pi3k  (Echelon Biosciences)

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: SchB treatment inhibits ATR kinase activity. Kinase activities of ATR ( A ), ATM ( B ), Chk1 ( C ), PI3K ( D ), DNA-PK ( E ) and mTOR ( F ) were measured in vitro as phosphorylation activity in the presence of SchB. A flag-tagged ATR-wt plasmid was transfected into HEK293T cells. ATR protein kinases were purified by immunoprecipitation using anti-Flag M2 antibody and Protein-G agarose. The endogeneous ATM protein was purified from A549 cell lysates with anti-ATM antibody. Kinase activity was monitored for 20 min at 30°C. Chk1 (Upstate), PI3K (Echelon Biosciences), DNA-PK (Promega) and mTOR (Calbiochem) activities were measured using respective assay kits according to the manufacturer ’ s instructions. Values are presented as mean ± SD ( n = 3).

Article Snippet: Moreover, the activities of Chk1, PI3K, DNA-PK and mTOR were not affected by SchB treatment ( C–F).

Techniques: Activity Assay, In Vitro, Plasmid Preparation, Transfection, Purification, Immunoprecipitation

Journal: Respiratory Research

Article Title: Non-neuronal cholinergic system contributes to corticosteroid resistance in chronic obstructive pulmonary disease patients

doi: 10.1186/s12931-016-0467-8

Figure Lengend Snippet: Effects of aclidinium bromide on glucocorticoid response element (GRE) signal and phosphatidylinositol-3-kinase delta activation (PI3Kδ). a – d Bronchial epithelial Beas2B cells were transfected with a GRE reporter gene and stimulated with different combinations of aclidinium bromide (Acl), atropine (Atr), fluticasone propionate (Flut) and the inhibitor of PI3K LY294002. d Beas2b transfected with silencing (siRNA) negative control, and siRNA for muscarinic receptors M2 and M3 receptors were verified by western blot analysis. e Beas2b cells transfected with the GRE reporter gene and subjected to siRNA of the genes encoding muscarinic receptors M2 and M3 were stimulated with Flut. The results are expressed as the mean ± SD of three independent experiments run in triplicate. One-way ANOVA was followed by a post hoc Bonferroni test. * p

Article Snippet: PI3K activity was measured using the PI3-kinase activity ELISA (cat. no. k-1000s; Echelon Bioscience, Salt Lake City, UT, USA), in accordance with the manufacturer’s protocol.

Techniques: Activation Assay, Transfection, Negative Control, Western Blot

Journal: Oncology Reports

Article Title: Bmal1 suppresses cancer cell invasion by blocking the phosphoinositide 3-kinase-Akt-MMP-2 signaling pathway

doi: 10.3892/or.2013.2381

Figure Lengend Snippet: Bmal1 suppresses the PI3K-Akt-MMP-2 pathway. (A) Cell lysates from control or Bmal1-knockdown A549 cells were analyzed for PI3K activity by competitive ELISA. (B) The levels of Bmal1, the p85 subunits of PI3K, PTEN, Akt, phospho-Akt, and β-actin in the lysates were analyzed by western blotting. Alternatively, conditioned media were prepared using Bmal1-knockdown cells, and MMP-2 levels were analyzed by western blotting. Protein loading was verified by Ponceau S staining. (C) Control and Bmal1-knockdown cells were incubated in the presence or absence of LY294002 (5 μM), Akt inhibitor (5 μM), or OA-Hy (10 μM) for 24 h, and cellular invasiveness was compared. * P

Article Snippet: PI3K activity assay The PI3K assay was conducted using a PI3K ELISA kit (Echelon Biosciences, Salt Lake City, UT, USA) according to the manufacturer’s protocol.

Techniques: Activity Assay, Competitive ELISA, Western Blot, Staining, Incubation

Journal: Journal of the Endocrine Society

Article Title: Thyroid Hormone Receptor Beta Inhibits PI3K-Akt-mTOR Signaling Axis in Anaplastic Thyroid Cancer via Genomic Mechanisms

doi: 10.1210/jendso/bvab102

Figure Lengend Snippet: Short-term exposure to 3,5,3′-triiodothyronine (T 3 ) is insufficient to induce thyroid hormone receptor beta (TRβ)-mediated phosphoinositide 3 kinase (PI3K) suppression. A, TRβ protein was assessed in SW1736-EV (empty vector; EV) and SW1736-TRβ (TRβ) cells to ensure successful lentiviral transduction. EV and TRβ cells were treated with 10-nM T 3 or vehicle (10-µM NaOH) for 30 minutes before protein levels were determined B, by immunoblot, or C, incubated with anti-p85⍺ antibody for PI3K catalysis enzyme-linked immunosorbent assay (ELISA). ELISA signal in C was standardized to protein concentration as determined by a Bradford assay. Significance in C was calculated by 2-way analysis of variance followed by Tukey multiple comparisons test. NS, no significance ( P ≥ .05) across treatment groups.

Article Snippet: PI3K activity in the immunoprecipitates was then assayed by PI3K ELISA according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Transduction, Incubation, Enzyme-linked Immunosorbent Assay, Protein Concentration, Bradford Assay

Journal: Journal of the Endocrine Society

Article Title: Thyroid Hormone Receptor Beta Inhibits PI3K-Akt-mTOR Signaling Axis in Anaplastic Thyroid Cancer via Genomic Mechanisms

doi: 10.1210/jendso/bvab102

Figure Lengend Snippet: Liganded thyroid hormone receptor beta (TRβ) decreases expression of oncogenic genes and increases tumor-suppressive genes in the phosphoinositide 3 kinase–protein kinase B–mechanistic target of rapamycin (PI3K-Akt-mTOR) signaling axis. A, Receptor tyrosine kinases (RTKs) dimerize in response to ligands to allow for IRS2 to dock and recruit PI3K [ 33 ]. PI3K phosphorylates PI(4,5)P 2 to PIP 3 . Phosphatase and tensin homolog (PTEN) and phosphatidylinositol 4,5-bisphosphate 5-phosphatase A (INPP5J) dephosphorylate PIP 3 to PI(4,5)P 2 and PI(3,4)P 2 , respectively. Inositol polyphosphate 4-phosphatase type II (INPP4B) dephosphorylates PI(3,4)P 2 to PI(3)P. PIP 3 recruits PDK1 (not shown) and Akt to the plasma membrane for PDK1 phosphorylation of Akt T308. NIP7-activated mechanistic target of rapamycin complex 2 (mTORC2) phosphorylates Akt S473, which is dephosphorylated by PH domain and leucine-rich repeat-protein phosphatase 1 (PHLPP1) [ 34 ]. ING5 has been shown to dephosphorylate Akt in hormone-dependent cancers [ 35 ]. The 2R5B subunit of protein phosphatase 2 (PPP2) directs the complex to dephosphorylate Akt T308 and S473 [ 36 ]. TNK2 phosphorylates Akt Y176 to enhance plasma membrane recruitment [ 37 ]. Akt leads to the activation of mTORC1 by TSC 1/2-Rheb (not shown). Nix and PML destabilize Rheb-mTORC1 binding [ 38 ]. Galectin-8 inhibits and delocalizes mTORC1 [ 39 ]. Glycogen synthase kinase 3 beta (GSK3β) is inhibited by Akt and inhibits multiple substrates relevant to glucose and glycogen metabolism, survival signaling, and cell cycle progression. mTORC1 phosphorylates and inhibits 4E-BP1, which inhibits EIF4E. mTORC1 also activates P70S6K, which inhibits GSK3β and PDCD4 and activates EIF4B and RPS6. EIF4B and PDCD4 regulate EIF4A [ 38 ]. B, Ligand-bound TRβ decreased expression of RTKs. C, Ligand-bound TRβ increased expression of PI3K-Akt phosphatases. Significance and fold-change values between empty vector (EV) + T 3 and TRβ + T 3 are located in Supplementary Table 2. Data were previously generated via RNA-sequencing [ 16 ].

Article Snippet: PI3K activity in the immunoprecipitates was then assayed by PI3K ELISA according to the manufacturer’s instructions.

Techniques: Expressing, Activation Assay, Binding Assay, Plasmid Preparation, Generated, RNA Sequencing Assay

Journal: Journal of the Endocrine Society

Article Title: Thyroid Hormone Receptor Beta Inhibits PI3K-Akt-mTOR Signaling Axis in Anaplastic Thyroid Cancer via Genomic Mechanisms

doi: 10.1210/jendso/bvab102

Figure Lengend Snippet: Short-term or no exposure to 3,5,3′-triiodothyronine (T 3 ) is insufficient for thyroid hormone receptor beta (TRβ) to enhance LY294002 (LY) suppression of phosphoinositide 3 kinase (PI3K). SW1736-EV (empty vector; EV) and SW1736-TRβ (TRβ) cells were treated with 10-nM T 3 or vehicle (10 µM NaOH) for 30 minutes before 1 hour of 10-µM LY treatment. Protein levels were determined by A, immunoblot or B, incubated with anti-p85⍺ antibody for PI3K catalysis enzyme-linked immunosorbent assay (ELISA). ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B was calculated by 2-way analysis of variance followed by a Tukey multiple comparisons test. NS, no significance ( P ≥ .05) across treatment groups.

Article Snippet: PI3K activity in the immunoprecipitates was then assayed by PI3K ELISA according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Incubation, Enzyme-linked Immunosorbent Assay, Protein Concentration, Bradford Assay