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  • 90
    Name:
    Akt Antibody
    Description:
    Akt also referred to as PKB or Rac plays a critical role in controlling survival and apoptosis 1 3 This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin sensitive pathway involving PI3 kinase 2 3 Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 4 and by phosphorylation within the carboxy terminus at Ser473 The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin mTOR in a rapamycin insensitive complex with rictor and Sin1 5 6 Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets including Bad 7 forkhead transcription factors 8 c Raf 9 and caspase 9 PTEN phosphatase is a major negative regulator of the PI3 kinase Akt signaling pathway 10 LY294002 is a specific PI3 kinase inhibitor 11 Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK 3α and β 12 13 Akt may also play a role in insulin stimulation of glucose transport 12 In addition to its role in survival and glycogen synthesis Akt is involved in cell cycle regulation by preventing GSK 3β mediated phosphorylation and degradation of cyclin D1 14 and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 15 and p21 Waf1 Cip1 16 Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin sensitive complex containing raptor 17 More importantly Akt phosphorylates and inactivates tuberin TSC2 an inhibitor of mTOR within the mTOR raptor complex 18 19
    Catalog Number:
    9272
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation, Immunofluorescence, Flow Cytometry
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the carboxy-terminal sequence of mouse Akt. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Hamster Monkey Chicken D melanogaster Bovine Dog Pig Guinea Pig
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc akt
    Feedback loop in TKI therapy IL-6 and VEGF activate <t>AKT-mTOR</t> and the STAT3 signaling cascade, consequently leading to enhanced secretion of IL-6 and VEGF. Secreted IL-6 and VEGF can again re-activate tumor cells and thus trigger tumor progression. A combination therapy of TKI together with an anti-IL-6R antibody could inhibit this amplifier circle.
    Akt also referred to as PKB or Rac plays a critical role in controlling survival and apoptosis 1 3 This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin sensitive pathway involving PI3 kinase 2 3 Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 4 and by phosphorylation within the carboxy terminus at Ser473 The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin mTOR in a rapamycin insensitive complex with rictor and Sin1 5 6 Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets including Bad 7 forkhead transcription factors 8 c Raf 9 and caspase 9 PTEN phosphatase is a major negative regulator of the PI3 kinase Akt signaling pathway 10 LY294002 is a specific PI3 kinase inhibitor 11 Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK 3α and β 12 13 Akt may also play a role in insulin stimulation of glucose transport 12 In addition to its role in survival and glycogen synthesis Akt is involved in cell cycle regulation by preventing GSK 3β mediated phosphorylation and degradation of cyclin D1 14 and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 15 and p21 Waf1 Cip1 16 Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin sensitive complex containing raptor 17 More importantly Akt phosphorylates and inactivates tuberin TSC2 an inhibitor of mTOR within the mTOR raptor complex 18 19
    https://www.bioz.com/result/akt/product/Cell Signaling Technology Inc
    Average 90 stars, based on 3353 article reviews
    Price from $9.99 to $1999.99
    akt - by Bioz Stars, 2020-02
    90/100 stars

    Images

    1) Product Images from "Overriding TKI resistance of renal cell carcinoma by combination therapy with IL-6 receptor blockade"

    Article Title: Overriding TKI resistance of renal cell carcinoma by combination therapy with IL-6 receptor blockade

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19420

    Feedback loop in TKI therapy IL-6 and VEGF activate AKT-mTOR and the STAT3 signaling cascade, consequently leading to enhanced secretion of IL-6 and VEGF. Secreted IL-6 and VEGF can again re-activate tumor cells and thus trigger tumor progression. A combination therapy of TKI together with an anti-IL-6R antibody could inhibit this amplifier circle.
    Figure Legend Snippet: Feedback loop in TKI therapy IL-6 and VEGF activate AKT-mTOR and the STAT3 signaling cascade, consequently leading to enhanced secretion of IL-6 and VEGF. Secreted IL-6 and VEGF can again re-activate tumor cells and thus trigger tumor progression. A combination therapy of TKI together with an anti-IL-6R antibody could inhibit this amplifier circle.

    Techniques Used:

    Effects of IL-6 signaling blockade on activity of the AKT-mTOR pathway after ( A ) sorafenib, ( B ) sunitinib or ( C ) pazopanib treatment. Influence of tocilizumab (50 μg/ml) on activation of AKT-mTOR pathway in 786-O cells after treatment with TKI was determined by Western blot. The concentrations of TKIs are indicated on the X axis of each graph. In combinational treatment (right part of each graph) tocilizumab was used in a concentration of 50 μg/ml. The column labeled by 0 μg/ml in the right part of each graph represents treatment with tocilizumab alone. Phosphorylation of AKT, mTOR, 4EBP1, S6RP, NFκB, and STAT3 was enhanced after TKI treatment at concentration of 0.5 μM. HIF-2α was enhanced by treatment with TKIs in all concentrations used. Tocilizumab neutralized these effects. The results are presented as the relative mean value ± standard deviation of three independent analyses, each in triplicate, related to untreated control cells. * p
    Figure Legend Snippet: Effects of IL-6 signaling blockade on activity of the AKT-mTOR pathway after ( A ) sorafenib, ( B ) sunitinib or ( C ) pazopanib treatment. Influence of tocilizumab (50 μg/ml) on activation of AKT-mTOR pathway in 786-O cells after treatment with TKI was determined by Western blot. The concentrations of TKIs are indicated on the X axis of each graph. In combinational treatment (right part of each graph) tocilizumab was used in a concentration of 50 μg/ml. The column labeled by 0 μg/ml in the right part of each graph represents treatment with tocilizumab alone. Phosphorylation of AKT, mTOR, 4EBP1, S6RP, NFκB, and STAT3 was enhanced after TKI treatment at concentration of 0.5 μM. HIF-2α was enhanced by treatment with TKIs in all concentrations used. Tocilizumab neutralized these effects. The results are presented as the relative mean value ± standard deviation of three independent analyses, each in triplicate, related to untreated control cells. * p

    Techniques Used: Activity Assay, Activation Assay, Western Blot, Concentration Assay, Labeling, Standard Deviation

    2) Product Images from "Rhus coriaria increases protein ubiquitination, proteasomal degradation and triggers non-canonical Beclin-1-independent autophagy and apoptotic cell death in colon cancer cells"

    Article Title: Rhus coriaria increases protein ubiquitination, proteasomal degradation and triggers non-canonical Beclin-1-independent autophagy and apoptotic cell death in colon cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-11202-3

    Rhus coriaria stimulates the activation of the ubiquitin proteasome system and its inhibition rescue blocks autophagy and apoptosis and rescues cell viability in HT-29 cells. ( A ) Inactivation of mTOR through proteasome degradation precedes autophagy. Time-course analysis, by Western blotting, of phospho-mTOR, total mTOR, phospho-AKT, total AKT, mutant p53 and procaspase-3 in RCE-treated HT-29 cells. Cells were treated with 450 μg/mL RCE and proteins were extracted at the indicated time-points (3, 6, 12, 24 and 48 h) as previously described. ( B ) Inhibition of the proteasome rescue phospho-mTOR and block autophagy and apoptosis induced by RCE. HT-29 cells were pre-treated for 2 h with or without MG-132 (15 µM) prior to treatment with RCE (450 µg/mL). Whole cell lysate was resolved on 6 and 15% SDS-PAGE for phospho-mTOR and LC3 and active caspase-7, respectively. ( C ) Inhibition of proteasome reduces cell death induced by RCE. HT-29 cells were pre-treated for 2 h with or without MG-132 (15 µM) prior to treatment with RCE (300 and 450 µg/mL) for 48 h. Cell viability was determined as described in Material and Methods. Data are representative of three independent experiments carried out in triplicate. Statistical analysis for cell viability data on control or treated cells were performed using one-way ANOVA followed by LSD Post-Hoc test to determine the significance (***p
    Figure Legend Snippet: Rhus coriaria stimulates the activation of the ubiquitin proteasome system and its inhibition rescue blocks autophagy and apoptosis and rescues cell viability in HT-29 cells. ( A ) Inactivation of mTOR through proteasome degradation precedes autophagy. Time-course analysis, by Western blotting, of phospho-mTOR, total mTOR, phospho-AKT, total AKT, mutant p53 and procaspase-3 in RCE-treated HT-29 cells. Cells were treated with 450 μg/mL RCE and proteins were extracted at the indicated time-points (3, 6, 12, 24 and 48 h) as previously described. ( B ) Inhibition of the proteasome rescue phospho-mTOR and block autophagy and apoptosis induced by RCE. HT-29 cells were pre-treated for 2 h with or without MG-132 (15 µM) prior to treatment with RCE (450 µg/mL). Whole cell lysate was resolved on 6 and 15% SDS-PAGE for phospho-mTOR and LC3 and active caspase-7, respectively. ( C ) Inhibition of proteasome reduces cell death induced by RCE. HT-29 cells were pre-treated for 2 h with or without MG-132 (15 µM) prior to treatment with RCE (300 and 450 µg/mL) for 48 h. Cell viability was determined as described in Material and Methods. Data are representative of three independent experiments carried out in triplicate. Statistical analysis for cell viability data on control or treated cells were performed using one-way ANOVA followed by LSD Post-Hoc test to determine the significance (***p

    Techniques Used: Activation Assay, Inhibition, Western Blot, Mutagenesis, Blocking Assay, SDS Page

    Rhus coriaria inhibits the AKT/mTOR pathway through targeting of mTOR and AKT proteins to proteasome-dependent degradation. ( A ) Concentration-dependent decrease of phospho-mTOR, total mTOR, phospho-AKT and total AKT protein in RCE-treated HT-29 cells. Cells were treated with or without increasing concentrations of RCE for 48 h, then whole-cell extracts were subjected to Western blot analysis for the phosphorylated and non-phosphorylated form of mTOR and AKT and for β-actin (loading control). ( B ) Downregulation of mTOR is transcription-independent. Total RNA from RCE-treated and untreated cells were used to amplify by qRT-PCR the mTOR transcripts using mTOR specific primers. GAPDH was used as internal normalization control. ( C,D ) RCE-mediated decrease in the protein level of mTOR and AKT is autophagy-independent. Cells were pretreated with or without CQ (50 µM) ( C ) or 3-MA (5 mM) ( D ) for 1 h and then RCE was added at the indicated concentration for 48 h. Proteins were extracted and mTOR and AKT protein level was determined by western blot. ( E ) RCE targets mTOR and AKT to proteasome degradation. HT-29 cells were pre-treated for 2 h with or without MG-132 (15 µM) prior to treatment with RCE (450 µg/mL). Whole cell lysate was resolved on 6% SDS-PAGE and analyzed for the two proteins.
    Figure Legend Snippet: Rhus coriaria inhibits the AKT/mTOR pathway through targeting of mTOR and AKT proteins to proteasome-dependent degradation. ( A ) Concentration-dependent decrease of phospho-mTOR, total mTOR, phospho-AKT and total AKT protein in RCE-treated HT-29 cells. Cells were treated with or without increasing concentrations of RCE for 48 h, then whole-cell extracts were subjected to Western blot analysis for the phosphorylated and non-phosphorylated form of mTOR and AKT and for β-actin (loading control). ( B ) Downregulation of mTOR is transcription-independent. Total RNA from RCE-treated and untreated cells were used to amplify by qRT-PCR the mTOR transcripts using mTOR specific primers. GAPDH was used as internal normalization control. ( C,D ) RCE-mediated decrease in the protein level of mTOR and AKT is autophagy-independent. Cells were pretreated with or without CQ (50 µM) ( C ) or 3-MA (5 mM) ( D ) for 1 h and then RCE was added at the indicated concentration for 48 h. Proteins were extracted and mTOR and AKT protein level was determined by western blot. ( E ) RCE targets mTOR and AKT to proteasome degradation. HT-29 cells were pre-treated for 2 h with or without MG-132 (15 µM) prior to treatment with RCE (450 µg/mL). Whole cell lysate was resolved on 6% SDS-PAGE and analyzed for the two proteins.

    Techniques Used: Concentration Assay, Western Blot, Quantitative RT-PCR, SDS Page

    3) Product Images from "Cyclocarya paliurus extract activates insulin signaling via Sirtuin1 in C2C12 myotubes and decreases blood glucose level in mice with impaired insulin secretion"

    Article Title: Cyclocarya paliurus extract activates insulin signaling via Sirtuin1 in C2C12 myotubes and decreases blood glucose level in mice with impaired insulin secretion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0183988

    Oral administration of CPE decreased blood glucose level in STZ-induced diabetic mice. ICR mice were injected intraperitoneally with a single dose of STZ (200 mg/kg) and randomly divided into 2 groups, a control group (Cont) and a CPE administration group (CPE). Mice injected with an equal volume of saline were maintained as normal groups (Nor). Blood glucose levels at 1 and 2 h after oral administration of CPE (1 g/kg) to STZ-induced diabetic mice (A), and the change from before administration (B). Blood insulin concentrations (C) and phosphorylation level of AS160 and Akt in the skeletal muscles (D) at 1 h after administration. Data are expressed as the mean ± SEM ( n = 5–9) * p
    Figure Legend Snippet: Oral administration of CPE decreased blood glucose level in STZ-induced diabetic mice. ICR mice were injected intraperitoneally with a single dose of STZ (200 mg/kg) and randomly divided into 2 groups, a control group (Cont) and a CPE administration group (CPE). Mice injected with an equal volume of saline were maintained as normal groups (Nor). Blood glucose levels at 1 and 2 h after oral administration of CPE (1 g/kg) to STZ-induced diabetic mice (A), and the change from before administration (B). Blood insulin concentrations (C) and phosphorylation level of AS160 and Akt in the skeletal muscles (D) at 1 h after administration. Data are expressed as the mean ± SEM ( n = 5–9) * p

    Techniques Used: Mouse Assay, Injection

    CPE affected insulin signaling via Sirt1 in C2C12 cells. Human recombinant Sirt1 were incubated with CPE (0.5, 2.5, and 5 μg/mL) or resveratrol (5 μM), a Sirt1 activator, as well as substrate peptide, NAD, and developer, and Sirt1 activity was measured (A). C2C12 cells were incubated with CPE (100 μg/mL) with or without treatment with EX527 (40 μM), and 2NBDG uptake (B), Akt phosphorylation at Ser473, and tyrosine phosphorylation of IRS (C) were measured. Sirt1 expression was determined after treatment with Sirt1 siRNA (D). Cells were incubated with CPE (100 μg/mL) with or without Sirt1 knockdown, and 2NBDG uptake (E) and Akt phosphorylation level at Ser473 (F) were determined. The effects of CPE on the phosphorylation level of AMPK (G). Values are expressed as the mean ± SEM ( n = 3–4). * p
    Figure Legend Snippet: CPE affected insulin signaling via Sirt1 in C2C12 cells. Human recombinant Sirt1 were incubated with CPE (0.5, 2.5, and 5 μg/mL) or resveratrol (5 μM), a Sirt1 activator, as well as substrate peptide, NAD, and developer, and Sirt1 activity was measured (A). C2C12 cells were incubated with CPE (100 μg/mL) with or without treatment with EX527 (40 μM), and 2NBDG uptake (B), Akt phosphorylation at Ser473, and tyrosine phosphorylation of IRS (C) were measured. Sirt1 expression was determined after treatment with Sirt1 siRNA (D). Cells were incubated with CPE (100 μg/mL) with or without Sirt1 knockdown, and 2NBDG uptake (E) and Akt phosphorylation level at Ser473 (F) were determined. The effects of CPE on the phosphorylation level of AMPK (G). Values are expressed as the mean ± SEM ( n = 3–4). * p

    Techniques Used: Recombinant, Incubation, Activity Assay, Expressing

    CPE activated PI3K-Akt-AS160 signaling in C2C12 cells. C2C12 cells were exposed to CPE (50 or 100 μg/mL) or insulin (100 nM) for 30 min. The phosphorylation level of AS160 at Thr642 (A), Akt at Ser473 and Thr308 (B), and effects of CPE on the 2NBDG uptake in C2C12 cells treated with 50 μM Perifosine, Akt inhibitor (C). IRS1 and p85 subunit of PI3K complex (D) was determined by immunoprecipitation. Data are expressed as the mean ± SEM of three independent experiments. ** p
    Figure Legend Snippet: CPE activated PI3K-Akt-AS160 signaling in C2C12 cells. C2C12 cells were exposed to CPE (50 or 100 μg/mL) or insulin (100 nM) for 30 min. The phosphorylation level of AS160 at Thr642 (A), Akt at Ser473 and Thr308 (B), and effects of CPE on the 2NBDG uptake in C2C12 cells treated with 50 μM Perifosine, Akt inhibitor (C). IRS1 and p85 subunit of PI3K complex (D) was determined by immunoprecipitation. Data are expressed as the mean ± SEM of three independent experiments. ** p

    Techniques Used: Immunoprecipitation

    4) Product Images from "The dual PI3K/mTOR inhibitor GSK2126458 is effective for treating solid renal tumours in Tsc2+/- mice through suppression of cell proliferation and induction of apoptosis"

    Article Title: The dual PI3K/mTOR inhibitor GSK2126458 is effective for treating solid renal tumours in Tsc2+/- mice through suppression of cell proliferation and induction of apoptosis

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17215

    Effect of GSK2126458 and rapamycin on mTOR/MAPK/apoptotic signalling of renal tumours in Tsc2 +/- mice Western blot was used to analyse mTOR/MAKP/apoptotic signalling. Proteins were prepared from solid renal tumours dissected from Tsc2 +/- mice treated for one month with vehicle, GSK2126458 or rapamycin. Beta-actin was used as a loading control. Representative Western blots were presented to show phosphorylation of mTOR at S2481, Akt at T308 and S473, S6 at S235/236, 4E-BP1 at T37/46, RAF1 at S256, Erk1/2 at T202/Y204 and MDM2 at S166 as well as expression of 4E-BP1 and p53.
    Figure Legend Snippet: Effect of GSK2126458 and rapamycin on mTOR/MAPK/apoptotic signalling of renal tumours in Tsc2 +/- mice Western blot was used to analyse mTOR/MAKP/apoptotic signalling. Proteins were prepared from solid renal tumours dissected from Tsc2 +/- mice treated for one month with vehicle, GSK2126458 or rapamycin. Beta-actin was used as a loading control. Representative Western blots were presented to show phosphorylation of mTOR at S2481, Akt at T308 and S473, S6 at S235/236, 4E-BP1 at T37/46, RAF1 at S256, Erk1/2 at T202/Y204 and MDM2 at S166 as well as expression of 4E-BP1 and p53.

    Techniques Used: Mouse Assay, Western Blot, Expressing

    PI3K/Akt/mTOR signalling in renal tumours of Tsc2 +/- mice Kidney sections prepared from 14 months old Tsc2 +/- mice were used for IHC analysis. Representative IHC-stained sections were presented to show phosphorylation of mTOR at S2448 and S2481, S6 at S235/236, 4E-BP1 at T37/46 and S65, and Akt at T308 and S473 in renal tumours. Twenty cystic and 20 solid renal lesions from 10 Tsc2 +/- mice were analysed consistently showing similar protein phosphorylation levels. Black lines are scale bars.
    Figure Legend Snippet: PI3K/Akt/mTOR signalling in renal tumours of Tsc2 +/- mice Kidney sections prepared from 14 months old Tsc2 +/- mice were used for IHC analysis. Representative IHC-stained sections were presented to show phosphorylation of mTOR at S2448 and S2481, S6 at S235/236, 4E-BP1 at T37/46 and S65, and Akt at T308 and S473 in renal tumours. Twenty cystic and 20 solid renal lesions from 10 Tsc2 +/- mice were analysed consistently showing similar protein phosphorylation levels. Black lines are scale bars.

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining

    Effect of GSK2126458 and rapamycin on mTOR signalling of renal tumours in Tsc2 +/- mice Kidney sections prepared from 14 months old Tsc2 +/- mice treated with vehicle, GSK2126458 or rapamycin were used for IHC analysis. Representative IHC-stained sections were presented to show phosphorylation of mTOR at S2481, S6 at S235/236 and Akt at S473 in renal tumours. Black lines are scale bars.
    Figure Legend Snippet: Effect of GSK2126458 and rapamycin on mTOR signalling of renal tumours in Tsc2 +/- mice Kidney sections prepared from 14 months old Tsc2 +/- mice treated with vehicle, GSK2126458 or rapamycin were used for IHC analysis. Representative IHC-stained sections were presented to show phosphorylation of mTOR at S2481, S6 at S235/236 and Akt at S473 in renal tumours. Black lines are scale bars.

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining

    Effect of GSK2126458 and rapamycin on mTOR/MAPK/apoptotic signalling of renal tumours in Tsc2 +/- mice Western blot was used to analyse mTOR/MAKP/apoptotic signalling. Proteins were prepared from solid renal tumours dissected from Tsc2 +/- mice treated for one month with vehicle, GSK2126458 or rapamycin. Beta-actin was used as a loading control. Representative Western blots were presented to show phosphorylation of mTOR at S2481, Akt at T308 and S473, S6 at S235/236, 4E-BP1 at T37/46, RAF1 at S256, Erk1/2 at T202/Y204 and MDM2 at S166 as well as expression of 4E-BP1 and p53.
    Figure Legend Snippet: Effect of GSK2126458 and rapamycin on mTOR/MAPK/apoptotic signalling of renal tumours in Tsc2 +/- mice Western blot was used to analyse mTOR/MAKP/apoptotic signalling. Proteins were prepared from solid renal tumours dissected from Tsc2 +/- mice treated for one month with vehicle, GSK2126458 or rapamycin. Beta-actin was used as a loading control. Representative Western blots were presented to show phosphorylation of mTOR at S2481, Akt at T308 and S473, S6 at S235/236, 4E-BP1 at T37/46, RAF1 at S256, Erk1/2 at T202/Y204 and MDM2 at S166 as well as expression of 4E-BP1 and p53.

    Techniques Used: Mouse Assay, Western Blot, Expressing

    PI3K/Akt/mTOR signalling in renal tumours of Tsc2 +/- mice Kidney sections prepared from 14 months old Tsc2 +/- mice were used for IHC analysis. Representative IHC-stained sections were presented to show phosphorylation of mTOR at S2448 and S2481, S6 at S235/236, 4E-BP1 at T37/46 and S65, and Akt at T308 and S473 in renal tumours. Twenty cystic and 20 solid renal lesions from 10 Tsc2 +/- mice were analysed consistently showing similar protein phosphorylation levels. Black lines are scale bars.
    Figure Legend Snippet: PI3K/Akt/mTOR signalling in renal tumours of Tsc2 +/- mice Kidney sections prepared from 14 months old Tsc2 +/- mice were used for IHC analysis. Representative IHC-stained sections were presented to show phosphorylation of mTOR at S2448 and S2481, S6 at S235/236, 4E-BP1 at T37/46 and S65, and Akt at T308 and S473 in renal tumours. Twenty cystic and 20 solid renal lesions from 10 Tsc2 +/- mice were analysed consistently showing similar protein phosphorylation levels. Black lines are scale bars.

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining

    Effect of GSK2126458 and rapamycin on mTOR signalling of renal tumours in Tsc2 +/- mice Kidney sections prepared from 14 months old Tsc2 +/- mice treated with vehicle, GSK2126458 or rapamycin were used for IHC analysis. Representative IHC-stained sections were presented to show phosphorylation of mTOR at S2481, S6 at S235/236 and Akt at S473 in renal tumours. Black lines are scale bars.
    Figure Legend Snippet: Effect of GSK2126458 and rapamycin on mTOR signalling of renal tumours in Tsc2 +/- mice Kidney sections prepared from 14 months old Tsc2 +/- mice treated with vehicle, GSK2126458 or rapamycin were used for IHC analysis. Representative IHC-stained sections were presented to show phosphorylation of mTOR at S2481, S6 at S235/236 and Akt at S473 in renal tumours. Black lines are scale bars.

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining

    5) Product Images from "Folliculin Interacts with Rab35 to Regulate EGF-Induced EGFR Degradation"

    Article Title: Folliculin Interacts with Rab35 to Regulate EGF-Induced EGFR Degradation

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00688

    Rab35 activation promotes EGFR degradation and attenuates EGFR signaling. (A) HeLa cells were starved overnight and then treated with cycloheximide for 30 min, then stimulated with 10 ng/mL EGF for the indicated time. EGFR, p-Akt and p-ERK1/2 levels were detected by western blot. Data were presented as mean ± SD of 3 independent determinations. (B) Overexpression of different plasmids of Rab35 (Rab35 DN, Rab35 WT, and Rab35 CA) to detect EGF-induced EGFR degradation and its downstream signaling. * P
    Figure Legend Snippet: Rab35 activation promotes EGFR degradation and attenuates EGFR signaling. (A) HeLa cells were starved overnight and then treated with cycloheximide for 30 min, then stimulated with 10 ng/mL EGF for the indicated time. EGFR, p-Akt and p-ERK1/2 levels were detected by western blot. Data were presented as mean ± SD of 3 independent determinations. (B) Overexpression of different plasmids of Rab35 (Rab35 DN, Rab35 WT, and Rab35 CA) to detect EGF-induced EGFR degradation and its downstream signaling. * P

    Techniques Used: Activation Assay, Western Blot, Over Expression

    6) Product Images from "Effect of a rosmarinic acid supplemented hemodialysis fluid on inflammation of human vascular endothelial cells"

    Article Title: Effect of a rosmarinic acid supplemented hemodialysis fluid on inflammation of human vascular endothelial cells

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20176145

    Hemodialysis fluid (HDF) supplemented with rosmarinic acid (RA) increased phosphorylation of cytosolic AKT and IκBα and diminished nuclear NF-κB in lipopolysaccharides (LPS)-stimulated human umbilical vein endothelial cells. Phosphorylation of cytosolic AKT and IκB-α, and the level of nuclear NF-κB were determined by immunoblot using specific antibodies and chemiluminescence development. Levels of GAPDH and histone H1 were used as cytosolic and nuclear control.
    Figure Legend Snippet: Hemodialysis fluid (HDF) supplemented with rosmarinic acid (RA) increased phosphorylation of cytosolic AKT and IκBα and diminished nuclear NF-κB in lipopolysaccharides (LPS)-stimulated human umbilical vein endothelial cells. Phosphorylation of cytosolic AKT and IκB-α, and the level of nuclear NF-κB were determined by immunoblot using specific antibodies and chemiluminescence development. Levels of GAPDH and histone H1 were used as cytosolic and nuclear control.

    Techniques Used:

    7) Product Images from "Apatinib has anti-tumor effects and induces autophagy in colon cancer cells"

    Article Title: Apatinib has anti-tumor effects and induces autophagy in colon cancer cells

    Journal: Iranian Journal of Basic Medical Sciences

    doi: 10.22038/IJBMS.2017.9263

    Apatinib induced apoptosis and autophagy through inhibition of AKT/mTOR pathway (a) HCT116 and SW480 cells were treated with apatinib (0, 20, 40 μM, respectively) for 24 hr, and the alterations of phosphorylated AKT and mTOR were detected. (b) HCT116 and SW480 cells were treated with apatinib (0, 20, 40 μM, respectively) for 24 hr, and the protein level of LC3 was detected. GAPDH was included as a loading control
    Figure Legend Snippet: Apatinib induced apoptosis and autophagy through inhibition of AKT/mTOR pathway (a) HCT116 and SW480 cells were treated with apatinib (0, 20, 40 μM, respectively) for 24 hr, and the alterations of phosphorylated AKT and mTOR were detected. (b) HCT116 and SW480 cells were treated with apatinib (0, 20, 40 μM, respectively) for 24 hr, and the protein level of LC3 was detected. GAPDH was included as a loading control

    Techniques Used: Inhibition

    8) Product Images from "Effect of weekly or daily dosing regimen of Gefitinib in mouse models of lung cancer"

    Article Title: Effect of weekly or daily dosing regimen of Gefitinib in mouse models of lung cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19785

    Weekly dosing with Gefitinib exhibits greater inhibition of phospho-EGFR, phospho-ERK and phospho-AKT compared with daily dosing (A) Western blot analyses of phospho-EGFR, phospho-ERK and phospho-AKT in tumor samples. Mice were treated with Gefitinib with the daily and weekly dosing protocols. β-actin was used as the internal control. (B) Immunohistochemical staining of phospho-EGFR, phospho-ERK and phospho-AKT in xenograft tumor sections from each group indicating inhibition with daily and weekly treatments.
    Figure Legend Snippet: Weekly dosing with Gefitinib exhibits greater inhibition of phospho-EGFR, phospho-ERK and phospho-AKT compared with daily dosing (A) Western blot analyses of phospho-EGFR, phospho-ERK and phospho-AKT in tumor samples. Mice were treated with Gefitinib with the daily and weekly dosing protocols. β-actin was used as the internal control. (B) Immunohistochemical staining of phospho-EGFR, phospho-ERK and phospho-AKT in xenograft tumor sections from each group indicating inhibition with daily and weekly treatments.

    Techniques Used: Inhibition, Western Blot, Mouse Assay, Immunohistochemistry, Staining

    9) Product Images from "Preclinical activity of DCZ3301, a novel aryl-guanidino compound in the therapy of multiple myeloma"

    Article Title: Preclinical activity of DCZ3301, a novel aryl-guanidino compound in the therapy of multiple myeloma

    Journal: Theranostics

    doi: 10.7150/thno.18345

    Mechanisms of anti-myeloma activity of DCZ3301. (A) MM.1S cells were treated with indicated concentrations of DCZ3301 for 48h. Whole cell lysates were subjected to western blotting using ERK1/2, p-ERK1/2, AKT, p-AKT, STAT3, p-STAT3 and β-actin. (B) NCI-H929 and U266 cells were treated with 4 μM DCZ3301 for 48 h. Whole cell lysates were subjected to western blotting using p-ERK1/2, p-AKT, p-STAT3 and β-actin. (C) MM.1S cells were treated with indicated concentrations of DCZ3301 for 48 h. Whole cell lysates were subjected to western blotting using IKBα, p-IKBα(Ser32), p-p65(S536) and β-actin. (D) MM.1S cells transfected with STAT3 siRNA or scrambled sequence were treated with 4 μM DCZ3301 for 48 h. After that, cell apoptosis was monitored by Annexin V/PI staining. Inset: Relative expression of STAT3 protein in MM.1S transfected with STAT3 siRNA and scrambled sequence. (E) MM.1S cells transfected with plasmid DNA containing STAT3 or empty vector were treated as in D, then cell apoptosis was monitored by Annexin V/PI staining. Inset: Relative expression of STAT3 protein in MM.1S transfected with plasmid DNA containing STAT3 or empty vector. MM.1S cells were grown in co-culture with bone marrow stromal cells (BMSCs) and then treated with DCZ3301 (0-1 μM) for 12, 24 and 48 h. After that, VEGF (F) or IL-6 (G) levels in the culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA). * represent p
    Figure Legend Snippet: Mechanisms of anti-myeloma activity of DCZ3301. (A) MM.1S cells were treated with indicated concentrations of DCZ3301 for 48h. Whole cell lysates were subjected to western blotting using ERK1/2, p-ERK1/2, AKT, p-AKT, STAT3, p-STAT3 and β-actin. (B) NCI-H929 and U266 cells were treated with 4 μM DCZ3301 for 48 h. Whole cell lysates were subjected to western blotting using p-ERK1/2, p-AKT, p-STAT3 and β-actin. (C) MM.1S cells were treated with indicated concentrations of DCZ3301 for 48 h. Whole cell lysates were subjected to western blotting using IKBα, p-IKBα(Ser32), p-p65(S536) and β-actin. (D) MM.1S cells transfected with STAT3 siRNA or scrambled sequence were treated with 4 μM DCZ3301 for 48 h. After that, cell apoptosis was monitored by Annexin V/PI staining. Inset: Relative expression of STAT3 protein in MM.1S transfected with STAT3 siRNA and scrambled sequence. (E) MM.1S cells transfected with plasmid DNA containing STAT3 or empty vector were treated as in D, then cell apoptosis was monitored by Annexin V/PI staining. Inset: Relative expression of STAT3 protein in MM.1S transfected with plasmid DNA containing STAT3 or empty vector. MM.1S cells were grown in co-culture with bone marrow stromal cells (BMSCs) and then treated with DCZ3301 (0-1 μM) for 12, 24 and 48 h. After that, VEGF (F) or IL-6 (G) levels in the culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA). * represent p

    Techniques Used: Activity Assay, Western Blot, Transfection, Sequencing, Staining, Expressing, Plasmid Preparation, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

    10) Product Images from "Data on the expression and insulin-stimulated phosphorylation of IRS-1 by miR-96 in L6-GLUT4myc myocytes"

    Article Title: Data on the expression and insulin-stimulated phosphorylation of IRS-1 by miR-96 in L6-GLUT4myc myocytes

    Journal: Data in Brief

    doi: 10.1016/j.dib.2017.10.054

    Effect of miR-96 on the expression and phosphorylation of insulin signaling molecules. L6-GLUT4myc myocytes were transfected with the scRNA (200 nM) or miR-96 (200 nM) mimic. After 48 h transfection, the cells were incubated in the presence or absence of insulin (100 nM) for 30 min and subjected to immunoblotting. (A) Representative immunoblots obtained from L6-GLUT4myc myocytes are shown in A. (B) The expression and phosphorylation of INSR (pINSR) were normalized to the amount of INSR. (C) The expression and phosphorylation of IRS-1 (pIRS-1) were normalized to the amount of β-Actin. (D) The protein expression of Akt was normalized to the amount of β-Actin. The level of Akt phosphorylation (pAkt2) was normalized to the amount of Akt. The values are the relative ratio, where the intensity of the scRNA control was set to one, and expressed as the means ± SEM from three independent experiments. *, P
    Figure Legend Snippet: Effect of miR-96 on the expression and phosphorylation of insulin signaling molecules. L6-GLUT4myc myocytes were transfected with the scRNA (200 nM) or miR-96 (200 nM) mimic. After 48 h transfection, the cells were incubated in the presence or absence of insulin (100 nM) for 30 min and subjected to immunoblotting. (A) Representative immunoblots obtained from L6-GLUT4myc myocytes are shown in A. (B) The expression and phosphorylation of INSR (pINSR) were normalized to the amount of INSR. (C) The expression and phosphorylation of IRS-1 (pIRS-1) were normalized to the amount of β-Actin. (D) The protein expression of Akt was normalized to the amount of β-Actin. The level of Akt phosphorylation (pAkt2) was normalized to the amount of Akt. The values are the relative ratio, where the intensity of the scRNA control was set to one, and expressed as the means ± SEM from three independent experiments. *, P

    Techniques Used: Expressing, Transfection, Incubation, Western Blot

    11) Product Images from "EZH2-mediated upregulation of ROS1 oncogene promotes oral cancer metastasis"

    Article Title: EZH2-mediated upregulation of ROS1 oncogene promotes oral cancer metastasis

    Journal: Oncogene

    doi: 10.1038/onc.2017.262

    Schematic model of how ROS1 promotes oral cancer progression. Higher level of miR-200a-3p correlates with low EZH2 and enhanced OSCC invasion. The reduction of EZH2 level in the highly invasive OSCC cells relieves H3K27m3 modification and opens chromatin for transcriptional activation of ROS1 as well as a number of novel ROS1 target genes. Elevated ROS1 activates the MEK-ERK1/2 and PI3K-AKT pathways to increase STAT1 occupancy at the regulatory regions of GLI1 and CXCL1 to promote cell proliferation and invasion.
    Figure Legend Snippet: Schematic model of how ROS1 promotes oral cancer progression. Higher level of miR-200a-3p correlates with low EZH2 and enhanced OSCC invasion. The reduction of EZH2 level in the highly invasive OSCC cells relieves H3K27m3 modification and opens chromatin for transcriptional activation of ROS1 as well as a number of novel ROS1 target genes. Elevated ROS1 activates the MEK-ERK1/2 and PI3K-AKT pathways to increase STAT1 occupancy at the regulatory regions of GLI1 and CXCL1 to promote cell proliferation and invasion.

    Techniques Used: Modification, Activation Assay

    Effect of increased ROS1 on MAPK, PI3K-AKT signaling pathways, cell proliferation, migration, and invasion. ( a ) Western blotting was carried out with lysates of OC3 and OC3-IV2 cells using anti-ROS1 and anti-pROS1(Y2274). The value for pROS1(Y2274) was normalized to total ROS1. ( b and c ) Relative protein levels of pAKT(S473), AKT, pERK1/2, ERK1/2, pSTAT3(Y705), and STAT3 in OC3, OC3-IV2, OC3-IV2-Scr, and OC3-IV2-shROS1#1 cells as determined with Western blotting. Levels of pERK1/2, pAKT(S473), and pSTAT3(Y705) were normalized to those of total ERK1/2, AKT, and STAT3, respectively, and then to OC3 and OC3-IV2-Scr cells. ( d ) pAKT(S473), AKT, pERK1/2, and ERK1/2 levels in lysates from OC3 and OC3-IV2 cells treated with 20 μ M U0126 or LY294002 for 24 h. ( e and f ) Relative proliferation and migration of OC3 and OC3-IV2 cells treated with 20 μ M U0126 or LY294002 for 24 or 48 h were assessed using MTT and wound-healing assays, respectively. * , # Compared with DMSO treatment. ( g ) Migration and invasion of OC3-IV2 cells treated with 20 μ M U0126 or LY294002 for 26 h were assessed with the Boyden chamber assay. Data from at least three independent experiments are presented as mean±SEM (* , # P
    Figure Legend Snippet: Effect of increased ROS1 on MAPK, PI3K-AKT signaling pathways, cell proliferation, migration, and invasion. ( a ) Western blotting was carried out with lysates of OC3 and OC3-IV2 cells using anti-ROS1 and anti-pROS1(Y2274). The value for pROS1(Y2274) was normalized to total ROS1. ( b and c ) Relative protein levels of pAKT(S473), AKT, pERK1/2, ERK1/2, pSTAT3(Y705), and STAT3 in OC3, OC3-IV2, OC3-IV2-Scr, and OC3-IV2-shROS1#1 cells as determined with Western blotting. Levels of pERK1/2, pAKT(S473), and pSTAT3(Y705) were normalized to those of total ERK1/2, AKT, and STAT3, respectively, and then to OC3 and OC3-IV2-Scr cells. ( d ) pAKT(S473), AKT, pERK1/2, and ERK1/2 levels in lysates from OC3 and OC3-IV2 cells treated with 20 μ M U0126 or LY294002 for 24 h. ( e and f ) Relative proliferation and migration of OC3 and OC3-IV2 cells treated with 20 μ M U0126 or LY294002 for 24 or 48 h were assessed using MTT and wound-healing assays, respectively. * , # Compared with DMSO treatment. ( g ) Migration and invasion of OC3-IV2 cells treated with 20 μ M U0126 or LY294002 for 26 h were assessed with the Boyden chamber assay. Data from at least three independent experiments are presented as mean±SEM (* , # P

    Techniques Used: Migration, Western Blot, MTT Assay, Boyden Chamber Assay

    12) Product Images from "Enhancement of the anti-tumor activity of FGFR1 inhibition in squamous cell lung cancer by targeting downstream signaling involved in glucose metabolism"

    Article Title: Enhancement of the anti-tumor activity of FGFR1 inhibition in squamous cell lung cancer by targeting downstream signaling involved in glucose metabolism

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19279

    Role of AKT/mTOR and PKM2 in the regulation of FGF2-mediated glucose metabolism (a) H1703 cells, cultured in -FCS for 24h, were pre-incubated for 1h with 2μM U0126, 0.1μM NVP-BEZ235, 0.1μM RAD001, 1μM NVP-BGJ398 or a combination of NVP-BGJ398 with NVP-BEZ235. The cells were then stimulated with FGF2 and glucose uptake was assessed after 6h. (b) H1703 cells, cultured in -FCS for 24h, were pre-incubated for 1h with 1μM dovitinib, 1μM NVP-BGJ398 or 10μM DASA, and then treated with FGF2. PKM2 activity was measured after 4h. (c) H1703 and H520 cells, cultured in -FCS for 24h, were pre-incubated for 1h with 1μM NVP-BGJ398 or 10μM DASA, and then treated with FGF2. Glucose uptake was measured after 16h. Data are expressed as percent versus -FCS control cells and are mean values ±SD of three independent experiments. * P
    Figure Legend Snippet: Role of AKT/mTOR and PKM2 in the regulation of FGF2-mediated glucose metabolism (a) H1703 cells, cultured in -FCS for 24h, were pre-incubated for 1h with 2μM U0126, 0.1μM NVP-BEZ235, 0.1μM RAD001, 1μM NVP-BGJ398 or a combination of NVP-BGJ398 with NVP-BEZ235. The cells were then stimulated with FGF2 and glucose uptake was assessed after 6h. (b) H1703 cells, cultured in -FCS for 24h, were pre-incubated for 1h with 1μM dovitinib, 1μM NVP-BGJ398 or 10μM DASA, and then treated with FGF2. PKM2 activity was measured after 4h. (c) H1703 and H520 cells, cultured in -FCS for 24h, were pre-incubated for 1h with 1μM NVP-BGJ398 or 10μM DASA, and then treated with FGF2. Glucose uptake was measured after 16h. Data are expressed as percent versus -FCS control cells and are mean values ±SD of three independent experiments. * P

    Techniques Used: Cell Culture, Incubation, Activity Assay

    Effects of the combination of NVP-BGJ398 with AKT/mTOR inhibitors on H1703 and LENTI-4 cells (a) H1703 cells were treated with 1μM NVP-BGJ398, 0.1μM NVP-BEZ235, and 0.1μM RAD001 alone or with NVP-BGJ398 in combination with NVP-BEZ235 or RAD001. After 24h protein expression was assessed by Western Blot analysis. H1703 cells were treated with 1μM NVP-BGJ398 or 0.01μM NVP-BEZ235 alone or in combination (b) and with 1μM NVP-BGJ398 or 0.01μM RAD001 alone or in combination (c) . After 72h cell survival/proliferation was evaluated by CV assay. (d) LENTI-4 cells were treated with 1μM NVP-BGJ398 or 0.1μM NVP-BEZ235 alone or in combination. After 24h protein expression was assessed by Western Blot analysis. LENTI-4 cells were treated with 1μM NVP-BGJ398, 0.01μM NVP-BEZ235 alone or in combination (e) and with 1μM NVP-BGJ398 or 0.01μM RAD001 alone or in combination (f) . After 72h cell survival/proliferation was evaluated by CV assay. Results in (a and d) are representative of three independent experiments. Data in (b, c, e , and f) are expressed as percent inhibition of cell proliferation vs control cells (C) and are mean values ±SD of three independent experiments. *** P
    Figure Legend Snippet: Effects of the combination of NVP-BGJ398 with AKT/mTOR inhibitors on H1703 and LENTI-4 cells (a) H1703 cells were treated with 1μM NVP-BGJ398, 0.1μM NVP-BEZ235, and 0.1μM RAD001 alone or with NVP-BGJ398 in combination with NVP-BEZ235 or RAD001. After 24h protein expression was assessed by Western Blot analysis. H1703 cells were treated with 1μM NVP-BGJ398 or 0.01μM NVP-BEZ235 alone or in combination (b) and with 1μM NVP-BGJ398 or 0.01μM RAD001 alone or in combination (c) . After 72h cell survival/proliferation was evaluated by CV assay. (d) LENTI-4 cells were treated with 1μM NVP-BGJ398 or 0.1μM NVP-BEZ235 alone or in combination. After 24h protein expression was assessed by Western Blot analysis. LENTI-4 cells were treated with 1μM NVP-BGJ398, 0.01μM NVP-BEZ235 alone or in combination (e) and with 1μM NVP-BGJ398 or 0.01μM RAD001 alone or in combination (f) . After 72h cell survival/proliferation was evaluated by CV assay. Results in (a and d) are representative of three independent experiments. Data in (b, c, e , and f) are expressed as percent inhibition of cell proliferation vs control cells (C) and are mean values ±SD of three independent experiments. *** P

    Techniques Used: Expressing, Western Blot, Inhibition

    13) Product Images from "Follicular Stimulating Hormone Accelerates Atherogenesis by Increasing Endothelial VCAM-1 Expression"

    Article Title: Follicular Stimulating Hormone Accelerates Atherogenesis by Increasing Endothelial VCAM-1 Expression

    Journal: Theranostics

    doi: 10.7150/thno.21216

    PI3K/Akt/mTORC1 pathway was upstream of NF-κB signaling. (A). HUVECs were treated with control vehicle (CON) or FSH (50 mIU/mL) for 24 h in the presence or absence of PI3K inhibitor LY294002 (5 μM) and NF-κB inhibitor PDTC (10 μM). Phosphorylated Akt (p-Akt), phosphorylated p65 (p-p65) and β-actin were detected by Western blotting. (B). HUVECs were transiently transfected with constitutively active p85α (WT p85α) or dominant-negative p85α (△p85α) plasmid (both 15 μg) for 48 h. Next, cells were treated with vehicle or FSH (50 mIU/mL) for another 24 h. Cell contents of p85α, VCAM-1 and β-actin were detected by Western blotting. (C). HUVECs were transfected with scrambled siRNA (100 nM) or Akt siRNA (100 nM) for 48 h followed by treatment with vehicle or FSH (50 mIU/mL) for another 24 h. Western blot of proteins is shown. (D) . HUVECs were treated with vehicle or 50 mIU/mL FSH for 24 h in the presence or absence of mTOR inhibitor KU 0063794 (KU, 1 μM). Western blot of proteins is shown. (E) . HUVECs were treated with control vehicle (CON) or FSH (50 mIU/mL) for 24 h in the presence or absence of PI3K inhibitor LY294002 (5 μM). Total rictor or raptor, phosphorylated rictor (p-rictor) or phosphorylated raptor (p-raptor) and β-actin were detected by Western blotting. (F). HUVECs were transfected with scrambled siRNA, raptor siRNA or rictor siRNA (all 100 nM) for 48 h. Cells were then treated with vehicle or FSH (50 mIU/mL) for another 24 h and the proteins were detected by Western blotting.
    Figure Legend Snippet: PI3K/Akt/mTORC1 pathway was upstream of NF-κB signaling. (A). HUVECs were treated with control vehicle (CON) or FSH (50 mIU/mL) for 24 h in the presence or absence of PI3K inhibitor LY294002 (5 μM) and NF-κB inhibitor PDTC (10 μM). Phosphorylated Akt (p-Akt), phosphorylated p65 (p-p65) and β-actin were detected by Western blotting. (B). HUVECs were transiently transfected with constitutively active p85α (WT p85α) or dominant-negative p85α (△p85α) plasmid (both 15 μg) for 48 h. Next, cells were treated with vehicle or FSH (50 mIU/mL) for another 24 h. Cell contents of p85α, VCAM-1 and β-actin were detected by Western blotting. (C). HUVECs were transfected with scrambled siRNA (100 nM) or Akt siRNA (100 nM) for 48 h followed by treatment with vehicle or FSH (50 mIU/mL) for another 24 h. Western blot of proteins is shown. (D) . HUVECs were treated with vehicle or 50 mIU/mL FSH for 24 h in the presence or absence of mTOR inhibitor KU 0063794 (KU, 1 μM). Western blot of proteins is shown. (E) . HUVECs were treated with control vehicle (CON) or FSH (50 mIU/mL) for 24 h in the presence or absence of PI3K inhibitor LY294002 (5 μM). Total rictor or raptor, phosphorylated rictor (p-rictor) or phosphorylated raptor (p-raptor) and β-actin were detected by Western blotting. (F). HUVECs were transfected with scrambled siRNA, raptor siRNA or rictor siRNA (all 100 nM) for 48 h. Cells were then treated with vehicle or FSH (50 mIU/mL) for another 24 h and the proteins were detected by Western blotting.

    Techniques Used: Western Blot, Transfection, Dominant Negative Mutation, Plasmid Preparation

    PI3K/Akt and NF-κB signaling were implicated in FSH-enhanced VCAM-1 expression. (A-C). HUVECs were treated with control vehicle (CON) or FSH (50 mIU/mL) for 24 h in the presence or absence of PI3K inhibitor LY294002 (5 μM), ERK1/2 inhibitor PD98059 (2.5 μM), 17β-estradiol (E2, 10 nM), c-Src inhibitor PP2 (5 μM), Gαi protein inhibitor PTX (50 ng/mL), NF-κB inhibitor PDTC (10 μM). VCAM-1 was detected by Western blotting. β-actin was used as an internal control. (D-E) . HUVECs were treated with different concentrations of FSH for 24 h, or treated with 50 mIU/mL of FSH over 48 h. Total Akt, phosphorylated Akt (p-Akt), total mTOR, phosphorylated mTOR (p-mTOR), total p65, phosphorylated p65 (p-p65) and β-actin were detected by Western blotting. * = P
    Figure Legend Snippet: PI3K/Akt and NF-κB signaling were implicated in FSH-enhanced VCAM-1 expression. (A-C). HUVECs were treated with control vehicle (CON) or FSH (50 mIU/mL) for 24 h in the presence or absence of PI3K inhibitor LY294002 (5 μM), ERK1/2 inhibitor PD98059 (2.5 μM), 17β-estradiol (E2, 10 nM), c-Src inhibitor PP2 (5 μM), Gαi protein inhibitor PTX (50 ng/mL), NF-κB inhibitor PDTC (10 μM). VCAM-1 was detected by Western blotting. β-actin was used as an internal control. (D-E) . HUVECs were treated with different concentrations of FSH for 24 h, or treated with 50 mIU/mL of FSH over 48 h. Total Akt, phosphorylated Akt (p-Akt), total mTOR, phosphorylated mTOR (p-mTOR), total p65, phosphorylated p65 (p-p65) and β-actin were detected by Western blotting. * = P

    Techniques Used: Expressing, Western Blot

    14) Product Images from "TRAF3 negatively regulates platelet activation and thrombosis"

    Article Title: TRAF3 negatively regulates platelet activation and thrombosis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17189-1

    Akt and p38 MAPK phosphorylation and fibrinogen binding in response to thrombin in TRAF3 +/+ and TRAF3 −/− platelets. ( a–c ) Washed platelets from TRAF3 +/+ and TRAF3 −/− mice were incubated with increasing concentrations of thrombin or collagen at 37 °C in a platelet aggregometer for 2 or 5 min, and solubilized with SDS-PAGE sample buffer. Phosphorylation of Akt and p38 MAPK was detected by Western blotting with rabbit monoclonal antibodies specifically recognizing the phosphorylated Akt residue Ser473 or phosphorylated p38 residues Thr180/Tyr182. Statistical data of densitometric analysis from at least four experiments were shown in ( b ) and ( c ). ( d and e ) Washed platelets from TRAF3 +/+ and TRAF3 −/− mice (3 × 10 8 /ml) were incubated with Oregon Green-labeled fibrinogen in the presence of various concentrations of thrombin at 22 °C for 30 min. Platelets were also incubated with Oregon Green-labeled fibrinogen in the absence of thrombin at 22 °C for 30 min as a control. Fibrinogen binding to platelets was analyzed by flow cytometry. Quantitative results were expressed as fibrinogen binding indices (geometric mean (MFI) mean of fluorescence intensity of stimulated platelets/ geometric mean of fluorescence intensity of unstimulated platelets; geometric mean ± SD; n = 3; * P
    Figure Legend Snippet: Akt and p38 MAPK phosphorylation and fibrinogen binding in response to thrombin in TRAF3 +/+ and TRAF3 −/− platelets. ( a–c ) Washed platelets from TRAF3 +/+ and TRAF3 −/− mice were incubated with increasing concentrations of thrombin or collagen at 37 °C in a platelet aggregometer for 2 or 5 min, and solubilized with SDS-PAGE sample buffer. Phosphorylation of Akt and p38 MAPK was detected by Western blotting with rabbit monoclonal antibodies specifically recognizing the phosphorylated Akt residue Ser473 or phosphorylated p38 residues Thr180/Tyr182. Statistical data of densitometric analysis from at least four experiments were shown in ( b ) and ( c ). ( d and e ) Washed platelets from TRAF3 +/+ and TRAF3 −/− mice (3 × 10 8 /ml) were incubated with Oregon Green-labeled fibrinogen in the presence of various concentrations of thrombin at 22 °C for 30 min. Platelets were also incubated with Oregon Green-labeled fibrinogen in the absence of thrombin at 22 °C for 30 min as a control. Fibrinogen binding to platelets was analyzed by flow cytometry. Quantitative results were expressed as fibrinogen binding indices (geometric mean (MFI) mean of fluorescence intensity of stimulated platelets/ geometric mean of fluorescence intensity of unstimulated platelets; geometric mean ± SD; n = 3; * P

    Techniques Used: Binding Assay, Mouse Assay, Incubation, SDS Page, Western Blot, Labeling, Flow Cytometry, Cytometry, Fluorescence

    15) Product Images from "Ozone oil promotes wound healing by increasing the migration of fibroblasts via PI3K/Akt/mTOR signaling pathway"

    Article Title: Ozone oil promotes wound healing by increasing the migration of fibroblasts via PI3K/Akt/mTOR signaling pathway

    Journal: Bioscience Reports

    doi: 10.1042/BSR20170658

    Ozone oil activated the PI3K/Akt/mTOR signaling pathway ( A ) Western blotting results show that the expression of important proteins for PI3K/Akt/mTOR signaling pathway is increased in the fibroblasts. Ozone oil further increases the expression of these proteins. ( B ) Summary of Western blotting results. Error bars represent the mean ± S.D.; * P
    Figure Legend Snippet: Ozone oil activated the PI3K/Akt/mTOR signaling pathway ( A ) Western blotting results show that the expression of important proteins for PI3K/Akt/mTOR signaling pathway is increased in the fibroblasts. Ozone oil further increases the expression of these proteins. ( B ) Summary of Western blotting results. Error bars represent the mean ± S.D.; * P

    Techniques Used: Western Blot, Expressing

    16) Product Images from "Glucose-derived AGEs enhance human gastric cancer metastasis through RAGE/ERK/Sp1/MMP2 cascade"

    Article Title: Glucose-derived AGEs enhance human gastric cancer metastasis through RAGE/ERK/Sp1/MMP2 cascade

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22185

    AGEs upregulated Sp1 expression through ERK activation (A) The level of p-AKT, p-ERK, p-JUK, p-p38, and Sp1 protein expression. (B) Incubation of MEK1/2 inhibitor, U0126 (10μM), for 48 hours reduced AGEs-induced Sp1 expression. ( ** p
    Figure Legend Snippet: AGEs upregulated Sp1 expression through ERK activation (A) The level of p-AKT, p-ERK, p-JUK, p-p38, and Sp1 protein expression. (B) Incubation of MEK1/2 inhibitor, U0126 (10μM), for 48 hours reduced AGEs-induced Sp1 expression. ( ** p

    Techniques Used: Expressing, Activation Assay, Incubation

    17) Product Images from "ZNF259 inhibits non-small cell lung cancer cells proliferation and invasion by FAK-AKT signaling"

    Article Title: ZNF259 inhibits non-small cell lung cancer cells proliferation and invasion by FAK-AKT signaling

    Journal: Cancer Management and Research

    doi: 10.2147/CMAR.S150614

    ZNF259 reduced cyclin D1 and MMP2 levels by inhibiting Fak/Akt phosphorylation. Notes: ( A ) The levels of Akt (Thr308), Akt, Fak (Tyr397), Fak, p-JNK (Thr183/Tyr185), JNK, p-P38 (Thr180/Tyr182), and P38 were determined after ZNF259 overexpression or knockdown in A549 cells. ( B ) PF562271, a Fak inhibitor, was added to the culture media of A549 cells with or without ZNF259 knockdown. DMSO was used as negative control. ( C ) LY294002, an inhibitor of Akt, was added to the culture media of A549 cells with or without ZNF259 depletion. DMSO was used as negative control. All studies were performed three times. Abbreviations: NC, negative control; NSCLC, non-small cell lung cancer; si, small interfering; ZNF259, zinc finger protein 259.
    Figure Legend Snippet: ZNF259 reduced cyclin D1 and MMP2 levels by inhibiting Fak/Akt phosphorylation. Notes: ( A ) The levels of Akt (Thr308), Akt, Fak (Tyr397), Fak, p-JNK (Thr183/Tyr185), JNK, p-P38 (Thr180/Tyr182), and P38 were determined after ZNF259 overexpression or knockdown in A549 cells. ( B ) PF562271, a Fak inhibitor, was added to the culture media of A549 cells with or without ZNF259 knockdown. DMSO was used as negative control. ( C ) LY294002, an inhibitor of Akt, was added to the culture media of A549 cells with or without ZNF259 depletion. DMSO was used as negative control. All studies were performed three times. Abbreviations: NC, negative control; NSCLC, non-small cell lung cancer; si, small interfering; ZNF259, zinc finger protein 259.

    Techniques Used: Over Expression, Negative Control

    18) Product Images from "Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype"

    Article Title: Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-017-1024-1

    TCLPA5 and CRT0066101 (CRT) inhibit LPA-mediated downstream signaling. a PMM were preincubated overnight with TCLPA5 (5 μM) or CRT (1 μM) before treatment with LPA (1 μM) or LPA (1 μM) plus each inhibitor for the indicated times. Cells incubated with 0.1% BSA or DMSO were used as controls. The phosphorylation states of PKDs, JNK, AKT, ERK1/ERK2, and p38 were detected using western blotting. One representative blot is shown. b Densitometric analysis of western blots ( N = 3). Results represent mean values + SEM (* p
    Figure Legend Snippet: TCLPA5 and CRT0066101 (CRT) inhibit LPA-mediated downstream signaling. a PMM were preincubated overnight with TCLPA5 (5 μM) or CRT (1 μM) before treatment with LPA (1 μM) or LPA (1 μM) plus each inhibitor for the indicated times. Cells incubated with 0.1% BSA or DMSO were used as controls. The phosphorylation states of PKDs, JNK, AKT, ERK1/ERK2, and p38 were detected using western blotting. One representative blot is shown. b Densitometric analysis of western blots ( N = 3). Results represent mean values + SEM (* p

    Techniques Used: Incubation, Western Blot

    19) Product Images from "Myricetin Inhibits Angiogenesis by Inducing Apoptosis and Suppressing PI3K/Akt/mTOR Signaling in Endothelial Cells"

    Article Title: Myricetin Inhibits Angiogenesis by Inducing Apoptosis and Suppressing PI3K/Akt/mTOR Signaling in Endothelial Cells

    Journal: Journal of Cancer Prevention

    doi: 10.15430/JCP.2017.22.4.219

    Effects of myricetin on the PI3K/Akt/mTOR signaling pathway in human umbilical vascular endothelial cells (HUVECs). (A) HUVECs were treated with myricetin (0.25, 0.5, and 1 μM) for 24 hours. Protein samples (40 μg) were subjected to 6% to 15% SDS-PAGE and the levels of PI3K, PDK1, Akt, mTOR, and their phosphorylated forms were detected by Western blotting. (B) The cells were treated with myricetin in combination with an Akt inhibitor, LY294002, and the level of p-Akt was detected by Western blotting; β-actin was used as the internal control. Values represent the mean ± SD of three separate experiments. * P
    Figure Legend Snippet: Effects of myricetin on the PI3K/Akt/mTOR signaling pathway in human umbilical vascular endothelial cells (HUVECs). (A) HUVECs were treated with myricetin (0.25, 0.5, and 1 μM) for 24 hours. Protein samples (40 μg) were subjected to 6% to 15% SDS-PAGE and the levels of PI3K, PDK1, Akt, mTOR, and their phosphorylated forms were detected by Western blotting. (B) The cells were treated with myricetin in combination with an Akt inhibitor, LY294002, and the level of p-Akt was detected by Western blotting; β-actin was used as the internal control. Values represent the mean ± SD of three separate experiments. * P

    Techniques Used: SDS Page, Western Blot

    20) Product Images from "miR-99a-5p acts as tumor suppressor via targeting to mTOR and enhances RAD001-induced apoptosis in human urinary bladder urothelial carcinoma cells"

    Article Title: miR-99a-5p acts as tumor suppressor via targeting to mTOR and enhances RAD001-induced apoptosis in human urinary bladder urothelial carcinoma cells

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S114276

    Dual inhibition of mTORC1 and mTORC2 in miR-99a-5p-expressing cells compared to RAD001-treated cells. Notes: ( A ) The expression level of phospho-mTOR (Ser2448), rpS6, phospho-rpS6, 4EBP1, phospho-4EBP1 (Ser65), phospho-4EBP1 (Thr37/46), phospho-mTOR (Ser2481), total AKT and phospho-AKT (Ser473) was detected in cells transfected with pSM, pSM-99a or pSM-28-5p. EGFP, which co-expressed with miRNA transcripts in the pSM vector, was detected, and β-actin was used as a loading control. The expression level of phospho-AKT in each treatment was quantified as shown in the lower panel. ( B ) The protein lysates from cells transfected with pSM, pSM-99a or pSM-28-5p were subjected to IP with anti-mTOR antibody overnight, and then, mTOR immunoprecipitates (IP: mTOR) were subjected to Western blot analysis for mTOR, Raptor and Rictor. ( C ) The expression level of phospho-AKT (Ser473) in 24 h control (dimethyl sulfoxide[DMSO])-treated) or RAD001-treated cells was determined by Western blot. The expression of β-actin served as a loading control. The relative protein levels in each treatment were quantified as shown in the lower panel. * P
    Figure Legend Snippet: Dual inhibition of mTORC1 and mTORC2 in miR-99a-5p-expressing cells compared to RAD001-treated cells. Notes: ( A ) The expression level of phospho-mTOR (Ser2448), rpS6, phospho-rpS6, 4EBP1, phospho-4EBP1 (Ser65), phospho-4EBP1 (Thr37/46), phospho-mTOR (Ser2481), total AKT and phospho-AKT (Ser473) was detected in cells transfected with pSM, pSM-99a or pSM-28-5p. EGFP, which co-expressed with miRNA transcripts in the pSM vector, was detected, and β-actin was used as a loading control. The expression level of phospho-AKT in each treatment was quantified as shown in the lower panel. ( B ) The protein lysates from cells transfected with pSM, pSM-99a or pSM-28-5p were subjected to IP with anti-mTOR antibody overnight, and then, mTOR immunoprecipitates (IP: mTOR) were subjected to Western blot analysis for mTOR, Raptor and Rictor. ( C ) The expression level of phospho-AKT (Ser473) in 24 h control (dimethyl sulfoxide[DMSO])-treated) or RAD001-treated cells was determined by Western blot. The expression of β-actin served as a loading control. The relative protein levels in each treatment were quantified as shown in the lower panel. * P

    Techniques Used: Inhibition, Expressing, Transfection, Plasmid Preparation, Western Blot

    Schematic presentation of a mechanistic model showing miR-99a-5p blocking mTORC1 and mTORC2 signaling. Note: Targeting mTOR by miR-99a-5p facilitates mTORC1 and mTORC2 blockage, thus without AKT activation. Abbreviations: mTORC1, mTOR complex 1; mTORC2, mTOR complex 2; mTOR, mammalian target of rapamycin.
    Figure Legend Snippet: Schematic presentation of a mechanistic model showing miR-99a-5p blocking mTORC1 and mTORC2 signaling. Note: Targeting mTOR by miR-99a-5p facilitates mTORC1 and mTORC2 blockage, thus without AKT activation. Abbreviations: mTORC1, mTOR complex 1; mTORC2, mTOR complex 2; mTOR, mammalian target of rapamycin.

    Techniques Used: Blocking Assay, Activation Assay

    21) Product Images from "Thioredoxin 1 is associated with the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synoviocytes"

    Article Title: Thioredoxin 1 is associated with the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synoviocytes

    Journal: Clinical Rheumatology

    doi: 10.1007/s10067-017-3832-1

    Trx1 modulates PI3K-Akt activation and the expression apoptosis-related proteins in RA-FLSs. a , c RA-FLSs were transfected with Trx1-siRNAs (si-Trx1-1 and si-Trx1-2) or control siRNA (NC) and cultured for 48 h under 3% O 2 condition. Western blot and grayscale ratio analyses for the activation of PI3K-Akt and the expression of apoptosis-related proteins in RA-FLSs are shown. * P
    Figure Legend Snippet: Trx1 modulates PI3K-Akt activation and the expression apoptosis-related proteins in RA-FLSs. a , c RA-FLSs were transfected with Trx1-siRNAs (si-Trx1-1 and si-Trx1-2) or control siRNA (NC) and cultured for 48 h under 3% O 2 condition. Western blot and grayscale ratio analyses for the activation of PI3K-Akt and the expression of apoptosis-related proteins in RA-FLSs are shown. * P

    Techniques Used: Activation Assay, Expressing, Transfection, Cell Culture, Western Blot

    22) Product Images from "Notoginsenoside R7 suppresses cervical cancer via PI3K/PTEN/Akt/mTOR signaling"

    Article Title: Notoginsenoside R7 suppresses cervical cancer via PI3K/PTEN/Akt/mTOR signaling

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22721

    In vivo R7 anti-tumor efficacy HeLa cell mouse xenograft model. (A) BALB/c male athymic mice were inoculated with 1×10 7 cells subcutaneously on d 0. R7 (5 or 10 mg/kg/day) or vehicle was administered intraperitoneally once every 2 d from d 9–21, and mice were sacrificed on d 21. Tumor volumes were monitored and tumor volume curves recorded once every 2 d. (B) Representative tumors removed from the indicated treatment groups on day 21. (C) Average tumor mass at sacrifice after R7 treatment. (D) Bcl-2, Akt, and p-Akt Thr308 levelsin xenograft tissues were evaluated via western blotting (E) .
    Figure Legend Snippet: In vivo R7 anti-tumor efficacy HeLa cell mouse xenograft model. (A) BALB/c male athymic mice were inoculated with 1×10 7 cells subcutaneously on d 0. R7 (5 or 10 mg/kg/day) or vehicle was administered intraperitoneally once every 2 d from d 9–21, and mice were sacrificed on d 21. Tumor volumes were monitored and tumor volume curves recorded once every 2 d. (B) Representative tumors removed from the indicated treatment groups on day 21. (C) Average tumor mass at sacrifice after R7 treatment. (D) Bcl-2, Akt, and p-Akt Thr308 levelsin xenograft tissues were evaluated via western blotting (E) .

    Techniques Used: In Vivo, Mouse Assay, Western Blot

    R7 inhibits cancer-related PI3K/PTEN/Akt/mTOR activation in HeLa cells Following R7 treatment, p-PTEN, Akt, p-Akt Ser473 , p-Akt Thr308 , mTOR, p-mTOR Ser2448 , and raptor were evaluated via western blotting. (A) Cells were treated with R7 with or without LY294002 (5 μM) pretreatment, and cell viability was determined by MTT assay. (B) Apoptosis induction was analyzed via flow cytometry. (C) p-Akt Ser473 and p-Akt Thr308 levels were assessed using fluorescence microscopy. (D) Original magnification: 400×.
    Figure Legend Snippet: R7 inhibits cancer-related PI3K/PTEN/Akt/mTOR activation in HeLa cells Following R7 treatment, p-PTEN, Akt, p-Akt Ser473 , p-Akt Thr308 , mTOR, p-mTOR Ser2448 , and raptor were evaluated via western blotting. (A) Cells were treated with R7 with or without LY294002 (5 μM) pretreatment, and cell viability was determined by MTT assay. (B) Apoptosis induction was analyzed via flow cytometry. (C) p-Akt Ser473 and p-Akt Thr308 levels were assessed using fluorescence microscopy. (D) Original magnification: 400×.

    Techniques Used: Activation Assay, Western Blot, MTT Assay, Flow Cytometry, Cytometry, Fluorescence, Microscopy

    Diagram illustrating the proposed effects of R7 on PI3K/PTEN/Akt/mTOR signaling
    Figure Legend Snippet: Diagram illustrating the proposed effects of R7 on PI3K/PTEN/Akt/mTOR signaling

    Techniques Used:

    23) Product Images from "Tentacle extract from the jellyfish Cyanea capillata increases proliferation and migration of human umbilical vein endothelial cells through the ERK1/2 signaling pathway"

    Article Title: Tentacle extract from the jellyfish Cyanea capillata increases proliferation and migration of human umbilical vein endothelial cells through the ERK1/2 signaling pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189920

    Effects of TE on the phosphorylation of signaling pathways in the presence or absence of inhibitors. A: HUVECs were pretreated with TE (1 μg/ml) for 15 min in the presence or absence of an inhibitor for the PI3K/Akt signaling pathway. B: HUVECs were pretreated with TE (1 μg/ml) for 15 min in the presence or absence of an inhibitor for the ERK1/2 MAPK signaling pathway. C: HUVECs were pretreated with TE (1 μg/ml) for 15 min in the presence or absence of an inhibitor for the JNK MAPK signaling pathway. The images were obtained by a fluorescence microscope at 600 × magnification, scale = 30 μm. *** P
    Figure Legend Snippet: Effects of TE on the phosphorylation of signaling pathways in the presence or absence of inhibitors. A: HUVECs were pretreated with TE (1 μg/ml) for 15 min in the presence or absence of an inhibitor for the PI3K/Akt signaling pathway. B: HUVECs were pretreated with TE (1 μg/ml) for 15 min in the presence or absence of an inhibitor for the ERK1/2 MAPK signaling pathway. C: HUVECs were pretreated with TE (1 μg/ml) for 15 min in the presence or absence of an inhibitor for the JNK MAPK signaling pathway. The images were obtained by a fluorescence microscope at 600 × magnification, scale = 30 μm. *** P

    Techniques Used: Fluorescence, Microscopy

    Effects of signaling pathway inhibitors on the TE-induced expression of CyclinB1 and CyclinD1. A: The expression of CyclinB1. B: The expression of CyclinD1. HUVECs were pretreated with TE (1 μg/ml) for 30 min in the presence or absence of inhibitors for Akt, ERK1/2 and JNK MAPK. Images were obtained by a fluorescence microscope at 600 × magnification, scale = 30 μm. *** P
    Figure Legend Snippet: Effects of signaling pathway inhibitors on the TE-induced expression of CyclinB1 and CyclinD1. A: The expression of CyclinB1. B: The expression of CyclinD1. HUVECs were pretreated with TE (1 μg/ml) for 30 min in the presence or absence of inhibitors for Akt, ERK1/2 and JNK MAPK. Images were obtained by a fluorescence microscope at 600 × magnification, scale = 30 μm. *** P

    Techniques Used: Expressing, Fluorescence, Microscopy

    Effects of signaling pathway inhibitors on the TE-induced expression of CyclinB1 and CyclinD1. HUVECs were pretreated with TE (1 μg/ml) for 30 min in the presence or absence of inhibitors of Akt, ERK1/2 and JNK MAPK. LY: LY294002, PD: PD98059, SP: SP600125, Bay: Bay11-7082; ** P
    Figure Legend Snippet: Effects of signaling pathway inhibitors on the TE-induced expression of CyclinB1 and CyclinD1. HUVECs were pretreated with TE (1 μg/ml) for 30 min in the presence or absence of inhibitors of Akt, ERK1/2 and JNK MAPK. LY: LY294002, PD: PD98059, SP: SP600125, Bay: Bay11-7082; ** P

    Techniques Used: Expressing

    24) Product Images from "Protein Kinases Type II (PKG II) Combined with L-Arginine Significantly Ameliorated Xenograft Tumor Development: Is L-Arginine a Potential Alternative in PKG II Activation?"

    Article Title: Protein Kinases Type II (PKG II) Combined with L-Arginine Significantly Ameliorated Xenograft Tumor Development: Is L-Arginine a Potential Alternative in PKG II Activation?

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.906213

    PKG II combined with L-Arg effectively ameliorated the development of breast and colon cancers via inhibition of EGF/EGFR activation inducing Erk1/2 or Akt pathways. The interventions of cancer bearing mice and the operation are shown in Figure 1 . The lysates from tumor tissues were used to analysis the levels of p-EGFR, p-ERK1/2, and p-Akt. β-actin was also examined as a loading control. The experiment was repeated 3 times and similar data were obtained. Representative blots are shown.
    Figure Legend Snippet: PKG II combined with L-Arg effectively ameliorated the development of breast and colon cancers via inhibition of EGF/EGFR activation inducing Erk1/2 or Akt pathways. The interventions of cancer bearing mice and the operation are shown in Figure 1 . The lysates from tumor tissues were used to analysis the levels of p-EGFR, p-ERK1/2, and p-Akt. β-actin was also examined as a loading control. The experiment was repeated 3 times and similar data were obtained. Representative blots are shown.

    Techniques Used: Inhibition, Activation Assay, Mouse Assay

    25) Product Images from "Epigallocatechin-3-gallate confers protection against corticosterone-induced neuron injuries via restoring extracellular signal-regulated kinase 1/2 and phosphatidylinositol-3 kinase/protein kinase B signaling pathways"

    Article Title: Epigallocatechin-3-gallate confers protection against corticosterone-induced neuron injuries via restoring extracellular signal-regulated kinase 1/2 and phosphatidylinositol-3 kinase/protein kinase B signaling pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0192083

    The effect of EGCG on CORT-induced morphological changes of hippocampal neurons by Hoechst 33342 staining. Primary hippocampal neuron cultures were treated with CORT for 24 h in the absence or presence of EGCG pre-treatment (1 μmol/L, 2 h prior to CORT stimulation), ERK1/2 inhibitor (U0126, 10 μmol/L) or PI3K/AKT inhibitor (LY294002, 10 μmol/L) pre-treatment (30 min before CORT exposure). A) Normal control neurons; B) CORT treatment; C) EGCG and CORT co-treatment; D) EGCG+CORT+U0126 treatment; E) EGCG+CORT+ LY294002 treatment; F) The apoptotic index of hippocampal neurons. a P
    Figure Legend Snippet: The effect of EGCG on CORT-induced morphological changes of hippocampal neurons by Hoechst 33342 staining. Primary hippocampal neuron cultures were treated with CORT for 24 h in the absence or presence of EGCG pre-treatment (1 μmol/L, 2 h prior to CORT stimulation), ERK1/2 inhibitor (U0126, 10 μmol/L) or PI3K/AKT inhibitor (LY294002, 10 μmol/L) pre-treatment (30 min before CORT exposure). A) Normal control neurons; B) CORT treatment; C) EGCG and CORT co-treatment; D) EGCG+CORT+U0126 treatment; E) EGCG+CORT+ LY294002 treatment; F) The apoptotic index of hippocampal neurons. a P

    Techniques Used: Staining

    The effect of CORT and EGCG on ERK1/2 and AKT mRNA in primary hippocampal neurons. Primary hippocampal neuron cultures were pre-treated with EGCG for 2 h at indicated concentrations before stimulation with CORT for 24 h. The total RNA was isolated to measure the expression of ERK1/2 and AKT mRNA by RT-PCR, and expressed as mean ± SD of GAPDH mRNA levels. A) The expression of ERK1/2 mRNA; B) The expression of AKT mRNA. a P
    Figure Legend Snippet: The effect of CORT and EGCG on ERK1/2 and AKT mRNA in primary hippocampal neurons. Primary hippocampal neuron cultures were pre-treated with EGCG for 2 h at indicated concentrations before stimulation with CORT for 24 h. The total RNA was isolated to measure the expression of ERK1/2 and AKT mRNA by RT-PCR, and expressed as mean ± SD of GAPDH mRNA levels. A) The expression of ERK1/2 mRNA; B) The expression of AKT mRNA. a P

    Techniques Used: Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction

    26) Product Images from "The inhibition of PLCγ1 protects chondrocytes against osteoarthritis, implicating its binding to Akt"

    Article Title: The inhibition of PLCγ1 protects chondrocytes against osteoarthritis, implicating its binding to Akt

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23286

    Effect of Akt inhibitor on PLCγ1 expression in a rat OA model Specimens were longitudinally cut into 3 μm sections and the levels of Aggrecan expression level were detected by immunohistochemisty technique (original magnification ×200). As described in Material and Methods, the positive chondrocytes were counted and analyzed using Image-Pro Plus 6.0 Software and GraphPad Prism version 5. ( A ) Representative images from rats treated by Akt inhibitor for 1 month after ACLT+MMx (original magnification ×200). Graph indicating the percentage of positive chondrocytes expressing PLCγ1 ( **** P
    Figure Legend Snippet: Effect of Akt inhibitor on PLCγ1 expression in a rat OA model Specimens were longitudinally cut into 3 μm sections and the levels of Aggrecan expression level were detected by immunohistochemisty technique (original magnification ×200). As described in Material and Methods, the positive chondrocytes were counted and analyzed using Image-Pro Plus 6.0 Software and GraphPad Prism version 5. ( A ) Representative images from rats treated by Akt inhibitor for 1 month after ACLT+MMx (original magnification ×200). Graph indicating the percentage of positive chondrocytes expressing PLCγ1 ( **** P

    Techniques Used: Expressing, Software

    Schematic diagram of PLCγ1 and Akt regulating ECM synthesis of OA chondrocyte
    Figure Legend Snippet: Schematic diagram of PLCγ1 and Akt regulating ECM synthesis of OA chondrocyte

    Techniques Used:

    Relationship between PLCγ1 and Akt in OA chondrocytes ( A and B ) Rat OA model chondrocytes by IL-1β pre-treatment were treated with U73122 (2 μM) and TCN (10 μM) for 3, 6, 9, 12 h, respectively. P-PLCγ1, p-Akt, and β-actin levels were detected by western blotting analysis using anti-p-PLCγ1, p-Akt, and β-actin antibodies. ( C ) Rat OA model chondrocytes by IL-1β pre-treatment were transfected with HA-PLCγ1 and HA-PLCγ1 Y783A vectors for 48 h, respectively, and PLCγ1, p-PLCγ1, Col 2, Aggrecan, and β-actin levels were detected by western blotting analysis using anti-PLCγ1, p-PLCγ1, Col 2, Aggrecan, and β-actin antibodies. ( D ) Rat OA model chondrocytes by IL-1β pre-treatment were transfected with myc-Akt and myc-Akt S473A for 48 h, respectively, and Akt, p-Akt, Col 2, Aggrecan, and β-actin levels were detected by western blotting using anti-Akt, p-Akt, Col2, Aggrecan, and β-actin antibodies. ( E ) Human OA Chondrocytes were transfected with HA-PLCγ1 and HA-PLCγ1 Y783A vectors for 48 h, respectively. Cell lysates were immunoprecipitated with PLCγ1 antibody, and then subjected to western blotting analysis with PLCγ1 and Akt antibodies. ( F ) Human OA Chondrocytes were transfected with myc-Akt and myc-Akt S473A for 48 h, respectively. Cell lysates were immunoprecipitated with Akt antibody, and then subjected to western blotting analysis with PLCγ1 and Akt antibodies. The same lysates were applied to ascertain the position and expression of PLCγ1 and Akt by western blotting analysis (Input). Data is representative of three independent experiments.
    Figure Legend Snippet: Relationship between PLCγ1 and Akt in OA chondrocytes ( A and B ) Rat OA model chondrocytes by IL-1β pre-treatment were treated with U73122 (2 μM) and TCN (10 μM) for 3, 6, 9, 12 h, respectively. P-PLCγ1, p-Akt, and β-actin levels were detected by western blotting analysis using anti-p-PLCγ1, p-Akt, and β-actin antibodies. ( C ) Rat OA model chondrocytes by IL-1β pre-treatment were transfected with HA-PLCγ1 and HA-PLCγ1 Y783A vectors for 48 h, respectively, and PLCγ1, p-PLCγ1, Col 2, Aggrecan, and β-actin levels were detected by western blotting analysis using anti-PLCγ1, p-PLCγ1, Col 2, Aggrecan, and β-actin antibodies. ( D ) Rat OA model chondrocytes by IL-1β pre-treatment were transfected with myc-Akt and myc-Akt S473A for 48 h, respectively, and Akt, p-Akt, Col 2, Aggrecan, and β-actin levels were detected by western blotting using anti-Akt, p-Akt, Col2, Aggrecan, and β-actin antibodies. ( E ) Human OA Chondrocytes were transfected with HA-PLCγ1 and HA-PLCγ1 Y783A vectors for 48 h, respectively. Cell lysates were immunoprecipitated with PLCγ1 antibody, and then subjected to western blotting analysis with PLCγ1 and Akt antibodies. ( F ) Human OA Chondrocytes were transfected with myc-Akt and myc-Akt S473A for 48 h, respectively. Cell lysates were immunoprecipitated with Akt antibody, and then subjected to western blotting analysis with PLCγ1 and Akt antibodies. The same lysates were applied to ascertain the position and expression of PLCγ1 and Akt by western blotting analysis (Input). Data is representative of three independent experiments.

    Techniques Used: Western Blot, Transfection, Immunoprecipitation, Expressing

    Histopathological evaluation of OA in a rat OA model treated with different inhibitors of PLCγ1 and Akt for 2 month ( A ) Representatives images of Safranin O –Fast green staining from the rats treated by different inhibitors of PLCγ1 and Akt after ACLT+MMx (original magnification ×100). ( B ) Graph indicating the relative cartilage thickness of each femur condyle (from superficial zone to tidemark). ( C ) Graph indicating the OARSI scores ( * P
    Figure Legend Snippet: Histopathological evaluation of OA in a rat OA model treated with different inhibitors of PLCγ1 and Akt for 2 month ( A ) Representatives images of Safranin O –Fast green staining from the rats treated by different inhibitors of PLCγ1 and Akt after ACLT+MMx (original magnification ×100). ( B ) Graph indicating the relative cartilage thickness of each femur condyle (from superficial zone to tidemark). ( C ) Graph indicating the OARSI scores ( * P

    Techniques Used: Staining

    Effect of PLCγ1 inhibitor on Akt expression in a rat OA model Specimens were longitudinally cut into 3 μm sections and the levels of Aggrecan expression level were detected by immunohistochemisty technique (original magnification ×200). As described in Material and Methods, the positive chondrocytes were counted and analyzed using Image-Pro Plus 6.0 Software and GraphPad Prism version 5. ( A ) Representative images from rats treated by PLCγ1 inhibitor for 1 month after ACLT+MMx (original magnification ×200). Graph indicating the percentage of positive chondrocytes expressing Akt. ( B ) Representative images from rats treated by PLCγ1 inhibitor for 2 month after ACLT+MMx (original magnification ×200). Graph indicating the percentage of positive chondrocytes expressing Akt.
    Figure Legend Snippet: Effect of PLCγ1 inhibitor on Akt expression in a rat OA model Specimens were longitudinally cut into 3 μm sections and the levels of Aggrecan expression level were detected by immunohistochemisty technique (original magnification ×200). As described in Material and Methods, the positive chondrocytes were counted and analyzed using Image-Pro Plus 6.0 Software and GraphPad Prism version 5. ( A ) Representative images from rats treated by PLCγ1 inhibitor for 1 month after ACLT+MMx (original magnification ×200). Graph indicating the percentage of positive chondrocytes expressing Akt. ( B ) Representative images from rats treated by PLCγ1 inhibitor for 2 month after ACLT+MMx (original magnification ×200). Graph indicating the percentage of positive chondrocytes expressing Akt.

    Techniques Used: Expressing, Software

    Histopathological evaluation of OA in a rat OA model treated with different inhibitors of PLCγ1 and Akt for 1 month ( A ) Representatives images of Safranin O –Fast green staining from the rats treated by different inhibitors of PLCγ1 and Akt after ACLT+MMx (original magnification ×100). ( B ) Graph indicating the relative cartilage thickness of each femur condyle (from superficial zone to tidemark). ( C ) Graph indicating the OARSI scores ( * P
    Figure Legend Snippet: Histopathological evaluation of OA in a rat OA model treated with different inhibitors of PLCγ1 and Akt for 1 month ( A ) Representatives images of Safranin O –Fast green staining from the rats treated by different inhibitors of PLCγ1 and Akt after ACLT+MMx (original magnification ×100). ( B ) Graph indicating the relative cartilage thickness of each femur condyle (from superficial zone to tidemark). ( C ) Graph indicating the OARSI scores ( * P

    Techniques Used: Staining

    27) Product Images from "A synthetic cell-penetrating peptide derived from nuclear localization signal of EPS8 exerts anticancer activity against acute myeloid leukemia"

    Article Title: A synthetic cell-penetrating peptide derived from nuclear localization signal of EPS8 exerts anticancer activity against acute myeloid leukemia

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0682-x

    The inhibitory effects of CP-EPS8-NLS on EPS8 associated signaling. a U937, KG1α, HL-60 and TF1α cells were treated with increasing concentrations of CP-EPS8-NLS for 12 h, and analyzed by western blot to determine EPS8, Akt, p-Akt (473), p-STAT3, Erk and p-Erk levels. GAPDH expression was assessed to confirm equal protein loading. Densitometric analysis was performed using ImageJ software (NIH, Bethesda, MD; http://imagej.nih.gov/ij ). b Western blot analyses of U937 and KG1α cells treated with 0, 35 or 105 μM CP-EPS8-NLS for 12 and 24 h were performed to examine mTOR, p-mTOR, p-Akt (308) and p-Akt (450) expression
    Figure Legend Snippet: The inhibitory effects of CP-EPS8-NLS on EPS8 associated signaling. a U937, KG1α, HL-60 and TF1α cells were treated with increasing concentrations of CP-EPS8-NLS for 12 h, and analyzed by western blot to determine EPS8, Akt, p-Akt (473), p-STAT3, Erk and p-Erk levels. GAPDH expression was assessed to confirm equal protein loading. Densitometric analysis was performed using ImageJ software (NIH, Bethesda, MD; http://imagej.nih.gov/ij ). b Western blot analyses of U937 and KG1α cells treated with 0, 35 or 105 μM CP-EPS8-NLS for 12 and 24 h were performed to examine mTOR, p-mTOR, p-Akt (308) and p-Akt (450) expression

    Techniques Used: Western Blot, Expressing, Software

    The ability of EPS8 to influence the AML cells survival. a Western blot assay of EPS8 in six AML cell lines and an adriamycin-resistant AML cell line. b Cell survival assay in EPS8 shRNA-infected U937cells compared with NC shRNA-infected and parental cells on days 0 to 5. U937/NC and U937 cells nearly filled the well by 5th day. c Chemosensitivity of U937 cells transfected with shRNA2 after ADR treatment for 24 and 48 h. d U937 cells transfected with shRNA1, shRNA2 and NC shRNA were subjected to western blot analysis for EPS8, Akt/pAkt, Erk/p-Erk, p38 MAPK, p-GSK3β, p-cRaf, Caspase-9 and GAPDH expression. e Representative images of tumors extracted from nude mice following injection of 5 × 10 6 NC shRNA-infected cells and Eps8 shRNA-infected cells. The weights of the excised tumors
    Figure Legend Snippet: The ability of EPS8 to influence the AML cells survival. a Western blot assay of EPS8 in six AML cell lines and an adriamycin-resistant AML cell line. b Cell survival assay in EPS8 shRNA-infected U937cells compared with NC shRNA-infected and parental cells on days 0 to 5. U937/NC and U937 cells nearly filled the well by 5th day. c Chemosensitivity of U937 cells transfected with shRNA2 after ADR treatment for 24 and 48 h. d U937 cells transfected with shRNA1, shRNA2 and NC shRNA were subjected to western blot analysis for EPS8, Akt/pAkt, Erk/p-Erk, p38 MAPK, p-GSK3β, p-cRaf, Caspase-9 and GAPDH expression. e Representative images of tumors extracted from nude mice following injection of 5 × 10 6 NC shRNA-infected cells and Eps8 shRNA-infected cells. The weights of the excised tumors

    Techniques Used: Western Blot, Clonogenic Cell Survival Assay, shRNA, Infection, Transfection, Expressing, Mouse Assay, Injection

    28) Product Images from "Oleic acid stimulates HC11 mammary epithelial cells proliferation and mammary gland development in peripubertal mice through activation of CD36-Ca2+ and PI3K/Akt signaling pathway"

    Article Title: Oleic acid stimulates HC11 mammary epithelial cells proliferation and mammary gland development in peripubertal mice through activation of CD36-Ca2+ and PI3K/Akt signaling pathway

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24204

    Inhibition of PI3K/Akt totally blocked the promotion of HC11 proliferation induced by OA ( A ) Effect of Wortmannin (WT), an inhibitor of PI3K, on the proliferation of HC11 after a 4-day incubation was determined by MTT assay. ( B ) The relative mRNA expression level of Cyclin D1, Cyclin D3, and PCNA in response to 100 μM OA and/or 100 nM WT. ( C ) Representative immunofluorescence staining of Cyclin D1 and p21 in the presence of 100 μM OA and/or 100 nM WT. Scale bar = 100 μm. ( D ) Analysis of the relative fluorescence intensity in panel ( C). (E ) Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, Cyclin D1 and p21 in HC11 after a 4-day culture in the presence of 100 μM OA and/or 100 nM WT. β-actin was used as the loading control. ( F ) Mean ± SEM of immunoblotting bands of p-PI3K/PI3K, p-Akt/Akt, Cyclin D1, and p21, and the intensities of the bands were expressed as the arbitrary units. ** P
    Figure Legend Snippet: Inhibition of PI3K/Akt totally blocked the promotion of HC11 proliferation induced by OA ( A ) Effect of Wortmannin (WT), an inhibitor of PI3K, on the proliferation of HC11 after a 4-day incubation was determined by MTT assay. ( B ) The relative mRNA expression level of Cyclin D1, Cyclin D3, and PCNA in response to 100 μM OA and/or 100 nM WT. ( C ) Representative immunofluorescence staining of Cyclin D1 and p21 in the presence of 100 μM OA and/or 100 nM WT. Scale bar = 100 μm. ( D ) Analysis of the relative fluorescence intensity in panel ( C). (E ) Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, Cyclin D1 and p21 in HC11 after a 4-day culture in the presence of 100 μM OA and/or 100 nM WT. β-actin was used as the loading control. ( F ) Mean ± SEM of immunoblotting bands of p-PI3K/PI3K, p-Akt/Akt, Cyclin D1, and p21, and the intensities of the bands were expressed as the arbitrary units. ** P

    Techniques Used: Inhibition, Incubation, MTT Assay, Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot

    Effects of peripubertal exposure to diet containing 2% OA on the serum level of IGF-1 and E2, expression of CD36 and Cyclin D1, and activation of PI3K/Akt in the right side of the mammary gland of mice ( A – B ) Effects of dietary 2% OA on the serum levels of IGF-1 (A) and E2 (B) ( n = 10). ( C ) Western blot analysis of CD36, p-PI3K, PI3K, p-Akt, Akt and cyclin D1 in the mammary gland of pubertal mice ( n = 6). β-actin was used as the loading control. ( D ) Mean ± SEM of immunoblotting bands of CD36, p-PI3K/PI3K, p-Akt/Akt and Cyclin D1, and the intensities of the bands were expressed as the arbitrary units. ** P
    Figure Legend Snippet: Effects of peripubertal exposure to diet containing 2% OA on the serum level of IGF-1 and E2, expression of CD36 and Cyclin D1, and activation of PI3K/Akt in the right side of the mammary gland of mice ( A – B ) Effects of dietary 2% OA on the serum levels of IGF-1 (A) and E2 (B) ( n = 10). ( C ) Western blot analysis of CD36, p-PI3K, PI3K, p-Akt, Akt and cyclin D1 in the mammary gland of pubertal mice ( n = 6). β-actin was used as the loading control. ( D ) Mean ± SEM of immunoblotting bands of CD36, p-PI3K/PI3K, p-Akt/Akt and Cyclin D1, and the intensities of the bands were expressed as the arbitrary units. ** P

    Techniques Used: Expressing, Activation Assay, Mouse Assay, Western Blot

    Chelation of [Ca 2+ ] i reversed the OA-induced activation of PI3K/Akt and promotion of HC11 proliferation ( A ) Effects of 100 μM OA and/or 2 μM BAPTA-AM on HC11 proliferation by using MTT analysis. ( B ) Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, Cyclin D1, and p21 in HC11 after a 4-day culture in the presence of 100 μM OA and/or 2 μM BAPTA-AM. β-actin was used as the loading control. ( C ) Mean ± SEM of immunoblotting bands of p-PI3K/PI3K, p-Akt/Akt, Cyclin D1 and p21. The intensities of the bands were expressed as the arbitrary units. ** P
    Figure Legend Snippet: Chelation of [Ca 2+ ] i reversed the OA-induced activation of PI3K/Akt and promotion of HC11 proliferation ( A ) Effects of 100 μM OA and/or 2 μM BAPTA-AM on HC11 proliferation by using MTT analysis. ( B ) Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, Cyclin D1, and p21 in HC11 after a 4-day culture in the presence of 100 μM OA and/or 2 μM BAPTA-AM. β-actin was used as the loading control. ( C ) Mean ± SEM of immunoblotting bands of p-PI3K/PI3K, p-Akt/Akt, Cyclin D1 and p21. The intensities of the bands were expressed as the arbitrary units. ** P

    Techniques Used: Activation Assay, MTT Assay, Western Blot

    29) Product Images from "BAG3 promotes tumour cell proliferation by regulating EGFR signal transduction pathways in triple negative breast cancer"

    Article Title: BAG3 promotes tumour cell proliferation by regulating EGFR signal transduction pathways in triple negative breast cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24590

    Silencing BAG3 reduces activation of the AKT and FAK signalling pathways which regulate proliferation in TNBC cell lines ( A ) MDA-MB-468 cells were treated with siControl or siBAG3 and EGF pathway analysis performed on lysates using an EGF phospho antibody array. Quantitative histograms of PI3K/AKT signalling components from the EGF array are displayed. The histograms represent average protein expression (± SD) ( n = 6). ( B ) The protein expression of pAktSer473, pAktThr308, pmTORSer2441, pIKKα/βSer180/181, Akt, mTOR, IKKα/β and BAG3 was confirmed by immunoblotting. ( C ) Quantitative histograms of FAK/Src pathway components. The histograms represent average protein expression (± SD) ( n = 6). ( D ) The protein expression of pSrcTyr418, Src, pFAKTyr397, pFAKTyr576, pFAKTyr925, FAK, pSTAT1Ser727, STAT1 was confirmed by immunoblotting. ( E ) A hypothetical model of potential regulation of EGFR downstream signaling pathways by BAG3. The diagram was produced using Servier Medical Art ( F ) The protein expression of pFAKTyr397, FAK, pAKTSer473, AKT in BT-549 cells treated with siBAG3 and siControl was confirmed by immunoblotting. ( G ) The protein expression of pFAKTyr397, pAKTSer473 and GAPDH in MDA-MB-468 and BT-549 cell lines treated with an additional siRNA sequence (S2) targeting BAG3 and was confirmed by immunoblotting. ( H ) The protein expression of pFAKTyr397, pAKTSer473 and GAPDH in HCC1937 cells treated with FlagBAG3 and siControl was confirmed by immunoblotting. ( I ) BT-549 and MDA-MB-468 cells were treated with 5 uM MK-2206 and FAK14. Reduced activation of pFAKTyr397 after FAK14 treatment was confirmed by immunoblotting. ( J ) Reduced activation of pAKTSer473 after MK-2206 treatment was confirmed by immunoblotting. ( K ) A quantitative graph of proliferation relative to the control in MDA-MB-468, and BT-549 cells after treating with FAK and MK-2206 inhibitors. The histograms represent mean ( ± SD) Brdu incorporation relative to the control ( n = 3). An asterisk represents p
    Figure Legend Snippet: Silencing BAG3 reduces activation of the AKT and FAK signalling pathways which regulate proliferation in TNBC cell lines ( A ) MDA-MB-468 cells were treated with siControl or siBAG3 and EGF pathway analysis performed on lysates using an EGF phospho antibody array. Quantitative histograms of PI3K/AKT signalling components from the EGF array are displayed. The histograms represent average protein expression (± SD) ( n = 6). ( B ) The protein expression of pAktSer473, pAktThr308, pmTORSer2441, pIKKα/βSer180/181, Akt, mTOR, IKKα/β and BAG3 was confirmed by immunoblotting. ( C ) Quantitative histograms of FAK/Src pathway components. The histograms represent average protein expression (± SD) ( n = 6). ( D ) The protein expression of pSrcTyr418, Src, pFAKTyr397, pFAKTyr576, pFAKTyr925, FAK, pSTAT1Ser727, STAT1 was confirmed by immunoblotting. ( E ) A hypothetical model of potential regulation of EGFR downstream signaling pathways by BAG3. The diagram was produced using Servier Medical Art ( F ) The protein expression of pFAKTyr397, FAK, pAKTSer473, AKT in BT-549 cells treated with siBAG3 and siControl was confirmed by immunoblotting. ( G ) The protein expression of pFAKTyr397, pAKTSer473 and GAPDH in MDA-MB-468 and BT-549 cell lines treated with an additional siRNA sequence (S2) targeting BAG3 and was confirmed by immunoblotting. ( H ) The protein expression of pFAKTyr397, pAKTSer473 and GAPDH in HCC1937 cells treated with FlagBAG3 and siControl was confirmed by immunoblotting. ( I ) BT-549 and MDA-MB-468 cells were treated with 5 uM MK-2206 and FAK14. Reduced activation of pFAKTyr397 after FAK14 treatment was confirmed by immunoblotting. ( J ) Reduced activation of pAKTSer473 after MK-2206 treatment was confirmed by immunoblotting. ( K ) A quantitative graph of proliferation relative to the control in MDA-MB-468, and BT-549 cells after treating with FAK and MK-2206 inhibitors. The histograms represent mean ( ± SD) Brdu incorporation relative to the control ( n = 3). An asterisk represents p

    Techniques Used: Activation Assay, Multiple Displacement Amplification, Ab Array, Expressing, Produced, Sequencing, BrdU Incorporation Assay

    30) Product Images from "LDL cholesterol counteracts the antitumour effect of tyrosine kinase inhibitors against renal cell carcinoma"

    Article Title: LDL cholesterol counteracts the antitumour effect of tyrosine kinase inhibitors against renal cell carcinoma

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2017.77

    Inhibition of PI3K/AKT signalling enhances the antitumour efficacy of TKIs in the presence of LDL. ( A ) SK-45 cells were pre-treated with sorafenib (1 μ M ) for 3 h followed by the addition of LDL (100 μ g ml −1 ), or in the reverse order. Cell viability was analysed by CellTiter Blue assay. ( B ) Sorafenib does not affect LDL-induced activation of AKT. SK-45 cells were pre-treated with sorafenib (2.5 μ M ) for 1 h followed by the addition of LDL (100 μ g ml −1 ) for 1 h. ( C ) SK-45 and PNX0010 cells were pre-incubated with AKT inhibitor IV (1 μ M ) or PI3K inhibitor LY294002 (10 μ M ) for 1 h followed by incubation with sorafenib (1 μ M ) and/or LDL (100 μ g ml −1 ) for 72 h. Cell viability was analysed by CellTiter Blue assay. ( D ) The addition of LDL activates MEK/ERK signalling. SK-45 and PNX0010 cells were pre-incubated with AKT inhibitor IV (1 μ M ) or LY294002 (10 μ M ) followed by the addition of LDL (100 μ g ml −1 ) for 1 h. ( E ) The effect of HPCD on LDL-mediated AKT activation in RCC cells. SK-45 and PNX0010 cells were pre-treated with LDL (100 μ g ml −1 ) for 1 h followed by the addition of HPCD (10 m M ) for 2 h. LY294002 (10 μ M ) was used as a control in this experiment.
    Figure Legend Snippet: Inhibition of PI3K/AKT signalling enhances the antitumour efficacy of TKIs in the presence of LDL. ( A ) SK-45 cells were pre-treated with sorafenib (1 μ M ) for 3 h followed by the addition of LDL (100 μ g ml −1 ), or in the reverse order. Cell viability was analysed by CellTiter Blue assay. ( B ) Sorafenib does not affect LDL-induced activation of AKT. SK-45 cells were pre-treated with sorafenib (2.5 μ M ) for 1 h followed by the addition of LDL (100 μ g ml −1 ) for 1 h. ( C ) SK-45 and PNX0010 cells were pre-incubated with AKT inhibitor IV (1 μ M ) or PI3K inhibitor LY294002 (10 μ M ) for 1 h followed by incubation with sorafenib (1 μ M ) and/or LDL (100 μ g ml −1 ) for 72 h. Cell viability was analysed by CellTiter Blue assay. ( D ) The addition of LDL activates MEK/ERK signalling. SK-45 and PNX0010 cells were pre-incubated with AKT inhibitor IV (1 μ M ) or LY294002 (10 μ M ) followed by the addition of LDL (100 μ g ml −1 ) for 1 h. ( E ) The effect of HPCD on LDL-mediated AKT activation in RCC cells. SK-45 and PNX0010 cells were pre-treated with LDL (100 μ g ml −1 ) for 1 h followed by the addition of HPCD (10 m M ) for 2 h. LY294002 (10 μ M ) was used as a control in this experiment.

    Techniques Used: Inhibition, CtB Assay, Activation Assay, Incubation

    31) Product Images from "Radiotherapy-induced cell death activates paracrine HMGB1-TLR2 signaling and accelerates pancreatic carcinoma metastasis"

    Article Title: Radiotherapy-induced cell death activates paracrine HMGB1-TLR2 signaling and accelerates pancreatic carcinoma metastasis

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0726-2

    A schematic of radiotherapy induced cell death activates paracrine HMGB1-TLR2 signaling and accelerates pancreatic carcinoma metastasis. Radiotherapy induced the cancer cells’ death. HMGB1 is released by dying cells and binds specific receptor TLR2. HMGB1/TLR2 stimulates phosphorylation of PI3K/Akt and EMT in a paracrine manner, which promotes the metastasis of cancer cells to the lung
    Figure Legend Snippet: A schematic of radiotherapy induced cell death activates paracrine HMGB1-TLR2 signaling and accelerates pancreatic carcinoma metastasis. Radiotherapy induced the cancer cells’ death. HMGB1 is released by dying cells and binds specific receptor TLR2. HMGB1/TLR2 stimulates phosphorylation of PI3K/Akt and EMT in a paracrine manner, which promotes the metastasis of cancer cells to the lung

    Techniques Used:

    PI3K/Akt pathway and EMT program was responsible for the dying cell derived HMGB1 mediating metastasis. a Western blot analyzing the expression of PI3K-p85α, p-Akt, N-cadherin, vimentin, and E-cadherin in Panc-1 cells treated with PBS, N-S, HMGB1 −/− -S, N-S + EP, and rhHMGB1 (150 ng/mL). β-Tubulin was a loading control. b RT-PCR analyzing the expression of EMT-related transcriptional factors Zeb1, Slug, and Snail in Panc-1 cells treated with PBS, N-S, HMGB1 −/− -S, N-S + EP, and rhHMGB1 (150 ng/mL). GAPDH was a loading control. c Western blot showing the shRNA-knockdown efficiency of Zeb1. β-Tubulin was a loading control. d The invasion ability of Panc-1 cells following Zeb1 knockdown or treated with DMSO: MK2206 (AKT phosphorylation inhibitor) in the presence of rhHMGB1 (150 ng/mL) was detected by transwell assay. Magnification: × 20. Experiments were repeated three times and the data were expressed as mean ± SEM. * p
    Figure Legend Snippet: PI3K/Akt pathway and EMT program was responsible for the dying cell derived HMGB1 mediating metastasis. a Western blot analyzing the expression of PI3K-p85α, p-Akt, N-cadherin, vimentin, and E-cadherin in Panc-1 cells treated with PBS, N-S, HMGB1 −/− -S, N-S + EP, and rhHMGB1 (150 ng/mL). β-Tubulin was a loading control. b RT-PCR analyzing the expression of EMT-related transcriptional factors Zeb1, Slug, and Snail in Panc-1 cells treated with PBS, N-S, HMGB1 −/− -S, N-S + EP, and rhHMGB1 (150 ng/mL). GAPDH was a loading control. c Western blot showing the shRNA-knockdown efficiency of Zeb1. β-Tubulin was a loading control. d The invasion ability of Panc-1 cells following Zeb1 knockdown or treated with DMSO: MK2206 (AKT phosphorylation inhibitor) in the presence of rhHMGB1 (150 ng/mL) was detected by transwell assay. Magnification: × 20. Experiments were repeated three times and the data were expressed as mean ± SEM. * p

    Techniques Used: Derivative Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, shRNA, Transwell Assay

    32) Product Images from "Stimulation of medulloblastoma stem cells differentiation by a peptidomimetic targeting neuropilin-1"

    Article Title: Stimulation of medulloblastoma stem cells differentiation by a peptidomimetic targeting neuropilin-1

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24521

    Effects of MR438 or Tuftsin on the key proteins involved in the neuropilin pathways ( A ) Representative blots of p-ERK, ERK, p-AKT, AKT, p-SAMAD2/3, β-actin and β-tublin by Western blot. ( B ) Ratio of p-ERK to ERK for MB stem cells treated by MR438 or Tuftsin. ( C ) Ratio of p-AKT to AKT for MB stem cells treated MR438 or Tuftsin. ( D ) Ratio of p-SMAD to β-actin protein for MB stem cells treated by MR438 or Tuftsin. * p
    Figure Legend Snippet: Effects of MR438 or Tuftsin on the key proteins involved in the neuropilin pathways ( A ) Representative blots of p-ERK, ERK, p-AKT, AKT, p-SAMAD2/3, β-actin and β-tublin by Western blot. ( B ) Ratio of p-ERK to ERK for MB stem cells treated by MR438 or Tuftsin. ( C ) Ratio of p-AKT to AKT for MB stem cells treated MR438 or Tuftsin. ( D ) Ratio of p-SMAD to β-actin protein for MB stem cells treated by MR438 or Tuftsin. * p

    Techniques Used: Western Blot

    33) Product Images from "Salidroside inhibits steroid-induced avascular necrosis of the femoral head via the PI3K/Akt signaling pathway: In vitro and in vivo studies"

    Article Title: Salidroside inhibits steroid-induced avascular necrosis of the femoral head via the PI3K/Akt signaling pathway: In vitro and in vivo studies

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.8349

    Sal alleviates Dex-induced osteoblasts apoptosis via activating PI3K/Akt-mediated downregulation of caspase-3 expression. (A) The protein expression of Akt, p-Akt, cleaved caspase-9 and cleaved caspase-3 in osteoblasts was determined by western blot analysis. The results of the western blot were quantified and the protein expression of (B) p-Akt, (C) cleaved caspase-3 and (D) cleaved caspase-9 were determined. Data represent the average ± SD. Significant differences between two are indicated as **P
    Figure Legend Snippet: Sal alleviates Dex-induced osteoblasts apoptosis via activating PI3K/Akt-mediated downregulation of caspase-3 expression. (A) The protein expression of Akt, p-Akt, cleaved caspase-9 and cleaved caspase-3 in osteoblasts was determined by western blot analysis. The results of the western blot were quantified and the protein expression of (B) p-Akt, (C) cleaved caspase-3 and (D) cleaved caspase-9 were determined. Data represent the average ± SD. Significant differences between two are indicated as **P

    Techniques Used: Expressing, Western Blot

    34) Product Images from "High Glucose-Mediated Tyrosine Nitration of PI3-Kinase: A Molecular Switch of Survival and Apoptosis in Endothelial Cells"

    Article Title: High Glucose-Mediated Tyrosine Nitration of PI3-Kinase: A Molecular Switch of Survival and Apoptosis in Endothelial Cells

    Journal: Antioxidants

    doi: 10.3390/antiox7040047

    Overexpression of Myr-Akt attenuates proapoptotic effect of high glucose and peroxynitrite. ( A ) Representative Western blot showing selective expression of Akt in retinal EC cultures transfected with constitutively active Akt (Myr-Akt), but not in cultures transfected with adenovirus construct of β-galactosidase (β-Gal). Overexpression of Myr-Akt decreased expression of p38 MAPK. These observations were consistent in ECs cultured in normal glucose (NG, 5 mM), high glucose (HG, 25 mM) for 3 days, or peroxynitrite (PN, 0.5 mM) for overnight. ( B ) Statistical analysis of caspase-3 activity using two-way ANOVA showed significant effect of apoptotic insult and for treatment. Overexpression of Myr-Akt significantly reduced HG- or PN-induced increase in caspase-3 activity compared to EC cultures transfected with β-Gal (* p
    Figure Legend Snippet: Overexpression of Myr-Akt attenuates proapoptotic effect of high glucose and peroxynitrite. ( A ) Representative Western blot showing selective expression of Akt in retinal EC cultures transfected with constitutively active Akt (Myr-Akt), but not in cultures transfected with adenovirus construct of β-galactosidase (β-Gal). Overexpression of Myr-Akt decreased expression of p38 MAPK. These observations were consistent in ECs cultured in normal glucose (NG, 5 mM), high glucose (HG, 25 mM) for 3 days, or peroxynitrite (PN, 0.5 mM) for overnight. ( B ) Statistical analysis of caspase-3 activity using two-way ANOVA showed significant effect of apoptotic insult and for treatment. Overexpression of Myr-Akt significantly reduced HG- or PN-induced increase in caspase-3 activity compared to EC cultures transfected with β-Gal (* p

    Techniques Used: Over Expression, Western Blot, Expressing, Transfection, Construct, Cell Culture, Activity Assay

    35) Product Images from "Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation"

    Article Title: Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA117.001254

    Mitochondrial oxidants induces selective insulin resistance. MitoPQ generates oxidants (O 2 ˙̄ and H 2 O 2 ; denoted by O 2 ˙̄ ) in the mitochondrial matrix without impairing oxidative phosphorylation. Mitochondrial oxidants inhibit insulin-stimulated GLUT4 translocation and glucose uptake in adipocytes and myocytes, but AMPK-stimulated GLUT4 translocation is not impaired. MitoPQ did not impair insulin signaling to TBC1D4 or Akt-mediated activation of protein synthesis and inhibition of lipolysis, indicating that mitochondrial oxidants impair processes downstream of TBC1D4 phosphorylation in the insulin-regulated GLUT4 trafficking pathway.
    Figure Legend Snippet: Mitochondrial oxidants induces selective insulin resistance. MitoPQ generates oxidants (O 2 ˙̄ and H 2 O 2 ; denoted by O 2 ˙̄ ) in the mitochondrial matrix without impairing oxidative phosphorylation. Mitochondrial oxidants inhibit insulin-stimulated GLUT4 translocation and glucose uptake in adipocytes and myocytes, but AMPK-stimulated GLUT4 translocation is not impaired. MitoPQ did not impair insulin signaling to TBC1D4 or Akt-mediated activation of protein synthesis and inhibition of lipolysis, indicating that mitochondrial oxidants impair processes downstream of TBC1D4 phosphorylation in the insulin-regulated GLUT4 trafficking pathway.

    Techniques Used: Translocation Assay, Activation Assay, Inhibition

    Mitochondria-targeted paraquat induces insulin resistance downstream of Akt. A and B , 3T3-L1 adipocytes ( A ) or L6 myotubes ( B ) overexpressing HA-GLUT4 and PDGFR were serum-starved, treated with DMSO or the indicated doses of MitoPQ ( mPQ ) for 2 h, and assessed for plasma membrane–localized HA-GLUT4 following incubation without ( basal ) or with 100 n m insulin, 20 ng/ml PDGF, or 2 m m AICAR and 100 μ m A-769662 ( A769 ) for 20 min where indicated ( n = 4, mean ± S.E. ( error bars ), two-sample t test corrected for multiple comparisons; *, p
    Figure Legend Snippet: Mitochondria-targeted paraquat induces insulin resistance downstream of Akt. A and B , 3T3-L1 adipocytes ( A ) or L6 myotubes ( B ) overexpressing HA-GLUT4 and PDGFR were serum-starved, treated with DMSO or the indicated doses of MitoPQ ( mPQ ) for 2 h, and assessed for plasma membrane–localized HA-GLUT4 following incubation without ( basal ) or with 100 n m insulin, 20 ng/ml PDGF, or 2 m m AICAR and 100 μ m A-769662 ( A769 ) for 20 min where indicated ( n = 4, mean ± S.E. ( error bars ), two-sample t test corrected for multiple comparisons; *, p

    Techniques Used: Incubation

    36) Product Images from "Sulforaphane prevents angiotensin II-induced cardiomyopathy by activation of Nrf2 via stimulating the Akt/GSK-3ß/Fyn pathway"

    Article Title: Sulforaphane prevents angiotensin II-induced cardiomyopathy by activation of Nrf2 via stimulating the Akt/GSK-3ß/Fyn pathway

    Journal: Redox Biology

    doi: 10.1016/j.redox.2017.12.016

    Up-regulation of Nrf2 by SFN is partially achieved through the AKT/GSK-3β/Fyn pathway. Cardiac tissues were collected as described in Fig. 1 . Cardiac Akt ( A ) and GSK-3β ( B ) phosphorylation was examined by Western blot. The nuclear translocation of Fyn was determined by immunofluorescent staining of Fyn nuclear accumulation (red) on heart tissue sections ( C , bar = 100μm) and Western blot assay of Fyn expression in cardiac nuclear fraction ( D ). Data are presented as the mean ± SD (n = 7). *, p
    Figure Legend Snippet: Up-regulation of Nrf2 by SFN is partially achieved through the AKT/GSK-3β/Fyn pathway. Cardiac tissues were collected as described in Fig. 1 . Cardiac Akt ( A ) and GSK-3β ( B ) phosphorylation was examined by Western blot. The nuclear translocation of Fyn was determined by immunofluorescent staining of Fyn nuclear accumulation (red) on heart tissue sections ( C , bar = 100μm) and Western blot assay of Fyn expression in cardiac nuclear fraction ( D ). Data are presented as the mean ± SD (n = 7). *, p

    Techniques Used: Western Blot, Translocation Assay, Staining, Expressing

    37) Product Images from "Cigarette smoke enhances oncogene addiction to c‐ MET and desensitizes EGFR‐expressing non‐small cell lung cancer to EGFR TKIs"

    Article Title: Cigarette smoke enhances oncogene addiction to c‐ MET and desensitizes EGFR‐expressing non‐small cell lung cancer to EGFR TKIs

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12193

    EGFR TKI failed to inhibit activation of Akt and Erk in H292/B[α]P cells but not in HCC 827/ B[α]P cells. H292 (A, B) and HCC 827 (C) cells selected with B[α]P or NNK were pretreated with 1 μ m EGFR TKI s for 2 h followed by 50 ng· mL −1 EGF stimulation as indicated. Whole‐cell extracts were prepared and subjected to western blot analysis to determine the expression of EGFR signaling pathway with indicated antibodies.
    Figure Legend Snippet: EGFR TKI failed to inhibit activation of Akt and Erk in H292/B[α]P cells but not in HCC 827/ B[α]P cells. H292 (A, B) and HCC 827 (C) cells selected with B[α]P or NNK were pretreated with 1 μ m EGFR TKI s for 2 h followed by 50 ng· mL −1 EGF stimulation as indicated. Whole‐cell extracts were prepared and subjected to western blot analysis to determine the expression of EGFR signaling pathway with indicated antibodies.

    Techniques Used: Activation Assay, Western Blot, Expressing

    Activities of Akt and Erk were higher in CSE ‐ and B[α]P‐selected H292 cells. The activities and protein levels of EGFR and its downstream signaling Akt and ERK were detected in H292 (A) and HCC 827 (B) by western blot analysis with indicated antibodies.
    Figure Legend Snippet: Activities of Akt and Erk were higher in CSE ‐ and B[α]P‐selected H292 cells. The activities and protein levels of EGFR and its downstream signaling Akt and ERK were detected in H292 (A) and HCC 827 (B) by western blot analysis with indicated antibodies.

    Techniques Used: Western Blot

    Current hypothetic model of this study. Cigarette smoke de‐represses c‐ MET expression through reduction of promoter methylation. The induced c‐ MET induced drug resistance to EGFR TKI s by maintaining Akt activity in wt EGFR ‐expressing lung cancer cells.
    Figure Legend Snippet: Current hypothetic model of this study. Cigarette smoke de‐represses c‐ MET expression through reduction of promoter methylation. The induced c‐ MET induced drug resistance to EGFR TKI s by maintaining Akt activity in wt EGFR ‐expressing lung cancer cells.

    Techniques Used: Expressing, Methylation, Activity Assay

    The activity of Akt was reduced by inhibition of c‐ MET in H292/ CSE and H292/B[α]P cells. c‐ MET expression was knocked down by sh RNA in H292/ CSE (A) and H292/B[α]P (B) cells for 3 days. (C,D) These stable clones were treated with 1 μ m crizotinib for 3 days. Total lysates were collected and subjected to western blots with indicated antibodies (C) and the cell viability was analyzed in MTT assay (D).
    Figure Legend Snippet: The activity of Akt was reduced by inhibition of c‐ MET in H292/ CSE and H292/B[α]P cells. c‐ MET expression was knocked down by sh RNA in H292/ CSE (A) and H292/B[α]P (B) cells for 3 days. (C,D) These stable clones were treated with 1 μ m crizotinib for 3 days. Total lysates were collected and subjected to western blots with indicated antibodies (C) and the cell viability was analyzed in MTT assay (D).

    Techniques Used: Activity Assay, Inhibition, Expressing, Clone Assay, Western Blot, MTT Assay

    38) Product Images from "6-Gingerol Activates PI3K/Akt and Inhibits Apoptosis to Attenuate Myocardial Ischemia/Reperfusion Injury"

    Article Title: 6-Gingerol Activates PI3K/Akt and Inhibits Apoptosis to Attenuate Myocardial Ischemia/Reperfusion Injury

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2018/9024034

    Pretreatment with 6-G (6 mg/kg) activated the PI3K/Akt signaling pathway. ((a) and (b)) PI3K and Akt activities in myocardial homogenates detected by ELISA. ((c) and (d)) The expression level of PI3K, p-Akt, and Akt in the myocardial tissues detected using western blot and quantitative analyses ((e), (f), and (g)). Note that A P
    Figure Legend Snippet: Pretreatment with 6-G (6 mg/kg) activated the PI3K/Akt signaling pathway. ((a) and (b)) PI3K and Akt activities in myocardial homogenates detected by ELISA. ((c) and (d)) The expression level of PI3K, p-Akt, and Akt in the myocardial tissues detected using western blot and quantitative analyses ((e), (f), and (g)). Note that A P

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

    39) Product Images from "Biphasic regulation of tumorigenesis by PTK7 expression level in esophageal squamous cell carcinoma"

    Article Title: Biphasic regulation of tumorigenesis by PTK7 expression level in esophageal squamous cell carcinoma

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26957-6

    Biphasic regulation of protein phosphorylation in ESCC TE-5 and TE-10 cells by PTK7 expression. Representative Western blots from PTK7-low TE-5 cells 48 h after transfections with increasing amounts of the PTK7 expression vector (pcDNA3-PTK7-FLAG; PTK7) ( a ), and PTK7-high TE-10 cells transfected with various amounts of a PTK7 knockdown vector (pLKO.1-shRNA-PTK7-6434; PTK7-KD) or the PTK7 expression vector ( b ). Levels of tyrosine-phosphorylated cellular proteins (pY), as well as phosphorylated Src, Akt, and ERK, are shown. Numbers to the left of the blots indicate the molecular mass of the marker proteins (kDa). Samples derived from the same experiment and gels/blots were processed in parallel. The blots were cropped to focus upon the specific proteins indicated. Uncropped images of blots are shown in Supplementary Fig. S4 .
    Figure Legend Snippet: Biphasic regulation of protein phosphorylation in ESCC TE-5 and TE-10 cells by PTK7 expression. Representative Western blots from PTK7-low TE-5 cells 48 h after transfections with increasing amounts of the PTK7 expression vector (pcDNA3-PTK7-FLAG; PTK7) ( a ), and PTK7-high TE-10 cells transfected with various amounts of a PTK7 knockdown vector (pLKO.1-shRNA-PTK7-6434; PTK7-KD) or the PTK7 expression vector ( b ). Levels of tyrosine-phosphorylated cellular proteins (pY), as well as phosphorylated Src, Akt, and ERK, are shown. Numbers to the left of the blots indicate the molecular mass of the marker proteins (kDa). Samples derived from the same experiment and gels/blots were processed in parallel. The blots were cropped to focus upon the specific proteins indicated. Uncropped images of blots are shown in Supplementary Fig. S4 .

    Techniques Used: Expressing, Western Blot, Transfection, Plasmid Preparation, shRNA, Marker, Derivative Assay

    40) Product Images from "Decreased expression of ARHGAP15 promotes the development of colorectal cancer through PTEN/AKT/FOXO1 axis"

    Article Title: Decreased expression of ARHGAP15 promotes the development of colorectal cancer through PTEN/AKT/FOXO1 axis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0707-6

    ARHGAP15 influenced the development of CRC via modulating PTEN/AKT/FOXO1-signaling pathway. ARHGAP15-silenced LoVo cells were transfected with pLVX-PTEN or treated with MK2206. Cell proliferation ( a ) was detected by CCK-8 assay. Cell migration ( b ) and invasion ( c ) was detected by transwell assay. Scale bar: 100 μm. Protein levels of related molecules ( d ) was detected by western blot. Experiments were repeated three times independently. NS: no significant difference; ** p
    Figure Legend Snippet: ARHGAP15 influenced the development of CRC via modulating PTEN/AKT/FOXO1-signaling pathway. ARHGAP15-silenced LoVo cells were transfected with pLVX-PTEN or treated with MK2206. Cell proliferation ( a ) was detected by CCK-8 assay. Cell migration ( b ) and invasion ( c ) was detected by transwell assay. Scale bar: 100 μm. Protein levels of related molecules ( d ) was detected by western blot. Experiments were repeated three times independently. NS: no significant difference; ** p

    Techniques Used: Transfection, CCK-8 Assay, Migration, Transwell Assay, Western Blot

    ARHGAP15 modulated PTEN/AKT/FOXO1-signaling pathway. a , b The protein levels of several pivotal molecules in HT29 ( a ) and RKO ( b ) cells after transfected with pLVX-ARHGAP15 or pLVS-NC. c The protein levels of several pivotal molecules in LoVo cells after transfected with ARHGAP15 shRNA (sh-ARHGAP15) or control shRNA (sh-NC). Experiments were repeated three times independently. NS: no significant difference; ** P
    Figure Legend Snippet: ARHGAP15 modulated PTEN/AKT/FOXO1-signaling pathway. a , b The protein levels of several pivotal molecules in HT29 ( a ) and RKO ( b ) cells after transfected with pLVX-ARHGAP15 or pLVS-NC. c The protein levels of several pivotal molecules in LoVo cells after transfected with ARHGAP15 shRNA (sh-ARHGAP15) or control shRNA (sh-NC). Experiments were repeated three times independently. NS: no significant difference; ** P

    Techniques Used: Transfection, shRNA

    41) Product Images from "Cytoprotective Effect of Epigallocatechin Gallate (EGCG)-5′-O-α-Glucopyranoside, a Novel EGCG Derivative"

    Article Title: Cytoprotective Effect of Epigallocatechin Gallate (EGCG)-5′-O-α-Glucopyranoside, a Novel EGCG Derivative

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19051466

    Anti-apoptotic effect of EGCG-5′Glu under SNP-induced apoptosis. ( a ) EGCG-5′Glu was applied to HaCaT cells for 24 h. Cell viability was tested by MTT assay. ( b ) EGCG-5′Glu was pre-treated on RAW264.7 cells for 30 min, and SNP (1.5 mM) was added for 24 h. SNP-derived NO was measured by Griess assay. ( c ) Under SNP treatment, cell viability of HaCaT cells with or without EGCG-5′Glu was identified by MTT assay. ( d ) Caspase levels of EGCG-5′Glu and SNP-treated HaCaT cells were analyzed by immunoblotting. Antibodies against total or cleaved caspase-3, -8, and -9 and β-actin were used. ( e ) Phosphorylated levels of PI3K, PDK1, and AKT in EGCG-5′Glu- and SNP-treated HaCaT cells were analyzed by immunoblotting. Antibodies against phospho- or total forms PI3K, PDK1, AKT, and β-actin were used. # p
    Figure Legend Snippet: Anti-apoptotic effect of EGCG-5′Glu under SNP-induced apoptosis. ( a ) EGCG-5′Glu was applied to HaCaT cells for 24 h. Cell viability was tested by MTT assay. ( b ) EGCG-5′Glu was pre-treated on RAW264.7 cells for 30 min, and SNP (1.5 mM) was added for 24 h. SNP-derived NO was measured by Griess assay. ( c ) Under SNP treatment, cell viability of HaCaT cells with or without EGCG-5′Glu was identified by MTT assay. ( d ) Caspase levels of EGCG-5′Glu and SNP-treated HaCaT cells were analyzed by immunoblotting. Antibodies against total or cleaved caspase-3, -8, and -9 and β-actin were used. ( e ) Phosphorylated levels of PI3K, PDK1, and AKT in EGCG-5′Glu- and SNP-treated HaCaT cells were analyzed by immunoblotting. Antibodies against phospho- or total forms PI3K, PDK1, AKT, and β-actin were used. # p

    Techniques Used: MTT Assay, Derivative Assay, Griess Assay

    Summary of the cytoprotective effect of EGCG-5′Glu. EGCG-5′Glu cleared various free radicals and downregulated caspase activities. Survival signal pathway (PI3K/AKT/NF-κB) improved with EGCG-5′Glu, and cell proliferation increased. → stimulation, ⊥ inhibition.
    Figure Legend Snippet: Summary of the cytoprotective effect of EGCG-5′Glu. EGCG-5′Glu cleared various free radicals and downregulated caspase activities. Survival signal pathway (PI3K/AKT/NF-κB) improved with EGCG-5′Glu, and cell proliferation increased. → stimulation, ⊥ inhibition.

    Techniques Used: Inhibition

    Anti-oxidant effect of EGCG-5′Glu against UVB-induced damage. ( a ) Images of HaCaT cells treated with EGCG-5′Glu (0–25 μM) and UVB (30 mJ/cm 2 ) irradiation for 48 h were captured with a camera attached to the microscope. ( b ) Under UVB irradiation, viability of HaCaT cells with and without EGCG-5′Glu was measured by MTT assay. ( c ) Under UVB irradiation, HaCaT cell were incubated with EGCG-5′Glu for 48 h. Phospho- and total PI3K, AKT, and PDK1 expression was detected by immunoblotting. β-Actin was used as an immunoblotting loading control. ** p
    Figure Legend Snippet: Anti-oxidant effect of EGCG-5′Glu against UVB-induced damage. ( a ) Images of HaCaT cells treated with EGCG-5′Glu (0–25 μM) and UVB (30 mJ/cm 2 ) irradiation for 48 h were captured with a camera attached to the microscope. ( b ) Under UVB irradiation, viability of HaCaT cells with and without EGCG-5′Glu was measured by MTT assay. ( c ) Under UVB irradiation, HaCaT cell were incubated with EGCG-5′Glu for 48 h. Phospho- and total PI3K, AKT, and PDK1 expression was detected by immunoblotting. β-Actin was used as an immunoblotting loading control. ** p

    Techniques Used: Irradiation, Microscopy, MTT Assay, Incubation, Expressing

    42) Product Images from "Silibinin Inhibits NSCLC Metastasis by Targeting the EGFR/LOX Pathway"

    Article Title: Silibinin Inhibits NSCLC Metastasis by Targeting the EGFR/LOX Pathway

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.00021

    Lysyl oxidase expression is regulated by the PI3K/AKT, MEK/ERK, and SAPK/JNK signaling pathways. (A,C) NCI-H1975 was exposed to LY294002 (PI3K inhibitor), U0126 (MEK inhibitor), SB203580 (P-38 inhibitor) and SP600125 (JNK inhibitor) for 48 h, LOX mRNA and protein expression were analyzed by PCR and Western Blot. (B,D) HCC827 was exposed to LY294002 (PI3K inhibitor), U0126 (MEK inhibitor), SB203580 (P-38 inhibitor), and SP600125 (JNK inhibitor) for 48 h, LOX mRNA, and protein expression were analyzed by PCR and Western Blot. The data are represented as the mean ± SD. of three independent experiments. The P -values
    Figure Legend Snippet: Lysyl oxidase expression is regulated by the PI3K/AKT, MEK/ERK, and SAPK/JNK signaling pathways. (A,C) NCI-H1975 was exposed to LY294002 (PI3K inhibitor), U0126 (MEK inhibitor), SB203580 (P-38 inhibitor) and SP600125 (JNK inhibitor) for 48 h, LOX mRNA and protein expression were analyzed by PCR and Western Blot. (B,D) HCC827 was exposed to LY294002 (PI3K inhibitor), U0126 (MEK inhibitor), SB203580 (P-38 inhibitor), and SP600125 (JNK inhibitor) for 48 h, LOX mRNA, and protein expression were analyzed by PCR and Western Blot. The data are represented as the mean ± SD. of three independent experiments. The P -values

    Techniques Used: Expressing, Polymerase Chain Reaction, Western Blot

    Silibinin inhibits the migration of the NSCLC cell line NCI-H1975 through the EGFR/PI3K/LOX pathway in vitro . (A) LOX mRNA expression after exposed to silibinin in NCI-H1975. (B) EGFR, P-EGFR, P-AKT and LOX expression were inhibited after the NCI-H1975 cells were treated with silibinin. (C,D) Pretreating the NCI-H1975 cells with the LOX inhibitor (BAPN 200 μM) decreased the anti-metastasis effect of silibinin. (E) LOX inhibition inhibited epithelial-to-mesenchymal transition of NCI-H1975. (F) LOX inhibition decreased the phosphorylation of EGFR but not the total expression of EGFR. The data is represented as the mean ± SD of three independent experiments. The P -values
    Figure Legend Snippet: Silibinin inhibits the migration of the NSCLC cell line NCI-H1975 through the EGFR/PI3K/LOX pathway in vitro . (A) LOX mRNA expression after exposed to silibinin in NCI-H1975. (B) EGFR, P-EGFR, P-AKT and LOX expression were inhibited after the NCI-H1975 cells were treated with silibinin. (C,D) Pretreating the NCI-H1975 cells with the LOX inhibitor (BAPN 200 μM) decreased the anti-metastasis effect of silibinin. (E) LOX inhibition inhibited epithelial-to-mesenchymal transition of NCI-H1975. (F) LOX inhibition decreased the phosphorylation of EGFR but not the total expression of EGFR. The data is represented as the mean ± SD of three independent experiments. The P -values

    Techniques Used: Migration, In Vitro, Expressing, Inhibition

    43) Product Images from "3,5-Dicaffeoylquinic acid protects H9C2 cells against oxidative stress-induced apoptosis via activation of the PI3K/Akt signaling pathway"

    Article Title: 3,5-Dicaffeoylquinic acid protects H9C2 cells against oxidative stress-induced apoptosis via activation of the PI3K/Akt signaling pathway

    Journal: Food & Nutrition Research

    doi: 10.29219/fnr.v62.1423

    Effects of 3,5-diCQA on the expression of activated PI3K / Akt signaling mediators in H9C2 cells. H9C2 cells were incubated with 3,5-diCQA (5, 10, and 20 μM) for 24 h. LY294002 was used in inhibitory experiments and H9C2 cells were pre-incubated with 25 μM LY294002 for 1 h, and 20 μM 3,5-diCQA for another 24 h. The indicated proteins were detected by Western blot. (a) Representative images of p-PI3K and p-Akt expression and their quantification (b and c); (d) expression of p-PI3K and p-Akt in presence of LY294002 and their quantification (e and f). The results were expressed as means ± SD ( n = 3). # p
    Figure Legend Snippet: Effects of 3,5-diCQA on the expression of activated PI3K / Akt signaling mediators in H9C2 cells. H9C2 cells were incubated with 3,5-diCQA (5, 10, and 20 μM) for 24 h. LY294002 was used in inhibitory experiments and H9C2 cells were pre-incubated with 25 μM LY294002 for 1 h, and 20 μM 3,5-diCQA for another 24 h. The indicated proteins were detected by Western blot. (a) Representative images of p-PI3K and p-Akt expression and their quantification (b and c); (d) expression of p-PI3K and p-Akt in presence of LY294002 and their quantification (e and f). The results were expressed as means ± SD ( n = 3). # p

    Techniques Used: Expressing, Incubation, Western Blot

    Effects of 3,5-diCQA on TBHP-induced injury of H9C2 cells under inhibition of the PI3K / Akt signaling pathway. LY294002 was applied to inhibit the activation of the PI3K/Akt signaling pathway in this section. H9C2 cells were pre-incubated with 25 μM LY294002 for 1 h, and 20 μM 3,5-diCQA for another 24 h and then 75 μM TBHP. (a) Western blotting of the PI3K/Akt signaling and the fold activation data analysis (b and c) ( n = 3); (d) cell viability of H9C2 determined by MTT assay ( n = 6); (e and f) Percentage of apoptotic cells of H9C2 determined by Hoechst 33342/PI staining ( n = 3); (g) Western blotting of apoptosis-related proteins including cleaved caspase-3, Bax and Bcl-2 and the fold activation data analysis (h through j) ( n = 3). Data were shown as mean ± SD. # p
    Figure Legend Snippet: Effects of 3,5-diCQA on TBHP-induced injury of H9C2 cells under inhibition of the PI3K / Akt signaling pathway. LY294002 was applied to inhibit the activation of the PI3K/Akt signaling pathway in this section. H9C2 cells were pre-incubated with 25 μM LY294002 for 1 h, and 20 μM 3,5-diCQA for another 24 h and then 75 μM TBHP. (a) Western blotting of the PI3K/Akt signaling and the fold activation data analysis (b and c) ( n = 3); (d) cell viability of H9C2 determined by MTT assay ( n = 6); (e and f) Percentage of apoptotic cells of H9C2 determined by Hoechst 33342/PI staining ( n = 3); (g) Western blotting of apoptosis-related proteins including cleaved caspase-3, Bax and Bcl-2 and the fold activation data analysis (h through j) ( n = 3). Data were shown as mean ± SD. # p

    Techniques Used: Inhibition, Activation Assay, Incubation, Western Blot, MTT Assay, Staining

    Effects of 3,5-diCQA on phosphatidylinositol 3-kinase (PI3K) / Akt signaling pathway in H9C2 cells exposed to TBHP. H9C2 cells were pre-incubated with the indicated dose of 3,5-diCQA (5, 10, and 20 μM) for 24 h and then stimulated with TBHP (75 μM) for 4 h. (a) Western blot was performed to demonstrate the expression of p-PI3K, p-Akt, and p-PTEN, and densities of the bands were quantified by densitometry analysis (b through d) ( n = 3). Data were shown as mean ± SD. # p
    Figure Legend Snippet: Effects of 3,5-diCQA on phosphatidylinositol 3-kinase (PI3K) / Akt signaling pathway in H9C2 cells exposed to TBHP. H9C2 cells were pre-incubated with the indicated dose of 3,5-diCQA (5, 10, and 20 μM) for 24 h and then stimulated with TBHP (75 μM) for 4 h. (a) Western blot was performed to demonstrate the expression of p-PI3K, p-Akt, and p-PTEN, and densities of the bands were quantified by densitometry analysis (b through d) ( n = 3). Data were shown as mean ± SD. # p

    Techniques Used: Incubation, Western Blot, Expressing

    44) Product Images from "Capsaicin and sorafenib combination treatment exerts synergistic anti-hepatocellular carcinoma activity by suppressing EGFR and PI3K/Akt/mTOR signaling"

    Article Title: Capsaicin and sorafenib combination treatment exerts synergistic anti-hepatocellular carcinoma activity by suppressing EGFR and PI3K/Akt/mTOR signaling

    Journal: Oncology Reports

    doi: 10.3892/or.2018.6754

    Capsaicin and sorafenib reduce the expression of EGFR, PI3K/Akt/mTOR pathway components. Capsaicin and sorafenib downregulate EGFR, PI3K/Akt/mTOR signaling and the client protein P-p70s6k in LM3 cells. The combination of capsaicin and sorafenib exerted a synergistic effect on this pathway. Cells were serum-starved for 24 h and then treated with capsaicin, sorafenib and their combination for 12 h. Protein lysates were analyzed by western blotting with the indicated antibodies. GAPDH served as a loading control. Values represented the mean ± standard error of the mean from three independent experiments. #, Capsaicin vs. combination; +, sorafenib vs combination. ++ and ###/+++, P
    Figure Legend Snippet: Capsaicin and sorafenib reduce the expression of EGFR, PI3K/Akt/mTOR pathway components. Capsaicin and sorafenib downregulate EGFR, PI3K/Akt/mTOR signaling and the client protein P-p70s6k in LM3 cells. The combination of capsaicin and sorafenib exerted a synergistic effect on this pathway. Cells were serum-starved for 24 h and then treated with capsaicin, sorafenib and their combination for 12 h. Protein lysates were analyzed by western blotting with the indicated antibodies. GAPDH served as a loading control. Values represented the mean ± standard error of the mean from three independent experiments. #, Capsaicin vs. combination; +, sorafenib vs combination. ++ and ###/+++, P

    Techniques Used: Expressing, Western Blot

    45) Product Images from "Supplemental Oxygen Protects Heart Against Acute Myocardial Infarction"

    Article Title: Supplemental Oxygen Protects Heart Against Acute Myocardial Infarction

    Journal: Frontiers in Cardiovascular Medicine

    doi: 10.3389/fcvm.2018.00114

    Key signaling proteins in the MI heart subjected to OxCy. The levels of Akt, pAkt, eNOS, p53, and Bax were probed in hearts subjected to daily oxygen cycling 3 days after induction of MI for 5 days. Representative western blot images are shown on the left and densitometric analyses (band intensity) of Akt, pAkt, eNOS, p53, and Bax are shown on the right. Results are plotted as Mean ± SD for each group, expressed as a percent of the respective Control group. The number of hearts (N) used for densitometric analysis: Control, 5–7; MI, 11–15; all other groups, 3–10. Statistical significance between Control and MI group is indicated (NS denotes not significant). * p
    Figure Legend Snippet: Key signaling proteins in the MI heart subjected to OxCy. The levels of Akt, pAkt, eNOS, p53, and Bax were probed in hearts subjected to daily oxygen cycling 3 days after induction of MI for 5 days. Representative western blot images are shown on the left and densitometric analyses (band intensity) of Akt, pAkt, eNOS, p53, and Bax are shown on the right. Results are plotted as Mean ± SD for each group, expressed as a percent of the respective Control group. The number of hearts (N) used for densitometric analysis: Control, 5–7; MI, 11–15; all other groups, 3–10. Statistical significance between Control and MI group is indicated (NS denotes not significant). * p

    Techniques Used: Western Blot

    46) Product Images from "EpCAM ectodomain EpEX is a ligand of EGFR that counteracts EGF-mediated epithelial-mesenchymal transition through modulation of phospho-ERK1/2 in head and neck cancers"

    Article Title: EpCAM ectodomain EpEX is a ligand of EGFR that counteracts EGF-mediated epithelial-mesenchymal transition through modulation of phospho-ERK1/2 in head and neck cancers

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2006624

    Soluble EpEX-Fc binds to EGFR and induces ERK1/2 and AKT. (A) Bidirectional co-immunoprecipitation (“IP”) of EGFR and EpCAM in whole-cell lysates of FaDu, Cal27, and HCT8 cells using EGFR- and EpCAM-specific antibodies. Isotype control antibody (“IgG”) served as control. Coimmunoprecipitated EGFR and EpCAM were visualized in immunoblotting with specific antibodies (“IB”), with whole-cell lysates as control (“lysate”). Shown are representative results from n = 3 independent experiments. (B) SNs of Cal27, Kyse30, FaDu, and HCT8 cells were immunoprecipitated with EpEX-specific antibodies and separated under reducing (left) and nonreducing native conditions (right), and EpEX was detected with specific antibodies. Antibody HCs and EpEX mono-, di-, and oligomers are indicated. Shown are representative results from n = 3 independent experiments. (C) EpEX-Fc or Fc were incubated with whole-cell lysates of FaDu and Cal27 and immobilized on protein A agarose beads, and protein complexes were separated on SDS-PAGE. Immunoprecipitated proteins were detected by immunoblotting (“IB”) with Fc- and EGFR-specific antibodies. Shown are representative results from n = 3 independent experiments. (D) EGFR ex and EpEX were incubated in the presence or absence of cross-linker (BS3). Where indicated, EGF was added. Monomers, dimers, and EGFR ex /EpEX complexes are marked. Shown are representative results from n = 3 independent experiments. (E, F) FaDu, Cal27, and Kyse30 cells were kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for the indicated time points. Where indicated, cells were additionally treated with Cetuximab (“Cet.”). Phosphorylation of ERK1/2 (E) and AKT (F) was assessed by immunoblotting with specific antibodies. Levels of ERK1/2 and AKT were assessed in parallel. Shown are representative results from n = 3 independent experiments. (G) FaDu and Cal27 cells were kept untreated or were treated with EGF (9 nM), Fc, or EpEX-Fc (10 nM) for 30 min, and phosphorylation of ERK1/2 and AKT was detected by immunofluorescence laser scanning confocal microscopy (ERK1/2 or AKT: green, nuclei: blue [DAPI]). Shown are representative results from n = 3 independent experiments. (H) FaDu, Cal27, and Kyse30 cells were kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for the indicated time points (“EGF 30 min”). Where indicated, MEK1 inhibitor AZD6244 or EGFR inhibitor AG1478 were added. Levels of ERK1/2 and AKT were assessed in parallel. Shown are representative results from n = 3 independent experiments. (I) HEK293 cells were transiently transfected with GFP or EGFR expression plasmids and were either kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for 30 min. Expression of EGFR and activation of ERK1/2 were assessed by immunoblotting. Levels of ERK1/2 were assessed in parallel. Shown are representative results from n = 3 independent experiments. (J) Expression of EpCAM and EGFR was assessed in HCT8WT and CRISPR-Cas9 EpCAM K.O.1 [ 48 ] by immunoblotting. Levels of actin were assessed in parallel. Shown are representative results from n = 3 independent experiments. (K) HCT8WT and CRISPR-Cas9 EpCAM K.O.1 cells were either kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for 30 min. Activation of ERK1/2 was assessed by immunoblotting. Levels of ERK1/2 were assessed in parallel. Shown are representative results from n = 3 independent experiments. BS3, bisulfosuccinimidyl suberate; CRISPR-Cas9, clustered regularly interspaced short palindromic repeat/CRISPR-associated 9; EGF, epidermal growth factor; EGFR, EGF receptor; EGFR ex , extracellular domain of EGFR; EpCAM, epithelial cell adhesion molecule; EpCAM K.O.1, EPCAM -knockout clone 1; EpEX, extracellular domain of EpCAM; EpICD, intracellular domain of EpCAM; ERK1/2, extracellular signal–regulated kinase 1/2; Fc, fragment crystallizable region; GFP, green fluorescent protein; HC, heavy chain; HCT8WT, HCT8 wild type; HEK293, human embryonic kidney 293; IgG, immunoglobulin G; MW, molecular mass; pAKT, phosphorylated AKT; pERK, phosphorylated ERK; SN, supernatant.
    Figure Legend Snippet: Soluble EpEX-Fc binds to EGFR and induces ERK1/2 and AKT. (A) Bidirectional co-immunoprecipitation (“IP”) of EGFR and EpCAM in whole-cell lysates of FaDu, Cal27, and HCT8 cells using EGFR- and EpCAM-specific antibodies. Isotype control antibody (“IgG”) served as control. Coimmunoprecipitated EGFR and EpCAM were visualized in immunoblotting with specific antibodies (“IB”), with whole-cell lysates as control (“lysate”). Shown are representative results from n = 3 independent experiments. (B) SNs of Cal27, Kyse30, FaDu, and HCT8 cells were immunoprecipitated with EpEX-specific antibodies and separated under reducing (left) and nonreducing native conditions (right), and EpEX was detected with specific antibodies. Antibody HCs and EpEX mono-, di-, and oligomers are indicated. Shown are representative results from n = 3 independent experiments. (C) EpEX-Fc or Fc were incubated with whole-cell lysates of FaDu and Cal27 and immobilized on protein A agarose beads, and protein complexes were separated on SDS-PAGE. Immunoprecipitated proteins were detected by immunoblotting (“IB”) with Fc- and EGFR-specific antibodies. Shown are representative results from n = 3 independent experiments. (D) EGFR ex and EpEX were incubated in the presence or absence of cross-linker (BS3). Where indicated, EGF was added. Monomers, dimers, and EGFR ex /EpEX complexes are marked. Shown are representative results from n = 3 independent experiments. (E, F) FaDu, Cal27, and Kyse30 cells were kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for the indicated time points. Where indicated, cells were additionally treated with Cetuximab (“Cet.”). Phosphorylation of ERK1/2 (E) and AKT (F) was assessed by immunoblotting with specific antibodies. Levels of ERK1/2 and AKT were assessed in parallel. Shown are representative results from n = 3 independent experiments. (G) FaDu and Cal27 cells were kept untreated or were treated with EGF (9 nM), Fc, or EpEX-Fc (10 nM) for 30 min, and phosphorylation of ERK1/2 and AKT was detected by immunofluorescence laser scanning confocal microscopy (ERK1/2 or AKT: green, nuclei: blue [DAPI]). Shown are representative results from n = 3 independent experiments. (H) FaDu, Cal27, and Kyse30 cells were kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for the indicated time points (“EGF 30 min”). Where indicated, MEK1 inhibitor AZD6244 or EGFR inhibitor AG1478 were added. Levels of ERK1/2 and AKT were assessed in parallel. Shown are representative results from n = 3 independent experiments. (I) HEK293 cells were transiently transfected with GFP or EGFR expression plasmids and were either kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for 30 min. Expression of EGFR and activation of ERK1/2 were assessed by immunoblotting. Levels of ERK1/2 were assessed in parallel. Shown are representative results from n = 3 independent experiments. (J) Expression of EpCAM and EGFR was assessed in HCT8WT and CRISPR-Cas9 EpCAM K.O.1 [ 48 ] by immunoblotting. Levels of actin were assessed in parallel. Shown are representative results from n = 3 independent experiments. (K) HCT8WT and CRISPR-Cas9 EpCAM K.O.1 cells were either kept untreated (control) or were treated with EpEX-Fc, Fc (10 nM), or EGF (1.8 nM) for 30 min. Activation of ERK1/2 was assessed by immunoblotting. Levels of ERK1/2 were assessed in parallel. Shown are representative results from n = 3 independent experiments. BS3, bisulfosuccinimidyl suberate; CRISPR-Cas9, clustered regularly interspaced short palindromic repeat/CRISPR-associated 9; EGF, epidermal growth factor; EGFR, EGF receptor; EGFR ex , extracellular domain of EGFR; EpCAM, epithelial cell adhesion molecule; EpCAM K.O.1, EPCAM -knockout clone 1; EpEX, extracellular domain of EpCAM; EpICD, intracellular domain of EpCAM; ERK1/2, extracellular signal–regulated kinase 1/2; Fc, fragment crystallizable region; GFP, green fluorescent protein; HC, heavy chain; HCT8WT, HCT8 wild type; HEK293, human embryonic kidney 293; IgG, immunoglobulin G; MW, molecular mass; pAKT, phosphorylated AKT; pERK, phosphorylated ERK; SN, supernatant.

    Techniques Used: Immunoprecipitation, Incubation, SDS Page, Immunofluorescence, Confocal Microscopy, Transfection, Expressing, Activation Assay, CRISPR, Knock-Out

    47) Product Images from "Curcumin suppresses the progression of laryngeal squamous cell carcinoma through the upregulation of miR-145 and inhibition of the PI3K/Akt/mTOR pathway"

    Article Title: Curcumin suppresses the progression of laryngeal squamous cell carcinoma through the upregulation of miR-145 and inhibition of the PI3K/Akt/mTOR pathway

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S159236

    Curcumin exacerbated miR-145-induced inhibition of the PI3K/Akt/mTOR pathway in LSCC cells. Notes: TU212 and AMC-HN-8 cells were treated with miR-145, miR-con, miR-145 + 20 μM curcumin, anti-miR-145, anti-miR-con or anti-miR-145 + 20 μM curcumin. Western blot analysis was conducted to evaluate the protein levels of phosphoinositol 1,3 kinase (PI3K), protein kinase B (Akt), p-Akt, mammalian target of rapamycin (mTOR) and p-mTOR in treated AMC-HN-8 ( A ) and TU212 cells ( B ). *.
    Figure Legend Snippet: Curcumin exacerbated miR-145-induced inhibition of the PI3K/Akt/mTOR pathway in LSCC cells. Notes: TU212 and AMC-HN-8 cells were treated with miR-145, miR-con, miR-145 + 20 μM curcumin, anti-miR-145, anti-miR-con or anti-miR-145 + 20 μM curcumin. Western blot analysis was conducted to evaluate the protein levels of phosphoinositol 1,3 kinase (PI3K), protein kinase B (Akt), p-Akt, mammalian target of rapamycin (mTOR) and p-mTOR in treated AMC-HN-8 ( A ) and TU212 cells ( B ). *.

    Techniques Used: Inhibition, Western Blot

    48) Product Images from "Biocompatible, Purified VEGF-A mRNA Improves Cardiac Function after Intracardiac Injection 1 Week Post-myocardial Infarction in Swine"

    Article Title: Biocompatible, Purified VEGF-A mRNA Improves Cardiac Function after Intracardiac Injection 1 Week Post-myocardial Infarction in Swine

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2018.04.003

    Optimized VEGF-A mRNA Produces Functional Protein In Vitro (A) Purified mRNA with 1-methylpseudouridine (m1Ψ) produces higher levels of luciferase protein when compared to first generation (pseudouridine [Ψ]) in 20,000 HeLa cells with 200 ng of mRNA and (B) less innate immune reactivity measured as interferon-alpha secretion in 500,000 primary human PBMC cells after transfection with 500 ng of mRNA. (C) Time course of VEGF-A protein production in human aortic smooth muscle cells (hAoSMC) and in human iPS-derived cardiomyocytes (hiPS-CM) after transfection with purified VEGF mRNA, showing that relevant primary cardiac cell types can be transfected. VEGF protein transcribed from VEGF mRNA is biologically active and activates VEGFR2 and downstream signaling pathways, shown by phosphorylation of (D) VEGF-Receptor 2 in human endothelial cells (HUVECs), (E) endothelial nitric oxide synthase in HUVECs, and (F) AKT in mouse cardiac fibroblasts cells in culture (representative western blot images shown; n = 2). (G) VEGF-A protein produced from VEGF mRNA and recombinant VEGF-A (recVEGF-A) induce similar responses and increase endothelial cell proliferation (n = 3). (H) Endothelial cell migration (n = 2; VEGF-A dose: 10 ng/mL) and (I) angiogenic sprouting in endothelial cells (n = 3; VEGF-A dose: 10 ng/mL) compared to controls. (J) Representative images on angiogenic sprouting in control cells and cells stimulated with VEGF-A protein produced from VEGF mRNA. Data are presented as mean ± SEM. Control is medium only without VEGF-A protein. Asterisks represent: *p
    Figure Legend Snippet: Optimized VEGF-A mRNA Produces Functional Protein In Vitro (A) Purified mRNA with 1-methylpseudouridine (m1Ψ) produces higher levels of luciferase protein when compared to first generation (pseudouridine [Ψ]) in 20,000 HeLa cells with 200 ng of mRNA and (B) less innate immune reactivity measured as interferon-alpha secretion in 500,000 primary human PBMC cells after transfection with 500 ng of mRNA. (C) Time course of VEGF-A protein production in human aortic smooth muscle cells (hAoSMC) and in human iPS-derived cardiomyocytes (hiPS-CM) after transfection with purified VEGF mRNA, showing that relevant primary cardiac cell types can be transfected. VEGF protein transcribed from VEGF mRNA is biologically active and activates VEGFR2 and downstream signaling pathways, shown by phosphorylation of (D) VEGF-Receptor 2 in human endothelial cells (HUVECs), (E) endothelial nitric oxide synthase in HUVECs, and (F) AKT in mouse cardiac fibroblasts cells in culture (representative western blot images shown; n = 2). (G) VEGF-A protein produced from VEGF mRNA and recombinant VEGF-A (recVEGF-A) induce similar responses and increase endothelial cell proliferation (n = 3). (H) Endothelial cell migration (n = 2; VEGF-A dose: 10 ng/mL) and (I) angiogenic sprouting in endothelial cells (n = 3; VEGF-A dose: 10 ng/mL) compared to controls. (J) Representative images on angiogenic sprouting in control cells and cells stimulated with VEGF-A protein produced from VEGF mRNA. Data are presented as mean ± SEM. Control is medium only without VEGF-A protein. Asterisks represent: *p

    Techniques Used: Functional Assay, In Vitro, Purification, Luciferase, Transfection, Derivative Assay, Western Blot, Produced, Recombinant, Migration

    49) Product Images from "Alamandine attenuates hypertension and cardiac hypertrophy in hypertensive rats"

    Article Title: Alamandine attenuates hypertension and cardiac hypertrophy in hypertensive rats

    Journal: Amino Acids

    doi: 10.1007/s00726-018-2583-x

    Interaction of alamandine with the protein kinase A (PKA) signaling pathway in spontaneously hypertensive rats (SHRs). PKA and phosphorylated protein kinase B (p-Akt) levels were higher in the hearts of SHRs than in those of Wistar–Kyoto (WKY) rats, and alamandine decreased the expression of PKA but not p-Akt in SHRs. * p
    Figure Legend Snippet: Interaction of alamandine with the protein kinase A (PKA) signaling pathway in spontaneously hypertensive rats (SHRs). PKA and phosphorylated protein kinase B (p-Akt) levels were higher in the hearts of SHRs than in those of Wistar–Kyoto (WKY) rats, and alamandine decreased the expression of PKA but not p-Akt in SHRs. * p

    Techniques Used: Expressing

    50) Product Images from "Wogonin Attenuates Isoprenaline-Induced Myocardial Hypertrophy in Mice by Suppressing the PI3K/Akt Pathway"

    Article Title: Wogonin Attenuates Isoprenaline-Induced Myocardial Hypertrophy in Mice by Suppressing the PI3K/Akt Pathway

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.00896

    Wogonin inhibits the Akt signaling pathway initiated by isoprenaline treatment. H9c2 cells were cultured in DMEM containing 1% FBS and 1 μM RA for 5-day differentiation. (A) Differentiated H9c2 cells were treated with isoprenaline (10 μM) and/or wogonin (10 μM) with indicated dose for 24 h. Cell extracts were blotted by antibodies against pAkt, Akt, pCREB, CREB, pJNK, JNK, pERK1/2, ERK1/2, pP38 and P38. (B) Quantitation of phosphoprotein to total protein ( n = 3). (C) Differentiated H9c2 cells, pretreated with wogonin (10 μM) for 15 min, then were incubated with DBcAMP (10 μM) for another 15 min. Cell extracts were blotted by antibodies against p-CREB and CREB. (D) Quantitation of p-CREB to CREB ( n = 3). (E) Differentiated H9c2 cells were treated with wogonin (10 μM) and PDBU (5 μM) for 24 h. The mRNA levels of ANP and BNP were determined by RT-qPCR ( n = 5). Data are given as mean ± SEM; ∗ p
    Figure Legend Snippet: Wogonin inhibits the Akt signaling pathway initiated by isoprenaline treatment. H9c2 cells were cultured in DMEM containing 1% FBS and 1 μM RA for 5-day differentiation. (A) Differentiated H9c2 cells were treated with isoprenaline (10 μM) and/or wogonin (10 μM) with indicated dose for 24 h. Cell extracts were blotted by antibodies against pAkt, Akt, pCREB, CREB, pJNK, JNK, pERK1/2, ERK1/2, pP38 and P38. (B) Quantitation of phosphoprotein to total protein ( n = 3). (C) Differentiated H9c2 cells, pretreated with wogonin (10 μM) for 15 min, then were incubated with DBcAMP (10 μM) for another 15 min. Cell extracts were blotted by antibodies against p-CREB and CREB. (D) Quantitation of p-CREB to CREB ( n = 3). (E) Differentiated H9c2 cells were treated with wogonin (10 μM) and PDBU (5 μM) for 24 h. The mRNA levels of ANP and BNP were determined by RT-qPCR ( n = 5). Data are given as mean ± SEM; ∗ p

    Techniques Used: Cell Culture, Quantitation Assay, Incubation, Aqueous Normal-phase Chromatography, Quantitative RT-PCR

    51) Product Images from "Acquired resistance to BRAFi reverses senescence-like phenotype in mutant BRAF melanoma"

    Article Title: Acquired resistance to BRAFi reverses senescence-like phenotype in mutant BRAF melanoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25879

    Drug addiction/dependency phenomenon in acquired resistance cells to vemurafenib ( A ) Western blots illustrating the evaluation of pERK, ERK, pAKT, AKT, PTEN, cyclin D1, pRB, p53, p21 and V600E BRAF (VE1) in parental sensitive line (MM074) and in line with acquired resistance (MM074-R) line treated with 2 µM vemurafenib (vemu) or after 14 days of washing out vemurafenib (w/o). β-actin is used as loading control. ( B ) Growth curves for tumors grafted in mice with parental and resistant cells untreated as control and ( C ) treated with vemurafenib (45 mg/kg). Data are presented as means tumor volumes (mm 3 ) ± SEM. ( D ) Senescence and proliferation markers in xenograft tumors. Tumors were analyzed for their histological appearance of from hematoxylin and eosin staining, the activity of β-Gal in frozen tissues, and the expression of the proliferation marker Ki67 in paraffin-embedded tissues. Positive cells were counted for each group and means ± SEM.
    Figure Legend Snippet: Drug addiction/dependency phenomenon in acquired resistance cells to vemurafenib ( A ) Western blots illustrating the evaluation of pERK, ERK, pAKT, AKT, PTEN, cyclin D1, pRB, p53, p21 and V600E BRAF (VE1) in parental sensitive line (MM074) and in line with acquired resistance (MM074-R) line treated with 2 µM vemurafenib (vemu) or after 14 days of washing out vemurafenib (w/o). β-actin is used as loading control. ( B ) Growth curves for tumors grafted in mice with parental and resistant cells untreated as control and ( C ) treated with vemurafenib (45 mg/kg). Data are presented as means tumor volumes (mm 3 ) ± SEM. ( D ) Senescence and proliferation markers in xenograft tumors. Tumors were analyzed for their histological appearance of from hematoxylin and eosin staining, the activity of β-Gal in frozen tissues, and the expression of the proliferation marker Ki67 in paraffin-embedded tissues. Positive cells were counted for each group and means ± SEM.

    Techniques Used: Western Blot, Mouse Assay, Staining, Activity Assay, Expressing, Marker

    52) Product Images from "Alpha-lipoic acid preserves skeletal muscle mass in type 2 diabetic OLETF rats"

    Article Title: Alpha-lipoic acid preserves skeletal muscle mass in type 2 diabetic OLETF rats

    Journal: Nutrition & Metabolism

    doi: 10.1186/s12986-018-0302-y

    ALA was upregulated protein expression levels of AKT and p70S6K in the skeletal muscles of OLETF rats. The phosphorylation of AKT, FOXO1, mTOR, p70S6K and AKT, FOXO1, mTOR, p706SK expression levels in rat skeletal muscle were determined after 8 weeks of ALA treatment using a western blot analyses, b scanning densitometry. Data presented as mean ± SEM ( n = 6 rats per group)
    Figure Legend Snippet: ALA was upregulated protein expression levels of AKT and p70S6K in the skeletal muscles of OLETF rats. The phosphorylation of AKT, FOXO1, mTOR, p70S6K and AKT, FOXO1, mTOR, p706SK expression levels in rat skeletal muscle were determined after 8 weeks of ALA treatment using a western blot analyses, b scanning densitometry. Data presented as mean ± SEM ( n = 6 rats per group)

    Techniques Used: Expressing, Western Blot

    53) Product Images from "In wound repair vimentin mediates the transition of mesenchymal leader cells to a myofibroblast phenotype"

    Article Title: In wound repair vimentin mediates the transition of mesenchymal leader cells to a myofibroblast phenotype

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-06-0364

    Extracellular vimentin is released in response to injury, associated with the cell surface of leader cells and localized along the ECM substrate beneath the cells. (A) Conditioned media was collected from ex vivo MCS cultures at 1 d postinjury and immunoblotted for vimentin. The results show that an extracellular form of vimentin is released in the media. No vimentin is present in culture media prior to exposure to the cells. (B) Cell surface proteins in the ECZ were biotinylated at culture D2 and fixed and the biotin label was tagged with fluorescent-conjugated streptavidin. The results showed that biotin labeling is restricted to the cell surface of cells in the ECZ. (C) Cell surface proteins in the ECZ were biotinylated at culture D2, extracted, and immunoblotted for vimentin, N-cadherin (positive control), and GAPDH and Akt (negative controls). The results demonstrated that extracellular vimentin is associated with the surface of cells in the ECZ. (D–H) Paraformaldehyde fixed but not permeabilized ex vivo wounded cultures were immunolabeled for vimentin to examine cell type specificity for extracellular-surface–associated vimentin at both the MCS wound edge (D, arrow) and the leading edge of the ECZ (D, arrowhead). Cell-surface vimentin localized exclusively to leader cells of both the native BM capsule (E) and in the ECZ (F). Boxed region in F is shown at higher magnification along apicolateral (G) and basal (H) aspects of the cells. While extracellular vimentin was most prominent on cellular protrusions at the apicolateral surface of the leader cells in the ECZ, the extracellular vimentin pool localized all along the cells’ basal surfaces of these cells where they contact the extracellular matrix and to basal membrane extensions between cells. (I) To determine whether basal extracellular vimentin also was associated with the substrate beneath the cells, cell-free extracts of substrate-associated proteins were examined by Western blot. Extracts of isolated ex vivo explants were included as a control (WCL). Results show that extracellular vimentin is associated with the proteins on the substrate. Immunoblotting for GAPDH confirmed that extracellular lysates were largely cell free. Results are representative of three independent experiments. (J–L) To directly view the substrate-linked extracellular vimentin, extracellular proteins in the ECZ were cross-linked with the membrane-impermeable BS 3 cross-linker, cells extracted with RIPA, and the cross-linked proteins fixed and labeled for tenascin-C (TN-C; J), vimentin (K), and F-actin (L). The results show that extracellular vimentin was associated with the extracellular matrix substrate organized beneath the leader cells of the ECZ (J, K). No staining for F-actin was detected in the cross-linked/extracted cultures (L) confirming effective removal of the cells. Mag bars B, E = 20 µm; F = 50 µm; G, H, J–L = 10 µm. Images in B, E, F, G, and H are presented as single optical sections. Images in J–L are presented as projections.
    Figure Legend Snippet: Extracellular vimentin is released in response to injury, associated with the cell surface of leader cells and localized along the ECM substrate beneath the cells. (A) Conditioned media was collected from ex vivo MCS cultures at 1 d postinjury and immunoblotted for vimentin. The results show that an extracellular form of vimentin is released in the media. No vimentin is present in culture media prior to exposure to the cells. (B) Cell surface proteins in the ECZ were biotinylated at culture D2 and fixed and the biotin label was tagged with fluorescent-conjugated streptavidin. The results showed that biotin labeling is restricted to the cell surface of cells in the ECZ. (C) Cell surface proteins in the ECZ were biotinylated at culture D2, extracted, and immunoblotted for vimentin, N-cadherin (positive control), and GAPDH and Akt (negative controls). The results demonstrated that extracellular vimentin is associated with the surface of cells in the ECZ. (D–H) Paraformaldehyde fixed but not permeabilized ex vivo wounded cultures were immunolabeled for vimentin to examine cell type specificity for extracellular-surface–associated vimentin at both the MCS wound edge (D, arrow) and the leading edge of the ECZ (D, arrowhead). Cell-surface vimentin localized exclusively to leader cells of both the native BM capsule (E) and in the ECZ (F). Boxed region in F is shown at higher magnification along apicolateral (G) and basal (H) aspects of the cells. While extracellular vimentin was most prominent on cellular protrusions at the apicolateral surface of the leader cells in the ECZ, the extracellular vimentin pool localized all along the cells’ basal surfaces of these cells where they contact the extracellular matrix and to basal membrane extensions between cells. (I) To determine whether basal extracellular vimentin also was associated with the substrate beneath the cells, cell-free extracts of substrate-associated proteins were examined by Western blot. Extracts of isolated ex vivo explants were included as a control (WCL). Results show that extracellular vimentin is associated with the proteins on the substrate. Immunoblotting for GAPDH confirmed that extracellular lysates were largely cell free. Results are representative of three independent experiments. (J–L) To directly view the substrate-linked extracellular vimentin, extracellular proteins in the ECZ were cross-linked with the membrane-impermeable BS 3 cross-linker, cells extracted with RIPA, and the cross-linked proteins fixed and labeled for tenascin-C (TN-C; J), vimentin (K), and F-actin (L). The results show that extracellular vimentin was associated with the extracellular matrix substrate organized beneath the leader cells of the ECZ (J, K). No staining for F-actin was detected in the cross-linked/extracted cultures (L) confirming effective removal of the cells. Mag bars B, E = 20 µm; F = 50 µm; G, H, J–L = 10 µm. Images in B, E, F, G, and H are presented as single optical sections. Images in J–L are presented as projections.

    Techniques Used: Ex Vivo, Labeling, Positive Control, Immunolabeling, Western Blot, Isolation, Staining

    54) Product Images from "Meisoindigo, but not its core chemical structure indirubin, inhibits zebrafish interstitial leukocyte chemotactic migration"

    Article Title: Meisoindigo, but not its core chemical structure indirubin, inhibits zebrafish interstitial leukocyte chemotactic migration

    Journal: Pharmaceutical Biology

    doi: 10.1080/13880209.2016.1238949

    Indirubin, the core chemical structure of meisoindigo, did not inhibit leukocyte chemotactic migration, and meisoindigo does not regulate pAKT/AKT or pERK/ERK level. (a) Chemical structure of indirubin (I) and meisoindigo (M). Meisoindigo is a synthetic derivative of indirubin with a methyl substitution at N element. (b,c) Tg:zlyz-EGFP embryos (3dpf) with tail transection were treated with vehicle ( n = 15), indirubin (I, 100 μM, n = 14) or meisoindigo (M, 100 μM, n = 11), respectively. Leukocytes that migrated to the highly inflamed regions [white boxes in (b)] at 6 hptt were quantitatively analyzed (c). The p values were annotated as obtained from a two-tailed t test. (d) Westernblot analysis showing the effect of meisoindigo on the selected signaling pathway components. 20 zebrafish embryos (3dpf, n = 20) with tail transection were treated with vehicle, meisoindigo or indirubin at the indicated concentration from 0 hppt to 6 hptt, respectively. Then, the lysate of whole zebrafish embryos was subjected to westernblot analysis for detecting pAKT/AKT or pERK/ERK level.
    Figure Legend Snippet: Indirubin, the core chemical structure of meisoindigo, did not inhibit leukocyte chemotactic migration, and meisoindigo does not regulate pAKT/AKT or pERK/ERK level. (a) Chemical structure of indirubin (I) and meisoindigo (M). Meisoindigo is a synthetic derivative of indirubin with a methyl substitution at N element. (b,c) Tg:zlyz-EGFP embryos (3dpf) with tail transection were treated with vehicle ( n = 15), indirubin (I, 100 μM, n = 14) or meisoindigo (M, 100 μM, n = 11), respectively. Leukocytes that migrated to the highly inflamed regions [white boxes in (b)] at 6 hptt were quantitatively analyzed (c). The p values were annotated as obtained from a two-tailed t test. (d) Westernblot analysis showing the effect of meisoindigo on the selected signaling pathway components. 20 zebrafish embryos (3dpf, n = 20) with tail transection were treated with vehicle, meisoindigo or indirubin at the indicated concentration from 0 hppt to 6 hptt, respectively. Then, the lysate of whole zebrafish embryos was subjected to westernblot analysis for detecting pAKT/AKT or pERK/ERK level.

    Techniques Used: Migration, Two Tailed Test, Concentration Assay

    55) Product Images from "An Osteoblast-Derived Proteinase Controls Tumor Cell Survival via TGF-beta Activation in the Bone Microenvironment"

    Article Title: An Osteoblast-Derived Proteinase Controls Tumor Cell Survival via TGF-beta Activation in the Bone Microenvironment

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029862

    Osteoblast-derived MMP-2 impacts TGFβ activation and tumor survival in the in vivo tumor-bone microenvironment. A , The levels of TGFβ in normalized tumor-bone lysates derived from WT and MMP-2 −/− tumor or sham injected tibias was assessed by ELISA. B , Representative immunoblots for phospho-SMAD (pSMAD2), total smad2 (SMAD2), phospho-AKT (pAKT), total AKT (AKT) and actin (loading control) in the tumor-bone lysates derived from WT and MMP-2 −/− mice. Densitometry on immunoblots generated from tumor bone lysates of at least 5 animals per group was used to generate graphs of the ratio of pSMAD2/SMAD2 and pAKT/AKT. Data are mean ± SD. *, p
    Figure Legend Snippet: Osteoblast-derived MMP-2 impacts TGFβ activation and tumor survival in the in vivo tumor-bone microenvironment. A , The levels of TGFβ in normalized tumor-bone lysates derived from WT and MMP-2 −/− tumor or sham injected tibias was assessed by ELISA. B , Representative immunoblots for phospho-SMAD (pSMAD2), total smad2 (SMAD2), phospho-AKT (pAKT), total AKT (AKT) and actin (loading control) in the tumor-bone lysates derived from WT and MMP-2 −/− mice. Densitometry on immunoblots generated from tumor bone lysates of at least 5 animals per group was used to generate graphs of the ratio of pSMAD2/SMAD2 and pAKT/AKT. Data are mean ± SD. *, p

    Techniques Used: Derivative Assay, Activation Assay, In Vivo, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Mouse Assay, Generated

    56) Product Images from "Intravenous injection of neural progenitor cells improved depression-like behavior after cerebral ischemia"

    Article Title: Intravenous injection of neural progenitor cells improved depression-like behavior after cerebral ischemia

    Journal: Translational Psychiatry

    doi: 10.1038/tp.2011.32

    Effect of neural progenitor cell (NPC) injection on the levels of brain-derived neurotrophic factor (BDNF), phosphorylated CREB, TrkB, phosphorylated ERK and phosphorylated AKT on Day 28 after the embolism. Samples obtained from sham-operated (sham), vehicle-injected ischemic (vehicle) and NPC-injected (NPC) ischemic rats (ME) were solubilized and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bands corresponding to BDNF ( a ), phospho-CREB ( b ), and TrkB ( c ), phosphorylated ERK ( d ) and phosphorylated Akt ( e ) were scanned, and scanned bands were normalized by actin ( a and c ), total CREB ( b ), total ERK ( d ) or total AKT ( e ) on the same blot. All results are presented as mean percentages of non-operated naïve rats±s.e.m. (BDNF and phospho-CREB: n =9 rats per group; TrkB: n =5–6 rats per group; and phospho-ERK and -AKT: n =6 rats per group). * Statistically significant difference from sham-operated rats ( P
    Figure Legend Snippet: Effect of neural progenitor cell (NPC) injection on the levels of brain-derived neurotrophic factor (BDNF), phosphorylated CREB, TrkB, phosphorylated ERK and phosphorylated AKT on Day 28 after the embolism. Samples obtained from sham-operated (sham), vehicle-injected ischemic (vehicle) and NPC-injected (NPC) ischemic rats (ME) were solubilized and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bands corresponding to BDNF ( a ), phospho-CREB ( b ), and TrkB ( c ), phosphorylated ERK ( d ) and phosphorylated Akt ( e ) were scanned, and scanned bands were normalized by actin ( a and c ), total CREB ( b ), total ERK ( d ) or total AKT ( e ) on the same blot. All results are presented as mean percentages of non-operated naïve rats±s.e.m. (BDNF and phospho-CREB: n =9 rats per group; TrkB: n =5–6 rats per group; and phospho-ERK and -AKT: n =6 rats per group). * Statistically significant difference from sham-operated rats ( P

    Techniques Used: Injection, Derivative Assay, Polyacrylamide Gel Electrophoresis, SDS Page

    57) Product Images from "MicroRNA-20b and ERK1/2 pathway independently regulate the expression of tissue factor in hematopoietic and trophoblastic differentiation of human embryonic stem cells"

    Article Title: MicroRNA-20b and ERK1/2 pathway independently regulate the expression of tissue factor in hematopoietic and trophoblastic differentiation of human embryonic stem cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt332

    Erk1/2 signaling pathway involved in regulating tissue factor expression in trophoblastic and hematopoietic differentiation of human embryonic stem cells. (A) Western blot analysis of phosphorylated Erk1/2 and Akt in various types of cells showing that Erk1/2 signaling pathway is active in granulocyte–macrophage (G-M) cells and trophoblasts. p-Erk1/2, phosphorylated Erk1/2; t-Erk1/2, total Erk1/2; pAkt, phosphorylated Akt; t-Akt, total Akt. (B),(C) Decreased (B) mRNA and (C) protein levels of tissue factor (TF) in G-M cells or trophoblasts treated with Erk1/2-specific inhibitor, U0126. Cells were treated with 10 μM U0126 or dimethylsulfoxide (control) for 4-6 or 7-9 days before harvest for quantitative real-time polymerase chain reaction for CDX2, PU.1, and TF mRNA levels. Data were reported as the mean ± standard error of the percentage of the mRNA levels of CDX2, PU.1, and TF in cells from the control group. * P
    Figure Legend Snippet: Erk1/2 signaling pathway involved in regulating tissue factor expression in trophoblastic and hematopoietic differentiation of human embryonic stem cells. (A) Western blot analysis of phosphorylated Erk1/2 and Akt in various types of cells showing that Erk1/2 signaling pathway is active in granulocyte–macrophage (G-M) cells and trophoblasts. p-Erk1/2, phosphorylated Erk1/2; t-Erk1/2, total Erk1/2; pAkt, phosphorylated Akt; t-Akt, total Akt. (B),(C) Decreased (B) mRNA and (C) protein levels of tissue factor (TF) in G-M cells or trophoblasts treated with Erk1/2-specific inhibitor, U0126. Cells were treated with 10 μM U0126 or dimethylsulfoxide (control) for 4-6 or 7-9 days before harvest for quantitative real-time polymerase chain reaction for CDX2, PU.1, and TF mRNA levels. Data were reported as the mean ± standard error of the percentage of the mRNA levels of CDX2, PU.1, and TF in cells from the control group. * P

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction

    58) Product Images from "Vascular endothelial cells facilitated HCC invasion and metastasis through the Akt and NF-?B pathways induced by paracrine cytokines"

    Article Title: Vascular endothelial cells facilitated HCC invasion and metastasis through the Akt and NF-?B pathways induced by paracrine cytokines

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-32-51

    Effects of CM on PI3K/Akt and ERK pathway activation in HCC cells. (A) Expression of p-Akt and p-ERK in MHCC97H cells under CM or EBM stimulation were detected by Western blot. (B) Expression of p-Akt and p-ERK in subcutaneous tumors derived from a mixture of MHCC97H cells and HUVECs were analyzed by immunohistochemistry.
    Figure Legend Snippet: Effects of CM on PI3K/Akt and ERK pathway activation in HCC cells. (A) Expression of p-Akt and p-ERK in MHCC97H cells under CM or EBM stimulation were detected by Western blot. (B) Expression of p-Akt and p-ERK in subcutaneous tumors derived from a mixture of MHCC97H cells and HUVECs were analyzed by immunohistochemistry.

    Techniques Used: Activation Assay, Expressing, Western Blot, Derivative Assay, Immunohistochemistry

    Effects of CCL2, IL-8, and CXCL16 on the activation of the Akt, ERK, and NF-κB pathways in HCC cells. The levels of phosphorylated Akt and ERK in MHCC97H (A) Cells after exposure to CCL2, IL-8, or CXCL16 at different concentrations. (B) Activation of NF-κB in MHCC97H cells were measured using a specific TransAM NF-κB p65 kit under CCL2, IL-8, or CXCL16 stimulation (* P
    Figure Legend Snippet: Effects of CCL2, IL-8, and CXCL16 on the activation of the Akt, ERK, and NF-κB pathways in HCC cells. The levels of phosphorylated Akt and ERK in MHCC97H (A) Cells after exposure to CCL2, IL-8, or CXCL16 at different concentrations. (B) Activation of NF-κB in MHCC97H cells were measured using a specific TransAM NF-κB p65 kit under CCL2, IL-8, or CXCL16 stimulation (* P

    Techniques Used: Activation Assay

    59) Product Images from "Syntenin increases the invasiveness of small cell lung cancer cells by activating p38, AKT, focal adhesion kinase and SP1"

    Article Title: Syntenin increases the invasiveness of small cell lung cancer cells by activating p38, AKT, focal adhesion kinase and SP1

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2014.1

    Activation of p38, AKT and focal adhesion kinase by forced expression of syntenin in small cell lung cancer cells. ( a ) Western blot analysis revealed enhanced phosphorylation of p38 MAPK and AKT after syntenin overexpression in NCI-H187 cells, compared with empty-vector transfection. In contrast, phosphorylated AKT and p38 MAPK were shown to be decreased by syntenin inhibition in NCI-H69 cells. Activation of focal adhesion kinase (FAK) by syntenin overexpression and suppression of FAK by syntenin inhibition was also observed without stimulation of any extracellular matrix proteins. ( b ) Western blot analysis after administration of SB203580 (10 μ M ) revealed the inhibition of p38 activation in syntenin-transfected cells compared with the p38 level in empty vector-transfected cells, although the basal level itself was upregulated, due to the role of SB203580 as not only an inhibitor but also as a partial agonist (left). In RT–PCR analysis, the inhibition of p38 MAPK induced the inhibition of both MMP2 and MT1-MMP mRNA (right). ( c ) The administration of LY294002 (1 μ M ) suppressed AKT activation in syntenin-transfected cells (left), which led to the downregulation of MMP2 and MT1-MMP, despite syntenin overexpression (right). ( d ) The activation of FAK was inhibited by administration of PF-573228 (1 μ M ) (left), which antagonized the syntenin-mediated upregulation of MMP2 and MT1-MMP (right). ( e ) When FAK, P38 and PI3K/AKT were inhibited by pharmacological inhibitors in NCI-H187 cells, the number of invaded cells in the Matrigel invasion assay did not increase in syntenin-transfected cells. ( f ) In NCI-H69 cells, the number of invaded cells decreased after inhibition of p38 and PI3K/AKT. The invasiveness of NCI-H69 cells was suppressed by syntenin inhibition. MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MT1, membrane type 1.
    Figure Legend Snippet: Activation of p38, AKT and focal adhesion kinase by forced expression of syntenin in small cell lung cancer cells. ( a ) Western blot analysis revealed enhanced phosphorylation of p38 MAPK and AKT after syntenin overexpression in NCI-H187 cells, compared with empty-vector transfection. In contrast, phosphorylated AKT and p38 MAPK were shown to be decreased by syntenin inhibition in NCI-H69 cells. Activation of focal adhesion kinase (FAK) by syntenin overexpression and suppression of FAK by syntenin inhibition was also observed without stimulation of any extracellular matrix proteins. ( b ) Western blot analysis after administration of SB203580 (10 μ M ) revealed the inhibition of p38 activation in syntenin-transfected cells compared with the p38 level in empty vector-transfected cells, although the basal level itself was upregulated, due to the role of SB203580 as not only an inhibitor but also as a partial agonist (left). In RT–PCR analysis, the inhibition of p38 MAPK induced the inhibition of both MMP2 and MT1-MMP mRNA (right). ( c ) The administration of LY294002 (1 μ M ) suppressed AKT activation in syntenin-transfected cells (left), which led to the downregulation of MMP2 and MT1-MMP, despite syntenin overexpression (right). ( d ) The activation of FAK was inhibited by administration of PF-573228 (1 μ M ) (left), which antagonized the syntenin-mediated upregulation of MMP2 and MT1-MMP (right). ( e ) When FAK, P38 and PI3K/AKT were inhibited by pharmacological inhibitors in NCI-H187 cells, the number of invaded cells in the Matrigel invasion assay did not increase in syntenin-transfected cells. ( f ) In NCI-H69 cells, the number of invaded cells decreased after inhibition of p38 and PI3K/AKT. The invasiveness of NCI-H69 cells was suppressed by syntenin inhibition. MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MT1, membrane type 1.

    Techniques Used: Activation Assay, Expressing, Western Blot, Over Expression, Plasmid Preparation, Transfection, Inhibition, Reverse Transcription Polymerase Chain Reaction, Invasion Assay

    Schematic model of signaling pathways activated by syntenin activation in small cell lung cancer cells. Without extracellular matrix interactions, focal adhesion kinase (FAK) is phosphorylated by syntenin activation. The activated signal is then transduced via p38 MAPK and AKT phosphorylation. Through action of the gene-regulating element SP1, signal transduction progressed from the cytoplasm to nucleus. Finally, expression of MT1-MMP and MMP2 was induced, and these products then enhanced cell motility, invasion and tumor growth. MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MT1, membrane type 1.
    Figure Legend Snippet: Schematic model of signaling pathways activated by syntenin activation in small cell lung cancer cells. Without extracellular matrix interactions, focal adhesion kinase (FAK) is phosphorylated by syntenin activation. The activated signal is then transduced via p38 MAPK and AKT phosphorylation. Through action of the gene-regulating element SP1, signal transduction progressed from the cytoplasm to nucleus. Finally, expression of MT1-MMP and MMP2 was induced, and these products then enhanced cell motility, invasion and tumor growth. MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MT1, membrane type 1.

    Techniques Used: Activation Assay, Transduction, Expressing

    60) Product Images from "Scavenger receptor class B type I regulates cellular cholesterol metabolism and cell signaling associated with breast cancer development"

    Article Title: Scavenger receptor class B type I regulates cellular cholesterol metabolism and cell signaling associated with breast cancer development

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr3483

    Inhibition of PI3K, not MEK1/2, prevents proliferation of MDA-MB-231 cells. (A) LY294002 and U0126 effectively inhibit Akt and Erk1/2 activation in MDA-MB-231 cells. Serum-starved shCTL and shSRBI MDA-MB-231 cells were incubated with or without the inhibitors LY294002 (15 μ M ) or U0126 (10 μ M ) for 2 hours. Medium containing 10% FBS was added for 30 minutes, cells were lysed, and whole-cell lysates were analyzed by Western blot for the indicated proteins. GAPDH was used as a loading control. (B) PI3K inhibition reduces cellular proliferation of shCTL MDA-MB-231 cells. shCTL and shSRBI MDA-MB-231 cells were incubated with culture media containing either LY294002 (15 μ M ) or U0126 (10 μ M ) and 3 H-thymidine for 6 hours, at which time, the assay was stopped, and lysates were collected. Columns represent the mean [ 3 H]thymidine incorporation (cpm/μg protein); bars represent ± SD. Results obtained from vehicle-treated (CTL) shCTL MDA-MB-231 cells are significantly different from those obtained with shSRBI MDA-MB-231 cells (** P
    Figure Legend Snippet: Inhibition of PI3K, not MEK1/2, prevents proliferation of MDA-MB-231 cells. (A) LY294002 and U0126 effectively inhibit Akt and Erk1/2 activation in MDA-MB-231 cells. Serum-starved shCTL and shSRBI MDA-MB-231 cells were incubated with or without the inhibitors LY294002 (15 μ M ) or U0126 (10 μ M ) for 2 hours. Medium containing 10% FBS was added for 30 minutes, cells were lysed, and whole-cell lysates were analyzed by Western blot for the indicated proteins. GAPDH was used as a loading control. (B) PI3K inhibition reduces cellular proliferation of shCTL MDA-MB-231 cells. shCTL and shSRBI MDA-MB-231 cells were incubated with culture media containing either LY294002 (15 μ M ) or U0126 (10 μ M ) and 3 H-thymidine for 6 hours, at which time, the assay was stopped, and lysates were collected. Columns represent the mean [ 3 H]thymidine incorporation (cpm/μg protein); bars represent ± SD. Results obtained from vehicle-treated (CTL) shCTL MDA-MB-231 cells are significantly different from those obtained with shSRBI MDA-MB-231 cells (** P

    Techniques Used: Inhibition, Multiple Displacement Amplification, Activation Assay, Incubation, Western Blot, CTL Assay

    61) Product Images from "AP-2?: a regulator of EGF receptor signaling and proliferation in skin epidermis"

    Article Title: AP-2?: a regulator of EGF receptor signaling and proliferation in skin epidermis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200510002

    In vitro and in vivo superactivation of the PI3K–Akt pathway in an EGF-dependent and AP-2α null–dependent fashion. (A) Primary WT and KO keratinocytes were cultured ± the EGFR-specific protein kinase inhibitor AG1478 without the addition of fibroblast feeder cells after the original plating. Growth curves represents three independent experiments performed in duplicate (see Separation of epidermis…transfections). Error bars represent SD for these experiments. (B) Keratinocytes were serum-starved for 24 h and at t = 0, 50 ng/ml EGF was added. At the times indicated, cell extracts were prepared, proteins were resolved by SDS-PAGE, and immunoblot analyses were conducted with the antibodies indicated at right. The antibodies against the active forms of Erk1/2 and Akt (p-Erk and p-Akt, respectively) are phosphospecific antibodies and do not recognize the inactive states of the kinases. β-tubulin antibodies are used as a loading control, along with ponseau red staining of the blots (not depicted). Note EGF-dependent superactivation of Akt and Erk1/2 in AP-2α –null keratinocytes. (C) Same experiment as in B, except 10× increments of EGF concentrations (0–50 ng/ml) were used, and extracts were harvested at t = 2 min. (D) Same experiment as in B, but insulin growth factor 1 (IGF1) was added at 50 ng/ml. Note that in contrast to EGF, IGF1 did not generate enhanced activated Akt in KO relative to WT cells. (E) Same experiment as in B, but the PI3K-specific inhibitor LY294002 was added at 20 μM. Note that the activation of Akt, but not Erk1/2, is dependent on PI3K activation. (F) Antiphospho-Akt staining of frozen sections (8 μm) from (top) lesional KO and WT thoracic skins and (bottom) TPA-treated KO and WT back skins. Note the superactivation of Akt in KO skin regions that are lesional and that correlate with elevated EGFR ligand expression. De, dermis; epi, epidermis; hf, hair follicle. Dotted lines denote dermo–epidermal borders. Bars, 20 μm.
    Figure Legend Snippet: In vitro and in vivo superactivation of the PI3K–Akt pathway in an EGF-dependent and AP-2α null–dependent fashion. (A) Primary WT and KO keratinocytes were cultured ± the EGFR-specific protein kinase inhibitor AG1478 without the addition of fibroblast feeder cells after the original plating. Growth curves represents three independent experiments performed in duplicate (see Separation of epidermis…transfections). Error bars represent SD for these experiments. (B) Keratinocytes were serum-starved for 24 h and at t = 0, 50 ng/ml EGF was added. At the times indicated, cell extracts were prepared, proteins were resolved by SDS-PAGE, and immunoblot analyses were conducted with the antibodies indicated at right. The antibodies against the active forms of Erk1/2 and Akt (p-Erk and p-Akt, respectively) are phosphospecific antibodies and do not recognize the inactive states of the kinases. β-tubulin antibodies are used as a loading control, along with ponseau red staining of the blots (not depicted). Note EGF-dependent superactivation of Akt and Erk1/2 in AP-2α –null keratinocytes. (C) Same experiment as in B, except 10× increments of EGF concentrations (0–50 ng/ml) were used, and extracts were harvested at t = 2 min. (D) Same experiment as in B, but insulin growth factor 1 (IGF1) was added at 50 ng/ml. Note that in contrast to EGF, IGF1 did not generate enhanced activated Akt in KO relative to WT cells. (E) Same experiment as in B, but the PI3K-specific inhibitor LY294002 was added at 20 μM. Note that the activation of Akt, but not Erk1/2, is dependent on PI3K activation. (F) Antiphospho-Akt staining of frozen sections (8 μm) from (top) lesional KO and WT thoracic skins and (bottom) TPA-treated KO and WT back skins. Note the superactivation of Akt in KO skin regions that are lesional and that correlate with elevated EGFR ligand expression. De, dermis; epi, epidermis; hf, hair follicle. Dotted lines denote dermo–epidermal borders. Bars, 20 μm.

    Techniques Used: In Vitro, In Vivo, Cell Culture, SDS Page, Staining, Activation Assay, Expressing

    62) Product Images from "Reciprocal regulation of IL-23 and IL-12 following co-activation of Dectin-1 and TLR signaling pathways"

    Article Title: Reciprocal regulation of IL-23 and IL-12 following co-activation of Dectin-1 and TLR signaling pathways

    Journal: European Journal of Immunology

    doi: 10.1002/eji.200838543

    Down-regulation of IL-12 is dependent on signaling through Dectin-1. (A) Phosphorylation of Syk and Akt after stimulation of Balb/C BMDC with 10 μg/mL soluble β-glucan and 1 μg/mL Pam 3 CSK 4 , as indicated. Production of IL-10 (B) and IL-12p70 (C) from Balb/C Syk −/− or WT thioglycollate-elicited macrophages pretreated with vehicle, 20 μM U0126 or 20 μM Ly294002, and stimulated with 10 μg/mL β-glucan and 10 ng/mL Pam 3 CSK 4 , as indicated. (D) Production of IL-12p70 from Balb/C WT or 129Sv Dectin-1 −/− BMDC pretreated with vehicle, 20 μM U0126 or 20 μM Ly294002, and stimulated with 10 μg/mL β-glucan and 100 ng/mL Pam 3 CSK 4 , as indicated. (E) Production of IL-12p70 from Balb/C WT or IL-10 −/− BMDC stimulated with 10 μg/mL β-glucan and 100 ng/mL Pam 3 CSK 4 , as indicated. Data shown are mean±SD and are representative of two independent experiments.
    Figure Legend Snippet: Down-regulation of IL-12 is dependent on signaling through Dectin-1. (A) Phosphorylation of Syk and Akt after stimulation of Balb/C BMDC with 10 μg/mL soluble β-glucan and 1 μg/mL Pam 3 CSK 4 , as indicated. Production of IL-10 (B) and IL-12p70 (C) from Balb/C Syk −/− or WT thioglycollate-elicited macrophages pretreated with vehicle, 20 μM U0126 or 20 μM Ly294002, and stimulated with 10 μg/mL β-glucan and 10 ng/mL Pam 3 CSK 4 , as indicated. (D) Production of IL-12p70 from Balb/C WT or 129Sv Dectin-1 −/− BMDC pretreated with vehicle, 20 μM U0126 or 20 μM Ly294002, and stimulated with 10 μg/mL β-glucan and 100 ng/mL Pam 3 CSK 4 , as indicated. (E) Production of IL-12p70 from Balb/C WT or IL-10 −/− BMDC stimulated with 10 μg/mL β-glucan and 100 ng/mL Pam 3 CSK 4 , as indicated. Data shown are mean±SD and are representative of two independent experiments.

    Techniques Used:

    63) Product Images from "Akt regulates the expression of MafK, synaptotagmin I, and syntenin-1, which play roles in neuronal function"

    Article Title: Akt regulates the expression of MafK, synaptotagmin I, and syntenin-1, which play roles in neuronal function

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-17-18

    Analyses of MafK , SytI , and Syn-1 mRNA levels in parental, WT-Akt-expressing, and DN-Akt-expressing PC12 cells . (A) Quantitative RT-PCR. mRNA level of the gene in each group of cells was normalized to the level of GAPDH mRNA and the transcript level of each gene in PC12 (WT-Akt) and PC12 (DN-Akt) cells is presented as a fold change from that of the PC12 (parental) cells. Parental, PC12 (parental) cells. * P
    Figure Legend Snippet: Analyses of MafK , SytI , and Syn-1 mRNA levels in parental, WT-Akt-expressing, and DN-Akt-expressing PC12 cells . (A) Quantitative RT-PCR. mRNA level of the gene in each group of cells was normalized to the level of GAPDH mRNA and the transcript level of each gene in PC12 (WT-Akt) and PC12 (DN-Akt) cells is presented as a fold change from that of the PC12 (parental) cells. Parental, PC12 (parental) cells. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of the Akt inhibitor AKTi-1/2 on mRNA levels for the MafK , SytI , and Syn-1 genes . (A) Western blot analysis of pAkt(Ser-473) protein level in PC12 (WT-Akt) cells treated with various concentrations of AKTi-1/2 prior to 10 min treatment with NGF. (B) The percentage of neuritogenic PC12 (WT-Akt) cells treated with or without AKTi-1/2. Averages and standard deviations are derived from more than six fields of view. * P
    Figure Legend Snippet: Effect of the Akt inhibitor AKTi-1/2 on mRNA levels for the MafK , SytI , and Syn-1 genes . (A) Western blot analysis of pAkt(Ser-473) protein level in PC12 (WT-Akt) cells treated with various concentrations of AKTi-1/2 prior to 10 min treatment with NGF. (B) The percentage of neuritogenic PC12 (WT-Akt) cells treated with or without AKTi-1/2. Averages and standard deviations are derived from more than six fields of view. * P

    Techniques Used: Western Blot, Derivative Assay

    64) Product Images from "EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT"

    Article Title: EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT

    Journal: Scientific Reports

    doi: 10.1038/srep11494

    EGCG regulates the neutrophil elastase-induced migration of A549 cells. EGCG inhibits the activity of neutrophil elastase by directly binding to neutrophil elastase. EGCG enhances the expression of AAT by up-regulating the Akt/PI3K signaling pathway.
    Figure Legend Snippet: EGCG regulates the neutrophil elastase-induced migration of A549 cells. EGCG inhibits the activity of neutrophil elastase by directly binding to neutrophil elastase. EGCG enhances the expression of AAT by up-regulating the Akt/PI3K signaling pathway.

    Techniques Used: Migration, Activity Assay, Binding Assay, Expressing

    EGCG attenuates the neutrophil elastase-induced cell migration via the PI3K/Akt pathway. ( A ) Western blot for AAT, IRS-1, pAkt (phosphorylated at Thy308 or Ser473), Akt, p-PI3K, PI3K and GAPDH in A549 cells treated with EGCG or sivelestat sodium with or without HNE. ( B - F ) The statistical data are presented as histograms of the Western blot results for AAT (B), IRS-1 ( C ), the pAkt (Thy308)/Akt ratio ( D ), the pAkt (Ser473)/Akt ratio ( E ) and the pPI3K/PI3K ratio ( F ) in A549 cells treated with neutrophil elastase, EGCG and sivelestat sodium. The data are presented as the means ± SEM; n = 3. **P
    Figure Legend Snippet: EGCG attenuates the neutrophil elastase-induced cell migration via the PI3K/Akt pathway. ( A ) Western blot for AAT, IRS-1, pAkt (phosphorylated at Thy308 or Ser473), Akt, p-PI3K, PI3K and GAPDH in A549 cells treated with EGCG or sivelestat sodium with or without HNE. ( B - F ) The statistical data are presented as histograms of the Western blot results for AAT (B), IRS-1 ( C ), the pAkt (Thy308)/Akt ratio ( D ), the pAkt (Ser473)/Akt ratio ( E ) and the pPI3K/PI3K ratio ( F ) in A549 cells treated with neutrophil elastase, EGCG and sivelestat sodium. The data are presented as the means ± SEM; n = 3. **P

    Techniques Used: Migration, Western Blot

    65) Product Images from "Hypoxia Promotes Uveal Melanoma Invasion through Enhanced Notch and MAPK Activation"

    Article Title: Hypoxia Promotes Uveal Melanoma Invasion through Enhanced Notch and MAPK Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105372

    Hypoxia activates MAPK and Akt pathways. A, Western blot analysis reveals that exposure to hypoxia for 24 hours activates Erk1-2 and Akt proteins in all the uveal melanoma lines, as found using antibodies specific for phospho-Erk1-2 Thr202/Tyr204 and phospho-Akt Ser473 ; total Erk1-2 and Akt were used as loading controls. B , Transwell invasion assay was performed in 92.1 and OCM1 cell lines treated with the Erk1-2 inhibitor SCH772984 at 500 nM, while exposed to normoxia or hypoxia for 24 hours. The microphotographs of the right panels show the invading cells on the lower surface of a Matrigel-coated filter after 24 hours of incubation in normal (21%) or low (1%) oxygen tension in the presence of the Erk1-2 inhibitor (***p
    Figure Legend Snippet: Hypoxia activates MAPK and Akt pathways. A, Western blot analysis reveals that exposure to hypoxia for 24 hours activates Erk1-2 and Akt proteins in all the uveal melanoma lines, as found using antibodies specific for phospho-Erk1-2 Thr202/Tyr204 and phospho-Akt Ser473 ; total Erk1-2 and Akt were used as loading controls. B , Transwell invasion assay was performed in 92.1 and OCM1 cell lines treated with the Erk1-2 inhibitor SCH772984 at 500 nM, while exposed to normoxia or hypoxia for 24 hours. The microphotographs of the right panels show the invading cells on the lower surface of a Matrigel-coated filter after 24 hours of incubation in normal (21%) or low (1%) oxygen tension in the presence of the Erk1-2 inhibitor (***p

    Techniques Used: Western Blot, Transwell Invasion Assay, Incubation

    66) Product Images from "Multi-scale, whole-system models of liver metabolic adaptation to fat and sugar in non-alcoholic fatty liver disease"

    Article Title: Multi-scale, whole-system models of liver metabolic adaptation to fat and sugar in non-alcoholic fatty liver disease

    Journal: NPJ Systems Biology and Applications

    doi: 10.1038/s41540-018-0070-3

    Insulin sensitivity and verification of sugar consumption in vitro and in silico. a Immunoblot analyses of pAKT/AKT expression (both ~60 kDa) in HepG2 cells stimulated with insulin ( n = 3–4), analysed by one-way ANOVA with Dunnett’s test post hoc between doses and vehicle. b The change in monosaccharide concentration of culture medium in vitro over the first and second 24 h period after treatments of glucose or fructose with (+) and without (−) 100 nM insulin ( n = 4–5), analysed within timepoints between treatment by one-way ANOVA with Tukey’s test post hoc. c – f The objective function was set as either the glucose or fructose transport flux between the external space (medium) and sinusoid space. Maximisation was the uptake of monosaccharide, and minimisation was the production and export into the medium (external space). c Model predictions of glucose concentration in the medium with (+) or without (−) the presence of 100 nM insulin over time alongside experimental data from HepG2 cells ( n = 3–5). d Predicted glucose transport rate over time. e Predictions of fructose concentration over time alongside experimental data from HepG2 cells ( n = 3–5). f Predicted fructose transport rate over time. Data shown as mean ± SEM. Statistical differences are indicated as * P
    Figure Legend Snippet: Insulin sensitivity and verification of sugar consumption in vitro and in silico. a Immunoblot analyses of pAKT/AKT expression (both ~60 kDa) in HepG2 cells stimulated with insulin ( n = 3–4), analysed by one-way ANOVA with Dunnett’s test post hoc between doses and vehicle. b The change in monosaccharide concentration of culture medium in vitro over the first and second 24 h period after treatments of glucose or fructose with (+) and without (−) 100 nM insulin ( n = 4–5), analysed within timepoints between treatment by one-way ANOVA with Tukey’s test post hoc. c – f The objective function was set as either the glucose or fructose transport flux between the external space (medium) and sinusoid space. Maximisation was the uptake of monosaccharide, and minimisation was the production and export into the medium (external space). c Model predictions of glucose concentration in the medium with (+) or without (−) the presence of 100 nM insulin over time alongside experimental data from HepG2 cells ( n = 3–5). d Predicted glucose transport rate over time. e Predictions of fructose concentration over time alongside experimental data from HepG2 cells ( n = 3–5). f Predicted fructose transport rate over time. Data shown as mean ± SEM. Statistical differences are indicated as * P

    Techniques Used: In Vitro, In Silico, Expressing, Concentration Assay

    67) Product Images from "Oxidative stress and extracellular matrices after hepatectomy and liver transplantation in rats"

    Article Title: Oxidative stress and extracellular matrices after hepatectomy and liver transplantation in rats

    Journal: World Journal of Hepatology

    doi: 10.4254/wjh.v6.i2.72

    Protein expression of malondialdehyde, 4-hydroxynonenal, ataxia-telangiectasia mutated kinas/H2AX, phosphatidylinositol 3-kinase/Akt and antioxidant enzymes. A: Actual intensities of malondialdehyde (MDA) in western blotting; B: Normalized MDA; C: Normalized
    Figure Legend Snippet: Protein expression of malondialdehyde, 4-hydroxynonenal, ataxia-telangiectasia mutated kinas/H2AX, phosphatidylinositol 3-kinase/Akt and antioxidant enzymes. A: Actual intensities of malondialdehyde (MDA) in western blotting; B: Normalized MDA; C: Normalized

    Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot

    68) Product Images from "Role of the E3 ubiquitin ligase RNF157 as a novel downstream effector linking PI3K and MAPK signaling pathways to the cell cycle"

    Article Title: Role of the E3 ubiquitin ligase RNF157 as a novel downstream effector linking PI3K and MAPK signaling pathways to the cell cycle

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.792754

    CDK2 promotes RNF157 phosphorylation. A , Western blot of FLAG-RNF157 co-immunoprecipitated with Myc-CDK2 from 624MEL melanoma cells transfected with the indicated vector combinations. B , analysis of the phosphorylation levels of FLAG-RNF157 by using the phosphospecific RNF157 antibody (pRNF157 S660–663 ) upon treatment with different inhibitors. RNF157 was co-transfected with control vector or Myc-CDK2 and where indicated was treated with DMSO as a control, CDK2 inhibitors CDK2i III (4.2 μ m ) and roscovitine (20 μ m ), or PI3K inhibitor/MEK inhibitor ( PI3Ki/MEKi ) in combination ( Combo ) for 6 h. C , HeLa cells were transfected with FLAG-RNF157 and serum-starved at 24 h post-transfection for 16 h. The cells were then treated with the inhibitors as indicated and lysed after EGF treatment (100 ng/ml for 5 min). SS lane , serum-starved unstimulated cells. Phosphorylation of RNF157 was analyzed by immunoblotting with the phosphospecific antibody. D , lysates of HeLa cells transfected with wild-type RNF157 or different phosphomutant plasmids together with control vector ( EV ) or Myc-CDK2 vector. Immunoblotting was performed with pRNF157 S660–663 , FLAG, Myc, and actin antibodies. E , lysates of HeLa cells transfected with FLAG-RNF157 and Myc-CDH1 expression plasmids and treated with the indicated inhibitors were subjected to immunoprecipitation with the Myc antibody followed by immunoblotting with FLAG, Myc, pRNF157 S660–663 , pRNF157 T170 , phospho-ERK ( pERK ), and actin antibodies. CDK2 inhibitor was used in two different concentrations, 2.1 and 4.2 μ m , for 8 h. F , lysates of HeLa cells transfected with control vector ( EV ), FLAG-RNF157, FLAG-RNF157(4SA), and Myc-CDH1 expression plasmids were subjected to immunoprecipitation with the Myc antibody followed by immunoblotting with FLAG, Myc, and actin antibodies. IP , immunoprecipitation, CoIP , co-immunoprecipitation; pAkt , phospho-AKT; pERK1/2 , phospho-ERK1/2; pCDK2 , phospho-CDK2.
    Figure Legend Snippet: CDK2 promotes RNF157 phosphorylation. A , Western blot of FLAG-RNF157 co-immunoprecipitated with Myc-CDK2 from 624MEL melanoma cells transfected with the indicated vector combinations. B , analysis of the phosphorylation levels of FLAG-RNF157 by using the phosphospecific RNF157 antibody (pRNF157 S660–663 ) upon treatment with different inhibitors. RNF157 was co-transfected with control vector or Myc-CDK2 and where indicated was treated with DMSO as a control, CDK2 inhibitors CDK2i III (4.2 μ m ) and roscovitine (20 μ m ), or PI3K inhibitor/MEK inhibitor ( PI3Ki/MEKi ) in combination ( Combo ) for 6 h. C , HeLa cells were transfected with FLAG-RNF157 and serum-starved at 24 h post-transfection for 16 h. The cells were then treated with the inhibitors as indicated and lysed after EGF treatment (100 ng/ml for 5 min). SS lane , serum-starved unstimulated cells. Phosphorylation of RNF157 was analyzed by immunoblotting with the phosphospecific antibody. D , lysates of HeLa cells transfected with wild-type RNF157 or different phosphomutant plasmids together with control vector ( EV ) or Myc-CDK2 vector. Immunoblotting was performed with pRNF157 S660–663 , FLAG, Myc, and actin antibodies. E , lysates of HeLa cells transfected with FLAG-RNF157 and Myc-CDH1 expression plasmids and treated with the indicated inhibitors were subjected to immunoprecipitation with the Myc antibody followed by immunoblotting with FLAG, Myc, pRNF157 S660–663 , pRNF157 T170 , phospho-ERK ( pERK ), and actin antibodies. CDK2 inhibitor was used in two different concentrations, 2.1 and 4.2 μ m , for 8 h. F , lysates of HeLa cells transfected with control vector ( EV ), FLAG-RNF157, FLAG-RNF157(4SA), and Myc-CDH1 expression plasmids were subjected to immunoprecipitation with the Myc antibody followed by immunoblotting with FLAG, Myc, and actin antibodies. IP , immunoprecipitation, CoIP , co-immunoprecipitation; pAkt , phospho-AKT; pERK1/2 , phospho-ERK1/2; pCDK2 , phospho-CDK2.

    Techniques Used: Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Co-Immunoprecipitation Assay

    69) Product Images from "Piperine induces autophagy by enhancing protein phosphotase 2A activity in a rotenone-induced Parkinson's disease model"

    Article Title: Piperine induces autophagy by enhancing protein phosphotase 2A activity in a rotenone-induced Parkinson's disease model

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11661

    OKA reverses PIP-induced autophagy and increase in cell viability via PP2A inactivation in SK-N-SH cells A. S6K, p-S6K1, AKT, p-AKT, PP2A, p-PP2A, LC3 I, and LC3 II expression was determined by western blotting following OKA treatment; β-actin was used as a loading control. B. - E. Quantification of the ratios of p-S6K1/S6K1 (B), p-AKT/AKT (C), p-PP2A/PP2A (D), and LC3 II /I (E). F. , G. Detection of cell viability (F) and cytotoxicity (G) with the MTT and LDH assays, respectively. Data are expressed as the mean ± SD (one-way analysis of variance). ### P
    Figure Legend Snippet: OKA reverses PIP-induced autophagy and increase in cell viability via PP2A inactivation in SK-N-SH cells A. S6K, p-S6K1, AKT, p-AKT, PP2A, p-PP2A, LC3 I, and LC3 II expression was determined by western blotting following OKA treatment; β-actin was used as a loading control. B. - E. Quantification of the ratios of p-S6K1/S6K1 (B), p-AKT/AKT (C), p-PP2A/PP2A (D), and LC3 II /I (E). F. , G. Detection of cell viability (F) and cytotoxicity (G) with the MTT and LDH assays, respectively. Data are expressed as the mean ± SD (one-way analysis of variance). ### P

    Techniques Used: Expressing, Western Blot, MTT Assay

    OKA reverses PIP-induced autophagy and increase in cell viability via PP2A inactivation in primary neurons A. S6K, p-S6K1, AKT, p-AKT, PP2A, p-PP2A, LC3 I, and LC3 II expression was determined by western blotting following OKA treatment; β-actin was used as a loading control. B. - E. Quantification of the ratios of p-S6K1/S6K1 (B), p-AKT/AKT (C), p-PP2A/PP2A (D), and LC3 II /I (E). F. , G. Detection of cell viability (F) and cytotoxicity (G) with the MTT and LDH assays, respectively. Data are expressed as the mean ± SD (one-way analysis of variance). ### P
    Figure Legend Snippet: OKA reverses PIP-induced autophagy and increase in cell viability via PP2A inactivation in primary neurons A. S6K, p-S6K1, AKT, p-AKT, PP2A, p-PP2A, LC3 I, and LC3 II expression was determined by western blotting following OKA treatment; β-actin was used as a loading control. B. - E. Quantification of the ratios of p-S6K1/S6K1 (B), p-AKT/AKT (C), p-PP2A/PP2A (D), and LC3 II /I (E). F. , G. Detection of cell viability (F) and cytotoxicity (G) with the MTT and LDH assays, respectively. Data are expressed as the mean ± SD (one-way analysis of variance). ### P

    Techniques Used: Expressing, Western Blot, MTT Assay

    PIP induces autophagy via PP2Aactivation in a mouse model of rotenone-induced PD A. S6K, p-S6K1, AKT, p-AKT, PP2A, p-PP2A, LC3 I and LC3 II expression was determined by western blotting of SN tissue lysates; β-actin was used as a loading control. B. - E. Quantification of the ratios of p-S6K1/S6K1 (B), p-AKT/AKT (C), p-PP2A/PP2A (D), and LC3 II /I (E). Data are expressed as the mean ± SD (one-way analysis of variance). ### P
    Figure Legend Snippet: PIP induces autophagy via PP2Aactivation in a mouse model of rotenone-induced PD A. S6K, p-S6K1, AKT, p-AKT, PP2A, p-PP2A, LC3 I and LC3 II expression was determined by western blotting of SN tissue lysates; β-actin was used as a loading control. B. - E. Quantification of the ratios of p-S6K1/S6K1 (B), p-AKT/AKT (C), p-PP2A/PP2A (D), and LC3 II /I (E). Data are expressed as the mean ± SD (one-way analysis of variance). ### P

    Techniques Used: Expressing, Western Blot

    70) Product Images from "UBE2T promotes nasopharyngeal carcinoma cell proliferation, invasion, and metastasis by activating the AKT/GSK3β/β-catenin pathway"

    Article Title: UBE2T promotes nasopharyngeal carcinoma cell proliferation, invasion, and metastasis by activating the AKT/GSK3β/β-catenin pathway

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7805

    UBE2T promotes NPC cell proliferation and metastasis probably by activating the AKT/GSK3β/β-catenin pathway A. Western blot detected the effects of UBE2T on β-catenin, its downstream proliferation/metastasis-related target proteins (Cyclin D1, C-MYC, C-JUN, MMP2, and MMP9), and its upstream pathway proteins (p-AKT, p-GSK3β). B. Immunofluorescence determined the effects of UBE2T overexpression on nuclear translocation of β-catenin. Scales indicate 40 μm. (up; ×1000 field), and separate nuclear and cytoplasmic protein western blot verified the effects of UBE2T on nuclear translocation of β-catenin (down). C. and D. Transwell and matrix-coated transwell analysis detected the effects of AKT inhibitor (MK-2206 2HCl) on the pro-migration and invasion abilities. Representative images of the transwell (C) and matrix-coated transwell (D) assay from indicated groups at 6h (migration) and 24h (invasion). The bar chart represents mean ±SEM number of migration and invasive cells from 5 random 20X objective fields (analysis of variance [ANOVA] of factorial design, *** P
    Figure Legend Snippet: UBE2T promotes NPC cell proliferation and metastasis probably by activating the AKT/GSK3β/β-catenin pathway A. Western blot detected the effects of UBE2T on β-catenin, its downstream proliferation/metastasis-related target proteins (Cyclin D1, C-MYC, C-JUN, MMP2, and MMP9), and its upstream pathway proteins (p-AKT, p-GSK3β). B. Immunofluorescence determined the effects of UBE2T overexpression on nuclear translocation of β-catenin. Scales indicate 40 μm. (up; ×1000 field), and separate nuclear and cytoplasmic protein western blot verified the effects of UBE2T on nuclear translocation of β-catenin (down). C. and D. Transwell and matrix-coated transwell analysis detected the effects of AKT inhibitor (MK-2206 2HCl) on the pro-migration and invasion abilities. Representative images of the transwell (C) and matrix-coated transwell (D) assay from indicated groups at 6h (migration) and 24h (invasion). The bar chart represents mean ±SEM number of migration and invasive cells from 5 random 20X objective fields (analysis of variance [ANOVA] of factorial design, *** P

    Techniques Used: Western Blot, Immunofluorescence, Over Expression, Translocation Assay, Migration

    71) Product Images from "c-MYC responds to glucose deprivation in a cell-type-dependent manner"

    Article Title: c-MYC responds to glucose deprivation in a cell-type-dependent manner

    Journal: Cell Death Discovery

    doi: 10.1038/cddiscovery.2015.57

    Glucose deprivation increases c-MYC transcription partially through ERK signaling pathway in MDA-MB-231 cells. Quantitative RT-PCR ( a ) and Western blot ( b and c ) detection of c-MYC in MDA-MB-231 cells treated with different chemical inhibitors for 12 h in the medium with or without glucose. The indicated chemical inhibitors are AMPK inhibitor P5499 (10 μ M), p38/MAPK inhibitor SB 203580 (10 μ M), PI3K/AKT inhibitor Wortmannin (10 μ M), ERK/MEK inhibitor U0126 (10 μ M), SIRT inhibitor NAM (1 mM) and HDAC inhibitor VPA (1 mM). ( d–f ) HeLa cells were treated and detected as that of MDA-MB-231 in a–c . Quantitative RT-PCR values were relative to the DMSO group with 25 mM Glc and normalized to 18S. Data of three independent experiments are shown.
    Figure Legend Snippet: Glucose deprivation increases c-MYC transcription partially through ERK signaling pathway in MDA-MB-231 cells. Quantitative RT-PCR ( a ) and Western blot ( b and c ) detection of c-MYC in MDA-MB-231 cells treated with different chemical inhibitors for 12 h in the medium with or without glucose. The indicated chemical inhibitors are AMPK inhibitor P5499 (10 μ M), p38/MAPK inhibitor SB 203580 (10 μ M), PI3K/AKT inhibitor Wortmannin (10 μ M), ERK/MEK inhibitor U0126 (10 μ M), SIRT inhibitor NAM (1 mM) and HDAC inhibitor VPA (1 mM). ( d–f ) HeLa cells were treated and detected as that of MDA-MB-231 in a–c . Quantitative RT-PCR values were relative to the DMSO group with 25 mM Glc and normalized to 18S. Data of three independent experiments are shown.

    Techniques Used: Multiple Displacement Amplification, Quantitative RT-PCR, Western Blot, Gas Chromatography

    72) Product Images from "Activation of AKT signaling promotes cell growth and survival in ?7?1 integrin-mediated alleviation of muscular dystrophy"

    Article Title: Activation of AKT signaling promotes cell growth and survival in ?7?1 integrin-mediated alleviation of muscular dystrophy

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbadis.2011.01.002

    Signaling pathways by which the α7 integrin decreases pathology in dystrophic mice. The α7 integrin–ILK complex is activated in response to extracellular matrix attachment, resulting in phosphorylation of AKT and p70S6K in an mTOR-dependent
    Figure Legend Snippet: Signaling pathways by which the α7 integrin decreases pathology in dystrophic mice. The α7 integrin–ILK complex is activated in response to extracellular matrix attachment, resulting in phosphorylation of AKT and p70S6K in an mTOR-dependent

    Techniques Used: Mouse Assay

    73) Product Images from "OSU-CG5, a novel energy restriction mimetic agent, targets human colorectal cancer cells in vitro"

    Article Title: OSU-CG5, a novel energy restriction mimetic agent, targets human colorectal cancer cells in vitro

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2013.183

    The antiproliferative activity of OSU-CG5 in CRC cells is associated with energy restriction cellular responses. Western blot analysis of the expression levels of p-Akt, p-AMPK, β-TrCP, Sp1, Cyclin D1, p-mTOR, and p-p70S6K in HCT-116 cells after
    Figure Legend Snippet: The antiproliferative activity of OSU-CG5 in CRC cells is associated with energy restriction cellular responses. Western blot analysis of the expression levels of p-Akt, p-AMPK, β-TrCP, Sp1, Cyclin D1, p-mTOR, and p-p70S6K in HCT-116 cells after

    Techniques Used: Activity Assay, Western Blot, Expressing

    74) Product Images from "Muscle Mechanics and Ventricular Function: Structural and functional cardiac profile after prolonged duration of mechanical unloading: potential implications for myocardial recovery"

    Article Title: Muscle Mechanics and Ventricular Function: Structural and functional cardiac profile after prolonged duration of mechanical unloading: potential implications for myocardial recovery

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00187.2018

    Stress- and growth-related proteins and left ventricular assist device (LVAD) support. A−G : total and phosphorylated (p-)ERK42 ( A ), total and p-ERK44 ( B ), total and p-p38 MAPK ( C ), total and p-Akt ( D ), total and p-mechanistic target of rapamycin (mTOR; E ), corticotropin-releasing factor 1 (CRF1; F ), and CRF2 ( G ) in normal hearts ( n = 4) and samples from failing hearts before (pre-LVAD; n = 15) and after (post-LVAD; n = 15) LVAD implantation with representative blots. Molecular variables were compared using one-way ANOVA with post hoc Bonferroni analysis. Simple linear regression analysis was used to determine the effect of LVAD time as a continuous variable in clinical and molecular measurements. A t -test was used to compare the effect on LVAD duration as a categorical variable in the change in molecular measurement with LVAD. * P
    Figure Legend Snippet: Stress- and growth-related proteins and left ventricular assist device (LVAD) support. A−G : total and phosphorylated (p-)ERK42 ( A ), total and p-ERK44 ( B ), total and p-p38 MAPK ( C ), total and p-Akt ( D ), total and p-mechanistic target of rapamycin (mTOR; E ), corticotropin-releasing factor 1 (CRF1; F ), and CRF2 ( G ) in normal hearts ( n = 4) and samples from failing hearts before (pre-LVAD; n = 15) and after (post-LVAD; n = 15) LVAD implantation with representative blots. Molecular variables were compared using one-way ANOVA with post hoc Bonferroni analysis. Simple linear regression analysis was used to determine the effect of LVAD time as a continuous variable in clinical and molecular measurements. A t -test was used to compare the effect on LVAD duration as a categorical variable in the change in molecular measurement with LVAD. * P

    Techniques Used:

    75) Product Images from "VSIG4 inhibits proinflammatory macrophage activation by reprogramming mitochondrial pyruvate metabolism"

    Article Title: VSIG4 inhibits proinflammatory macrophage activation by reprogramming mitochondrial pyruvate metabolism

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01327-4

    Vsig4 −/− mice are more susceptible to HFD-induced obesity with insulin resistance. Eight-week-old male Vsig4 −/− mice and age-matched C57BL/6 WT controls were fed a HFD. a Body weight was measured and compared. The obese mice were sacrificed after 10 weeks of HFD feeding. b Fat distribution was detected by μCT. Yellow indicates subcutaneous fat and brown indicates that visceral fat. c Measurement of abdominal wall fat, perirenal fat, serum triglyceride, cholesterol, and free fatty acid. d Representative liver H E staining (left), and intrahepatic triglyceride contents (right), scale bar = 20 μm, n = 10 per group. e Representative the architecture of adipose tissues stained by H E (left), adipocyte size and cell numbers was calculated (right), scale bar = 20 μm, n = 10 per group. f The 15-h-fasting blood glucose levels and 5-h-fasting serum insulin levels. g GTT and ITT were performed in theses obese mice, n = 6 per group. h Western blot of the AKT, p-Akt ser473 , and p-IRS-1 in VAT, muscle and liver tissues of obese mice after 4 min of insulin administration, n = 4 per group. ATMs were isolated from obese mice. i Flow cytometry analyzing pro-IL-1β, IFN-γ, and TNF. Cytokines in VAT were detected by j qRT-PCR and k western blot. Error bar, s.e.m. * p
    Figure Legend Snippet: Vsig4 −/− mice are more susceptible to HFD-induced obesity with insulin resistance. Eight-week-old male Vsig4 −/− mice and age-matched C57BL/6 WT controls were fed a HFD. a Body weight was measured and compared. The obese mice were sacrificed after 10 weeks of HFD feeding. b Fat distribution was detected by μCT. Yellow indicates subcutaneous fat and brown indicates that visceral fat. c Measurement of abdominal wall fat, perirenal fat, serum triglyceride, cholesterol, and free fatty acid. d Representative liver H E staining (left), and intrahepatic triglyceride contents (right), scale bar = 20 μm, n = 10 per group. e Representative the architecture of adipose tissues stained by H E (left), adipocyte size and cell numbers was calculated (right), scale bar = 20 μm, n = 10 per group. f The 15-h-fasting blood glucose levels and 5-h-fasting serum insulin levels. g GTT and ITT were performed in theses obese mice, n = 6 per group. h Western blot of the AKT, p-Akt ser473 , and p-IRS-1 in VAT, muscle and liver tissues of obese mice after 4 min of insulin administration, n = 4 per group. ATMs were isolated from obese mice. i Flow cytometry analyzing pro-IL-1β, IFN-γ, and TNF. Cytokines in VAT were detected by j qRT-PCR and k western blot. Error bar, s.e.m. * p

    Techniques Used: Mouse Assay, Staining, Western Blot, Isolation, Flow Cytometry, Cytometry, Quantitative RT-PCR

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    Centrifugation:

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    Article Snippet: The cells were lysed with lysis buffer for 15 min at 4 °C and the supernatant was harvested after centrifugation. .. The dilution ratios for the primary antibodies used in the present study are listed as followed: caspase 3 antibody (Proteintech, Rosemont, IL, USA; 1:1000); caspase 7 antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); PARP antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); Akt antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-Akt (Ser473) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); PI3K antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-PI3K (Tyr458 + Tyr199) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-EGFR antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-p38 (Thr180 + Tyr182) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-ERK1/2 (Thr202 + Tyr204) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000), and GAPDH antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000).

    Synthesized:

    Article Title: Grb2-SH3 ligand inhibits the growth of HER2+ cancer cells and has antitumor effects in human cancer xenografts alone and in combination with docetaxel
    Article Snippet: Grb2-SH3 inhibitor conjugated to penetratin and peptide binding Mona SH3 domain (sequence: PPPVNRNLKPGRKSRPPPLD-Aha-penetratin) were synthesized by Fmoc chemistry as described in Cussac et al. . .. ERK1/2 antibody, Phosphorylated-ERK1/2 (p42/44 MAP kinase) (Thr 202/Tyr204) antibody, AKT antibody, phosphorylated-AKT (Ser473) antibody, phosphorylated-Shc (Tyr 239/240) antibody and Shc antibody were purchased from Cell Signaling Technology Inc. (Beverly, MA).

    Blocking Assay:

    Article Title: Metabolic effects of leptin receptor knockdown or reconstitution in adipose tissues
    Article Snippet: After protein transfer to polyvinylidene fluoride membranes, membranes were blocked (Odyssey blocking buffer, Li-Cor, Lincoln, NE, USA) and incubated overnight at 4 °C with phospho-Akt (Ser473) antibody (Cell Signaling Technology, Danvers, MA, USA; Catalogue #4060, 1:2000 dilution). .. Afterwards, membranes were stripped (NewBlot PVDF stripping buffer, Li-Cor), blocked, and incubated overnight at 4 °C with an Akt antibody that detects total Akt protein (Cell Signaling Technology; Catalogue #2920, 1:2000 dilution).

    Article Title: Sleep-Disordered Breathing, Circulating Exosomes, and Insulin Sensitivity in Adipocytes
    Article Snippet: .. The lysates were separated on 12% SDS-acrylamide gel and transferred to nitrocellulose membranes, incubated in blocking buffer (5% nonfat dry milk in TBST) followed by phosphoAkt (Ser473) antibody (Cell Signaling Technology, Danvers, MA) or Akt antibody (Cell Signaling Technology) overnight at 4°C. .. Immune-reactive bands were visualized using an enhanced chemiluminescence detection system (Chemidoc XRS+; Bio-Rad, Hercules, CA), and quantified by the Image Lab software (Bio-Rad, Hercules, CA).

    Electrophoresis:

    Article Title: Metformin and sulodexide restore cardiac microvascular perfusion capacity in diet-induced obese rats
    Article Snippet: After electrophoresis samples were transferred to a PVDF membrane (Bio-Rad) and, after washing, incubated with a phospho-Akt (Ser473) rabbit antibody (Cell Signalling Technology, Beverly MA) and subsequently with anti-rabbit secondary antibody (Cell Signalling Technology) and with HRP substrate (SuperSignal West Femto Chemiluminescent Substrate, Pierce). .. After stripping (Restore Western Blot Stripping Buffer, Pierce) membranes were incubated with an Akt antibody (Cell Signalling Technology).

    Incubation:

    Article Title: Structural and functional abnormalities of penile cavernous endothelial cells result in erectile dysfunction at experimental autoimmune prostatitis rat
    Article Snippet: The PVDF membrane was then blocked with 5% milk for 2 h at room temperature and incubated with the primary antibody overnight. .. Phosphorylated eNOS (ab184154,1:500) and anti-eNOS (ab76198, 1:1000), anti- inducible nitric oxide synthase (iNOS) (ab3523, 1:500), anti-nNOS (ab5586, 1:500), Anti-CD90/Thy1(ab92574,1:1000),anti-Desmin(ab32362, 1:5000) were obtained from Abeam; anti-AR (sc-7305,1:200) was obtained from Santa Cruz Biotechnology; phospho-Akt (4060,1:1000) and Akt antibody (9272,1:1000) were obtained from Cell Signaling Technology.

    Article Title: Metformin and sulodexide restore cardiac microvascular perfusion capacity in diet-induced obese rats
    Article Snippet: .. After stripping (Restore Western Blot Stripping Buffer, Pierce) membranes were incubated with an Akt antibody (Cell Signalling Technology). .. Chemiluminescent signals were detected and quantified using the ChemiDoc XRS system (Bio-Rad).

    Article Title: Metabolic effects of leptin receptor knockdown or reconstitution in adipose tissues
    Article Snippet: .. Afterwards, membranes were stripped (NewBlot PVDF stripping buffer, Li-Cor), blocked, and incubated overnight at 4 °C with an Akt antibody that detects total Akt protein (Cell Signaling Technology; Catalogue #2920, 1:2000 dilution). .. The rest of the steps taken for imaging have been described above, except the secondary antibody was IRDye 680RD goat anti-mouse IgG (Li-Cor; Catalogue #925-68070, 1:10000 dilution).

    Article Title: A Novel Mutant of rLj-RGD3 (rLj-112) Suppressed the Proliferation and Metastasis of B16 Cells through the EGFR Signaling Pathway
    Article Snippet: The dilution ratios for the primary antibodies used in the present study are listed as followed: caspase 3 antibody (Proteintech, Rosemont, IL, USA; 1:1000); caspase 7 antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); PARP antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); Akt antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-Akt (Ser473) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); PI3K antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-PI3K (Tyr458 + Tyr199) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-EGFR antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-p38 (Thr180 + Tyr182) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-ERK1/2 (Thr202 + Tyr204) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000), and GAPDH antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000). .. After washing with PBS five times, the membranes were also incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: Sleep-Disordered Breathing, Circulating Exosomes, and Insulin Sensitivity in Adipocytes
    Article Snippet: .. The lysates were separated on 12% SDS-acrylamide gel and transferred to nitrocellulose membranes, incubated in blocking buffer (5% nonfat dry milk in TBST) followed by phosphoAkt (Ser473) antibody (Cell Signaling Technology, Danvers, MA) or Akt antibody (Cell Signaling Technology) overnight at 4°C. .. Immune-reactive bands were visualized using an enhanced chemiluminescence detection system (Chemidoc XRS+; Bio-Rad, Hercules, CA), and quantified by the Image Lab software (Bio-Rad, Hercules, CA).

    Stripping Membranes:

    Article Title: Metformin and sulodexide restore cardiac microvascular perfusion capacity in diet-induced obese rats
    Article Snippet: .. After stripping (Restore Western Blot Stripping Buffer, Pierce) membranes were incubated with an Akt antibody (Cell Signalling Technology). .. Chemiluminescent signals were detected and quantified using the ChemiDoc XRS system (Bio-Rad).

    Article Title: Metabolic effects of leptin receptor knockdown or reconstitution in adipose tissues
    Article Snippet: .. Afterwards, membranes were stripped (NewBlot PVDF stripping buffer, Li-Cor), blocked, and incubated overnight at 4 °C with an Akt antibody that detects total Akt protein (Cell Signaling Technology; Catalogue #2920, 1:2000 dilution). .. The rest of the steps taken for imaging have been described above, except the secondary antibody was IRDye 680RD goat anti-mouse IgG (Li-Cor; Catalogue #925-68070, 1:10000 dilution).

    Expressing:

    Article Title: Structural and functional abnormalities of penile cavernous endothelial cells result in erectile dysfunction at experimental autoimmune prostatitis rat
    Article Snippet: Paragraph title: Western blot analysis of AR, eNOS and AKt expression in EAP rat corpus cavernosum endothelial cells ... Phosphorylated eNOS (ab184154,1:500) and anti-eNOS (ab76198, 1:1000), anti- inducible nitric oxide synthase (iNOS) (ab3523, 1:500), anti-nNOS (ab5586, 1:500), Anti-CD90/Thy1(ab92574,1:1000),anti-Desmin(ab32362, 1:5000) were obtained from Abeam; anti-AR (sc-7305,1:200) was obtained from Santa Cruz Biotechnology; phospho-Akt (4060,1:1000) and Akt antibody (9272,1:1000) were obtained from Cell Signaling Technology.

    Article Title: Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KII? as anti-tumor strategy
    Article Snippet: Human HSP90AB1 plasmid was kindly provided by Professor Fei Sun (Institute of Biophysics, Chinese Academy of Sciences, China) and was ligated into pcDNA3.1 (Invitrogen, Paisley, UK) vector for expression. .. AKT antibody, p-AKT antibody, ERK antibody and p-ERK antibody were from Cell Signaling Technology (Herts, UK).

    BIA-KA:

    Article Title: Structural and functional abnormalities of penile cavernous endothelial cells result in erectile dysfunction at experimental autoimmune prostatitis rat
    Article Snippet: Phosphorylated eNOS (ab184154,1:500) and anti-eNOS (ab76198, 1:1000), anti- inducible nitric oxide synthase (iNOS) (ab3523, 1:500), anti-nNOS (ab5586, 1:500), Anti-CD90/Thy1(ab92574,1:1000),anti-Desmin(ab32362, 1:5000) were obtained from Abeam; anti-AR (sc-7305,1:200) was obtained from Santa Cruz Biotechnology; phospho-Akt (4060,1:1000) and Akt antibody (9272,1:1000) were obtained from Cell Signaling Technology. .. Phosphorylated eNOS (ab184154,1:500) and anti-eNOS (ab76198, 1:1000), anti- inducible nitric oxide synthase (iNOS) (ab3523, 1:500), anti-nNOS (ab5586, 1:500), Anti-CD90/Thy1(ab92574,1:1000),anti-Desmin(ab32362, 1:5000) were obtained from Abeam; anti-AR (sc-7305,1:200) was obtained from Santa Cruz Biotechnology; phospho-Akt (4060,1:1000) and Akt antibody (9272,1:1000) were obtained from Cell Signaling Technology.

    Article Title: Metformin and sulodexide restore cardiac microvascular perfusion capacity in diet-induced obese rats
    Article Snippet: Protein concentrations of the supernatants were determined using a BCA Protein Assay (Micro BCA Protein Assay Kit, Pierce). .. After stripping (Restore Western Blot Stripping Buffer, Pierce) membranes were incubated with an Akt antibody (Cell Signalling Technology).

    Article Title: Sleep-Disordered Breathing, Circulating Exosomes, and Insulin Sensitivity in Adipocytes
    Article Snippet: Protein concentrations of the cell lysates were determined using the BCA Kit (Life Technologies, Grand Island, NY). .. The lysates were separated on 12% SDS-acrylamide gel and transferred to nitrocellulose membranes, incubated in blocking buffer (5% nonfat dry milk in TBST) followed by phosphoAkt (Ser473) antibody (Cell Signaling Technology, Danvers, MA) or Akt antibody (Cell Signaling Technology) overnight at 4°C.

    Modification:

    Article Title: Structural and functional abnormalities of penile cavernous endothelial cells result in erectile dysfunction at experimental autoimmune prostatitis rat
    Article Snippet: Next, 250 mL of ice-cold modified RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) was used to contain protease and phosphatase inhibitors using a cell scraper, and the cells were collected into EP tubes. .. Phosphorylated eNOS (ab184154,1:500) and anti-eNOS (ab76198, 1:1000), anti- inducible nitric oxide synthase (iNOS) (ab3523, 1:500), anti-nNOS (ab5586, 1:500), Anti-CD90/Thy1(ab92574,1:1000),anti-Desmin(ab32362, 1:5000) were obtained from Abeam; anti-AR (sc-7305,1:200) was obtained from Santa Cruz Biotechnology; phospho-Akt (4060,1:1000) and Akt antibody (9272,1:1000) were obtained from Cell Signaling Technology.

    Western Blot:

    Article Title: Structural and functional abnormalities of penile cavernous endothelial cells result in erectile dysfunction at experimental autoimmune prostatitis rat
    Article Snippet: Paragraph title: Western blot analysis of AR, eNOS and AKt expression in EAP rat corpus cavernosum endothelial cells ... Phosphorylated eNOS (ab184154,1:500) and anti-eNOS (ab76198, 1:1000), anti- inducible nitric oxide synthase (iNOS) (ab3523, 1:500), anti-nNOS (ab5586, 1:500), Anti-CD90/Thy1(ab92574,1:1000),anti-Desmin(ab32362, 1:5000) were obtained from Abeam; anti-AR (sc-7305,1:200) was obtained from Santa Cruz Biotechnology; phospho-Akt (4060,1:1000) and Akt antibody (9272,1:1000) were obtained from Cell Signaling Technology.

    Article Title: Blockade of leukemia inhibitory factor as a therapeutic approach to KRAS driven pancreatic cancer
    Article Snippet: .. AKT antibody (Cell Signaling, #4691) for western blotting (1:1,000 dilution). .. P44/42MAPK (Erk1/2) antibody (Cell Signaling, #4695) for western blotting (1:2,000 dilution).

    Article Title: Stromal cell-derived factor-1 (SDF-1)/chemokine (C-X-C motif) receptor 4 (CXCR4) axis activation induces intra-islet glucagon-like peptide-1 (GLP-1) production and enhances beta cell survival
    Article Snippet: Protein density on immunoblots was quantified by densitometric analysis using a Kodak Image Station 440 CF (Eastman Kodak, Rochester, NY, USA). .. The primary antibodies used were phospho-Akt (Ser473) (587F11) monoclonal antibody (catalogue no. 4051; Cell Signaling Technologies, Beverly, MA, USA) and Akt antibody, which recognises Akt1, 2 and 3 (catalogue no. 9272; Cell Signaling Technologies).

    Article Title: Vagal nerve stimulation protects cardiac injury by attenuating mitochondrial dysfunction in a murine burn injury model
    Article Snippet: Paragraph title: Western blot analysis ... Equal amount of protein was loaded and subjected to 8–16% SDS-PAGE gels and immunoblotted by using the following antibodies: cytochrome C antibody (1:500; abcam, Cambridge, MA, USA), AIF antibody (1:1000; abcam), Bcl-2 antibody (1:100; abcam), phosphorylated Bad136 antibody (1:500; Cell Signaling, Danvers, MA, USA), Bad antibody (1:500; Cell Signaling), phosphorylated Akt308 antibody (1:1000; Cell Signaling), Akt antibody (1:1000; Cell Signaling), GAPDH antibody (1:1000; Cell Signaling) and ANT antibody (1:1000; abcam) respectively.

    Article Title: Metformin and sulodexide restore cardiac microvascular perfusion capacity in diet-induced obese rats
    Article Snippet: .. After stripping (Restore Western Blot Stripping Buffer, Pierce) membranes were incubated with an Akt antibody (Cell Signalling Technology). .. Chemiluminescent signals were detected and quantified using the ChemiDoc XRS system (Bio-Rad).

    Article Title: Metabolic effects of leptin receptor knockdown or reconstitution in adipose tissues
    Article Snippet: Paragraph title: Western blot analysis and hepatic glycogen content ... Afterwards, membranes were stripped (NewBlot PVDF stripping buffer, Li-Cor), blocked, and incubated overnight at 4 °C with an Akt antibody that detects total Akt protein (Cell Signaling Technology; Catalogue #2920, 1:2000 dilution).

    Article Title: A Novel Mutant of rLj-RGD3 (rLj-112) Suppressed the Proliferation and Metastasis of B16 Cells through the EGFR Signaling Pathway
    Article Snippet: Paragraph title: 4.5. Western Blotting ... The dilution ratios for the primary antibodies used in the present study are listed as followed: caspase 3 antibody (Proteintech, Rosemont, IL, USA; 1:1000); caspase 7 antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); PARP antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); Akt antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-Akt (Ser473) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); PI3K antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-PI3K (Tyr458 + Tyr199) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-EGFR antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-p38 (Thr180 + Tyr182) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-ERK1/2 (Thr202 + Tyr204) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000), and GAPDH antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000).

    Kinase Assay:

    Article Title: Perifosine-induced inhibition of Akt attenuates BDNF/TrkB-induced chemoresistance in neuroblastoma in vivo
    Article Snippet: .. Akt antibody, phospho-Akt (P-Akt, Ser473, Thr308) antibody, S6 antibody, phospho-S6 (P-S6, Ser235/236) antibody, GAPDH antibody and Akt kinase assay kit were obtained from Cell signaling technology (Beverly, MA). .. TB3 cells were cultured in the presence of tetracycline (1μg/ml) for 3 days in order to repress the TrkB expression.

    Transfection:

    Article Title: MiR-4465 directly targets PTEN to inhibit AKT/mTOR pathway–mediated autophagy
    Article Snippet: The cells were collected at the time indicated after transfection and were lysed in cell lysis buffer containing 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, and protease inhibitor cocktail (Roche, Mannheim, Germany). .. The phospho-p38 antibody, p38 antibody, phospho-AKT antibody, AKT antibody, phospho-mTOR antibody, mTOR antibody, phospho-p70S6K antibody, p70S6K antibody, phospho-ERK1/2 antibody, and anti-ERK1/2 antibody were purchased from CST.

    Protease Inhibitor:

    Article Title: MiR-4465 directly targets PTEN to inhibit AKT/mTOR pathway–mediated autophagy
    Article Snippet: The cells were collected at the time indicated after transfection and were lysed in cell lysis buffer containing 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, and protease inhibitor cocktail (Roche, Mannheim, Germany). .. The phospho-p38 antibody, p38 antibody, phospho-AKT antibody, AKT antibody, phospho-mTOR antibody, mTOR antibody, phospho-p70S6K antibody, p70S6K antibody, phospho-ERK1/2 antibody, and anti-ERK1/2 antibody were purchased from CST.

    Article Title: Metformin and sulodexide restore cardiac microvascular perfusion capacity in diet-induced obese rats
    Article Snippet: Western blotting Tissues were homogenized, sonicated, and centrifuged in ice-cold SET buffer in the presence of phosphatase inhibitor (PhosSTOP, Roche) and protease inhibitor (Complete, Roche). .. After stripping (Restore Western Blot Stripping Buffer, Pierce) membranes were incubated with an Akt antibody (Cell Signalling Technology).

    Cell Culture:

    Article Title: CD164 regulates the tumorigenesis of ovarian surface epithelial cells through the SDF-1?/CXCR4 axis
    Article Snippet: The HEYA8, OVCAR3, ES-2 and SKOV3 human ovarian cancer cell lines were from the American type Culture Collection (Rockville, MD, USA) and were cultured with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C in a humidified atmosphere consisting of 5% CO2. .. Akt antibody, the phospho-Akt antibody, histone H3 antibody and Oct4 antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). p53 (DO-1) and p21 (C-19) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Imaging:

    Article Title: Metabolic effects of leptin receptor knockdown or reconstitution in adipose tissues
    Article Snippet: Membranes were washed before imaging (Odyssey Classic Infrared Imaging System, Li-Cor). .. Afterwards, membranes were stripped (NewBlot PVDF stripping buffer, Li-Cor), blocked, and incubated overnight at 4 °C with an Akt antibody that detects total Akt protein (Cell Signaling Technology; Catalogue #2920, 1:2000 dilution).

    Protein Concentration:

    Article Title: Structural and functional abnormalities of penile cavernous endothelial cells result in erectile dysfunction at experimental autoimmune prostatitis rat
    Article Snippet: Phosphorylated eNOS (ab184154,1:500) and anti-eNOS (ab76198, 1:1000), anti- inducible nitric oxide synthase (iNOS) (ab3523, 1:500), anti-nNOS (ab5586, 1:500), Anti-CD90/Thy1(ab92574,1:1000),anti-Desmin(ab32362, 1:5000) were obtained from Abeam; anti-AR (sc-7305,1:200) was obtained from Santa Cruz Biotechnology; phospho-Akt (4060,1:1000) and Akt antibody (9272,1:1000) were obtained from Cell Signaling Technology. .. Phosphorylated eNOS (ab184154,1:500) and anti-eNOS (ab76198, 1:1000), anti- inducible nitric oxide synthase (iNOS) (ab3523, 1:500), anti-nNOS (ab5586, 1:500), Anti-CD90/Thy1(ab92574,1:1000),anti-Desmin(ab32362, 1:5000) were obtained from Abeam; anti-AR (sc-7305,1:200) was obtained from Santa Cruz Biotechnology; phospho-Akt (4060,1:1000) and Akt antibody (9272,1:1000) were obtained from Cell Signaling Technology.

    Sequencing:

    Article Title: Grb2-SH3 ligand inhibits the growth of HER2+ cancer cells and has antitumor effects in human cancer xenografts alone and in combination with docetaxel
    Article Snippet: Grb2-SH3 inhibitor conjugated to penetratin and peptide binding Mona SH3 domain (sequence: PPPVNRNLKPGRKSRPPPLD-Aha-penetratin) were synthesized by Fmoc chemistry as described in Cussac et al. . .. ERK1/2 antibody, Phosphorylated-ERK1/2 (p42/44 MAP kinase) (Thr 202/Tyr204) antibody, AKT antibody, phosphorylated-AKT (Ser473) antibody, phosphorylated-Shc (Tyr 239/240) antibody and Shc antibody were purchased from Cell Signaling Technology Inc. (Beverly, MA).

    Sonication:

    Article Title: Metformin and sulodexide restore cardiac microvascular perfusion capacity in diet-induced obese rats
    Article Snippet: Western blotting Tissues were homogenized, sonicated, and centrifuged in ice-cold SET buffer in the presence of phosphatase inhibitor (PhosSTOP, Roche) and protease inhibitor (Complete, Roche). .. After stripping (Restore Western Blot Stripping Buffer, Pierce) membranes were incubated with an Akt antibody (Cell Signalling Technology).

    Injection:

    Article Title: Metabolic effects of leptin receptor knockdown or reconstitution in adipose tissues
    Article Snippet: Afterwards, membranes were stripped (NewBlot PVDF stripping buffer, Li-Cor), blocked, and incubated overnight at 4 °C with an Akt antibody that detects total Akt protein (Cell Signaling Technology; Catalogue #2920, 1:2000 dilution). .. ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to quantify bands; quantification of bands for insulin- or vehicle-injected mice were normalized to bands of AdipoqCre − Lepr flox/flox mice that received the same type of injection.

    Recombinant:

    Article Title: Perifosine-induced inhibition of Akt attenuates BDNF/TrkB-induced chemoresistance in neuroblastoma in vivo
    Article Snippet: Recombinant human BDNF was obtained from PeproTech, Inc (Rocky Hill, NJ). .. Akt antibody, phospho-Akt (P-Akt, Ser473, Thr308) antibody, S6 antibody, phospho-S6 (P-S6, Ser235/236) antibody, GAPDH antibody and Akt kinase assay kit were obtained from Cell signaling technology (Beverly, MA).

    Mouse Assay:

    Article Title: Metabolic effects of leptin receptor knockdown or reconstitution in adipose tissues
    Article Snippet: Afterwards, membranes were stripped (NewBlot PVDF stripping buffer, Li-Cor), blocked, and incubated overnight at 4 °C with an Akt antibody that detects total Akt protein (Cell Signaling Technology; Catalogue #2920, 1:2000 dilution). .. ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to quantify bands; quantification of bands for insulin- or vehicle-injected mice were normalized to bands of AdipoqCre − Lepr flox/flox mice that received the same type of injection.

    Stripping:

    Article Title: Metformin and sulodexide restore cardiac microvascular perfusion capacity in diet-induced obese rats
    Article Snippet: .. After stripping (Restore Western Blot Stripping Buffer, Pierce) membranes were incubated with an Akt antibody (Cell Signalling Technology). .. Chemiluminescent signals were detected and quantified using the ChemiDoc XRS system (Bio-Rad).

    SDS Page:

    Article Title: Structural and functional abnormalities of penile cavernous endothelial cells result in erectile dysfunction at experimental autoimmune prostatitis rat
    Article Snippet: Approximately 40 mg of protein lysate was separated on a 10–12% SDS-PAGE gel and transferred to a PVDF membrane. .. Phosphorylated eNOS (ab184154,1:500) and anti-eNOS (ab76198, 1:1000), anti- inducible nitric oxide synthase (iNOS) (ab3523, 1:500), anti-nNOS (ab5586, 1:500), Anti-CD90/Thy1(ab92574,1:1000),anti-Desmin(ab32362, 1:5000) were obtained from Abeam; anti-AR (sc-7305,1:200) was obtained from Santa Cruz Biotechnology; phospho-Akt (4060,1:1000) and Akt antibody (9272,1:1000) were obtained from Cell Signaling Technology.

    Article Title: MiR-4465 directly targets PTEN to inhibit AKT/mTOR pathway–mediated autophagy
    Article Snippet: Approximately 20 μg of the cell lysates were separated by SDS-PAGE and was then transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). .. The phospho-p38 antibody, p38 antibody, phospho-AKT antibody, AKT antibody, phospho-mTOR antibody, mTOR antibody, phospho-p70S6K antibody, p70S6K antibody, phospho-ERK1/2 antibody, and anti-ERK1/2 antibody were purchased from CST.

    Article Title: Vagal nerve stimulation protects cardiac injury by attenuating mitochondrial dysfunction in a murine burn injury model
    Article Snippet: .. Equal amount of protein was loaded and subjected to 8–16% SDS-PAGE gels and immunoblotted by using the following antibodies: cytochrome C antibody (1:500; abcam, Cambridge, MA, USA), AIF antibody (1:1000; abcam), Bcl-2 antibody (1:100; abcam), phosphorylated Bad136 antibody (1:500; Cell Signaling, Danvers, MA, USA), Bad antibody (1:500; Cell Signaling), phosphorylated Akt308 antibody (1:1000; Cell Signaling), Akt antibody (1:1000; Cell Signaling), GAPDH antibody (1:1000; Cell Signaling) and ANT antibody (1:1000; abcam) respectively. .. The horseradish peroxidase-linked antirabbit IgG or horseradish peroxidase-linked antimouse IgG (1:2000; Cell Signaling) was used as the secondary antibody.

    Article Title: A Novel Mutant of rLj-RGD3 (rLj-112) Suppressed the Proliferation and Metastasis of B16 Cells through the EGFR Signaling Pathway
    Article Snippet: The cell lysates were subsequently resolved on SDS-PAGE and then transferred onto the nitrocellulose membranes (Millipore, Darmstadt, Germany), respectively. .. The dilution ratios for the primary antibodies used in the present study are listed as followed: caspase 3 antibody (Proteintech, Rosemont, IL, USA; 1:1000); caspase 7 antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); PARP antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); Akt antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-Akt (Ser473) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); PI3K antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-PI3K (Tyr458 + Tyr199) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-EGFR antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-p38 (Thr180 + Tyr182) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-ERK1/2 (Thr202 + Tyr204) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000), and GAPDH antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000).

    Plasmid Preparation:

    Article Title: Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KII? as anti-tumor strategy
    Article Snippet: Human HSP90AB1 plasmid was kindly provided by Professor Fei Sun (Institute of Biophysics, Chinese Academy of Sciences, China) and was ligated into pcDNA3.1 (Invitrogen, Paisley, UK) vector for expression. .. AKT antibody, p-AKT antibody, ERK antibody and p-ERK antibody were from Cell Signaling Technology (Herts, UK).

    Software:

    Article Title: Vagal nerve stimulation protects cardiac injury by attenuating mitochondrial dysfunction in a murine burn injury model
    Article Snippet: Equal amount of protein was loaded and subjected to 8–16% SDS-PAGE gels and immunoblotted by using the following antibodies: cytochrome C antibody (1:500; abcam, Cambridge, MA, USA), AIF antibody (1:1000; abcam), Bcl-2 antibody (1:100; abcam), phosphorylated Bad136 antibody (1:500; Cell Signaling, Danvers, MA, USA), Bad antibody (1:500; Cell Signaling), phosphorylated Akt308 antibody (1:1000; Cell Signaling), Akt antibody (1:1000; Cell Signaling), GAPDH antibody (1:1000; Cell Signaling) and ANT antibody (1:1000; abcam) respectively. .. The membrane was processed with Pierce Supersignal West Pico Chemiluminescent Kit and images were obtained with a Xenogen IVIS Lumina imagine system using the program Living Image 3.1 (Caliper Life Sciences, Hopkinton, MA, USA) and quantified by using UN-SCAN-IT gel Digitizing software (Silk Scientific, Orem, UT, USA).

    Article Title: Sleep-Disordered Breathing, Circulating Exosomes, and Insulin Sensitivity in Adipocytes
    Article Snippet: The lysates were separated on 12% SDS-acrylamide gel and transferred to nitrocellulose membranes, incubated in blocking buffer (5% nonfat dry milk in TBST) followed by phosphoAkt (Ser473) antibody (Cell Signaling Technology, Danvers, MA) or Akt antibody (Cell Signaling Technology) overnight at 4°C. .. Immune-reactive bands were visualized using an enhanced chemiluminescence detection system (Chemidoc XRS+; Bio-Rad, Hercules, CA), and quantified by the Image Lab software (Bio-Rad, Hercules, CA).

    Binding Assay:

    Article Title: Grb2-SH3 ligand inhibits the growth of HER2+ cancer cells and has antitumor effects in human cancer xenografts alone and in combination with docetaxel
    Article Snippet: Grb2-SH3 inhibitor conjugated to penetratin and peptide binding Mona SH3 domain (sequence: PPPVNRNLKPGRKSRPPPLD-Aha-penetratin) were synthesized by Fmoc chemistry as described in Cussac et al. . .. ERK1/2 antibody, Phosphorylated-ERK1/2 (p42/44 MAP kinase) (Thr 202/Tyr204) antibody, AKT antibody, phosphorylated-AKT (Ser473) antibody, phosphorylated-Shc (Tyr 239/240) antibody and Shc antibody were purchased from Cell Signaling Technology Inc. (Beverly, MA).

    Lysis:

    Article Title: MiR-4465 directly targets PTEN to inhibit AKT/mTOR pathway–mediated autophagy
    Article Snippet: The cells were collected at the time indicated after transfection and were lysed in cell lysis buffer containing 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, and protease inhibitor cocktail (Roche, Mannheim, Germany). .. The phospho-p38 antibody, p38 antibody, phospho-AKT antibody, AKT antibody, phospho-mTOR antibody, mTOR antibody, phospho-p70S6K antibody, p70S6K antibody, phospho-ERK1/2 antibody, and anti-ERK1/2 antibody were purchased from CST.

    Article Title: A Novel Mutant of rLj-RGD3 (rLj-112) Suppressed the Proliferation and Metastasis of B16 Cells through the EGFR Signaling Pathway
    Article Snippet: The cells were lysed with lysis buffer for 15 min at 4 °C and the supernatant was harvested after centrifugation. .. The dilution ratios for the primary antibodies used in the present study are listed as followed: caspase 3 antibody (Proteintech, Rosemont, IL, USA; 1:1000); caspase 7 antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); PARP antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); Akt antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-Akt (Ser473) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); PI3K antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-PI3K (Tyr458 + Tyr199) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-EGFR antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-p38 (Thr180 + Tyr182) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000); phospho-ERK1/2 (Thr202 + Tyr204) antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000), and GAPDH antibody (Cell Signalling Technology, Danvers, MA, USA; 1:1000).

    Article Title: Sleep-Disordered Breathing, Circulating Exosomes, and Insulin Sensitivity in Adipocytes
    Article Snippet: Exosomes were added for 24 hours and adipocytes cells were treated with 0 or 5nm insulin (Sigma-Aldrich, St. Louis, MO) at 37°C for 30min prior to lysis. .. The lysates were separated on 12% SDS-acrylamide gel and transferred to nitrocellulose membranes, incubated in blocking buffer (5% nonfat dry milk in TBST) followed by phosphoAkt (Ser473) antibody (Cell Signaling Technology, Danvers, MA) or Akt antibody (Cell Signaling Technology) overnight at 4°C.

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    Cell Signaling Technology Inc p akt
    PUMA induction by anti-EGFR antibodies is mediated by <t>p73</t> (A) DiFi cells transfected with control scrambled or p73 siRNA for 24 hr were re-plated and treated with 10 nM cetuximab (Cmab). Expression of p73 at 8 hr, and PUMA and cleaved (C) caspase-3 at 24 hr after cetuximab treatment was analyzed by western blotting. Cells without siRNA transfection and re-plating were used as the control for analyzing p73 at 8r after treatment. (B) Western blotting of indicated proteins in DiFi cells treated 10 nM cetuximab at the indicated time points. Phospho-p73 (p-p73, Y99); <t>phospho-AKT</t> (p-AKT, S473); phospho-ERK1/2 (p-ERK1/2, T202/Y204). (C) DiFi cells transfected with either a control empty vector or a HA-p73α construct were treated with 10 nM Cmab for the indicated times. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody with IgG as control, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (D) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected with siRNA as in (A) and treated with Cmab at the indicated doses for 72 hr. (E) Western blotting of indicated proteins in DiFi cells transfected with control empty vector or constitutively active AKT for 6 hr, and then treated with 10 nM cetuximab for 8 or 24 hr. (F) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected as in (E) and treated with Cmab at the indicated doses for 72 hr. (G) A model of PUMA induction by anti-EGFR antibodies.
    P Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1505 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phosphot akt ser473
    Immunohistochemistry of Qdot-GRP78 antibody in MDA-MB-231/GFP breast cells and LNCaP prostate cancer cells. ( A ) and ( B ) Fluorescence images of cells staining with Qdot625-GRP78 antibody. ( A ) control cells staining with unlabeled nanobeads; ( B ) MDA-MB-231/GFP cells staining with Qdot-GRP78 antibody; ( C ) Scanned confocal microscopy imaging in LNCaP prostate cancer cell, stained with anti-GRP78 mouse monoclonal antibody with secondary Goat anti-mouse IgG labeled with Alexa488(green)and Qdot-GRP78(red dots) and overlapped with two probes (yellow); ( D ) and ( E ) Internalization of Qdot-GRP78 antibody by MDA-MB-231/GFP cells. Cells were incubated with unlabelled nanobeads. ( D ) or Qdot-GRP78 antibody; ( E ) at 37 °C for 16 h. Cells were then washed with PBS and analyzed by fluorescence microscopy; ( F ) Scanned confocal microscopy imaging in MDA-MB-231/GFP cell (green), treated with control unlabelled beads; ( G ) Qdot-GRP78(red dots) was detected inside MDA-MB-231/GFP cell; ( H ) 3D reconstruction of confocal Z stack with 0.8-μM, GFP cell showing in green channel, while Qdot-GRP78 showing in red channel. Scale bar represents 20 μm; ( I ) Western blot analysis of GRP78 protein in Qdot-GRP78 antibody treated cells. Cells either treated with control (unlabelled Qdot) or Qdot-labeled antibody. Phosphorylated <t>Akt-ser473</t> protein was detected by an anti-anti-Akt-se473 antibody and Pan-Akt antibody was used as a loading control. The western blot represents three independent experiments. ( J ) MDA-MB-231/GFP cells were incubated with various concentrations of Qot-GRP78 antibody for 24h. Apoptotic nuclei or nuclear DNA strand breaks were visualized using Hoechst DNA dye H33342 (10). A minimum of 200 cells were counted in each sample and condensed or fragmented nuclei were expressed as a percentage of the total number of nuclei. Values are presented as mean ± S.D. of three determinations. * indicates significant difference ( p
    Anti Phosphot Akt Ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PUMA induction by anti-EGFR antibodies is mediated by p73 (A) DiFi cells transfected with control scrambled or p73 siRNA for 24 hr were re-plated and treated with 10 nM cetuximab (Cmab). Expression of p73 at 8 hr, and PUMA and cleaved (C) caspase-3 at 24 hr after cetuximab treatment was analyzed by western blotting. Cells without siRNA transfection and re-plating were used as the control for analyzing p73 at 8r after treatment. (B) Western blotting of indicated proteins in DiFi cells treated 10 nM cetuximab at the indicated time points. Phospho-p73 (p-p73, Y99); phospho-AKT (p-AKT, S473); phospho-ERK1/2 (p-ERK1/2, T202/Y204). (C) DiFi cells transfected with either a control empty vector or a HA-p73α construct were treated with 10 nM Cmab for the indicated times. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody with IgG as control, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (D) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected with siRNA as in (A) and treated with Cmab at the indicated doses for 72 hr. (E) Western blotting of indicated proteins in DiFi cells transfected with control empty vector or constitutively active AKT for 6 hr, and then treated with 10 nM cetuximab for 8 or 24 hr. (F) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected as in (E) and treated with Cmab at the indicated doses for 72 hr. (G) A model of PUMA induction by anti-EGFR antibodies.

    Journal: Oncogene

    Article Title: Restoring PUMA induction overcomes KRAS-mediated resistance to anti-EGFR antibodies in colorectal cancer

    doi: 10.1038/s41388-018-0289-x

    Figure Lengend Snippet: PUMA induction by anti-EGFR antibodies is mediated by p73 (A) DiFi cells transfected with control scrambled or p73 siRNA for 24 hr were re-plated and treated with 10 nM cetuximab (Cmab). Expression of p73 at 8 hr, and PUMA and cleaved (C) caspase-3 at 24 hr after cetuximab treatment was analyzed by western blotting. Cells without siRNA transfection and re-plating were used as the control for analyzing p73 at 8r after treatment. (B) Western blotting of indicated proteins in DiFi cells treated 10 nM cetuximab at the indicated time points. Phospho-p73 (p-p73, Y99); phospho-AKT (p-AKT, S473); phospho-ERK1/2 (p-ERK1/2, T202/Y204). (C) DiFi cells transfected with either a control empty vector or a HA-p73α construct were treated with 10 nM Cmab for the indicated times. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody with IgG as control, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (D) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected with siRNA as in (A) and treated with Cmab at the indicated doses for 72 hr. (E) Western blotting of indicated proteins in DiFi cells transfected with control empty vector or constitutively active AKT for 6 hr, and then treated with 10 nM cetuximab for 8 or 24 hr. (F) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected as in (E) and treated with Cmab at the indicated doses for 72 hr. (G) A model of PUMA induction by anti-EGFR antibodies.

    Article Snippet: Western blotting Western blotting was performed as previously described [ ] using antibodies against: β-Actin (A5441, Sigma), cleaved caspase-3 (#9661, Cell Signaling, Danvers, MA, USA), cleaved caspase-9 (#9502, Cell Signaling), Mcl-1 (#559027, BD Biosciences, San Jose, CA, USA), Bax (#610983, BD Biosciences), Bid (#2002, Cell Signaling), Bcl-xL (#610212, BD Biosciences), Bim (#2819, Cell Signaling), Bcl-2 (#M0887, Agilent DAKO, Santa Clara, CA, USA), cytochrome c (sc-7159, Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX IV (A21348, Invitrogen), PUMA [ ], Bak (#06–536, EMD Millipore), Noxa (#OP180, EMD Millipore), p73 (A300–126A, Bethyl Laboratories, Montgomery, TX, USA), p-p73 (#4665, Cell Signaling), p-AKT (#4058, Cell Signaling), total AKT (#9272, Cell Signaling), p-ERK1/2 (#4376, Cell Signaling), total ERK1/2 (#9102, Cell Signaling), p-FoxO3A (#9464, Cell Signaling), total FoxO3A (07–702, EMD Millipore), p53 (sc-126, Santa Cruz), p-EGFR (#2234, Cell Signaling), total EGFR (#610016, BD Biosciences), KRAS (sc-30, Santa Cruz), p-Aurora A/B/C (#2914, Cell Signaling), total Aurora A (#4718, Cell Signaling), total Aurora B (#3094, Cell Signaling), and HA (#12CA5, Roche, Indianapolis, IN, USA).

    Techniques: Transfection, Expressing, Western Blot, Plasmid Preparation, Construct, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

    KRAS -mediated resistance to anti-EGFR antibodies abrogates PUMA induction (A) MTS analysis of parental (red) and cetuximab-resistant (Cmab-R, black) DiFi cells treated with cetuximab (Cmab) or panitumumab (Pmab) at the indicated doses for 72 hr. (B) Western blotting of cleaved (C) caspase-3 in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab or Pmab for 24 hr. (C) Western blotting of indicated proteins in the parental and Cmab-R DiFi cells. Lysates of Cmab-R DiFi cells were prepared from cells cultured in medium with 10 nM cetuximab (Cmab+), or without cetuximab (Cmab-) for 6 days. p-EGFR (Y1068); p-AKT (S473); p-ERK1/2 (T202/Y204). (D) Western blotting of indicated Bcl-2 family proteins in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab for 24 hr. (E) Western blotting of phosphorylated (p-p73, Y99) and total p73 in the parental and Cmab-R DiFi cells treated with 10 nM Cmab for 8 hr. (F) Parental and Cmab-R DiFi cells transfected with control empty or HA-p73α-expressing vector were treated with 10 nM cetuximab for 8 hr. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (G) Parental and Cmab-R DiFi cells were infected with EGFP-PUMA-expressing adenovirus (Ad-PUMA) at the indicated MOI for 24 hr. Upper, analysis of apoptosis by nuclear fragmentation; lower , western blotting of PUMA. Results were expressed as means ± s.e.m. of triplicates in two independent experiments. *** P

    Journal: Oncogene

    Article Title: Restoring PUMA induction overcomes KRAS-mediated resistance to anti-EGFR antibodies in colorectal cancer

    doi: 10.1038/s41388-018-0289-x

    Figure Lengend Snippet: KRAS -mediated resistance to anti-EGFR antibodies abrogates PUMA induction (A) MTS analysis of parental (red) and cetuximab-resistant (Cmab-R, black) DiFi cells treated with cetuximab (Cmab) or panitumumab (Pmab) at the indicated doses for 72 hr. (B) Western blotting of cleaved (C) caspase-3 in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab or Pmab for 24 hr. (C) Western blotting of indicated proteins in the parental and Cmab-R DiFi cells. Lysates of Cmab-R DiFi cells were prepared from cells cultured in medium with 10 nM cetuximab (Cmab+), or without cetuximab (Cmab-) for 6 days. p-EGFR (Y1068); p-AKT (S473); p-ERK1/2 (T202/Y204). (D) Western blotting of indicated Bcl-2 family proteins in the parental and Cmab-R DiFi cells treated with 10 nM of Cmab for 24 hr. (E) Western blotting of phosphorylated (p-p73, Y99) and total p73 in the parental and Cmab-R DiFi cells treated with 10 nM Cmab for 8 hr. (F) Parental and Cmab-R DiFi cells transfected with control empty or HA-p73α-expressing vector were treated with 10 nM cetuximab for 8 hr. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (G) Parental and Cmab-R DiFi cells were infected with EGFP-PUMA-expressing adenovirus (Ad-PUMA) at the indicated MOI for 24 hr. Upper, analysis of apoptosis by nuclear fragmentation; lower , western blotting of PUMA. Results were expressed as means ± s.e.m. of triplicates in two independent experiments. *** P

    Article Snippet: Western blotting Western blotting was performed as previously described [ ] using antibodies against: β-Actin (A5441, Sigma), cleaved caspase-3 (#9661, Cell Signaling, Danvers, MA, USA), cleaved caspase-9 (#9502, Cell Signaling), Mcl-1 (#559027, BD Biosciences, San Jose, CA, USA), Bax (#610983, BD Biosciences), Bid (#2002, Cell Signaling), Bcl-xL (#610212, BD Biosciences), Bim (#2819, Cell Signaling), Bcl-2 (#M0887, Agilent DAKO, Santa Clara, CA, USA), cytochrome c (sc-7159, Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX IV (A21348, Invitrogen), PUMA [ ], Bak (#06–536, EMD Millipore), Noxa (#OP180, EMD Millipore), p73 (A300–126A, Bethyl Laboratories, Montgomery, TX, USA), p-p73 (#4665, Cell Signaling), p-AKT (#4058, Cell Signaling), total AKT (#9272, Cell Signaling), p-ERK1/2 (#4376, Cell Signaling), total ERK1/2 (#9102, Cell Signaling), p-FoxO3A (#9464, Cell Signaling), total FoxO3A (07–702, EMD Millipore), p53 (sc-126, Santa Cruz), p-EGFR (#2234, Cell Signaling), total EGFR (#610016, BD Biosciences), KRAS (sc-30, Santa Cruz), p-Aurora A/B/C (#2914, Cell Signaling), total Aurora A (#4718, Cell Signaling), total Aurora B (#3094, Cell Signaling), and HA (#12CA5, Roche, Indianapolis, IN, USA).

    Techniques: Western Blot, Cell Culture, Transfection, Expressing, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Infection

    The downregulation of CDC20 inhibited the phenylephrine (PE)-induced hypertrophic response in cultured cardiomyocytes. (A) Immunoblotting analysis of CDC20 in neonatal rat cardiomyocytes infected with adenovirus siCDC20 or siControl (n=3). (B) Representative images of double immunostaining (red for α-actinin, blue for DAPI) of NRCMs after 24 hours of PE (100 nM) (left). Scale bar: 50 μm. Quantification of the myocyte surface area (n=3, 150 cells counted per experiment; right). (C) qPCR analysis of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) mRNA levels in NRCMs treated as described in (B) (n=3). (D) Immunoblot analysis of AKT, ERK1/2, p38 and GAPDH in NRCMs treated as described in (B) (n=3); * P

    Journal: Theranostics

    Article Title: CDC20 regulates cardiac hypertrophy via targeting LC3-dependent autophagy

    doi: 10.7150/thno.27706

    Figure Lengend Snippet: The downregulation of CDC20 inhibited the phenylephrine (PE)-induced hypertrophic response in cultured cardiomyocytes. (A) Immunoblotting analysis of CDC20 in neonatal rat cardiomyocytes infected with adenovirus siCDC20 or siControl (n=3). (B) Representative images of double immunostaining (red for α-actinin, blue for DAPI) of NRCMs after 24 hours of PE (100 nM) (left). Scale bar: 50 μm. Quantification of the myocyte surface area (n=3, 150 cells counted per experiment; right). (C) qPCR analysis of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) mRNA levels in NRCMs treated as described in (B) (n=3). (D) Immunoblot analysis of AKT, ERK1/2, p38 and GAPDH in NRCMs treated as described in (B) (n=3); * P

    Article Snippet: Antibodies and reagents Antibodies: LC3B (Sigma; L7532), ATG5 (Cell Signalling; 12994), AKT (Cell Signalling; 9272), p-AKT (Cell Signalling; 4060S), ERK1/2(Cell Signalling; 9102), p-ERK1/2 (Cell Signalling; 9101), Beclin-1 (Cell Signalling; 3738), horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (Cell Signalling; 7074S).

    Techniques: Cell Culture, Infection, Double Immunostaining, Real-time Polymerase Chain Reaction, Aqueous Normal-phase Chromatography

    The knockdown of CDC20 suppresses hypertrophy in vivo . (A) Wild-type (WT) mice injected with rAAV9-siZsGreen or rAAV9-siCDC20 were subjected to sham operation or TAC for 5 weeks. Echocardiographic assessment of ejection fraction (EF%) and fractional shortening (FS%) (n=6). (B) H E staining of heart sections (upper). Scale bar 0.5 cm. The ratios of heart weight to tibia length (HW/TL) and body weight (HW/BW) (lower) (n=6). (C) Heart cross-sections were stained with TRITC-labeled wheat germ agglutinin (WGA, upper panel). Scale bar: 10 μm. Quantification of the relative myocyte cross-sectional area (n=6, 200 cells counted per heart; lower panel). (D) qPCR analysis of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) mRNA levels in the hearts of rAAV9-siZsGreen- and rAAV9-siCDC20-treated mice following sham operation or TAC (n=6). (E) Representative images of Masson's Trichrome (upper) and DHE staining (middle) of ventricular sections. Scale bar: 50 μm. Quantification of the relative fibrotic area and ROS fluorescence intensity (n=6, lower). (F) Immunoblotting analysis of p-AKT, AKT, p-ERK1/2, ERK1/2, NOX4 and GAPDH in heart samples (n=6). * P

    Journal: Theranostics

    Article Title: CDC20 regulates cardiac hypertrophy via targeting LC3-dependent autophagy

    doi: 10.7150/thno.27706

    Figure Lengend Snippet: The knockdown of CDC20 suppresses hypertrophy in vivo . (A) Wild-type (WT) mice injected with rAAV9-siZsGreen or rAAV9-siCDC20 were subjected to sham operation or TAC for 5 weeks. Echocardiographic assessment of ejection fraction (EF%) and fractional shortening (FS%) (n=6). (B) H E staining of heart sections (upper). Scale bar 0.5 cm. The ratios of heart weight to tibia length (HW/TL) and body weight (HW/BW) (lower) (n=6). (C) Heart cross-sections were stained with TRITC-labeled wheat germ agglutinin (WGA, upper panel). Scale bar: 10 μm. Quantification of the relative myocyte cross-sectional area (n=6, 200 cells counted per heart; lower panel). (D) qPCR analysis of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) mRNA levels in the hearts of rAAV9-siZsGreen- and rAAV9-siCDC20-treated mice following sham operation or TAC (n=6). (E) Representative images of Masson's Trichrome (upper) and DHE staining (middle) of ventricular sections. Scale bar: 50 μm. Quantification of the relative fibrotic area and ROS fluorescence intensity (n=6, lower). (F) Immunoblotting analysis of p-AKT, AKT, p-ERK1/2, ERK1/2, NOX4 and GAPDH in heart samples (n=6). * P

    Article Snippet: Antibodies and reagents Antibodies: LC3B (Sigma; L7532), ATG5 (Cell Signalling; 12994), AKT (Cell Signalling; 9272), p-AKT (Cell Signalling; 4060S), ERK1/2(Cell Signalling; 9102), p-ERK1/2 (Cell Signalling; 9101), Beclin-1 (Cell Signalling; 3738), horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (Cell Signalling; 7074S).

    Techniques: In Vivo, Mouse Assay, Injection, Staining, Labeling, Whole Genome Amplification, Real-time Polymerase Chain Reaction, Aqueous Normal-phase Chromatography, Fluorescence

    Knockdown of LC3 reversed the reduction of cardiomyocyte size after deletion of CDC20 in vitro . (A) Representative images of double immunostaining (green for α-actinin) of NRCMs transfected with siRNA-CDC20 or siRNA-control with or without Ad-siLC3 treatment after 24 hours of Ang II stimulation (left). Scale bar 50 μm. Quantification of the myocyte surface area (right) (n=3). (B) Representative Western blot analyses of p-AKT, AKT, p-ERK1/2, ERK1/2, NOX4, LC3 I/II and GAPDH in NRCMs infected with siRNA-control or siRNA-CDC20 with or without Ad-siLC3 treatment after 24 hours of Ang II stimulation (n=3). C, A functional link between CDC20 and LC3-dependent autophagy in hypertrophy. ** P

    Journal: Theranostics

    Article Title: CDC20 regulates cardiac hypertrophy via targeting LC3-dependent autophagy

    doi: 10.7150/thno.27706

    Figure Lengend Snippet: Knockdown of LC3 reversed the reduction of cardiomyocyte size after deletion of CDC20 in vitro . (A) Representative images of double immunostaining (green for α-actinin) of NRCMs transfected with siRNA-CDC20 or siRNA-control with or without Ad-siLC3 treatment after 24 hours of Ang II stimulation (left). Scale bar 50 μm. Quantification of the myocyte surface area (right) (n=3). (B) Representative Western blot analyses of p-AKT, AKT, p-ERK1/2, ERK1/2, NOX4, LC3 I/II and GAPDH in NRCMs infected with siRNA-control or siRNA-CDC20 with or without Ad-siLC3 treatment after 24 hours of Ang II stimulation (n=3). C, A functional link between CDC20 and LC3-dependent autophagy in hypertrophy. ** P

    Article Snippet: Antibodies and reagents Antibodies: LC3B (Sigma; L7532), ATG5 (Cell Signalling; 12994), AKT (Cell Signalling; 9272), p-AKT (Cell Signalling; 4060S), ERK1/2(Cell Signalling; 9102), p-ERK1/2 (Cell Signalling; 9101), Beclin-1 (Cell Signalling; 3738), horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (Cell Signalling; 7074S).

    Techniques: In Vitro, Double Immunostaining, Transfection, Western Blot, Infection, Functional Assay

    The effect of MDHB on AKT, GSK3β and β-catenin. MDHB inhibited phosphorylation of AKT in the differentiation of neural stem cells, activated phosphorylation of GSK3β at tyrosine 216 (Y216), and downregulated transcription factor β-catenin. (A,B) Western blot analyses of proteins extracted from MDHB-treated neural stem cell in differentiation, (C–H) quantification of protein blots is shown, GAPDH serves as protein loading control. Each point represents the mean relative protein level of each group ( ∗ P

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Methyl 3,4-Dihydroxybenzoate Induces Neural Stem Cells to Differentiate Into Cholinergic Neurons in vitro

    doi: 10.3389/fncel.2018.00478

    Figure Lengend Snippet: The effect of MDHB on AKT, GSK3β and β-catenin. MDHB inhibited phosphorylation of AKT in the differentiation of neural stem cells, activated phosphorylation of GSK3β at tyrosine 216 (Y216), and downregulated transcription factor β-catenin. (A,B) Western blot analyses of proteins extracted from MDHB-treated neural stem cell in differentiation, (C–H) quantification of protein blots is shown, GAPDH serves as protein loading control. Each point represents the mean relative protein level of each group ( ∗ P

    Article Snippet: The expressions of β-III-tubulin, GFAP, AKT (CST), p-AKT (CST), GSK3β (CST), p-ser9-GSK3β (CST), p-try-GSK3β (Thermofisher) and β-catenin (CST) were determined via calculating their density ratio to the GAPDH band.

    Techniques: Western Blot

    Immunohistochemistry of Qdot-GRP78 antibody in MDA-MB-231/GFP breast cells and LNCaP prostate cancer cells. ( A ) and ( B ) Fluorescence images of cells staining with Qdot625-GRP78 antibody. ( A ) control cells staining with unlabeled nanobeads; ( B ) MDA-MB-231/GFP cells staining with Qdot-GRP78 antibody; ( C ) Scanned confocal microscopy imaging in LNCaP prostate cancer cell, stained with anti-GRP78 mouse monoclonal antibody with secondary Goat anti-mouse IgG labeled with Alexa488(green)and Qdot-GRP78(red dots) and overlapped with two probes (yellow); ( D ) and ( E ) Internalization of Qdot-GRP78 antibody by MDA-MB-231/GFP cells. Cells were incubated with unlabelled nanobeads. ( D ) or Qdot-GRP78 antibody; ( E ) at 37 °C for 16 h. Cells were then washed with PBS and analyzed by fluorescence microscopy; ( F ) Scanned confocal microscopy imaging in MDA-MB-231/GFP cell (green), treated with control unlabelled beads; ( G ) Qdot-GRP78(red dots) was detected inside MDA-MB-231/GFP cell; ( H ) 3D reconstruction of confocal Z stack with 0.8-μM, GFP cell showing in green channel, while Qdot-GRP78 showing in red channel. Scale bar represents 20 μm; ( I ) Western blot analysis of GRP78 protein in Qdot-GRP78 antibody treated cells. Cells either treated with control (unlabelled Qdot) or Qdot-labeled antibody. Phosphorylated Akt-ser473 protein was detected by an anti-anti-Akt-se473 antibody and Pan-Akt antibody was used as a loading control. The western blot represents three independent experiments. ( J ) MDA-MB-231/GFP cells were incubated with various concentrations of Qot-GRP78 antibody for 24h. Apoptotic nuclei or nuclear DNA strand breaks were visualized using Hoechst DNA dye H33342 (10). A minimum of 200 cells were counted in each sample and condensed or fragmented nuclei were expressed as a percentage of the total number of nuclei. Values are presented as mean ± S.D. of three determinations. * indicates significant difference ( p

    Journal: Molecules

    Article Title: Quantum Dot- Conjugated Anti-GRP78 scFv Inhibits Cancer Growth in Mice

    doi: 10.3390/molecules17010796

    Figure Lengend Snippet: Immunohistochemistry of Qdot-GRP78 antibody in MDA-MB-231/GFP breast cells and LNCaP prostate cancer cells. ( A ) and ( B ) Fluorescence images of cells staining with Qdot625-GRP78 antibody. ( A ) control cells staining with unlabeled nanobeads; ( B ) MDA-MB-231/GFP cells staining with Qdot-GRP78 antibody; ( C ) Scanned confocal microscopy imaging in LNCaP prostate cancer cell, stained with anti-GRP78 mouse monoclonal antibody with secondary Goat anti-mouse IgG labeled with Alexa488(green)and Qdot-GRP78(red dots) and overlapped with two probes (yellow); ( D ) and ( E ) Internalization of Qdot-GRP78 antibody by MDA-MB-231/GFP cells. Cells were incubated with unlabelled nanobeads. ( D ) or Qdot-GRP78 antibody; ( E ) at 37 °C for 16 h. Cells were then washed with PBS and analyzed by fluorescence microscopy; ( F ) Scanned confocal microscopy imaging in MDA-MB-231/GFP cell (green), treated with control unlabelled beads; ( G ) Qdot-GRP78(red dots) was detected inside MDA-MB-231/GFP cell; ( H ) 3D reconstruction of confocal Z stack with 0.8-μM, GFP cell showing in green channel, while Qdot-GRP78 showing in red channel. Scale bar represents 20 μm; ( I ) Western blot analysis of GRP78 protein in Qdot-GRP78 antibody treated cells. Cells either treated with control (unlabelled Qdot) or Qdot-labeled antibody. Phosphorylated Akt-ser473 protein was detected by an anti-anti-Akt-se473 antibody and Pan-Akt antibody was used as a loading control. The western blot represents three independent experiments. ( J ) MDA-MB-231/GFP cells were incubated with various concentrations of Qot-GRP78 antibody for 24h. Apoptotic nuclei or nuclear DNA strand breaks were visualized using Hoechst DNA dye H33342 (10). A minimum of 200 cells were counted in each sample and condensed or fragmented nuclei were expressed as a percentage of the total number of nuclei. Values are presented as mean ± S.D. of three determinations. * indicates significant difference ( p

    Article Snippet: Anti-phosphot-Akt (ser473) and anti-Akt (pan) were from the Cell Signaling Technology, Inc (Danvers, USA).

    Techniques: Immunohistochemistry, Multiple Displacement Amplification, Fluorescence, Staining, Confocal Microscopy, Imaging, Labeling, Incubation, Microscopy, Western Blot