: Permeability, In Vivo

    Akt1 deficiency affects real-time changes in the expression of proteins in endothelial-barrier AJs and TJs in response to VEGF and Ang-1 treatments

    Journal: Cellular and molecular life sciences : CMLS

    Article Title: Akt1 promotes stimuli-induced endothelial-barrier protection through FoxO-mediated tight-junction protein turnover

    doi: 10.1007/s00018-016-2232-z

    Figure Lengend Snippet: Akt1 deficiency affects real-time changes in the expression of proteins in endothelial-barrier AJs and TJs in response to VEGF and Ang-1 treatments

    Article Snippet: For tissue immunofluorescent staining, sections were blocked with 10% goat serum followed by incubation with primary antibodies against Akt1 (1:100, rabbit anti-mouse, Cell Signaling, Danvers, MA) and CD31 (1:100, mouse anti-mouse, Abcam, Cambridge, MA) at 4 °C overnight.

    Techniques: Expressing

    Long-term, not short-term changes in claudin-5 expression in HMEC is regulated by Akt1

    Journal: Cellular and molecular life sciences : CMLS

    Article Title: Akt1 promotes stimuli-induced endothelial-barrier protection through FoxO-mediated tight-junction protein turnover

    doi: 10.1007/s00018-016-2232-z

    Figure Lengend Snippet: Long-term, not short-term changes in claudin-5 expression in HMEC is regulated by Akt1

    Article Snippet: For tissue immunofluorescent staining, sections were blocked with 10% goat serum followed by incubation with primary antibodies against Akt1 (1:100, rabbit anti-mouse, Cell Signaling, Danvers, MA) and CD31 (1:100, mouse anti-mouse, Abcam, Cambridge, MA) at 4 °C overnight.

    Techniques: Expressing

    Akt1 deficiency compromises endothelial-barrier integrity and modulates VEGF and Ang-1-mediated endothelial-barrier function

    Journal: Cellular and molecular life sciences : CMLS

    Article Title: Akt1 promotes stimuli-induced endothelial-barrier protection through FoxO-mediated tight-junction protein turnover

    doi: 10.1007/s00018-016-2232-z

    Figure Lengend Snippet: Akt1 deficiency compromises endothelial-barrier integrity and modulates VEGF and Ang-1-mediated endothelial-barrier function

    Article Snippet: For tissue immunofluorescent staining, sections were blocked with 10% goat serum followed by incubation with primary antibodies against Akt1 (1:100, rabbit anti-mouse, Cell Signaling, Danvers, MA) and CD31 (1:100, mouse anti-mouse, Abcam, Cambridge, MA) at 4 °C overnight.

    Techniques:

    Akt and GSK-3β protein and phosphorylation levels in 6-days denervated hypertrophic hemidiaphragm muscle. Expression of Akt1, Akt2 and GSK-3β total protein (t-Akt1, a; t-Akt2, c and t-GSK-3β, e) and phosphorylated Akt1 protein (p-Akt1) at S473 (b), phosphorylated Akt2 protein (p-Akt2) at S474 (d) and phosphorylated GSK-3β protein (p-GSK-3β) at S9 (f) in 6-days denervated hypertrophic hemidiaphragm muscle (Den) compared to innervated (Inn) controls. Representative Western blots are shown together with densitometric quantifications. One innervated hemidiaphragm muscle sample was used as a reference sample and was included in all gels. All other samples were measured relative to this reference. The data were normalized to give an average signal of 100.0 in innervated muscles. Mean values ± standard error of the mean. **p

    Journal: Journal of Molecular Signaling

    Article Title: Akt (protein kinase B) isoform phosphorylation and signaling downstream of mTOR (mammalian target of rapamycin) in denervated atrophic and hypertrophic mouse skeletal muscle

    doi: 10.1186/1750-2187-7-7

    Figure Lengend Snippet: Akt and GSK-3β protein and phosphorylation levels in 6-days denervated hypertrophic hemidiaphragm muscle. Expression of Akt1, Akt2 and GSK-3β total protein (t-Akt1, a; t-Akt2, c and t-GSK-3β, e) and phosphorylated Akt1 protein (p-Akt1) at S473 (b), phosphorylated Akt2 protein (p-Akt2) at S474 (d) and phosphorylated GSK-3β protein (p-GSK-3β) at S9 (f) in 6-days denervated hypertrophic hemidiaphragm muscle (Den) compared to innervated (Inn) controls. Representative Western blots are shown together with densitometric quantifications. One innervated hemidiaphragm muscle sample was used as a reference sample and was included in all gels. All other samples were measured relative to this reference. The data were normalized to give an average signal of 100.0 in innervated muscles. Mean values ± standard error of the mean. **p

    Article Snippet: Primary antibody for detecting phospho-Akt1 (S473) [07-310] was from Upstate Cell Signaling Solutions (Lake Placid, NY), primary antibody for detecting phospho-Akt2 (S474) [ab38513] was from Abcam (Cambridge, UK) and primary antibody for detecting total GSK-3β [610202] was from BD Transduction Laboratories (San Diego, CA).

    Techniques: Expressing, Western Blot

    Akt and GSK-3β protein and phosphorylation levels in 6-days denervated atrophic anterior tibial muscle . Expression of Akt1, Akt2 and GSK-3β total protein (t-Akt1, a; t-Akt2, c and t-GSK-3β, e) and phosphorylated Akt1 protein (p-Akt1) at S473 (b), phosphorylated Akt2 protein (p-Akt2) at S474 (d) and phosphorylated GSK-3β protein (p-GSK-3β) at S9 (f) in 6-days denervated atrophic anterior tibial (Den) muscle compared to innervated (Inn) controls. Representative Western blots are shown together with densitometric quantifications. One innervated anterior tibial muscle sample was used as a reference sample and was included in all gels. All other samples were measured relative to this reference. The data were normalized to give an average signal of 100.0 in innervated muscles. Mean values ± standard error of the mean. *p

    Journal: Journal of Molecular Signaling

    Article Title: Akt (protein kinase B) isoform phosphorylation and signaling downstream of mTOR (mammalian target of rapamycin) in denervated atrophic and hypertrophic mouse skeletal muscle

    doi: 10.1186/1750-2187-7-7

    Figure Lengend Snippet: Akt and GSK-3β protein and phosphorylation levels in 6-days denervated atrophic anterior tibial muscle . Expression of Akt1, Akt2 and GSK-3β total protein (t-Akt1, a; t-Akt2, c and t-GSK-3β, e) and phosphorylated Akt1 protein (p-Akt1) at S473 (b), phosphorylated Akt2 protein (p-Akt2) at S474 (d) and phosphorylated GSK-3β protein (p-GSK-3β) at S9 (f) in 6-days denervated atrophic anterior tibial (Den) muscle compared to innervated (Inn) controls. Representative Western blots are shown together with densitometric quantifications. One innervated anterior tibial muscle sample was used as a reference sample and was included in all gels. All other samples were measured relative to this reference. The data were normalized to give an average signal of 100.0 in innervated muscles. Mean values ± standard error of the mean. *p

    Article Snippet: Primary antibody for detecting phospho-Akt1 (S473) [07-310] was from Upstate Cell Signaling Solutions (Lake Placid, NY), primary antibody for detecting phospho-Akt2 (S474) [ab38513] was from Abcam (Cambridge, UK) and primary antibody for detecting total GSK-3β [610202] was from BD Transduction Laboratories (San Diego, CA).

    Techniques: Expressing, Western Blot

    Akt1-knockout mice have increased sensitivity to noise-induced hearing loss. A Western blot analysis of total cochlear homogenates shows a significant decrease in the expression of total Akt1/2 in Akt1-knockout mice. Data are presented as means + SD;

    Journal: JARO: Journal of the Association for Research in Otolaryngology

    Article Title: Increased Sensitivity to Noise-Induced Hearing Loss by Blockade of Endogenous PI3K/Akt Signaling

    doi: 10.1007/s10162-015-0508-x

    Figure Lengend Snippet: Akt1-knockout mice have increased sensitivity to noise-induced hearing loss. A Western blot analysis of total cochlear homogenates shows a significant decrease in the expression of total Akt1/2 in Akt1-knockout mice. Data are presented as means + SD;

    Article Snippet: The membranes were incubated with anti-p85α (1:1000), anti-p110α (1:1000), anti-p-Akt (S473) (1:1000), anti-total Akt1/2 (Cell Signaling Technology #9272, 1:1000), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Millipore #ABS16, 1:10,000) at 4 °C overnight, and then washed three times (10 min each) with PBS-T buffer.

    Techniques: Knock-Out, Mouse Assay, Western Blot, Expressing

    siRNA knocking-down efficacy. Differentiated 3T3-L1-GLUT4myc adipocytes were transfected with siRNAs for negative control (NC) and IR ( A ), PI3K ( B ), PDK1 ( C ), Akt1/2 ( D ), PKCλ/ι ( E ), PKCζ ( F ), PKCε ( G ) or PKCγ ( H ), and 48 h later Western blotting was carried out. The signal intensity for each protein was normalized by that for β-actin. In the graphs, each column represents the mean (±SEM) normalized intensity (n = 4 independent experiments). P values, unpaired t -test.

    Journal: Scientific Reports

    Article Title: The phosphatidylethanolamine derivative diDCP-LA-PE mimics intracellular insulin signaling

    doi: 10.1038/srep27267

    Figure Lengend Snippet: siRNA knocking-down efficacy. Differentiated 3T3-L1-GLUT4myc adipocytes were transfected with siRNAs for negative control (NC) and IR ( A ), PI3K ( B ), PDK1 ( C ), Akt1/2 ( D ), PKCλ/ι ( E ), PKCζ ( F ), PKCε ( G ) or PKCγ ( H ), and 48 h later Western blotting was carried out. The signal intensity for each protein was normalized by that for β-actin. In the graphs, each column represents the mean (±SEM) normalized intensity (n = 4 independent experiments). P values, unpaired t -test.

    Article Snippet: In the cell-free Akt2 assay, human recombinant Akt2 (Active Motif, Carlsbad, CA, USA) was reacted diDCP-LA-PE in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgCl2 , 12.5 mM glycerol 2-phosphate, 5 mM EGTA, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP containing PKCγ, -λ/ι, -ζ or -ε at 30 °C for 20 min. Phosphorylated Akt1/2 was quantified by Western blotting using antibodies against pT308(9) (Cell Signaling Technology), pS473(4) (Cell Signaling Technology), and Akt1/2 (Cell Signaling Technology).

    Techniques: Transfection, Negative Control, Western Blot

    PI3K, PDK1, Akt1/2, PKCζ and PKCε are implicated in the regulation of diDCP-LA-PE-induced GLUT4 translocation. Differentiated 3T3-L1-GLUT4myc adipocytes, which were transfected with siRNAs for IR ( A ), PI3K ( B ), PDK1 ( C ), Akt1/2 ( D ), PKCζ ( E ), PKCε ( F ), PKCλ/ι ( G ), PKCγ ( H ) or mTOR ( I ), were treated with diDCP-LA-PE (1 μM) for 20 min. In the graphs, each column represents the mean (±SEM) signal intensity for GLUT4 on the plasma membrane relative to that for whole cells (n = 4–6 independent experiments). P values, ANOVA followed by a Bonferroni correction.

    Journal: Scientific Reports

    Article Title: The phosphatidylethanolamine derivative diDCP-LA-PE mimics intracellular insulin signaling

    doi: 10.1038/srep27267

    Figure Lengend Snippet: PI3K, PDK1, Akt1/2, PKCζ and PKCε are implicated in the regulation of diDCP-LA-PE-induced GLUT4 translocation. Differentiated 3T3-L1-GLUT4myc adipocytes, which were transfected with siRNAs for IR ( A ), PI3K ( B ), PDK1 ( C ), Akt1/2 ( D ), PKCζ ( E ), PKCε ( F ), PKCλ/ι ( G ), PKCγ ( H ) or mTOR ( I ), were treated with diDCP-LA-PE (1 μM) for 20 min. In the graphs, each column represents the mean (±SEM) signal intensity for GLUT4 on the plasma membrane relative to that for whole cells (n = 4–6 independent experiments). P values, ANOVA followed by a Bonferroni correction.

    Article Snippet: In the cell-free Akt2 assay, human recombinant Akt2 (Active Motif, Carlsbad, CA, USA) was reacted diDCP-LA-PE in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgCl2 , 12.5 mM glycerol 2-phosphate, 5 mM EGTA, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP containing PKCγ, -λ/ι, -ζ or -ε at 30 °C for 20 min. Phosphorylated Akt1/2 was quantified by Western blotting using antibodies against pT308(9) (Cell Signaling Technology), pS473(4) (Cell Signaling Technology), and Akt1/2 (Cell Signaling Technology).

    Techniques: Translocation Assay, Transfection

    diDCP-LA-PE promotes GLUT4 translocation towards the cell surface in a PI3K-, PDK1-, Akt1/2- and PKC-dependent manner. Differentiated 3T3-L1-GLUT4myc adipocytes were treated with drugs as indicated for 20 min. Then, cells were separated into the cytosolic and plasma membrane fractions, followed by Western blotting. ( A ) Insulin (100 nM) and diDCP-LA-PE (1 μM). ( B ) diDCP-LA-PE at concentrations as indicated. ( C ) diDCP-LA-PS, diDCP-LA-PC, diDCP-LA-PI, DCP-LA and DL-PE at a concentration of 1 μM. diDCP-LA-PE (1 μM) in the presence of GS (50 μM) ( D ), WM (1 μM) ( E ), BX (100 nM) ( F ), MK (5 μM) ( G ) or GF (100 nM) ( H ). In the graphs, each column represents the mean (±SEM) signal intensity for GLUT4 on the plasma membrane relative to that for whole cells (n = 4 independent experiments). P values, ANOVA followed by a Bonferroni correction.

    Journal: Scientific Reports

    Article Title: The phosphatidylethanolamine derivative diDCP-LA-PE mimics intracellular insulin signaling

    doi: 10.1038/srep27267

    Figure Lengend Snippet: diDCP-LA-PE promotes GLUT4 translocation towards the cell surface in a PI3K-, PDK1-, Akt1/2- and PKC-dependent manner. Differentiated 3T3-L1-GLUT4myc adipocytes were treated with drugs as indicated for 20 min. Then, cells were separated into the cytosolic and plasma membrane fractions, followed by Western blotting. ( A ) Insulin (100 nM) and diDCP-LA-PE (1 μM). ( B ) diDCP-LA-PE at concentrations as indicated. ( C ) diDCP-LA-PS, diDCP-LA-PC, diDCP-LA-PI, DCP-LA and DL-PE at a concentration of 1 μM. diDCP-LA-PE (1 μM) in the presence of GS (50 μM) ( D ), WM (1 μM) ( E ), BX (100 nM) ( F ), MK (5 μM) ( G ) or GF (100 nM) ( H ). In the graphs, each column represents the mean (±SEM) signal intensity for GLUT4 on the plasma membrane relative to that for whole cells (n = 4 independent experiments). P values, ANOVA followed by a Bonferroni correction.

    Article Snippet: In the cell-free Akt2 assay, human recombinant Akt2 (Active Motif, Carlsbad, CA, USA) was reacted diDCP-LA-PE in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgCl2 , 12.5 mM glycerol 2-phosphate, 5 mM EGTA, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP containing PKCγ, -λ/ι, -ζ or -ε at 30 °C for 20 min. Phosphorylated Akt1/2 was quantified by Western blotting using antibodies against pT308(9) (Cell Signaling Technology), pS473(4) (Cell Signaling Technology), and Akt1/2 (Cell Signaling Technology).

    Techniques: Translocation Assay, Western Blot, Concentration Assay

    diDCP-LA-PE activates Akt1/2 in a PKCζ- or PKCε-dependent manner. Differentiated 3T3-L1-GLUT4myc adipocytes, which were non-transfected ( A ) and transfected with siRNAs for IR ( B ), PI3K ( C ), PDK1 ( D ), PKCζ ( E ), PKCε ( F ), PKCλ/ι ( G ), PKCγ ( H ) or mTOR ( I ) were treated with diDCP-LA-PE (1 μM) for 10 min followed by Western blotting. In the graphs, each column represents the mean (±SEM) signal intensity for phosphorylation at Thr308/309 [pT308(9)] or Ser473/474 [pS473(4)] relative to that for Akt1/2 (n = 4–6 independent experiments). P values, ANOVA followed by a Bonferroni correction. NS , not significant. NC, negative control; KD, knock-down.

    Journal: Scientific Reports

    Article Title: The phosphatidylethanolamine derivative diDCP-LA-PE mimics intracellular insulin signaling

    doi: 10.1038/srep27267

    Figure Lengend Snippet: diDCP-LA-PE activates Akt1/2 in a PKCζ- or PKCε-dependent manner. Differentiated 3T3-L1-GLUT4myc adipocytes, which were non-transfected ( A ) and transfected with siRNAs for IR ( B ), PI3K ( C ), PDK1 ( D ), PKCζ ( E ), PKCε ( F ), PKCλ/ι ( G ), PKCγ ( H ) or mTOR ( I ) were treated with diDCP-LA-PE (1 μM) for 10 min followed by Western blotting. In the graphs, each column represents the mean (±SEM) signal intensity for phosphorylation at Thr308/309 [pT308(9)] or Ser473/474 [pS473(4)] relative to that for Akt1/2 (n = 4–6 independent experiments). P values, ANOVA followed by a Bonferroni correction. NS , not significant. NC, negative control; KD, knock-down.

    Article Snippet: In the cell-free Akt2 assay, human recombinant Akt2 (Active Motif, Carlsbad, CA, USA) was reacted diDCP-LA-PE in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgCl2 , 12.5 mM glycerol 2-phosphate, 5 mM EGTA, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP containing PKCγ, -λ/ι, -ζ or -ε at 30 °C for 20 min. Phosphorylated Akt1/2 was quantified by Western blotting using antibodies against pT308(9) (Cell Signaling Technology), pS473(4) (Cell Signaling Technology), and Akt1/2 (Cell Signaling Technology).

    Techniques: Transfection, Western Blot, Negative Control

    Insulin stimulates phosphorylation of Akt1/2 in a PI3K-dependent manner in 3T3-L1-GLUT4myc adipocytes. Cells were left untreated or treated with insulin (Ins) (100 nM) for 10 min in the presence and absence of wortmannin (+WM) (20 nM) (A) or BX912 (+BX) (100 nM) (B). Western blotting was carried out using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graphs, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

    Journal: The Journal of Endocrinology

    Article Title: PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

    doi: 10.1530/JOE-13-0172

    Figure Lengend Snippet: Insulin stimulates phosphorylation of Akt1/2 in a PI3K-dependent manner in 3T3-L1-GLUT4myc adipocytes. Cells were left untreated or treated with insulin (Ins) (100 nM) for 10 min in the presence and absence of wortmannin (+WM) (20 nM) (A) or BX912 (+BX) (100 nM) (B). Western blotting was carried out using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graphs, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

    Article Snippet: Blotting membranes were blocked with TBS-T containing 5% (w/v) BSA and subsequently reacted with antibodies against peroxisome proliferator-activated receptor γ (PPARγ) (Cell Signaling Technology, Inc., Danvers, MA, USA), phospho-Thr308/309-Akt1/2 (pT308(9)), phospho-Ser473/474-Akt1/2 (pS473(4)), Akt1/2 (Cell Signaling Technology), Akt1 (Cell Signaling Technology), Akt2 (Cell Signaling Technology), PI3K (Sigma), PDK1 (Sigma), or β-actin (Sigma).

    Techniques: Western Blot

    Insulin-induced Akt1/2 phosphorylation at Thr308 and Ser473 was prevented by knocking-down PI3K, but not PDK1, in 3T3-L1-GLUT4myc adipocytes. (A) In the left panel, cells were transfected with the NC siRNA or the PI3K siRNA, and 48 h after transfection western blotting was carried out using antibodies against PI3K or β-actin. Signal intensities for PI3K were normalized to those for β-actin. In the graph, each column represents the mean (± s.e.m .) normalized expression of PI3K ( n =4 independent experiments). P value, unpaired t -test. In the right panel, cells transfected with the NC siRNA (NC) or the PI3K siRNA (PI3K KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. (B) In the left panel, cells were transfected with the NC siRNA or the PDK1 siRNA, and 48 h after transfection western blotting was carried out using antibodies against PDK1 or β-actin. Signal intensities for PDK1 were normalized to those for β-actin. In the graph, each column represents the mean (± s.e.m .) normalized expression of PDK1 ( n =4 independent experiments). P value, unpaired t -test. In the right panel, cells transfected with the NC siRNA (NC) or the PDK1 siRNA (PDK1 KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

    Journal: The Journal of Endocrinology

    Article Title: PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

    doi: 10.1530/JOE-13-0172

    Figure Lengend Snippet: Insulin-induced Akt1/2 phosphorylation at Thr308 and Ser473 was prevented by knocking-down PI3K, but not PDK1, in 3T3-L1-GLUT4myc adipocytes. (A) In the left panel, cells were transfected with the NC siRNA or the PI3K siRNA, and 48 h after transfection western blotting was carried out using antibodies against PI3K or β-actin. Signal intensities for PI3K were normalized to those for β-actin. In the graph, each column represents the mean (± s.e.m .) normalized expression of PI3K ( n =4 independent experiments). P value, unpaired t -test. In the right panel, cells transfected with the NC siRNA (NC) or the PI3K siRNA (PI3K KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. (B) In the left panel, cells were transfected with the NC siRNA or the PDK1 siRNA, and 48 h after transfection western blotting was carried out using antibodies against PDK1 or β-actin. Signal intensities for PDK1 were normalized to those for β-actin. In the graph, each column represents the mean (± s.e.m .) normalized expression of PDK1 ( n =4 independent experiments). P value, unpaired t -test. In the right panel, cells transfected with the NC siRNA (NC) or the PDK1 siRNA (PDK1 KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

    Article Snippet: Blotting membranes were blocked with TBS-T containing 5% (w/v) BSA and subsequently reacted with antibodies against peroxisome proliferator-activated receptor γ (PPARγ) (Cell Signaling Technology, Inc., Danvers, MA, USA), phospho-Thr308/309-Akt1/2 (pT308(9)), phospho-Ser473/474-Akt1/2 (pS473(4)), Akt1/2 (Cell Signaling Technology), Akt1 (Cell Signaling Technology), Akt2 (Cell Signaling Technology), PI3K (Sigma), PDK1 (Sigma), or β-actin (Sigma).

    Techniques: Transfection, Western Blot, Expressing

    Insulin stimulates GLUT4 translocation toward the plasma membrane in an Akt1/2-dependent manner. (A) The Akt1 or Akt2 concentration/intensity standard curves were made using a human recombinant Akt1 or Akt2 respectively. 3T3-L1-GLUT4myc fibroblasts on day 14 after differentiation induction were lysed followed by western blotting using antibodies against Akt1 and Akt2, and the amount of Akt1 and Akt2 was calculated from each standard curve ( n =4 independent experiments). (B) 3T3-L1-GLUT4myc adipocytes were treated with insulin (100 nM) in the presence and absence of MK2206 (+MK) (5 μM). Then, cells were lysed and separated into the cytosolic (C) and plasma membrane fractions (M), followed by western blotting using an antibody against c-myc. In the graph, each column represents the mean (± s.e.m .) ratio of signal intensity for c-myc in the plasma membrane fraction to that in the total cell ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant. (C) In the left panel, 3T3-L1-GLUT4myc adipocytes were transfected with the NC siRNA or the Akt1/2 siRNA, and 48 h after transfection western blotting was carried out using antibodies against Akt1/2 or β-actin. Signal intensities for Akt1/2 were normalized to those for β-actin. In the graph, each column represents the mean (± s.e.m .) normalized expression of Akt1/2 ( n =4 independent experiments). P value, unpaired t -test. In the right panel, cells transfected with the NC siRNA (NC) or the Akt1/2 siRNA (Akt1/2 KD) were left untreated or treated with insulin (100 nM) for 20 min. Then, cells were lysed and separated into the cytosolic (C) and plasma membrane fractions (M), followed by western blotting using an antibody against c-myc. In the graph, each column represents the mean (± s.e.m .) ratio of signal intensity for c-myc in the plasma membrane fraction to that in the total cell ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

    Journal: The Journal of Endocrinology

    Article Title: PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

    doi: 10.1530/JOE-13-0172

    Figure Lengend Snippet: Insulin stimulates GLUT4 translocation toward the plasma membrane in an Akt1/2-dependent manner. (A) The Akt1 or Akt2 concentration/intensity standard curves were made using a human recombinant Akt1 or Akt2 respectively. 3T3-L1-GLUT4myc fibroblasts on day 14 after differentiation induction were lysed followed by western blotting using antibodies against Akt1 and Akt2, and the amount of Akt1 and Akt2 was calculated from each standard curve ( n =4 independent experiments). (B) 3T3-L1-GLUT4myc adipocytes were treated with insulin (100 nM) in the presence and absence of MK2206 (+MK) (5 μM). Then, cells were lysed and separated into the cytosolic (C) and plasma membrane fractions (M), followed by western blotting using an antibody against c-myc. In the graph, each column represents the mean (± s.e.m .) ratio of signal intensity for c-myc in the plasma membrane fraction to that in the total cell ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant. (C) In the left panel, 3T3-L1-GLUT4myc adipocytes were transfected with the NC siRNA or the Akt1/2 siRNA, and 48 h after transfection western blotting was carried out using antibodies against Akt1/2 or β-actin. Signal intensities for Akt1/2 were normalized to those for β-actin. In the graph, each column represents the mean (± s.e.m .) normalized expression of Akt1/2 ( n =4 independent experiments). P value, unpaired t -test. In the right panel, cells transfected with the NC siRNA (NC) or the Akt1/2 siRNA (Akt1/2 KD) were left untreated or treated with insulin (100 nM) for 20 min. Then, cells were lysed and separated into the cytosolic (C) and plasma membrane fractions (M), followed by western blotting using an antibody against c-myc. In the graph, each column represents the mean (± s.e.m .) ratio of signal intensity for c-myc in the plasma membrane fraction to that in the total cell ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

    Article Snippet: Blotting membranes were blocked with TBS-T containing 5% (w/v) BSA and subsequently reacted with antibodies against peroxisome proliferator-activated receptor γ (PPARγ) (Cell Signaling Technology, Inc., Danvers, MA, USA), phospho-Thr308/309-Akt1/2 (pT308(9)), phospho-Ser473/474-Akt1/2 (pS473(4)), Akt1/2 (Cell Signaling Technology), Akt1 (Cell Signaling Technology), Akt2 (Cell Signaling Technology), PI3K (Sigma), PDK1 (Sigma), or β-actin (Sigma).

    Techniques: Translocation Assay, Concentration Assay, Recombinant, Western Blot, Transfection, Expressing

    PI3K-dependent and PDK1-independent Akt1 phosphorylation and PI3K-/PDK1-dependent Akt2 phosphorylation under cell-free conditions. Akt1 or Akt2 was reacted with (+) and without (−) PI3K (1 μg/ml) (A and C) or PDK1 (1 μg/ml) (B and D) in the presence and absence of wortmannin (WM) (20 nM) or BX912 (BX) (100 nM), and western blotting was carried out using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1 (pAkt1) or Akt2 (pAkt2) were normalized to those for Akt1 or Akt2. In the graphs, each value represents the mean (± s.e.m .) intensity for pAkt1 or pAkt2 at each site ( n =4). P values, Dunnett's test. NS, not significant.

    Journal: The Journal of Endocrinology

    Article Title: PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

    doi: 10.1530/JOE-13-0172

    Figure Lengend Snippet: PI3K-dependent and PDK1-independent Akt1 phosphorylation and PI3K-/PDK1-dependent Akt2 phosphorylation under cell-free conditions. Akt1 or Akt2 was reacted with (+) and without (−) PI3K (1 μg/ml) (A and C) or PDK1 (1 μg/ml) (B and D) in the presence and absence of wortmannin (WM) (20 nM) or BX912 (BX) (100 nM), and western blotting was carried out using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1 (pAkt1) or Akt2 (pAkt2) were normalized to those for Akt1 or Akt2. In the graphs, each value represents the mean (± s.e.m .) intensity for pAkt1 or pAkt2 at each site ( n =4). P values, Dunnett's test. NS, not significant.

    Article Snippet: Blotting membranes were blocked with TBS-T containing 5% (w/v) BSA and subsequently reacted with antibodies against peroxisome proliferator-activated receptor γ (PPARγ) (Cell Signaling Technology, Inc., Danvers, MA, USA), phospho-Thr308/309-Akt1/2 (pT308(9)), phospho-Ser473/474-Akt1/2 (pS473(4)), Akt1/2 (Cell Signaling Technology), Akt1 (Cell Signaling Technology), Akt2 (Cell Signaling Technology), PI3K (Sigma), PDK1 (Sigma), or β-actin (Sigma).

    Techniques: Western Blot

    Effects of Cr(VI) on pro-apoptotic and pro-survival signaling pathways in TM3 and TM4 cells. ( a , b ) Western blot analysis of mitochondria-dependent apoptotic pathways in TM3 cells after treatment with different concentrations of Cr(VI) for 24 h. ( c ) Western blot analysis of phospho and total AKT1, MAPK and P53 proteins in TM3 cells after treatment with different concentrations of Cr(VI) for 24 h. ( d – e ) Western blot analysis of mitochondria-dependent apoptotic pathways in TM4 cells after treatment with different concentrations of Cr(VI) for 24 h. ( f ) Western blot analysis of phospho and total AKT1, MAPK and P53 proteins in TM4 cells after treatment with different concentrations of Cr(VI) for 24 h. BAX, BCL2, CASP9, CASP3, PARP, AKT1, ERK 1/2, JNK 1/2, P38, and P53 proteins were analyzed in the whole cell protein lysate. CYCS was analyzed in the cytosolic fraction. For the BCL2/BAX ratio, values are expressed as mean ± S.E.M. ( n = 3 ). * P

    Journal: Scientific Reports

    Article Title: Hexavalent chromium induces apoptosis in male somatic and spermatogonial stem cells via redox imbalance

    doi: 10.1038/srep13921

    Figure Lengend Snippet: Effects of Cr(VI) on pro-apoptotic and pro-survival signaling pathways in TM3 and TM4 cells. ( a , b ) Western blot analysis of mitochondria-dependent apoptotic pathways in TM3 cells after treatment with different concentrations of Cr(VI) for 24 h. ( c ) Western blot analysis of phospho and total AKT1, MAPK and P53 proteins in TM3 cells after treatment with different concentrations of Cr(VI) for 24 h. ( d – e ) Western blot analysis of mitochondria-dependent apoptotic pathways in TM4 cells after treatment with different concentrations of Cr(VI) for 24 h. ( f ) Western blot analysis of phospho and total AKT1, MAPK and P53 proteins in TM4 cells after treatment with different concentrations of Cr(VI) for 24 h. BAX, BCL2, CASP9, CASP3, PARP, AKT1, ERK 1/2, JNK 1/2, P38, and P53 proteins were analyzed in the whole cell protein lysate. CYCS was analyzed in the cytosolic fraction. For the BCL2/BAX ratio, values are expressed as mean ± S.E.M. ( n = 3 ). * P

    Article Snippet: The antibodies used for immunoblotting were against phospho P53, phospho ERK1/2, total ERK1/2, total AKT1 (Cell Signaling Technology, Beverly, MA), phospho P38 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phospho AKT1, phospho JNK1/2, total P53, total JNK1/2, total P38, BAX, BCL2, CASP9, CASP3, PARP, beta-actin (Abcam, Cambridge, MA), and cytochrome c (ENZO Diagnostics Inc., Farmingdale, NY).

    Techniques: Western Blot

    The PI3Kγ signaling is enhanced in the human heart tissue with Chagas disease. a Transcriptome analysis of family I PIK3 genes expression ( PIK3CA , PIK3CB , PIK3CD , and PIK3CG ) in healthy control (Ctl; n = 7) and patients with idiopathic dilated cardiomyopathy (DCM; n = 14) or chronic chagasic cardiomyopathy (CCC; n = 10). b RT-PCR analysis of the mRNA expression of PIK3CA , PIK3CB , PIK3CD , and PIK3CG genes in the heart tissue of Ctl ( n = 5); DCM ( n = 10); and CCC ( n = 10) patients. GAPDH was used as a housekeeping gene. c Representative western blots and analysis of phosphorylated (p) and total (t) AKT1 expression in the heart tissue of Ctl ( n = 5), DCM ( n = 10), and CCC ( n = 10) patients. GAPDH was used as a loading control. d Representative western blots and analysis of phosphorylated (p) and total (t) AKT2 expression in the heart tissue of Ctl ( n = 5), DCM ( n = 10), and CCC ( n = 10) patients. GAPDH was used as a loading control. AU refers to arbitrary units. ns = no statistical significance (one-way ANOVA with Tukey’s post hoc test in b – d ). * P

    Journal: Nature Communications

    Article Title: Canonical PI3Kγ signaling in myeloid cells restricts Trypanosoma cruzi infection and dampens chagasic myocarditis

    doi: 10.1038/s41467-018-03986-3

    Figure Lengend Snippet: The PI3Kγ signaling is enhanced in the human heart tissue with Chagas disease. a Transcriptome analysis of family I PIK3 genes expression ( PIK3CA , PIK3CB , PIK3CD , and PIK3CG ) in healthy control (Ctl; n = 7) and patients with idiopathic dilated cardiomyopathy (DCM; n = 14) or chronic chagasic cardiomyopathy (CCC; n = 10). b RT-PCR analysis of the mRNA expression of PIK3CA , PIK3CB , PIK3CD , and PIK3CG genes in the heart tissue of Ctl ( n = 5); DCM ( n = 10); and CCC ( n = 10) patients. GAPDH was used as a housekeeping gene. c Representative western blots and analysis of phosphorylated (p) and total (t) AKT1 expression in the heart tissue of Ctl ( n = 5), DCM ( n = 10), and CCC ( n = 10) patients. GAPDH was used as a loading control. d Representative western blots and analysis of phosphorylated (p) and total (t) AKT2 expression in the heart tissue of Ctl ( n = 5), DCM ( n = 10), and CCC ( n = 10) patients. GAPDH was used as a loading control. AU refers to arbitrary units. ns = no statistical significance (one-way ANOVA with Tukey’s post hoc test in b – d ). * P

    Article Snippet: The primary antibodies used in the study were: phospho-AKT1 (1:600; Cell Signaling, cat. 9018); total AKT1 (1:1000; Cell Signaling, cat. 2938); phospho AKT2 (1:600, Cell Signaling, cat. 8599); total AKT2 (1:1000; Cell Signaling, cat. 3063); and GAPDH (1: 5000; Sigma-Aldrich, cat. G9545).

    Techniques: Expressing, CTL Assay, Countercurrent Chromatography, Reverse Transcription Polymerase Chain Reaction, Western Blot

    AKT1 signaling in myeloid cells confers resistance to T . cruzi infection. a Survival rate of littermate control ( n = 7) and Akt1 −/− Lysm cre ( n = 7) mice after infection with 10 3 trypomastigote forms of T . cruzi Y strain. b Quantitative PCR analysis of picogram of T . cruzi DNA presents in 1 ng of heart tissue DNA isolated from WT ( n = 5) and Akt1 −/− Lysm cre ( n = 5) mice. c Survival rate of WT ( n = 7) and AKT2 −/− ( n = 7) mice infected with 10 3 trypomastigote forms of T . cruzi Y strain. * P

    Journal: Nature Communications

    Article Title: Canonical PI3Kγ signaling in myeloid cells restricts Trypanosoma cruzi infection and dampens chagasic myocarditis

    doi: 10.1038/s41467-018-03986-3

    Figure Lengend Snippet: AKT1 signaling in myeloid cells confers resistance to T . cruzi infection. a Survival rate of littermate control ( n = 7) and Akt1 −/− Lysm cre ( n = 7) mice after infection with 10 3 trypomastigote forms of T . cruzi Y strain. b Quantitative PCR analysis of picogram of T . cruzi DNA presents in 1 ng of heart tissue DNA isolated from WT ( n = 5) and Akt1 −/− Lysm cre ( n = 5) mice. c Survival rate of WT ( n = 7) and AKT2 −/− ( n = 7) mice infected with 10 3 trypomastigote forms of T . cruzi Y strain. * P

    Article Snippet: The primary antibodies used in the study were: phospho-AKT1 (1:600; Cell Signaling, cat. 9018); total AKT1 (1:1000; Cell Signaling, cat. 2938); phospho AKT2 (1:600, Cell Signaling, cat. 8599); total AKT2 (1:1000; Cell Signaling, cat. 3063); and GAPDH (1: 5000; Sigma-Aldrich, cat. G9545).

    Techniques: Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Isolation

    The PI3Kγ signaling is enhanced in the heart tissue of mice after experimental infection with T . cruzi . a Transcriptome analysis for the expression of Pik3ca , Pik3cd , and Pik3cg genes in non-infected C57BL/6J mice or 18 days post infection with T . cruzi . b RT-PCR analysis of the mRNA expression of Pik3ca , Pik3cb , Pik3cd , and Pik3cg genes in the heart tissue of C57BL/6 non-infected mice ( n = 7) or 18 days post infection with T . cruzi Y strain ( n = 11). Gapdh was used as a housekeeping gene. c Representative western blots and analysis of phosphorylated (p) and total (t) AKT1 expression in the heart tissue of non-infected C57BL/6 mice or 18 days post infection with T . cruzi ( n = 6). GAPDH was used as a loading control. d Representative western blots and analysis of phosphorylated (p) and total (t) AKT2 expression in the heart tissue of non-infected C57BL/6 mice or 18 days post infection with T . cruzi ( n = 6). GAPDH was used as a loading control. AU refers to arbitrary units ( c , d ). * P

    Journal: Nature Communications

    Article Title: Canonical PI3Kγ signaling in myeloid cells restricts Trypanosoma cruzi infection and dampens chagasic myocarditis

    doi: 10.1038/s41467-018-03986-3

    Figure Lengend Snippet: The PI3Kγ signaling is enhanced in the heart tissue of mice after experimental infection with T . cruzi . a Transcriptome analysis for the expression of Pik3ca , Pik3cd , and Pik3cg genes in non-infected C57BL/6J mice or 18 days post infection with T . cruzi . b RT-PCR analysis of the mRNA expression of Pik3ca , Pik3cb , Pik3cd , and Pik3cg genes in the heart tissue of C57BL/6 non-infected mice ( n = 7) or 18 days post infection with T . cruzi Y strain ( n = 11). Gapdh was used as a housekeeping gene. c Representative western blots and analysis of phosphorylated (p) and total (t) AKT1 expression in the heart tissue of non-infected C57BL/6 mice or 18 days post infection with T . cruzi ( n = 6). GAPDH was used as a loading control. d Representative western blots and analysis of phosphorylated (p) and total (t) AKT2 expression in the heart tissue of non-infected C57BL/6 mice or 18 days post infection with T . cruzi ( n = 6). GAPDH was used as a loading control. AU refers to arbitrary units ( c , d ). * P

    Article Snippet: The primary antibodies used in the study were: phospho-AKT1 (1:600; Cell Signaling, cat. 9018); total AKT1 (1:1000; Cell Signaling, cat. 2938); phospho AKT2 (1:600, Cell Signaling, cat. 8599); total AKT2 (1:1000; Cell Signaling, cat. 3063); and GAPDH (1: 5000; Sigma-Aldrich, cat. G9545).

    Techniques: Mouse Assay, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    AKT1 overexpression partially reverses miRNA-490-3p-induced apoptosis. The effect of AKT1 on (A) SNU-1 and (B) N87 cell growth was analyzed by cell counting kit-8 assay following miRNA-490-3p overexpression. The effect of AKT1 on the rate of apoptosis in (C) SNU-1 and (D) N87, following overexpression of miRNA-490-3p. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: MicroRNA-490-3p regulates cell proliferation and apoptosis in gastric cancer via direct targeting of AKT1

    doi: 10.3892/etm.2018.7042

    Figure Lengend Snippet: AKT1 overexpression partially reverses miRNA-490-3p-induced apoptosis. The effect of AKT1 on (A) SNU-1 and (B) N87 cell growth was analyzed by cell counting kit-8 assay following miRNA-490-3p overexpression. The effect of AKT1 on the rate of apoptosis in (C) SNU-1 and (D) N87, following overexpression of miRNA-490-3p. *P

    Article Snippet: The separated proteins were subsequently transferred onto a polyvinylidene difluoride membrane and blocked with 5% skimmed milk at 20°C for 2 h. The membranes were incubated with primary antibodies, including AKT1 (cat. no. 2938), GAPDH (cat. no. 5174; both 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C.

    Techniques: Over Expression, Cell Counting

    miRNA-490-3p directly targets AKT1 expression. Following miRNA-490-3p overexpression luciferase activity was measured in (A) SNU-1 and (B) N87 cells. The mRNA expression level of AKT1 was determined by reverse transcription-quantitative polymerase chain reaction in (C) SNU-1 and (D) N87 cells following transient transfection with miRNA-490-3p mimic, inhibitor and negative control. (E) The protein expression level of AKT1 was determined by western blot analysis. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: MicroRNA-490-3p regulates cell proliferation and apoptosis in gastric cancer via direct targeting of AKT1

    doi: 10.3892/etm.2018.7042

    Figure Lengend Snippet: miRNA-490-3p directly targets AKT1 expression. Following miRNA-490-3p overexpression luciferase activity was measured in (A) SNU-1 and (B) N87 cells. The mRNA expression level of AKT1 was determined by reverse transcription-quantitative polymerase chain reaction in (C) SNU-1 and (D) N87 cells following transient transfection with miRNA-490-3p mimic, inhibitor and negative control. (E) The protein expression level of AKT1 was determined by western blot analysis. *P

    Article Snippet: The separated proteins were subsequently transferred onto a polyvinylidene difluoride membrane and blocked with 5% skimmed milk at 20°C for 2 h. The membranes were incubated with primary antibodies, including AKT1 (cat. no. 2938), GAPDH (cat. no. 5174; both 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C.

    Techniques: Expressing, Over Expression, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Western Blot

    CMP treatment of SH-SY5Y cells affects intermediary cell metabolic responses to stimulatory ligands. Representative western blots and associated histograms depict the changes in ERK1/2 ( A ), c-Src ( B ) and Akt-1 ( C ) activation in response to β-methylcholine (MeCh, 10 nM) or brain-derived neurotrophic factor (BDNF, 10 ng/mL) stimulating ligands in both control (blue bars) or CMP-treated (red bars) SH-SY5Y cells. The time courses (0-60 minutes) for stimulation are denoted in the associated histograms depicting the mean ± SEM from at least three separate experiments. Statistical significance is indicated for changes in kinase activity in the CMP state relative to their time-matched control in vehicle-treated (control) cells. Statistical significance was measured using a Student's t-test (GraphPad Prism v.3): * - p

    Journal: PLoS ONE

    Article Title: Minimal Peroxide Exposure of Neuronal Cells Induces Multifaceted Adaptive Responses

    doi: 10.1371/journal.pone.0014352

    Figure Lengend Snippet: CMP treatment of SH-SY5Y cells affects intermediary cell metabolic responses to stimulatory ligands. Representative western blots and associated histograms depict the changes in ERK1/2 ( A ), c-Src ( B ) and Akt-1 ( C ) activation in response to β-methylcholine (MeCh, 10 nM) or brain-derived neurotrophic factor (BDNF, 10 ng/mL) stimulating ligands in both control (blue bars) or CMP-treated (red bars) SH-SY5Y cells. The time courses (0-60 minutes) for stimulation are denoted in the associated histograms depicting the mean ± SEM from at least three separate experiments. Statistical significance is indicated for changes in kinase activity in the CMP state relative to their time-matched control in vehicle-treated (control) cells. Statistical significance was measured using a Student's t-test (GraphPad Prism v.3): * - p

    Article Snippet: Specific primary antisera used were obtained from the following sources: lamin-A, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin, calreticulin, extracellular signal-regulated kinase (ERK), c-Src, 14-3-3 zeta and beta-actin - Santa Cruz Biotechnology, Santa Cruz, CA; GIT-2 - NeuroMab, San Jose CA; tyrosine-418 phosphorylated c-Src - Invitrogen, Carlsbad CA; phospho-ERK, phospho-Akt-1 and Akt-1 - Cell Signaling Technology, Danvers MA).

    Techniques: Western Blot, Activation Assay, Derivative Assay, Activity Assay

    NF-κB nuclear translocations and Akt phosphorylations were associated with O-GlcNAcylation. The activations of Akt, β-catenin and NF-kB were determined in 48 h treated siOGT; and siOGA cells. ( A ) Total Akt and phosphorylated-Akt were determined by western blot analysis. ( B ) Total cellular and nuclear β-catenin and ( C ) NF-κB were examined by western blotting. ( D ) Cellular localization of NF-κB (green) is demonstrated by immunocytofluorescent staining; nuclei (blue) are stained with Hoechst 33342. The quantitative analyses are compared in each graph by using the scramble control as 100%. The results (mean ± SEM) are the averages from three independent experiments; * P

    Journal: Scientific Reports

    Article Title: Mechanistic insights of O-GlcNAcylation that promote progression of cholangiocarcinoma cells via nuclear translocation of NF-κB

    doi: 10.1038/srep27853

    Figure Lengend Snippet: NF-κB nuclear translocations and Akt phosphorylations were associated with O-GlcNAcylation. The activations of Akt, β-catenin and NF-kB were determined in 48 h treated siOGT; and siOGA cells. ( A ) Total Akt and phosphorylated-Akt were determined by western blot analysis. ( B ) Total cellular and nuclear β-catenin and ( C ) NF-κB were examined by western blotting. ( D ) Cellular localization of NF-κB (green) is demonstrated by immunocytofluorescent staining; nuclei (blue) are stained with Hoechst 33342. The quantitative analyses are compared in each graph by using the scramble control as 100%. The results (mean ± SEM) are the averages from three independent experiments; * P

    Article Snippet: Immunodetection of particular proteins was performed using specific monoclonal antibodies as follow; 1:200 of anti-OGT and anti-MMP7 (Santa Cruz Biotechnology, Santa Cruz, CA); 1:400 of anti-β-catenin (BD Transduction Laboratories, New Jersey); 1:1000 of anti-NF-κB (p65) (Santa Cruz), anti-β-tubulin (Santa Cruz, CA), anti-O-GlcNAc (RL2, Pierce Biotechnology, IL), anti-Akt and anti-pAkt (Cell Signaling Technology, Danvers, MA).

    Techniques: Western Blot, Staining

    Hyperoxia-induced Nrf2-target gene expression in AKT1-depleted cells. (A) qRT-PCR analysis of hyperoxia-induced Nrf2-target gene expression in cells transfected with either scrambled siRNA (Scr-Si) or AKT1 siRNA. (A) Expression of AKT1 was calculated relative to Scr-Si-transfected room air-exposed samples. (B) Nrf2-target gene expression was calculated relative to Scr-Si-transfected room air-exposed samples. Values from the Scr-si transfected cells exposed to room air are considered as one unit. p ≤ 0.05, room air (RA) vs. hyperoxia (hyp); † p ≤ 0.05, Scr siRNA vs AKT1-siRNA. Data are expressed as mean ± SEM (n = 3–4).

    Journal: PLoS ONE

    Article Title: PI3K-AKT Signaling via Nrf2 Protects against Hyperoxia-Induced Acute Lung Injury, but Promotes Inflammation Post-Injury Independent of Nrf2 in Mice

    doi: 10.1371/journal.pone.0129676

    Figure Lengend Snippet: Hyperoxia-induced Nrf2-target gene expression in AKT1-depleted cells. (A) qRT-PCR analysis of hyperoxia-induced Nrf2-target gene expression in cells transfected with either scrambled siRNA (Scr-Si) or AKT1 siRNA. (A) Expression of AKT1 was calculated relative to Scr-Si-transfected room air-exposed samples. (B) Nrf2-target gene expression was calculated relative to Scr-Si-transfected room air-exposed samples. Values from the Scr-si transfected cells exposed to room air are considered as one unit. p ≤ 0.05, room air (RA) vs. hyperoxia (hyp); † p ≤ 0.05, Scr siRNA vs AKT1-siRNA. Data are expressed as mean ± SEM (n = 3–4).

    Article Snippet: Transient transfection of AKT1 siRNA Suppression of AKT1 function was performed by knockdown of AKT1 expression by AKT1 small interfering RNA (AKT1-siRNA) (cat# M-004364-00, Cell Signaling Technology).

    Techniques: Expressing, Quantitative RT-PCR, Transfection

    Phosphorylation of palladin at Ser507 by Akt1 inhibits cell migration A , MDA-MB-231 cells infected with palladin shRNA or empty lentiviral vectors were transfected with HA-Myr-Akt1 or control vector. 24 h after transfection cells were subjected to Transwell migration assays and lysates were immunoblotted with the indicated antibodies. B , MDA-MB-231 cells were infected with palladin shRNA lentiviral vector or empty vector. Forty-eight hours after infection cells were serum-starved overnight then stimulated with IGF-1 (100 ng ml −1 ) for 18 h, followed by Transwell migration assays. Total cell lysates were subjected to immunoblot analysis. C , MDA-MB-231 cells were infected with palladin shRNA and/or Akt1 shRNA lentiviral vectors, or empty vector. Forty-eight hours after infection, cells were subjected to Transwell migration assays and lysates were immunoblotted. D .

    Journal: Molecular cell

    Article Title: THE ACTIN BUNDLING PROTEIN PALLADIN IS AN AKT1-SPECIFIC SUBSTRATE THAT REGULATES BREAST CANCER CELL MIGRATION

    doi: 10.1016/j.molcel.2010.02.031

    Figure Lengend Snippet: Phosphorylation of palladin at Ser507 by Akt1 inhibits cell migration A , MDA-MB-231 cells infected with palladin shRNA or empty lentiviral vectors were transfected with HA-Myr-Akt1 or control vector. 24 h after transfection cells were subjected to Transwell migration assays and lysates were immunoblotted with the indicated antibodies. B , MDA-MB-231 cells were infected with palladin shRNA lentiviral vector or empty vector. Forty-eight hours after infection cells were serum-starved overnight then stimulated with IGF-1 (100 ng ml −1 ) for 18 h, followed by Transwell migration assays. Total cell lysates were subjected to immunoblot analysis. C , MDA-MB-231 cells were infected with palladin shRNA and/or Akt1 shRNA lentiviral vectors, or empty vector. Forty-eight hours after infection, cells were subjected to Transwell migration assays and lysates were immunoblotted. D .

    Article Snippet: Palladin was immunoprecipitated from cell extracts and incubated with 500 ng recombinant Akt1 (Cell Signaling Technology) or Akt2 (Cell Signaling Technology) in the presence of 250 μM cold ATP in a kinase buffer for 1 h at 30°C.

    Techniques: Migration, Multiple Displacement Amplification, Infection, shRNA, Transfection, Plasmid Preparation

    Palladin is an Akt1-specific substrate A , HeLa, SKBR3 and MDA-MB-231 cells were transfected with HA-Myr-Akt1, HA-Myr-Akt2, or empty vector, along with GFP-palladin. 24 h after transfection cells were lysed and immunoprecipitated with anti-GFP. Whole cell lysates and immunoprecipitates were subjected to immunoblotting. B , SKBR3 cells were transfected with HA-palladin WT in serum-free medium for 24 h. Anti-HA immunoprecipitates were used as substrates in in vitro kinase assays with recombinant active Akt1 or Akt2. The kinase reaction was terminated and samples were immunoblotted. C , HeLa and SKBR3 cells were co-transfected with HA-palladin and Akt1, Akt2 or control luciferase siRNA. 36 h after transfection, HeLa and SKBR3 cells were serum starved for 12 h then treated with IGF-1 (100 ng ml −1 ) for 20 min and EGF (20 ng ml −1 ) for 10 min, respectively. BT-549 cells were infected with Akt1 or Akt2 shRNA lentiviral vector or empty vector for 72 h, followed by serum starvation and then stimulation with EGF (20 ng ml −1 ) for 10 min. Lysates were subjected to immunoprecipitation and immunoblot analysis. D .

    Journal: Molecular cell

    Article Title: THE ACTIN BUNDLING PROTEIN PALLADIN IS AN AKT1-SPECIFIC SUBSTRATE THAT REGULATES BREAST CANCER CELL MIGRATION

    doi: 10.1016/j.molcel.2010.02.031

    Figure Lengend Snippet: Palladin is an Akt1-specific substrate A , HeLa, SKBR3 and MDA-MB-231 cells were transfected with HA-Myr-Akt1, HA-Myr-Akt2, or empty vector, along with GFP-palladin. 24 h after transfection cells were lysed and immunoprecipitated with anti-GFP. Whole cell lysates and immunoprecipitates were subjected to immunoblotting. B , SKBR3 cells were transfected with HA-palladin WT in serum-free medium for 24 h. Anti-HA immunoprecipitates were used as substrates in in vitro kinase assays with recombinant active Akt1 or Akt2. The kinase reaction was terminated and samples were immunoblotted. C , HeLa and SKBR3 cells were co-transfected with HA-palladin and Akt1, Akt2 or control luciferase siRNA. 36 h after transfection, HeLa and SKBR3 cells were serum starved for 12 h then treated with IGF-1 (100 ng ml −1 ) for 20 min and EGF (20 ng ml −1 ) for 10 min, respectively. BT-549 cells were infected with Akt1 or Akt2 shRNA lentiviral vector or empty vector for 72 h, followed by serum starvation and then stimulation with EGF (20 ng ml −1 ) for 10 min. Lysates were subjected to immunoprecipitation and immunoblot analysis. D .

    Article Snippet: Palladin was immunoprecipitated from cell extracts and incubated with 500 ng recombinant Akt1 (Cell Signaling Technology) or Akt2 (Cell Signaling Technology) in the presence of 250 μM cold ATP in a kinase buffer for 1 h at 30°C.

    Techniques: Multiple Displacement Amplification, Transfection, Plasmid Preparation, Immunoprecipitation, In Vitro, Recombinant, Luciferase, Infection, shRNA

    Figure 6. Knockdown of LAMP2A triggers AKT1 phosphorylation at Ser473. Proliferating T47D cells were either transfected with LAMP2A, or empty pcDNA3 vector for 48 h ( A, C and E ) and with LAMP2A siRNA or control siRNA for 72 h ( B, D and F ). ( A and B ) showing western blots analysis of total AKT1 and phospho AKT1 (Ser 473). ( C and D ) demonstrate AKT1 kinase activity using AKT1 substrate GSK3 fusion protein as a measure of AKT1 activation. ( E and F ) demonstrate the AKT1 substrate assay under oxidative stress condition rendered by 150 nM H 2 O 2 . Densitometric analyses from ( E and F ) are given under each panel. Data are mean ± SEMs, and significant changes are indicated by an asterisk (*p

    Journal: Autophagy

    Article Title: LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

    doi: 10.4161/auto.21654

    Figure Lengend Snippet: Figure 6. Knockdown of LAMP2A triggers AKT1 phosphorylation at Ser473. Proliferating T47D cells were either transfected with LAMP2A, or empty pcDNA3 vector for 48 h ( A, C and E ) and with LAMP2A siRNA or control siRNA for 72 h ( B, D and F ). ( A and B ) showing western blots analysis of total AKT1 and phospho AKT1 (Ser 473). ( C and D ) demonstrate AKT1 kinase activity using AKT1 substrate GSK3 fusion protein as a measure of AKT1 activation. ( E and F ) demonstrate the AKT1 substrate assay under oxidative stress condition rendered by 150 nM H 2 O 2 . Densitometric analyses from ( E and F ) are given under each panel. Data are mean ± SEMs, and significant changes are indicated by an asterisk (*p

    Article Snippet: Whole cell lysates from transfected/nontransfected cells were prepared after the indicated treatments as described previously., The primary antibodies were as follows: LAMP2A (Abcam, ab18528, 1 μg/ml); LAMP2B, (Abcam, ab18529 1 μg/ml); LAMP1 (Cell Signaling, C54H11, 1:1000); HSPA8 (Santa Cruz Biotechnology, sc1059, 1:200); CASP3 (Imgenex, IMG-144A, 5 μg/ml); BAX (Santa Cruz Biotechnology, sc526, 1:200); BCL2 (Santa Cruz Biotechnology, sc-7382, 1:200); GAPDH (Imgenex, IMG5143A, 1 μg/ml); PKM (Cell Signaling, 3186, 1:1000); AKT1 (Cell Signaling, 9272, 1:1000); phospho-AKT1 (ser473) (Cell Signaling, 4051S, 1:1000); TUBB (Imgenex, IMG-5810A, 1 μg/ml) and ACTB (Imgenex, IMG-5142A, 1 μg/ml).

    Techniques: Transfection, Plasmid Preparation, Western Blot, Activity Assay, Activation Assay

    Confirmation of PTENα deletion in mouse heart. (a) Tissue distribution of PTENα. Analysis of PTENα by immunoprecipitation with PTENα N- terminus-specific antibody (α-N) in various tissue homogenates from 2-months-old wild-type C57BL/6 mice. *, another isoform of PTEN. (b) Subcellular localization of PTENα in mouse liver and heart. C, cytosolic fraction; M, mitochondrial fraction. GAPDH is a cytosolic marker; CYCS is a mitochondrial marker. (c-d) Immunoblot analysis of PTENα, PTEN, AKT1 and p-AKT1 Ser473 in cardiac homogenates. Left ventricles were dissected from 3-months-old mice, and homogenates were immunoblotted with the indicated antibodies. ACTB was used as a loading control. Expression of PTEN, AKT1, and p-AKT1 Ser473 was quantified with ImageJ (n = 3). Data are presented as mean ± SEM; n.s., not significant, p > 0.05 (two-tailed paired Student’s t-test).

    Journal: Autophagy

    Article Title: PTENα regulates mitophagy and maintains mitochondrial quality control

    doi: 10.1080/15548627.2018.1489477

    Figure Lengend Snippet: Confirmation of PTENα deletion in mouse heart. (a) Tissue distribution of PTENα. Analysis of PTENα by immunoprecipitation with PTENα N- terminus-specific antibody (α-N) in various tissue homogenates from 2-months-old wild-type C57BL/6 mice. *, another isoform of PTEN. (b) Subcellular localization of PTENα in mouse liver and heart. C, cytosolic fraction; M, mitochondrial fraction. GAPDH is a cytosolic marker; CYCS is a mitochondrial marker. (c-d) Immunoblot analysis of PTENα, PTEN, AKT1 and p-AKT1 Ser473 in cardiac homogenates. Left ventricles were dissected from 3-months-old mice, and homogenates were immunoblotted with the indicated antibodies. ACTB was used as a loading control. Expression of PTEN, AKT1, and p-AKT1 Ser473 was quantified with ImageJ (n = 3). Data are presented as mean ± SEM; n.s., not significant, p > 0.05 (two-tailed paired Student’s t-test).

    Article Snippet: Immunoblotting was carried out using primary antibodies including: PTEN (Cell Signaling Technology, 9559); PTENα (established in our laboratory,αα-N); TOMM20 (Abclonal, A6774); PRKN (Cell Signaling Technology, 4211 and 2312); SQSTM1 (Abclonal, A7758); LC3B (Ruiyingbio, RLM3381); AKT1 (Cell Signaling Technology, 9272); p-AKT1 Ser473 (Cell Signaling Technology, 9271); TOMM40 (Santa Cruz Biotechnology, sc-11414); CYCS (Santa Cruz Biotechnology, sc-13156); MFN1 (Abclonal,A9880); MFN2 (Abclonal, ); TUBA1A (MBL, M175-3); ACTB (MBL, PM053); GAPDH (Sungene Biotech, KM9002); FLAG (Sigma-Aldrich, F3165); GFP (Ray Antibody Biotech, RM1008); HA (Sigma-Aldrich, H3663); His (Abcam, ab18184); GST (Abcam, ab19256); and GFP (Biodragon-immunotech, B1152).

    Techniques: Immunoprecipitation, Mouse Assay, Marker, Expressing, Two Tailed Test

    MG132 treatment depresses PDGF-stimulated phosphorylation of ERK and also reduces the activation of upstream signaling components. a ) Immunoblot results, representative of 3 independent experiments, showing the phosphorylation kinetics of PDGF β-receptor Tyr 751 (pPDGFR), Akt1/2/3 Ser 473 (pAkt), MEK1/2 Ser 217 /Ser 221 (pMEK), and ERK1/2 Thr 202 /Tyr 204 (pERK) in cells pretreated with either DMSO or 25 µM MG132 for 6 h and then stimulated with the indicated concentration of PDGF-BB. Stimulation times are 5, 15, 30, 60, and 120 minutes. Total ERK1/2 (tERK) serves as a loading control. For each antigen, the DMSO and MG132 bands are cropped from the same gel. At right it is shown that total Akt (tAkt) protein expression is not affected by MG132 treatment, whereas total MEK1/2 (tMEK) is only modestly increased in MG132-treated cells, relative to β-actin loading control. b-e ) Quantification of the phosphorylation kinetics represented in a . Each readout is normalized by total ERK and expressed as mean ± s.e.m. ( n = 3): b , pPDGFR; c , pAkt; d , pMEK; e , pERK. The indicated p value for each time course is from two-way ANOVA analysis comparing MG132-treated and control measurements.

    Journal: PLoS ONE

    Article Title: Systemic Perturbation of the ERK Signaling Pathway by the Proteasome Inhibitor, MG132

    doi: 10.1371/journal.pone.0050975

    Figure Lengend Snippet: MG132 treatment depresses PDGF-stimulated phosphorylation of ERK and also reduces the activation of upstream signaling components. a ) Immunoblot results, representative of 3 independent experiments, showing the phosphorylation kinetics of PDGF β-receptor Tyr 751 (pPDGFR), Akt1/2/3 Ser 473 (pAkt), MEK1/2 Ser 217 /Ser 221 (pMEK), and ERK1/2 Thr 202 /Tyr 204 (pERK) in cells pretreated with either DMSO or 25 µM MG132 for 6 h and then stimulated with the indicated concentration of PDGF-BB. Stimulation times are 5, 15, 30, 60, and 120 minutes. Total ERK1/2 (tERK) serves as a loading control. For each antigen, the DMSO and MG132 bands are cropped from the same gel. At right it is shown that total Akt (tAkt) protein expression is not affected by MG132 treatment, whereas total MEK1/2 (tMEK) is only modestly increased in MG132-treated cells, relative to β-actin loading control. b-e ) Quantification of the phosphorylation kinetics represented in a . Each readout is normalized by total ERK and expressed as mean ± s.e.m. ( n = 3): b , pPDGFR; c , pAkt; d , pMEK; e , pERK. The indicated p value for each time course is from two-way ANOVA analysis comparing MG132-treated and control measurements.

    Article Snippet: Antibodies against total ERK1/2, MEK1/2, Akt1/2/3 and MKP3 and phospho-specific antibodies against PDGF β-receptor pTyr751 , Akt pSer473 , ERK pThr202 /pTyr204 , and MEK pSer217 /pSer221 were from Cell Signaling Technology (Beverly, MA).

    Techniques: Activation Assay, Concentration Assay, Expressing

    Effects of PPP3CA siRNA on VEGF-induced AKT1 phosphorylation in OFPAE cells. Cells were transfected with the scrambled or PPP3CA siRNA for 48 h. After serum starvation, cells were treated with 10 ng/ml of VEGF. Proteins (15 μg/lane) were subjected

    Journal: Biology of Reproduction

    Article Title: Protein Phosphatase 3 Differentially Modulates Vascular Endothelial Growth Factor- and Fibroblast Growth Factor 2-Stimulated Cell Proliferation and Signaling in Ovine Fetoplacental Artery Endothelial Cells

    doi: 10.1095/biolreprod.108.068957

    Figure Lengend Snippet: Effects of PPP3CA siRNA on VEGF-induced AKT1 phosphorylation in OFPAE cells. Cells were transfected with the scrambled or PPP3CA siRNA for 48 h. After serum starvation, cells were treated with 10 ng/ml of VEGF. Proteins (15 μg/lane) were subjected

    Article Snippet: Proteins on the membranes were probed with an antibody against total or phospho-specific MAPK3/1 (1:2000; Cell Signaling Technology, Beverly, MA), total AKT1 (1:2000; Cell Signaling Technology), or phospho-specific AKT1 (1:1000; Cell Signaling Technology).

    Techniques: Transfection

    Effects of PPP3CA siRNA on FGF2-induced AKT1 phosphorylation in OFPAE cells. Cells were transfected with the scrambled or PPP3CA siRNA for 48 h. After serum starvation, cells were treated with 10 ng/ml of FGF2. Proteins (15 μg/lane) were subjected

    Journal: Biology of Reproduction

    Article Title: Protein Phosphatase 3 Differentially Modulates Vascular Endothelial Growth Factor- and Fibroblast Growth Factor 2-Stimulated Cell Proliferation and Signaling in Ovine Fetoplacental Artery Endothelial Cells

    doi: 10.1095/biolreprod.108.068957

    Figure Lengend Snippet: Effects of PPP3CA siRNA on FGF2-induced AKT1 phosphorylation in OFPAE cells. Cells were transfected with the scrambled or PPP3CA siRNA for 48 h. After serum starvation, cells were treated with 10 ng/ml of FGF2. Proteins (15 μg/lane) were subjected

    Article Snippet: Proteins on the membranes were probed with an antibody against total or phospho-specific MAPK3/1 (1:2000; Cell Signaling Technology, Beverly, MA), total AKT1 (1:2000; Cell Signaling Technology), or phospho-specific AKT1 (1:1000; Cell Signaling Technology).

    Techniques: Transfection

    Alteration of the insulin receptor signaling pathway in adipocytes cultured with amyloid‐β. ( A ) Insulin receptor substrate‐2 ( IRS2 ) gene expression in differentiated adipocytes incubated for 6 days with 100 or 1,000 pg/mL amyloid‐β40 ( n = 10–11 per condition). ( B ) IRS2 transcription in cultures treated with amyloid‐β42 at 10 or 100 pg/mL for 6 days ( n = 10–11 per condition). Representative immunoblot and quantification of ( C ) total Akt‐1 and ( D ), phosphor‐serine 473 Akt‐1 and ( E ) ratio of phospho/total Akt‐1 in adipocyte cultures grown at 5.5 mM glucose with 10 nM insulin with 100 pg/mL or 1,000 pg/mL amyloid‐β40; n = 3 per condition, log‐transformed data. * P

    Journal: Obesity (Silver Spring, Md.)

    Article Title: Effects of glucose and insulin on secretion of amyloid‐β by human adipose tissue cells

    doi: 10.1002/oby.21494

    Figure Lengend Snippet: Alteration of the insulin receptor signaling pathway in adipocytes cultured with amyloid‐β. ( A ) Insulin receptor substrate‐2 ( IRS2 ) gene expression in differentiated adipocytes incubated for 6 days with 100 or 1,000 pg/mL amyloid‐β40 ( n = 10–11 per condition). ( B ) IRS2 transcription in cultures treated with amyloid‐β42 at 10 or 100 pg/mL for 6 days ( n = 10–11 per condition). Representative immunoblot and quantification of ( C ) total Akt‐1 and ( D ), phosphor‐serine 473 Akt‐1 and ( E ) ratio of phospho/total Akt‐1 in adipocyte cultures grown at 5.5 mM glucose with 10 nM insulin with 100 pg/mL or 1,000 pg/mL amyloid‐β40; n = 3 per condition, log‐transformed data. * P

    Article Snippet: Samples were separated on 10% SDS‐PAGE, transferred to PVDF, probed with rabbit monoclonal Akt‐1 antibody (Cell Signaling), followed by goat anti‐rabbit‐HRP‐conjugated antibody (Bio‐Rad), and bands detected using ECL reagent (Amersham).

    Techniques: Cell Culture, Expressing, Incubation, Transformation Assay

    The mitochondrial cycle of Akt1 under redox stimuli. In (A) and (B), Akt1 requires to be phosphorylated in Ser 473 to enter mitochondria. Inactive Akt1-His tagged and mTORC2 were incubated in kinase buffer to allow phosphorylation. In absence of P-mTORC2 Akt1 remains outside mitochondria, In the presence of mTORC2, P-Akt Ser 473 translocates to mitochondria and becomes phosphorylated in Thr 308 ; approximately after 50 min biphosphorylated P-Akt Ser 473 /Thr 308 becomes detectable in the supernatant. These extracts were incubated with purified mitochondria in import buffer and the samples were centrifuged and prepared to run in SDS-PAGE. (C) Imaging of wb using a His-tag ab anti mouse conjugated to Cy3 and a P-Thr308 ab anti rabbit conjugated to Cy2 shows colocalization (yellow) in the presence of P-mTORC2. Addition of PDK1 alone is unable to phosphorylate Akt1 in Thr 308 .

    Journal: PLoS ONE

    Article Title: Akt1 Intramitochondrial Cycling Is a Crucial Step in the Redox Modulation of Cell Cycle Progression

    doi: 10.1371/journal.pone.0007523

    Figure Lengend Snippet: The mitochondrial cycle of Akt1 under redox stimuli. In (A) and (B), Akt1 requires to be phosphorylated in Ser 473 to enter mitochondria. Inactive Akt1-His tagged and mTORC2 were incubated in kinase buffer to allow phosphorylation. In absence of P-mTORC2 Akt1 remains outside mitochondria, In the presence of mTORC2, P-Akt Ser 473 translocates to mitochondria and becomes phosphorylated in Thr 308 ; approximately after 50 min biphosphorylated P-Akt Ser 473 /Thr 308 becomes detectable in the supernatant. These extracts were incubated with purified mitochondria in import buffer and the samples were centrifuged and prepared to run in SDS-PAGE. (C) Imaging of wb using a His-tag ab anti mouse conjugated to Cy3 and a P-Thr308 ab anti rabbit conjugated to Cy2 shows colocalization (yellow) in the presence of P-mTORC2. Addition of PDK1 alone is unable to phosphorylate Akt1 in Thr 308 .

    Article Snippet: Membranes were incubated with antibodies anti Akt1, P-Akt1 Ser473 , P-Akt1 Thr308 (Cell Signaling), cytochrome c , complex I, His (Molecular Probes), Bcl-xL , cyclin D1, β-actin, RNA POL RPB6 (Santa Cruz) or caspase 3 and HA (Sigma).

    Techniques: Incubation, Purification, SDS Page, Imaging, Western Blot

    ZINC4085554 inhibits pressure-stimulated FAK-Tyr-397 yet not Akt1-Ser-473 phosphorylation, and Akt-Thr-308 remains unaffected by pressure and/or ZINC4085554. SW620 cells were treated with vehicle (0.1% DMSO) or 50 µM ZINC4085554 and subjected to ambient or 15 mmHg increased extracellular pressure for 30 min. (A) Representative blots probed for FAK-Tyr-397 and total FAK. (B) Densitometric quantitation of the blots in A. (C) Representative blots probed for Akt1-Ser-473 and total Akt1. (D) Densitometric quantitation of the blots in C. (E) Representative blots probed for Akt-Thr-308 and total Akt1. (F) Densitometric quantitation of the blots in E. (n=5-8) *P

    Journal: Oncology Letters

    Article Title: ZINC4085554 inhibits cancer cell adhesion by interfering with the interaction of Akt1 and FAK

    doi: 10.3892/ol.2019.10192

    Figure Lengend Snippet: ZINC4085554 inhibits pressure-stimulated FAK-Tyr-397 yet not Akt1-Ser-473 phosphorylation, and Akt-Thr-308 remains unaffected by pressure and/or ZINC4085554. SW620 cells were treated with vehicle (0.1% DMSO) or 50 µM ZINC4085554 and subjected to ambient or 15 mmHg increased extracellular pressure for 30 min. (A) Representative blots probed for FAK-Tyr-397 and total FAK. (B) Densitometric quantitation of the blots in A. (C) Representative blots probed for Akt1-Ser-473 and total Akt1. (D) Densitometric quantitation of the blots in C. (E) Representative blots probed for Akt-Thr-308 and total Akt1. (F) Densitometric quantitation of the blots in E. (n=5-8) *P

    Article Snippet: Membranes were blocked with blocking buffer (Odyssey Blocking Buffer; LI-COR Biosciences, Lincoln, NE, USA) at room temperature for 1 h and immunoblotted at 4°C overnight with primary antibodies as follows: FAK (1:1,000; cat. no. 05-537, clone 4.47; mouse monoclonal; Merck KGaA, Darmstadt, Germany), FAK-Try-397 (1:1,000; cat. no. ab81298; rabbit monoclonal; Abcam, Cambridge, UK), Akt1 (1:1,000; cat. no. 2967, clone 2H10; mouse monoclonal; Cell Signaling Technology, Inc., Danvers, MA, USA), Akt1-Ser-473 (1:1,000; cat. no. 9276; mouse monoclonal; Cell Signaling Technology, Inc.) and Akt-Thr-308 (1:1,000; cat. no. 9275; Cell Signaling Technology, Inc.).

    Techniques: Quantitation Assay

    Akt1 sequentially phosphorylates p27 at S10 and a neighboring serine . A , Akt1 does not phosphorylate hp27S10A. Upper panel: Recombinant Akt1 was incubated in a kinase reaction with the indicated forms of p27 as described. Samples were separated by SDS/PAGE and visualized by autoradiography. Bottom panel: His-tagged cyclin-E-CDK2 was purified from transfected HEK293 cells (see Methods), then incubated with indicated forms of p27 in the presence of [ 32 P]-γ-ATP. Samples were separated by SDS-PAGE and visualized by autoradiography. Commassie staining shows equal amounts of hwtp27 and p27S10A were present in the reactions. B , Akt1 phosphorylates hp27S10T. Kinase reaction was performed with recombinant Akt1 and indicated substrates. Upper panel: autoradiograph showing phosphorylated p27. Bottom panel: western blot showing similar levels of p27 in the reaction. C , Phospho-amino acid analysis comparing hwtp27 and hp27S10T. Kinase reaction described in B was repeated and radiolabeled p27 was subjected to PAA as in Figure 3C. Top panel: scheme representing migration of phospho-amino acid standards. Second panel: Phospho-amino acid standards separated by electrophoresis and visualized by 0.25% ninhydrin. Third panel: phospho-amino acid analysis of radiolabeled hwtp27. Bottom panel: phospho-amino acid analysis of hp27S10T. Radiolabeled peptides and amino acids were detected by phosphoimager.

    Journal: Cell Division

    Article Title: Akt1 sequentially phosphorylates p27kip1 within a conserved but non-canonical region

    doi: 10.1186/1747-1028-1-11

    Figure Lengend Snippet: Akt1 sequentially phosphorylates p27 at S10 and a neighboring serine . A , Akt1 does not phosphorylate hp27S10A. Upper panel: Recombinant Akt1 was incubated in a kinase reaction with the indicated forms of p27 as described. Samples were separated by SDS/PAGE and visualized by autoradiography. Bottom panel: His-tagged cyclin-E-CDK2 was purified from transfected HEK293 cells (see Methods), then incubated with indicated forms of p27 in the presence of [ 32 P]-γ-ATP. Samples were separated by SDS-PAGE and visualized by autoradiography. Commassie staining shows equal amounts of hwtp27 and p27S10A were present in the reactions. B , Akt1 phosphorylates hp27S10T. Kinase reaction was performed with recombinant Akt1 and indicated substrates. Upper panel: autoradiograph showing phosphorylated p27. Bottom panel: western blot showing similar levels of p27 in the reaction. C , Phospho-amino acid analysis comparing hwtp27 and hp27S10T. Kinase reaction described in B was repeated and radiolabeled p27 was subjected to PAA as in Figure 3C. Top panel: scheme representing migration of phospho-amino acid standards. Second panel: Phospho-amino acid standards separated by electrophoresis and visualized by 0.25% ninhydrin. Third panel: phospho-amino acid analysis of radiolabeled hwtp27. Bottom panel: phospho-amino acid analysis of hp27S10T. Radiolabeled peptides and amino acids were detected by phosphoimager.

    Article Snippet: Purified recombinant Akt1 protein was obtained from Cell Signaling Technology (prep1:#7502; and prep3: #9274) and from Calbiochem (prep2).

    Techniques: Recombinant, Incubation, SDS Page, Autoradiography, Purification, Transfection, Staining, Western Blot, Migration, Electrophoresis

    Full length Akt1 phosphorylates p27 at serine 10 in vitro and in cells . A , Expression and activation of Akt1 in HEK293 cells. Increasing amounts of HA-tagged wtAkt1 were transfected in HEK293 cells as described in the Methods. After 36 hours active and total levels of Akt1 were determined by western blot analysis. B , Full length wild type Akt1 phosphorylates p27. Overexpressed HA-Akt1 was purified by immunoprecipitation (IP-Akt1) and incubated alone or with indicated forms of His-p27 in the presence of [ 32 p]-γ-ATP as described in Methods. Autoradiograph shows full length Akt1 phosphorylates human and mouse p27 as well as the hp27T157A mutant. C , Wild type Akt1 targets S10. The kinase reaction was performed by incubating IP-Akt1 with non-radiolabeled ATP and indicated substrates. Western blot shows full length Akt phosphorylates S10. D , Specific Akt inhibitor blocks S10 phosphorylation. A kinase reaction was performed as in C incubating full length His-purified Akt1 with p27 in the presence or absence of Akt inhibitor. S10 phosphorylation was determined by phospho-specific antibody. E , Full length Akt1 fails to phosphorylate hp27S10A. Full length His-Akt1 was incubated in a kinase reaction with the indicated forms of p27 in the presence of radiolabeled ATP. Samples were separated by SDS/PAGE and visualized by autoradiography. Akt inhibitor was added where indicated.

    Journal: Cell Division

    Article Title: Akt1 sequentially phosphorylates p27kip1 within a conserved but non-canonical region

    doi: 10.1186/1747-1028-1-11

    Figure Lengend Snippet: Full length Akt1 phosphorylates p27 at serine 10 in vitro and in cells . A , Expression and activation of Akt1 in HEK293 cells. Increasing amounts of HA-tagged wtAkt1 were transfected in HEK293 cells as described in the Methods. After 36 hours active and total levels of Akt1 were determined by western blot analysis. B , Full length wild type Akt1 phosphorylates p27. Overexpressed HA-Akt1 was purified by immunoprecipitation (IP-Akt1) and incubated alone or with indicated forms of His-p27 in the presence of [ 32 p]-γ-ATP as described in Methods. Autoradiograph shows full length Akt1 phosphorylates human and mouse p27 as well as the hp27T157A mutant. C , Wild type Akt1 targets S10. The kinase reaction was performed by incubating IP-Akt1 with non-radiolabeled ATP and indicated substrates. Western blot shows full length Akt phosphorylates S10. D , Specific Akt inhibitor blocks S10 phosphorylation. A kinase reaction was performed as in C incubating full length His-purified Akt1 with p27 in the presence or absence of Akt inhibitor. S10 phosphorylation was determined by phospho-specific antibody. E , Full length Akt1 fails to phosphorylate hp27S10A. Full length His-Akt1 was incubated in a kinase reaction with the indicated forms of p27 in the presence of radiolabeled ATP. Samples were separated by SDS/PAGE and visualized by autoradiography. Akt inhibitor was added where indicated.

    Article Snippet: Purified recombinant Akt1 protein was obtained from Cell Signaling Technology (prep1:#7502; and prep3: #9274) and from Calbiochem (prep2).

    Techniques: In Vitro, Expressing, Activation Assay, Transfection, Western Blot, Purification, Immunoprecipitation, Incubation, Autoradiography, Mutagenesis, SDS Page

    Stress-activated Akt phosphorylates p27 at serine 10 in cells . A , Overexpressed Akt1 phosphorylates S10 in HEK293 cells. hwtp27 was transiently overexpressed in HEK293 alone or in the presence of co-transfected Akt1. S10 phosphorylation was determined by western blot analysis after 36 hrs. B , CuSO 4 -dependent activation of Akt leads to p27S10 phosphorylation. Cells were transiently transfected as in A and treated with vehicle or CuSO 4 for 2 hours prior to harvest. Samples were analyzed by western blot. Akt inhibitor was added 30 minutes prior to CuSO 4 . C , Endogenous Akt1 activated by oxidative stress phosphorylates p27S10. HEK293 cells were transfected with p27 and treated with CuSO 4 or FBS for 2 hours prior to harvest. Inhibitors were added 30 minutes before treatment. Samples were analyzed by western blot. D , H 2 O 2 -activated endogenous Akt phosphorylates p27S10. Experiment was performed as in C treating the cells with H 2 O 2 . E , Growth factor withdrawal leads to an Akt-dependent phosphorylation of p27S10. Hela cells were grown in 10% FBS (prolif.) or serum starved (G0) in the presence or absence of Akt inhibitor. Cells were harvested and phosphorylation of S10 analyzed by western blot. F , Mitogen-activated Akt does not target p27S10. HDF cells were synchronized in G0 by serum deprivation and re-fed with 20% FBS. Cells were lysed at the indicated time points and analyzed by western blot.

    Journal: Cell Division

    Article Title: Akt1 sequentially phosphorylates p27kip1 within a conserved but non-canonical region

    doi: 10.1186/1747-1028-1-11

    Figure Lengend Snippet: Stress-activated Akt phosphorylates p27 at serine 10 in cells . A , Overexpressed Akt1 phosphorylates S10 in HEK293 cells. hwtp27 was transiently overexpressed in HEK293 alone or in the presence of co-transfected Akt1. S10 phosphorylation was determined by western blot analysis after 36 hrs. B , CuSO 4 -dependent activation of Akt leads to p27S10 phosphorylation. Cells were transiently transfected as in A and treated with vehicle or CuSO 4 for 2 hours prior to harvest. Samples were analyzed by western blot. Akt inhibitor was added 30 minutes prior to CuSO 4 . C , Endogenous Akt1 activated by oxidative stress phosphorylates p27S10. HEK293 cells were transfected with p27 and treated with CuSO 4 or FBS for 2 hours prior to harvest. Inhibitors were added 30 minutes before treatment. Samples were analyzed by western blot. D , H 2 O 2 -activated endogenous Akt phosphorylates p27S10. Experiment was performed as in C treating the cells with H 2 O 2 . E , Growth factor withdrawal leads to an Akt-dependent phosphorylation of p27S10. Hela cells were grown in 10% FBS (prolif.) or serum starved (G0) in the presence or absence of Akt inhibitor. Cells were harvested and phosphorylation of S10 analyzed by western blot. F , Mitogen-activated Akt does not target p27S10. HDF cells were synchronized in G0 by serum deprivation and re-fed with 20% FBS. Cells were lysed at the indicated time points and analyzed by western blot.

    Article Snippet: Purified recombinant Akt1 protein was obtained from Cell Signaling Technology (prep1:#7502; and prep3: #9274) and from Calbiochem (prep2).

    Techniques: Transfection, Western Blot, Activation Assay

    Akt1 phosphorylates human and mouse p27 . A , Purification of His-tagged p27 from E. coli. Upper panel: Aliquots from indicated steps of the purification protocol (see Methods) were separated by SDS-PAGE and visualized by Coomassie blue staining. Bottom panel: Indicated forms of His-tagged p27 were purified as described in Methods and their concentration compared by SDS-PAGE followed by Coomassie staining. B , Constitutively active Akt1 phosphorylates p27 lacking T157. An in vitro kinase assay was performed as described in the Methods by incubating commercial recombinant Akt1(prep1) (see Methods) and [ 32 P]ATP with the indicated substrates. Samples were then separated by SDS-PAGE and visualized by autoradiography. Lane 1 is a negative control lacking Akt1 substrate, while lane 2 shows Akt1 phosphorylates its well-known substrate GSK. Lane 3–5 show Akt1 phosphorylates human wild type p27, mouse p27, and hp27T157A equally well.

    Journal: Cell Division

    Article Title: Akt1 sequentially phosphorylates p27kip1 within a conserved but non-canonical region

    doi: 10.1186/1747-1028-1-11

    Figure Lengend Snippet: Akt1 phosphorylates human and mouse p27 . A , Purification of His-tagged p27 from E. coli. Upper panel: Aliquots from indicated steps of the purification protocol (see Methods) were separated by SDS-PAGE and visualized by Coomassie blue staining. Bottom panel: Indicated forms of His-tagged p27 were purified as described in Methods and their concentration compared by SDS-PAGE followed by Coomassie staining. B , Constitutively active Akt1 phosphorylates p27 lacking T157. An in vitro kinase assay was performed as described in the Methods by incubating commercial recombinant Akt1(prep1) (see Methods) and [ 32 P]ATP with the indicated substrates. Samples were then separated by SDS-PAGE and visualized by autoradiography. Lane 1 is a negative control lacking Akt1 substrate, while lane 2 shows Akt1 phosphorylates its well-known substrate GSK. Lane 3–5 show Akt1 phosphorylates human wild type p27, mouse p27, and hp27T157A equally well.

    Article Snippet: Purified recombinant Akt1 protein was obtained from Cell Signaling Technology (prep1:#7502; and prep3: #9274) and from Calbiochem (prep2).

    Techniques: Purification, SDS Page, Staining, Concentration Assay, In Vitro, Kinase Assay, Recombinant, Autoradiography, Negative Control

    Akt1 phosphorylates p27 at multiple serines within the N-terminus . A , Phospho-peptide maps comparing hwtp27 to hp27T157A. The kinase assay was performed as described with indicated forms of p27. After SDS/PAGE separation (upper panel) radiolabeled p27 was subjected to two-dimensional phospho-peptide analysis (lower panel) (See Methods for details). Direction of electrophoresis and chromatography are indicated by arrows. B , Schematic representation of phospho-peptide pattern. Ellipses 1–3 correspond to phospho-peptides in 3A, while number 4 represents [ 32 P]. C , Akt1 phosphorylates p27 at serines. Radiolabeled human wtp27 was hydrolyzed to constituent amino acids and then separated by electrophoresis on a TLC plate as described in Methods. Top panel: scheme representing migration of the phospho-amino acid standards (phospho-S and phospho-T). Middle panel: Phospho-amino acid standards separated by electrophoresis and visualized by 0.25% ninhydrin (see Methods). Bottom panel: phospho-amino acid analysis of [ 32 P]hwtp27 visualized by phosphoimager. D , Akt1 phosphorylates p27(1–86). Kinase assay was performed as described with His-purified p27(1–86) and p27(87–198). Samples were separated by SDS/PAGE and visualized by autoradiography. Commassie staining shows the levels of deletion mutants were similar. E , Phospho-peptide maps comparing hwtp27 to hp27(1–86). Radiolabeled hwtp27 and p27(1–86) were analyzed as in A .

    Journal: Cell Division

    Article Title: Akt1 sequentially phosphorylates p27kip1 within a conserved but non-canonical region

    doi: 10.1186/1747-1028-1-11

    Figure Lengend Snippet: Akt1 phosphorylates p27 at multiple serines within the N-terminus . A , Phospho-peptide maps comparing hwtp27 to hp27T157A. The kinase assay was performed as described with indicated forms of p27. After SDS/PAGE separation (upper panel) radiolabeled p27 was subjected to two-dimensional phospho-peptide analysis (lower panel) (See Methods for details). Direction of electrophoresis and chromatography are indicated by arrows. B , Schematic representation of phospho-peptide pattern. Ellipses 1–3 correspond to phospho-peptides in 3A, while number 4 represents [ 32 P]. C , Akt1 phosphorylates p27 at serines. Radiolabeled human wtp27 was hydrolyzed to constituent amino acids and then separated by electrophoresis on a TLC plate as described in Methods. Top panel: scheme representing migration of the phospho-amino acid standards (phospho-S and phospho-T). Middle panel: Phospho-amino acid standards separated by electrophoresis and visualized by 0.25% ninhydrin (see Methods). Bottom panel: phospho-amino acid analysis of [ 32 P]hwtp27 visualized by phosphoimager. D , Akt1 phosphorylates p27(1–86). Kinase assay was performed as described with His-purified p27(1–86) and p27(87–198). Samples were separated by SDS/PAGE and visualized by autoradiography. Commassie staining shows the levels of deletion mutants were similar. E , Phospho-peptide maps comparing hwtp27 to hp27(1–86). Radiolabeled hwtp27 and p27(1–86) were analyzed as in A .

    Article Snippet: Purified recombinant Akt1 protein was obtained from Cell Signaling Technology (prep1:#7502; and prep3: #9274) and from Calbiochem (prep2).

    Techniques: Kinase Assay, SDS Page, Electrophoresis, Chromatography, Thin Layer Chromatography, Migration, Purification, Autoradiography, Staining

    Akt1 phosphorylates p27S10 . A , Determination of S10 phosphorylation by specific antibody. Recombinant Akt1 was incubated with the indicated forms of His-tagged p27 in the presence of non-labeled ATP. Samples were separated by SDS/PAGE and analyzed by western blot with the indicated antibodies. B , Alkaline phosphatase abolishes recognition of phosphorylated p27. hwtp27 was phosphorylated as in A and treated with or without alkaline phosphatase. Samples were then analyzed by western blot with the indicated antibodies. C , Akt1 phosphorylates serine 10 in mouse p27. Akt1 was incubated alone or with the indicated forms of p27 in the presence of non-labeled ATP. Total and phospho-S10 levels were analyzed by western blot as indicated. D , Phospho-peptide maps of human and mouse p27. Radiolabeled samples were analyzed as described in Figure 3A.

    Journal: Cell Division

    Article Title: Akt1 sequentially phosphorylates p27kip1 within a conserved but non-canonical region

    doi: 10.1186/1747-1028-1-11

    Figure Lengend Snippet: Akt1 phosphorylates p27S10 . A , Determination of S10 phosphorylation by specific antibody. Recombinant Akt1 was incubated with the indicated forms of His-tagged p27 in the presence of non-labeled ATP. Samples were separated by SDS/PAGE and analyzed by western blot with the indicated antibodies. B , Alkaline phosphatase abolishes recognition of phosphorylated p27. hwtp27 was phosphorylated as in A and treated with or without alkaline phosphatase. Samples were then analyzed by western blot with the indicated antibodies. C , Akt1 phosphorylates serine 10 in mouse p27. Akt1 was incubated alone or with the indicated forms of p27 in the presence of non-labeled ATP. Total and phospho-S10 levels were analyzed by western blot as indicated. D , Phospho-peptide maps of human and mouse p27. Radiolabeled samples were analyzed as described in Figure 3A.

    Article Snippet: Purified recombinant Akt1 protein was obtained from Cell Signaling Technology (prep1:#7502; and prep3: #9274) and from Calbiochem (prep2).

    Techniques: Recombinant, Incubation, Labeling, SDS Page, Western Blot

    Kinetic analysis of p27 phosphorylation . A , Time course of p27 phosphorylation. Kinase reaction was performed as described with hwtp27 or p27T157A and stopped at indicated times. Phosphorylation is shown by autoradiograph and total p27 by western blot. The amount of [ 32 P] incorporated was measured by densitometry, expressed as OD, and plotted vs . reaction time. [ 32 P] incorporation was normalized against the highest value arbitrarily set at 1. B , Phosphorylation rate as a function of p27 concentration. Indicated dilutions of hwtp27 and p27T157A were utilized in the kinase reaction. Autoradiographs show [ 32 P] incorporation. Graph shows radiolabeled p27 quantitated as in A vs . protein dilution. C , Rate of S10 phosphorylation. Indicated forms of His-tagged p27 were incubated with Akt1 and non-labeled ATP. Samples were separated by SDS-PAGE and visualized by western blotting with the indicated antibodies. D , p27(87–198) inhibits Akt1 phosphorylation of p27. Kinase reaction was performed with hwtp27, Akt1 and [ 32 P]-γ-ATP in the presence of indicated amounts of p27(87–198). Upper panel: autoradiograph. Bottom panel: western blot showing levels of full length and C-terminus of p27. E , p27(87–198) inhibits Akt1 phosphorylation of p27S10. Kinase reaction was performed as described in D with non-labeled ATP in the presence of p27(87–198) or GSK. S10 phosphorylation was determined by western blot analysis.

    Journal: Cell Division

    Article Title: Akt1 sequentially phosphorylates p27kip1 within a conserved but non-canonical region

    doi: 10.1186/1747-1028-1-11

    Figure Lengend Snippet: Kinetic analysis of p27 phosphorylation . A , Time course of p27 phosphorylation. Kinase reaction was performed as described with hwtp27 or p27T157A and stopped at indicated times. Phosphorylation is shown by autoradiograph and total p27 by western blot. The amount of [ 32 P] incorporated was measured by densitometry, expressed as OD, and plotted vs . reaction time. [ 32 P] incorporation was normalized against the highest value arbitrarily set at 1. B , Phosphorylation rate as a function of p27 concentration. Indicated dilutions of hwtp27 and p27T157A were utilized in the kinase reaction. Autoradiographs show [ 32 P] incorporation. Graph shows radiolabeled p27 quantitated as in A vs . protein dilution. C , Rate of S10 phosphorylation. Indicated forms of His-tagged p27 were incubated with Akt1 and non-labeled ATP. Samples were separated by SDS-PAGE and visualized by western blotting with the indicated antibodies. D , p27(87–198) inhibits Akt1 phosphorylation of p27. Kinase reaction was performed with hwtp27, Akt1 and [ 32 P]-γ-ATP in the presence of indicated amounts of p27(87–198). Upper panel: autoradiograph. Bottom panel: western blot showing levels of full length and C-terminus of p27. E , p27(87–198) inhibits Akt1 phosphorylation of p27S10. Kinase reaction was performed as described in D with non-labeled ATP in the presence of p27(87–198) or GSK. S10 phosphorylation was determined by western blot analysis.

    Article Snippet: Purified recombinant Akt1 protein was obtained from Cell Signaling Technology (prep1:#7502; and prep3: #9274) and from Calbiochem (prep2).

    Techniques: Autoradiography, Western Blot, Concentration Assay, Incubation, Labeling, SDS Page

    p27 is highly conserved between human and mouse . Comparison of human and mouse p27 sequences reveals they are 92% identical. Highlighted amino acids show absolute differences. Underlined amino acids indicate functional motifs: Cyclin E (RXL), CDK2 (FNF), Grb2 (PXXP) binding sites and the bipartite Nuclear Localization Signal (aa 152–166). Solid arrows show conserved phosphorylation sites. Blow up of Akt1 consensus site shows T157 is not maintained between species.

    Journal: Cell Division

    Article Title: Akt1 sequentially phosphorylates p27kip1 within a conserved but non-canonical region

    doi: 10.1186/1747-1028-1-11

    Figure Lengend Snippet: p27 is highly conserved between human and mouse . Comparison of human and mouse p27 sequences reveals they are 92% identical. Highlighted amino acids show absolute differences. Underlined amino acids indicate functional motifs: Cyclin E (RXL), CDK2 (FNF), Grb2 (PXXP) binding sites and the bipartite Nuclear Localization Signal (aa 152–166). Solid arrows show conserved phosphorylation sites. Blow up of Akt1 consensus site shows T157 is not maintained between species.

    Article Snippet: Purified recombinant Akt1 protein was obtained from Cell Signaling Technology (prep1:#7502; and prep3: #9274) and from Calbiochem (prep2).

    Techniques: Functional Assay, Binding Assay

    AKT1 and AKT3 are phosphorylated in different types of spermatogonia. (A) Whole-mount wild-type or Tg(Gdnf) tubules co-immunostained for phosphorylated AKT1 Ser473 (P-AKT1) and GFRA1. GFRA + cells in wild-type testes (arrows) and large clusters of GFRA + cells in Tg(GDNF) testes (asterisks) are P-AKT1 negative. (B) Whole-mount wild-type or Tg(Gdnf) tubules co-immunostained for phosphorylated AKT3 Ser472 (P-AKT3) and GFRA1. Arrows indicate GFRA1 + P-AKT3 + A s and A pr spermatogonia. Arrowheads indicate GFRA1 + P-AKT3 + cluster in Tg(Gdnf) . Scale bars: 100 µm.

    Journal: Development (Cambridge, England)

    Article Title: Cyclical expression of GDNF is required for spermatogonial stem cell homeostasis

    doi: 10.1242/dev.151555

    Figure Lengend Snippet: AKT1 and AKT3 are phosphorylated in different types of spermatogonia. (A) Whole-mount wild-type or Tg(Gdnf) tubules co-immunostained for phosphorylated AKT1 Ser473 (P-AKT1) and GFRA1. GFRA + cells in wild-type testes (arrows) and large clusters of GFRA + cells in Tg(GDNF) testes (asterisks) are P-AKT1 negative. (B) Whole-mount wild-type or Tg(Gdnf) tubules co-immunostained for phosphorylated AKT3 Ser472 (P-AKT3) and GFRA1. Arrows indicate GFRA1 + P-AKT3 + A s and A pr spermatogonia. Arrowheads indicate GFRA1 + P-AKT3 + cluster in Tg(Gdnf) . Scale bars: 100 µm.

    Article Snippet: Antibodies used were: mouse PLZF (Santa Cruz Biotech); rat GFRA1 (R & D Systems); rat LIN28A (a gift from Dr Eric G. Moss, UMDNJ, NJ, USA); rabbit SOHLH1 (Abcam); rabbit phospho-S6 (Ser240/244; #2215) and rabbit phospho-AKT1 (S473; D7F10) (Cell Signaling Technology); rabbit phospho-Akt3 (S472, #AP3468a) (Abgent); and mouse KIT (#CBL1360, Millipore) all at 1:200.

    Techniques:

    Effect of germ cell-specific deletion of Akt1 on germ cell development. (A-C) No significant change in body weight (A), testis weight (B) or sperm count (C) in 2-month-old mice when Akt1 is deleted in germ cells ( n =3). (D) Paraffin wax-embedded sections of testis from control and mutant testis stained with PSA and probed with PLZF antibody. (E) Average PLZF + cells per tubule (200-250 tubules counted) in control and mutant testis ( n =3). (F) Akt1 transcript is significantly (*** P =0.002) downregulated in mutant testis ( n =3) as determined by qRT-PCR. (G) Absence of P-AKT1 staining in the mutant germ cells. Arrows indicate GFRA1 + P-AKT1 + (double-positive) cells. (H) Detection of P-AKT3 in Akt1 -deleted germ cells. Arrows indicate GFRA1 + P-AKT3 + (double-positive) cells. Scale bars: 100 µm.

    Journal: Development (Cambridge, England)

    Article Title: Cyclical expression of GDNF is required for spermatogonial stem cell homeostasis

    doi: 10.1242/dev.151555

    Figure Lengend Snippet: Effect of germ cell-specific deletion of Akt1 on germ cell development. (A-C) No significant change in body weight (A), testis weight (B) or sperm count (C) in 2-month-old mice when Akt1 is deleted in germ cells ( n =3). (D) Paraffin wax-embedded sections of testis from control and mutant testis stained with PSA and probed with PLZF antibody. (E) Average PLZF + cells per tubule (200-250 tubules counted) in control and mutant testis ( n =3). (F) Akt1 transcript is significantly (*** P =0.002) downregulated in mutant testis ( n =3) as determined by qRT-PCR. (G) Absence of P-AKT1 staining in the mutant germ cells. Arrows indicate GFRA1 + P-AKT1 + (double-positive) cells. (H) Detection of P-AKT3 in Akt1 -deleted germ cells. Arrows indicate GFRA1 + P-AKT3 + (double-positive) cells. Scale bars: 100 µm.

    Article Snippet: Antibodies used were: mouse PLZF (Santa Cruz Biotech); rat GFRA1 (R & D Systems); rat LIN28A (a gift from Dr Eric G. Moss, UMDNJ, NJ, USA); rabbit SOHLH1 (Abcam); rabbit phospho-S6 (Ser240/244; #2215) and rabbit phospho-AKT1 (S473; D7F10) (Cell Signaling Technology); rabbit phospho-Akt3 (S472, #AP3468a) (Abgent); and mouse KIT (#CBL1360, Millipore) all at 1:200.

    Techniques: Mouse Assay, Mutagenesis, Staining, Quantitative RT-PCR

    Deficit in BDNF-stimulated protein translation in cultured cortical primary neurons from APP/PS1 mice is rescued by myristoylated Akt1 overexpression. Increased protein translation was observed in both soma and neurites of primary cortical neurons isolated from WT mice after BDNF stimulation. Stimulation of protein translation at soma and neurites after BDNF treatment was not observed in primary cortical neurons isolated from APP/PS1 mice. Overexpression of myristoylated Akt1 (myr-Akt1), which is constitutively active, rescues the compromised stimulated protein translation in APP/PS1 neurons. Values are mean ± SEM ( n = 31–36 neurons) and * denotes values significantly different from corresponding controls ( p

    Journal: Antioxidants & Redox Signaling

    Article Title: Reactive Oxygen Species-Mediated Loss of Synaptic Akt1 Signaling Leads to Deficient Activity-Dependent Protein Translation Early in Alzheimer's Disease

    doi: 10.1089/ars.2016.6860

    Figure Lengend Snippet: Deficit in BDNF-stimulated protein translation in cultured cortical primary neurons from APP/PS1 mice is rescued by myristoylated Akt1 overexpression. Increased protein translation was observed in both soma and neurites of primary cortical neurons isolated from WT mice after BDNF stimulation. Stimulation of protein translation at soma and neurites after BDNF treatment was not observed in primary cortical neurons isolated from APP/PS1 mice. Overexpression of myristoylated Akt1 (myr-Akt1), which is constitutively active, rescues the compromised stimulated protein translation in APP/PS1 neurons. Values are mean ± SEM ( n = 31–36 neurons) and * denotes values significantly different from corresponding controls ( p

    Article Snippet: Akt1 kinase assay Akt1 kinase assay was performed according to the manufacturer's instructions (Cell Signaling Technology).

    Techniques: Cell Culture, Mouse Assay, Over Expression, Isolation

    Akt1 phosphorylation and its kinase activity are decreased in synapses of APP/PS1 mice early in the disease pathogenesis. Levels of both the phosphorylated forms of Akt1 [Thr308; (A) and Ser473; (B) ], which are critical for its kinase activity, are reduced in synaptosomes isolated from ADL and YA APP/PS1 mice compared with those from age-matched WT controls. Akt1 kinase activity was measured by immunoblot-based assay of phosphorylation of the Akt1 substrate, recombinant-truncated GSK [rec GSK; (C) ]. Cortical synaptosomes from APP/PS1 mice have reduced Akt1 activity when compared with those from WT controls at all ages examined. Values are mean ± SEM ( n = 6 mice) and * denotes values significantly different from corresponding controls ( p

    Journal: Antioxidants & Redox Signaling

    Article Title: Reactive Oxygen Species-Mediated Loss of Synaptic Akt1 Signaling Leads to Deficient Activity-Dependent Protein Translation Early in Alzheimer's Disease

    doi: 10.1089/ars.2016.6860

    Figure Lengend Snippet: Akt1 phosphorylation and its kinase activity are decreased in synapses of APP/PS1 mice early in the disease pathogenesis. Levels of both the phosphorylated forms of Akt1 [Thr308; (A) and Ser473; (B) ], which are critical for its kinase activity, are reduced in synaptosomes isolated from ADL and YA APP/PS1 mice compared with those from age-matched WT controls. Akt1 kinase activity was measured by immunoblot-based assay of phosphorylation of the Akt1 substrate, recombinant-truncated GSK [rec GSK; (C) ]. Cortical synaptosomes from APP/PS1 mice have reduced Akt1 activity when compared with those from WT controls at all ages examined. Values are mean ± SEM ( n = 6 mice) and * denotes values significantly different from corresponding controls ( p

    Article Snippet: Akt1 kinase assay Akt1 kinase assay was performed according to the manufacturer's instructions (Cell Signaling Technology).

    Techniques: Activity Assay, Mouse Assay, Isolation, Recombinant

    Increased oxidation of Akt1 and its increased affinity for PP2A are observed in synaptosomes of APP/PS1 mice. Aβ42 levels in cortical homogenates from different ages of WT and APP/PS1 mice measured using ELISA is depicted for ADL (1–1.5 months old) and YA (3–4 months old) mice (A) . Immunoblotting provides evidence for increased accumulation of oligomeric Aβ in synaptosomes from ADL and YA APP/PS1 mice (B) . Immunoreactive signals for PSD95 were used to normalize the blots. The leftmost panel with the synthetic Aβ standard is shown as a reference. Treatment of synaptosomes isolated from cortices of ADL and YA APP/PS1 mice with DCFH-DA leads to increased generation of DCF fluorescence when compared with that from age-matched controls (C) . See Supplementary Fig. S4 also. Primary cortical neurons were treated with DCFH-DA and the resultant DCF fluorescence ( green ) was observed along with immunostaining for Homer1 ( red ; D) . Increased colocalization of DCF with Homer1 is observed, indicating extensive ROS levels in neurites and terminals of APP/PS1 cortical neurons, whereas WT cortical neurons show very little staining for ROS ( n = 7–11independent cultures). Scale bar is 20 μm. See Supplementary Fig. S5 . Reduced Akt1 levels assayed as AMS derivatized protein were decreased in synaptosomes isolated from ADL (1 month old) APP/PS1 mice when compared with those from age-matched controls (E) . Immunoprecipitated Akt1 from synaptosomes of 1 month (F) and 9 months (G) old APP/PS1 mice had increased affinity for the phosphatase, PP2A. Values are mean ± SEM ( n = 4 mice) and * denotes values significantly different from corresponding controls ( p

    Journal: Antioxidants & Redox Signaling

    Article Title: Reactive Oxygen Species-Mediated Loss of Synaptic Akt1 Signaling Leads to Deficient Activity-Dependent Protein Translation Early in Alzheimer's Disease

    doi: 10.1089/ars.2016.6860

    Figure Lengend Snippet: Increased oxidation of Akt1 and its increased affinity for PP2A are observed in synaptosomes of APP/PS1 mice. Aβ42 levels in cortical homogenates from different ages of WT and APP/PS1 mice measured using ELISA is depicted for ADL (1–1.5 months old) and YA (3–4 months old) mice (A) . Immunoblotting provides evidence for increased accumulation of oligomeric Aβ in synaptosomes from ADL and YA APP/PS1 mice (B) . Immunoreactive signals for PSD95 were used to normalize the blots. The leftmost panel with the synthetic Aβ standard is shown as a reference. Treatment of synaptosomes isolated from cortices of ADL and YA APP/PS1 mice with DCFH-DA leads to increased generation of DCF fluorescence when compared with that from age-matched controls (C) . See Supplementary Fig. S4 also. Primary cortical neurons were treated with DCFH-DA and the resultant DCF fluorescence ( green ) was observed along with immunostaining for Homer1 ( red ; D) . Increased colocalization of DCF with Homer1 is observed, indicating extensive ROS levels in neurites and terminals of APP/PS1 cortical neurons, whereas WT cortical neurons show very little staining for ROS ( n = 7–11independent cultures). Scale bar is 20 μm. See Supplementary Fig. S5 . Reduced Akt1 levels assayed as AMS derivatized protein were decreased in synaptosomes isolated from ADL (1 month old) APP/PS1 mice when compared with those from age-matched controls (E) . Immunoprecipitated Akt1 from synaptosomes of 1 month (F) and 9 months (G) old APP/PS1 mice had increased affinity for the phosphatase, PP2A. Values are mean ± SEM ( n = 4 mice) and * denotes values significantly different from corresponding controls ( p

    Article Snippet: Akt1 kinase assay Akt1 kinase assay was performed according to the manufacturer's instructions (Cell Signaling Technology).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Isolation, Fluorescence, Immunostaining, Staining, Affinity Magnetic Separation, Immunoprecipitation