pi3k activity elisa kit  (Echelon Biosciences)


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    Echelon Biosciences pi3k activity elisa kit
    Expression and activation of IGF-IR and <t>PI3K</t> in MDA-MB-231 and MCF7 cells. (a) After stimulation with IGF-I for 0, 0.5, or 6 h, cells were lysed and processed for immunoblotting with anti-IGF-IR antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (b) After stimulation with IGF-I, cells were immunostained with antibody to phospho-IGF-IR (Tyr1135/1135). Scale Bars 20 μ m. (c) After stimulation with IGF-I, cells were lysed and processed for immunoblotting with anti-PI3K (p85) antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (d) Cells after stimulation with IGF-I were lysed and immunoprecipitated with anti-PI3K (p85) antibody. PI3K activity was assayed for the immunoprecipitates as described in Materials and Methods. The mean (SD) values of triplicate assays are given as activity relative to that in unstimulated control MDA-MB-231 cells.
    Pi3k Activity Elisa Kit, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Rac1 and Stathmin but Not EB1 Are Required for Invasion of Breast Cancer Cells in Response to IGF-I"

    Article Title: Rac1 and Stathmin but Not EB1 Are Required for Invasion of Breast Cancer Cells in Response to IGF-I

    Journal: International Journal of Cell Biology

    doi: 10.1155/2011/615912

    Expression and activation of IGF-IR and PI3K in MDA-MB-231 and MCF7 cells. (a) After stimulation with IGF-I for 0, 0.5, or 6 h, cells were lysed and processed for immunoblotting with anti-IGF-IR antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (b) After stimulation with IGF-I, cells were immunostained with antibody to phospho-IGF-IR (Tyr1135/1135). Scale Bars 20 μ m. (c) After stimulation with IGF-I, cells were lysed and processed for immunoblotting with anti-PI3K (p85) antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (d) Cells after stimulation with IGF-I were lysed and immunoprecipitated with anti-PI3K (p85) antibody. PI3K activity was assayed for the immunoprecipitates as described in Materials and Methods. The mean (SD) values of triplicate assays are given as activity relative to that in unstimulated control MDA-MB-231 cells.
    Figure Legend Snippet: Expression and activation of IGF-IR and PI3K in MDA-MB-231 and MCF7 cells. (a) After stimulation with IGF-I for 0, 0.5, or 6 h, cells were lysed and processed for immunoblotting with anti-IGF-IR antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (b) After stimulation with IGF-I, cells were immunostained with antibody to phospho-IGF-IR (Tyr1135/1135). Scale Bars 20 μ m. (c) After stimulation with IGF-I, cells were lysed and processed for immunoblotting with anti-PI3K (p85) antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (d) Cells after stimulation with IGF-I were lysed and immunoprecipitated with anti-PI3K (p85) antibody. PI3K activity was assayed for the immunoprecipitates as described in Materials and Methods. The mean (SD) values of triplicate assays are given as activity relative to that in unstimulated control MDA-MB-231 cells.

    Techniques Used: Expressing, Activation Assay, Western Blot, Immunoprecipitation, Activity Assay

    pi3k activity elisa kit  (Echelon Biosciences)


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    Echelon Biosciences pi3k activity elisa kit
    Expression and activation of IGF-IR and <t>PI3K</t> in MDA-MB-231 and MCF7 cells. (a) After stimulation with IGF-I for 0, 0.5, or 6 h, cells were lysed and processed for immunoblotting with anti-IGF-IR antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (b) After stimulation with IGF-I, cells were immunostained with antibody to phospho-IGF-IR (Tyr1135/1135). Scale Bars 20 μ m. (c) After stimulation with IGF-I, cells were lysed and processed for immunoblotting with anti-PI3K (p85) antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (d) Cells after stimulation with IGF-I were lysed and immunoprecipitated with anti-PI3K (p85) antibody. PI3K activity was assayed for the immunoprecipitates as described in Materials and Methods. The mean (SD) values of triplicate assays are given as activity relative to that in unstimulated control MDA-MB-231 cells.
    Pi3k Activity Elisa Kit, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Rac1 and Stathmin but Not EB1 Are Required for Invasion of Breast Cancer Cells in Response to IGF-I"

    Article Title: Rac1 and Stathmin but Not EB1 Are Required for Invasion of Breast Cancer Cells in Response to IGF-I

    Journal: International Journal of Cell Biology

    doi: 10.1155/2011/615912

    Expression and activation of IGF-IR and PI3K in MDA-MB-231 and MCF7 cells. (a) After stimulation with IGF-I for 0, 0.5, or 6 h, cells were lysed and processed for immunoblotting with anti-IGF-IR antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (b) After stimulation with IGF-I, cells were immunostained with antibody to phospho-IGF-IR (Tyr1135/1135). Scale Bars 20 μ m. (c) After stimulation with IGF-I, cells were lysed and processed for immunoblotting with anti-PI3K (p85) antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (d) Cells after stimulation with IGF-I were lysed and immunoprecipitated with anti-PI3K (p85) antibody. PI3K activity was assayed for the immunoprecipitates as described in Materials and Methods. The mean (SD) values of triplicate assays are given as activity relative to that in unstimulated control MDA-MB-231 cells.
    Figure Legend Snippet: Expression and activation of IGF-IR and PI3K in MDA-MB-231 and MCF7 cells. (a) After stimulation with IGF-I for 0, 0.5, or 6 h, cells were lysed and processed for immunoblotting with anti-IGF-IR antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (b) After stimulation with IGF-I, cells were immunostained with antibody to phospho-IGF-IR (Tyr1135/1135). Scale Bars 20 μ m. (c) After stimulation with IGF-I, cells were lysed and processed for immunoblotting with anti-PI3K (p85) antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (d) Cells after stimulation with IGF-I were lysed and immunoprecipitated with anti-PI3K (p85) antibody. PI3K activity was assayed for the immunoprecipitates as described in Materials and Methods. The mean (SD) values of triplicate assays are given as activity relative to that in unstimulated control MDA-MB-231 cells.

    Techniques Used: Expressing, Activation Assay, Western Blot, Immunoprecipitation, Activity Assay

    pi3 kinase activity elisa pico  (Echelon Biosciences)


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    Echelon Biosciences pi3 kinase activity elisa pico
    Pi3 Kinase Activity Elisa Pico, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pi3k activity  (Echelon Biosciences)


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    Echelon Biosciences pi3k activity
    Slit2N inhibits VEGF-C-enhanced <t>PI3K</t> activity and Akt phosphorylation in L-LECs. (A) Representative Western blot analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Total ERK1/2 used as loading control. (B) Quantitative analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4A was determined by densitometry. The ratio of p-ERK1/2 to total ERK1/2 of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments. (C) PI3K activity by ELISA in L-LECs incubated with various concentrations of Slit2N and/or VEGF-C [100 ng/ml]. Data represent the mean ± SD of 3 independent experiments (**p < 0.01, ***p < 0.001). (D) Representative Western blot analysis of phosphorylated Akt in L-LECs incubated with a control, VEGF-C [100 ng/ml]; or preincubated with various concentrations of Slit2N, then VEGF-C. Total Akt used as loading control. (E) Quantitative analysis of phosphorylated Akt in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4D was determined by densitometry. The ratio of p-Akt to total Akt of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments (**p < 0.01).
    Pi3k Activity, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Slit2N and Robo4 regulate lymphangiogenesis through the VEGF-C/VEGFR-3 pathway"

    Article Title: Slit2N and Robo4 regulate lymphangiogenesis through the VEGF-C/VEGFR-3 pathway

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-12-25

    Slit2N inhibits VEGF-C-enhanced PI3K activity and Akt phosphorylation in L-LECs. (A) Representative Western blot analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Total ERK1/2 used as loading control. (B) Quantitative analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4A was determined by densitometry. The ratio of p-ERK1/2 to total ERK1/2 of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments. (C) PI3K activity by ELISA in L-LECs incubated with various concentrations of Slit2N and/or VEGF-C [100 ng/ml]. Data represent the mean ± SD of 3 independent experiments (**p < 0.01, ***p < 0.001). (D) Representative Western blot analysis of phosphorylated Akt in L-LECs incubated with a control, VEGF-C [100 ng/ml]; or preincubated with various concentrations of Slit2N, then VEGF-C. Total Akt used as loading control. (E) Quantitative analysis of phosphorylated Akt in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4D was determined by densitometry. The ratio of p-Akt to total Akt of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments (**p < 0.01).
    Figure Legend Snippet: Slit2N inhibits VEGF-C-enhanced PI3K activity and Akt phosphorylation in L-LECs. (A) Representative Western blot analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Total ERK1/2 used as loading control. (B) Quantitative analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4A was determined by densitometry. The ratio of p-ERK1/2 to total ERK1/2 of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments. (C) PI3K activity by ELISA in L-LECs incubated with various concentrations of Slit2N and/or VEGF-C [100 ng/ml]. Data represent the mean ± SD of 3 independent experiments (**p < 0.01, ***p < 0.001). (D) Representative Western blot analysis of phosphorylated Akt in L-LECs incubated with a control, VEGF-C [100 ng/ml]; or preincubated with various concentrations of Slit2N, then VEGF-C. Total Akt used as loading control. (E) Quantitative analysis of phosphorylated Akt in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4D was determined by densitometry. The ratio of p-Akt to total Akt of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments (**p < 0.01).

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

    Slit2N inhibition of VEGF-C-enhanced PI3K activity and Akt phosphorylation in L-LECs is Robo4-dependent. (A) PI3K activity by ELISA in L-LECs transfected with control siRNAs or Robo4-specific siRNAs, incubated with control or VEGF-C [100 ng/ml]; or after pretreatment with 10 nM Slit2N, then VEGF-C. Data represent the mean ± SD of 3 independent experiments (*p < 0.05; NS: not statistically significant). (B) Representative Western blot analysis of Akt phosphorylation in L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs, and subsequently incubated with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after pretreatment with Slit2N, then VEGF-C. Total Akt used as loading control. (C) Quantitative analysis of Akt phosphorylation in L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs, and subsequently incubated with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after pretreatment with Slit2N, then VEGF-C. Band intensity of each lane from Figure 6B was determined by densitometry. The ratio of p-Akt/total Akt in control siRNA-transfected L-LECs incubated with VEGF-C alone was set to “1” and all other ratios were determined vs. this control. Data represent the mean ± SD of 3 independent experiments (***p < 0.001).
    Figure Legend Snippet: Slit2N inhibition of VEGF-C-enhanced PI3K activity and Akt phosphorylation in L-LECs is Robo4-dependent. (A) PI3K activity by ELISA in L-LECs transfected with control siRNAs or Robo4-specific siRNAs, incubated with control or VEGF-C [100 ng/ml]; or after pretreatment with 10 nM Slit2N, then VEGF-C. Data represent the mean ± SD of 3 independent experiments (*p < 0.05; NS: not statistically significant). (B) Representative Western blot analysis of Akt phosphorylation in L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs, and subsequently incubated with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after pretreatment with Slit2N, then VEGF-C. Total Akt used as loading control. (C) Quantitative analysis of Akt phosphorylation in L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs, and subsequently incubated with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after pretreatment with Slit2N, then VEGF-C. Band intensity of each lane from Figure 6B was determined by densitometry. The ratio of p-Akt/total Akt in control siRNA-transfected L-LECs incubated with VEGF-C alone was set to “1” and all other ratios were determined vs. this control. Data represent the mean ± SD of 3 independent experiments (***p < 0.001).

    Techniques Used: Inhibition, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Incubation, Western Blot

    immunosorbent assay elisa k  (Echelon Biosciences)


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    Echelon Biosciences immunosorbent assay elisa k
    Immunosorbent Assay Elisa K, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pi3k activity assay  (Echelon Biosciences)


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    Echelon Biosciences pi3k activity assay
    (A) The cellular content of PIP3 was quantified using a competitive PIP3 ELISA; *p<0.05; **p<0.01 versus 2008-scb control; # p<0.05 versus CLDN3KD; ## p<0.01 versus CLDN4KD. (B) Activity of <t>PI3K</t> immunoprecipitated with antibody to p85 regulatory subunit of class IA PI3K; *p<0.01; **p<0.01 versus 2008-scb control; # p<0.01 versus CLDN3KD; ## p<0.01 versus CLDN4KD. (C) PIP3 levels; *p<0.001 versus CLDN3KD; **p<0.001 versus CLDN4KD. (D) Activity of PI3K; *p<0.001 versus CLDN3KD; **p<0.001 versus CLDN4KD. Values are mean ± SEM, n = 3. PI3K activity is expressed as percent of control.
    Pi3k Activity Assay, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Regulation of the Epithelial-Mesenchymal Transition by Claudin-3 and Claudin-4"

    Article Title: Regulation of the Epithelial-Mesenchymal Transition by Claudin-3 and Claudin-4

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067496

    (A) The cellular content of PIP3 was quantified using a competitive PIP3 ELISA; *p<0.05; **p<0.01 versus 2008-scb control; # p<0.05 versus CLDN3KD; ## p<0.01 versus CLDN4KD. (B) Activity of PI3K immunoprecipitated with antibody to p85 regulatory subunit of class IA PI3K; *p<0.01; **p<0.01 versus 2008-scb control; # p<0.01 versus CLDN3KD; ## p<0.01 versus CLDN4KD. (C) PIP3 levels; *p<0.001 versus CLDN3KD; **p<0.001 versus CLDN4KD. (D) Activity of PI3K; *p<0.001 versus CLDN3KD; **p<0.001 versus CLDN4KD. Values are mean ± SEM, n = 3. PI3K activity is expressed as percent of control.
    Figure Legend Snippet: (A) The cellular content of PIP3 was quantified using a competitive PIP3 ELISA; *p<0.05; **p<0.01 versus 2008-scb control; # p<0.05 versus CLDN3KD; ## p<0.01 versus CLDN4KD. (B) Activity of PI3K immunoprecipitated with antibody to p85 regulatory subunit of class IA PI3K; *p<0.01; **p<0.01 versus 2008-scb control; # p<0.01 versus CLDN3KD; ## p<0.01 versus CLDN4KD. (C) PIP3 levels; *p<0.001 versus CLDN3KD; **p<0.001 versus CLDN4KD. (D) Activity of PI3K; *p<0.001 versus CLDN3KD; **p<0.001 versus CLDN4KD. Values are mean ± SEM, n = 3. PI3K activity is expressed as percent of control.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Activity Assay, Immunoprecipitation

    pi3k activity elisa kit  (Echelon Biosciences)


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    Echelon Biosciences pi3k activity elisa kit
    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 activity of RGS20-depleted 149RM and Penl1 cells. The PI3K activity in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001. (f) PI3K activity of 149RCa and LM156 cells with or without the RGS20 overexpression. The PI3K activity in EV control was regards as 100%. n = 4, ∗∗∗ P < 0.001. (g) PI3K activity of RGS20-depleted Penl1 cells after EGF stimulation. After serum deprivation for 36 hours, Penl1 cells (Scr or shRGS20) were stimulated with EGF (50 ng/mL) for 30 min, and then PI3K activity was analyzed. n = 4, ∗∗∗ P < 0.001. (h) Western blotting analysis on PI3K/AKT signaling proteins following RGS20 depletion in 149RM and Penl1 cells. (i) Western blotting analysis on PI3K/AKT signaling proteins following the overexpression of RGS20 in 149RCa and LM156 cells. (j) Western blotting analysis on PI3K/AKT signaling proteins in RGS20-depleted Penl1 cells after EGF stimulation. After serum deprivation for 36 hours, Penl1 cells (Scr or shRGS20) were stimulated with EGF (50 ng/mL) for 30 min. Western blotting was conducted to analyze PI3K/AKT signaling proteins.
    Pi3k Activity Elisa Kit, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "RGS20 Promotes Tumor Progression through Modulating PI3K/AKT Signaling Activation in Penile Cancer"

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

    Journal: Journal of Oncology

    doi: 10.1155/2022/1293622

    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 < 0.001. (f) PI3K activity of 149RCa and LM156 cells with or without the RGS20 overexpression. The PI3K activity in EV control was regards as 100%. n = 4, ∗∗∗ P < 0.001. (g) PI3K activity of RGS20-depleted Penl1 cells after EGF stimulation. After serum deprivation for 36 hours, Penl1 cells (Scr or shRGS20) were stimulated with EGF (50 ng/mL) for 30 min, and then PI3K activity was analyzed. n = 4, ∗∗∗ P < 0.001. (h) Western blotting analysis on PI3K/AKT signaling proteins following RGS20 depletion in 149RM and Penl1 cells. (i) Western blotting analysis on PI3K/AKT signaling proteins following the overexpression of RGS20 in 149RCa and LM156 cells. (j) Western blotting analysis on PI3K/AKT signaling proteins in RGS20-depleted Penl1 cells after EGF stimulation. After serum deprivation for 36 hours, Penl1 cells (Scr or shRGS20) were stimulated with EGF (50 ng/mL) for 30 min. Western blotting was conducted to analyze PI3K/AKT signaling proteins.
    Figure Legend 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 < 0.001. (f) PI3K activity of 149RCa and LM156 cells with or without the RGS20 overexpression. The PI3K activity in EV control was regards as 100%. n = 4, ∗∗∗ P < 0.001. (g) PI3K activity of RGS20-depleted Penl1 cells after EGF stimulation. After serum deprivation for 36 hours, Penl1 cells (Scr or shRGS20) were stimulated with EGF (50 ng/mL) for 30 min, and then PI3K activity was analyzed. n = 4, ∗∗∗ P < 0.001. (h) Western blotting analysis on PI3K/AKT signaling proteins following RGS20 depletion in 149RM and Penl1 cells. (i) Western blotting analysis on PI3K/AKT signaling proteins following the overexpression of RGS20 in 149RCa and LM156 cells. (j) Western blotting analysis on PI3K/AKT signaling proteins in RGS20-depleted Penl1 cells after EGF stimulation. After serum deprivation for 36 hours, Penl1 cells (Scr or shRGS20) were stimulated with EGF (50 ng/mL) for 30 min. Western blotting was conducted to analyze PI3K/AKT signaling proteins.

    Techniques Used: Activation Assay, RNA Sequencing Assay, Activity Assay, Over Expression, Western Blot

    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 < 0.001. (c) BrdU incorporation analysis on cell proliferation following p85 α or p110 α knockdown in 149RM and Penl1 cells. The BrdU incorporation in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001. (d) Soft agar clonogenesis of PC cells following p85 α or p110 α knockdown. The soft agar clonogenesis in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001. Bars: 100 μ m. (e) Caspase-3 activity of PC cells following p85 α or p110 α knockdown. The caspase-3 activity in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001. (f) Wound healing assay on PC cells following p85 α or p110 α knockdown. Micrographs showed the results of Penl1 cells. Bars: 100 μ m. The cell migration in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001. (g) Transwell invasion assay on PC cells following p85 α or p110 α knockdown. Bars: 50 μ m. The cell invasion in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001.
    Figure Legend 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 < 0.001. (c) BrdU incorporation analysis on cell proliferation following p85 α or p110 α knockdown in 149RM and Penl1 cells. The BrdU incorporation in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001. (d) Soft agar clonogenesis of PC cells following p85 α or p110 α knockdown. The soft agar clonogenesis in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001. Bars: 100 μ m. (e) Caspase-3 activity of PC cells following p85 α or p110 α knockdown. The caspase-3 activity in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001. (f) Wound healing assay on PC cells following p85 α or p110 α knockdown. Micrographs showed the results of Penl1 cells. Bars: 100 μ m. The cell migration in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001. (g) Transwell invasion assay on PC cells following p85 α or p110 α knockdown. Bars: 50 μ m. The cell invasion in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001.

    Techniques Used: Migration, Western Blot, Expressing, Transfection, CCK-8 Assay, BrdU Incorporation Assay, Activity Assay, Wound Healing Assay, Transwell Invasion Assay

    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 < 0.001, as compared with EV. (c) Soft agar assay on clonogenesis after PI3K p110 α Myr and KD plasmids transfection in RGS20-depleted 149RM and Penl1 cells. The soft agar clonogenesis in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001, as compared with EV. (d) Caspase-3 activity after PI3K p110 α Myr and KD plasmid transfection in RGS20-depleted 149RM and Penl1 cells. The caspase-3 activity in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001, as compared with EV. (e) Wound healing assay on PC cells following PI3K p110 α Myr and KD plasmid transfection in RGS20-depleted 149RM and Penl1 cells. Bars: 100 μ m. The cell migration in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001, as compared with EV. (f) Transwell invasion assay on PC cells following PI3K p110 α Myr and KD plasmid transfection in RGS20-depleted 149RM and Penl1 cells. Bars: 50 μ m. The cell invasion in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001, as compared with EV.
    Figure Legend 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 < 0.001, as compared with EV. (c) Soft agar assay on clonogenesis after PI3K p110 α Myr and KD plasmids transfection in RGS20-depleted 149RM and Penl1 cells. The soft agar clonogenesis in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001, as compared with EV. (d) Caspase-3 activity after PI3K p110 α Myr and KD plasmid transfection in RGS20-depleted 149RM and Penl1 cells. The caspase-3 activity in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001, as compared with EV. (e) Wound healing assay on PC cells following PI3K p110 α Myr and KD plasmid transfection in RGS20-depleted 149RM and Penl1 cells. Bars: 100 μ m. The cell migration in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001, as compared with EV. (f) Transwell invasion assay on PC cells following PI3K p110 α Myr and KD plasmid transfection in RGS20-depleted 149RM and Penl1 cells. Bars: 50 μ m. The cell invasion in Scr control was regards as 100%. n = 4, ∗∗∗ P < 0.001, as compared with EV.

    Techniques Used: Over Expression, Western Blot, Expressing, Transfection, Plasmid Preparation, CCK-8 Assay, Soft Agar Assay, Activity Assay, Wound Healing Assay, Migration, Transwell Invasion 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 < 0.05, shRGS20 vs. Scr control. (b) Tumor weight of Penl1 xenografts with or without RGS20 knockdown. ∗ P < 0.001. (c) Western blotting analysis on protein lysates extracted from Penl1 xenografts with or without RGS20 depletion ( n = 3). (d) IHC staining on Penl1 xenografts with or without RGS20 depletion at day 26 after inoculation. The tissue sections were incubated with antibodies against indicated antibodies (RGS20, p-AKT (T308), p-AKT (S473), Ki-67, and cleaved caspase-3). Bar, 50 μ m.
    Figure Legend 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 < 0.05, shRGS20 vs. Scr control. (b) Tumor weight of Penl1 xenografts with or without RGS20 knockdown. ∗ P < 0.001. (c) Western blotting analysis on protein lysates extracted from Penl1 xenografts with or without RGS20 depletion ( n = 3). (d) IHC staining on Penl1 xenografts with or without RGS20 depletion at day 26 after inoculation. The tissue sections were incubated with antibodies against indicated antibodies (RGS20, p-AKT (T308), p-AKT (S473), Ki-67, and cleaved caspase-3). Bar, 50 μ m.

    Techniques Used: In Vivo, Activation Assay, Western Blot, Immunohistochemistry, Incubation

    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).
    Figure Legend 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).

    Techniques Used: Expressing, Activation Assay

    pi3k activity elisa kit k 1000s  (Echelon Biosciences)


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    Echelon Biosciences pi3k activity elisa kit k 1000s
    In vitro cell invasion assays were performed with a Fluorimetric Cell Invasion Assay kit (Chemicon; Millipore) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase <t>(PI3K)</t> inhibitor BKM120 (50 µM) was used as a positive control. Cell invasion was determined by fluorescence and shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.
    Pi3k Activity Elisa Kit K 1000s, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Endothelin A Receptor Antagonism Enhances Inhibitory Effects of Anti-Ganglioside GD2 Monoclonal Antibody on Invasiveness and Viability of Human Osteosarcoma Cells"

    Article Title: Endothelin A Receptor Antagonism Enhances Inhibitory Effects of Anti-Ganglioside GD2 Monoclonal Antibody on Invasiveness and Viability of Human Osteosarcoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093576

    In vitro cell invasion assays were performed with a Fluorimetric Cell Invasion Assay kit (Chemicon; Millipore) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. Cell invasion was determined by fluorescence and shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.
    Figure Legend Snippet: In vitro cell invasion assays were performed with a Fluorimetric Cell Invasion Assay kit (Chemicon; Millipore) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. Cell invasion was determined by fluorescence and shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Techniques Used: In Vitro, Invasion Assay, shRNA, Positive Control, Fluorescence

    MMP-2 mRNA levels were determined by real-time RT-PCR in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. The MMP-2 mRNA level was shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.
    Figure Legend Snippet: MMP-2 mRNA levels were determined by real-time RT-PCR in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. The MMP-2 mRNA level was shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Techniques Used: Quantitative RT-PCR, shRNA, Positive Control

    MMP-2 protein levels were determined by Western blot analyses in in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) cells treated with control IgG (50 µg/mL, lane 2), selective ETAR antagonist BQ123 (5 µM, lane 3), stably-transduced ETAR-shRNA (lane 4), 14G2a mAb (50 µg/mL, lane 5), 14G2a+BQ123 (lane 6), 14G2a+ETAR-shRNA (lane 7), and selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM, lane 8). The untreated control was in lane 1 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blotting was used as a loading control. Density of the MMP-2 blot was normalized against that of GAPDH to obtain a relative blot density, which was expressed as fold changes to the relative MMP-2 blot density of the untreated control cells (designated as 1). Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.
    Figure Legend Snippet: MMP-2 protein levels were determined by Western blot analyses in in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) cells treated with control IgG (50 µg/mL, lane 2), selective ETAR antagonist BQ123 (5 µM, lane 3), stably-transduced ETAR-shRNA (lane 4), 14G2a mAb (50 µg/mL, lane 5), 14G2a+BQ123 (lane 6), 14G2a+ETAR-shRNA (lane 7), and selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM, lane 8). The untreated control was in lane 1 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blotting was used as a loading control. Density of the MMP-2 blot was normalized against that of GAPDH to obtain a relative blot density, which was expressed as fold changes to the relative MMP-2 blot density of the untreated control cells (designated as 1). Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Techniques Used: Western Blot, Stable Transfection, shRNA

    MMP-2 activities were determined with a SensoLyte 520 MMP-2 Assay Kit (AnaSpec) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. The MMP-2 activity was shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.
    Figure Legend Snippet: MMP-2 activities were determined with a SensoLyte 520 MMP-2 Assay Kit (AnaSpec) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. The MMP-2 activity was shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Techniques Used: shRNA, Positive Control, Activity Assay

    Methlythiazoletetrazolium (MTT) cell viability assays were performed in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 24 or 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. Viability of the control cells was designated as 100%. The inhibition rate of cell viability was calculated and shown as a percentage of the control cell viability. Each experiment was repeated for three times in triplicates. Data values were expressed as Mean+SD.
    Figure Legend Snippet: Methlythiazoletetrazolium (MTT) cell viability assays were performed in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 24 or 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. Viability of the control cells was designated as 100%. The inhibition rate of cell viability was calculated and shown as a percentage of the control cell viability. Each experiment was repeated for three times in triplicates. Data values were expressed as Mean+SD.

    Techniques Used: shRNA, Positive Control, Inhibition

    PI3K activities were determined with a PI3K Activity ELISA kit (Echelon Biosciences) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. The PI3K activity was shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.
    Figure Legend Snippet: PI3K activities were determined with a PI3K Activity ELISA kit (Echelon Biosciences) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. The PI3K activity was shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, shRNA, Positive Control

    Levels of total Akt and P-Akt at serine 473 (ser473) were determined by Western blot analyses in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) cells treated with control IgG (50 µg/mL, lane 2), selective ETAR antagonist BQ123 (5 µM, lane 3), stably-transduced ETAR-shRNA (lane 4), 14G2a mAb (50 µg/mL, lane 5), 14G2a+BQ123 (lane 6), 14G2a+ETAR-shRNA (lane 7), and selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM, lane 8). The untreated control was in lane 1 . Density of the P-Akt (ser473) blot was normalized against that of total Akt to obtain a relative blot density, which was expressed as fold changes to the relative P-Akt (ser473) blot density of the untreated control cells (designated as 1). Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.
    Figure Legend Snippet: Levels of total Akt and P-Akt at serine 473 (ser473) were determined by Western blot analyses in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) cells treated with control IgG (50 µg/mL, lane 2), selective ETAR antagonist BQ123 (5 µM, lane 3), stably-transduced ETAR-shRNA (lane 4), 14G2a mAb (50 µg/mL, lane 5), 14G2a+BQ123 (lane 6), 14G2a+ETAR-shRNA (lane 7), and selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM, lane 8). The untreated control was in lane 1 . Density of the P-Akt (ser473) blot was normalized against that of total Akt to obtain a relative blot density, which was expressed as fold changes to the relative P-Akt (ser473) blot density of the untreated control cells (designated as 1). Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Techniques Used: Western Blot, Stable Transfection, shRNA

    pi3 kinase activity elisa pico  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences pi3 kinase activity elisa pico
    Neutrophils were treated as indicated, lysed, and <t>PI3-kinase</t> was immunoprecipitated. Immunoprecipitated PI3-kinase was incubated with PIP , 2 substrate and the amount of resultant PIP3 was measured by <t>ELISA.</t> Graph represents the mean ± SEM of 3 independent experiments ( ** P<0.01, *** P<0.001, unpaired t test).
    Pi3 Kinase Activity Elisa Pico, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Treponema denticola Major Outer Sheath Protein Impairs the Cellular Phosphoinositide Balance That Regulates Neutrophil Chemotaxis"

    Article Title: Treponema denticola Major Outer Sheath Protein Impairs the Cellular Phosphoinositide Balance That Regulates Neutrophil Chemotaxis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0066209

    Neutrophils were treated as indicated, lysed, and PI3-kinase was immunoprecipitated. Immunoprecipitated PI3-kinase was incubated with PIP , 2 substrate and the amount of resultant PIP3 was measured by ELISA. Graph represents the mean ± SEM of 3 independent experiments ( ** P<0.01, *** P<0.001, unpaired t test).
    Figure Legend Snippet: Neutrophils were treated as indicated, lysed, and PI3-kinase was immunoprecipitated. Immunoprecipitated PI3-kinase was incubated with PIP , 2 substrate and the amount of resultant PIP3 was measured by ELISA. Graph represents the mean ± SEM of 3 independent experiments ( ** P<0.01, *** P<0.001, unpaired t test).

    Techniques Used: Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay

    pi3 kinase activity  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences pi3 kinase activity
    Neutrophils were treated as indicated, lysed, and <t>PI3-kinase</t> was immunoprecipitated. Immunoprecipitated PI3-kinase was incubated with PIP , 2 substrate and the amount of resultant PIP3 was measured by ELISA. Graph represents the mean ± SEM of 3 independent experiments ( ** P<0.01, *** P<0.001, unpaired t test).
    Pi3 Kinase Activity, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Treponema denticola Major Outer Sheath Protein Impairs the Cellular Phosphoinositide Balance That Regulates Neutrophil Chemotaxis"

    Article Title: Treponema denticola Major Outer Sheath Protein Impairs the Cellular Phosphoinositide Balance That Regulates Neutrophil Chemotaxis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0066209

    Neutrophils were treated as indicated, lysed, and PI3-kinase was immunoprecipitated. Immunoprecipitated PI3-kinase was incubated with PIP , 2 substrate and the amount of resultant PIP3 was measured by ELISA. Graph represents the mean ± SEM of 3 independent experiments ( ** P<0.01, *** P<0.001, unpaired t test).
    Figure Legend Snippet: Neutrophils were treated as indicated, lysed, and PI3-kinase was immunoprecipitated. Immunoprecipitated PI3-kinase was incubated with PIP , 2 substrate and the amount of resultant PIP3 was measured by ELISA. Graph represents the mean ± SEM of 3 independent experiments ( ** P<0.01, *** P<0.001, unpaired t test).

    Techniques Used: Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay

    Akt is a downstream target of PI3-kinase. Akt localization and activation can be considered indirect measures of PIP3 generation. A) Localization of Akt and actin in neutrophils visualized by indirect immunofluorescence. Alexa 594-phalloidin F-actin label in red and Akt antibody label in green. Asterix indicates leading edge of neutrophil. B) Graph represents the percentage of cells in which Akt and actin fluorescence are localized together or mislocalized (mean ± SEM, Msp + fMLP treatment (white bars), fMLP treatment alone (black bars). C) Phosphorylation of Akt in cell lysates measured by immunoblotting, following Msp pretreatment and fMLP stimulation. D) PTEN inhibition with the inhibitor VO-OHpic increased PIP3 production and downstream signaling (left panel graph). Treatment of neutrophils with VO-OHpic prior to Msp treatment restored PIP3 production and Akt signaling in response to fMLP stimulation (right panel graph). Representative phospho-Akt immunoblots are shown in panels B and C. Graphs represent the mean ± SEM densitometry of 3 independent experiments (* P<0.05, ** P<0.01, *** P<0.001, ns = not significant, unpaired t test).
    Figure Legend Snippet: Akt is a downstream target of PI3-kinase. Akt localization and activation can be considered indirect measures of PIP3 generation. A) Localization of Akt and actin in neutrophils visualized by indirect immunofluorescence. Alexa 594-phalloidin F-actin label in red and Akt antibody label in green. Asterix indicates leading edge of neutrophil. B) Graph represents the percentage of cells in which Akt and actin fluorescence are localized together or mislocalized (mean ± SEM, Msp + fMLP treatment (white bars), fMLP treatment alone (black bars). C) Phosphorylation of Akt in cell lysates measured by immunoblotting, following Msp pretreatment and fMLP stimulation. D) PTEN inhibition with the inhibitor VO-OHpic increased PIP3 production and downstream signaling (left panel graph). Treatment of neutrophils with VO-OHpic prior to Msp treatment restored PIP3 production and Akt signaling in response to fMLP stimulation (right panel graph). Representative phospho-Akt immunoblots are shown in panels B and C. Graphs represent the mean ± SEM densitometry of 3 independent experiments (* P<0.05, ** P<0.01, *** P<0.001, ns = not significant, unpaired t test).

    Techniques Used: Activation Assay, Immunofluorescence, Fluorescence, Western Blot, Inhibition

    pi3k activity  (Echelon Biosciences)


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    Echelon Biosciences pi3k activity
    (A): Doxycycline inhibited the VEGF-C-induced proliferation of HDLECs in a dose-dependent manner. (B): The effect of different concentrations of doxycycline on cell viability. (C): The nitrite content in the supernatants was measured using the Griess reaction. (D): The expression of (phosphorylated) eNOS was determined using Western blotting. <t>PI3K</t> activity (E) and (phosphorylated) Akt (F) levels were determined using ELISA and Western blotting, respectively. * p <0.05, ** p <0.01, *** p <0.001.
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    Images

    1) Product Images from "Doxycycline Inhibits Inflammation-Induced Lymphangiogenesis in Mouse Cornea by Multiple Mechanisms"

    Article Title: Doxycycline Inhibits Inflammation-Induced Lymphangiogenesis in Mouse Cornea by Multiple Mechanisms

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0108931

    (A): Doxycycline inhibited the VEGF-C-induced proliferation of HDLECs in a dose-dependent manner. (B): The effect of different concentrations of doxycycline on cell viability. (C): The nitrite content in the supernatants was measured using the Griess reaction. (D): The expression of (phosphorylated) eNOS was determined using Western blotting. PI3K activity (E) and (phosphorylated) Akt (F) levels were determined using ELISA and Western blotting, respectively. * p <0.05, ** p <0.01, *** p <0.001.
    Figure Legend Snippet: (A): Doxycycline inhibited the VEGF-C-induced proliferation of HDLECs in a dose-dependent manner. (B): The effect of different concentrations of doxycycline on cell viability. (C): The nitrite content in the supernatants was measured using the Griess reaction. (D): The expression of (phosphorylated) eNOS was determined using Western blotting. PI3K activity (E) and (phosphorylated) Akt (F) levels were determined using ELISA and Western blotting, respectively. * p <0.05, ** p <0.01, *** p <0.001.

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

    The levels of IL-1β (A) and TNF-α (B) in the supernatants were measured using ELISA. The expression of VEGF-C mRNA (C) was determined using real-time PCR. Doxycycline treatment significantly reduced PI3K activity (D). The levels of (phosphorylated) NF-κBp65 (E), IκB-α (F) and (phosphorylated) Akt (G) were determined using Western blotting. * p <0.05, ** p <0.01.
    Figure Legend Snippet: The levels of IL-1β (A) and TNF-α (B) in the supernatants were measured using ELISA. The expression of VEGF-C mRNA (C) was determined using real-time PCR. Doxycycline treatment significantly reduced PI3K activity (D). The levels of (phosphorylated) NF-κBp65 (E), IκB-α (F) and (phosphorylated) Akt (G) were determined using Western blotting. * p <0.05, ** p <0.01.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Western Blot

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    Echelon Biosciences pi3k activity elisa kit
    Expression and activation of IGF-IR and <t>PI3K</t> in MDA-MB-231 and MCF7 cells. (a) After stimulation with IGF-I for 0, 0.5, or 6 h, cells were lysed and processed for immunoblotting with anti-IGF-IR antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (b) After stimulation with IGF-I, cells were immunostained with antibody to phospho-IGF-IR (Tyr1135/1135). Scale Bars 20 μ m. (c) After stimulation with IGF-I, cells were lysed and processed for immunoblotting with anti-PI3K (p85) antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (d) Cells after stimulation with IGF-I were lysed and immunoprecipitated with anti-PI3K (p85) antibody. PI3K activity was assayed for the immunoprecipitates as described in Materials and Methods. The mean (SD) values of triplicate assays are given as activity relative to that in unstimulated control MDA-MB-231 cells.
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    Echelon Biosciences pi3 kinase activity elisa pico
    Expression and activation of IGF-IR and <t>PI3K</t> in MDA-MB-231 and MCF7 cells. (a) After stimulation with IGF-I for 0, 0.5, or 6 h, cells were lysed and processed for immunoblotting with anti-IGF-IR antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (b) After stimulation with IGF-I, cells were immunostained with antibody to phospho-IGF-IR (Tyr1135/1135). Scale Bars 20 μ m. (c) After stimulation with IGF-I, cells were lysed and processed for immunoblotting with anti-PI3K (p85) antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (d) Cells after stimulation with IGF-I were lysed and immunoprecipitated with anti-PI3K (p85) antibody. PI3K activity was assayed for the immunoprecipitates as described in Materials and Methods. The mean (SD) values of triplicate assays are given as activity relative to that in unstimulated control MDA-MB-231 cells.
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    Echelon Biosciences pi3k activity
    Slit2N inhibits VEGF-C-enhanced <t>PI3K</t> activity and Akt phosphorylation in L-LECs. (A) Representative Western blot analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Total ERK1/2 used as loading control. (B) Quantitative analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4A was determined by densitometry. The ratio of p-ERK1/2 to total ERK1/2 of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments. (C) PI3K activity by ELISA in L-LECs incubated with various concentrations of Slit2N and/or VEGF-C [100 ng/ml]. Data represent the mean ± SD of 3 independent experiments (**p < 0.01, ***p < 0.001). (D) Representative Western blot analysis of phosphorylated Akt in L-LECs incubated with a control, VEGF-C [100 ng/ml]; or preincubated with various concentrations of Slit2N, then VEGF-C. Total Akt used as loading control. (E) Quantitative analysis of phosphorylated Akt in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4D was determined by densitometry. The ratio of p-Akt to total Akt of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments (**p < 0.01).
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    Echelon Biosciences immunosorbent assay elisa k
    Slit2N inhibits VEGF-C-enhanced <t>PI3K</t> activity and Akt phosphorylation in L-LECs. (A) Representative Western blot analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Total ERK1/2 used as loading control. (B) Quantitative analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4A was determined by densitometry. The ratio of p-ERK1/2 to total ERK1/2 of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments. (C) PI3K activity by ELISA in L-LECs incubated with various concentrations of Slit2N and/or VEGF-C [100 ng/ml]. Data represent the mean ± SD of 3 independent experiments (**p < 0.01, ***p < 0.001). (D) Representative Western blot analysis of phosphorylated Akt in L-LECs incubated with a control, VEGF-C [100 ng/ml]; or preincubated with various concentrations of Slit2N, then VEGF-C. Total Akt used as loading control. (E) Quantitative analysis of phosphorylated Akt in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4D was determined by densitometry. The ratio of p-Akt to total Akt of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments (**p < 0.01).
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    Echelon Biosciences pi3k activity assay
    (A) The cellular content of PIP3 was quantified using a competitive PIP3 ELISA; *p<0.05; **p<0.01 versus 2008-scb control; # p<0.05 versus CLDN3KD; ## p<0.01 versus CLDN4KD. (B) Activity of <t>PI3K</t> immunoprecipitated with antibody to p85 regulatory subunit of class IA PI3K; *p<0.01; **p<0.01 versus 2008-scb control; # p<0.01 versus CLDN3KD; ## p<0.01 versus CLDN4KD. (C) PIP3 levels; *p<0.001 versus CLDN3KD; **p<0.001 versus CLDN4KD. (D) Activity of PI3K; *p<0.001 versus CLDN3KD; **p<0.001 versus CLDN4KD. Values are mean ± SEM, n = 3. PI3K activity is expressed as percent of control.
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    Echelon Biosciences pi3k activity elisa kit k 1000s
    In vitro cell invasion assays were performed with a Fluorimetric Cell Invasion Assay kit (Chemicon; Millipore) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase <t>(PI3K)</t> inhibitor BKM120 (50 µM) was used as a positive control. Cell invasion was determined by fluorescence and shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.
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    Echelon Biosciences pi3 kinase activity
    Neutrophils were treated as indicated, lysed, and <t>PI3-kinase</t> was immunoprecipitated. Immunoprecipitated PI3-kinase was incubated with PIP , 2 substrate and the amount of resultant PIP3 was measured by ELISA. Graph represents the mean ± SEM of 3 independent experiments ( ** P<0.01, *** P<0.001, unpaired t test).
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    Expression and activation of IGF-IR and PI3K in MDA-MB-231 and MCF7 cells. (a) After stimulation with IGF-I for 0, 0.5, or 6 h, cells were lysed and processed for immunoblotting with anti-IGF-IR antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (b) After stimulation with IGF-I, cells were immunostained with antibody to phospho-IGF-IR (Tyr1135/1135). Scale Bars 20 μ m. (c) After stimulation with IGF-I, cells were lysed and processed for immunoblotting with anti-PI3K (p85) antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (d) Cells after stimulation with IGF-I were lysed and immunoprecipitated with anti-PI3K (p85) antibody. PI3K activity was assayed for the immunoprecipitates as described in Materials and Methods. The mean (SD) values of triplicate assays are given as activity relative to that in unstimulated control MDA-MB-231 cells.

    Journal: International Journal of Cell Biology

    Article Title: Rac1 and Stathmin but Not EB1 Are Required for Invasion of Breast Cancer Cells in Response to IGF-I

    doi: 10.1155/2011/615912

    Figure Lengend Snippet: Expression and activation of IGF-IR and PI3K in MDA-MB-231 and MCF7 cells. (a) After stimulation with IGF-I for 0, 0.5, or 6 h, cells were lysed and processed for immunoblotting with anti-IGF-IR antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (b) After stimulation with IGF-I, cells were immunostained with antibody to phospho-IGF-IR (Tyr1135/1135). Scale Bars 20 μ m. (c) After stimulation with IGF-I, cells were lysed and processed for immunoblotting with anti-PI3K (p85) antibody. Values are given as band intensity relative to that in unstimulated control MDA-MB-231 cells. (d) Cells after stimulation with IGF-I were lysed and immunoprecipitated with anti-PI3K (p85) antibody. PI3K activity was assayed for the immunoprecipitates as described in Materials and Methods. The mean (SD) values of triplicate assays are given as activity relative to that in unstimulated control MDA-MB-231 cells.

    Article Snippet: PI3K activity was measured by immunoprecipitation of PI3K p85 with anti-p85 antibody, followed by the kinase reaction of the immunoprecipitates in a PI3K activity ELISA kit (Echelon Bioscience, Salt Lake City, UT), according to the manufacturer's instructions.

    Techniques: Expressing, Activation Assay, Western Blot, Immunoprecipitation, Activity Assay

    Slit2N inhibits VEGF-C-enhanced PI3K activity and Akt phosphorylation in L-LECs. (A) Representative Western blot analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Total ERK1/2 used as loading control. (B) Quantitative analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4A was determined by densitometry. The ratio of p-ERK1/2 to total ERK1/2 of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments. (C) PI3K activity by ELISA in L-LECs incubated with various concentrations of Slit2N and/or VEGF-C [100 ng/ml]. Data represent the mean ± SD of 3 independent experiments (**p < 0.01, ***p < 0.001). (D) Representative Western blot analysis of phosphorylated Akt in L-LECs incubated with a control, VEGF-C [100 ng/ml]; or preincubated with various concentrations of Slit2N, then VEGF-C. Total Akt used as loading control. (E) Quantitative analysis of phosphorylated Akt in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4D was determined by densitometry. The ratio of p-Akt to total Akt of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments (**p < 0.01).

    Journal: Cell Communication and Signaling : CCS

    Article Title: Slit2N and Robo4 regulate lymphangiogenesis through the VEGF-C/VEGFR-3 pathway

    doi: 10.1186/1478-811X-12-25

    Figure Lengend Snippet: Slit2N inhibits VEGF-C-enhanced PI3K activity and Akt phosphorylation in L-LECs. (A) Representative Western blot analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Total ERK1/2 used as loading control. (B) Quantitative analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4A was determined by densitometry. The ratio of p-ERK1/2 to total ERK1/2 of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments. (C) PI3K activity by ELISA in L-LECs incubated with various concentrations of Slit2N and/or VEGF-C [100 ng/ml]. Data represent the mean ± SD of 3 independent experiments (**p < 0.01, ***p < 0.001). (D) Representative Western blot analysis of phosphorylated Akt in L-LECs incubated with a control, VEGF-C [100 ng/ml]; or preincubated with various concentrations of Slit2N, then VEGF-C. Total Akt used as loading control. (E) Quantitative analysis of phosphorylated Akt in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4D was determined by densitometry. The ratio of p-Akt to total Akt of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments (**p < 0.01).

    Article Snippet: PI3K activity was assayed with the PI3-Kinase Activity ELISA: Pico (Echelon Biosciences, Inc., Salt Lake City, UT).

    Techniques: Activity Assay, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay

    Slit2N inhibition of VEGF-C-enhanced PI3K activity and Akt phosphorylation in L-LECs is Robo4-dependent. (A) PI3K activity by ELISA in L-LECs transfected with control siRNAs or Robo4-specific siRNAs, incubated with control or VEGF-C [100 ng/ml]; or after pretreatment with 10 nM Slit2N, then VEGF-C. Data represent the mean ± SD of 3 independent experiments (*p < 0.05; NS: not statistically significant). (B) Representative Western blot analysis of Akt phosphorylation in L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs, and subsequently incubated with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after pretreatment with Slit2N, then VEGF-C. Total Akt used as loading control. (C) Quantitative analysis of Akt phosphorylation in L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs, and subsequently incubated with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after pretreatment with Slit2N, then VEGF-C. Band intensity of each lane from Figure 6B was determined by densitometry. The ratio of p-Akt/total Akt in control siRNA-transfected L-LECs incubated with VEGF-C alone was set to “1” and all other ratios were determined vs. this control. Data represent the mean ± SD of 3 independent experiments (***p < 0.001).

    Journal: Cell Communication and Signaling : CCS

    Article Title: Slit2N and Robo4 regulate lymphangiogenesis through the VEGF-C/VEGFR-3 pathway

    doi: 10.1186/1478-811X-12-25

    Figure Lengend Snippet: Slit2N inhibition of VEGF-C-enhanced PI3K activity and Akt phosphorylation in L-LECs is Robo4-dependent. (A) PI3K activity by ELISA in L-LECs transfected with control siRNAs or Robo4-specific siRNAs, incubated with control or VEGF-C [100 ng/ml]; or after pretreatment with 10 nM Slit2N, then VEGF-C. Data represent the mean ± SD of 3 independent experiments (*p < 0.05; NS: not statistically significant). (B) Representative Western blot analysis of Akt phosphorylation in L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs, and subsequently incubated with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after pretreatment with Slit2N, then VEGF-C. Total Akt used as loading control. (C) Quantitative analysis of Akt phosphorylation in L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs, and subsequently incubated with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after pretreatment with Slit2N, then VEGF-C. Band intensity of each lane from Figure 6B was determined by densitometry. The ratio of p-Akt/total Akt in control siRNA-transfected L-LECs incubated with VEGF-C alone was set to “1” and all other ratios were determined vs. this control. Data represent the mean ± SD of 3 independent experiments (***p < 0.001).

    Article Snippet: PI3K activity was assayed with the PI3-Kinase Activity ELISA: Pico (Echelon Biosciences, Inc., Salt Lake City, UT).

    Techniques: Inhibition, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Incubation, Western Blot

    (A) The cellular content of PIP3 was quantified using a competitive PIP3 ELISA; *p<0.05; **p<0.01 versus 2008-scb control; # p<0.05 versus CLDN3KD; ## p<0.01 versus CLDN4KD. (B) Activity of PI3K immunoprecipitated with antibody to p85 regulatory subunit of class IA PI3K; *p<0.01; **p<0.01 versus 2008-scb control; # p<0.01 versus CLDN3KD; ## p<0.01 versus CLDN4KD. (C) PIP3 levels; *p<0.001 versus CLDN3KD; **p<0.001 versus CLDN4KD. (D) Activity of PI3K; *p<0.001 versus CLDN3KD; **p<0.001 versus CLDN4KD. Values are mean ± SEM, n = 3. PI3K activity is expressed as percent of control.

    Journal: PLoS ONE

    Article Title: Regulation of the Epithelial-Mesenchymal Transition by Claudin-3 and Claudin-4

    doi: 10.1371/journal.pone.0067496

    Figure Lengend Snippet: (A) The cellular content of PIP3 was quantified using a competitive PIP3 ELISA; *p<0.05; **p<0.01 versus 2008-scb control; # p<0.05 versus CLDN3KD; ## p<0.01 versus CLDN4KD. (B) Activity of PI3K immunoprecipitated with antibody to p85 regulatory subunit of class IA PI3K; *p<0.01; **p<0.01 versus 2008-scb control; # p<0.01 versus CLDN3KD; ## p<0.01 versus CLDN4KD. (C) PIP3 levels; *p<0.001 versus CLDN3KD; **p<0.001 versus CLDN4KD. (D) Activity of PI3K; *p<0.001 versus CLDN3KD; **p<0.001 versus CLDN4KD. Values are mean ± SEM, n = 3. PI3K activity is expressed as percent of control.

    Article Snippet: PI3K activity assay was performed with an enzyme-linked immunosorbent assay (ELISA) K-1000s-PI3-kinase activity (ELISA:Pico, Echelon Biosciences Inc., Salt Lake City, UT,) according the manufacturer's instructions.

    Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Immunoprecipitation

    In vitro cell invasion assays were performed with a Fluorimetric Cell Invasion Assay kit (Chemicon; Millipore) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. Cell invasion was determined by fluorescence and shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Journal: PLoS ONE

    Article Title: Endothelin A Receptor Antagonism Enhances Inhibitory Effects of Anti-Ganglioside GD2 Monoclonal Antibody on Invasiveness and Viability of Human Osteosarcoma Cells

    doi: 10.1371/journal.pone.0093576

    Figure Lengend Snippet: In vitro cell invasion assays were performed with a Fluorimetric Cell Invasion Assay kit (Chemicon; Millipore) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. Cell invasion was determined by fluorescence and shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Article Snippet: The PI3K Activity ELISA kit (K-1000s) was purchased from Echelon Biosciences (Salt Lake City, UT, USA).

    Techniques: In Vitro, Invasion Assay, shRNA, Positive Control, Fluorescence

    MMP-2 mRNA levels were determined by real-time RT-PCR in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. The MMP-2 mRNA level was shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Journal: PLoS ONE

    Article Title: Endothelin A Receptor Antagonism Enhances Inhibitory Effects of Anti-Ganglioside GD2 Monoclonal Antibody on Invasiveness and Viability of Human Osteosarcoma Cells

    doi: 10.1371/journal.pone.0093576

    Figure Lengend Snippet: MMP-2 mRNA levels were determined by real-time RT-PCR in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. The MMP-2 mRNA level was shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Article Snippet: The PI3K Activity ELISA kit (K-1000s) was purchased from Echelon Biosciences (Salt Lake City, UT, USA).

    Techniques: Quantitative RT-PCR, shRNA, Positive Control

    MMP-2 protein levels were determined by Western blot analyses in in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) cells treated with control IgG (50 µg/mL, lane 2), selective ETAR antagonist BQ123 (5 µM, lane 3), stably-transduced ETAR-shRNA (lane 4), 14G2a mAb (50 µg/mL, lane 5), 14G2a+BQ123 (lane 6), 14G2a+ETAR-shRNA (lane 7), and selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM, lane 8). The untreated control was in lane 1 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blotting was used as a loading control. Density of the MMP-2 blot was normalized against that of GAPDH to obtain a relative blot density, which was expressed as fold changes to the relative MMP-2 blot density of the untreated control cells (designated as 1). Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Journal: PLoS ONE

    Article Title: Endothelin A Receptor Antagonism Enhances Inhibitory Effects of Anti-Ganglioside GD2 Monoclonal Antibody on Invasiveness and Viability of Human Osteosarcoma Cells

    doi: 10.1371/journal.pone.0093576

    Figure Lengend Snippet: MMP-2 protein levels were determined by Western blot analyses in in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) cells treated with control IgG (50 µg/mL, lane 2), selective ETAR antagonist BQ123 (5 µM, lane 3), stably-transduced ETAR-shRNA (lane 4), 14G2a mAb (50 µg/mL, lane 5), 14G2a+BQ123 (lane 6), 14G2a+ETAR-shRNA (lane 7), and selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM, lane 8). The untreated control was in lane 1 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blotting was used as a loading control. Density of the MMP-2 blot was normalized against that of GAPDH to obtain a relative blot density, which was expressed as fold changes to the relative MMP-2 blot density of the untreated control cells (designated as 1). Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Article Snippet: The PI3K Activity ELISA kit (K-1000s) was purchased from Echelon Biosciences (Salt Lake City, UT, USA).

    Techniques: Western Blot, Stable Transfection, shRNA

    MMP-2 activities were determined with a SensoLyte 520 MMP-2 Assay Kit (AnaSpec) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. The MMP-2 activity was shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Journal: PLoS ONE

    Article Title: Endothelin A Receptor Antagonism Enhances Inhibitory Effects of Anti-Ganglioside GD2 Monoclonal Antibody on Invasiveness and Viability of Human Osteosarcoma Cells

    doi: 10.1371/journal.pone.0093576

    Figure Lengend Snippet: MMP-2 activities were determined with a SensoLyte 520 MMP-2 Assay Kit (AnaSpec) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. The MMP-2 activity was shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Article Snippet: The PI3K Activity ELISA kit (K-1000s) was purchased from Echelon Biosciences (Salt Lake City, UT, USA).

    Techniques: shRNA, Positive Control, Activity Assay

    Methlythiazoletetrazolium (MTT) cell viability assays were performed in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 24 or 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. Viability of the control cells was designated as 100%. The inhibition rate of cell viability was calculated and shown as a percentage of the control cell viability. Each experiment was repeated for three times in triplicates. Data values were expressed as Mean+SD.

    Journal: PLoS ONE

    Article Title: Endothelin A Receptor Antagonism Enhances Inhibitory Effects of Anti-Ganglioside GD2 Monoclonal Antibody on Invasiveness and Viability of Human Osteosarcoma Cells

    doi: 10.1371/journal.pone.0093576

    Figure Lengend Snippet: Methlythiazoletetrazolium (MTT) cell viability assays were performed in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 24 or 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. Viability of the control cells was designated as 100%. The inhibition rate of cell viability was calculated and shown as a percentage of the control cell viability. Each experiment was repeated for three times in triplicates. Data values were expressed as Mean+SD.

    Article Snippet: The PI3K Activity ELISA kit (K-1000s) was purchased from Echelon Biosciences (Salt Lake City, UT, USA).

    Techniques: shRNA, Positive Control, Inhibition

    PI3K activities were determined with a PI3K Activity ELISA kit (Echelon Biosciences) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. The PI3K activity was shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Journal: PLoS ONE

    Article Title: Endothelin A Receptor Antagonism Enhances Inhibitory Effects of Anti-Ganglioside GD2 Monoclonal Antibody on Invasiveness and Viability of Human Osteosarcoma Cells

    doi: 10.1371/journal.pone.0093576

    Figure Lengend Snippet: PI3K activities were determined with a PI3K Activity ELISA kit (Echelon Biosciences) in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) OS cells treated with control IgG (PK136 mAb, 50 µg/mL), 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), and 14G2a (50 µg/mL)+BQ123 (5 µM) for 48 hours. Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) was used as a positive control. The PI3K activity was shown as fold changes to that of the untreated control cells (designated as 1). Each experiment was repeated for three times in duplicates. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Article Snippet: The PI3K Activity ELISA kit (K-1000s) was purchased from Echelon Biosciences (Salt Lake City, UT, USA).

    Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, shRNA, Positive Control

    Levels of total Akt and P-Akt at serine 473 (ser473) were determined by Western blot analyses in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) cells treated with control IgG (50 µg/mL, lane 2), selective ETAR antagonist BQ123 (5 µM, lane 3), stably-transduced ETAR-shRNA (lane 4), 14G2a mAb (50 µg/mL, lane 5), 14G2a+BQ123 (lane 6), 14G2a+ETAR-shRNA (lane 7), and selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM, lane 8). The untreated control was in lane 1 . Density of the P-Akt (ser473) blot was normalized against that of total Akt to obtain a relative blot density, which was expressed as fold changes to the relative P-Akt (ser473) blot density of the untreated control cells (designated as 1). Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Journal: PLoS ONE

    Article Title: Endothelin A Receptor Antagonism Enhances Inhibitory Effects of Anti-Ganglioside GD2 Monoclonal Antibody on Invasiveness and Viability of Human Osteosarcoma Cells

    doi: 10.1371/journal.pone.0093576

    Figure Lengend Snippet: Levels of total Akt and P-Akt at serine 473 (ser473) were determined by Western blot analyses in Saos-2 ( A ), MG-63 ( B ) and SJSA-1 ( C ) cells treated with control IgG (50 µg/mL, lane 2), selective ETAR antagonist BQ123 (5 µM, lane 3), stably-transduced ETAR-shRNA (lane 4), 14G2a mAb (50 µg/mL, lane 5), 14G2a+BQ123 (lane 6), 14G2a+ETAR-shRNA (lane 7), and selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM, lane 8). The untreated control was in lane 1 . Density of the P-Akt (ser473) blot was normalized against that of total Akt to obtain a relative blot density, which was expressed as fold changes to the relative P-Akt (ser473) blot density of the untreated control cells (designated as 1). Three independent experiments were performed for each Western blot analysis. Data values were expressed as Mean+SD. a p <0.05 vs. control or control IgG; b p <0.05 vs. BQ123; c p <0.05 vs. ETAR-shRNA; d p <0.05 vs. 14G2a; e p <0.05 vs. 14G2a+BQ123; f p <0.05 vs. 14G2a+ETAR-shRNA.

    Article Snippet: The PI3K Activity ELISA kit (K-1000s) was purchased from Echelon Biosciences (Salt Lake City, UT, USA).

    Techniques: Western Blot, Stable Transfection, shRNA

    Neutrophils were treated as indicated, lysed, and PI3-kinase was immunoprecipitated. Immunoprecipitated PI3-kinase was incubated with PIP , 2 substrate and the amount of resultant PIP3 was measured by ELISA. Graph represents the mean ± SEM of 3 independent experiments ( ** P<0.01, *** P<0.001, unpaired t test).

    Journal: PLoS ONE

    Article Title: Treponema denticola Major Outer Sheath Protein Impairs the Cellular Phosphoinositide Balance That Regulates Neutrophil Chemotaxis

    doi: 10.1371/journal.pone.0066209

    Figure Lengend Snippet: Neutrophils were treated as indicated, lysed, and PI3-kinase was immunoprecipitated. Immunoprecipitated PI3-kinase was incubated with PIP , 2 substrate and the amount of resultant PIP3 was measured by ELISA. Graph represents the mean ± SEM of 3 independent experiments ( ** P<0.01, *** P<0.001, unpaired t test).

    Article Snippet: PI3-kinase activity was measured using a PI3-Kinase activity ELISA: Pico (Echelon) according to the manufacturer's protocol.

    Techniques: Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay

    Akt is a downstream target of PI3-kinase. Akt localization and activation can be considered indirect measures of PIP3 generation. A) Localization of Akt and actin in neutrophils visualized by indirect immunofluorescence. Alexa 594-phalloidin F-actin label in red and Akt antibody label in green. Asterix indicates leading edge of neutrophil. B) Graph represents the percentage of cells in which Akt and actin fluorescence are localized together or mislocalized (mean ± SEM, Msp + fMLP treatment (white bars), fMLP treatment alone (black bars). C) Phosphorylation of Akt in cell lysates measured by immunoblotting, following Msp pretreatment and fMLP stimulation. D) PTEN inhibition with the inhibitor VO-OHpic increased PIP3 production and downstream signaling (left panel graph). Treatment of neutrophils with VO-OHpic prior to Msp treatment restored PIP3 production and Akt signaling in response to fMLP stimulation (right panel graph). Representative phospho-Akt immunoblots are shown in panels B and C. Graphs represent the mean ± SEM densitometry of 3 independent experiments (* P<0.05, ** P<0.01, *** P<0.001, ns = not significant, unpaired t test).

    Journal: PLoS ONE

    Article Title: Treponema denticola Major Outer Sheath Protein Impairs the Cellular Phosphoinositide Balance That Regulates Neutrophil Chemotaxis

    doi: 10.1371/journal.pone.0066209

    Figure Lengend Snippet: Akt is a downstream target of PI3-kinase. Akt localization and activation can be considered indirect measures of PIP3 generation. A) Localization of Akt and actin in neutrophils visualized by indirect immunofluorescence. Alexa 594-phalloidin F-actin label in red and Akt antibody label in green. Asterix indicates leading edge of neutrophil. B) Graph represents the percentage of cells in which Akt and actin fluorescence are localized together or mislocalized (mean ± SEM, Msp + fMLP treatment (white bars), fMLP treatment alone (black bars). C) Phosphorylation of Akt in cell lysates measured by immunoblotting, following Msp pretreatment and fMLP stimulation. D) PTEN inhibition with the inhibitor VO-OHpic increased PIP3 production and downstream signaling (left panel graph). Treatment of neutrophils with VO-OHpic prior to Msp treatment restored PIP3 production and Akt signaling in response to fMLP stimulation (right panel graph). Representative phospho-Akt immunoblots are shown in panels B and C. Graphs represent the mean ± SEM densitometry of 3 independent experiments (* P<0.05, ** P<0.01, *** P<0.001, ns = not significant, unpaired t test).

    Article Snippet: PI3-kinase activity was measured using a PI3-Kinase activity ELISA: Pico (Echelon) according to the manufacturer's protocol.

    Techniques: Activation Assay, Immunofluorescence, Fluorescence, Western Blot, Inhibition