pp2a specific reaction buffer  (Millipore)


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    Millipore pp2a specific reaction buffer
    Untransformed hepatocytes showed lower I2PP2A and constitutive NF-κB activity than those in HCC cells and were resistant to isolie A. Basal levels of I2PP2A, <t>PP2A-C</t> and p65 phosphorylation at Ser536 in malignant and untransformed hepatocytes were compared. B. mRNA levels of NF-κB-responsive genes and I2PP2A in malignant and untransformed hepatocytes were compared. C, D. HL-7702 cells and PMHs were incubated with vehicle or 10 μg/mL isolie. After 24 h, transcription of NF-κB target genes and the binding between p65 and Bcl-2 promoter were detected by real-time PCR (C) and ChIP (D), respectively. E. PMH and HL-7702 cells were treated with vehicle or 10 μg/mL isolie. After 48 h, cell apoptosis was assessed using FACS (left) and caspase-3 activity assay (right).
    Pp2a Specific Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pp2a specific reaction buffer/product/Millipore
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pp2a specific reaction buffer - by Bioz Stars, 2020-03
    79/100 stars

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    1) Product Images from "Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction"

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9603

    Untransformed hepatocytes showed lower I2PP2A and constitutive NF-κB activity than those in HCC cells and were resistant to isolie A. Basal levels of I2PP2A, PP2A-C and p65 phosphorylation at Ser536 in malignant and untransformed hepatocytes were compared. B. mRNA levels of NF-κB-responsive genes and I2PP2A in malignant and untransformed hepatocytes were compared. C, D. HL-7702 cells and PMHs were incubated with vehicle or 10 μg/mL isolie. After 24 h, transcription of NF-κB target genes and the binding between p65 and Bcl-2 promoter were detected by real-time PCR (C) and ChIP (D), respectively. E. PMH and HL-7702 cells were treated with vehicle or 10 μg/mL isolie. After 48 h, cell apoptosis was assessed using FACS (left) and caspase-3 activity assay (right).
    Figure Legend Snippet: Untransformed hepatocytes showed lower I2PP2A and constitutive NF-κB activity than those in HCC cells and were resistant to isolie A. Basal levels of I2PP2A, PP2A-C and p65 phosphorylation at Ser536 in malignant and untransformed hepatocytes were compared. B. mRNA levels of NF-κB-responsive genes and I2PP2A in malignant and untransformed hepatocytes were compared. C, D. HL-7702 cells and PMHs were incubated with vehicle or 10 μg/mL isolie. After 24 h, transcription of NF-κB target genes and the binding between p65 and Bcl-2 promoter were detected by real-time PCR (C) and ChIP (D), respectively. E. PMH and HL-7702 cells were treated with vehicle or 10 μg/mL isolie. After 48 h, cell apoptosis was assessed using FACS (left) and caspase-3 activity assay (right).

    Techniques Used: Activity Assay, Incubation, Binding Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, FACS, Caspase-3 Activity Assay

    Knockdown of PP2A-C abrogated isolie-induced p65 dephosphorylation and HCC cell apoptosis A. HCC cells were transfected with control or PP2A-C specific siRNA. After 48 h, protein levels of PP2A-C were detected. B, C, D, E. HepG2 and Huh-7 cells were transfected with control or PP2A-C siRNAs for 48 h. Then, cells were treated with 10 μg/mL isolie. After 24 h, NF-κB activities were detected by luciferase reporter assay (B). Levels of indicated proteins were examined by immunoblotting (C). mRNA levels of Bcl-2, Bcl-xL and MMP9 were detected by real-time PCR (D). Interactions between p65 and promoter regions of indicated genes were determined by ChIP (E). F. Indicated HCC cell transfectants were treated with 10 μg/mL isolie. After 48 h, cell apoptosis were quantified by FACS (left) and caspase-3 activity assay (right). ** p
    Figure Legend Snippet: Knockdown of PP2A-C abrogated isolie-induced p65 dephosphorylation and HCC cell apoptosis A. HCC cells were transfected with control or PP2A-C specific siRNA. After 48 h, protein levels of PP2A-C were detected. B, C, D, E. HepG2 and Huh-7 cells were transfected with control or PP2A-C siRNAs for 48 h. Then, cells were treated with 10 μg/mL isolie. After 24 h, NF-κB activities were detected by luciferase reporter assay (B). Levels of indicated proteins were examined by immunoblotting (C). mRNA levels of Bcl-2, Bcl-xL and MMP9 were detected by real-time PCR (D). Interactions between p65 and promoter regions of indicated genes were determined by ChIP (E). F. Indicated HCC cell transfectants were treated with 10 μg/mL isolie. After 48 h, cell apoptosis were quantified by FACS (left) and caspase-3 activity assay (right). ** p

    Techniques Used: De-Phosphorylation Assay, Transfection, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, FACS, Caspase-3 Activity Assay

    Isolie suppressed PP2A/I2PP2A interaction A. HepG2 cells were incubated with indicated concentrations of isolie for 24 h. Then, PP2A/I2PP2A interaction was detected by immunoprecipitation (left). Phosphatase activity of immunoprecipitated PP2A was quantified by phosphatase activity assay (right). B. Indicated dosages of isolie or vehicle were injected intraperitoneally into nude mice bearing Huh-7 xenograft tumors. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. C. Kunming mice bearing H22 transplanted tumors were treated with indicated dosages of isolie by gavage. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. D. Purified 6 × his-tagged I2PP2A proteins already associated with Ni-NTA agarose were incubated with purified PP2A-C proteins and indicated concentrations of isolie. Amounts of PP2A-C proteins bound to I2PP2A proteins in vitro were determined by immunoblotting. * p
    Figure Legend Snippet: Isolie suppressed PP2A/I2PP2A interaction A. HepG2 cells were incubated with indicated concentrations of isolie for 24 h. Then, PP2A/I2PP2A interaction was detected by immunoprecipitation (left). Phosphatase activity of immunoprecipitated PP2A was quantified by phosphatase activity assay (right). B. Indicated dosages of isolie or vehicle were injected intraperitoneally into nude mice bearing Huh-7 xenograft tumors. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. C. Kunming mice bearing H22 transplanted tumors were treated with indicated dosages of isolie by gavage. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. D. Purified 6 × his-tagged I2PP2A proteins already associated with Ni-NTA agarose were incubated with purified PP2A-C proteins and indicated concentrations of isolie. Amounts of PP2A-C proteins bound to I2PP2A proteins in vitro were determined by immunoblotting. * p

    Techniques Used: Incubation, Immunoprecipitation, Activity Assay, Phosphatase Assay, Injection, Mouse Assay, Purification, In Vitro

    Isolie-provoked p65 dephosphorylation was initiated by decreased PP2A/I2PP2A interaction A, B, C. HCC cells were transfected with control or I2PP2A specific siRNA. After 48 h, levels of I2PP2A and effect of I2PP2A knockdown on p65/PP2A association as well as p65 phosphorylation at Ser536 were determined (A). NF-κB activities were measured by luciferase reporter assay (B). After 72 h, cell apoptosis were quantified by FACS (C, left) and caspase-3 activity assay (C, right). D. The encoding gene of human I2PP2A was stably transfected into indicated HCC cells, using empty vectors as the negative control. Then, I2PP2A proteins were detected. E, F. HCC cell transfectants as indicated were treated with 10 μg/mL isolie. After 24 h, levels of p65 phosphorylation at Ser536 (E) and NF-κB activity (F) were detected. G. Indicated HCC cell transfectants were incubated with 10 μg/mL isolie. After 48 h, cell apoptosis was detected by FACS (left) and caspase-3 activity assay (right). H, I. Indicated Huh-7 HCC cell transfectants were subcutaneously transplanted into nude mice. When volumes of xenograft tumors reached about 200 mm 3 , mice were treated with 10 mg/kg isolie or vehicle once daily for 20 d by gavage. Tumor growth was monitored every 5 d (H). Finally, xenograft tumor lysates were prepared and subjected to caspase-3 activity assay (I). ** p
    Figure Legend Snippet: Isolie-provoked p65 dephosphorylation was initiated by decreased PP2A/I2PP2A interaction A, B, C. HCC cells were transfected with control or I2PP2A specific siRNA. After 48 h, levels of I2PP2A and effect of I2PP2A knockdown on p65/PP2A association as well as p65 phosphorylation at Ser536 were determined (A). NF-κB activities were measured by luciferase reporter assay (B). After 72 h, cell apoptosis were quantified by FACS (C, left) and caspase-3 activity assay (C, right). D. The encoding gene of human I2PP2A was stably transfected into indicated HCC cells, using empty vectors as the negative control. Then, I2PP2A proteins were detected. E, F. HCC cell transfectants as indicated were treated with 10 μg/mL isolie. After 24 h, levels of p65 phosphorylation at Ser536 (E) and NF-κB activity (F) were detected. G. Indicated HCC cell transfectants were incubated with 10 μg/mL isolie. After 48 h, cell apoptosis was detected by FACS (left) and caspase-3 activity assay (right). H, I. Indicated Huh-7 HCC cell transfectants were subcutaneously transplanted into nude mice. When volumes of xenograft tumors reached about 200 mm 3 , mice were treated with 10 mg/kg isolie or vehicle once daily for 20 d by gavage. Tumor growth was monitored every 5 d (H). Finally, xenograft tumor lysates were prepared and subjected to caspase-3 activity assay (I). ** p

    Techniques Used: De-Phosphorylation Assay, Transfection, Luciferase, Reporter Assay, FACS, Caspase-3 Activity Assay, Stable Transfection, Negative Control, Activity Assay, Incubation, Mouse Assay

    Summary by a schematic panel In untreated HCC cells, I2PP2A binds to PP2A. PP2A/I2PP2A association reduces the phosphatase activity of PP2A and prevents PP2A from binding to NF-κB p65 subunit. These events benefit p65 hyperphosphorylation at Ser536 and hyperactivation of NF-κB, which is important for the survival of HCC cells. Isolie disrupts PP2A/I2PP2A interaction, increases p65/PP2A association and upregulates the phosphatase activity of PP2A. In this way, p65 is dephosphorylated at Ser536, giving rise to the inhibition of NF-κB and subsequent apoptosis of HCC cells.
    Figure Legend Snippet: Summary by a schematic panel In untreated HCC cells, I2PP2A binds to PP2A. PP2A/I2PP2A association reduces the phosphatase activity of PP2A and prevents PP2A from binding to NF-κB p65 subunit. These events benefit p65 hyperphosphorylation at Ser536 and hyperactivation of NF-κB, which is important for the survival of HCC cells. Isolie disrupts PP2A/I2PP2A interaction, increases p65/PP2A association and upregulates the phosphatase activity of PP2A. In this way, p65 is dephosphorylated at Ser536, giving rise to the inhibition of NF-κB and subsequent apoptosis of HCC cells.

    Techniques Used: Activity Assay, Binding Assay, Inhibition

    PP2A participated in isolie-induced p65 dephosphorylation A. Indicated HCC cells were treated with different concentrations of isolie. After 24 h, protein levels of PP2A-C (left) and p65/PP2A interaction (right) were determined. B. Indicated dosages of isolie or vehicle were intraperitoneally injected into tumor-bearing nude mice over the course of 3 weeks. Finally, effects of isolie on p65/PP2A interaction in Huh-7 tumor tissues were determined. C. Kunming mice bearing H22 tumors were treated with indicated dosages of isolie by gavage for 10 d. Then, effects of isolie on p65/PP2A interaction in transplanted tumor tissues were detected. D, E. HCC cells were treated with 10 μg/mL isolie and indicated concentrations of OA. After 24 h, phosphorylation of p65 at Ser536 was determined (D), and NF-κB activity was measured by NF-κB-dependent luciferase reporter assay (E). F. Cells were subjected to the same treatment as described in D and E. Cell apoptosis was quantified by FACS (left) and caspase-3 activity assay (right) 48 h later. * p
    Figure Legend Snippet: PP2A participated in isolie-induced p65 dephosphorylation A. Indicated HCC cells were treated with different concentrations of isolie. After 24 h, protein levels of PP2A-C (left) and p65/PP2A interaction (right) were determined. B. Indicated dosages of isolie or vehicle were intraperitoneally injected into tumor-bearing nude mice over the course of 3 weeks. Finally, effects of isolie on p65/PP2A interaction in Huh-7 tumor tissues were determined. C. Kunming mice bearing H22 tumors were treated with indicated dosages of isolie by gavage for 10 d. Then, effects of isolie on p65/PP2A interaction in transplanted tumor tissues were detected. D, E. HCC cells were treated with 10 μg/mL isolie and indicated concentrations of OA. After 24 h, phosphorylation of p65 at Ser536 was determined (D), and NF-κB activity was measured by NF-κB-dependent luciferase reporter assay (E). F. Cells were subjected to the same treatment as described in D and E. Cell apoptosis was quantified by FACS (left) and caspase-3 activity assay (right) 48 h later. * p

    Techniques Used: De-Phosphorylation Assay, Injection, Mouse Assay, Activity Assay, Luciferase, Reporter Assay, FACS, Caspase-3 Activity Assay

    Related Articles

    Immunoprecipitation:

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction
    Article Snippet: .. The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate. .. After incubation at 30°C for 10 min, malachite dye was added, and levels of free phosphate were represented by the optical density of the reaction system at 650 nm.

    Incubation:

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction
    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate. .. After incubation at 30°C for 10 min, malachite dye was added, and levels of free phosphate were represented by the optical density of the reaction system at 650 nm.

    Activity Assay:

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction
    Article Snippet: Paragraph title: PP2A activity assay ... The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Phosphatase Assay:

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction
    Article Snippet: .. The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate. .. After incubation at 30°C for 10 min, malachite dye was added, and levels of free phosphate were represented by the optical density of the reaction system at 650 nm.

    Lysis:

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction
    Article Snippet: HCC cell or tumor lysates were prepared in a low-detergent lysis buffer (1% Nonidet P-40, 10 mM HEPES, 150 mM NaCl, 10% glycerol, 1 mM PMSF, 5 mM benzamidine, and 10 g/mL leupeptin). .. The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

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    Millipore pp2a specific reaction buffer
    Untransformed hepatocytes showed lower I2PP2A and constitutive NF-κB activity than those in HCC cells and were resistant to isolie A. Basal levels of I2PP2A, <t>PP2A-C</t> and p65 phosphorylation at Ser536 in malignant and untransformed hepatocytes were compared. B. mRNA levels of NF-κB-responsive genes and I2PP2A in malignant and untransformed hepatocytes were compared. C, D. HL-7702 cells and PMHs were incubated with vehicle or 10 μg/mL isolie. After 24 h, transcription of NF-κB target genes and the binding between p65 and Bcl-2 promoter were detected by real-time PCR (C) and ChIP (D), respectively. E. PMH and HL-7702 cells were treated with vehicle or 10 μg/mL isolie. After 48 h, cell apoptosis was assessed using FACS (left) and caspase-3 activity assay (right).
    Pp2a Specific Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pp2a specific reaction buffer/product/Millipore
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pp2a specific reaction buffer - by Bioz Stars, 2020-03
    79/100 stars
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    Untransformed hepatocytes showed lower I2PP2A and constitutive NF-κB activity than those in HCC cells and were resistant to isolie A. Basal levels of I2PP2A, PP2A-C and p65 phosphorylation at Ser536 in malignant and untransformed hepatocytes were compared. B. mRNA levels of NF-κB-responsive genes and I2PP2A in malignant and untransformed hepatocytes were compared. C, D. HL-7702 cells and PMHs were incubated with vehicle or 10 μg/mL isolie. After 24 h, transcription of NF-κB target genes and the binding between p65 and Bcl-2 promoter were detected by real-time PCR (C) and ChIP (D), respectively. E. PMH and HL-7702 cells were treated with vehicle or 10 μg/mL isolie. After 48 h, cell apoptosis was assessed using FACS (left) and caspase-3 activity assay (right).

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Untransformed hepatocytes showed lower I2PP2A and constitutive NF-κB activity than those in HCC cells and were resistant to isolie A. Basal levels of I2PP2A, PP2A-C and p65 phosphorylation at Ser536 in malignant and untransformed hepatocytes were compared. B. mRNA levels of NF-κB-responsive genes and I2PP2A in malignant and untransformed hepatocytes were compared. C, D. HL-7702 cells and PMHs were incubated with vehicle or 10 μg/mL isolie. After 24 h, transcription of NF-κB target genes and the binding between p65 and Bcl-2 promoter were detected by real-time PCR (C) and ChIP (D), respectively. E. PMH and HL-7702 cells were treated with vehicle or 10 μg/mL isolie. After 48 h, cell apoptosis was assessed using FACS (left) and caspase-3 activity assay (right).

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: Activity Assay, Incubation, Binding Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, FACS, Caspase-3 Activity Assay

    Knockdown of PP2A-C abrogated isolie-induced p65 dephosphorylation and HCC cell apoptosis A. HCC cells were transfected with control or PP2A-C specific siRNA. After 48 h, protein levels of PP2A-C were detected. B, C, D, E. HepG2 and Huh-7 cells were transfected with control or PP2A-C siRNAs for 48 h. Then, cells were treated with 10 μg/mL isolie. After 24 h, NF-κB activities were detected by luciferase reporter assay (B). Levels of indicated proteins were examined by immunoblotting (C). mRNA levels of Bcl-2, Bcl-xL and MMP9 were detected by real-time PCR (D). Interactions between p65 and promoter regions of indicated genes were determined by ChIP (E). F. Indicated HCC cell transfectants were treated with 10 μg/mL isolie. After 48 h, cell apoptosis were quantified by FACS (left) and caspase-3 activity assay (right). ** p

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Knockdown of PP2A-C abrogated isolie-induced p65 dephosphorylation and HCC cell apoptosis A. HCC cells were transfected with control or PP2A-C specific siRNA. After 48 h, protein levels of PP2A-C were detected. B, C, D, E. HepG2 and Huh-7 cells were transfected with control or PP2A-C siRNAs for 48 h. Then, cells were treated with 10 μg/mL isolie. After 24 h, NF-κB activities were detected by luciferase reporter assay (B). Levels of indicated proteins were examined by immunoblotting (C). mRNA levels of Bcl-2, Bcl-xL and MMP9 were detected by real-time PCR (D). Interactions between p65 and promoter regions of indicated genes were determined by ChIP (E). F. Indicated HCC cell transfectants were treated with 10 μg/mL isolie. After 48 h, cell apoptosis were quantified by FACS (left) and caspase-3 activity assay (right). ** p

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: De-Phosphorylation Assay, Transfection, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, FACS, Caspase-3 Activity Assay

    Isolie suppressed PP2A/I2PP2A interaction A. HepG2 cells were incubated with indicated concentrations of isolie for 24 h. Then, PP2A/I2PP2A interaction was detected by immunoprecipitation (left). Phosphatase activity of immunoprecipitated PP2A was quantified by phosphatase activity assay (right). B. Indicated dosages of isolie or vehicle were injected intraperitoneally into nude mice bearing Huh-7 xenograft tumors. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. C. Kunming mice bearing H22 transplanted tumors were treated with indicated dosages of isolie by gavage. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. D. Purified 6 × his-tagged I2PP2A proteins already associated with Ni-NTA agarose were incubated with purified PP2A-C proteins and indicated concentrations of isolie. Amounts of PP2A-C proteins bound to I2PP2A proteins in vitro were determined by immunoblotting. * p

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Isolie suppressed PP2A/I2PP2A interaction A. HepG2 cells were incubated with indicated concentrations of isolie for 24 h. Then, PP2A/I2PP2A interaction was detected by immunoprecipitation (left). Phosphatase activity of immunoprecipitated PP2A was quantified by phosphatase activity assay (right). B. Indicated dosages of isolie or vehicle were injected intraperitoneally into nude mice bearing Huh-7 xenograft tumors. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. C. Kunming mice bearing H22 transplanted tumors were treated with indicated dosages of isolie by gavage. In the end, tumor lysates were prepared and subjected to the same experiments as described in A. D. Purified 6 × his-tagged I2PP2A proteins already associated with Ni-NTA agarose were incubated with purified PP2A-C proteins and indicated concentrations of isolie. Amounts of PP2A-C proteins bound to I2PP2A proteins in vitro were determined by immunoblotting. * p

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: Incubation, Immunoprecipitation, Activity Assay, Phosphatase Assay, Injection, Mouse Assay, Purification, In Vitro

    Isolie-provoked p65 dephosphorylation was initiated by decreased PP2A/I2PP2A interaction A, B, C. HCC cells were transfected with control or I2PP2A specific siRNA. After 48 h, levels of I2PP2A and effect of I2PP2A knockdown on p65/PP2A association as well as p65 phosphorylation at Ser536 were determined (A). NF-κB activities were measured by luciferase reporter assay (B). After 72 h, cell apoptosis were quantified by FACS (C, left) and caspase-3 activity assay (C, right). D. The encoding gene of human I2PP2A was stably transfected into indicated HCC cells, using empty vectors as the negative control. Then, I2PP2A proteins were detected. E, F. HCC cell transfectants as indicated were treated with 10 μg/mL isolie. After 24 h, levels of p65 phosphorylation at Ser536 (E) and NF-κB activity (F) were detected. G. Indicated HCC cell transfectants were incubated with 10 μg/mL isolie. After 48 h, cell apoptosis was detected by FACS (left) and caspase-3 activity assay (right). H, I. Indicated Huh-7 HCC cell transfectants were subcutaneously transplanted into nude mice. When volumes of xenograft tumors reached about 200 mm 3 , mice were treated with 10 mg/kg isolie or vehicle once daily for 20 d by gavage. Tumor growth was monitored every 5 d (H). Finally, xenograft tumor lysates were prepared and subjected to caspase-3 activity assay (I). ** p

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Isolie-provoked p65 dephosphorylation was initiated by decreased PP2A/I2PP2A interaction A, B, C. HCC cells were transfected with control or I2PP2A specific siRNA. After 48 h, levels of I2PP2A and effect of I2PP2A knockdown on p65/PP2A association as well as p65 phosphorylation at Ser536 were determined (A). NF-κB activities were measured by luciferase reporter assay (B). After 72 h, cell apoptosis were quantified by FACS (C, left) and caspase-3 activity assay (C, right). D. The encoding gene of human I2PP2A was stably transfected into indicated HCC cells, using empty vectors as the negative control. Then, I2PP2A proteins were detected. E, F. HCC cell transfectants as indicated were treated with 10 μg/mL isolie. After 24 h, levels of p65 phosphorylation at Ser536 (E) and NF-κB activity (F) were detected. G. Indicated HCC cell transfectants were incubated with 10 μg/mL isolie. After 48 h, cell apoptosis was detected by FACS (left) and caspase-3 activity assay (right). H, I. Indicated Huh-7 HCC cell transfectants were subcutaneously transplanted into nude mice. When volumes of xenograft tumors reached about 200 mm 3 , mice were treated with 10 mg/kg isolie or vehicle once daily for 20 d by gavage. Tumor growth was monitored every 5 d (H). Finally, xenograft tumor lysates were prepared and subjected to caspase-3 activity assay (I). ** p

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: De-Phosphorylation Assay, Transfection, Luciferase, Reporter Assay, FACS, Caspase-3 Activity Assay, Stable Transfection, Negative Control, Activity Assay, Incubation, Mouse Assay

    Summary by a schematic panel In untreated HCC cells, I2PP2A binds to PP2A. PP2A/I2PP2A association reduces the phosphatase activity of PP2A and prevents PP2A from binding to NF-κB p65 subunit. These events benefit p65 hyperphosphorylation at Ser536 and hyperactivation of NF-κB, which is important for the survival of HCC cells. Isolie disrupts PP2A/I2PP2A interaction, increases p65/PP2A association and upregulates the phosphatase activity of PP2A. In this way, p65 is dephosphorylated at Ser536, giving rise to the inhibition of NF-κB and subsequent apoptosis of HCC cells.

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: Summary by a schematic panel In untreated HCC cells, I2PP2A binds to PP2A. PP2A/I2PP2A association reduces the phosphatase activity of PP2A and prevents PP2A from binding to NF-κB p65 subunit. These events benefit p65 hyperphosphorylation at Ser536 and hyperactivation of NF-κB, which is important for the survival of HCC cells. Isolie disrupts PP2A/I2PP2A interaction, increases p65/PP2A association and upregulates the phosphatase activity of PP2A. In this way, p65 is dephosphorylated at Ser536, giving rise to the inhibition of NF-κB and subsequent apoptosis of HCC cells.

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: Activity Assay, Binding Assay, Inhibition

    PP2A participated in isolie-induced p65 dephosphorylation A. Indicated HCC cells were treated with different concentrations of isolie. After 24 h, protein levels of PP2A-C (left) and p65/PP2A interaction (right) were determined. B. Indicated dosages of isolie or vehicle were intraperitoneally injected into tumor-bearing nude mice over the course of 3 weeks. Finally, effects of isolie on p65/PP2A interaction in Huh-7 tumor tissues were determined. C. Kunming mice bearing H22 tumors were treated with indicated dosages of isolie by gavage for 10 d. Then, effects of isolie on p65/PP2A interaction in transplanted tumor tissues were detected. D, E. HCC cells were treated with 10 μg/mL isolie and indicated concentrations of OA. After 24 h, phosphorylation of p65 at Ser536 was determined (D), and NF-κB activity was measured by NF-κB-dependent luciferase reporter assay (E). F. Cells were subjected to the same treatment as described in D and E. Cell apoptosis was quantified by FACS (left) and caspase-3 activity assay (right) 48 h later. * p

    Journal: Oncotarget

    Article Title: Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    doi: 10.18632/oncotarget.9603

    Figure Lengend Snippet: PP2A participated in isolie-induced p65 dephosphorylation A. Indicated HCC cells were treated with different concentrations of isolie. After 24 h, protein levels of PP2A-C (left) and p65/PP2A interaction (right) were determined. B. Indicated dosages of isolie or vehicle were intraperitoneally injected into tumor-bearing nude mice over the course of 3 weeks. Finally, effects of isolie on p65/PP2A interaction in Huh-7 tumor tissues were determined. C. Kunming mice bearing H22 tumors were treated with indicated dosages of isolie by gavage for 10 d. Then, effects of isolie on p65/PP2A interaction in transplanted tumor tissues were detected. D, E. HCC cells were treated with 10 μg/mL isolie and indicated concentrations of OA. After 24 h, phosphorylation of p65 at Ser536 was determined (D), and NF-κB activity was measured by NF-κB-dependent luciferase reporter assay (E). F. Cells were subjected to the same treatment as described in D and E. Cell apoptosis was quantified by FACS (left) and caspase-3 activity assay (right) 48 h later. * p

    Article Snippet: The lysates were then subjected to immunoprecipitation using the primary antibody specific to PP2A-C. Phosphatase activity assay was performed in a PP2A-specific reaction buffer from Millipore (Billerica, MA, USA) containing 750 mM phosphopeptide substrate.

    Techniques: De-Phosphorylation Assay, Injection, Mouse Assay, Activity Assay, Luciferase, Reporter Assay, FACS, Caspase-3 Activity Assay