class iii pi3 kinase kit  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences class iii pi3 kinase kit
    scDNAs induce autophagy by releasing Rubicon from the Beclin-1-phosphatidylinositol <t>3-kinase</t> <t>class</t> <t>III</t> (PI3KC3) complex. a , HEK293T-cGAS or HEK293T-Vector cells were transfected with Flag-Beclin-1 plasmid and stimulated with scDNAs or HT-DNA (1 µg/mL). The cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. b , RAW264.7 cells stably expressing p40PX-EGFP were stimulated with scDNAs (1 µg/mL) or mock-transfected. The number of p40PX-EGFP foci was determined using confocal microscopy. Data are expressed as mean ± s.e.m of three biological replicates, n ≥ 200 cells. Statistical analysis was performed using two-sided Student’s t -tests; ** indicates p
    Class Iii Pi3 Kinase Kit, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    class iii pi3 kinase kit - by Bioz Stars, 2022-12
    93/100 stars

    Images

    1) Product Images from "Small cytosolic dsDNAs repress cGAS activation and induce autophagy"

    Article Title: Small cytosolic dsDNAs repress cGAS activation and induce autophagy

    Journal: bioRxiv

    doi: 10.1101/2022.06.30.498186

    scDNAs induce autophagy by releasing Rubicon from the Beclin-1-phosphatidylinositol 3-kinase class III (PI3KC3) complex. a , HEK293T-cGAS or HEK293T-Vector cells were transfected with Flag-Beclin-1 plasmid and stimulated with scDNAs or HT-DNA (1 µg/mL). The cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. b , RAW264.7 cells stably expressing p40PX-EGFP were stimulated with scDNAs (1 µg/mL) or mock-transfected. The number of p40PX-EGFP foci was determined using confocal microscopy. Data are expressed as mean ± s.e.m of three biological replicates, n ≥ 200 cells. Statistical analysis was performed using two-sided Student’s t -tests; ** indicates p
    Figure Legend Snippet: scDNAs induce autophagy by releasing Rubicon from the Beclin-1-phosphatidylinositol 3-kinase class III (PI3KC3) complex. a , HEK293T-cGAS or HEK293T-Vector cells were transfected with Flag-Beclin-1 plasmid and stimulated with scDNAs or HT-DNA (1 µg/mL). The cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. b , RAW264.7 cells stably expressing p40PX-EGFP were stimulated with scDNAs (1 µg/mL) or mock-transfected. The number of p40PX-EGFP foci was determined using confocal microscopy. Data are expressed as mean ± s.e.m of three biological replicates, n ≥ 200 cells. Statistical analysis was performed using two-sided Student’s t -tests; ** indicates p

    Techniques Used: Plasmid Preparation, Transfection, Immunoprecipitation, Stable Transfection, Expressing, Confocal Microscopy

    2) Product Images from "Group V Secretory Phospholipase A2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line"

    Article Title: Group V Secretory Phospholipase A2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

    Journal: Journal of Atherosclerosis and Thrombosis

    doi: 10.5551/jat.62216

    A, Suppression of Beclin1 phosphorylation at S30 (p-Beclin1/Beclin1) by siRNA (#1 and #2)-mediated reduction of PGK1 expression. Values were normalized to that of control siRNA after incubation with PBS as a vehicle (=1). n =5 in each experiment. ** , P <0.01 vs. control siRNA. B, Representative immunoblots for panel A. C, Successful suppression of PGK1 expression by siRNA confirmed by immunoblotting. D, E, F, Comparison of PGK1 expression (D), Beclin1 phosphorylation at S30 (E), and PI3-kinase activity (F) after incubation for 2 hr with 20 µg/mL AcLDL or PBS as a vehicle in sPLA 2 -V-WT and KD cells, and sPLA 2 -V KD cells with re-constitutive expression of sPLA 2 -V or sPLA 2 -V-H48Q. G, Representative immunoblots showing PGK1 expression and Beclin1 phosphorylation in various types of RAW264.7 cells. Values in panels D and E were normalized to that of WT after incubation with PBS as a vehicle (=1). Each bar represents the mean±SEM of 5–6 independent experiments. * , P <0.05, ** , P <0.01 vs. WT, † , P <0.05, †† , P <0.01 vs. KD.
    Figure Legend Snippet: A, Suppression of Beclin1 phosphorylation at S30 (p-Beclin1/Beclin1) by siRNA (#1 and #2)-mediated reduction of PGK1 expression. Values were normalized to that of control siRNA after incubation with PBS as a vehicle (=1). n =5 in each experiment. ** , P <0.01 vs. control siRNA. B, Representative immunoblots for panel A. C, Successful suppression of PGK1 expression by siRNA confirmed by immunoblotting. D, E, F, Comparison of PGK1 expression (D), Beclin1 phosphorylation at S30 (E), and PI3-kinase activity (F) after incubation for 2 hr with 20 µg/mL AcLDL or PBS as a vehicle in sPLA 2 -V-WT and KD cells, and sPLA 2 -V KD cells with re-constitutive expression of sPLA 2 -V or sPLA 2 -V-H48Q. G, Representative immunoblots showing PGK1 expression and Beclin1 phosphorylation in various types of RAW264.7 cells. Values in panels D and E were normalized to that of WT after incubation with PBS as a vehicle (=1). Each bar represents the mean±SEM of 5–6 independent experiments. * , P <0.05, ** , P <0.01 vs. WT, † , P <0.05, †† , P <0.01 vs. KD.

    Techniques Used: Expressing, Activity Assay, Incubation, Western Blot

    3) Product Images from "Thyroid Hormone Receptor Beta Inhibits PI3K-Akt-mTOR Signaling Axis in Anaplastic Thyroid Cancer via Genomic Mechanisms"

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

    Journal: Journal of the Endocrine Society

    doi: 10.1210/jendso/bvab102

    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.
    Figure Legend 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.

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

    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 ].
    Figure Legend 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 ].

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

    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.
    Figure Legend 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.

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

    4) Product Images from "Thyroid Hormone Receptor Beta Inhibits PI3K-Akt-mTOR Signaling Axis in Anaplastic Thyroid Cancer via Genomic Mechanisms"

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

    Journal: Journal of the Endocrine Society

    doi: 10.1210/jendso/bvab102

    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.
    Figure Legend 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.

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

    Phosphoinositide 3 kinase (PI3K) signaling genes regulated by thyroid hormone receptor beta (TRβ) in SW1736 cells are aberrantly expressed in patient thyroid cancer samples. Patient thyroid cancer microarray data (GSE76039, GSE3467, GSE82208) were analyzed for genes encoding A, receptor tyrosine kinases; B, phosphoinositide and protein kinase B (Akt) phosphatases; and C, TRβ, enzymes, and transporters necessary for synthesizing thyroid hormones. Significance was determined by one-way analysis of variance followed by Dunnett multiple comparisons test NS, no significance ( P ≥ .05, * P
    Figure Legend Snippet: Phosphoinositide 3 kinase (PI3K) signaling genes regulated by thyroid hormone receptor beta (TRβ) in SW1736 cells are aberrantly expressed in patient thyroid cancer samples. Patient thyroid cancer microarray data (GSE76039, GSE3467, GSE82208) were analyzed for genes encoding A, receptor tyrosine kinases; B, phosphoinositide and protein kinase B (Akt) phosphatases; and C, TRβ, enzymes, and transporters necessary for synthesizing thyroid hormones. Significance was determined by one-way analysis of variance followed by Dunnett multiple comparisons test NS, no significance ( P ≥ .05, * P

    Techniques Used: Microarray

    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 ].
    Figure Legend 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 ].

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

    Long-term exposure to 3,5,3′-triiodothyronine (T 3 ) is required to induce thyroid hormone receptor beta (TRβ)-mediated suppression of the phosphoinositide 3 kinase–protein kinase B–mechanistic target of rapamycin (PI3K-Akt-mTOR) pathway. SW1736-EV (empty vector; EV) and SW1736-TRβ (TRβ) cells were treated with 10-nM T 3 for 24 hours before protein levels were determined by immunoblot (A and quantified in C) or B, subjugated to a pAkt Ser473 sandwich enzyme-linked immunosorbent assay (ELISA). The samples in A are biological replicates. Dashed lines in immunoblots indicate gap between 2 sets of lanes on the same membrane. ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B and C was calculated by t test. NS, no significance ( P ≥ .05, * P
    Figure Legend Snippet: Long-term exposure to 3,5,3′-triiodothyronine (T 3 ) is required to induce thyroid hormone receptor beta (TRβ)-mediated suppression of the phosphoinositide 3 kinase–protein kinase B–mechanistic target of rapamycin (PI3K-Akt-mTOR) pathway. SW1736-EV (empty vector; EV) and SW1736-TRβ (TRβ) cells were treated with 10-nM T 3 for 24 hours before protein levels were determined by immunoblot (A and quantified in C) or B, subjugated to a pAkt Ser473 sandwich enzyme-linked immunosorbent assay (ELISA). The samples in A are biological replicates. Dashed lines in immunoblots indicate gap between 2 sets of lanes on the same membrane. ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B and C was calculated by t test. NS, no significance ( P ≥ .05, * P

    Techniques Used: Plasmid Preparation, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Western Blot, Protein Concentration, Bradford Assay

    Thyroid hormone receptor beta (TRβ) enhances LY294002 (LY)-mediated inactivation of the phosphoinositide 3 kinase–protein kinase B–mechanistic target of rapamycin (PI3K-Akt-mTOR) axis. SW1736-EV (empty vector; EV) and SW1736-TRβ (TRβ) cells were treated with 10-nM 3,5,3′-triiodothyronine (T 3 ) for 24 hours before 1 hour of LY treatment (A, 10 µM; B, 0, 1, or 10 µM). Protein levels were determined by immunoblot (A and quantified in C) or B, subjugated to a pAkt Ser473 sandwich enzyme-linked immunosorbent assay (ELISA). Samples in A are biological replicates. Dashed lines in immunoblots indicate gap between 2 sets of lanes on the same membrane. ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B was calculated by one-way analysis of variance followed by a Sidak multiple comparison test. Significance in C was calculated by t test. NS, no significance ( P ≥ .05, * P
    Figure Legend Snippet: Thyroid hormone receptor beta (TRβ) enhances LY294002 (LY)-mediated inactivation of the phosphoinositide 3 kinase–protein kinase B–mechanistic target of rapamycin (PI3K-Akt-mTOR) axis. SW1736-EV (empty vector; EV) and SW1736-TRβ (TRβ) cells were treated with 10-nM 3,5,3′-triiodothyronine (T 3 ) for 24 hours before 1 hour of LY treatment (A, 10 µM; B, 0, 1, or 10 µM). Protein levels were determined by immunoblot (A and quantified in C) or B, subjugated to a pAkt Ser473 sandwich enzyme-linked immunosorbent assay (ELISA). Samples in A are biological replicates. Dashed lines in immunoblots indicate gap between 2 sets of lanes on the same membrane. ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B was calculated by one-way analysis of variance followed by a Sidak multiple comparison test. Significance in C was calculated by t test. NS, no significance ( P ≥ .05, * P

    Techniques Used: Plasmid Preparation, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Western Blot, Protein Concentration, Bradford Assay

    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.
    Figure Legend 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.

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

    5) Product Images from "Thyroid hormone receptor beta inhibits the PI3K-Akt-mTOR signaling axis in anaplastic thyroid cancer via genomic mechanisms"

    Article Title: Thyroid hormone receptor beta inhibits the PI3K-Akt-mTOR signaling axis in anaplastic thyroid cancer via genomic mechanisms

    Journal: bioRxiv

    doi: 10.1101/2020.11.12.379933

    TRβ enhances LY294002-mediated inactivation of the PI3K-Akt-mTOR axis. SW1736-EV (EV) and SW1736-TRβ (TRβ) cells were treated with 10 nM T 3 for 24 hours before 1 hour of LY294002 (LY) treatment ( A and C: 10 μM, B: 0, 1, or 10 μM). Protein levels were determined by immunoblot ( A and C ) or subjugated to a pAkt Ser473 sandwich ELISA ( B ). Samples in A are replicates. Dashed lines in immunoblots indicate gap between two sets of lanes on the same membrane. ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B was calculated by one-way ANOVA followed by Sidak’s multiple comparison test. (*p
    Figure Legend Snippet: TRβ enhances LY294002-mediated inactivation of the PI3K-Akt-mTOR axis. SW1736-EV (EV) and SW1736-TRβ (TRβ) cells were treated with 10 nM T 3 for 24 hours before 1 hour of LY294002 (LY) treatment ( A and C: 10 μM, B: 0, 1, or 10 μM). Protein levels were determined by immunoblot ( A and C ) or subjugated to a pAkt Ser473 sandwich ELISA ( B ). Samples in A are replicates. Dashed lines in immunoblots indicate gap between two sets of lanes on the same membrane. ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B was calculated by one-way ANOVA followed by Sidak’s multiple comparison test. (*p

    Techniques Used: Sandwich ELISA, Western Blot, Enzyme-linked Immunosorbent Assay, Protein Concentration, Bradford Assay

    Short-term or no exposure to T 3 is insufficient for TRβ to enhance LY294002 suppression of PI3K. SW1736-EV (EV) and SW1736-TRβ (TRβ) cells were treated with 10 nM T 3 or vehicle (1 N NaOH) for 30 min before 1 hour of 10 μM LY294002 (LY) treatment. Protein levels were determined by immunoblot ( A ) or incubated with anti-p85α antibody for PI3K catalysis ELISA ( B ). ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B was calculated by two-way ANOVA followed by Tukey multiple comparisons test. ns = no significance (p ≥ 0.05) across treatment groups.
    Figure Legend Snippet: Short-term or no exposure to T 3 is insufficient for TRβ to enhance LY294002 suppression of PI3K. SW1736-EV (EV) and SW1736-TRβ (TRβ) cells were treated with 10 nM T 3 or vehicle (1 N NaOH) for 30 min before 1 hour of 10 μM LY294002 (LY) treatment. Protein levels were determined by immunoblot ( A ) or incubated with anti-p85α antibody for PI3K catalysis ELISA ( B ). ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B was calculated by two-way ANOVA followed by Tukey multiple comparisons test. ns = no significance (p ≥ 0.05) across treatment groups.

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

    Short-term or no exposure to T 3 is insufficient for TRβ-mediated PI3K suppression. SW1736-EV (EV) and SW1736-TRβ (TRβ) cells were treated with 10 nM T 3 or vehicle (1 N NaOH) for 30 min before protein levels were determined by immunoblot ( A ) or incubated with anti-p85α antibody for PI3K catalysis ELISA ( B ). ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B was calculated by two-way ANOVA followed by Tukey multiple comparisons test. ns = no significance (p ≥ 0.05) across treatment groups.
    Figure Legend Snippet: Short-term or no exposure to T 3 is insufficient for TRβ-mediated PI3K suppression. SW1736-EV (EV) and SW1736-TRβ (TRβ) cells were treated with 10 nM T 3 or vehicle (1 N NaOH) for 30 min before protein levels were determined by immunoblot ( A ) or incubated with anti-p85α antibody for PI3K catalysis ELISA ( B ). ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B was calculated by two-way ANOVA followed by Tukey multiple comparisons test. ns = no significance (p ≥ 0.05) across treatment groups.

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

    Long-term T 3 with TRβ inhibits the PI3K-Akt-mTOR pathway in SW1736 cells. SW1736-EV (EV) and SW1736-TRβ (TRβ) cells were treated with 10 nM T 3 for 24 hours before protein levels were determined by immunoblot ( A and C ) or subjugated to a pAkt Ser473 sandwich ELISA ( B ). The samples in A are replicates. Dashed lines in immunoblots indicate gap between two sets of lanes on the same membrane. ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B was calculated by Student’s t-test. (*p
    Figure Legend Snippet: Long-term T 3 with TRβ inhibits the PI3K-Akt-mTOR pathway in SW1736 cells. SW1736-EV (EV) and SW1736-TRβ (TRβ) cells were treated with 10 nM T 3 for 24 hours before protein levels were determined by immunoblot ( A and C ) or subjugated to a pAkt Ser473 sandwich ELISA ( B ). The samples in A are replicates. Dashed lines in immunoblots indicate gap between two sets of lanes on the same membrane. ELISA signal in B was standardized to protein concentration as determined by a Bradford assay. Significance in B was calculated by Student’s t-test. (*p

    Techniques Used: Sandwich ELISA, Western Blot, Enzyme-linked Immunosorbent Assay, Protein Concentration, Bradford Assay

    6) Product Images from "The Ancient Phosphatidylinositol 3-Kinase Signaling System Is a Master Regulator of Energy and Carbon Metabolism in Algae 1The Ancient Phosphatidylinositol 3-Kinase Signaling System Is a Master Regulator of Energy and Carbon Metabolism in Algae 1 [OPEN]"

    Article Title: The Ancient Phosphatidylinositol 3-Kinase Signaling System Is a Master Regulator of Energy and Carbon Metabolism in Algae 1The Ancient Phosphatidylinositol 3-Kinase Signaling System Is a Master Regulator of Energy and Carbon Metabolism in Algae 1 [OPEN]

    Journal: Plant Physiology

    doi: 10.1104/pp.17.01780

    Phylogenetic analysis of class III PI3K enzymes from algae, plant, and animal kingdoms and screening of VPS34 knockdown mutants through physiological analyses. A, Phylogenetic tree of class III PI3K using amino acid sequences from algae, plant, and animal kingdoms. The tree was constructed using a maximum parsimony method, and the bootstrap values (1,000 replicates) are shown on each node. Heterodimeric class III PI3K (vacuolar protein sorting 34, VPS34 ) of C. reinhardtii was clustered with other green algal homologs (green). PI3Ks from plants are clustered together (orange) and are closely related to green algae. The animal PI3Ks are clustered together (purple), fungi, yeast (gray), and other algal PI3Ks are not related to plant and animal PI3Ks and may have different origins. B, C. reinhardtii cc124 (wild type [WT]), the mock and the KD lines were grown in photoautrophic and mixotrophic conditions in the presence and absence of N. C, The growth pattern of wild type, mock, and mutant lines. D, Principal component analyses (PCAs) of various morphological, physiological, and genetic analyses such as area and shape, total lipid content, TAG levels, and VPS34 .
    Figure Legend Snippet: Phylogenetic analysis of class III PI3K enzymes from algae, plant, and animal kingdoms and screening of VPS34 knockdown mutants through physiological analyses. A, Phylogenetic tree of class III PI3K using amino acid sequences from algae, plant, and animal kingdoms. The tree was constructed using a maximum parsimony method, and the bootstrap values (1,000 replicates) are shown on each node. Heterodimeric class III PI3K (vacuolar protein sorting 34, VPS34 ) of C. reinhardtii was clustered with other green algal homologs (green). PI3Ks from plants are clustered together (orange) and are closely related to green algae. The animal PI3Ks are clustered together (purple), fungi, yeast (gray), and other algal PI3Ks are not related to plant and animal PI3Ks and may have different origins. B, C. reinhardtii cc124 (wild type [WT]), the mock and the KD lines were grown in photoautrophic and mixotrophic conditions in the presence and absence of N. C, The growth pattern of wild type, mock, and mutant lines. D, Principal component analyses (PCAs) of various morphological, physiological, and genetic analyses such as area and shape, total lipid content, TAG levels, and VPS34 .

    Techniques Used: Construct, Mutagenesis

    7) Product Images from "ψ-Bufarenogin, a novel anti-tumor compound, suppresses liver cancer growth by inhibiting receptor tyrosine kinase-mediated signaling"

    Article Title: ψ-Bufarenogin, a novel anti-tumor compound, suppresses liver cancer growth by inhibiting receptor tyrosine kinase-mediated signaling

    Journal: Oncotarget

    doi:

    ψ-Bufarenogin suppresses Raf/MEK/ERKs and PI3-K/Akt cascades (A B) Hepatoma cells pretreated with ψ-Bufarenogin as indicated were exposed to EGF for 15 min followed by western blot assay. (C) H E staining and immunohistochemistry of p-MEK in the HCC xenografts of nude mice treated with ψ-Bufarenogin (i.v.). Representative pictures are shown. The extracts of HCC xenografts from nude mice described above were analyzed by western blot assay. (D) SMMC-7721 cells were infected by Ad-DN-Akt or Ad-GFP, and Mcl-1 and Sox2 expression was determined by western blot assay.
    Figure Legend Snippet: ψ-Bufarenogin suppresses Raf/MEK/ERKs and PI3-K/Akt cascades (A B) Hepatoma cells pretreated with ψ-Bufarenogin as indicated were exposed to EGF for 15 min followed by western blot assay. (C) H E staining and immunohistochemistry of p-MEK in the HCC xenografts of nude mice treated with ψ-Bufarenogin (i.v.). Representative pictures are shown. The extracts of HCC xenografts from nude mice described above were analyzed by western blot assay. (D) SMMC-7721 cells were infected by Ad-DN-Akt or Ad-GFP, and Mcl-1 and Sox2 expression was determined by western blot assay.

    Techniques Used: Western Blot, Staining, Immunohistochemistry, Mouse Assay, Infection, Expressing

    8) Product Images from "Tanshinone IIA promotes non-amyloidogenic processing of amyloid precursor protein in platelets via estrogen receptor signaling to phosphatidylinositol 3-kinase/Akt"

    Article Title: Tanshinone IIA promotes non-amyloidogenic processing of amyloid precursor protein in platelets via estrogen receptor signaling to phosphatidylinositol 3-kinase/Akt

    Journal: Biomedical Reports

    doi: 10.3892/br.2014.263

    Estrogenic effect of Tanshinone IIA (Tan IIA) on platelet phosphatidylinositol 3-kinase (PI3K) activity. The platelets were treated with Tan IIA in the presence or absence of the estrogen receptor α-specific antagonist methyl-piperidinopyrazole (MPP) at 37°C for 24 h. Subsequently, the platelets were collected and lysed. PI3K activity was then measured using a PI3K ELISA kit. Values are presented as means ± SD of five determinations. * P
    Figure Legend Snippet: Estrogenic effect of Tanshinone IIA (Tan IIA) on platelet phosphatidylinositol 3-kinase (PI3K) activity. The platelets were treated with Tan IIA in the presence or absence of the estrogen receptor α-specific antagonist methyl-piperidinopyrazole (MPP) at 37°C for 24 h. Subsequently, the platelets were collected and lysed. PI3K activity was then measured using a PI3K ELISA kit. Values are presented as means ± SD of five determinations. * P

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay

    9) Product Images from "Enhanced myeloid differentiation factor 88 promotes tumor metastasis via induction of epithelial-mesenchymal transition in human hepatocellular carcinoma"

    Article Title: Enhanced myeloid differentiation factor 88 promotes tumor metastasis via induction of epithelial-mesenchymal transition in human hepatocellular carcinoma

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.71

    MyD88 associates with p85 to activate PI3-K/Akt signaling. ( a ) PI3-K was immunoprecipitated and its activity was detected as described in Materials and Methods. ( b , c ) After overexpression or knockdown of MyD88, the cell lysates were subjected to coimmunoprecipitation and western blot analysis. ( d ) PLC/PRF/5 cells stably expressing MyD88 were transfected with Δp85, and the phosphorylation level of Akt was analyzed by western blot assay
    Figure Legend Snippet: MyD88 associates with p85 to activate PI3-K/Akt signaling. ( a ) PI3-K was immunoprecipitated and its activity was detected as described in Materials and Methods. ( b , c ) After overexpression or knockdown of MyD88, the cell lysates were subjected to coimmunoprecipitation and western blot analysis. ( d ) PLC/PRF/5 cells stably expressing MyD88 were transfected with Δp85, and the phosphorylation level of Akt was analyzed by western blot assay

    Techniques Used: Immunoprecipitation, Activity Assay, Over Expression, Western Blot, Planar Chromatography, Stable Transfection, Expressing, Transfection

    10) Product Images from "Potential role of ATM in hepatocyte endocytosis of ApoE-deficient, ApoB48-containing lipoprotein in ApoE-deficient mice"

    Article Title: Potential role of ATM in hepatocyte endocytosis of ApoE-deficient, ApoB48-containing lipoprotein in ApoE-deficient mice

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2013.1566

    Analysis of the interaction betweetn ataxia telangiectasia mutated (ATM) and class III phosphatidylinositol-3-kinases (PI3Ks) by co-immunoprecipitation and augmentation of class III PI3K activity induced by the activation of ATM protein by chloroquine. (A) Representative protein bands from co-immunoprecipitation analysis between and ATM and class III PI3K protein. Hepatocytes from apolipoprotein (Apo)E-deficient mice ( ApoE −/− mice) were pre-treated with 5 μmol/l chloroquine for 1 h and then treated with 40 μg/ml ApoE-deficient, ApoB48-containing (E − /B 48 ) lipoproteins for 8 h at 37°C. Cells were collected and homogenated. Protein in the cytoplasm was extracted and immunoprecipitated with ATM and class III PI3K proteins. Western blot analysis was performed to determine whether the immunoprecipitates contained ATM and class III PI3K proteins. (B) Class III PI3K activity was assessed by ELISA. Hepatocytes from ApoE −/− mice were incubated with 5 μmol/l chloroquine, 40 μg/ml E − /B 48 lipoproteins and 10 μmol/l KU55933 (an ATM inhibitor) for 8 h at 37°C. Cells were collected and centrifuged. Cytoplasmic proteins from the cells were extracted using the Nuclear-Cytosol Extraction kit. Class III PI3K protein in the cytoplasm was immunoprecipitated, and then its activity was determined using the class III PI3K activity ELISA kit. Values represent the means ± SEM of 5 mice. * P
    Figure Legend Snippet: Analysis of the interaction betweetn ataxia telangiectasia mutated (ATM) and class III phosphatidylinositol-3-kinases (PI3Ks) by co-immunoprecipitation and augmentation of class III PI3K activity induced by the activation of ATM protein by chloroquine. (A) Representative protein bands from co-immunoprecipitation analysis between and ATM and class III PI3K protein. Hepatocytes from apolipoprotein (Apo)E-deficient mice ( ApoE −/− mice) were pre-treated with 5 μmol/l chloroquine for 1 h and then treated with 40 μg/ml ApoE-deficient, ApoB48-containing (E − /B 48 ) lipoproteins for 8 h at 37°C. Cells were collected and homogenated. Protein in the cytoplasm was extracted and immunoprecipitated with ATM and class III PI3K proteins. Western blot analysis was performed to determine whether the immunoprecipitates contained ATM and class III PI3K proteins. (B) Class III PI3K activity was assessed by ELISA. Hepatocytes from ApoE −/− mice were incubated with 5 μmol/l chloroquine, 40 μg/ml E − /B 48 lipoproteins and 10 μmol/l KU55933 (an ATM inhibitor) for 8 h at 37°C. Cells were collected and centrifuged. Cytoplasmic proteins from the cells were extracted using the Nuclear-Cytosol Extraction kit. Class III PI3K protein in the cytoplasm was immunoprecipitated, and then its activity was determined using the class III PI3K activity ELISA kit. Values represent the means ± SEM of 5 mice. * P

    Techniques Used: Immunoprecipitation, Activity Assay, Activation Assay, Mouse Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation

    Class III phosphatidylinositol-3-kinase (PI3K) inhibitor attenuates intracellular total cholesterol accumulation induced by ataxia telangiectasia muta ted (ATM) activation. Hepatocytes were isolated from apolipoprotein (Apo)E-deficient mice ( ApoE −/− mice) by perfusion of the portal vein and pre-treated with 5 μM chloroquine, 10 μmol/l KU55933 (an ATM inhibitor), 10 μM LY290042 (a class III PI3K inhibitor) or 5 mM 3-MA (a class III PI3K inhibitor) for 2 h and then incubated with 40 μg/ml ApoE-deficient, ApoB48-containing (E − /B 48 ) lipoproteins for 22 h at 37°C. Cellular total cholesterol in hepatocytes was determined using an enzymatic kit. Values represent the means ± SEM of 8 mice. * P
    Figure Legend Snippet: Class III phosphatidylinositol-3-kinase (PI3K) inhibitor attenuates intracellular total cholesterol accumulation induced by ataxia telangiectasia muta ted (ATM) activation. Hepatocytes were isolated from apolipoprotein (Apo)E-deficient mice ( ApoE −/− mice) by perfusion of the portal vein and pre-treated with 5 μM chloroquine, 10 μmol/l KU55933 (an ATM inhibitor), 10 μM LY290042 (a class III PI3K inhibitor) or 5 mM 3-MA (a class III PI3K inhibitor) for 2 h and then incubated with 40 μg/ml ApoE-deficient, ApoB48-containing (E − /B 48 ) lipoproteins for 22 h at 37°C. Cellular total cholesterol in hepatocytes was determined using an enzymatic kit. Values represent the means ± SEM of 8 mice. * P

    Techniques Used: Activation Assay, Isolation, Mouse Assay, Incubation

    11) Product Images from "Potential role of ATM in hepatocyte endocytosis of ApoE-deficient, ApoB48-containing lipoprotein in ApoE-deficient mice"

    Article Title: Potential role of ATM in hepatocyte endocytosis of ApoE-deficient, ApoB48-containing lipoprotein in ApoE-deficient mice

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2013.1566

    Analysis of the interaction betweetn ataxia telangiectasia mutated (ATM) and class III phosphatidylinositol-3-kinases (PI3Ks) by co-immunoprecipitation and augmentation of class III PI3K activity induced by the activation of ATM protein by chloroquine. (A) Representative protein bands from co-immunoprecipitation analysis between and ATM and class III PI3K protein. Hepatocytes from apolipoprotein (Apo)E-deficient mice ( ApoE −/− mice) were pre-treated with 5 μmol/l chloroquine for 1 h and then treated with 40 μg/ml ApoE-deficient, ApoB48-containing (E − /B 48 ) lipoproteins for 8 h at 37°C. Cells were collected and homogenated. Protein in the cytoplasm was extracted and immunoprecipitated with ATM and class III PI3K proteins. Western blot analysis was performed to determine whether the immunoprecipitates contained ATM and class III PI3K proteins. (B) Class III PI3K activity was assessed by ELISA. Hepatocytes from ApoE −/− mice were incubated with 5 μmol/l chloroquine, 40 μg/ml E − /B 48 lipoproteins and 10 μmol/l KU55933 (an ATM inhibitor) for 8 h at 37°C. Cells were collected and centrifuged. Cytoplasmic proteins from the cells were extracted using the Nuclear-Cytosol Extraction kit. Class III PI3K protein in the cytoplasm was immunoprecipitated, and then its activity was determined using the class III PI3K activity ELISA kit. Values represent the means ± SEM of 5 mice. * P
    Figure Legend Snippet: Analysis of the interaction betweetn ataxia telangiectasia mutated (ATM) and class III phosphatidylinositol-3-kinases (PI3Ks) by co-immunoprecipitation and augmentation of class III PI3K activity induced by the activation of ATM protein by chloroquine. (A) Representative protein bands from co-immunoprecipitation analysis between and ATM and class III PI3K protein. Hepatocytes from apolipoprotein (Apo)E-deficient mice ( ApoE −/− mice) were pre-treated with 5 μmol/l chloroquine for 1 h and then treated with 40 μg/ml ApoE-deficient, ApoB48-containing (E − /B 48 ) lipoproteins for 8 h at 37°C. Cells were collected and homogenated. Protein in the cytoplasm was extracted and immunoprecipitated with ATM and class III PI3K proteins. Western blot analysis was performed to determine whether the immunoprecipitates contained ATM and class III PI3K proteins. (B) Class III PI3K activity was assessed by ELISA. Hepatocytes from ApoE −/− mice were incubated with 5 μmol/l chloroquine, 40 μg/ml E − /B 48 lipoproteins and 10 μmol/l KU55933 (an ATM inhibitor) for 8 h at 37°C. Cells were collected and centrifuged. Cytoplasmic proteins from the cells were extracted using the Nuclear-Cytosol Extraction kit. Class III PI3K protein in the cytoplasm was immunoprecipitated, and then its activity was determined using the class III PI3K activity ELISA kit. Values represent the means ± SEM of 5 mice. * P

    Techniques Used: Immunoprecipitation, Activity Assay, Activation Assay, Mouse Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation

    Class III phosphatidylinositol-3-kinase (PI3K) inhibitor attenuates intracellular total cholesterol accumulation induced by ataxia telangiectasia muta ted (ATM) activation. Hepatocytes were isolated from apolipoprotein (Apo)E-deficient mice ( ApoE −/− mice) by perfusion of the portal vein and pre-treated with 5 μM chloroquine, 10 μmol/l KU55933 (an ATM inhibitor), 10 μM LY290042 (a class III PI3K inhibitor) or 5 mM 3-MA (a class III PI3K inhibitor) for 2 h and then incubated with 40 μg/ml ApoE-deficient, ApoB48-containing (E − /B 48 ) lipoproteins for 22 h at 37°C. Cellular total cholesterol in hepatocytes was determined using an enzymatic kit. Values represent the means ± SEM of 8 mice. * P
    Figure Legend Snippet: Class III phosphatidylinositol-3-kinase (PI3K) inhibitor attenuates intracellular total cholesterol accumulation induced by ataxia telangiectasia muta ted (ATM) activation. Hepatocytes were isolated from apolipoprotein (Apo)E-deficient mice ( ApoE −/− mice) by perfusion of the portal vein and pre-treated with 5 μM chloroquine, 10 μmol/l KU55933 (an ATM inhibitor), 10 μM LY290042 (a class III PI3K inhibitor) or 5 mM 3-MA (a class III PI3K inhibitor) for 2 h and then incubated with 40 μg/ml ApoE-deficient, ApoB48-containing (E − /B 48 ) lipoproteins for 22 h at 37°C. Cellular total cholesterol in hepatocytes was determined using an enzymatic kit. Values represent the means ± SEM of 8 mice. * P

    Techniques Used: Activation Assay, Isolation, Mouse Assay, Incubation

    12) Product Images from "Bmal1 suppresses cancer cell invasion by blocking the phosphoinositide 3-kinase-Akt-MMP-2 signaling pathway"

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

    Journal: Oncology Reports

    doi: 10.3892/or.2013.2381

    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
    Figure Legend 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

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

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    Echelon Biosciences class iii pi3 kinase kit
    scDNAs induce autophagy by releasing Rubicon from the Beclin-1-phosphatidylinositol <t>3-kinase</t> <t>class</t> <t>III</t> (PI3KC3) complex. a , HEK293T-cGAS or HEK293T-Vector cells were transfected with Flag-Beclin-1 plasmid and stimulated with scDNAs or HT-DNA (1 µg/mL). The cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. b , RAW264.7 cells stably expressing p40PX-EGFP were stimulated with scDNAs (1 µg/mL) or mock-transfected. The number of p40PX-EGFP foci was determined using confocal microscopy. Data are expressed as mean ± s.e.m of three biological replicates, n ≥ 200 cells. Statistical analysis was performed using two-sided Student’s t -tests; ** indicates p
    Class Iii Pi3 Kinase Kit, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/class iii pi3 kinase kit/product/Echelon Biosciences
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    Echelon Biosciences pi3k activity
    RGS20 regulates <t>PI3K/AKT</t> signaling activation in PC cells. ( a) RNA-Seq analysis identified differentially expressed genes in Penl1 cells with or without RGS20 knockdown. (b) Top 5 enriched pathways for downregulated genes ( n = 118) following RGS20 knockdown in Penl1 cells. (c) GSEA analysis revealed that RGS20-high PC cases exhibited highly enriched PI3K/AKT signaling in GSE57955 dataset. (d) Coimmunoprecitation analysis on RGS20 interaction with PI3K p85 α subunit in PC cells. IgG was used as coimmunoprecitation control. (e) PI3K <t>activity</t> of RGS20-depleted 149RM and Penl1 cells. The PI3K activity in Scr control was regards as 100%. n = 4, ∗∗∗ P
    Pi3k Activity, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    scDNAs induce autophagy by releasing Rubicon from the Beclin-1-phosphatidylinositol 3-kinase class III (PI3KC3) complex. a , HEK293T-cGAS or HEK293T-Vector cells were transfected with Flag-Beclin-1 plasmid and stimulated with scDNAs or HT-DNA (1 µg/mL). The cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. b , RAW264.7 cells stably expressing p40PX-EGFP were stimulated with scDNAs (1 µg/mL) or mock-transfected. The number of p40PX-EGFP foci was determined using confocal microscopy. Data are expressed as mean ± s.e.m of three biological replicates, n ≥ 200 cells. Statistical analysis was performed using two-sided Student’s t -tests; ** indicates p

    Journal: bioRxiv

    Article Title: Small cytosolic dsDNAs repress cGAS activation and induce autophagy

    doi: 10.1101/2022.06.30.498186

    Figure Lengend Snippet: scDNAs induce autophagy by releasing Rubicon from the Beclin-1-phosphatidylinositol 3-kinase class III (PI3KC3) complex. a , HEK293T-cGAS or HEK293T-Vector cells were transfected with Flag-Beclin-1 plasmid and stimulated with scDNAs or HT-DNA (1 µg/mL). The cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. b , RAW264.7 cells stably expressing p40PX-EGFP were stimulated with scDNAs (1 µg/mL) or mock-transfected. The number of p40PX-EGFP foci was determined using confocal microscopy. Data are expressed as mean ± s.e.m of three biological replicates, n ≥ 200 cells. Statistical analysis was performed using two-sided Student’s t -tests; ** indicates p

    Article Snippet: The PI3KC3 kinase activity assay and the production of phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2] were measured using a Class III PI3-Kinase Kit according to the manufacturer’s protocols (Echelon Biosciences Inc. Salt Lake City, UT, USA).

    Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Stable Transfection, Expressing, Confocal Microscopy

    A, Suppression of Beclin1 phosphorylation at S30 (p-Beclin1/Beclin1) by siRNA (#1 and #2)-mediated reduction of PGK1 expression. Values were normalized to that of control siRNA after incubation with PBS as a vehicle (=1). n =5 in each experiment. ** , P <0.01 vs. control siRNA. B, Representative immunoblots for panel A. C, Successful suppression of PGK1 expression by siRNA confirmed by immunoblotting. D, E, F, Comparison of PGK1 expression (D), Beclin1 phosphorylation at S30 (E), and PI3-kinase activity (F) after incubation for 2 hr with 20 µg/mL AcLDL or PBS as a vehicle in sPLA 2 -V-WT and KD cells, and sPLA 2 -V KD cells with re-constitutive expression of sPLA 2 -V or sPLA 2 -V-H48Q. G, Representative immunoblots showing PGK1 expression and Beclin1 phosphorylation in various types of RAW264.7 cells. Values in panels D and E were normalized to that of WT after incubation with PBS as a vehicle (=1). Each bar represents the mean±SEM of 5–6 independent experiments. * , P <0.05, ** , P <0.01 vs. WT, † , P <0.05, †† , P <0.01 vs. KD.

    Journal: Journal of Atherosclerosis and Thrombosis

    Article Title: Group V Secretory Phospholipase A2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

    doi: 10.5551/jat.62216

    Figure Lengend Snippet: A, Suppression of Beclin1 phosphorylation at S30 (p-Beclin1/Beclin1) by siRNA (#1 and #2)-mediated reduction of PGK1 expression. Values were normalized to that of control siRNA after incubation with PBS as a vehicle (=1). n =5 in each experiment. ** , P <0.01 vs. control siRNA. B, Representative immunoblots for panel A. C, Successful suppression of PGK1 expression by siRNA confirmed by immunoblotting. D, E, F, Comparison of PGK1 expression (D), Beclin1 phosphorylation at S30 (E), and PI3-kinase activity (F) after incubation for 2 hr with 20 µg/mL AcLDL or PBS as a vehicle in sPLA 2 -V-WT and KD cells, and sPLA 2 -V KD cells with re-constitutive expression of sPLA 2 -V or sPLA 2 -V-H48Q. G, Representative immunoblots showing PGK1 expression and Beclin1 phosphorylation in various types of RAW264.7 cells. Values in panels D and E were normalized to that of WT after incubation with PBS as a vehicle (=1). Each bar represents the mean±SEM of 5–6 independent experiments. * , P <0.05, ** , P <0.01 vs. WT, † , P <0.05, †† , P <0.01 vs. KD.

    Article Snippet: The enzyme activity of the VPS34 protein complex was determined using Class III PI3-Kinase Kit (K-3000; Echelon BioSciences, Salt Lake City, UT, USA) according to the manufacturer’s instructions.

    Techniques: Expressing, Activity Assay, Incubation, Western Blot

    RGS20 regulates PI3K/AKT signaling activation in PC cells. ( a) RNA-Seq analysis identified differentially expressed genes in Penl1 cells with or without RGS20 knockdown. (b) Top 5 enriched pathways for downregulated genes ( n = 118) following RGS20 knockdown in Penl1 cells. (c) GSEA analysis revealed that RGS20-high PC cases exhibited highly enriched PI3K/AKT signaling in GSE57955 dataset. (d) Coimmunoprecitation analysis on RGS20 interaction with PI3K p85 α subunit in PC cells. IgG was used as coimmunoprecitation control. (e) PI3K activity of RGS20-depleted 149RM and Penl1 cells. The PI3K activity in Scr control was regards as 100%. n = 4, ∗∗∗ P

    Journal: Journal of Oncology

    Article Title: RGS20 Promotes Tumor Progression through Modulating PI3K/AKT Signaling Activation in Penile Cancer

    doi: 10.1155/2022/1293622

    Figure Lengend Snippet: RGS20 regulates PI3K/AKT signaling activation in PC cells. ( a) RNA-Seq analysis identified differentially expressed genes in Penl1 cells with or without RGS20 knockdown. (b) Top 5 enriched pathways for downregulated genes ( n = 118) following RGS20 knockdown in Penl1 cells. (c) GSEA analysis revealed that RGS20-high PC cases exhibited highly enriched PI3K/AKT signaling in GSE57955 dataset. (d) Coimmunoprecitation analysis on RGS20 interaction with PI3K p85 α subunit in PC cells. IgG was used as coimmunoprecitation control. (e) PI3K activity of RGS20-depleted 149RM and Penl1 cells. The PI3K activity in Scr control was regards as 100%. n = 4, ∗∗∗ P

    Article Snippet: The PI3K activity was determined by measuring the level of PI (3, 4, 5) and P3 (PIP3) converted from PI3K substrate PI (4, 5) and P2 (PIP2).

    Techniques: Activation Assay, RNA Sequencing Assay, Activity Assay

    Knockdown of PI3K p85 α or p110 α suppresses cell proliferation, soft agar clonogenesis, and migration/invasion in PC cell lines. (a) Western blotting on PI3K p85 α or p110 α expression in 149RM and Penl1 cells transfected with scramble (Scr) control or shRNAs targeting p85 α or p110 α . β -Actin was used as loading control. (b) CCK-8 analysis on cell viability after p85 α or p110 α knockdown in 149RM and Penl1 cells. The cell viability in Scr control was regards as 100%. n = 4, ∗∗∗ P

    Journal: Journal of Oncology

    Article Title: RGS20 Promotes Tumor Progression through Modulating PI3K/AKT Signaling Activation in Penile Cancer

    doi: 10.1155/2022/1293622

    Figure Lengend Snippet: Knockdown of PI3K p85 α or p110 α suppresses cell proliferation, soft agar clonogenesis, and migration/invasion in PC cell lines. (a) Western blotting on PI3K p85 α or p110 α expression in 149RM and Penl1 cells transfected with scramble (Scr) control or shRNAs targeting p85 α or p110 α . β -Actin was used as loading control. (b) CCK-8 analysis on cell viability after p85 α or p110 α knockdown in 149RM and Penl1 cells. The cell viability in Scr control was regards as 100%. n = 4, ∗∗∗ P

    Article Snippet: The PI3K activity was determined by measuring the level of PI (3, 4, 5) and P3 (PIP3) converted from PI3K substrate PI (4, 5) and P2 (PIP2).

    Techniques: Migration, Western Blot, Expressing, Transfection, CCK-8 Assay

    RGS20 knockdown suppressed in vivo tumor growth and disrupted PI3K/AKT signaling activation in the Penl1 xenograft model . ( a). Nude mice xenograft study showed that RGS20 depletion attenuated subcutaneous tumor growth in nude mice. Tumor volume was measured every two days after Penl1 inoculation. n = 6, ∗ P

    Journal: Journal of Oncology

    Article Title: RGS20 Promotes Tumor Progression through Modulating PI3K/AKT Signaling Activation in Penile Cancer

    doi: 10.1155/2022/1293622

    Figure Lengend Snippet: RGS20 knockdown suppressed in vivo tumor growth and disrupted PI3K/AKT signaling activation in the Penl1 xenograft model . ( a). Nude mice xenograft study showed that RGS20 depletion attenuated subcutaneous tumor growth in nude mice. Tumor volume was measured every two days after Penl1 inoculation. n = 6, ∗ P

    Article Snippet: The PI3K activity was determined by measuring the level of PI (3, 4, 5) and P3 (PIP3) converted from PI3K substrate PI (4, 5) and P2 (PIP2).

    Techniques: In Vivo, Activation Assay, Mouse Assay

    The high RGS20 expression is associated with PI3K/AKT signaling activation and unfavorable patient survival in PC. (a) IHC micrographs showed consistent expression of RGS20 and p-AKT (T308, S473) in RGS20-high or RGS20-low PCs. Bars: 100 μ m. (b) The RGS20 expression was significantly correlated with p-AKT (T308) and p-AKT (S473) in our PC cohort ( n = 94). (c, d) Log-rank survival analysis showed that the high RGS20 expression was associated with progression-free survival and overall survival in our PC cohort ( n = 94).

    Journal: Journal of Oncology

    Article Title: RGS20 Promotes Tumor Progression through Modulating PI3K/AKT Signaling Activation in Penile Cancer

    doi: 10.1155/2022/1293622

    Figure Lengend Snippet: The high RGS20 expression is associated with PI3K/AKT signaling activation and unfavorable patient survival in PC. (a) IHC micrographs showed consistent expression of RGS20 and p-AKT (T308, S473) in RGS20-high or RGS20-low PCs. Bars: 100 μ m. (b) The RGS20 expression was significantly correlated with p-AKT (T308) and p-AKT (S473) in our PC cohort ( n = 94). (c, d) Log-rank survival analysis showed that the high RGS20 expression was associated with progression-free survival and overall survival in our PC cohort ( n = 94).

    Article Snippet: The PI3K activity was determined by measuring the level of PI (3, 4, 5) and P3 (PIP3) converted from PI3K substrate PI (4, 5) and P2 (PIP2).

    Techniques: Expressing, Activation Assay, Immunohistochemistry

    The overexpression of constitutively active PI3K p110 α rescued malignant phenotypes impaired by RGS20 depletion. (a) Western blotting analysis on the expression of PI3K/AKT signaling pathway proteins after transfection with empty vector (EV), PI3K p110 α Myr, or PI3K p110 α KD plasmids in RGS20-depleted 149RM and Penl1cells. (b) CCK-8 analysis on cell viability after PI3K p110 α Myr and KD plasmids transfection in RGS20-depleted 149RM and Penl1 cells. The cell viability in Scr control was regards as 100%. n = 4, ∗∗∗ P

    Journal: Journal of Oncology

    Article Title: RGS20 Promotes Tumor Progression through Modulating PI3K/AKT Signaling Activation in Penile Cancer

    doi: 10.1155/2022/1293622

    Figure Lengend Snippet: The overexpression of constitutively active PI3K p110 α rescued malignant phenotypes impaired by RGS20 depletion. (a) Western blotting analysis on the expression of PI3K/AKT signaling pathway proteins after transfection with empty vector (EV), PI3K p110 α Myr, or PI3K p110 α KD plasmids in RGS20-depleted 149RM and Penl1cells. (b) CCK-8 analysis on cell viability after PI3K p110 α Myr and KD plasmids transfection in RGS20-depleted 149RM and Penl1 cells. The cell viability in Scr control was regards as 100%. n = 4, ∗∗∗ P

    Article Snippet: The PI3K activity was determined by measuring the level of PI (3, 4, 5) and P3 (PIP3) converted from PI3K substrate PI (4, 5) and P2 (PIP2).

    Techniques: Over Expression, Western Blot, Expressing, Transfection, Plasmid Preparation, CCK-8 Assay