anti-phospho-akt Search Results


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  • 99
    Cell Signaling Technology Inc anti phospho akt
    <t>NCOA5</t> upregulates Cyclin D1 and MMP9 as well as downreuglates P27 in CRC cells via the <t>PI3K/AKT</t> pathway (A) Western blot analysis of NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27 and MMP9. The lysates derived from SW620-shNCOA5 2 # , SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells were immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27, MMP9 and GAPDH (a loading control), respectively. The representative figures of Western blot assay were shown. (B) Western blot analysis after PI3K inhibition. The SW480-LV-NCOA5 CRC cells were pretreated with different concentrations (0, 5, 10 and 20 μM) of PI3K inhibitor LY294002. The lysates of LY294002-treated and -untreated SW480-LV-NCOA5 CRC cells were harvested and then immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, Cyclin D1, P27, MMP9 and GAPDH (a loading control). The representative figures of Western blot assay were shown. (C) ELISA analysis of MMP9 secretion.SW620-shNCOA5 2 # and SW620-shNCOA5 3 # compared with SW620-shNTC group: ** , P
    Anti Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 10483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho akt ser473
    Insulin-stimulated cell signaling in skeletal muscle. Phosphorylation of insulin receptor substrate 1 (IRS1) on Tyr632 ( A ), <t>Akt</t> on Thr308 ( B ), and <t>Ser473</t> ( C ), and PAS160 ( D ) and relative total protein levels of Akt1, Akt2, and AS160 ( E ) were determined
    Phospho Akt Ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho akt ser473
    Total and phosphorylated <t>Akt</t> in WT and Akt3 −/− platelets. (A,D) Washed Akt3 −/− and WT mouse platelets were stimulated with thrombin (0.018 U/mL) for 1, 3, and 5 minutes, solubilized, and immunoblotted with antibodies directed against: (A) Akt3, total Akt (Akt1, Akt2, and Akt3), phosphorylated <t>Ser473</t> of Akt, and GSK-3β (loading control), and (D) phosphorylated Thr 308 of Akt, total Akt and α-tubulin (loading control). (B,C) Western blot results from each of 3 experiments as shown in panel A were scanned and quantitated using NIH ImageJ for uncalibrated optical density. The relative quantity of total Akt (B) and phosphorylated Ser473 of Akt (C) in wild-type and Akt3 −/− platelets are shown (mean ± SE). (E) Washed Akt3 −/− and WT mouse platelets were solubilized and immunoblotted with antibodies directed against Akt1, Akt2, and Akt3 and α-tubulin (loading control).
    Anti Phospho Akt Ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3865 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti phospho akt
    Immunofluorescent detection of Ki-67 ( panels a, b ), <t>phospho-PDGFR-β</t> ( panels c, d ), and <t>phospho-Akt</t> ( e,f ) in control ( a,c,e ) and injured carotid arteries ( b,d,f ) of smLrp1 −/− mice 14 days after endothelial denudation. The elastic
    Rabbit Anti Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2008 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho akt thr308
    LS-induced upregulation of KCa3.1 and KCa2.3 expression is mediated by CaMKK-dependent <t>Akt</t> activation. A and B : HCAECs were exposed to LS for 24 h in the absence or presence of compound C (an AMPK inhibitor), KN-62 (a CaMK inhibitor), or Akt inhibitor IV. Compound C tended to reduce LS-induced upregulation of KCa3.1 mRNA expression ( A ) but did not change LS induction of KCa2.3 expression ( B ) ( n = 9). KN-62 had no effect on the upregulation of KCa3.1 and KCa2.3 mRNA expression ( n = 7–8). Akt inhibition completely blocked LS-induced upregulation of both KCa3.1 and KCa2.3 expression. C : LS-induced increase in KCa2.3 and KCa3.1 protein expression levels was suppressed by Akt inhibition. Top : a representative image of immunoblots. Bottom : the summaries of densitometric analysis of KCa3.1 and KCa2.3 signals ( n = 3). D : Akt inhibitor IV abolished the phosphorylation of Akt at Ser473 in HCAECs exposed to 60-min LS ( top ). Levels of phospho-Akt (Ser473) were suppressed after inhibiting CaMKK with STO-609 in ECs exposed to 15 min of LS ( bottom ). Phosphorylation of Akt at <t>Thr308</t> was not observed in ECs under ST or LS condition. #Signficant difference vs. LS ( P
    Phospho Akt Thr308, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho akt antibody
    Effect of MEK inhibitor on <t>ERK1/2,</t> <t>Akt</t> and NF-κB activation. Cells were administrated with ( A , B ) trametinib or ( C , D ) PD0325901 for 3 days. Control cells (0 μM) were administrated with 0.5% dimethyl sulfoxide (DMSO) for 3 days. ( A , C ) Cell lysates were examined by western blotting assay using indicated antibodies. ( B , D ) Quantification of phosphorylated protein expression, normalized corresponding protein, respectively. The results showed the 5 independent experiments. * p
    Anti Phospho Akt Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti phospho akt
    Mdm20 regulates the phosphorylation status of <t>Akt.</t> A. Western blots for Akt with a focus on phosphorylated Akt on Ser473 and Thr308. HEK293 cells were transfected either with mock, Flag-Mdm20 and mCherry-Nat5 or control, Mdm20 and Nat5 siRNA. At 48 hrs or 72hrs post-transfection, respectively, the cells were harvested, and cell lysates were prepared and processed for western blot analysis. The antibodies used are indicated. For details, see Materials and Methods. B. Western blots were used to determine the phosphorylation status of GSK3β, PTEN, PDK-1, mTOR, and <t>PP1.</t> The antibodies used were as indicated. Note that the phosphorylation of mTOR (Ser-2481) is consistently reduced in Mdm20-KD cells. C. Akt inhibition increases the levels of LC3II. Shown is western blots of HEK293 cellular extracts treated or untreated with siRNAs for Mdm20 or Nat5 in the absence (DMSO control) or presence of Akt inhibitor (Akt-I-VIII). Levels of Akt, and phospho-Akt (pAkt-Ser473), and LC3 levels are shown with the loading control of actin blot.
    Anti Phospho Akt, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p akt  (Abcam)
    99
    Abcam p akt
    Knockdown of CHRF reduces the protein expression level of <t>PI3K/Akt</t> pathway in LAD. Western blotting revealed that knockdown of CHRF decreased the protein expression levels of p-PI3K and p-Akt in (A) SPCA-1 and (B) NCI-H441 cells. Error bars represented the mean ± standard deviation of ≥3 independent experiments. **P
    P Akt, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2037 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    NCOA5 upregulates Cyclin D1 and MMP9 as well as downreuglates P27 in CRC cells via the PI3K/AKT pathway (A) Western blot analysis of NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27 and MMP9. The lysates derived from SW620-shNCOA5 2 # , SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells were immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27, MMP9 and GAPDH (a loading control), respectively. The representative figures of Western blot assay were shown. (B) Western blot analysis after PI3K inhibition. The SW480-LV-NCOA5 CRC cells were pretreated with different concentrations (0, 5, 10 and 20 μM) of PI3K inhibitor LY294002. The lysates of LY294002-treated and -untreated SW480-LV-NCOA5 CRC cells were harvested and then immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, Cyclin D1, P27, MMP9 and GAPDH (a loading control). The representative figures of Western blot assay were shown. (C) ELISA analysis of MMP9 secretion.SW620-shNCOA5 2 # and SW620-shNCOA5 3 # compared with SW620-shNTC group: ** , P

    Journal: Oncotarget

    Article Title: NCOA5 promotes proliferation, migration and invasion of colorectal cancer cells via activation of PI3K/AKT pathway

    doi: 10.18632/oncotarget.22429

    Figure Lengend Snippet: NCOA5 upregulates Cyclin D1 and MMP9 as well as downreuglates P27 in CRC cells via the PI3K/AKT pathway (A) Western blot analysis of NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27 and MMP9. The lysates derived from SW620-shNCOA5 2 # , SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells were immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27, MMP9 and GAPDH (a loading control), respectively. The representative figures of Western blot assay were shown. (B) Western blot analysis after PI3K inhibition. The SW480-LV-NCOA5 CRC cells were pretreated with different concentrations (0, 5, 10 and 20 μM) of PI3K inhibitor LY294002. The lysates of LY294002-treated and -untreated SW480-LV-NCOA5 CRC cells were harvested and then immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, Cyclin D1, P27, MMP9 and GAPDH (a loading control). The representative figures of Western blot assay were shown. (C) ELISA analysis of MMP9 secretion.SW620-shNCOA5 2 # and SW620-shNCOA5 3 # compared with SW620-shNTC group: ** , P

    Article Snippet: The membranes transferred by cell lysates were incubated with primary antibodies including anti-NCOA5 (1:1000; Cat. No. A300-789A, Bethyl, Montgomery, TX, USA), anti-phospho-AKT (anti-p-AKT) (Ser473) (clone No. D9E) (1:1000; Cat. No. 4060S, Cell Signaling Technology, Danvers, MA, USA), anti-AKT (1:1000; Cat. No. AP52689-100, ABGENT, San Diego, CA, USA), anti-phospho-extracellular signal-regulated kinase 1/2 (anti-p-ERK1/2) (Thr202/Tyr204) (clone No. 20G11) (1:1000; Cat. No. 4376S, Cell Signaling Technology, Danvers, MA, USA), anti-ERK1/2 (clone No. 137F5) (1:1000; Cat. No. 4695S, Cell Signaling Technology, Danvers, MA, USA), anti-Cyclin D1 (clone No. 92G2) (1:1000; Cat. No. 2978S, Cell Signaling Technology, Danvers, MA, USA), anti-P27 (clone No. D69C12) (1:1000; Cat. No. 3686S, Cell Signaling Technology, Danvers, MA, USA), anti-MMP9 (clone No. 5G3) (1:1000; Cat. No. GTX60482, GeneTex, Irvine, CA, USA) and anti-GAPDH (1:3000; Cat. No. AP0063, Bioworld, St. Louis Park, MN, USA) (a loading control), respectively.

    Techniques: Western Blot, Derivative Assay, Inhibition, Enzyme-linked Immunosorbent Assay

    Autophagy, ER stress, and p-AKT change after CQ (Q), Rapa (R) and TMZ (T) in different combination treatment GBM8401 cells and U87MG cells were treated with CQ, Rapa and TMZ in different combinations, harvested at 24 hours and immunoblotted for LC3-I, LC3-II and ( A ) p62, ( B ) p-AKT, ( C ) p-mTOR and p-S6K, ( D ) GRP78 and CHOP.

    Journal: Oncotarget

    Article Title: Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells

    doi: 10.18632/oncotarget.23855

    Figure Lengend Snippet: Autophagy, ER stress, and p-AKT change after CQ (Q), Rapa (R) and TMZ (T) in different combination treatment GBM8401 cells and U87MG cells were treated with CQ, Rapa and TMZ in different combinations, harvested at 24 hours and immunoblotted for LC3-I, LC3-II and ( A ) p62, ( B ) p-AKT, ( C ) p-mTOR and p-S6K, ( D ) GRP78 and CHOP.

    Article Snippet: The membrane was then incubated with antibodies against β-actin (Sigma, 1:10000), GAPDH (Sigma, 1:10000), LC3 (Novus Biologicals Inc., Littleton, CO, 1:10000), SQSTM1/p62 (MBL international, 1:1000), PARP (Cell Signaling Technologies, 1:1000), phospho-mTOR, phospho-Akt (Ser 473) (Cell Signaling Technologies, 1:1000), phospho-p70S6K (Cell Signaling Technologies, 1:1000), ABCA1 (Abcam, 1:1000), LDLR (Abcam, 1:5000), SREBP1 (BD, 1:1000), SREBP2 (BD, 1:1000), CHOP (Cell Signaling Technologies, 1:1000) and GRP78 (Cell Signaling Technologies, 1:1000) overnight at 4°C in PBS containing 0.05% Tween 20 and 5% nonfat milk, followed by incubation for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in the same buffer.

    Techniques:

    Activation of mTOR in jejunum. (Up) Representative western blots of total 4EBP1(T-4EBP1), phosphorylated 4EBP1 (Ser65) (P-4EBP1), total Akt (T-Akt), phosphorylated Akt (Ser473) (P-Akt), total mTOR (T- mTOR), phosphorylated mTOR (Ser2448) (P- mTOR) in jejunum of piglets fed the various dietary treatments. β-actin was the loading control. (Down) Quantification by image analysis of 4EBP1, Akt and mTOR phosphorylation. Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4 mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% (g/g) glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% (g/g) glutamic acid supplementation. n = 7 for treatments.

    Journal: PLoS ONE

    Article Title: Therapeutic Effects of Glutamic Acid in Piglets Challenged with Deoxynivalenol

    doi: 10.1371/journal.pone.0100591

    Figure Lengend Snippet: Activation of mTOR in jejunum. (Up) Representative western blots of total 4EBP1(T-4EBP1), phosphorylated 4EBP1 (Ser65) (P-4EBP1), total Akt (T-Akt), phosphorylated Akt (Ser473) (P-Akt), total mTOR (T- mTOR), phosphorylated mTOR (Ser2448) (P- mTOR) in jejunum of piglets fed the various dietary treatments. β-actin was the loading control. (Down) Quantification by image analysis of 4EBP1, Akt and mTOR phosphorylation. Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4 mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% (g/g) glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% (g/g) glutamic acid supplementation. n = 7 for treatments.

    Article Snippet: The following primary antibodies were incubated overnight at 4°C with gentle rocking: β-actin (Santa Vruz, 1∶400), total 4EBP1 (T-4EBP1, Cell Signaling, 1∶1000), phosphorylated 4EBP1 (Ser 65) (Cell Signaling, 1∶1000), total Akt (T-Akt, Cell Signaling, 1∶1000), phosphorylated Akt (Ser 473) (Cell Signaling, 1∶1000), total mTOR (T-mTOR, Cell Signaling, 1∶1000) or phosphorylated mTOR (Ser 2448) (Cell Signaling, 1∶1000).

    Techniques: Activation Assay, Western Blot

    Activation of mTOR in ileum. (Up) Representative western blots of total 4EBP1(T-4EBP1), phosphorylated 4EBP1 (Ser65) (P-4EBP1), total Akt (T-Akt), phosphorylated Akt (Ser473) (P-Akt), total mTOR (T- mTOR), phosphorylated mTOR (Ser2448) (P- mTOR) in ileal of piglets fed the various dietary treatments. β-actin was the loading control. (Down) Quantification by image analysis of 4EBP1, Akt and mTOR phosphorylation. Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4 mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% (g/g) glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% (g/g) glutamic acid supplementation. n = 7 for treatments.

    Journal: PLoS ONE

    Article Title: Therapeutic Effects of Glutamic Acid in Piglets Challenged with Deoxynivalenol

    doi: 10.1371/journal.pone.0100591

    Figure Lengend Snippet: Activation of mTOR in ileum. (Up) Representative western blots of total 4EBP1(T-4EBP1), phosphorylated 4EBP1 (Ser65) (P-4EBP1), total Akt (T-Akt), phosphorylated Akt (Ser473) (P-Akt), total mTOR (T- mTOR), phosphorylated mTOR (Ser2448) (P- mTOR) in ileal of piglets fed the various dietary treatments. β-actin was the loading control. (Down) Quantification by image analysis of 4EBP1, Akt and mTOR phosphorylation. Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4 mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% (g/g) glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% (g/g) glutamic acid supplementation. n = 7 for treatments.

    Article Snippet: The following primary antibodies were incubated overnight at 4°C with gentle rocking: β-actin (Santa Vruz, 1∶400), total 4EBP1 (T-4EBP1, Cell Signaling, 1∶1000), phosphorylated 4EBP1 (Ser 65) (Cell Signaling, 1∶1000), total Akt (T-Akt, Cell Signaling, 1∶1000), phosphorylated Akt (Ser 473) (Cell Signaling, 1∶1000), total mTOR (T-mTOR, Cell Signaling, 1∶1000) or phosphorylated mTOR (Ser 2448) (Cell Signaling, 1∶1000).

    Techniques: Activation Assay, Western Blot

    Draxin treatment of cortical neurons inhibits Akt activity and activates GSK-3β. Immunoblot analyses of cortical neurons from newborn wild-type ( A ) or MAP1B-/- ( B ) mice cultured for 60 h, treated with draxin for the indicated times, lysed and probed using the indicated antibodies. The GSK-3β doublets represent the GSK-3β1 and GSK-3β2 isoforms [ 33 ].The relative levels of GSK-3β phosphorylated at Ser9 (P-GSK-3β) and Akt phosphorylated on Ser473 (P-Akt) were determined by normalizing the signals for the phosphorylated proteins to the corresponding signals for the total proteins in 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Repulsive Axon Guidance by Draxin Is Mediated by Protein Kinase B (Akt), Glycogen Synthase Kinase-3β (GSK-3β) and Microtubule-Associated Protein 1B

    doi: 10.1371/journal.pone.0119524

    Figure Lengend Snippet: Draxin treatment of cortical neurons inhibits Akt activity and activates GSK-3β. Immunoblot analyses of cortical neurons from newborn wild-type ( A ) or MAP1B-/- ( B ) mice cultured for 60 h, treated with draxin for the indicated times, lysed and probed using the indicated antibodies. The GSK-3β doublets represent the GSK-3β1 and GSK-3β2 isoforms [ 33 ].The relative levels of GSK-3β phosphorylated at Ser9 (P-GSK-3β) and Akt phosphorylated on Ser473 (P-Akt) were determined by normalizing the signals for the phosphorylated proteins to the corresponding signals for the total proteins in 3 independent experiments.

    Article Snippet: Primary antibodies: mouse monoclonal antibodies against phospho-MAP1B (SMI31; 1:1000; Covance) and neurofilament H (1:1000; Cell Signaling); rabbit polyclonal antibodies against total-MAP1B raised against peptides ATVVVEATEPEPSGC and ETVTEEHLRRAIGN [ ], phospho Ser473 Akt (1:1000; Cell Signaling), total Akt (1:1000; Cell Signaling) and GAPDH (1:3000; Sigma-Aldrich); rabbit monoclonal antibodies against phospho Ser9-GSK-3β (1:1000; Cell Signaling) and total GSK-3β (1:1000; Cell Signaling).

    Techniques: Activity Assay, Mouse Assay, Cell Culture

    Effects of CO-EtOAc on the regulation of insulin receptor-mediated signaling. Relative protein levels of hepatic: ( A ) IRS-1 (phosphorylated IRS-1 at Ser-307 and total IRS-1); and ( B ) Akt (phosphorylated Akt at Ser-473 and total Akt) were detected by Western blot analysis. Data are represented as mean ± SEM ( n = 8–10). Values with different letters are statistically different ( p

    Journal: Nutrients

    Article Title: Chinese Olive (Canarium album L.) Fruit Extract Attenuates Metabolic Dysfunction in Diabetic Rats

    doi: 10.3390/nu9101123

    Figure Lengend Snippet: Effects of CO-EtOAc on the regulation of insulin receptor-mediated signaling. Relative protein levels of hepatic: ( A ) IRS-1 (phosphorylated IRS-1 at Ser-307 and total IRS-1); and ( B ) Akt (phosphorylated Akt at Ser-473 and total Akt) were detected by Western blot analysis. Data are represented as mean ± SEM ( n = 8–10). Values with different letters are statistically different ( p

    Article Snippet: The blots were blocked with 5% (w /v ) skim milk and probed with antibodies against CPT-1 (Abcam, Cambridge, MA, USA), AMPKα, phospho-AMPKα (Thr-172), LDLR, ABCA1 (Gene Tex, Irvine, CA, USA), IRS-1, phospho-IRS-1 (Ser-307), Akt, and phospho-Akt (Ser-473) (Cell Signaling Technology, Beverly, MA, USA) separately, followed by goat anti-rabbit or mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibodies.

    Techniques: Western Blot

    Insulin-stimulated cell signaling in skeletal muscle. Phosphorylation of insulin receptor substrate 1 (IRS1) on Tyr632 ( A ), Akt on Thr308 ( B ), and Ser473 ( C ), and PAS160 ( D ) and relative total protein levels of Akt1, Akt2, and AS160 ( E ) were determined

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

    Article Title: Low intrinsic running capacity is associated with reduced skeletal muscle substrate oxidation and lower mitochondrial content in white skeletal muscle

    doi: 10.1152/ajpregu.00659.2010

    Figure Lengend Snippet: Insulin-stimulated cell signaling in skeletal muscle. Phosphorylation of insulin receptor substrate 1 (IRS1) on Tyr632 ( A ), Akt on Thr308 ( B ), and Ser473 ( C ), and PAS160 ( D ) and relative total protein levels of Akt1, Akt2, and AS160 ( E ) were determined

    Article Snippet: The membranes were then blocked (5% nonfat dry milk), and incubated overnight at 4°C with primary antibodies specific for either phospho-Akt Ser473, phospho-Akt Thr308, Akt1, Akt2, phospho-Akt substrate (1:1,000; Cell Signaling, Beverly, MA), phospho-insulin receptor substrate 1 (IRS1) Tyr632, CPTI (1:200–500; Santa Cruz Biotechnology, Santa Cruz, CA), or AS160 (TBCD14), which was produced as previously described , using a region of human AS160 from amino acids 621–766 fused with glutathione S -transferase (1:1,000, a gift from Prof. David James, Garvan Institute, Sydney, Australia).

    Techniques:

    MiR-222-3p overexpression reduces ovarian cancer cell proliferation by inhibiting phosphorylation of AKT ( A ) Tara R182 cell line was transfected with miR-222-3p mimic, Western blot analysis of AKT phosphorylation levels at both Ser473- and Thr308- residues and total AKT were detected. Expression of β-actin was used as a loading control. ( B ) The relative expressions of GNAI2, pAKT (ser473), pAKT (thr308) and total AKT proteins were normalized to β-actin. ( C ) SKOV3 cells were transfected with miR-222-3p inhibitor, Western blot analysis of AKT phosphorylation levels at both Ser473- and Thr308- residues and total AKT were detected. Expression of GAPDH was used as a loading control. ( D ) The relative expressions of GNAI2, pAKT (ser473), pAKT (thr308) and total AKT proteins were normalized to GAPDH.

    Journal: Oncotarget

    Article Title: MicroRNA-222-3p/GNAI2/AKT axis inhibits epithelial ovarian cancer cell growth and associates with good overall survival

    doi: 10.18632/oncotarget.13017

    Figure Lengend Snippet: MiR-222-3p overexpression reduces ovarian cancer cell proliferation by inhibiting phosphorylation of AKT ( A ) Tara R182 cell line was transfected with miR-222-3p mimic, Western blot analysis of AKT phosphorylation levels at both Ser473- and Thr308- residues and total AKT were detected. Expression of β-actin was used as a loading control. ( B ) The relative expressions of GNAI2, pAKT (ser473), pAKT (thr308) and total AKT proteins were normalized to β-actin. ( C ) SKOV3 cells were transfected with miR-222-3p inhibitor, Western blot analysis of AKT phosphorylation levels at both Ser473- and Thr308- residues and total AKT were detected. Expression of GAPDH was used as a loading control. ( D ) The relative expressions of GNAI2, pAKT (ser473), pAKT (thr308) and total AKT proteins were normalized to GAPDH.

    Article Snippet: Immunoblotting was performed using the human anti-G protein alpha inhibitor 2 antibody (ab20392, 1:500; abcam, UK), the Phospho-AKT (Thr308) Antibody (9275, 1:1,000; Cell Signaling, USA), the Phospho-AKT (Ser473) Antibody (9271, 4060, 1:1,000; Cell Signaling, USA), the AKT antibody (9292, 1:1,000; Cell Signaling, USA), and the PTEN Antibody (9552, 1:1,000; Cell Signaling, USA); The GAPDH (14C10) Rabbit mAb (2118, 1:1,000; Cell Signaling, USA), the β-Actin (13E5) Rabbit mAb (4970, 1:1,000; Cell Signaling, USA), and the β-Tubulin (9F3) Rabbit mAb (2128, 1:1,000; Cell Signaling, USA) were used as internal control proteins.

    Techniques: Over Expression, Transfection, Western Blot, Expressing

    BRAF , pERKS, ERKS, phospho-AKT Ser473, AKT, phospho-S6 Ser235/236, and S6 expression in the K1 cell line after BRAF silencing. Western blot for BRAF , pERKS, ERKS, phospho-AKT Ser473, AKT, phospho-S6 Ser235/236, S6 expression and actin in K1 cell line treated with BRAF -C2 siRNA (50 nM) after 24 and 72 h. The levels of BRAF were analyzed for control of silencing efficiency and the levels of pERKS as a readout of MAPK pathway activity. Representative actin expression pattern is shown. Protein level, in scramble siRNA treated cells, was evaluated in duplicate (Scr), whereas in BRAF siRNA treated cells, it was analyzed in triplicate. The graphics depicts the mean fold change of protein expression observed in K1 cell line treated with BRAF -C2 siRNA in comparison to cells treated with scramble siRNA. Phosphorylated proteins were normalized by the levels of their correspondent total proteins, all others were normalized by the levels of control protein (actin). Results are shown as mean expression value of two independent experiments ±SEM. * p

    Journal: International Journal of Molecular Sciences

    Article Title: mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression

    doi: 10.3390/ijms19051448

    Figure Lengend Snippet: BRAF , pERKS, ERKS, phospho-AKT Ser473, AKT, phospho-S6 Ser235/236, and S6 expression in the K1 cell line after BRAF silencing. Western blot for BRAF , pERKS, ERKS, phospho-AKT Ser473, AKT, phospho-S6 Ser235/236, S6 expression and actin in K1 cell line treated with BRAF -C2 siRNA (50 nM) after 24 and 72 h. The levels of BRAF were analyzed for control of silencing efficiency and the levels of pERKS as a readout of MAPK pathway activity. Representative actin expression pattern is shown. Protein level, in scramble siRNA treated cells, was evaluated in duplicate (Scr), whereas in BRAF siRNA treated cells, it was analyzed in triplicate. The graphics depicts the mean fold change of protein expression observed in K1 cell line treated with BRAF -C2 siRNA in comparison to cells treated with scramble siRNA. Phosphorylated proteins were normalized by the levels of their correspondent total proteins, all others were normalized by the levels of control protein (actin). Results are shown as mean expression value of two independent experiments ±SEM. * p

    Article Snippet: Sections were then incubated overnight at 4 °C with anti-phospho-AKT Ser473 antibody (clone 736E11) (Cell Signaling Technology, Danvers, MA, USA) (1:50).

    Techniques: Expressing, Western Blot, Activity Assay

    RAD001 and Torin2 effect on TPC1 and K1 cell lines. ( A ) Cells were treated with 20 nM of RAD001 and 450 nM of Torin2 for 72 h. Western blot analysis of RAD001 and Torin2 effect on the activation status of mTORC1 and mTORC2 complexes was evaluated by phospho-S6 Ser235/236 and phospho-AKT Ser473 expression, respectively. Representative actin expression is shown. Protein level in treated cells was evaluated in duplicate. ( B ) Mean fold change of protein expression observed in TPC1 cell line treated with 20 nM of RAD001 and 450 nM of Torin2 in comparison to cells treated with DMSO. Phosphorylated proteins were normalized by the levels of their correspondent total proteins. Results are shown as mean expression value of three independent experiments ±SEM. * p

    Journal: International Journal of Molecular Sciences

    Article Title: mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression

    doi: 10.3390/ijms19051448

    Figure Lengend Snippet: RAD001 and Torin2 effect on TPC1 and K1 cell lines. ( A ) Cells were treated with 20 nM of RAD001 and 450 nM of Torin2 for 72 h. Western blot analysis of RAD001 and Torin2 effect on the activation status of mTORC1 and mTORC2 complexes was evaluated by phospho-S6 Ser235/236 and phospho-AKT Ser473 expression, respectively. Representative actin expression is shown. Protein level in treated cells was evaluated in duplicate. ( B ) Mean fold change of protein expression observed in TPC1 cell line treated with 20 nM of RAD001 and 450 nM of Torin2 in comparison to cells treated with DMSO. Phosphorylated proteins were normalized by the levels of their correspondent total proteins. Results are shown as mean expression value of three independent experiments ±SEM. * p

    Article Snippet: Sections were then incubated overnight at 4 °C with anti-phospho-AKT Ser473 antibody (clone 736E11) (Cell Signaling Technology, Danvers, MA, USA) (1:50).

    Techniques: Western Blot, Activation Assay, Expressing

    ( A – C ) Intensification of the immunostaining and phospho-AKT Ser473 nuclear expression in the invasive front of a classic papillary thyroid carcinoma (cPTC); (A) 0.44×, (B) 10×, and (C) 40× magnification; ( D – F ) Preferential phospho-AKT Ser473 expression in the tumor periphery, another example in a cPTC. Notice that, in this case, the nuclear translocation was not so intense compared to the previous one; (D) 0.44×, (E) 4×, and (F) 40× magnification; ( G – I ) Strong and disseminated phospho-AKT Ser473 nuclear expression in a hobnail variant of papillary thyroid carcinoma (PTC); (G) 0.44×, (H) 10×, and (I) 40× magnification. The drawn lines, at 0.44× magnification (Figure 1A,D,G), circumscribe the tumor.

    Journal: International Journal of Molecular Sciences

    Article Title: mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression

    doi: 10.3390/ijms19051448

    Figure Lengend Snippet: ( A – C ) Intensification of the immunostaining and phospho-AKT Ser473 nuclear expression in the invasive front of a classic papillary thyroid carcinoma (cPTC); (A) 0.44×, (B) 10×, and (C) 40× magnification; ( D – F ) Preferential phospho-AKT Ser473 expression in the tumor periphery, another example in a cPTC. Notice that, in this case, the nuclear translocation was not so intense compared to the previous one; (D) 0.44×, (E) 4×, and (F) 40× magnification; ( G – I ) Strong and disseminated phospho-AKT Ser473 nuclear expression in a hobnail variant of papillary thyroid carcinoma (PTC); (G) 0.44×, (H) 10×, and (I) 40× magnification. The drawn lines, at 0.44× magnification (Figure 1A,D,G), circumscribe the tumor.

    Article Snippet: Sections were then incubated overnight at 4 °C with anti-phospho-AKT Ser473 antibody (clone 736E11) (Cell Signaling Technology, Danvers, MA, USA) (1:50).

    Techniques: Immunostaining, Expressing, Translocation Assay, Variant Assay

    Total and phosphorylated Akt in WT and Akt3 −/− platelets. (A,D) Washed Akt3 −/− and WT mouse platelets were stimulated with thrombin (0.018 U/mL) for 1, 3, and 5 minutes, solubilized, and immunoblotted with antibodies directed against: (A) Akt3, total Akt (Akt1, Akt2, and Akt3), phosphorylated Ser473 of Akt, and GSK-3β (loading control), and (D) phosphorylated Thr 308 of Akt, total Akt and α-tubulin (loading control). (B,C) Western blot results from each of 3 experiments as shown in panel A were scanned and quantitated using NIH ImageJ for uncalibrated optical density. The relative quantity of total Akt (B) and phosphorylated Ser473 of Akt (C) in wild-type and Akt3 −/− platelets are shown (mean ± SE). (E) Washed Akt3 −/− and WT mouse platelets were solubilized and immunoblotted with antibodies directed against Akt1, Akt2, and Akt3 and α-tubulin (loading control).

    Journal: Blood

    Article Title: An important role for Akt3 in platelet activation and thrombosis

    doi: 10.1182/blood-2010-12-323204

    Figure Lengend Snippet: Total and phosphorylated Akt in WT and Akt3 −/− platelets. (A,D) Washed Akt3 −/− and WT mouse platelets were stimulated with thrombin (0.018 U/mL) for 1, 3, and 5 minutes, solubilized, and immunoblotted with antibodies directed against: (A) Akt3, total Akt (Akt1, Akt2, and Akt3), phosphorylated Ser473 of Akt, and GSK-3β (loading control), and (D) phosphorylated Thr 308 of Akt, total Akt and α-tubulin (loading control). (B,C) Western blot results from each of 3 experiments as shown in panel A were scanned and quantitated using NIH ImageJ for uncalibrated optical density. The relative quantity of total Akt (B) and phosphorylated Ser473 of Akt (C) in wild-type and Akt3 −/− platelets are shown (mean ± SE). (E) Washed Akt3 −/− and WT mouse platelets were solubilized and immunoblotted with antibodies directed against Akt1, Akt2, and Akt3 and α-tubulin (loading control).

    Article Snippet: The membranes were immunoblotted with an anti-Akt3 rabbit monoclonal antibody, anti–GSK-3β, anti–phosphoGSK-3β, anti-phosphoAkt Thr308 , anti-phospho Akt Ser473, anti-Akt1, anti-Akt2 (Cell Signaling Technology), and total Akt (recognizing Akt1, Akt2 and Akt3; Santa Cruz Biotechnology Inc).

    Techniques: Western Blot

    Shc and STAT3 Signaling Pathways Are Constitutively Activated in Cells Expressing the Mutant EGFR (A) Cells expressing the wild-type or L858R mutant EGFR were placed in 0.5% calf serum for 24 h and left unstimulated or stimulated with 20 ng/ml EGF (“+EGF”) for 8 min. EGFR was immunoprecipitated from 200 μg of cell lysate, and eluted immune complexes were separated by SDS-PAGE and immunoblotted with anti-Shc. Shc constitutively coimmunoprecipitates with the L858R EGFR but not the wild-type EGFR. IP, immunoprecipitation; NL, no-lysate control immunoprecipitation. (B) Anti-phospho-Shc immunoblots (upper row of blots) and anti-Shc immunoblots (lower row) of whole cell lysates from the experiment in (A). All three Shc isoforms are constitutively phosphorylated in cells expressing the L858R EGFR . (C) Immunoblots of whole cell lysates with anti-phospho-STAT3 Y705 (upper row), total STAT3 (middle row), or actin as a loading control (lower row). STAT3 is constitutively phosphorylated in cells expressing any of the four mutant EGFR proteins, representative of the four classes of EGFR mutations observed in lung adenocarcinoma tumor DNA. del, L747_E749del A750P; ins, D770_N771insNPG; pBp, pBabe-Puro vector control; wt, wild-type EGFR. (D) M67 STAT3 luciferase reporter assay. NIH-3T3 cells expressing the indicated EGFR were transfected with a STAT-dependent reporter (m67-firefly luciferase) [ 25 ] and a control reporter expressing Renilla luciferase. STAT-dependent luciferase production was measured after 48 h and normalized to Renilla luciferase. The normalized luciferase values were divided by the values for cells expressing the wild-type EGFR to produce relative luciferase units. NIH-3T3 expressing mutant forms of EGFR exhibited elevated levels of STAT-dependent transcriptional activity relative to wild-type. ins, D770_N771insNPG EGFR; wt, wild-type EGFR. (E) Immunoblots of whole cell lysates with anti-phospho-Akt S473 (upper row), total Akt (middle row), or actin as a loading control (lower row). Akt is constitutively phosphorylated in cells expressing mutant EGFR. del, L747_E749del A750P; ins, D770_N771insNPG; wt, wild-type EGFR.

    Journal: PLoS Medicine

    Article Title: Oncogenic Transformation by Inhibitor-Sensitive and -Resistant EGFR MutantsEGFR Mutations and Lung CancerInhibition of EGFR Signaling: All Mutations are not Created Equal

    doi: 10.1371/journal.pmed.0020313

    Figure Lengend Snippet: Shc and STAT3 Signaling Pathways Are Constitutively Activated in Cells Expressing the Mutant EGFR (A) Cells expressing the wild-type or L858R mutant EGFR were placed in 0.5% calf serum for 24 h and left unstimulated or stimulated with 20 ng/ml EGF (“+EGF”) for 8 min. EGFR was immunoprecipitated from 200 μg of cell lysate, and eluted immune complexes were separated by SDS-PAGE and immunoblotted with anti-Shc. Shc constitutively coimmunoprecipitates with the L858R EGFR but not the wild-type EGFR. IP, immunoprecipitation; NL, no-lysate control immunoprecipitation. (B) Anti-phospho-Shc immunoblots (upper row of blots) and anti-Shc immunoblots (lower row) of whole cell lysates from the experiment in (A). All three Shc isoforms are constitutively phosphorylated in cells expressing the L858R EGFR . (C) Immunoblots of whole cell lysates with anti-phospho-STAT3 Y705 (upper row), total STAT3 (middle row), or actin as a loading control (lower row). STAT3 is constitutively phosphorylated in cells expressing any of the four mutant EGFR proteins, representative of the four classes of EGFR mutations observed in lung adenocarcinoma tumor DNA. del, L747_E749del A750P; ins, D770_N771insNPG; pBp, pBabe-Puro vector control; wt, wild-type EGFR. (D) M67 STAT3 luciferase reporter assay. NIH-3T3 cells expressing the indicated EGFR were transfected with a STAT-dependent reporter (m67-firefly luciferase) [ 25 ] and a control reporter expressing Renilla luciferase. STAT-dependent luciferase production was measured after 48 h and normalized to Renilla luciferase. The normalized luciferase values were divided by the values for cells expressing the wild-type EGFR to produce relative luciferase units. NIH-3T3 expressing mutant forms of EGFR exhibited elevated levels of STAT-dependent transcriptional activity relative to wild-type. ins, D770_N771insNPG EGFR; wt, wild-type EGFR. (E) Immunoblots of whole cell lysates with anti-phospho-Akt S473 (upper row), total Akt (middle row), or actin as a loading control (lower row). Akt is constitutively phosphorylated in cells expressing mutant EGFR. del, L747_E749del A750P; ins, D770_N771insNPG; wt, wild-type EGFR.

    Article Snippet: Antibodies used for immunoblotting were: anti-EGFR (#2232, Cell Signaling Technologies), anti-phospho-EGFR Y1173 (#05–483, Upstate), anti-phospho-EGFR Y1068 (#2234, Cell Signaling Technologies), anti-phospho-EGFR Y845 (Cell Signaling Technologies, Beverly, Massachusetts, United States; #2231), anti-phospho-EGFR Y1045 (Cell Signaling Technologies; #2237), anti-actin (Santa Cruz Biotechnology, Santa Cruz, California, United States; #sc-1615), anti-Shc (Upstate; #06–203), anti-phospho-Shc Y317 (Upstate; #05–668), anti-Stat3 (Cell Signaling Technologies; #9132), and anti-phospho-Stat3 Y705 (Cell Signaling Technologies; #9131), anti-phospho-Akt S473 (Cell Signaling Technologies; #9271), and anti-Akt (Cell Signaling Technologies; #9272).

    Techniques: Expressing, Mutagenesis, Immunoprecipitation, SDS Page, Western Blot, Plasmid Preparation, Luciferase, Reporter Assay, Transfection, Activity Assay

    Validation of up-regulated genes from genes array analysis of genes differently expressed in cisplatin resistant UMSCC cells via western blot and tissue array. a RNAs from UMSCC 14A, 14B and 17A, 17B cells were purified and requested for the gene array analysis. Up-regulated genes in both UMSCC 14B and 17B cells (associated with drug resistant) were listed. b Whole-cell lysate samples from UMSCC 14A, 14B and 17A, 17B cells were used for western blot and probed with antibodies for phosphorylated AKT, BAG-1, and BCL-xL. Each membrane was stripped and re-probed with GAPDH for loading control. c Immunohistochemistry staining of phosphorylated AKT, BAG-1, and BCL-xL on HNSCC tissue array. The imagines show representative staining patterns of AKT, BAG-1, and BCL-xL, in the primary HNSCC (Case 1) and metastatic HNSCC (Case 2), the imagines of HE staining were downloaded from online data of IMGENEX

    Journal: Journal of Translational Medicine

    Article Title: Over-expression of BAG-1 in head and neck squamous cell carcinomas (HNSCC) is associated with cisplatin-resistance

    doi: 10.1186/s12967-017-1289-2

    Figure Lengend Snippet: Validation of up-regulated genes from genes array analysis of genes differently expressed in cisplatin resistant UMSCC cells via western blot and tissue array. a RNAs from UMSCC 14A, 14B and 17A, 17B cells were purified and requested for the gene array analysis. Up-regulated genes in both UMSCC 14B and 17B cells (associated with drug resistant) were listed. b Whole-cell lysate samples from UMSCC 14A, 14B and 17A, 17B cells were used for western blot and probed with antibodies for phosphorylated AKT, BAG-1, and BCL-xL. Each membrane was stripped and re-probed with GAPDH for loading control. c Immunohistochemistry staining of phosphorylated AKT, BAG-1, and BCL-xL on HNSCC tissue array. The imagines show representative staining patterns of AKT, BAG-1, and BCL-xL, in the primary HNSCC (Case 1) and metastatic HNSCC (Case 2), the imagines of HE staining were downloaded from online data of IMGENEX

    Article Snippet: The membranes were blocked for 1 h at room temperature with 5% bovine serum albumin in 0.1% Tween 20 in Tris-buffered saline and incubated overnight at 4 °C with anti-BAG-1 (sc-939 1:500), anti-BCL-xL (sc-7195 1:1000), anti-BCL-2 (sc-7382, 1:1000), anti-Akt (Cell Signaling; 1:1000), anti-phosphorylated Akt (Ser473; Cell Signaling; 1:1000), anti-mitogen–activated protein kinase (MAPK, Cell signaling, 1:1000) in 5% non-fat milk in 0.1% Tween 20 tris-buffered saline.

    Techniques: Western Blot, Purification, Immunohistochemistry, Staining

    Sustained high expression of BAG-1, and its associated proteins in response to cisplatin resistance. Whole cell lysates of UMSCC 14A, 14B and 17A, 17B cells were treated with indicated concentration of cisplatin or absence for 24, for western blot analysis. a Membranes were probed with antibodies for BAG-1, BCL-xL, BCL-2, BID, and phosphor γH2AX. b Whole cell lysates were also probed for pro-survival pathway, PI3K/AKT, Jak/STAT3, and MAKP/ERK. Each membrane was stripped and re-probed with GAPDH for loading control

    Journal: Journal of Translational Medicine

    Article Title: Over-expression of BAG-1 in head and neck squamous cell carcinomas (HNSCC) is associated with cisplatin-resistance

    doi: 10.1186/s12967-017-1289-2

    Figure Lengend Snippet: Sustained high expression of BAG-1, and its associated proteins in response to cisplatin resistance. Whole cell lysates of UMSCC 14A, 14B and 17A, 17B cells were treated with indicated concentration of cisplatin or absence for 24, for western blot analysis. a Membranes were probed with antibodies for BAG-1, BCL-xL, BCL-2, BID, and phosphor γH2AX. b Whole cell lysates were also probed for pro-survival pathway, PI3K/AKT, Jak/STAT3, and MAKP/ERK. Each membrane was stripped and re-probed with GAPDH for loading control

    Article Snippet: The membranes were blocked for 1 h at room temperature with 5% bovine serum albumin in 0.1% Tween 20 in Tris-buffered saline and incubated overnight at 4 °C with anti-BAG-1 (sc-939 1:500), anti-BCL-xL (sc-7195 1:1000), anti-BCL-2 (sc-7382, 1:1000), anti-Akt (Cell Signaling; 1:1000), anti-phosphorylated Akt (Ser473; Cell Signaling; 1:1000), anti-mitogen–activated protein kinase (MAPK, Cell signaling, 1:1000) in 5% non-fat milk in 0.1% Tween 20 tris-buffered saline.

    Techniques: Expressing, Concentration Assay, Western Blot

    CTAB-coated GNRs induced autophagy in cells. Cells were treated with 0.25–2 nM CTAB-coated GNRs for 24 h. The autophagy of HCT116 cells induced by CTAB-coated GNRs was Akt independent. ( a ) Western blot analysis of LC3 protein levels in cancer cell lines as well as in immortalized nonmalignant cell lines. Cropping lines are used in the figure. Full-length blots are presented in Supplementary Figure 2 . The gels have been run under the same experimental conditions. ( b ) Representative GFP-LC3 fluorescent punctate dot images in HCT116. ( c ) Detection of acidic vesicular organelles with acridine orange staining. ( d ) Western blot analysis of Akt in cells treated with 0.25–2 nM CTAB-coated GNRs for 24 h. Cropping lines are used in the figure. Full-length blots are presented in Supplementary Figure 2 . The gels have been run under the same experimental conditions. ( e ) Cells transfected with vehicle plasmid (pUSE) or constitutively active Akt (CA-Akt) were incubated with or without 2 nM CTAB-coated GNRs for 24 h and then analyzed for LC3 and Akt by Western blot. Cropping lines are used in the figure. Full-length blots are presented in Supplementary Figure 2 . The gels have been run under the same experimental conditions.

    Journal: Scientific Reports

    Article Title: Surface chemistry but not aspect ratio mediates the biological toxicity of gold nanorods in vitro and in vivo

    doi: 10.1038/srep11398

    Figure Lengend Snippet: CTAB-coated GNRs induced autophagy in cells. Cells were treated with 0.25–2 nM CTAB-coated GNRs for 24 h. The autophagy of HCT116 cells induced by CTAB-coated GNRs was Akt independent. ( a ) Western blot analysis of LC3 protein levels in cancer cell lines as well as in immortalized nonmalignant cell lines. Cropping lines are used in the figure. Full-length blots are presented in Supplementary Figure 2 . The gels have been run under the same experimental conditions. ( b ) Representative GFP-LC3 fluorescent punctate dot images in HCT116. ( c ) Detection of acidic vesicular organelles with acridine orange staining. ( d ) Western blot analysis of Akt in cells treated with 0.25–2 nM CTAB-coated GNRs for 24 h. Cropping lines are used in the figure. Full-length blots are presented in Supplementary Figure 2 . The gels have been run under the same experimental conditions. ( e ) Cells transfected with vehicle plasmid (pUSE) or constitutively active Akt (CA-Akt) were incubated with or without 2 nM CTAB-coated GNRs for 24 h and then analyzed for LC3 and Akt by Western blot. Cropping lines are used in the figure. Full-length blots are presented in Supplementary Figure 2 . The gels have been run under the same experimental conditions.

    Article Snippet: Rhodamine 123 (Rh123), cyclosporin A (CsA), acridine orange, GAPDH antibody and HRP-conjugated secondary antibodies (goat anti-rabbit and goat anti-mouse) were purchased from Beyotime (Nantong, China).The antibody against microtubule-associated protein 1 light chain 3 (LC3) was purchased from Sigma, and antibodies against caspase-9, PARP, total-Akt, phospho-Akt (Ser473), total MEK, phospho-MEK, total-ERK, phospho-ERK (Thr202/Tyr204), total-p38, phospho-p38 (Thr180/Tyr182), mTOR, phospho-p70s6k, and phospho-rps6 were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Western Blot, Staining, Transfection, Plasmid Preparation, Incubation

    Differential effects of Akt inhibition at low and high concentrations of isoproterenol. (A) Floxed Akt2 fibroblasts stably expressing PPARγ were infected with either Adeno-GFP or Adeno-Cre and then differentiated into adipocytes. These cells were serum starved and treated with 1 nM insulin and analyzed by immunoblotting using Licor Odyssey for the excision of Akt2 and the loss of phospho-Akt Ser473 signal (left panel; representative of two experiments). The quantification of immunoblot analysis of phospho-Akt Ser473 of Akt2 lox/lox adipocytes infected with Ad-GFP or Ad-Cre is shown (middle panel). A glycerol release assay (right panel) was performed with increasing doses of isoproterenol in the presence and absence of 1 nM insulin for 2 h. Data are expressed as means ± standard deviations (SD) from two experiments performed in duplicate. (B) RNA inteference-mediated reduction in Akt2 does not affect insulin inhibition of glycerol release. 3T3-L1 preadipocytes were infected with an shRNA lentiviral construct that targets Akt2 and sorted for the low and high expression of GFP. Adipocytes were treated with the indicated concentrations of insulin and subjected to the immunoblot analysis of Akt2 and phospho-Akt Thr308, confirming the efficiency of knockdown (left). Highly expressing cells were differentiated into adipocytes, and a glycerol release assay was performed using 2 nM isoproterenol with increasing doses of insulin (upper right). Data are expressed as means ± SD from two experiments performed in duplicate. A glucose uptake assay also was performed on differentiated control, highly, and lowly expressing cells (bottom right).

    Journal: Molecular and Cellular Biology

    Article Title: Insulin Regulates Adipocyte Lipolysis via an Akt-Independent Signaling Pathway ▿

    doi: 10.1128/MCB.00797-10

    Figure Lengend Snippet: Differential effects of Akt inhibition at low and high concentrations of isoproterenol. (A) Floxed Akt2 fibroblasts stably expressing PPARγ were infected with either Adeno-GFP or Adeno-Cre and then differentiated into adipocytes. These cells were serum starved and treated with 1 nM insulin and analyzed by immunoblotting using Licor Odyssey for the excision of Akt2 and the loss of phospho-Akt Ser473 signal (left panel; representative of two experiments). The quantification of immunoblot analysis of phospho-Akt Ser473 of Akt2 lox/lox adipocytes infected with Ad-GFP or Ad-Cre is shown (middle panel). A glycerol release assay (right panel) was performed with increasing doses of isoproterenol in the presence and absence of 1 nM insulin for 2 h. Data are expressed as means ± standard deviations (SD) from two experiments performed in duplicate. (B) RNA inteference-mediated reduction in Akt2 does not affect insulin inhibition of glycerol release. 3T3-L1 preadipocytes were infected with an shRNA lentiviral construct that targets Akt2 and sorted for the low and high expression of GFP. Adipocytes were treated with the indicated concentrations of insulin and subjected to the immunoblot analysis of Akt2 and phospho-Akt Thr308, confirming the efficiency of knockdown (left). Highly expressing cells were differentiated into adipocytes, and a glycerol release assay was performed using 2 nM isoproterenol with increasing doses of insulin (upper right). Data are expressed as means ± SD from two experiments performed in duplicate. A glucose uptake assay also was performed on differentiated control, highly, and lowly expressing cells (bottom right).

    Article Snippet: The pan-Akt, Akt1, phospho-HSL Ser660, phospho-Akt Thr308 and Ser473, phospho-PKA substrate, phospho-Akt substrate, and PKA-C antibodies were obtained from Cell Signaling Technology (Beverly, MA).

    Techniques: Inhibition, Stable Transfection, Expressing, Infection, Glucose Assay, shRNA, Construct

    Critical role of Akt in cardiomyocyte protection and Akt down-regulation by Akt siRNA at the protein level. ( A ) Critical role of Akt in cardiomyocyte protection. Small interfering RNA targeting Akt1 or ERK1, or negative control (scrambled) siRNA was transfected

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

    Article Title: Cardioprotection by a nonerythropoietic, tissue-protective peptide mimicking the 3D structure of erythropoietin

    doi: 10.1073/pnas.1003019107

    Figure Lengend Snippet: Critical role of Akt in cardiomyocyte protection and Akt down-regulation by Akt siRNA at the protein level. ( A ) Critical role of Akt in cardiomyocyte protection. Small interfering RNA targeting Akt1 or ERK1, or negative control (scrambled) siRNA was transfected

    Article Snippet: The blots were incubated overnight at 4 °C or for 4 h at room temperature with a primary antibody against phospho-Akt, phospho-ERK1/2 or phospho-STAT3, followed by incubation for 1 h with a secondary, horseradish peroxidase-conjugated antibody.

    Techniques: Small Interfering RNA, Negative Control, Transfection

    Potential mechanism of action of soy peptides in HepG2 cells. Upon cell penetration, they act as competitive inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoAR) leading to an intracellular cholesterol synthesis reduction. The consequence is the activation of the transcription factor sterol regulatory element-binding proteins (SREBP) 2, which leads to low density lipoprotein receptor (LDLR) and HMGCoAR genes transcription, with subsequent increase of LDLR and HMGCoAR protein levels and LDLR localization in plasma membrane ( a ); In parallel, soy peptides reduce cholesterol production also by activation of the monophosphate-activated protein kinase (AMPK) pathway through an increase of phosphorylation at Thr-172, which in turn inactivates its target substrate HMGCoAR through phosphorylation at serine-872 and produces an increase of the activity of glucose transporters ( b ); IAVPGEVA, IAVPTGVA and LPYP enhance Akt activation, through an increase of phosphorylation at Ser473, which in turn inactivates glycogen synthase kinase 3 αβ (GSK3αβ), a kinase that contributes to proteasomal degradation of SREBP2 [ 10 , 28 , 29 ]. Moreover, GSK3αβ, inactivated by Akt phosphorylation at Ser21/9, does not inhibit glycogen synthase (its substrate) that can convert the increased up-taken glucose in intracellular glycogen ( c ); Finally, extracellular-signal-regulated kinases (ERK) 1/2 pathway activation [ 10 ] could lead to stabilization of mRNA levels of LDLR contributing to increase LDLR protein levels in plasma membrane ( d ). Thus, the distinct modulation of four pathways leads to an increased LDLR activity, which can bind and carry extracellular LDL in HepG2 cells with final hypocholesterolemic effects. Moreover, Akt and AMPK pathway activations are correlated with glucose metabolism modulation, which leads to an increase of glucose-uptake by human hepatic cells.

    Journal: International Journal of Molecular Sciences

    Article Title: Three Peptides from Soy Glycinin Modulate Glucose Metabolism in Human Hepatic HepG2 Cells

    doi: 10.3390/ijms161126029

    Figure Lengend Snippet: Potential mechanism of action of soy peptides in HepG2 cells. Upon cell penetration, they act as competitive inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoAR) leading to an intracellular cholesterol synthesis reduction. The consequence is the activation of the transcription factor sterol regulatory element-binding proteins (SREBP) 2, which leads to low density lipoprotein receptor (LDLR) and HMGCoAR genes transcription, with subsequent increase of LDLR and HMGCoAR protein levels and LDLR localization in plasma membrane ( a ); In parallel, soy peptides reduce cholesterol production also by activation of the monophosphate-activated protein kinase (AMPK) pathway through an increase of phosphorylation at Thr-172, which in turn inactivates its target substrate HMGCoAR through phosphorylation at serine-872 and produces an increase of the activity of glucose transporters ( b ); IAVPGEVA, IAVPTGVA and LPYP enhance Akt activation, through an increase of phosphorylation at Ser473, which in turn inactivates glycogen synthase kinase 3 αβ (GSK3αβ), a kinase that contributes to proteasomal degradation of SREBP2 [ 10 , 28 , 29 ]. Moreover, GSK3αβ, inactivated by Akt phosphorylation at Ser21/9, does not inhibit glycogen synthase (its substrate) that can convert the increased up-taken glucose in intracellular glycogen ( c ); Finally, extracellular-signal-regulated kinases (ERK) 1/2 pathway activation [ 10 ] could lead to stabilization of mRNA levels of LDLR contributing to increase LDLR protein levels in plasma membrane ( d ). Thus, the distinct modulation of four pathways leads to an increased LDLR activity, which can bind and carry extracellular LDL in HepG2 cells with final hypocholesterolemic effects. Moreover, Akt and AMPK pathway activations are correlated with glucose metabolism modulation, which leads to an increase of glucose-uptake by human hepatic cells.

    Article Snippet: The antibodies against phospho-Akt (Ser473) and GSK3 α/β (Ser21/9) were purchased from Cell Signaling Technologies (Danvers, MA, USA); whereas the antibodies against GLUT1 and GLUT4 were purchased from GeneTex (Irvine, CA, USA).

    Techniques: Activated Clotting Time Assay, Activation Assay, Binding Assay, Activity Assay

    Arsenic growth inhibition correlates with decreased temsirolimus-induced phospho-AKT. MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10 -6 N NaOH and 2.5 x 10 -5 % EtOH in PBS), 1-2 µM ATO, 0.5-5 ng/ml temsirolimus and the combinations for 24 hours. Whole cell extracts were used in immunoblotting experiments for phospho-S473-AKT, phospho-T308-AKT, AKT, and β-actin. Immunoblots shown are representative of experiments performed at least 3 times.

    Journal: PLoS ONE

    Article Title: Arsenic Trioxide Overcomes Rapamycin-Induced Feedback Activation of AKT and ERK Signaling to Enhance the Anti-Tumor Effects in Breast Cancer

    doi: 10.1371/journal.pone.0085995

    Figure Lengend Snippet: Arsenic growth inhibition correlates with decreased temsirolimus-induced phospho-AKT. MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10 -6 N NaOH and 2.5 x 10 -5 % EtOH in PBS), 1-2 µM ATO, 0.5-5 ng/ml temsirolimus and the combinations for 24 hours. Whole cell extracts were used in immunoblotting experiments for phospho-S473-AKT, phospho-T308-AKT, AKT, and β-actin. Immunoblots shown are representative of experiments performed at least 3 times.

    Article Snippet: Membranes were incubated overnight with primary antibodies: phospho-AKT (S473) (1:500, Cell Signaling), phospho-AKT (T308) (1:750, Cell Signaling), AKT (1:750 Cell Signaling), phospho-p44/42 MAPK (T202/Y204) (1:1000, Cell Signaling), ERK2 (1:2000, SantaCruz), phospho-S6 (1:1500, Cell Signaling), S6 (1:1000, Cell Signaling), and β–actin (1:5000, Sigma).

    Techniques: Inhibition, Multiple Displacement Amplification, Western Blot

    Addition of ATO decreases rapamycin-induced AKT activation in vivo . Tumor samples were stained with antibody against phospho-S473-AKT. Representative pictures of each treatment are shown (A). Quantification of staining intensity was performed using algorithms provided with the ImageScope software (B). Individual animals are represented with mean and standard error bars (n=4-5 mice). (C-D) Tumors at the end of the experiment were analyzed in immunoblotting experiments with antibodies against phospho-S473-AKT (C), phospho-T308-AKT (D), AKT, and β-actin. Band densitometry was performed and the relative intensity of phospho-AKT/AKT/β-actin was calculated. Individual animals are represented with mean and standard error bars (n= 8-9 mice).

    Journal: PLoS ONE

    Article Title: Arsenic Trioxide Overcomes Rapamycin-Induced Feedback Activation of AKT and ERK Signaling to Enhance the Anti-Tumor Effects in Breast Cancer

    doi: 10.1371/journal.pone.0085995

    Figure Lengend Snippet: Addition of ATO decreases rapamycin-induced AKT activation in vivo . Tumor samples were stained with antibody against phospho-S473-AKT. Representative pictures of each treatment are shown (A). Quantification of staining intensity was performed using algorithms provided with the ImageScope software (B). Individual animals are represented with mean and standard error bars (n=4-5 mice). (C-D) Tumors at the end of the experiment were analyzed in immunoblotting experiments with antibodies against phospho-S473-AKT (C), phospho-T308-AKT (D), AKT, and β-actin. Band densitometry was performed and the relative intensity of phospho-AKT/AKT/β-actin was calculated. Individual animals are represented with mean and standard error bars (n= 8-9 mice).

    Article Snippet: Membranes were incubated overnight with primary antibodies: phospho-AKT (S473) (1:500, Cell Signaling), phospho-AKT (T308) (1:750, Cell Signaling), AKT (1:750 Cell Signaling), phospho-p44/42 MAPK (T202/Y204) (1:1000, Cell Signaling), ERK2 (1:2000, SantaCruz), phospho-S6 (1:1500, Cell Signaling), S6 (1:1000, Cell Signaling), and β–actin (1:5000, Sigma).

    Techniques: Activation Assay, In Vivo, Staining, Software, Mouse Assay

    Immunofluorescent detection of Ki-67 ( panels a, b ), phospho-PDGFR-β ( panels c, d ), and phospho-Akt ( e,f ) in control ( a,c,e ) and injured carotid arteries ( b,d,f ) of smLrp1 −/− mice 14 days after endothelial denudation. The elastic

    Journal:

    Article Title: Smooth Muscle LDL Receptor-related Protein-1 Inactivation Reduces Vascular Reactivity and Promotes Injury-induced Neointima Formation

    doi: 10.1161/ATVBAHA.109.194357

    Figure Lengend Snippet: Immunofluorescent detection of Ki-67 ( panels a, b ), phospho-PDGFR-β ( panels c, d ), and phospho-Akt ( e,f ) in control ( a,c,e ) and injured carotid arteries ( b,d,f ) of smLrp1 −/− mice 14 days after endothelial denudation. The elastic

    Article Snippet: Cell proliferation and PDGFR signaling were assessed by incubating deparaffinized tissue sections overnight at 4°C with rabbit anti-Ki67 (Vector Laboratories), goat anti-phospho-PDGFR (Santa Cruz Biotechnology), or rabbit anti-phospho-Akt (Cell Signaling) at a 1:1000 dilution in Antibody Diluent (Zymed).

    Techniques: Mouse Assay

    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

    LS-induced upregulation of KCa3.1 and KCa2.3 expression is mediated by CaMKK-dependent Akt activation. A and B : HCAECs were exposed to LS for 24 h in the absence or presence of compound C (an AMPK inhibitor), KN-62 (a CaMK inhibitor), or Akt inhibitor IV. Compound C tended to reduce LS-induced upregulation of KCa3.1 mRNA expression ( A ) but did not change LS induction of KCa2.3 expression ( B ) ( n = 9). KN-62 had no effect on the upregulation of KCa3.1 and KCa2.3 mRNA expression ( n = 7–8). Akt inhibition completely blocked LS-induced upregulation of both KCa3.1 and KCa2.3 expression. C : LS-induced increase in KCa2.3 and KCa3.1 protein expression levels was suppressed by Akt inhibition. Top : a representative image of immunoblots. Bottom : the summaries of densitometric analysis of KCa3.1 and KCa2.3 signals ( n = 3). D : Akt inhibitor IV abolished the phosphorylation of Akt at Ser473 in HCAECs exposed to 60-min LS ( top ). Levels of phospho-Akt (Ser473) were suppressed after inhibiting CaMKK with STO-609 in ECs exposed to 15 min of LS ( bottom ). Phosphorylation of Akt at Thr308 was not observed in ECs under ST or LS condition. #Signficant difference vs. LS ( P

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Laminar shear stress upregulates endothelial Ca2+-activated K+ channels KCa2.3 and KCa3.1 via a Ca2+/calmodulin-dependent protein kinase kinase/Akt/p300 cascade

    doi: 10.1152/ajpheart.00642.2012

    Figure Lengend Snippet: LS-induced upregulation of KCa3.1 and KCa2.3 expression is mediated by CaMKK-dependent Akt activation. A and B : HCAECs were exposed to LS for 24 h in the absence or presence of compound C (an AMPK inhibitor), KN-62 (a CaMK inhibitor), or Akt inhibitor IV. Compound C tended to reduce LS-induced upregulation of KCa3.1 mRNA expression ( A ) but did not change LS induction of KCa2.3 expression ( B ) ( n = 9). KN-62 had no effect on the upregulation of KCa3.1 and KCa2.3 mRNA expression ( n = 7–8). Akt inhibition completely blocked LS-induced upregulation of both KCa3.1 and KCa2.3 expression. C : LS-induced increase in KCa2.3 and KCa3.1 protein expression levels was suppressed by Akt inhibition. Top : a representative image of immunoblots. Bottom : the summaries of densitometric analysis of KCa3.1 and KCa2.3 signals ( n = 3). D : Akt inhibitor IV abolished the phosphorylation of Akt at Ser473 in HCAECs exposed to 60-min LS ( top ). Levels of phospho-Akt (Ser473) were suppressed after inhibiting CaMKK with STO-609 in ECs exposed to 15 min of LS ( bottom ). Phosphorylation of Akt at Thr308 was not observed in ECs under ST or LS condition. #Signficant difference vs. LS ( P

    Article Snippet: The primary antibodies used were KCa2.3 (Santa Cruz), KCa3.1 (obtained from rabbit sera) ( ); phospho-Akt (Thr308), phospho-Akt (Ser473), and total Akt (Cell Signaling); phospho-p300 (Ser1834) and total p300 (Thermo Scientific); phospho-CREB (Ser133) and total CREB (Cell Signaling); and β-actin (Santa Cruz).

    Techniques: Expressing, Activation Assay, Inhibition, Western Blot

    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

    Journal: Molecular Cancer

    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

    doi: 10.1186/1476-4598-7-88

    Figure Lengend 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

    Article Snippet: Proteins were transferred onto nitrocellulose membranes and blotted with rabbit polyclonal anti-PI-3K p85 (Upstate) (1:1000 dilution), rabbit polyclonal anti-phospho-Akt Ser473, anti-phospho-Akt Thr308 or anti-Akt (total) (Cell Signaling) (1:500 dilution), rabbit polyclonal anti-Fas-L (Q20, Santa Cruz) (1:200 dilution), phospho- and total Bad antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000 dilution).

    Techniques: De-Phosphorylation Assay, Western Blot

    Effect of MEK inhibitor on ERK1/2, Akt and NF-κB activation. Cells were administrated with ( A , B ) trametinib or ( C , D ) PD0325901 for 3 days. Control cells (0 μM) were administrated with 0.5% dimethyl sulfoxide (DMSO) for 3 days. ( A , C ) Cell lysates were examined by western blotting assay using indicated antibodies. ( B , D ) Quantification of phosphorylated protein expression, normalized corresponding protein, respectively. The results showed the 5 independent experiments. * p

    Journal: Cancers

    Article Title: Overactivation of Akt Contributes to MEK Inhibitor Primary and Acquired Resistance in Colorectal Cancer Cells

    doi: 10.3390/cancers11121866

    Figure Lengend Snippet: Effect of MEK inhibitor on ERK1/2, Akt and NF-κB activation. Cells were administrated with ( A , B ) trametinib or ( C , D ) PD0325901 for 3 days. Control cells (0 μM) were administrated with 0.5% dimethyl sulfoxide (DMSO) for 3 days. ( A , C ) Cell lysates were examined by western blotting assay using indicated antibodies. ( B , D ) Quantification of phosphorylated protein expression, normalized corresponding protein, respectively. The results showed the 5 independent experiments. * p

    Article Snippet: The membranes were reacted with following primary antibodies according to the manufacturer’s instruction: anti-phospho-p44/42 MAPK (ERK1/2) antibody, anti-p44/42 MAPK (ERK1/2) antibody, anti-phospho-Akt antibody, anti-Akt antibody, anti-phospho-STAT3 antibody, anti-STAT3 antibody, anti-phospho-p38MAPK antibody, anti-p38MAPK antibody, anti-phospho-NF-κB antibody, anti- NF-κB antibody (Cell Signaling Technology, Beverly, MA, USA); anti-Bax antibody, anti-Bim antibody, anti-Bcl-xL antibody, anti-Bcl-2 antibody (Santa Cruz Biotechnologies, CA, USA); and anti-β-actin antibody (Sigma).

    Techniques: Activation Assay, Western Blot, Expressing

    hMena is differently distributed among breast cancer subtypes and correlates with HER2 overexpression, MAPK, AKT activity and Ki67. A. Relationship between hMena staining and HER2 positivity in human breast carcinomas. Histograms represent the percentage of the hMena staining (score 0; 1; 2; 3) in 82 HER2 positive breast cancer samples (2+ with gene amplification and 3+) (p = 0.02). B. hMena distribution within 286 human breast carcinomas grouped according to molecular subtypes. hMena expression (0, 1, 2, 3 scores) in Luminal A, Luminal B, HER2 subtypes and Triple Negative. The percentage of hMena-positive cases (score 3) is significantly higher in the HER2 subtype and Triple-Negative in comparison to Luminal A and Luminal B breast cancer (p = 0.032). C. Correlation between hMena expression and HER2, P-AKT, P-MAPK and Ki67 in a subgroup of 178 breast cancer patients. Percentage of HER2 positivity, high Ki67 proliferation index, elevated P-AKT and P-MAPK expression for each hMena score. The percentage of HER2 positivity (p = 0.016), P-AKT (p

    Journal: PLoS ONE

    Article Title: The Cooperation between hMena Overexpression and HER2 Signalling in Breast Cancer

    doi: 10.1371/journal.pone.0015852

    Figure Lengend Snippet: hMena is differently distributed among breast cancer subtypes and correlates with HER2 overexpression, MAPK, AKT activity and Ki67. A. Relationship between hMena staining and HER2 positivity in human breast carcinomas. Histograms represent the percentage of the hMena staining (score 0; 1; 2; 3) in 82 HER2 positive breast cancer samples (2+ with gene amplification and 3+) (p = 0.02). B. hMena distribution within 286 human breast carcinomas grouped according to molecular subtypes. hMena expression (0, 1, 2, 3 scores) in Luminal A, Luminal B, HER2 subtypes and Triple Negative. The percentage of hMena-positive cases (score 3) is significantly higher in the HER2 subtype and Triple-Negative in comparison to Luminal A and Luminal B breast cancer (p = 0.032). C. Correlation between hMena expression and HER2, P-AKT, P-MAPK and Ki67 in a subgroup of 178 breast cancer patients. Percentage of HER2 positivity, high Ki67 proliferation index, elevated P-AKT and P-MAPK expression for each hMena score. The percentage of HER2 positivity (p = 0.016), P-AKT (p

    Article Snippet: Anti estrogen (6F11 MoAb) and progesterone (1A6 MoAb) receptors were purchased from Novocastra (Menarini, Florence, Italy); HER-2 (A0485 pAb) and Ki67 (MIB-1 MoAb) antibodies from DAKO (Milan, Italy); phospho-p44/42 MAPK and phospho-AKT antibodies from Cell Signaling (SIAL, Rome, Italy).

    Techniques: Over Expression, Activity Assay, Staining, Amplification, Expressing

    hMena overexpression correlates with the phosphorylated status of MAPK and AKT. A. The table evidences that in 60 HER2 positive and 118 HER2 negative breast tumors the percentage of P-MAPK and P-AKT positive breast tumors is significantly higher in hMena positive (score 2/3) than in hMena negative breast tumors (score 0/1). B. A representative, although not a paradigmatic case, where P-MAPK displays a heterogeneous immunostaining (40% tumor cells). P-MAPK positivity is mostly confined in the tumor area (positive area) concomitantly showing a strong hMena (score 3) immunoreactivity.

    Journal: PLoS ONE

    Article Title: The Cooperation between hMena Overexpression and HER2 Signalling in Breast Cancer

    doi: 10.1371/journal.pone.0015852

    Figure Lengend Snippet: hMena overexpression correlates with the phosphorylated status of MAPK and AKT. A. The table evidences that in 60 HER2 positive and 118 HER2 negative breast tumors the percentage of P-MAPK and P-AKT positive breast tumors is significantly higher in hMena positive (score 2/3) than in hMena negative breast tumors (score 0/1). B. A representative, although not a paradigmatic case, where P-MAPK displays a heterogeneous immunostaining (40% tumor cells). P-MAPK positivity is mostly confined in the tumor area (positive area) concomitantly showing a strong hMena (score 3) immunoreactivity.

    Article Snippet: Anti estrogen (6F11 MoAb) and progesterone (1A6 MoAb) receptors were purchased from Novocastra (Menarini, Florence, Italy); HER-2 (A0485 pAb) and Ki67 (MIB-1 MoAb) antibodies from DAKO (Milan, Italy); phospho-p44/42 MAPK and phospho-AKT antibodies from Cell Signaling (SIAL, Rome, Italy).

    Techniques: Over Expression, Immunostaining

    hMena knock-down affects HER2 signalling and inhibits the EGF/NRG1-mediated mitogenic effects in MCF7-HER2 cells. A. Western blot analysis of MCF7-HER2 breast cancer cell line after 72 h transfection with control and hMena/hMena 11a -specific siRNAs, untreated or treated with EGF (100 ng/ml) or NRG1 (10 ng/ml) for the last 24 h of transfection. hMena/hMena 11a expression (evaluated by pan-hMena and hMena 11a specific antibodies) and phosphorylation status of HER2, EGFR, HER3, AKT and MAPK (p44/42) from whole cell lysates were assessed. Membranes were sequentially stripped and reprobed with the indicated total and phospho-specific antibodies. Densitometric quantitation of anti-P-HER2, anti-P-AKT and anti-P-MAPK immunoreactivity was determined by Quantity One software (Biorad) and normalized in comparison with the Actin immunoreactivity. Densitometric quantitation of anti-P-HER3 immunoreactivity was not determined due to the fact that the total HER3 expression level is not unchanged following treatments. B–C. Silencing of hMena/hMena 11a reduces EGF and NRG1-mediated cell proliferation of HER2 overexpressing MCF7-HER2 (B) and MDA-MB-361 (C) cell lines, but has no significant effect in MCF7 cells (B). Proliferation assays were conducted 72 h after the siRNA transfection by measuring [3H]thymidine incorporation as described in Materials and Methods . Si-CNTR: MCF7, MCF7-HER2 and MDA-MB-361 cells transfected with non-targeting siRNA; Si-hMena: MCF7, MCF7-HER2 and MDA-MB-361 cells transfected with specific hMena/hMena 11a siRNA. Histograms represent the mean of three different experiments. Bars, Standard deviations. P , according to Student's t test (two tailed).

    Journal: PLoS ONE

    Article Title: The Cooperation between hMena Overexpression and HER2 Signalling in Breast Cancer

    doi: 10.1371/journal.pone.0015852

    Figure Lengend Snippet: hMena knock-down affects HER2 signalling and inhibits the EGF/NRG1-mediated mitogenic effects in MCF7-HER2 cells. A. Western blot analysis of MCF7-HER2 breast cancer cell line after 72 h transfection with control and hMena/hMena 11a -specific siRNAs, untreated or treated with EGF (100 ng/ml) or NRG1 (10 ng/ml) for the last 24 h of transfection. hMena/hMena 11a expression (evaluated by pan-hMena and hMena 11a specific antibodies) and phosphorylation status of HER2, EGFR, HER3, AKT and MAPK (p44/42) from whole cell lysates were assessed. Membranes were sequentially stripped and reprobed with the indicated total and phospho-specific antibodies. Densitometric quantitation of anti-P-HER2, anti-P-AKT and anti-P-MAPK immunoreactivity was determined by Quantity One software (Biorad) and normalized in comparison with the Actin immunoreactivity. Densitometric quantitation of anti-P-HER3 immunoreactivity was not determined due to the fact that the total HER3 expression level is not unchanged following treatments. B–C. Silencing of hMena/hMena 11a reduces EGF and NRG1-mediated cell proliferation of HER2 overexpressing MCF7-HER2 (B) and MDA-MB-361 (C) cell lines, but has no significant effect in MCF7 cells (B). Proliferation assays were conducted 72 h after the siRNA transfection by measuring [3H]thymidine incorporation as described in Materials and Methods . Si-CNTR: MCF7, MCF7-HER2 and MDA-MB-361 cells transfected with non-targeting siRNA; Si-hMena: MCF7, MCF7-HER2 and MDA-MB-361 cells transfected with specific hMena/hMena 11a siRNA. Histograms represent the mean of three different experiments. Bars, Standard deviations. P , according to Student's t test (two tailed).

    Article Snippet: Anti estrogen (6F11 MoAb) and progesterone (1A6 MoAb) receptors were purchased from Novocastra (Menarini, Florence, Italy); HER-2 (A0485 pAb) and Ki67 (MIB-1 MoAb) antibodies from DAKO (Milan, Italy); phospho-p44/42 MAPK and phospho-AKT antibodies from Cell Signaling (SIAL, Rome, Italy).

    Techniques: Western Blot, Transfection, Expressing, Quantitation Assay, Software, Multiple Displacement Amplification, Two Tailed Test

    Mdm20 regulates the phosphorylation status of Akt. A. Western blots for Akt with a focus on phosphorylated Akt on Ser473 and Thr308. HEK293 cells were transfected either with mock, Flag-Mdm20 and mCherry-Nat5 or control, Mdm20 and Nat5 siRNA. At 48 hrs or 72hrs post-transfection, respectively, the cells were harvested, and cell lysates were prepared and processed for western blot analysis. The antibodies used are indicated. For details, see Materials and Methods. B. Western blots were used to determine the phosphorylation status of GSK3β, PTEN, PDK-1, mTOR, and PP1. The antibodies used were as indicated. Note that the phosphorylation of mTOR (Ser-2481) is consistently reduced in Mdm20-KD cells. C. Akt inhibition increases the levels of LC3II. Shown is western blots of HEK293 cellular extracts treated or untreated with siRNAs for Mdm20 or Nat5 in the absence (DMSO control) or presence of Akt inhibitor (Akt-I-VIII). Levels of Akt, and phospho-Akt (pAkt-Ser473), and LC3 levels are shown with the loading control of actin blot.

    Journal: PLoS ONE

    Article Title: Mdm20 Stimulates PolyQ Aggregation via Inhibiting Autophagy Through Akt-Ser473 Phosphorylation

    doi: 10.1371/journal.pone.0082523

    Figure Lengend Snippet: Mdm20 regulates the phosphorylation status of Akt. A. Western blots for Akt with a focus on phosphorylated Akt on Ser473 and Thr308. HEK293 cells were transfected either with mock, Flag-Mdm20 and mCherry-Nat5 or control, Mdm20 and Nat5 siRNA. At 48 hrs or 72hrs post-transfection, respectively, the cells were harvested, and cell lysates were prepared and processed for western blot analysis. The antibodies used are indicated. For details, see Materials and Methods. B. Western blots were used to determine the phosphorylation status of GSK3β, PTEN, PDK-1, mTOR, and PP1. The antibodies used were as indicated. Note that the phosphorylation of mTOR (Ser-2481) is consistently reduced in Mdm20-KD cells. C. Akt inhibition increases the levels of LC3II. Shown is western blots of HEK293 cellular extracts treated or untreated with siRNAs for Mdm20 or Nat5 in the absence (DMSO control) or presence of Akt inhibitor (Akt-I-VIII). Levels of Akt, and phospho-Akt (pAkt-Ser473), and LC3 levels are shown with the loading control of actin blot.

    Article Snippet: The other antibodies used in this study were purchased as follows: anti-Flag, anti-β-actin and anti-MAP2 were purchased from Sigma; anti-ribosomal protein L3, anti-Myc, anti-vimentin and anti-PP1 antibody were purchased from Santa Cruz; anti-phospho-Akt (Ser473 and Thr308), anti-Akt, anti-GSK3β, anti-phospho-GSK3β, anti-mTOR, anti-phospho-mTOR (Ser2481), anti-phospho-PDK (Ser241) were purchased from Cell Signaling Technology; anti-LC3 was purchased from Medical and Biological Laboratories (MBL); anti-ubiquitin was purchased from Millipore; anti-GFP was purchased from Nacalai Tesque; MG132 and 3-methyladenine were purchased from Sigma, ammonium chloride was from Nacalai Tesque, rapamycin was from Cell Signaling Technology and Akt inhibitor VIII was from Merck.

    Techniques: Western Blot, Transfection, Inhibition

    Mdm20 affects the polyQ aggregate formation by the regulation of pAkt Ser473 level. A. A phospho-mimic mutant of Akt (Akt-S473D) increases polyQ aggregate formation. Left: Shown are western blots for Akt and phospho-Akt (at Ser473 and Thr308). HEK293 cells were co-transfected with GFP-polyQ81 and Flag-tagged wild type Akt (F-Akt) or various phospho- and none-phospho-mimic Akt mutants as indicated. Right: The relative levels of polyQ aggregate formation were evaluated in each of the transfectants and compared with the mock-transfected control. Experiments were performed in naïve HEK293 cells (green bars) and Mdm20-KD cells (red bars). The numbers of polyQ-bearing cells among the GFP-positive cells were calculated and normalized to the level of mock transfection. The data represent the mean +/- S.D. (n=3) ***P

    Journal: PLoS ONE

    Article Title: Mdm20 Stimulates PolyQ Aggregation via Inhibiting Autophagy Through Akt-Ser473 Phosphorylation

    doi: 10.1371/journal.pone.0082523

    Figure Lengend Snippet: Mdm20 affects the polyQ aggregate formation by the regulation of pAkt Ser473 level. A. A phospho-mimic mutant of Akt (Akt-S473D) increases polyQ aggregate formation. Left: Shown are western blots for Akt and phospho-Akt (at Ser473 and Thr308). HEK293 cells were co-transfected with GFP-polyQ81 and Flag-tagged wild type Akt (F-Akt) or various phospho- and none-phospho-mimic Akt mutants as indicated. Right: The relative levels of polyQ aggregate formation were evaluated in each of the transfectants and compared with the mock-transfected control. Experiments were performed in naïve HEK293 cells (green bars) and Mdm20-KD cells (red bars). The numbers of polyQ-bearing cells among the GFP-positive cells were calculated and normalized to the level of mock transfection. The data represent the mean +/- S.D. (n=3) ***P

    Article Snippet: The other antibodies used in this study were purchased as follows: anti-Flag, anti-β-actin and anti-MAP2 were purchased from Sigma; anti-ribosomal protein L3, anti-Myc, anti-vimentin and anti-PP1 antibody were purchased from Santa Cruz; anti-phospho-Akt (Ser473 and Thr308), anti-Akt, anti-GSK3β, anti-phospho-GSK3β, anti-mTOR, anti-phospho-mTOR (Ser2481), anti-phospho-PDK (Ser241) were purchased from Cell Signaling Technology; anti-LC3 was purchased from Medical and Biological Laboratories (MBL); anti-ubiquitin was purchased from Millipore; anti-GFP was purchased from Nacalai Tesque; MG132 and 3-methyladenine were purchased from Sigma, ammonium chloride was from Nacalai Tesque, rapamycin was from Cell Signaling Technology and Akt inhibitor VIII was from Merck.

    Techniques: Mutagenesis, Western Blot, Transfection

    Knockdown of CHRF reduces the protein expression level of PI3K/Akt pathway in LAD. Western blotting revealed that knockdown of CHRF decreased the protein expression levels of p-PI3K and p-Akt in (A) SPCA-1 and (B) NCI-H441 cells. Error bars represented the mean ± standard deviation of ≥3 independent experiments. **P

    Journal: Oncology Letters

    Article Title: Long non-coding RNA, CHRF, predicts poor prognosis of lung adenocarcinoma and promotes cell proliferation and migration

    doi: 10.3892/ol.2018.9425

    Figure Lengend Snippet: Knockdown of CHRF reduces the protein expression level of PI3K/Akt pathway in LAD. Western blotting revealed that knockdown of CHRF decreased the protein expression levels of p-PI3K and p-Akt in (A) SPCA-1 and (B) NCI-H441 cells. Error bars represented the mean ± standard deviation of ≥3 independent experiments. **P

    Article Snippet: The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 1% Tween (TBS) for 1 h, then incubated with the following primary antibodies overnight at 4°C: Phosphorylated (p-)PI3K (cat no. ab125633, 1:2,000), total PI3K (cat no. ab127617, 1:2,000), p-AKT (cat no. ab38449, 1:2,000), total Akt (cat no. ab126580; 1:2,000) and GAPDH (cat no. ab8245; 1:2,000) (all from Abcam, Cambridge, UK).

    Techniques: Expressing, Western Blot, Standard Deviation

    CSNK2A1 modulates the PI3K-Akt-mTOR signaling pathway. ( A ) The Western blot results show that the levels of p-Akt S473/T308 and p-mTOR were increased in CSNK2A1-overexpressing cells and decreased in CSNK2A1-knockdown cells. ( B ) The levels of p-Akt S473/T308 and p-mTOR were decreased after treatment with a PI3K inhibitor (ly294002) in the CSNK2A1-overexpressing GC cell line. ( C ) The migratory and invasive abilities were markedly reduced after PI3K inhibitor ly294002 treatment in the CSNK2A1-overexpressing GC cell line. ( D ) A CCK-8 assay was used to detect the viability of cells treated with empty vector, CSNK2A1 and CSNK2A1+ ly294002. (*P

    Journal: Cancer Management and Research

    Article Title: CSNK2A1 Promotes Gastric Cancer Invasion Through the PI3K-Akt-mTOR Signaling Pathway

    doi: 10.2147/CMAR.S222620

    Figure Lengend Snippet: CSNK2A1 modulates the PI3K-Akt-mTOR signaling pathway. ( A ) The Western blot results show that the levels of p-Akt S473/T308 and p-mTOR were increased in CSNK2A1-overexpressing cells and decreased in CSNK2A1-knockdown cells. ( B ) The levels of p-Akt S473/T308 and p-mTOR were decreased after treatment with a PI3K inhibitor (ly294002) in the CSNK2A1-overexpressing GC cell line. ( C ) The migratory and invasive abilities were markedly reduced after PI3K inhibitor ly294002 treatment in the CSNK2A1-overexpressing GC cell line. ( D ) A CCK-8 assay was used to detect the viability of cells treated with empty vector, CSNK2A1 and CSNK2A1+ ly294002. (*P

    Article Snippet: The following antibodies were used in our experiments: anti-CSNK2A1 (#ab76040, Abcam, Cambridge, UK; 1:100), anti-E-cadherin (#3195, Cell Signaling Technology, Shanghai, China; 1:1000), anti-N-cadherin (#13,116; Cell Signaling Technology, Shanghai, China; 1:1000), anti-vimentin (#5741; Cell Signaling Technology, Shanghai, China; 1:1000), anti-ALK (#ab16670, Abcam, Cambridge, UK; 1:100;), anti-AKT1 (phospho S473) (#ab81283, Abcam, Cambridge, UK; 1:6000), anti-pan-Akt (phospho T308) (#ab38449, Abcam, Cambridge, UK; 1:500), anti-mTOR (#ab134903, Abcam, Cambridge, UK; 1:10,000), and anti-mTOR (phospho S2448) (#ab109268, Abcam, Cambridge, UK; 1:5000).

    Techniques: Western Blot, CCK-8 Assay, Plasmid Preparation