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  • 93
    Thermo Fisher frap1 mtor assay buffer
    TFEB serine 122 is regulated by MTORC1. (A) Validation of the p-TFEB (S122) antibody. HeLa cells were transfected with the indicated MYC-tagged TFEB constructs followed by MYC-IP and western blot. λ, lambda-phosphatase treatment of immunoprecipitates. HeLa cells were treated with Torin1 (250 nM) (B), or starved for amino acids (C), serum (D), or glucose (E). (F, G) HeLa cells were transfected with active (RRAGB GTP /D GDP ) or inactive (RRAGB GDP /D GTP ) RRAG-GTPases and its effects on TFEB localization and phosphorylation were assessed. (H) <t>MTOR</t> in vitro kinase assay of epitope tag immunoprecipitates from cells transfected with empty vector (EV) vs. MYC-TFEB and incubated with or without recombinant MTOR. Scale bar: 10 μm.
    Frap1 Mtor Assay Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    WuXi AppTec mtor
    Down-regulation of <t>PIK3R3</t> and <t>mTOR</t> after inhibiting the expression of XBP1 . ( a , b ) The mRNA expression of PIK3R3, mTOR, and XBP1 between the scrambled and XBP1-knockdown group under both normoxic and hypoxic condition in MG63 and U2OS cells; ( c , d ) The protein expression of PIK3R3, mTOR, and XBP1 between the scrambled and XBP1-knockdown group under both normoxic and hypoxic condition in MG63 and U2OS cells. Transfection efficacy of XBP1-siRNA was confirmed by qRT-PCR and Western blot assays. The data are representative of three independent experiments. Error bars represent mean ± s.d. * p
    Mtor, supplied by WuXi AppTec, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Cell Signaling Technology Inc mtor
    The schematic signaling pathways involved in galangin inhibiting the MMP-9 expression in SK-N-SH cells challenged with thrombin. Galangin inhibits MMP-9 expression by attenuating c-Src, <t>Pyk2,</t> PKCα/β/δ, Akt, <t>mTOR,</t> p42/p44 MAPK, p38 MAPK, and JNK1/2 phosphorylation to decrease NF-κB, AP-1, and FoxO1 activation and ultimately reduces SK-N-SH cell migration.
    Mtor, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 10377 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti frap1
    <t>FRAP1</t> expression in Amhr2 tm3(cre)Bhr ; Ctnnb1 tm1Mmt uteri. A ) FRAP1 expression was detected by immunohistochemistry at high levels in the central areas of a leiomyoma (outlined in white) in a mutant uterus, the epithelial cells, the ES as well as in the circular myometrial layer (MC) and longitudinal myometrial layer (ML). B ) A higher magnification view of FRAP1 expression in the MC and ML of a mutant mouse. C ) In control uteri, strong FRAP1 expression is found in the EG, LE, and ES. D ) Expression in the control myometrium appears limited to the endothelial cells. LE, luminal epithelium; ES, endometrial stroma; M, myometrium; EG, endometrial glands. Bars = 50 μm ( A and C ), 100 μm ( B and D ).
    Anti Frap1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher frap1 mtor recombinant human protein
    Chemical inhibition of TRF1 binding to telomere by PI3K inhibitors. a Structures of ETP-47037, ETP-47228, and their corresponding “inactive analogs” ETP-51259 and ETP-50952. b <t>PI3K/mTOR</t> IC 50 data generated internally and reported for the inhibitors used in the study. c Time course Inhibition of AKT phosphorylation at Ser473 by ETP-47037, ETP-51259, ETP-47228, and ETP-50952 at 10 μM in CHA-9.3 cell line. d Percent inhibition of TRF1 foci by immunofluorescence in CHA-9.3 mouse lung tumor cell line treated with 10 μM of either ETP-47037, ETP-47228, or their corresponding inactive analogs (ETP-51259 and ETP-50952, respectively) relative to TRF1 levels with DMSO treatment. The ETP compound inhibitory activity on PI3K pathway is stated at the bottom of the graph. Error bars represent standard deviation. N/D not determined, n number of independent experiments
    Frap1 Mtor Recombinant Human Protein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Genecopoeia mtor frap1 reporter assay a frap1 reporter assay a frap1 mtor reporter construct
    Chemical inhibition of TRF1 binding to telomere by PI3K inhibitors. a Structures of ETP-47037, ETP-47228, and their corresponding “inactive analogs” ETP-51259 and ETP-50952. b <t>PI3K/mTOR</t> IC 50 data generated internally and reported for the inhibitors used in the study. c Time course Inhibition of AKT phosphorylation at Ser473 by ETP-47037, ETP-51259, ETP-47228, and ETP-50952 at 10 μM in CHA-9.3 cell line. d Percent inhibition of TRF1 foci by immunofluorescence in CHA-9.3 mouse lung tumor cell line treated with 10 μM of either ETP-47037, ETP-47228, or their corresponding inactive analogs (ETP-51259 and ETP-50952, respectively) relative to TRF1 levels with DMSO treatment. The ETP compound inhibitory activity on PI3K pathway is stated at the bottom of the graph. Error bars represent standard deviation. N/D not determined, n number of independent experiments
    Mtor Frap1 Reporter Assay A Frap1 Reporter Assay A Frap1 Mtor Reporter Construct, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GenScript plasmid puc57 frap1
    Chemical inhibition of TRF1 binding to telomere by PI3K inhibitors. a Structures of ETP-47037, ETP-47228, and their corresponding “inactive analogs” ETP-51259 and ETP-50952. b <t>PI3K/mTOR</t> IC 50 data generated internally and reported for the inhibitors used in the study. c Time course Inhibition of AKT phosphorylation at Ser473 by ETP-47037, ETP-51259, ETP-47228, and ETP-50952 at 10 μM in CHA-9.3 cell line. d Percent inhibition of TRF1 foci by immunofluorescence in CHA-9.3 mouse lung tumor cell line treated with 10 μM of either ETP-47037, ETP-47228, or their corresponding inactive analogs (ETP-51259 and ETP-50952, respectively) relative to TRF1 levels with DMSO treatment. The ETP compound inhibitory activity on PI3K pathway is stated at the bottom of the graph. Error bars represent standard deviation. N/D not determined, n number of independent experiments
    Plasmid Puc57 Frap1, supplied by GenScript, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson mouse monoclonal anti raft1 frap mtor
    Chemical inhibition of TRF1 binding to telomere by PI3K inhibitors. a Structures of ETP-47037, ETP-47228, and their corresponding “inactive analogs” ETP-51259 and ETP-50952. b <t>PI3K/mTOR</t> IC 50 data generated internally and reported for the inhibitors used in the study. c Time course Inhibition of AKT phosphorylation at Ser473 by ETP-47037, ETP-51259, ETP-47228, and ETP-50952 at 10 μM in CHA-9.3 cell line. d Percent inhibition of TRF1 foci by immunofluorescence in CHA-9.3 mouse lung tumor cell line treated with 10 μM of either ETP-47037, ETP-47228, or their corresponding inactive analogs (ETP-51259 and ETP-50952, respectively) relative to TRF1 levels with DMSO treatment. The ETP compound inhibitory activity on PI3K pathway is stated at the bottom of the graph. Error bars represent standard deviation. N/D not determined, n number of independent experiments
    Mouse Monoclonal Anti Raft1 Frap Mtor, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson anti raft1
    MAPKAP1 Binds to Ras and Modulates the Ras-mTORC2 Interaction. (A) Schematic of full length MAPKAP1 isoform 1 wild-type (WT) and the MAPKAP1 deletion (Del) proteins with domains highlighted. CRIM, Conserved Region In The Middle. RBD, Ras Binding Domain. PH, Pleckstrin Homology. (B) Microscale thermophoresis of labeled FHH:MAPKAP1 RBD (16.8nM) with a titration series of GDP or GTPγs-loaded Ras. The binding curve is positive as the MST signal of the complex is higher than MAPKAP1 RBD alone. MST-on time of 5s, n = 3 independent replicates. (C) BioID-western blot streptavidin pulldowns and input levels for birA* control, NRAS WT , NRAS Q61K and NRAS Q61K with FHH:eGFP, MAPKAP1 WT :FHH or MAPKAP1 Del :FHH expression in CHL-1 cells. PI3K p110α subunit, Raf-1, and HA protein pulldown are controls. Pulldown normalized signal relative to control birA*:NRAS Q61K shown below. (D) Quantification of <t>mTOR</t> and Rictor protein levels in the streptavidin pulldowns normalized to HA pulldown. All values and statistical tests relative to birA*:NRAS Q61K , n=6 (Welch’s two-sided t-test). (E) PLA with endogenous Pan-Ras and mTOR or Rictor in MT NRAS MM485 melanoma cells. Scale bar, 20 μm. (F) PLA quantification in (E). n=3 independent experiments, 6-8 fields analyzed per condition per experiment (unpaired two-sided t-test). EV, empty vector. (G) Quantification of LocaTOR2 experiments with FHH:eGFP, MAPKAP1 WT :FHH or MAPKAP1 Del :FHH expression. All values relative to average of FHH:eGFP and MAPKAP1 WT :FHH fold induction; n=3 (unpaired two-sided t-test). (H) Quantification of FHH:eGFP, MAPKAP1 WT :FHH and MAPKAP1 Del :FHH expression relative to MAPKAP1 WT .
    Anti Raft1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    TFEB serine 122 is regulated by MTORC1. (A) Validation of the p-TFEB (S122) antibody. HeLa cells were transfected with the indicated MYC-tagged TFEB constructs followed by MYC-IP and western blot. λ, lambda-phosphatase treatment of immunoprecipitates. HeLa cells were treated with Torin1 (250 nM) (B), or starved for amino acids (C), serum (D), or glucose (E). (F, G) HeLa cells were transfected with active (RRAGB GTP /D GDP ) or inactive (RRAGB GDP /D GTP ) RRAG-GTPases and its effects on TFEB localization and phosphorylation were assessed. (H) MTOR in vitro kinase assay of epitope tag immunoprecipitates from cells transfected with empty vector (EV) vs. MYC-TFEB and incubated with or without recombinant MTOR. Scale bar: 10 μm.

    Journal: Autophagy

    Article Title: Multistep regulation of TFEB by MTORC1

    doi: 10.1080/15548627.2016.1271514

    Figure Lengend Snippet: TFEB serine 122 is regulated by MTORC1. (A) Validation of the p-TFEB (S122) antibody. HeLa cells were transfected with the indicated MYC-tagged TFEB constructs followed by MYC-IP and western blot. λ, lambda-phosphatase treatment of immunoprecipitates. HeLa cells were treated with Torin1 (250 nM) (B), or starved for amino acids (C), serum (D), or glucose (E). (F, G) HeLa cells were transfected with active (RRAGB GTP /D GDP ) or inactive (RRAGB GDP /D GTP ) RRAG-GTPases and its effects on TFEB localization and phosphorylation were assessed. (H) MTOR in vitro kinase assay of epitope tag immunoprecipitates from cells transfected with empty vector (EV) vs. MYC-TFEB and incubated with or without recombinant MTOR. Scale bar: 10 μm.

    Article Snippet: MYC immunoprecipitates were washed twice with IP buffer, twice with FRAP kinase buffer (Invitrogen, PV4794), and resuspended in FRAP kinase buffer containing DTT (2 mM), ATP (10 μM; Invitrogen, PV3227) and MTOR kinase (250 ng; Calbiochem, 475987).

    Techniques: Transfection, Construct, Western Blot, In Vitro, Kinase Assay, Plasmid Preparation, Incubation, Recombinant

    ( A , available at Carcinogenesis Online. ( B ) DOA decreases mTOR and DNMT activities in cultured cells. Left. Representative western blotting analyses of phospho-p70 S6 Kinase (Thr389)/phospho-p85 S6 Kinase (Thr412) in SUM-159 cells cultured in the absence of presence of DOA or rapamycin for up to 6 h, as specified. Right. Nuclear extracts of SUM-159 were exposed to 20 μmol/L DOA for 2 h before assessing DNMT activity using the DNMT activity/Inhibition Assay Kit of Active Motif as per manufacturer’s instructions. The results are expressed as percentages of the means (columns) ± SD (bars); three technical replicates per n ; n = 2 biological replicates. (** P

    Journal: Carcinogenesis

    Article Title: Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells

    doi: 10.1093/carcin/bgy023

    Figure Lengend Snippet: ( A , available at Carcinogenesis Online. ( B ) DOA decreases mTOR and DNMT activities in cultured cells. Left. Representative western blotting analyses of phospho-p70 S6 Kinase (Thr389)/phospho-p85 S6 Kinase (Thr412) in SUM-159 cells cultured in the absence of presence of DOA or rapamycin for up to 6 h, as specified. Right. Nuclear extracts of SUM-159 were exposed to 20 μmol/L DOA for 2 h before assessing DNMT activity using the DNMT activity/Inhibition Assay Kit of Active Motif as per manufacturer’s instructions. The results are expressed as percentages of the means (columns) ± SD (bars); three technical replicates per n ; n = 2 biological replicates. (** P

    Article Snippet: To characterize the mTOR kinase potency of DOA, PP242 and Torin 2, IC50 determinations for FRAP1 (mTOR) were outsourced to Invitrogen (Life Technologies) using the FRET-based Z-LYTE™ SelectScreen Kinase Profiling Service.

    Techniques: Cell Culture, Western Blot, Activity Assay, Inhibition

    ( A ) DOA augments the capacity of mTOR inhibitor rapamycin and DNMT inhibitor 5-azacytidine to inhibit BC mammosphere formation activity. Cell2Sphere™ assays using BRCA1 -deficient MDA-MB-436 (left) and HER2-overexpressing BT-474 cell (right) were performed as per the manufacturer’s instructions. Drugs were added to sextuplicate sets of wells on days 1 and 4 without replenishing the medium. ImageJ was used to quantify the size (central lines indicate mean values) of 6-day-old mammospheres. Size bar = 2000 μm. ( B ) EVOO-derived oleoside DOA is a metabolo-epigenetic suppressor of CSC cellular states: A phenotypic drug discovery approach. EVOO is the juice from fruits of olive trees ( Olea europaea L.) obtained solely by mechanical means and consumed without further refining processes other than washing, filtration, decantation and centrifugation. Whereas well-known secoiridoids such as the glucoside oleuropein are characteristic and abundant constituents easily accessible from the drupes and other organs (leaves) of O. europaea L., DOA is an oleuropein-dialdehyde derivative confirmed only in EVOO in highly variable concentrations. We performed a holistic approach of phenotypic drug discovery coupled to mechanism-of-action (MOA) functional profiling and target deconvolution that identified DOA as an unforeseen EVOO bioactive phytochemical that operates as a dual TORKinhib/DNMTinhib molecule capable of specifically and potently suppressing the functional traits of CSC irrespective of the mutational landscape of BC populations.

    Journal: Carcinogenesis

    Article Title: Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells

    doi: 10.1093/carcin/bgy023

    Figure Lengend Snippet: ( A ) DOA augments the capacity of mTOR inhibitor rapamycin and DNMT inhibitor 5-azacytidine to inhibit BC mammosphere formation activity. Cell2Sphere™ assays using BRCA1 -deficient MDA-MB-436 (left) and HER2-overexpressing BT-474 cell (right) were performed as per the manufacturer’s instructions. Drugs were added to sextuplicate sets of wells on days 1 and 4 without replenishing the medium. ImageJ was used to quantify the size (central lines indicate mean values) of 6-day-old mammospheres. Size bar = 2000 μm. ( B ) EVOO-derived oleoside DOA is a metabolo-epigenetic suppressor of CSC cellular states: A phenotypic drug discovery approach. EVOO is the juice from fruits of olive trees ( Olea europaea L.) obtained solely by mechanical means and consumed without further refining processes other than washing, filtration, decantation and centrifugation. Whereas well-known secoiridoids such as the glucoside oleuropein are characteristic and abundant constituents easily accessible from the drupes and other organs (leaves) of O. europaea L., DOA is an oleuropein-dialdehyde derivative confirmed only in EVOO in highly variable concentrations. We performed a holistic approach of phenotypic drug discovery coupled to mechanism-of-action (MOA) functional profiling and target deconvolution that identified DOA as an unforeseen EVOO bioactive phytochemical that operates as a dual TORKinhib/DNMTinhib molecule capable of specifically and potently suppressing the functional traits of CSC irrespective of the mutational landscape of BC populations.

    Article Snippet: To characterize the mTOR kinase potency of DOA, PP242 and Torin 2, IC50 determinations for FRAP1 (mTOR) were outsourced to Invitrogen (Life Technologies) using the FRET-based Z-LYTE™ SelectScreen Kinase Profiling Service.

    Techniques: Activity Assay, Multiple Displacement Amplification, Derivative Assay, Refining, Filtration, Decantation Assay, Centrifugation, Functional Assay

    Down-regulation of PIK3R3 and mTOR after inhibiting the expression of XBP1 . ( a , b ) The mRNA expression of PIK3R3, mTOR, and XBP1 between the scrambled and XBP1-knockdown group under both normoxic and hypoxic condition in MG63 and U2OS cells; ( c , d ) The protein expression of PIK3R3, mTOR, and XBP1 between the scrambled and XBP1-knockdown group under both normoxic and hypoxic condition in MG63 and U2OS cells. Transfection efficacy of XBP1-siRNA was confirmed by qRT-PCR and Western blot assays. The data are representative of three independent experiments. Error bars represent mean ± s.d. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Overexpression of X-Box Binding Protein 1 (XBP1) Correlates to Poor Prognosis and Up-Regulation of PI3K/mTOR in Human Osteosarcoma

    doi: 10.3390/ijms161226123

    Figure Lengend Snippet: Down-regulation of PIK3R3 and mTOR after inhibiting the expression of XBP1 . ( a , b ) The mRNA expression of PIK3R3, mTOR, and XBP1 between the scrambled and XBP1-knockdown group under both normoxic and hypoxic condition in MG63 and U2OS cells; ( c , d ) The protein expression of PIK3R3, mTOR, and XBP1 between the scrambled and XBP1-knockdown group under both normoxic and hypoxic condition in MG63 and U2OS cells. Transfection efficacy of XBP1-siRNA was confirmed by qRT-PCR and Western blot assays. The data are representative of three independent experiments. Error bars represent mean ± s.d. * p

    Article Snippet: Following the standard procedure, the blocking proteins in the nitrocellulose filter membranes were incubated with primary antibodies against the following antigens: XBP1 (Santa Cruz Biotechnology, Dallas, TX, USA), HIF1α (Santa Cruz Biotechnology), PIK3R3 (Abgent, San Diego, CA, USA), mTOR (Abgent), β-Actin (Sigma-Aldrich, New York, NY, USA).

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot

    Effects of bpV(pic) on mTOR and autophagy-related protein analysis 1d post-SCI. A) Western blot profiles from tissue collected from experimental animals 1d post-SCI. B–E) Quantification of blots shown in A. B) Total PTEN protein expression does not significantly change following injury, though a mild increase in expression is observed. C) p-Akt levels significantly decrease following injury, and are significantly increased following bpV (pic) treatment. D) Downstream, p-S6 protein levels significantly increase following injury, and are enhanced further following bpV treatment. E) LC3 II ratio to LC3 I, an indicator of autophagic activity, is increased following injury, and is significantly reduced following bpV(pic) therapy. **, p

    Journal: PLoS ONE

    Article Title: Systemic Bisperoxovanadium Activates Akt/mTOR, Reduces Autophagy, and Enhances Recovery following Cervical Spinal Cord Injury

    doi: 10.1371/journal.pone.0030012

    Figure Lengend Snippet: Effects of bpV(pic) on mTOR and autophagy-related protein analysis 1d post-SCI. A) Western blot profiles from tissue collected from experimental animals 1d post-SCI. B–E) Quantification of blots shown in A. B) Total PTEN protein expression does not significantly change following injury, though a mild increase in expression is observed. C) p-Akt levels significantly decrease following injury, and are significantly increased following bpV (pic) treatment. D) Downstream, p-S6 protein levels significantly increase following injury, and are enhanced further following bpV treatment. E) LC3 II ratio to LC3 I, an indicator of autophagic activity, is increased following injury, and is significantly reduced following bpV(pic) therapy. **, p

    Article Snippet: The membranes were immunoblotted with the following primary antibodies: monoclonal mouse-anti PTEN (1∶200); monoclonal mouse anti-ribosomal protein S6 (1∶200) (Santa Cruz Biotechnologies); polyclonal rabbit anti-phospho Akt (Ser473 ) (1∶1,000), a marker used for assessing PI3K activation; monoclonal mouse anti-pan Akt (1∶1,500); polyclonal rabbit anti-phospho ribosomal protein S6 (Ser235/236 ) (1∶400) (Cell Signaling, Inc.), an indicator of mTOR activity; polyclonal rabbit anti-LC3B (1∶100; Abgent), to assess autophagy activity; monoclonal mouse anti-β-tubulin (1∶1000; Sigma-Aldrich) as a loading control.

    Techniques: Western Blot, Expressing, Activity Assay

    Potential mechanistic explanation for bpV(pic)-mediated neuroprotective effects. PTEN's phosphatase activity converts phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) into phosphatidylinositol (4,5)-bisphosphate (PIP 2 ), thus inhibiting downstream Akt and mTOR signaling. PI3K converts PIP 2 into PIP 3 , which can then activate Akt and mTOR, thus enhancing p-S6 expression and contributing to the decrease in cellular autophagic activity that may be involved in programmed cell death, and leading to neuroprotection.

    Journal: PLoS ONE

    Article Title: Systemic Bisperoxovanadium Activates Akt/mTOR, Reduces Autophagy, and Enhances Recovery following Cervical Spinal Cord Injury

    doi: 10.1371/journal.pone.0030012

    Figure Lengend Snippet: Potential mechanistic explanation for bpV(pic)-mediated neuroprotective effects. PTEN's phosphatase activity converts phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) into phosphatidylinositol (4,5)-bisphosphate (PIP 2 ), thus inhibiting downstream Akt and mTOR signaling. PI3K converts PIP 2 into PIP 3 , which can then activate Akt and mTOR, thus enhancing p-S6 expression and contributing to the decrease in cellular autophagic activity that may be involved in programmed cell death, and leading to neuroprotection.

    Article Snippet: The membranes were immunoblotted with the following primary antibodies: monoclonal mouse-anti PTEN (1∶200); monoclonal mouse anti-ribosomal protein S6 (1∶200) (Santa Cruz Biotechnologies); polyclonal rabbit anti-phospho Akt (Ser473 ) (1∶1,000), a marker used for assessing PI3K activation; monoclonal mouse anti-pan Akt (1∶1,500); polyclonal rabbit anti-phospho ribosomal protein S6 (Ser235/236 ) (1∶400) (Cell Signaling, Inc.), an indicator of mTOR activity; polyclonal rabbit anti-LC3B (1∶100; Abgent), to assess autophagy activity; monoclonal mouse anti-β-tubulin (1∶1000; Sigma-Aldrich) as a loading control.

    Techniques: Activity Assay, Expressing

    C96 delays myeloma tumor growth in a xenograft mouse model ( A ) JJN3 cells were injected subcutaneously into nude mice with a density of 30 million cells/site/mouse. When tumors were palpable, mice (n=5/group) were orally given C96 (100 mg/kg body weight) in PBS containing 10% Tween 80 and 10% DMSO daily for continuous 16 days. Tumor volumes were monitored every other day. ( B ) Mouse body weight was monitored every other day. ( C ) At the end of the experiment, tumor samples from each group were subjected to immunoblotting analysis for the expression levels of p-AKT, T-AKT, p-mTOR and mTOR with specific antibodies. GAPDH was used as a loading control.

    Journal: Oncotarget

    Article Title: A virtual screen identified C96 as a novel inhibitor of phosphatidylinositol 3-kinase that displays potent preclinical activity against multiple myeloma in vitro and in vivo

    doi:

    Figure Lengend Snippet: C96 delays myeloma tumor growth in a xenograft mouse model ( A ) JJN3 cells were injected subcutaneously into nude mice with a density of 30 million cells/site/mouse. When tumors were palpable, mice (n=5/group) were orally given C96 (100 mg/kg body weight) in PBS containing 10% Tween 80 and 10% DMSO daily for continuous 16 days. Tumor volumes were monitored every other day. ( B ) Mouse body weight was monitored every other day. ( C ) At the end of the experiment, tumor samples from each group were subjected to immunoblotting analysis for the expression levels of p-AKT, T-AKT, p-mTOR and mTOR with specific antibodies. GAPDH was used as a loading control.

    Article Snippet: Antibodies against mTOR, p-mTOR(S2448), p70S6K, p-p70S6K(S424), and GAPDH were obtained from Abgent (Suzhou, China).

    Techniques: Injection, Mouse Assay, Expressing

    C96 inhibits AKT and mTOR signaling ( A ) LP1 and OPM2 were starved overnight, then treated with C96 at the indicated concentrations, or 100 μM of S14161 for 2 hrs, followed by IGF-1 (100 ng/mL) for 15 min. Cells were collected for the analysis of the expression of p-AKT and total AKT (T-AKT) by immunoblotting. ( B ) LP1 and OPM2 cells were starved overnight, then treated with C96 (50 μM) for different time periods, or S14161 (100 (μM) for 2 hrs, followed by IGF-1 (100 ng/mL) for 15 min. Cells were for the analysis of the expression of p-AKT and T-AKT by immunoblotting. ( C ) LP1, OPM2, and JJN3 cells were treated with C96 at the indicated concentrations for 24 hrs. Cell lysates were prepared and subjected to immunoblotting assay against p-AKT, AKT, p-mTOR, mTOR, p-4E-BP1, and 4E-BP1. GAPDH was used as a loading control.

    Journal: Oncotarget

    Article Title: A virtual screen identified C96 as a novel inhibitor of phosphatidylinositol 3-kinase that displays potent preclinical activity against multiple myeloma in vitro and in vivo

    doi:

    Figure Lengend Snippet: C96 inhibits AKT and mTOR signaling ( A ) LP1 and OPM2 were starved overnight, then treated with C96 at the indicated concentrations, or 100 μM of S14161 for 2 hrs, followed by IGF-1 (100 ng/mL) for 15 min. Cells were collected for the analysis of the expression of p-AKT and total AKT (T-AKT) by immunoblotting. ( B ) LP1 and OPM2 cells were starved overnight, then treated with C96 (50 μM) for different time periods, or S14161 (100 (μM) for 2 hrs, followed by IGF-1 (100 ng/mL) for 15 min. Cells were for the analysis of the expression of p-AKT and T-AKT by immunoblotting. ( C ) LP1, OPM2, and JJN3 cells were treated with C96 at the indicated concentrations for 24 hrs. Cell lysates were prepared and subjected to immunoblotting assay against p-AKT, AKT, p-mTOR, mTOR, p-4E-BP1, and 4E-BP1. GAPDH was used as a loading control.

    Article Snippet: Antibodies against mTOR, p-mTOR(S2448), p70S6K, p-p70S6K(S424), and GAPDH were obtained from Abgent (Suzhou, China).

    Techniques: Expressing

    The schematic signaling pathways involved in galangin inhibiting the MMP-9 expression in SK-N-SH cells challenged with thrombin. Galangin inhibits MMP-9 expression by attenuating c-Src, Pyk2, PKCα/β/δ, Akt, mTOR, p42/p44 MAPK, p38 MAPK, and JNK1/2 phosphorylation to decrease NF-κB, AP-1, and FoxO1 activation and ultimately reduces SK-N-SH cell migration.

    Journal: International Journal of Molecular Sciences

    Article Title: Galangin Inhibits Thrombin-Induced MMP-9 Expression in SK-N-SH Cells via Protein Kinase-Dependent NF-κB Phosphorylation

    doi: 10.3390/ijms19124084

    Figure Lengend Snippet: The schematic signaling pathways involved in galangin inhibiting the MMP-9 expression in SK-N-SH cells challenged with thrombin. Galangin inhibits MMP-9 expression by attenuating c-Src, Pyk2, PKCα/β/δ, Akt, mTOR, p42/p44 MAPK, p38 MAPK, and JNK1/2 phosphorylation to decrease NF-κB, AP-1, and FoxO1 activation and ultimately reduces SK-N-SH cell migration.

    Article Snippet: The anti-phospho antibodies against p42/p44 MAPK (#9101), p38 MAPK (#9211), JNK1/2 (#4668), mTOR (#5536), Akt (#9271), c-Src (#2101), Pyk2 (#3291), PKCα/βII (#9375), PKCδ (#9374), p65 (#3031), FoxO1 (#9461), and mTOR (#2972) were from Cell Signaling (Danvers, MA, USA).

    Techniques: Expressing, Activation Assay, Migration

    Galangin inhibits thrombin-induced phosphorylation of Akt and mammalian target of rapamycin (mTOR). The cells were pretreated with galangin (3 μM) for 1 h and then challenged with thrombin (10 U/mL) for the indicated time intervals. The levels of ( A ) mTOR and ( B ) Akt phosphorylation and their respective protein levels were determined by western blot. The data are expressed as mean ± SEM of three independent experiments. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Galangin Inhibits Thrombin-Induced MMP-9 Expression in SK-N-SH Cells via Protein Kinase-Dependent NF-κB Phosphorylation

    doi: 10.3390/ijms19124084

    Figure Lengend Snippet: Galangin inhibits thrombin-induced phosphorylation of Akt and mammalian target of rapamycin (mTOR). The cells were pretreated with galangin (3 μM) for 1 h and then challenged with thrombin (10 U/mL) for the indicated time intervals. The levels of ( A ) mTOR and ( B ) Akt phosphorylation and their respective protein levels were determined by western blot. The data are expressed as mean ± SEM of three independent experiments. * p

    Article Snippet: The anti-phospho antibodies against p42/p44 MAPK (#9101), p38 MAPK (#9211), JNK1/2 (#4668), mTOR (#5536), Akt (#9271), c-Src (#2101), Pyk2 (#3291), PKCα/βII (#9375), PKCδ (#9374), p65 (#3031), FoxO1 (#9461), and mTOR (#2972) were from Cell Signaling (Danvers, MA, USA).

    Techniques: Western Blot

    LBP therapeutically ameliorated hepatic inflammatory responses and apoptosis through modulating autophagy and MAPK pathway. (a–d) Protein expressional changes of inflammatory and chemoattractive responses markers tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), cyclooxygenase-2 (COX-2) and monocyte chemotactic protein-1 (MCP-1) from each group of rats after 12-week experiment. (e) Representative Western blot results of inhibitor of NF-κB alpha (IκBα) formation in the liver of each group of rats from 3 repeated Western blot experiments. Full-length blots are presented in Supplementary Fig. 4 . (f) Changes of nuclear factor-kappa B (NF-κB) subunit p65 in each group of rats, as measured by EIA kit. (g) Representative Western blot results of cleaved caspase-3 and cytochrome c (cyto c) in rats with different treatments from 3 repeated Western blot experiments. Full-length blots are presented in Supplementary Fig. 4 . (h,i) Changes of mRNA expression level of intrinsic apoptotic markers Bcl-2 and Bax in each group of rats. (j) Representative Western blot results of key autophagic markers including phosphorylated mTOR, total mTOR, beclin 1, Atg5, LC3II, and p62 in each group of rats from 3 repeated Western blot experiments. Full-length blots are presented in Supplementary Fig. 4 . (k) Representative Western blot results of key MAPK markers including phosphorylated and total forms of p38 MAPK, JNK and ERK1/2 in each group of rats from 3 repeated Western blot experiments. Full-length blots are presented in Supplementary Fig. 4 . Beta-actin was used as the internal control. Data from each group were expressed as means ± SEMs (n = 5 or 6). Statistical comparisons between groups were done using the Kruskal–Wallis test followed by Dunn's post hoc test to detect differences in all groups. Different letters (e.g. a and b) mean a statistical significant change ( p

    Journal: Scientific Reports

    Article Title: Lycium barbarum polysaccharides therapeutically improve hepatic functions in non-alcoholic steatohepatitis rats and cellular steatosis model

    doi: 10.1038/srep05587

    Figure Lengend Snippet: LBP therapeutically ameliorated hepatic inflammatory responses and apoptosis through modulating autophagy and MAPK pathway. (a–d) Protein expressional changes of inflammatory and chemoattractive responses markers tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), cyclooxygenase-2 (COX-2) and monocyte chemotactic protein-1 (MCP-1) from each group of rats after 12-week experiment. (e) Representative Western blot results of inhibitor of NF-κB alpha (IκBα) formation in the liver of each group of rats from 3 repeated Western blot experiments. Full-length blots are presented in Supplementary Fig. 4 . (f) Changes of nuclear factor-kappa B (NF-κB) subunit p65 in each group of rats, as measured by EIA kit. (g) Representative Western blot results of cleaved caspase-3 and cytochrome c (cyto c) in rats with different treatments from 3 repeated Western blot experiments. Full-length blots are presented in Supplementary Fig. 4 . (h,i) Changes of mRNA expression level of intrinsic apoptotic markers Bcl-2 and Bax in each group of rats. (j) Representative Western blot results of key autophagic markers including phosphorylated mTOR, total mTOR, beclin 1, Atg5, LC3II, and p62 in each group of rats from 3 repeated Western blot experiments. Full-length blots are presented in Supplementary Fig. 4 . (k) Representative Western blot results of key MAPK markers including phosphorylated and total forms of p38 MAPK, JNK and ERK1/2 in each group of rats from 3 repeated Western blot experiments. Full-length blots are presented in Supplementary Fig. 4 . Beta-actin was used as the internal control. Data from each group were expressed as means ± SEMs (n = 5 or 6). Statistical comparisons between groups were done using the Kruskal–Wallis test followed by Dunn's post hoc test to detect differences in all groups. Different letters (e.g. a and b) mean a statistical significant change ( p

    Article Snippet: Antibodies of nitrotyrosine, phosphorylated IRS-1 at Ser307, total IRS-1, phosphorylated GSK3α at Ser21, total GSK3α, phosphorylated p38 MAPK at Thr180/Tyr182, total p38 MAPK, phosphorylated JNK at Thr183/Tyr185, total JNK, phosphorylated ERK1/2 at Thr202/Tyr204, total ERK1/2, IκBα, cleaved caspase-3, cytochrome c, p62, beclin-1, Atg5, phosphorylated mTOR at Ser2448, and total mTOR were purchased from Cell Signaling (Beverly, MA).

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

    Runx2 knockdown alters expression levels of mTORC-2 proteins. A) The MDA-MB-231 cells’ transient Runx2 suppression was analyzed for mTOR and Runx2 levels. B) A quantification of mTOR protein expression normalized to β-Actin is shown. C) The stable Runx2 knockdown MDA-MB-231 cells were serum-deprived, epidermal growth factor (EGF) stimulated and examined for pmTOR (Serine 2481) and mTOR (total) protein. D) The Runx2 knockdown cells were assayed for mTOR gene expression by RT-PCR (normalized to GAPDH ). E) The Runx2 knockdown and Ad-GFP- or WT-Runx2-treated MDA-MB-231 cells were tested for Runx2 recruitment on mTOR promoter by ChIP assays. A schematic diagram of the mTOR promoter region is bars indicating potential Runx binding sites (black bars) and location of PCR primers (small arrows). F) and G) The mRNA (F) and protein (G) expression levels of Rictor were analyzed in control or Runx2 knockdown MDA-MB-231 cells by RT-PCR and Western blotting, respectively. H) The MDA-MB-231 cells stably over-expressing WT-Runx2 were assayed for Runx2 and Rictor gene expression by RT-PCR. I) The MDA-MB-231 cells with stable Rictor knockdown were stimulated with EGF and were analyzed for the expression of pAkt (Serine 473), Akt (total) and Rictor protein levels by Western blotting. J) The stably Runx2 knockdown MDA-MB-231 cells together with Rictor suppression were stimulated with EGF and analyzed for the expression of pAkt (Serine 473), Akt (total) and Rictor proteins by Western blotting. K) The glucose and serum-deprived (24 h) MDA-MB-231 cells with Runx2 knockdown (Runx2-RNAi) together with Rictor knockdown (Rictor -shRNA) were stained for Annexin V and AAD by flow cytometry. L) A quantification of positive cells with or without Rictor knockdown is shown. M) A schematic diagram depicting the function of Runx2 in regulating Akt via mTORC2 complex and its effect on survival of invasive cancer cells.

    Journal: Breast Cancer Research : BCR

    Article Title: Runx2 activates PI3K/Akt signaling via mTORC2 regulation in invasive breast cancer cells

    doi: 10.1186/bcr3611

    Figure Lengend Snippet: Runx2 knockdown alters expression levels of mTORC-2 proteins. A) The MDA-MB-231 cells’ transient Runx2 suppression was analyzed for mTOR and Runx2 levels. B) A quantification of mTOR protein expression normalized to β-Actin is shown. C) The stable Runx2 knockdown MDA-MB-231 cells were serum-deprived, epidermal growth factor (EGF) stimulated and examined for pmTOR (Serine 2481) and mTOR (total) protein. D) The Runx2 knockdown cells were assayed for mTOR gene expression by RT-PCR (normalized to GAPDH ). E) The Runx2 knockdown and Ad-GFP- or WT-Runx2-treated MDA-MB-231 cells were tested for Runx2 recruitment on mTOR promoter by ChIP assays. A schematic diagram of the mTOR promoter region is bars indicating potential Runx binding sites (black bars) and location of PCR primers (small arrows). F) and G) The mRNA (F) and protein (G) expression levels of Rictor were analyzed in control or Runx2 knockdown MDA-MB-231 cells by RT-PCR and Western blotting, respectively. H) The MDA-MB-231 cells stably over-expressing WT-Runx2 were assayed for Runx2 and Rictor gene expression by RT-PCR. I) The MDA-MB-231 cells with stable Rictor knockdown were stimulated with EGF and were analyzed for the expression of pAkt (Serine 473), Akt (total) and Rictor protein levels by Western blotting. J) The stably Runx2 knockdown MDA-MB-231 cells together with Rictor suppression were stimulated with EGF and analyzed for the expression of pAkt (Serine 473), Akt (total) and Rictor proteins by Western blotting. K) The glucose and serum-deprived (24 h) MDA-MB-231 cells with Runx2 knockdown (Runx2-RNAi) together with Rictor knockdown (Rictor -shRNA) were stained for Annexin V and AAD by flow cytometry. L) A quantification of positive cells with or without Rictor knockdown is shown. M) A schematic diagram depicting the function of Runx2 in regulating Akt via mTORC2 complex and its effect on survival of invasive cancer cells.

    Article Snippet: The antibodies for pAkt (Serine 473 and Threonine 308), Akt (total), Akt1, Akt2, pPdk1 (Serine 241), pmTOR (Serine 2448 and 2481), mTOR (total), Rictor, Raptor, GβL, pGSK-3β (Serine 9) and FOXO1 were purchased from Cell Signaling Technology , Danvers, MA, USA.

    Techniques: Expressing, Multiple Displacement Amplification, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Western Blot, Stable Transfection, shRNA, Staining, Flow Cytometry, Cytometry

    Effects of HYP on the phosphorylation of PI3K, Akt, mTOR, and p70S6K induced by HG in the cultured mesangial cells in vitro . (A–D) WB analysis (upon) for the protein expressions of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, p70S6K, and p-p70S6K treated with normal glucose (Normal), MNT, DMSO and HG at 24, 48, and 72 h, respectively, with the quantification (below); (E–H) WB analysis (upon) for the protein expressions of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, p70S6K, and p-p70S6K after the treatment with normal glucose, HG, L-HYP, H-HYP, and RAP at 72 h, respectively, with the quantification (below). The data are expressed as mean ± S.E. (A–D) ** P

    Journal: Frontiers in Pharmacology

    Article Title: Inhibition of Akt/mTOR/p70S6K Signaling Activity With Huangkui Capsule Alleviates the Early Glomerular Pathological Changes in Diabetic Nephropathy

    doi: 10.3389/fphar.2018.00443

    Figure Lengend Snippet: Effects of HYP on the phosphorylation of PI3K, Akt, mTOR, and p70S6K induced by HG in the cultured mesangial cells in vitro . (A–D) WB analysis (upon) for the protein expressions of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, p70S6K, and p-p70S6K treated with normal glucose (Normal), MNT, DMSO and HG at 24, 48, and 72 h, respectively, with the quantification (below); (E–H) WB analysis (upon) for the protein expressions of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, p70S6K, and p-p70S6K after the treatment with normal glucose, HG, L-HYP, H-HYP, and RAP at 72 h, respectively, with the quantification (below). The data are expressed as mean ± S.E. (A–D) ** P

    Article Snippet: RAP and antibodies against Akt, phosphorylated Akt (p-Akt), p70S6K, p-p70S6K, mTOR, phosphorylated mTOR (p-mTOR), 4EBP1, phosphorylated 4EBP1 (p- 4EBP1), TGF-β1, Smad2, phosphorylated Smad2 (p-Smad2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Cell Culture, In Vitro, Western Blot

    Effects of HKC on the key signaling molecules of PI3K/Akt/mTOR and TGF-β1/Smad2 signaling pathways in the kidneys of the early DN model rats. (A–E) WB analysis (upon) for the protein expressions of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, p70S6K, p-p70S6K, 4EBP1, and p-4EBP1, with the quantification (below); (F,G) WB analysis (upon) for the protein expressions of TGF-β1, Smad, p-Smad2 and GAPDH, with the quantification (below). The data are expressed as mean ± S.E. ** P

    Journal: Frontiers in Pharmacology

    Article Title: Inhibition of Akt/mTOR/p70S6K Signaling Activity With Huangkui Capsule Alleviates the Early Glomerular Pathological Changes in Diabetic Nephropathy

    doi: 10.3389/fphar.2018.00443

    Figure Lengend Snippet: Effects of HKC on the key signaling molecules of PI3K/Akt/mTOR and TGF-β1/Smad2 signaling pathways in the kidneys of the early DN model rats. (A–E) WB analysis (upon) for the protein expressions of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, p70S6K, p-p70S6K, 4EBP1, and p-4EBP1, with the quantification (below); (F,G) WB analysis (upon) for the protein expressions of TGF-β1, Smad, p-Smad2 and GAPDH, with the quantification (below). The data are expressed as mean ± S.E. ** P

    Article Snippet: RAP and antibodies against Akt, phosphorylated Akt (p-Akt), p70S6K, p-p70S6K, mTOR, phosphorylated mTOR (p-mTOR), 4EBP1, phosphorylated 4EBP1 (p- 4EBP1), TGF-β1, Smad2, phosphorylated Smad2 (p-Smad2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Western Blot

    VSMC-SIRT1 is the main sensor of CR in aortas. (A) SIRT1, SIRT3, phosphorylated (p-AMPKα) and total AMPKα (t-AMPKα), and phosphorylated (p-mTOR) and total mTOR (t-mTOR) protein expression in aortas of Apoe −/− mice in the indicated group. Protein expression was detected by Western blotting (left) and quantified by densitometry (right). Quantitative results are normalized to the AL-Con group values. n = 5 per group. G, GAPDH. (B) SIRT1 activity in aortas of Apoe −/− mice from the indicated group. n = 5 per group. (C) Representative images of IF staining of SIRT1, VSMCs (αSMA), and nuclei (Hoechst) in the suprarenal aortas of Apoe −/− mice for the indicated groups. Anti-SIRT1 and anti-αSMA antibodies were replaced by normal IgG as a negative control. A, adventitia; L, lumen. Bars, 50 µm. (D) SIRT1, SIRT3, phosphorylated and total AMPKα, and phosphorylated and total mTOR protein expression in VSMCs (isolated from the suprarenal abdominal aortas of WT mice) that were incubated with serum from AL-Con mice or CR-Con mice for 48 h. Protein expression was detected by Western blotting (left) and quantified by densitometry (right). Quantitative results are normalized to the values of the AL-Con group. Experiments were performed in triplicate. (E and F) Relative mRNA expression of genes encoding mitochondrial respiratory chain subunits (E) and genes involved in glucose and lipid metabolism (F) in WT VSMCs that were incubated with serum from AL-Con mice or CR-Con mice for 48 h. Experiments were performed in triplicate. All values are shown as the means ± SEM. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Calorie restriction protects against experimental abdominal aortic aneurysms in mice

    doi: 10.1084/jem.20151794

    Figure Lengend Snippet: VSMC-SIRT1 is the main sensor of CR in aortas. (A) SIRT1, SIRT3, phosphorylated (p-AMPKα) and total AMPKα (t-AMPKα), and phosphorylated (p-mTOR) and total mTOR (t-mTOR) protein expression in aortas of Apoe −/− mice in the indicated group. Protein expression was detected by Western blotting (left) and quantified by densitometry (right). Quantitative results are normalized to the AL-Con group values. n = 5 per group. G, GAPDH. (B) SIRT1 activity in aortas of Apoe −/− mice from the indicated group. n = 5 per group. (C) Representative images of IF staining of SIRT1, VSMCs (αSMA), and nuclei (Hoechst) in the suprarenal aortas of Apoe −/− mice for the indicated groups. Anti-SIRT1 and anti-αSMA antibodies were replaced by normal IgG as a negative control. A, adventitia; L, lumen. Bars, 50 µm. (D) SIRT1, SIRT3, phosphorylated and total AMPKα, and phosphorylated and total mTOR protein expression in VSMCs (isolated from the suprarenal abdominal aortas of WT mice) that were incubated with serum from AL-Con mice or CR-Con mice for 48 h. Protein expression was detected by Western blotting (left) and quantified by densitometry (right). Quantitative results are normalized to the values of the AL-Con group. Experiments were performed in triplicate. (E and F) Relative mRNA expression of genes encoding mitochondrial respiratory chain subunits (E) and genes involved in glucose and lipid metabolism (F) in WT VSMCs that were incubated with serum from AL-Con mice or CR-Con mice for 48 h. Experiments were performed in triplicate. All values are shown as the means ± SEM. *, P

    Article Snippet: The primary antibodies used were SIRT1 (07-131; EMD Millipore), SIRT3 (5490; Cell Signaling Technology), AMPKα (9957; Cell Signaling Technology), phosphorylated AMPKα (9957; Cell Signaling Technology), mTOR (2983; Cell Signaling Technology), phosphorylated mTOR (2971; Cell Signaling Technology), GAPDH (5174; Cell Signaling Technology), MMP2 (sc13595; Santa Cruz Biotechnology, Inc.), MMP9 (ab38898; Abcam), H3K9ac (ab10812; Abcam), H3 (ab1791; Abcam), H4K16ac (07-329; EMD Millipore), and H4 (05-858; EMD Millipore).

    Techniques: Expressing, Mouse Assay, Western Blot, Activity Assay, Staining, Negative Control, Isolation, Incubation

    Both UV and IGF-I enhance mTOR-mediated responses on 4E-BP1 on cap-dependent translation and p70S6K activity . A , MCF7 cells were treated in the same fashion as described in but harvested 2 and 4 h after treatment. 300 μg of total cell

    Journal: The Journal of Biological Chemistry

    Article Title: Stress and IGF-I Differentially Control Cell Fate through Mammalian Target of Rapamycin (mTOR) and Retinoblastoma Protein (pRB) *

    doi: 10.1074/jbc.M805724200

    Figure Lengend Snippet: Both UV and IGF-I enhance mTOR-mediated responses on 4E-BP1 on cap-dependent translation and p70S6K activity . A , MCF7 cells were treated in the same fashion as described in but harvested 2 and 4 h after treatment. 300 μg of total cell

    Article Snippet: Antibodies and Reagents —mTOR, phospho-Akt (Ser-473), phospho-4E-BP1 (Thr-37/46), 4E-BP1, phospho-eIF2α (Ser-51), eIF2E, c-Myc, caspase 7, cleaved caspase 3, and phospho-p70S6K (Thr-389) antibodies were purchased from Cell Signaling Technologies (Beverly, MA).

    Techniques: Activity Assay

    Rpap3 deletion decreases expression of R2TP clients and leads to cytoplasmic accumulation of RNA polymerase II in intestinal crypts and TA compartment. (A) Western blot analysis of preparations enriched for epithelial crypt cells from the jejunum of animals, sacrificed 6 days after the first tamoxifen injection. NOP58, PRP8, ATR and mTOR and TRRAP were detected with specific antibodies. Tubulin and GAPDH were used as loading controls. Each lane was loaded with the lysate obtained from one animal of the indicated genotype (6 animals per gel). Molecular weights are indicated on the right. See Supplemental Figure 4 for an expanded view of the membranes and signal quantification. (B) Images are tissue sections of small intestines stained by immunohistochemistry (IHC) for Rpb1, the large subunit of RNA polymerase II, from control Rpap3 flox/flox mice (blue frame, left panel), or VilCreER T2; Rpap3 flox/flox animals at day 6 (red frame, right panel). Note that the staining in stromal cells is nuclear in both wild-type and VilCreER T2 ; Rpap3 flox/flox animals (stromal cells do not express the Cre), while it becomes cytoplasmic in the mutant epithelium. The scale is identical for both pictures. Panels are representative for n=6 animals from three independent experiments. (C) Magnification of crypts from (B). The scale bar is identical for both images. (D) Schematic interpretation of the IHC in (B, C). In control epithelial cells, R2TP incorporates Rpb1 into RNA PolII, which is then imported into the nucleus. In the absence of Rpap3, neo-synthesized Rpb1 accumulates in the cytoplasm.

    Journal: bioRxiv

    Article Title: The R2TP chaperone assembles cellular machineries in intestinal CBC stem cells and progenitors

    doi: 10.1101/2019.12.19.882712

    Figure Lengend Snippet: Rpap3 deletion decreases expression of R2TP clients and leads to cytoplasmic accumulation of RNA polymerase II in intestinal crypts and TA compartment. (A) Western blot analysis of preparations enriched for epithelial crypt cells from the jejunum of animals, sacrificed 6 days after the first tamoxifen injection. NOP58, PRP8, ATR and mTOR and TRRAP were detected with specific antibodies. Tubulin and GAPDH were used as loading controls. Each lane was loaded with the lysate obtained from one animal of the indicated genotype (6 animals per gel). Molecular weights are indicated on the right. See Supplemental Figure 4 for an expanded view of the membranes and signal quantification. (B) Images are tissue sections of small intestines stained by immunohistochemistry (IHC) for Rpb1, the large subunit of RNA polymerase II, from control Rpap3 flox/flox mice (blue frame, left panel), or VilCreER T2; Rpap3 flox/flox animals at day 6 (red frame, right panel). Note that the staining in stromal cells is nuclear in both wild-type and VilCreER T2 ; Rpap3 flox/flox animals (stromal cells do not express the Cre), while it becomes cytoplasmic in the mutant epithelium. The scale is identical for both pictures. Panels are representative for n=6 animals from three independent experiments. (C) Magnification of crypts from (B). The scale bar is identical for both images. (D) Schematic interpretation of the IHC in (B, C). In control epithelial cells, R2TP incorporates Rpb1 into RNA PolII, which is then imported into the nucleus. In the absence of Rpap3, neo-synthesized Rpb1 accumulates in the cytoplasm.

    Article Snippet: Primary antibodies from Cell Signaling: ATM D2E2 (CST 2873S) rabbit at 1/1000, ATR E1S3S (CST 13934S) rabbit at 1/1000, mTOR (CST 2972S) rabbit 1/1000, p53 1C12 (CST2524S) mouse 1/1000; PRP8 (sc-30207, Santa Cruz) rabbit 1/200; from ABCAM: EFTUD2 (ab72456) rabbit 1/2000, GAPDH (ab8245 6C5) mouse 1/10000; and from Sigma: NOP58 (HPA018472) rabbit 1/100, RPAP3 (SAB1411438) rabbit 1/1000, Tubulin (I2G10) mouse 1/500, TRRAP 2TRR-1B3 (MABE1008) mouse 1/1000.

    Techniques: Expressing, Western Blot, Injection, Staining, Immunohistochemistry, Mouse Assay, Mutagenesis, Synthesized

    FRAP1 expression in Amhr2 tm3(cre)Bhr ; Ctnnb1 tm1Mmt uteri. A ) FRAP1 expression was detected by immunohistochemistry at high levels in the central areas of a leiomyoma (outlined in white) in a mutant uterus, the epithelial cells, the ES as well as in the circular myometrial layer (MC) and longitudinal myometrial layer (ML). B ) A higher magnification view of FRAP1 expression in the MC and ML of a mutant mouse. C ) In control uteri, strong FRAP1 expression is found in the EG, LE, and ES. D ) Expression in the control myometrium appears limited to the endothelial cells. LE, luminal epithelium; ES, endometrial stroma; M, myometrium; EG, endometrial glands. Bars = 50 μm ( A and C ), 100 μm ( B and D ).

    Journal: Biology of Reproduction

    Article Title: Constitutive Activation of Beta-Catenin in Uterine Stroma and Smooth Muscle Leads to the Development of Mesenchymal Tumors in Mice 1

    doi: 10.1095/biolreprod.108.075648

    Figure Lengend Snippet: FRAP1 expression in Amhr2 tm3(cre)Bhr ; Ctnnb1 tm1Mmt uteri. A ) FRAP1 expression was detected by immunohistochemistry at high levels in the central areas of a leiomyoma (outlined in white) in a mutant uterus, the epithelial cells, the ES as well as in the circular myometrial layer (MC) and longitudinal myometrial layer (ML). B ) A higher magnification view of FRAP1 expression in the MC and ML of a mutant mouse. C ) In control uteri, strong FRAP1 expression is found in the EG, LE, and ES. D ) Expression in the control myometrium appears limited to the endothelial cells. LE, luminal epithelium; ES, endometrial stroma; M, myometrium; EG, endometrial glands. Bars = 50 μm ( A and C ), 100 μm ( B and D ).

    Article Snippet: Following are the primary and secondary antibodies used in this study: anti-β-catenin (1:250; BD Transduction Laboratories, San Jose, CA); anti-KIT (1:200; R & D Systems, Minneapolis, MN); anti-TGFB3 (1:200; Abcam, Cambridge, MA); anti-HMGA2 and anti-PECAM1 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA); anti-vimentin (1:200; Santa Cruz Biotechnology); anti-Ecadherin (1:100; Santa Cruz Biotechnology); anti-pH3 (1:200; BD Transduction Laboratories); anti-CD11B (1:100; eBiosciences, San Diego, CA); anti-CD140B (1:200; Ebiosciences); anti-ACTA2, CY3-conjugated (Sigma); anti- PCNA (ready to use; Zymed, San Francisco, CA); anti-FRAP1 (1:200; Cell Signaling, Danvers, MA); AlexaFluor secondary antibodies (1:500; Invitrogen, Carlsbad, CA); and biotinylated donkey anti-mouse or anti-rabbit antibody F(ab)2 (1:1000; Jackson ImmunoResearch Laboratories, West Grove, PA).

    Techniques: Expressing, Immunohistochemistry, Mutagenesis

    Chemical inhibition of TRF1 binding to telomere by PI3K inhibitors. a Structures of ETP-47037, ETP-47228, and their corresponding “inactive analogs” ETP-51259 and ETP-50952. b PI3K/mTOR IC 50 data generated internally and reported for the inhibitors used in the study. c Time course Inhibition of AKT phosphorylation at Ser473 by ETP-47037, ETP-51259, ETP-47228, and ETP-50952 at 10 μM in CHA-9.3 cell line. d Percent inhibition of TRF1 foci by immunofluorescence in CHA-9.3 mouse lung tumor cell line treated with 10 μM of either ETP-47037, ETP-47228, or their corresponding inactive analogs (ETP-51259 and ETP-50952, respectively) relative to TRF1 levels with DMSO treatment. The ETP compound inhibitory activity on PI3K pathway is stated at the bottom of the graph. Error bars represent standard deviation. N/D not determined, n number of independent experiments

    Journal: Nature Communications

    Article Title: Modulation of telomere protection by the PI3K/AKT pathway

    doi: 10.1038/s41467-017-01329-2

    Figure Lengend Snippet: Chemical inhibition of TRF1 binding to telomere by PI3K inhibitors. a Structures of ETP-47037, ETP-47228, and their corresponding “inactive analogs” ETP-51259 and ETP-50952. b PI3K/mTOR IC 50 data generated internally and reported for the inhibitors used in the study. c Time course Inhibition of AKT phosphorylation at Ser473 by ETP-47037, ETP-51259, ETP-47228, and ETP-50952 at 10 μM in CHA-9.3 cell line. d Percent inhibition of TRF1 foci by immunofluorescence in CHA-9.3 mouse lung tumor cell line treated with 10 μM of either ETP-47037, ETP-47228, or their corresponding inactive analogs (ETP-51259 and ETP-50952, respectively) relative to TRF1 levels with DMSO treatment. The ETP compound inhibitory activity on PI3K pathway is stated at the bottom of the graph. Error bars represent standard deviation. N/D not determined, n number of independent experiments

    Article Snippet: The kinase mTOR (FRAP1; ThermoFisher, PV4753), a GFP-labeled substrate (GFP-4E BP1, ThermoFisher, PV4759), and ATP were allowed to react.

    Techniques: Inhibition, Binding Assay, Generated, Immunofluorescence, Activity Assay, Standard Deviation

    TRF1 regulation by PI3K and AKT inhibitors. a Structurally diverse PI3K and PI3K/mTOR inhibitors used in the study. b PI3K and mTOR data generated internally and reported in literature. c Representative western blot images of phosphorylated AKT-Ser473 and total AKT in CHA-9.3 mouse lung tumor cell line at 24 h after treatment with PI3K, AKT, and mTOR inhibitors as indicated. d Percent inhibition of TRF1 foci by immunofluorescence and of AKT phosphorylation at S473 (pAKT) in CHA-9.3 mouse lung tumor cell line at 24 h after treatment with the indicated inhibitors relative to TRF1 levels and to pAKT levels in control cells treated with DMSO. The inhibitors were tested at 10 μM except GSK-2126458 that was used at 1.0 μM (in c and d ). Error bars represent standard deviation. The Student’s t test was used for statistical analysis; P values are shown. n number of independent experiments

    Journal: Nature Communications

    Article Title: Modulation of telomere protection by the PI3K/AKT pathway

    doi: 10.1038/s41467-017-01329-2

    Figure Lengend Snippet: TRF1 regulation by PI3K and AKT inhibitors. a Structurally diverse PI3K and PI3K/mTOR inhibitors used in the study. b PI3K and mTOR data generated internally and reported in literature. c Representative western blot images of phosphorylated AKT-Ser473 and total AKT in CHA-9.3 mouse lung tumor cell line at 24 h after treatment with PI3K, AKT, and mTOR inhibitors as indicated. d Percent inhibition of TRF1 foci by immunofluorescence and of AKT phosphorylation at S473 (pAKT) in CHA-9.3 mouse lung tumor cell line at 24 h after treatment with the indicated inhibitors relative to TRF1 levels and to pAKT levels in control cells treated with DMSO. The inhibitors were tested at 10 μM except GSK-2126458 that was used at 1.0 μM (in c and d ). Error bars represent standard deviation. The Student’s t test was used for statistical analysis; P values are shown. n number of independent experiments

    Article Snippet: The kinase mTOR (FRAP1; ThermoFisher, PV4753), a GFP-labeled substrate (GFP-4E BP1, ThermoFisher, PV4759), and ATP were allowed to react.

    Techniques: Generated, Western Blot, Inhibition, Immunofluorescence, Standard Deviation

    MAPKAP1 Binds to Ras and Modulates the Ras-mTORC2 Interaction. (A) Schematic of full length MAPKAP1 isoform 1 wild-type (WT) and the MAPKAP1 deletion (Del) proteins with domains highlighted. CRIM, Conserved Region In The Middle. RBD, Ras Binding Domain. PH, Pleckstrin Homology. (B) Microscale thermophoresis of labeled FHH:MAPKAP1 RBD (16.8nM) with a titration series of GDP or GTPγs-loaded Ras. The binding curve is positive as the MST signal of the complex is higher than MAPKAP1 RBD alone. MST-on time of 5s, n = 3 independent replicates. (C) BioID-western blot streptavidin pulldowns and input levels for birA* control, NRAS WT , NRAS Q61K and NRAS Q61K with FHH:eGFP, MAPKAP1 WT :FHH or MAPKAP1 Del :FHH expression in CHL-1 cells. PI3K p110α subunit, Raf-1, and HA protein pulldown are controls. Pulldown normalized signal relative to control birA*:NRAS Q61K shown below. (D) Quantification of mTOR and Rictor protein levels in the streptavidin pulldowns normalized to HA pulldown. All values and statistical tests relative to birA*:NRAS Q61K , n=6 (Welch’s two-sided t-test). (E) PLA with endogenous Pan-Ras and mTOR or Rictor in MT NRAS MM485 melanoma cells. Scale bar, 20 μm. (F) PLA quantification in (E). n=3 independent experiments, 6-8 fields analyzed per condition per experiment (unpaired two-sided t-test). EV, empty vector. (G) Quantification of LocaTOR2 experiments with FHH:eGFP, MAPKAP1 WT :FHH or MAPKAP1 Del :FHH expression. All values relative to average of FHH:eGFP and MAPKAP1 WT :FHH fold induction; n=3 (unpaired two-sided t-test). (H) Quantification of FHH:eGFP, MAPKAP1 WT :FHH and MAPKAP1 Del :FHH expression relative to MAPKAP1 WT .

    Journal: Molecular cell

    Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

    doi: 10.1016/j.molcel.2018.12.001

    Figure Lengend Snippet: MAPKAP1 Binds to Ras and Modulates the Ras-mTORC2 Interaction. (A) Schematic of full length MAPKAP1 isoform 1 wild-type (WT) and the MAPKAP1 deletion (Del) proteins with domains highlighted. CRIM, Conserved Region In The Middle. RBD, Ras Binding Domain. PH, Pleckstrin Homology. (B) Microscale thermophoresis of labeled FHH:MAPKAP1 RBD (16.8nM) with a titration series of GDP or GTPγs-loaded Ras. The binding curve is positive as the MST signal of the complex is higher than MAPKAP1 RBD alone. MST-on time of 5s, n = 3 independent replicates. (C) BioID-western blot streptavidin pulldowns and input levels for birA* control, NRAS WT , NRAS Q61K and NRAS Q61K with FHH:eGFP, MAPKAP1 WT :FHH or MAPKAP1 Del :FHH expression in CHL-1 cells. PI3K p110α subunit, Raf-1, and HA protein pulldown are controls. Pulldown normalized signal relative to control birA*:NRAS Q61K shown below. (D) Quantification of mTOR and Rictor protein levels in the streptavidin pulldowns normalized to HA pulldown. All values and statistical tests relative to birA*:NRAS Q61K , n=6 (Welch’s two-sided t-test). (E) PLA with endogenous Pan-Ras and mTOR or Rictor in MT NRAS MM485 melanoma cells. Scale bar, 20 μm. (F) PLA quantification in (E). n=3 independent experiments, 6-8 fields analyzed per condition per experiment (unpaired two-sided t-test). EV, empty vector. (G) Quantification of LocaTOR2 experiments with FHH:eGFP, MAPKAP1 WT :FHH or MAPKAP1 Del :FHH expression. All values relative to average of FHH:eGFP and MAPKAP1 WT :FHH fold induction; n=3 (unpaired two-sided t-test). (H) Quantification of FHH:eGFP, MAPKAP1 WT :FHH and MAPKAP1 Del :FHH expression relative to MAPKAP1 WT .

    Article Snippet: Primary antibodies were as follows: anti-Ki67 (1:250; Thermo Fisher Scientific, RM-9106), anti-Pan-Ras (C-4) (1:50; sc-166691, Santa Cruz Biotechnology), anti-Raf-1 (C12) (1:200; sc-133, Santa Cruz Biotechnology), anti-WASL (1:500; 14306-1-AP, ProteinTech), anti-MARK2 (1:200; HPA038790, Millipore Sigma), anti-NUMB (1:1000; ab14140, Abcam), anti-EHBP1 (1:500; 17637-1-AP, ProteinTech), anti-RAFT1 (1:100; 611132, BD Transduction Laboratories), anti-MTOR (1:2000; ab2732, Abcam), anti-Rictor (1:5000; A300-459A, Bethyl Laboratories), anti-SIN1 (1:1000; A300-910A, Bethyl Laboratories) and anti-Raptor (1:1000; 20984-1-AP, ProteinTech).

    Techniques: Binding Assay, Microscale Thermophoresis, Labeling, Titration, Western Blot, Expressing, Proximity Ligation Assay, Plasmid Preparation

    Multi-Cell Line CRISPR Screen of Ras Proximal Proteins. (A) ). The right heatmap relative enrichment MT vs WT Ras in mass spectrometry data for each Ras isoform; new proteins (red), known proteins (*). Ranked by combined FDR and log 2 (PSM MT/WT Ras) score. (B) Common Ras-proximal interacting protein-protein network based on candidates with ≥1 database interaction with another common interactor. Large squares are novel and small squares are known interactors. (C) PLA in MT NRAS MM415 melanoma cells with endogenous mTOR and Pan-Ras or Ras:GTP; interaction (red), nuclei (blue). Scale bar, 20 μm. (D) Quantification of PLA analysis in (C). n=8-10 fields/condition. (E) Western blot of HA co-immunoprecipitation of empty vector (EV), FLAG-HA-6xHIS tagged NRAS WT or FHH: NRAS Q61K with endogenous mTOR, p110α and Raf1 in wild-type RAS CHL-1 cells. (F) Quantification of HA co-IP experiments as in (E). Values are normalized to HA pulldown signal and relative to NRAS WT signal, n= 6 (Wilcoxon Signed Rank Test). (G) PLA in genotyped human colorectal adenocarcinomas with endogenous Pan-Ras and mTOR. Scale bar, 20 μm. (H) Quantification of PLA analysis in (G). Each dot represents median signal per patient. n=5 patients/genotype group, ≥7 images analyzed per patient (Mann-Whitney U Test). (I) Microscale thermophoresis with labeled FHH:Raf1 RBD (18.2nM) with a titration series of GDP or GTPγs-loaded Ras. The binding curve is negative as the MST signal of the complex is lower than that of Raf1 RBD alone. MST-on time of 15s, n = 3 independent replicates. (J) Microscale thermophoresis with labeled FHH:mTOR KinaseDomain and FHH:mTOR HEAT .

    Journal: Molecular cell

    Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

    doi: 10.1016/j.molcel.2018.12.001

    Figure Lengend Snippet: Multi-Cell Line CRISPR Screen of Ras Proximal Proteins. (A) ). The right heatmap relative enrichment MT vs WT Ras in mass spectrometry data for each Ras isoform; new proteins (red), known proteins (*). Ranked by combined FDR and log 2 (PSM MT/WT Ras) score. (B) Common Ras-proximal interacting protein-protein network based on candidates with ≥1 database interaction with another common interactor. Large squares are novel and small squares are known interactors. (C) PLA in MT NRAS MM415 melanoma cells with endogenous mTOR and Pan-Ras or Ras:GTP; interaction (red), nuclei (blue). Scale bar, 20 μm. (D) Quantification of PLA analysis in (C). n=8-10 fields/condition. (E) Western blot of HA co-immunoprecipitation of empty vector (EV), FLAG-HA-6xHIS tagged NRAS WT or FHH: NRAS Q61K with endogenous mTOR, p110α and Raf1 in wild-type RAS CHL-1 cells. (F) Quantification of HA co-IP experiments as in (E). Values are normalized to HA pulldown signal and relative to NRAS WT signal, n= 6 (Wilcoxon Signed Rank Test). (G) PLA in genotyped human colorectal adenocarcinomas with endogenous Pan-Ras and mTOR. Scale bar, 20 μm. (H) Quantification of PLA analysis in (G). Each dot represents median signal per patient. n=5 patients/genotype group, ≥7 images analyzed per patient (Mann-Whitney U Test). (I) Microscale thermophoresis with labeled FHH:Raf1 RBD (18.2nM) with a titration series of GDP or GTPγs-loaded Ras. The binding curve is negative as the MST signal of the complex is lower than that of Raf1 RBD alone. MST-on time of 15s, n = 3 independent replicates. (J) Microscale thermophoresis with labeled FHH:mTOR KinaseDomain and FHH:mTOR HEAT .

    Article Snippet: Primary antibodies were as follows: anti-Ki67 (1:250; Thermo Fisher Scientific, RM-9106), anti-Pan-Ras (C-4) (1:50; sc-166691, Santa Cruz Biotechnology), anti-Raf-1 (C12) (1:200; sc-133, Santa Cruz Biotechnology), anti-WASL (1:500; 14306-1-AP, ProteinTech), anti-MARK2 (1:200; HPA038790, Millipore Sigma), anti-NUMB (1:1000; ab14140, Abcam), anti-EHBP1 (1:500; 17637-1-AP, ProteinTech), anti-RAFT1 (1:100; 611132, BD Transduction Laboratories), anti-MTOR (1:2000; ab2732, Abcam), anti-Rictor (1:5000; A300-459A, Bethyl Laboratories), anti-SIN1 (1:1000; A300-910A, Bethyl Laboratories) and anti-Raptor (1:1000; 20984-1-AP, ProteinTech).

    Techniques: CRISPR, Mass Spectrometry, Proximity Ligation Assay, Western Blot, Immunoprecipitation, Plasmid Preparation, Co-Immunoprecipitation Assay, MANN-WHITNEY, Microscale Thermophoresis, Labeling, Titration, Binding Assay