anti phospho akt thr308  (Cell Signaling Technology Inc)

 
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    Name:
    Phospho Akt Thr308 244F9H2 Rabbit mAb IHC Specific
    Description:
    Akt also referred to as PKB or Rac plays a critical role in controlling survival and apoptosis 1 3 This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin sensitive pathway involving PI3 kinase 2 3 Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 4 and by phosphorylation within the carboxy terminus at Ser473 The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin mTOR in a rapamycin insensitive complex with rictor and Sin1 5 6 Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets including Bad 7 forkhead transcription factors 8 c Raf 9 and caspase 9 PTEN phosphatase is a major negative regulator of the PI3 kinase Akt signaling pathway 10 LY294002 is a specific PI3 kinase inhibitor 11 Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK 3α and β 12 13 Akt may also play a role in insulin stimulation of glucose transport 12 In addition to its role in survival and glycogen synthesis Akt is involved in cell cycle regulation by preventing GSK 3β mediated phosphorylation and degradation of cyclin D1 14 and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 15 and p21 Waf1 Cip1 16 Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin sensitive complex containing raptor 17 More importantly Akt phosphorylates and inactivates tuberin TSC2 an inhibitor of mTOR within the mTOR raptor complex 18 19
    Catalog Number:
    9266
    Price:
    None
    Applications:
    Immunohistochemistry
    Category:
    Primary Antibodies
    Source:
    Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Thr308 of mouse Akt.
    Reactivity:
    Human Mouse
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc anti phospho akt thr308
    Long-term dephosphorylation/inactivation of <t>Akt</t> kinase in DU145 cells . DU145 cells were exposed to 10 -7 M testosterone-BSA for the indicated time periods: (A: 0, 5, 15, 30, 60 and 120 min), (B: 0, 1, 2, 6 h) and (C: 0, 12 and 24 h). The ratio of the cellular content of the phosphorylated (at Ser473 or <t>Thr308</t> residues) versus the total isoform of Akt kinase was measured in cell lysates by Western blotting using specific antibodies for each form and was normalized to the corresponding ratio of control. Blots show a representative experiment, while the numbers below each lane correspond to the mean values ± SE from three independent experiments (*P
    Akt also referred to as PKB or Rac plays a critical role in controlling survival and apoptosis 1 3 This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin sensitive pathway involving PI3 kinase 2 3 Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 4 and by phosphorylation within the carboxy terminus at Ser473 The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin mTOR in a rapamycin insensitive complex with rictor and Sin1 5 6 Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets including Bad 7 forkhead transcription factors 8 c Raf 9 and caspase 9 PTEN phosphatase is a major negative regulator of the PI3 kinase Akt signaling pathway 10 LY294002 is a specific PI3 kinase inhibitor 11 Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK 3α and β 12 13 Akt may also play a role in insulin stimulation of glucose transport 12 In addition to its role in survival and glycogen synthesis Akt is involved in cell cycle regulation by preventing GSK 3β mediated phosphorylation and degradation of cyclin D1 14 and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 15 and p21 Waf1 Cip1 16 Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin sensitive complex containing raptor 17 More importantly Akt phosphorylates and inactivates tuberin TSC2 an inhibitor of mTOR within the mTOR raptor complex 18 19
    https://www.bioz.com/result/anti phospho akt thr308/product/Cell Signaling Technology Inc
    Average 99 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    anti phospho akt thr308 - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Membrane androgen receptor activation triggers down-regulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU145 prostate cancer cells"

    Article Title: Membrane androgen receptor activation triggers down-regulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU145 prostate cancer cells

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-7-88

    Long-term dephosphorylation/inactivation of Akt kinase in DU145 cells . DU145 cells were exposed to 10 -7 M testosterone-BSA for the indicated time periods: (A: 0, 5, 15, 30, 60 and 120 min), (B: 0, 1, 2, 6 h) and (C: 0, 12 and 24 h). The ratio of the cellular content of the phosphorylated (at Ser473 or Thr308 residues) versus the total isoform of Akt kinase was measured in cell lysates by Western blotting using specific antibodies for each form and was normalized to the corresponding ratio of control. Blots show a representative experiment, while the numbers below each lane correspond to the mean values ± SE from three independent experiments (*P
    Figure Legend Snippet: Long-term dephosphorylation/inactivation of Akt kinase in DU145 cells . DU145 cells were exposed to 10 -7 M testosterone-BSA for the indicated time periods: (A: 0, 5, 15, 30, 60 and 120 min), (B: 0, 1, 2, 6 h) and (C: 0, 12 and 24 h). The ratio of the cellular content of the phosphorylated (at Ser473 or Thr308 residues) versus the total isoform of Akt kinase was measured in cell lysates by Western blotting using specific antibodies for each form and was normalized to the corresponding ratio of control. Blots show a representative experiment, while the numbers below each lane correspond to the mean values ± SE from three independent experiments (*P

    Techniques Used: De-Phosphorylation Assay, Western Blot

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

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

    Journal: Redox Biology

    doi: 10.1016/j.redox.2018.04.021

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

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

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

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

    Journal: Redox Biology

    doi: 10.1016/j.redox.2018.04.021

    S-nitrosylation of Akt at Cys224 inhibits the phosphorylation of Akt at Ser473. ( A-B ), S-nitrosylation of Akt (SNO-Akt) in T cells examined by biotin switch assay in vivo ( A ) or in vitro ( B ). ( C-D ), Phosphorylation of Akt detected by western blot analysis in T cells purified from C57BL/6 J mice, in which isolated T cells were cultured with plate-bound anti-CD3 antibody and pretreated for 30 min with or without 50 μmol/L GSNO or the indicated S-nitrosylation inhibitor, 50 μmol/L N-acetyl- L -cysteine (NAc) ( C ) or 25 μmol/L dithiothreitol (DTT) ( D ), then incubated with or without 100 μmol/L Hcy for 24 h. ( E ), EL4 cells were transiently transfected with flag-labeled wild-type (WT) and mutated Akt (C224S or C296S). At 24 h after transfection, EL4 cells expressing Akt variants were pretreated for 30 min with or without 100 μmol/L GSNO, then incubated with or without 100 μmol/L Hcy for 24 h. S-nitrosylation of flag-labeled Akt was detected by biotin switch assay. Phosphorylation of flag-labeled Akt at Ser473 and Thr308 measured by western blot analysis. Flag-labeled Akt was an internal control. Data are mean ± SEM of at least three independent experiments (n = 3–6 mice in each group). * P ﹤.05 compared with the control. # P ﹤.05 compared with HHcy or Hcy. † P ﹤.05 compared with Hcy + GSNO. n.s. not significant.
    Figure Legend Snippet: S-nitrosylation of Akt at Cys224 inhibits the phosphorylation of Akt at Ser473. ( A-B ), S-nitrosylation of Akt (SNO-Akt) in T cells examined by biotin switch assay in vivo ( A ) or in vitro ( B ). ( C-D ), Phosphorylation of Akt detected by western blot analysis in T cells purified from C57BL/6 J mice, in which isolated T cells were cultured with plate-bound anti-CD3 antibody and pretreated for 30 min with or without 50 μmol/L GSNO or the indicated S-nitrosylation inhibitor, 50 μmol/L N-acetyl- L -cysteine (NAc) ( C ) or 25 μmol/L dithiothreitol (DTT) ( D ), then incubated with or without 100 μmol/L Hcy for 24 h. ( E ), EL4 cells were transiently transfected with flag-labeled wild-type (WT) and mutated Akt (C224S or C296S). At 24 h after transfection, EL4 cells expressing Akt variants were pretreated for 30 min with or without 100 μmol/L GSNO, then incubated with or without 100 μmol/L Hcy for 24 h. S-nitrosylation of flag-labeled Akt was detected by biotin switch assay. Phosphorylation of flag-labeled Akt at Ser473 and Thr308 measured by western blot analysis. Flag-labeled Akt was an internal control. Data are mean ± SEM of at least three independent experiments (n = 3–6 mice in each group). * P ﹤.05 compared with the control. # P ﹤.05 compared with HHcy or Hcy. † P ﹤.05 compared with Hcy + GSNO. n.s. not significant.

    Techniques Used: Biotin Switch Assay, In Vivo, In Vitro, Western Blot, Purification, Mouse Assay, Isolation, Cell Culture, Incubation, Transfection, Labeling, Expressing

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

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

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

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

    4) Product Images from "Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats"

    Article Title: Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    doi: 10.1152/ajpendo.00659.2010

    Top : Akt Thr 308 phosphorylation (pAkt Thr308 ) in epitrochlearis and soleus muscles with 0, 1.2, or 30 nM insulin. Middle : pAkt1 Thr308 in epitrochlearis and soleus muscles with 0, 1.2, or 30 nM insulin. Muscle lysate was immunoprecipitated with Akt1 antibody
    Figure Legend Snippet: Top : Akt Thr 308 phosphorylation (pAkt Thr308 ) in epitrochlearis and soleus muscles with 0, 1.2, or 30 nM insulin. Middle : pAkt1 Thr308 in epitrochlearis and soleus muscles with 0, 1.2, or 30 nM insulin. Muscle lysate was immunoprecipitated with Akt1 antibody

    Techniques Used: Immunoprecipitation

    5) Product Images from "Inhibition of Akt2 phosphorylation abolishes the calorie restriction-induced improvement in insulin-stimulated glucose uptake by rat soleus muscle"

    Article Title: Inhibition of Akt2 phosphorylation abolishes the calorie restriction-induced improvement in insulin-stimulated glucose uptake by rat soleus muscle

    Journal: Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme

    doi: 10.1139/apnm-2016-0326

    Immunoblot analysis of Akt phosphorylation in rat soleus muscle. (A) pan-Akt Thr308 phosphorylation (pAkt Thr308 ). (B) Akt2 Thr309 phosphorylation (pAkt2 Thr309 ), muscle lysates were immunoprecipitated with Akt2 antibody prior to immunoblotting with phospho-Akt Thr308 antibody, (C) pan-Akt Ser473 phosphorylation (pAkt Ser473 ), and (D) Akt2 Ser474 phosphorylation (pAkt2 Ser474 ), muscle lysates were immunoprecipitated with Akt2 antibody prior to immunoblotting with phospho-Akt Ser473 antibody. Filled bars are muscles from AL-fed rats, open bars are muscles from CR-fed rats, and hatched bar are muscles from CR-fed rats incubated with MK-2206. “ns” indicates no statistical difference between AL and CR groups in the absence of insulin. Insulin-stimulated groups with matching letters are not statistically different, and groups with differing letters are statistically (P
    Figure Legend Snippet: Immunoblot analysis of Akt phosphorylation in rat soleus muscle. (A) pan-Akt Thr308 phosphorylation (pAkt Thr308 ). (B) Akt2 Thr309 phosphorylation (pAkt2 Thr309 ), muscle lysates were immunoprecipitated with Akt2 antibody prior to immunoblotting with phospho-Akt Thr308 antibody, (C) pan-Akt Ser473 phosphorylation (pAkt Ser473 ), and (D) Akt2 Ser474 phosphorylation (pAkt2 Ser474 ), muscle lysates were immunoprecipitated with Akt2 antibody prior to immunoblotting with phospho-Akt Ser473 antibody. Filled bars are muscles from AL-fed rats, open bars are muscles from CR-fed rats, and hatched bar are muscles from CR-fed rats incubated with MK-2206. “ns” indicates no statistical difference between AL and CR groups in the absence of insulin. Insulin-stimulated groups with matching letters are not statistically different, and groups with differing letters are statistically (P

    Techniques Used: Immunoprecipitation, Incubation

    6) Product Images from "The novel miR-1269b-regulated protein SVEP1 induces hepatocellular carcinoma proliferation and metastasis likely through the PI3K/Akt pathway"

    Article Title: The novel miR-1269b-regulated protein SVEP1 induces hepatocellular carcinoma proliferation and metastasis likely through the PI3K/Akt pathway

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2535-8

    Decreased SVEP1 expression promotes HCC cell proliferation and metastasis in vivo. a Images of tumors from NOD-SCID nude mice in the SCR and SVEP1/KD groups. b , c The tumor volume and weight of the two groups. d HE and representative IHC staining of SVEP1, Akt Thr308 and PKCζ in tumor sections derived from SCR and SVEP1/KD HCC cell-derived models (the images shown are representative). e Statistical analyses of the bone invasion and multiple in situ metastases of the two groups. f, g Representative images and statistical analysis of the lung metastatic nodules in the two groups.
    Figure Legend Snippet: Decreased SVEP1 expression promotes HCC cell proliferation and metastasis in vivo. a Images of tumors from NOD-SCID nude mice in the SCR and SVEP1/KD groups. b , c The tumor volume and weight of the two groups. d HE and representative IHC staining of SVEP1, Akt Thr308 and PKCζ in tumor sections derived from SCR and SVEP1/KD HCC cell-derived models (the images shown are representative). e Statistical analyses of the bone invasion and multiple in situ metastases of the two groups. f, g Representative images and statistical analysis of the lung metastatic nodules in the two groups.

    Techniques Used: Expressing, In Vivo, Mouse Assay, Immunohistochemistry, Staining, Derivative Assay, In Situ

    7) Product Images from "GITR Intrinsically Sustains Early Type 1 and Late Follicular Helper CD4 T Cell Accumulation to Control a Chronic Viral Infection"

    Article Title: GITR Intrinsically Sustains Early Type 1 and Late Follicular Helper CD4 T Cell Accumulation to Control a Chronic Viral Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004517

    GITR co-stimulation activates classical NF-κB and the Akt-mTORC1 signaling axis to regulate CD4 T cell accumulation post-priming. (A, B) C57BL/6 mice received a 1:1 mixture of GITR +/+ and GITR -/- SMARTA, and were infected the following day with LCMV cl 13. At day eight p.i., proportions of GITR + and GITR- cells were evaluated, with gating strategy shown in B. (C) 10 6 or (D) 10 4 GITR +/+ and GITR -/- CFSE-labeled CD45.1 + SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice one day prior to LCMV cl 13 infection. The total numbers of GITR + and GITR- SMARTA cells in the spleen at different time points following LCMV cl 13 infection are shown. Each symbol in C shows mean ± SEM of at least two to three mice per group, representative of at least two experiments per time point. (E) Proportions of GITR + and GITR- of: total, CD44 hi , T-bet + Th1, and Tfh SMARTA were evaluated in the spleen at day eight p.i. (F) The proportions of IFNγ + or IFNγ + IL-2 + , and the quantity of IFNγ produced per cell were evaluated in GITR + and GITR - SMARTA CD4 T cells following five hours of GP 61–80 peptide restimulation, with representative staining shown in G. (H) 10 6 GITR +/+ and GITR -/- CFSE-labeled CD45.1 + SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice. At days three and five p.i., CFSE dilution was evaluated. (I) 10 6 GITR +/+ and GITR -/- CD45.1 + SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice as in A. At day three post-infection, cells were stained directly ex vivo for (p) p65 NF-κB pSer529 and (p)S6 ribosomal protein pSer235/236. (J) Activated GITR +/+ SMARTA were expanded for two days and then serum starved for 12 hours prior to engaging with 10μg/mL anti-GITR (cone DTA-1) or Rat IgG, followed by evaluation of phosphorylation at Thr308 of Akt. MFI of pThr308 following stimulation is shown, representative of three independent experiments. Numbers adjacent to the representative histograms of I and J are median MFI values. Each symbol in D–G represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two experiments with a total of eight mice. Data in H show a single representative experiment with two-three mice per group, with at least two independent repeats. Data in I are pooled from two independent experiments with a total of seven mice.
    Figure Legend Snippet: GITR co-stimulation activates classical NF-κB and the Akt-mTORC1 signaling axis to regulate CD4 T cell accumulation post-priming. (A, B) C57BL/6 mice received a 1:1 mixture of GITR +/+ and GITR -/- SMARTA, and were infected the following day with LCMV cl 13. At day eight p.i., proportions of GITR + and GITR- cells were evaluated, with gating strategy shown in B. (C) 10 6 or (D) 10 4 GITR +/+ and GITR -/- CFSE-labeled CD45.1 + SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice one day prior to LCMV cl 13 infection. The total numbers of GITR + and GITR- SMARTA cells in the spleen at different time points following LCMV cl 13 infection are shown. Each symbol in C shows mean ± SEM of at least two to three mice per group, representative of at least two experiments per time point. (E) Proportions of GITR + and GITR- of: total, CD44 hi , T-bet + Th1, and Tfh SMARTA were evaluated in the spleen at day eight p.i. (F) The proportions of IFNγ + or IFNγ + IL-2 + , and the quantity of IFNγ produced per cell were evaluated in GITR + and GITR - SMARTA CD4 T cells following five hours of GP 61–80 peptide restimulation, with representative staining shown in G. (H) 10 6 GITR +/+ and GITR -/- CFSE-labeled CD45.1 + SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice. At days three and five p.i., CFSE dilution was evaluated. (I) 10 6 GITR +/+ and GITR -/- CD45.1 + SMARTA from F2 littermates were co-transferred into naïve CD45.2 C57BL/6 mice as in A. At day three post-infection, cells were stained directly ex vivo for (p) p65 NF-κB pSer529 and (p)S6 ribosomal protein pSer235/236. (J) Activated GITR +/+ SMARTA were expanded for two days and then serum starved for 12 hours prior to engaging with 10μg/mL anti-GITR (cone DTA-1) or Rat IgG, followed by evaluation of phosphorylation at Thr308 of Akt. MFI of pThr308 following stimulation is shown, representative of three independent experiments. Numbers adjacent to the representative histograms of I and J are median MFI values. Each symbol in D–G represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two experiments with a total of eight mice. Data in H show a single representative experiment with two-three mice per group, with at least two independent repeats. Data in I are pooled from two independent experiments with a total of seven mice.

    Techniques Used: Mouse Assay, Infection, Labeling, Produced, Staining, Ex Vivo

    8) Product Images from "Valproic Acid Induces Monoamine Oxidase A via Akt/Forkhead Box O1 Activation S⃞"

    Article Title: Valproic Acid Induces Monoamine Oxidase A via Akt/Forkhead Box O1 Activation S⃞

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.111.072744

    Akt mediated the VPA induction of MAO A. A, Western blotting analysis of phospho-Akt level at both Ser473 and Thr308 sites in BE(2)C cells treated with 1 mM VPA for different times (3, 6, 12, and 24 h). Total Akt and β-actin were used as loading
    Figure Legend Snippet: Akt mediated the VPA induction of MAO A. A, Western blotting analysis of phospho-Akt level at both Ser473 and Thr308 sites in BE(2)C cells treated with 1 mM VPA for different times (3, 6, 12, and 24 h). Total Akt and β-actin were used as loading

    Techniques Used: Western Blot

    9) Product Images from "Insulin-induced Effects on the Subcellular Localization of AKT1, AKT2 and AS160 in Rat Skeletal Muscle"

    Article Title: Insulin-induced Effects on the Subcellular Localization of AKT1, AKT2 and AS160 in Rat Skeletal Muscle

    Journal: Scientific Reports

    doi: 10.1038/srep39230

    Effects of insulin and wortmannin on phosphorylation of AKT (pAKT) in cytosol and membrane fractions of muscle. ( a ) pAKT Ser473 in the cytosol fraction. ( b ) pAKT Ser473 in the membrane fraction. ( c ) pAKT Thr308 in the cytosol fraction. ( d ) pAKT Thr308 in the membrane fraction. † Significantly (P
    Figure Legend Snippet: Effects of insulin and wortmannin on phosphorylation of AKT (pAKT) in cytosol and membrane fractions of muscle. ( a ) pAKT Ser473 in the cytosol fraction. ( b ) pAKT Ser473 in the membrane fraction. ( c ) pAKT Thr308 in the cytosol fraction. ( d ) pAKT Thr308 in the membrane fraction. † Significantly (P

    Techniques Used:

    10) Product Images from "Greater insulin-mediated Akt phosphorylation concomitant with heterogeneous effects on phosphorylation of Akt substrates in soleus of calorie-restricted rats"

    Article Title: Greater insulin-mediated Akt phosphorylation concomitant with heterogeneous effects on phosphorylation of Akt substrates in soleus of calorie-restricted rats

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    doi: 10.1152/ajpregu.00457.2012

    Phosphorylation of Akt Thr308 ( A ), Akt Ser473 ( B ), Akt2 Thr308 ( C ), and Akt2 Ser473 ( D ). Main effects of diet, insulin, and diet × insulin interactions are reported from two-way ANOVA analysis. P ≤ 0.05 is considered statistically significant.
    Figure Legend Snippet: Phosphorylation of Akt Thr308 ( A ), Akt Ser473 ( B ), Akt2 Thr308 ( C ), and Akt2 Ser473 ( D ). Main effects of diet, insulin, and diet × insulin interactions are reported from two-way ANOVA analysis. P ≤ 0.05 is considered statistically significant.

    Techniques Used:

    11) Product Images from "Inflammation Improves Glucose Homeostasis Through IKKβ-XBP1s Interaction"

    Article Title: Inflammation Improves Glucose Homeostasis Through IKKβ-XBP1s Interaction

    Journal: Cell

    doi: 10.1016/j.cell.2016.10.015

    IKKβ Reduces ER Stress in the Liver of Ob/ob Mice Male ob/ob mice (8 weeks old) were injected with AAV-ca-IKKβ and AAV-GFP at a dose of 2.4 × 10 9 GC g -1 (n=5 in each group) through the tail vein. (A) Protein lysates were subjected to IR-IP and subsequently phospho-tyrosine and IR immunoblotting. Direct lysates were used to analyze phospho AKT Ser473 , AKT Thr308 and GSK3β Ser9 and the total protein levels of AKT and GSK3β. (B-E) Densitometric quantifications of the western blots bands in (A) . Graphs depict the ratio of the autoradiographical signals of (B) phospho-IR/total IR, (C) phospho-AKT Thr308 /total AKT, (D) phospho-AKT Ser473 /total AKT and (E) phospho-GSK3β Ser9 /total GSK3β. (F) Nuclear phospho-XBP1s Thr48 and total XBP1s protein levels in the liver of ob/ob mice. Nup98 was used as a loading control. (G) The graph depicts the ratio of the signals of phospho-XBP1s Thr48 protein to XBP1s protein shown in (F) . (H) Gene expression levels of Xbp1s, Dnajb9, Hspa5, Herpud1 and Pdia3 in livers of ob/ob mice. (I) PERK Thr980 phosphorylation and total PERK protein levels in livers of ob/ob mice. (J) Ddit3 mRNA levels in livers of ob/ob mice. 18S was used for normalization of gene expression. (K-L), (K) Plasma alanine transaminase (ALT; IU L -1 ) and (L) Aspartate transaminase (AST; IU L -1 ) of ob/ob mice 12 days after injection. Error bars are represented as mean ± SEM. Significance was determined by student’s t test. *P
    Figure Legend Snippet: IKKβ Reduces ER Stress in the Liver of Ob/ob Mice Male ob/ob mice (8 weeks old) were injected with AAV-ca-IKKβ and AAV-GFP at a dose of 2.4 × 10 9 GC g -1 (n=5 in each group) through the tail vein. (A) Protein lysates were subjected to IR-IP and subsequently phospho-tyrosine and IR immunoblotting. Direct lysates were used to analyze phospho AKT Ser473 , AKT Thr308 and GSK3β Ser9 and the total protein levels of AKT and GSK3β. (B-E) Densitometric quantifications of the western blots bands in (A) . Graphs depict the ratio of the autoradiographical signals of (B) phospho-IR/total IR, (C) phospho-AKT Thr308 /total AKT, (D) phospho-AKT Ser473 /total AKT and (E) phospho-GSK3β Ser9 /total GSK3β. (F) Nuclear phospho-XBP1s Thr48 and total XBP1s protein levels in the liver of ob/ob mice. Nup98 was used as a loading control. (G) The graph depicts the ratio of the signals of phospho-XBP1s Thr48 protein to XBP1s protein shown in (F) . (H) Gene expression levels of Xbp1s, Dnajb9, Hspa5, Herpud1 and Pdia3 in livers of ob/ob mice. (I) PERK Thr980 phosphorylation and total PERK protein levels in livers of ob/ob mice. (J) Ddit3 mRNA levels in livers of ob/ob mice. 18S was used for normalization of gene expression. (K-L), (K) Plasma alanine transaminase (ALT; IU L -1 ) and (L) Aspartate transaminase (AST; IU L -1 ) of ob/ob mice 12 days after injection. Error bars are represented as mean ± SEM. Significance was determined by student’s t test. *P

    Techniques Used: Mouse Assay, Injection, Western Blot, Expressing, AST Assay

    12) Product Images from "Heterogeneous Effects of Calorie Restriction on In Vivo Glucose Uptake and Insulin Signaling of Individual Rat Skeletal Muscles"

    Article Title: Heterogeneous Effects of Calorie Restriction on In Vivo Glucose Uptake and Insulin Signaling of Individual Rat Skeletal Muscles

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065118

    Akt Thr308 phosphorylation (pAkt Thr308 ) in predominantly fast-twitch and predominantly slow-twitch muscles. EPI, epitrochlearis; GN, gastrocnemius; TA, tibialis anterior; PLN, plantaris; SOL, soleus; ADL, adductor longus. Filled bars are the AL group and open bars are the CR group. Values are normalized to the average value of the AL samples on each blot. *P
    Figure Legend Snippet: Akt Thr308 phosphorylation (pAkt Thr308 ) in predominantly fast-twitch and predominantly slow-twitch muscles. EPI, epitrochlearis; GN, gastrocnemius; TA, tibialis anterior; PLN, plantaris; SOL, soleus; ADL, adductor longus. Filled bars are the AL group and open bars are the CR group. Values are normalized to the average value of the AL samples on each blot. *P

    Techniques Used:

    13) Product Images from "Growth hormone modulation of EGF-induced PI3K-Akt pathway in mice liver"

    Article Title: Growth hormone modulation of EGF-induced PI3K-Akt pathway in mice liver

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2011.10.001

    Kinetics of Akt phosphorylation at Ser473 and Thr308 related to protein content in normal and GH-overexpressing mice
    Figure Legend Snippet: Kinetics of Akt phosphorylation at Ser473 and Thr308 related to protein content in normal and GH-overexpressing mice

    Techniques Used: Mouse Assay

    Related Articles

    Western Blot:

    Article Title: Essential role for SphK1/S1P signaling to regulate hypoxia-inducible factor 2α expression and activity in cancer
    Article Snippet: .. Western-blot analysis and antibodies Rabbit anti-HIF-2α (Novus, Littleton, CO, USA), mouse anti-HIF-1α (BD, San Jose, CA, USA), rabbit anti-GLUT-1 (ThermoScientific, Villebon-sur-Yvette, France), mouse anti-cyclin D1, rabbit anti-p70S6K, rabbit anti-phospho-p70S6K (Thr389), rabbit anti-phospho-p70S6K (Thr421/Ser424), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-4E-BP1, rabbit anti-phospho 4E-BP1 (Ser65), rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Spns2 (Sigma), anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-puromycin (Millipore, 12D10) were used as primary antibodies. .. Proteins were visualized by an ECL detection system (Perbio, Villebon-sur-Yvette, France) using anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG (Bio-Rad, Marnes-la-Coquette, France).

    Incubation:

    Article Title: Hydrogen Sulfide Prevents Formation of Reactive Oxygen Species through PI3K/Akt Signaling and Limits Ventilator-Induced Lung Injury
    Article Snippet: .. Membranes were incubated with antibodies against NADPH oxidases 1 and 4 (Nox1; Nox4; Santa Cruz Biotechnology Inc., Heidelberg, Germany), Nox2 (Becton Dickinson GmbH, Heidelberg, Germany), phosphorylated Akt, or Akt (Cell Signaling, Leiden, The Netherlands). .. Normalization in order to control equal protein loading was performed by stripping and reblotting of the membranes with glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; Enzo Life Sciences GmbH, Lörrach, Germany).

    other:

    Article Title: Angiopoietin-1 protects myocardial endothelial cell function blunted by angiopoietin-2 and high glucose condition
    Article Snippet: The following primary antibodies were used: anti-phosphotyrosine (Upstate Co, NJ, USA), mouse anti-phospho-eNOS, mouse anti-eNOS (BD Co, CA, USA), rabbit anti-Ang-1 (Santa Cruz, CA, USA), rabbit anti-phospho-AKT, rabbit anti-Akt, mouse anti-cleaved caspase-3, mouse anti-Tie-2, and rabbit anti-β-actin (Cell Signaling, MA, USA).

    SDS Page:

    Article Title: Atorvastatin prevents Aβ oligomer-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting Tau cleavage
    Article Snippet: .. Equal amounts of protein were then separated by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with primary antibodies against the following proteins: mouse monoclonal anti-spectrin αII (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-vimentin (1:1000, Abcam, New Territories, HK, USA), mouse monoclonal anti-Tau (Tau-5, 1:500, Abcam, New Territories, HK, USA), rabbit polyclonal anti-p35 (C-19, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-phospho-GSK3β (Ser9, 1:1000, Cell Signaling Technology, Beverly, MA, USA), rabbit monoclonal anti-phospho-Erk1/2 (Thr202/Tyr204, 1:1000, Cell Signaling Technology, Beverly, MA, USA), and rabbit monoclonal anti-phospho-Akt (Ser473, 1:1000, Cell Signaling Technology, Beverly, MA, USA). .. After being washed with PBS, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2000, Cell Signaling Technology, Beverly, MA, USA).

    Article Title: High Density Lipoprotein Stimulated Migration of Macrophages Depends on the Scavenger Receptor Class B, Type I, PDZK1 and Akt1 and Is Blocked by Sphingosine 1 Phosphate Receptor Antagonists
    Article Snippet: .. After boiling, the samples were subjected to SDS-PAGE followed by immunoblotting with rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA). .. HRP-conjugated donkey-anti-rabbit and donkey-anti-mouse antibodies (Jackson ImmunoResearch Labs Inc, West Grove PA, USA) were used as secondary antibodies and were detected using Western Lightning ECL reagent kit (PerkinElmer Canada, Woodbridge ON, Canada).

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    Cell Signaling Technology Inc rabbit anti phospho akt ser473
    HDL stimulated macrophage migration involves Rho kinase and <t>PI3K-Akt</t> 1 signaling. A. RAW 264.7 cells were cultured in media containing 3% NCLPDS for 18 hrs. Cells were pre-incubated with 10 µM of either the Rho Kinase inhibitor, Y-27632, or the PI3K inhibitor LY294002, or with DMSO vehicle for 30 min and then the migration in response to HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured in the continued presence or absence of the indicated inhibitors. B. RAW 264.7 cells were serum starved for 18 hrs, washed and treated with or without HDL (100 µg protein/ml) for 10, 30 or 60 min. Equal amounts of proteins were analyzed by SDS-PAGE and immunoblotting for either <t>phospho-Ser473-</t> or total-Akt. C. MPM's were harvested from 6 independent WT, Akt1 KO, Akt2 KO or Akt3 KO mice. Migration in response to no stimulus, HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured. Data from A and C are means ± standard deviations of 6 replicates done over two independent experiments. Values identified with different letters are statistically significantly different (P
    Rabbit Anti Phospho Akt Ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti phosphorylated akt
    Activated <t>Akt</t> attenuates Aur-A inhibitory VX-680-induced apoptosis in TSCC cells . (a) Cells were incubated in serum-free media with indicated doses of VX-680 for 24 h, and subjected to Western blot analysis with pAkt (ser473), and Akt1 antibodies. (b) Myr-Akt1 or pUSE stable transfected cells were subjected to Western blot with pAkt and Akt1 antibodies, <t>GAPDH</t> was used as a control. (c) Myr-Akt1 or pUSE transfected cells were treated with VX-680 (5 nM or 10 nM) for 24 h. Cell survival rates were measured by MTT assay.
    Rabbit Anti Phosphorylated Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HDL stimulated macrophage migration involves Rho kinase and PI3K-Akt 1 signaling. A. RAW 264.7 cells were cultured in media containing 3% NCLPDS for 18 hrs. Cells were pre-incubated with 10 µM of either the Rho Kinase inhibitor, Y-27632, or the PI3K inhibitor LY294002, or with DMSO vehicle for 30 min and then the migration in response to HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured in the continued presence or absence of the indicated inhibitors. B. RAW 264.7 cells were serum starved for 18 hrs, washed and treated with or without HDL (100 µg protein/ml) for 10, 30 or 60 min. Equal amounts of proteins were analyzed by SDS-PAGE and immunoblotting for either phospho-Ser473- or total-Akt. C. MPM's were harvested from 6 independent WT, Akt1 KO, Akt2 KO or Akt3 KO mice. Migration in response to no stimulus, HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured. Data from A and C are means ± standard deviations of 6 replicates done over two independent experiments. Values identified with different letters are statistically significantly different (P

    Journal: PLoS ONE

    Article Title: High Density Lipoprotein Stimulated Migration of Macrophages Depends on the Scavenger Receptor Class B, Type I, PDZK1 and Akt1 and Is Blocked by Sphingosine 1 Phosphate Receptor Antagonists

    doi: 10.1371/journal.pone.0106487

    Figure Lengend Snippet: HDL stimulated macrophage migration involves Rho kinase and PI3K-Akt 1 signaling. A. RAW 264.7 cells were cultured in media containing 3% NCLPDS for 18 hrs. Cells were pre-incubated with 10 µM of either the Rho Kinase inhibitor, Y-27632, or the PI3K inhibitor LY294002, or with DMSO vehicle for 30 min and then the migration in response to HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured in the continued presence or absence of the indicated inhibitors. B. RAW 264.7 cells were serum starved for 18 hrs, washed and treated with or without HDL (100 µg protein/ml) for 10, 30 or 60 min. Equal amounts of proteins were analyzed by SDS-PAGE and immunoblotting for either phospho-Ser473- or total-Akt. C. MPM's were harvested from 6 independent WT, Akt1 KO, Akt2 KO or Akt3 KO mice. Migration in response to no stimulus, HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured. Data from A and C are means ± standard deviations of 6 replicates done over two independent experiments. Values identified with different letters are statistically significantly different (P

    Article Snippet: After boiling, the samples were subjected to SDS-PAGE followed by immunoblotting with rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Migration, Cell Culture, Incubation, SDS Page, Mouse Assay

    SphK1 silencing downregulates Akt/mTOR signaling pathway in VHL -defective A498 and 786-O ccRCC cells. A498 and 786-O cells were untransfected or transfected with 20 nmol/l of siSphK1 or siScr for 72 h before the experiments followed by 4 h under normoxic or hypoxic condition. Cell lysates were assayed for Ser2448 phosphorylated mTOR (p-mTOR Ser2448) and mTOR expression ( a ); Thr389 phosphorylated p70S6K (p-p70S6K Thr389), Thr421/Ser424 phosphorylated p70S6K (p-p70S6K Thr421/Ser424) and p70S6K expression ( b ); Ser65 phosphorylated 4E-BP1 (p-4E-BP1 Ser65) and 4E-BP1 expression ( c ); Ser473 phosphorylated Akt (P-Akt Ser473) and Akt expression ( d ) were analyzed by immunoblotting. Similar results were obtained in three independent experiments.

    Journal: Oncogenesis

    Article Title: Essential role for SphK1/S1P signaling to regulate hypoxia-inducible factor 2α expression and activity in cancer

    doi: 10.1038/oncsis.2016.13

    Figure Lengend Snippet: SphK1 silencing downregulates Akt/mTOR signaling pathway in VHL -defective A498 and 786-O ccRCC cells. A498 and 786-O cells were untransfected or transfected with 20 nmol/l of siSphK1 or siScr for 72 h before the experiments followed by 4 h under normoxic or hypoxic condition. Cell lysates were assayed for Ser2448 phosphorylated mTOR (p-mTOR Ser2448) and mTOR expression ( a ); Thr389 phosphorylated p70S6K (p-p70S6K Thr389), Thr421/Ser424 phosphorylated p70S6K (p-p70S6K Thr421/Ser424) and p70S6K expression ( b ); Ser65 phosphorylated 4E-BP1 (p-4E-BP1 Ser65) and 4E-BP1 expression ( c ); Ser473 phosphorylated Akt (P-Akt Ser473) and Akt expression ( d ) were analyzed by immunoblotting. Similar results were obtained in three independent experiments.

    Article Snippet: Western-blot analysis and antibodies Rabbit anti-HIF-2α (Novus, Littleton, CO, USA), mouse anti-HIF-1α (BD, San Jose, CA, USA), rabbit anti-GLUT-1 (ThermoScientific, Villebon-sur-Yvette, France), mouse anti-cyclin D1, rabbit anti-p70S6K, rabbit anti-phospho-p70S6K (Thr389), rabbit anti-phospho-p70S6K (Thr421/Ser424), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-4E-BP1, rabbit anti-phospho 4E-BP1 (Ser65), rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Spns2 (Sigma), anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-puromycin (Millipore, 12D10) were used as primary antibodies.

    Techniques: Transfection, Expressing

    Atorvastatin prevented AβO-induced downregulation of phospho-Akt and phospho-Erk1/2 expression. (A) Representative immunoblot comparing phospho-Akt to total Akt in hippocampal neurons after the treatments indicated. Group data showing the normalization

    Journal: Acta Pharmacologica Sinica

    Article Title: Atorvastatin prevents Aβ oligomer-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting Tau cleavage

    doi: 10.1038/aps.2014.161

    Figure Lengend Snippet: Atorvastatin prevented AβO-induced downregulation of phospho-Akt and phospho-Erk1/2 expression. (A) Representative immunoblot comparing phospho-Akt to total Akt in hippocampal neurons after the treatments indicated. Group data showing the normalization

    Article Snippet: Equal amounts of protein were then separated by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with primary antibodies against the following proteins: mouse monoclonal anti-spectrin αII (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-vimentin (1:1000, Abcam, New Territories, HK, USA), mouse monoclonal anti-Tau (Tau-5, 1:500, Abcam, New Territories, HK, USA), rabbit polyclonal anti-p35 (C-19, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-phospho-GSK3β (Ser9, 1:1000, Cell Signaling Technology, Beverly, MA, USA), rabbit monoclonal anti-phospho-Erk1/2 (Thr202/Tyr204, 1:1000, Cell Signaling Technology, Beverly, MA, USA), and rabbit monoclonal anti-phospho-Akt (Ser473, 1:1000, Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Expressing

    Activated Akt attenuates Aur-A inhibitory VX-680-induced apoptosis in TSCC cells . (a) Cells were incubated in serum-free media with indicated doses of VX-680 for 24 h, and subjected to Western blot analysis with pAkt (ser473), and Akt1 antibodies. (b) Myr-Akt1 or pUSE stable transfected cells were subjected to Western blot with pAkt and Akt1 antibodies, GAPDH was used as a control. (c) Myr-Akt1 or pUSE transfected cells were treated with VX-680 (5 nM or 10 nM) for 24 h. Cell survival rates were measured by MTT assay.

    Journal: Molecular Cancer

    Article Title: Aurora-A down-regulates IkappaB? via Akt activation and interacts with insulin-like growth factor-1 induced phosphatidylinositol 3-kinase pathway for cancer cell survival

    doi: 10.1186/1476-4598-8-95

    Figure Lengend Snippet: Activated Akt attenuates Aur-A inhibitory VX-680-induced apoptosis in TSCC cells . (a) Cells were incubated in serum-free media with indicated doses of VX-680 for 24 h, and subjected to Western blot analysis with pAkt (ser473), and Akt1 antibodies. (b) Myr-Akt1 or pUSE stable transfected cells were subjected to Western blot with pAkt and Akt1 antibodies, GAPDH was used as a control. (c) Myr-Akt1 or pUSE transfected cells were treated with VX-680 (5 nM or 10 nM) for 24 h. Cell survival rates were measured by MTT assay.

    Article Snippet: Antibodies used were mouse anti-GAPDH (Ambion), rabbit anti-Bcl-2, rabbit anti-cleaved caspase-3, mouse anti-cleaved PARP (Asp175), rabbit anti-phosphorylated Akt (pAkt, Ser473), mouse anti-phospho-GSK3α/β (Ser 21/9, Cell Signaling), mouse anti-IκBα (BD), rabbit anti-GSK3β, goat anti-Akt1, rabbit anti-Bcl-xL (Santa Cruz Biotechnology) and rabbit anti-Aur-A (Upstate).

    Techniques: Incubation, Western Blot, Transfection, MTT Assay