phospho akt  (Cell Signaling Technology Inc)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Phospho Akt Ser473 Blocking Peptide
    Description:
    This peptide is used to block Phospho Akt Ser473 D9W9U Mouse mAb
    Catalog Number:
    1140
    Price:
    None
    Category:
    Experimental Controls
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc phospho akt
    Profilin induces cellular responses and activates classical signaling cascades in rat VSMCs. ( A ) DNA-synthesis as assessed by measurement of BrdU incorporation. ( B ) Chemotaxis was evaluated utilizing modified Boyden chemotaxis chambers. ( C ) Quiescent VSMCs were stimulated with recombinant profilin-1 (1 µM) for various time points as indicated. The cells were lysed, and equal amounts of protein were subjected to SDS-PAGE and Western blot analyses, using phospho-specific antibodies recognizing phosphorylated p70 S6K , <t>Akt,</t> and Erk1/2. <t>RasGAP</t> served as a lysate control. ( D ) Cells were stimulated with recombinant profilin-1 (1 µM) for 15 min in the presence of pharmacological inhibitors as indicated. Data in C and D were quantified by densitometry and are expressed as fold increase compared to buffer-treated control cells. ( E ) DNA synthesis was assessed by measurement of BrdU incorporation, and recombinant profilin was added in the presence of pharmacological inhibitors as indicated. Data are expressed as the percentage of the maximal profilin-response. ( F ) Chemotaxis was evaluated utilizing modified Boyden chemotaxis chambers in the presence of pharmacological inhibitors. Data are expressed as fold increase compared to buffer-treated control cells. Pharmacological inhibitors: PI3K inhibitors wortmannin (W; 100 nM) and LY294002 (LY; 20 µM), MEK inhibitor PD98059 (PD; 30 µM), rapamycin (Rap; 10 nM), tyrosine kinase inhibitor genestein (Gen; 50 µM), Src inhibitors SU6656 (SU; 2.5 µM) or PP2 (50 nM), PLCγ inhibitor U73122 (U; 10 µM). All data represent means ± SEM from at least three independent experiments. * P
    This peptide is used to block Phospho Akt Ser473 D9W9U Mouse mAb
    https://www.bioz.com/result/phospho akt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 2368 article reviews
    Price from $9.99 to $1999.99
    phospho akt - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Profilin-1 Is Expressed in Human Atherosclerotic Plaques and Induces Atherogenic Effects on Vascular Smooth Muscle Cells"

    Article Title: Profilin-1 Is Expressed in Human Atherosclerotic Plaques and Induces Atherogenic Effects on Vascular Smooth Muscle Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013608

    Profilin induces cellular responses and activates classical signaling cascades in rat VSMCs. ( A ) DNA-synthesis as assessed by measurement of BrdU incorporation. ( B ) Chemotaxis was evaluated utilizing modified Boyden chemotaxis chambers. ( C ) Quiescent VSMCs were stimulated with recombinant profilin-1 (1 µM) for various time points as indicated. The cells were lysed, and equal amounts of protein were subjected to SDS-PAGE and Western blot analyses, using phospho-specific antibodies recognizing phosphorylated p70 S6K , Akt, and Erk1/2. RasGAP served as a lysate control. ( D ) Cells were stimulated with recombinant profilin-1 (1 µM) for 15 min in the presence of pharmacological inhibitors as indicated. Data in C and D were quantified by densitometry and are expressed as fold increase compared to buffer-treated control cells. ( E ) DNA synthesis was assessed by measurement of BrdU incorporation, and recombinant profilin was added in the presence of pharmacological inhibitors as indicated. Data are expressed as the percentage of the maximal profilin-response. ( F ) Chemotaxis was evaluated utilizing modified Boyden chemotaxis chambers in the presence of pharmacological inhibitors. Data are expressed as fold increase compared to buffer-treated control cells. Pharmacological inhibitors: PI3K inhibitors wortmannin (W; 100 nM) and LY294002 (LY; 20 µM), MEK inhibitor PD98059 (PD; 30 µM), rapamycin (Rap; 10 nM), tyrosine kinase inhibitor genestein (Gen; 50 µM), Src inhibitors SU6656 (SU; 2.5 µM) or PP2 (50 nM), PLCγ inhibitor U73122 (U; 10 µM). All data represent means ± SEM from at least three independent experiments. * P
    Figure Legend Snippet: Profilin induces cellular responses and activates classical signaling cascades in rat VSMCs. ( A ) DNA-synthesis as assessed by measurement of BrdU incorporation. ( B ) Chemotaxis was evaluated utilizing modified Boyden chemotaxis chambers. ( C ) Quiescent VSMCs were stimulated with recombinant profilin-1 (1 µM) for various time points as indicated. The cells were lysed, and equal amounts of protein were subjected to SDS-PAGE and Western blot analyses, using phospho-specific antibodies recognizing phosphorylated p70 S6K , Akt, and Erk1/2. RasGAP served as a lysate control. ( D ) Cells were stimulated with recombinant profilin-1 (1 µM) for 15 min in the presence of pharmacological inhibitors as indicated. Data in C and D were quantified by densitometry and are expressed as fold increase compared to buffer-treated control cells. ( E ) DNA synthesis was assessed by measurement of BrdU incorporation, and recombinant profilin was added in the presence of pharmacological inhibitors as indicated. Data are expressed as the percentage of the maximal profilin-response. ( F ) Chemotaxis was evaluated utilizing modified Boyden chemotaxis chambers in the presence of pharmacological inhibitors. Data are expressed as fold increase compared to buffer-treated control cells. Pharmacological inhibitors: PI3K inhibitors wortmannin (W; 100 nM) and LY294002 (LY; 20 µM), MEK inhibitor PD98059 (PD; 30 µM), rapamycin (Rap; 10 nM), tyrosine kinase inhibitor genestein (Gen; 50 µM), Src inhibitors SU6656 (SU; 2.5 µM) or PP2 (50 nM), PLCγ inhibitor U73122 (U; 10 µM). All data represent means ± SEM from at least three independent experiments. * P

    Techniques Used: DNA Synthesis, BrdU Incorporation Assay, Chemotaxis Assay, Modification, Recombinant, SDS Page, Western Blot

    2) Product Images from "Tick sialostatins L and L2 differentially influence dendritic cell responses to Borrelia spirochetes"

    Article Title: Tick sialostatins L and L2 differentially influence dendritic cell responses to Borrelia spirochetes

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-015-0887-1

    Effect of sialostatins on the signalling pathways activated by LTA in dendritic cells. Dendritic cells were seeded in 24-well plate. Next day DCs were incubated 2 h with tick cystatins (both 3 μM) prior to the addition of LTA (2 μg/ml) and further incubated for indicated times. Afterwards, cells were lysed and obtained protein extract was further analysed by immunoblotting using antibodies recognizing phosphorylated form of tested kinases. Membranes were stripped and reprobed with antibodies against total kinase protein, which served as a control ( a ). Proteins were visualized by enhanced chemiluminescence. Bands were quantified using scanning densitometry and phosphorylation/activities of Erk1/2 ( b ), Akt ( c ), and NF-κB ( d ) kinases were normalized by total kinase protein level (relative activity = phospho kinase/total kinase). Three independent experiments were performed and representative blots are shown. Graphs represent the average ± SD from 2-3 experiments, the relative phosphorylation of kinase achieved at 60 min upon LTA stimulation was set up to 1 to allow pooling of data
    Figure Legend Snippet: Effect of sialostatins on the signalling pathways activated by LTA in dendritic cells. Dendritic cells were seeded in 24-well plate. Next day DCs were incubated 2 h with tick cystatins (both 3 μM) prior to the addition of LTA (2 μg/ml) and further incubated for indicated times. Afterwards, cells were lysed and obtained protein extract was further analysed by immunoblotting using antibodies recognizing phosphorylated form of tested kinases. Membranes were stripped and reprobed with antibodies against total kinase protein, which served as a control ( a ). Proteins were visualized by enhanced chemiluminescence. Bands were quantified using scanning densitometry and phosphorylation/activities of Erk1/2 ( b ), Akt ( c ), and NF-κB ( d ) kinases were normalized by total kinase protein level (relative activity = phospho kinase/total kinase). Three independent experiments were performed and representative blots are shown. Graphs represent the average ± SD from 2-3 experiments, the relative phosphorylation of kinase achieved at 60 min upon LTA stimulation was set up to 1 to allow pooling of data

    Techniques Used: Incubation, Activity Assay

    3) Product Images from "The Epithelial αvβ3-Integrin Boosts the MYD88-Dependent TLR2 Signaling in Response to Viral and Bacterial Componentsαvβ6- and αvβ8-Integrins Serve As Interchangeable Receptors for HSV gH/gL to Promote Endocytosis and Activation of Membrane Fusion"

    Article Title: The Epithelial αvβ3-Integrin Boosts the MYD88-Dependent TLR2 Signaling in Response to Viral and Bacterial Componentsαvβ6- and αvβ8-Integrins Serve As Interchangeable Receptors for HSV gH/gL to Promote Endocytosis and Activation of Membrane Fusion

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004477

    HSV-induced Akt phosphorylation dependent on αvβ3-integrin, its C-tail, and TLR2. (A, B, D–F) Akt phosphorylation requires β3-integrin and TLR2. wt or sh-β3 cells 293T (A, B), HaCaT (D), HeLa (E), SK-N-SH (F) cells were transfected or not with plasmid encoding TLR2-Flag, silenced for β3-integrin as indicated (sh-β3), exposed to R7910 (60 PFU/cell) for 10, 20 and 30 min. Cells were lysed and total proteins were subjected to SDS-PAGE. Phosphorylated and total Akt were detected with PAb to phospho-Akt (Ser473) (P-Akt) or PAb to total Akt (T-Akt). (C) β3-integrin silenced (sh-β3) 293T were transfected with wt-β3-integrin or β3 Y747-759F plus αv-integrin, and TLR2. Cells were cultured in pre-exhausted medium [8] for 48–72 h from 12 after transfection, and then exposed to R7910 (60 PFU/cell) for 10, 20, 30 min, or uninfected (0′). Total cell lysates were subjected to SDS-PAGE and WB for P-Akt or T-Akt.
    Figure Legend Snippet: HSV-induced Akt phosphorylation dependent on αvβ3-integrin, its C-tail, and TLR2. (A, B, D–F) Akt phosphorylation requires β3-integrin and TLR2. wt or sh-β3 cells 293T (A, B), HaCaT (D), HeLa (E), SK-N-SH (F) cells were transfected or not with plasmid encoding TLR2-Flag, silenced for β3-integrin as indicated (sh-β3), exposed to R7910 (60 PFU/cell) for 10, 20 and 30 min. Cells were lysed and total proteins were subjected to SDS-PAGE. Phosphorylated and total Akt were detected with PAb to phospho-Akt (Ser473) (P-Akt) or PAb to total Akt (T-Akt). (C) β3-integrin silenced (sh-β3) 293T were transfected with wt-β3-integrin or β3 Y747-759F plus αv-integrin, and TLR2. Cells were cultured in pre-exhausted medium [8] for 48–72 h from 12 after transfection, and then exposed to R7910 (60 PFU/cell) for 10, 20, 30 min, or uninfected (0′). Total cell lysates were subjected to SDS-PAGE and WB for P-Akt or T-Akt.

    Techniques Used: Transfection, Plasmid Preparation, SDS Page, Cell Culture, Western Blot

    Simplified view of the concerted contribution of αvβ3-integrin and TLR2 to the immediate defensive response of the cell to an invader, exemplified by HSV. The virion envelope gH bind simultaneously αvβ3-integrin and TLR2, and thus cross-links them. Signaling by the C-tail of the β3 subunit (green curved arrow) results in boosting of the TLR2-dependent pathway – MAL, MYD88 recruitment, IRAK1 and 4 phosphorylation, and NK-κB activation. Akt serves as a hub, downstream of αvβ3-integrin and TLR2, possibly through the prior activation of FAK and PI3K. Src, and, downstream, syk, card9, IRF3 are activated by αvβ3-integrin, independently of TLR2. The major cellular targets of this pathway are IFN-α, IFN-β, and a specific set of cytokines, including IL2 and IL10.
    Figure Legend Snippet: Simplified view of the concerted contribution of αvβ3-integrin and TLR2 to the immediate defensive response of the cell to an invader, exemplified by HSV. The virion envelope gH bind simultaneously αvβ3-integrin and TLR2, and thus cross-links them. Signaling by the C-tail of the β3 subunit (green curved arrow) results in boosting of the TLR2-dependent pathway – MAL, MYD88 recruitment, IRAK1 and 4 phosphorylation, and NK-κB activation. Akt serves as a hub, downstream of αvβ3-integrin and TLR2, possibly through the prior activation of FAK and PI3K. Src, and, downstream, syk, card9, IRF3 are activated by αvβ3-integrin, independently of TLR2. The major cellular targets of this pathway are IFN-α, IFN-β, and a specific set of cytokines, including IL2 and IL10.

    Techniques Used: Activation Assay

    Interaction of gH with TLR2 and β3-integrin, and failure of gH −/− virions to elicit the innate response. (A) Co-immunoprecipitation of gH by TLR2 occurs in β3-integrin–silenced cells. 293T cells were transfected with plasmids encoding gH/gL (lane 1), TLR2-Flag (lane 2), gH/gL plus TLR2-Flag (lane 4). sh-β3 293T cells were transfected with plasmids encoding gH/gL plus TLR2-Flag (lane 3). TLR2-Flag was immunoprecipitated with anti-Flag MAb (IP: TLR2). The co-immunoprecipitated gH was revealed by WB with PAb to gH/gL. (B) 293T cells were transfected with plasmids encoding TLR2-Flag, αv-integrin+β3-integrin, gH+gL. Negative controls lacked TLR2. TLR2-Flag was immunoprecipitated with anti-Flag MAb (IP: TLR2). The co-immunoprecipitated gH and β3-integrin were revealed by WB with PAb to gH/gL, MAb to TLR2-Flag, and PAb to β3-integrin. Figures to the right indicate the migration position and M r of molecular weight markers. (C–E) Failure of gH −/− virions to induce the MYD88 recruitment to TLR2 (C), phosphorylation of Src (D), and phosphorylation of Akt (E). 293T cells, transfected or not with TLR2-Flag, were exposed to R7910 or gH −/− virions for 10, 20, 30 min, or unexposed (0′). (C) TLR2-Flag was immunoprecipitaed with anti-Flag MAb. Co-immunoprecipitated MYD88 was detected by WB, as detailed in the legend to fig. 2A . (D, E) Total cell lysates were subjected to SDS-PAGE and WB for P-Src and T-Src (D), or for P-Akt and T-Akt (E).
    Figure Legend Snippet: Interaction of gH with TLR2 and β3-integrin, and failure of gH −/− virions to elicit the innate response. (A) Co-immunoprecipitation of gH by TLR2 occurs in β3-integrin–silenced cells. 293T cells were transfected with plasmids encoding gH/gL (lane 1), TLR2-Flag (lane 2), gH/gL plus TLR2-Flag (lane 4). sh-β3 293T cells were transfected with plasmids encoding gH/gL plus TLR2-Flag (lane 3). TLR2-Flag was immunoprecipitated with anti-Flag MAb (IP: TLR2). The co-immunoprecipitated gH was revealed by WB with PAb to gH/gL. (B) 293T cells were transfected with plasmids encoding TLR2-Flag, αv-integrin+β3-integrin, gH+gL. Negative controls lacked TLR2. TLR2-Flag was immunoprecipitated with anti-Flag MAb (IP: TLR2). The co-immunoprecipitated gH and β3-integrin were revealed by WB with PAb to gH/gL, MAb to TLR2-Flag, and PAb to β3-integrin. Figures to the right indicate the migration position and M r of molecular weight markers. (C–E) Failure of gH −/− virions to induce the MYD88 recruitment to TLR2 (C), phosphorylation of Src (D), and phosphorylation of Akt (E). 293T cells, transfected or not with TLR2-Flag, were exposed to R7910 or gH −/− virions for 10, 20, 30 min, or unexposed (0′). (C) TLR2-Flag was immunoprecipitaed with anti-Flag MAb. Co-immunoprecipitated MYD88 was detected by WB, as detailed in the legend to fig. 2A . (D, E) Total cell lysates were subjected to SDS-PAGE and WB for P-Src and T-Src (D), or for P-Akt and T-Akt (E).

    Techniques Used: Immunoprecipitation, Transfection, Western Blot, Migration, Molecular Weight, SDS Page

    4) Product Images from "Interleukin-6-stimulated progranulin expression contributes to the malignancy of hepatocellular carcinoma cells by activating mTOR signaling"

    Article Title: Interleukin-6-stimulated progranulin expression contributes to the malignancy of hepatocellular carcinoma cells by activating mTOR signaling

    Journal: Scientific Reports

    doi: 10.1038/srep21260

    rhPGRN treatment promoted the activation of mammalian target of rapamycin (mTOR) signaling. ( A ) Western blot assay of Akt and Erk activation with 500 ng/mL rhPGRN treatment in HepG2 cells. Phosphorylation of mTORC1 downstream molecules ( B ) and mTORC2-dependent Akt at Ser473 ( C ) in HepG2 cells with rhPGRN treatment. Phosphorylation and acetylation of FoxO1 ( D ) and Sirt1 protein expression ( E ) in HepG2 cells with rhPGRN treatment. ( F ) Western blot assay of mTOR signaling in HepG2 cells transfected with PGRN siRNA (si-PGRN-2) or negative control siRNA (si-NC). GAPDH was a loading control.
    Figure Legend Snippet: rhPGRN treatment promoted the activation of mammalian target of rapamycin (mTOR) signaling. ( A ) Western blot assay of Akt and Erk activation with 500 ng/mL rhPGRN treatment in HepG2 cells. Phosphorylation of mTORC1 downstream molecules ( B ) and mTORC2-dependent Akt at Ser473 ( C ) in HepG2 cells with rhPGRN treatment. Phosphorylation and acetylation of FoxO1 ( D ) and Sirt1 protein expression ( E ) in HepG2 cells with rhPGRN treatment. ( F ) Western blot assay of mTOR signaling in HepG2 cells transfected with PGRN siRNA (si-PGRN-2) or negative control siRNA (si-NC). GAPDH was a loading control.

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

    5) Product Images from "Early Cellular Changes in the Ascending Aorta and Myocardium in a Swine Model of Metabolic Syndrome"

    Article Title: Early Cellular Changes in the Ascending Aorta and Myocardium in a Swine Model of Metabolic Syndrome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0146481

    Apoptosis in the myocardium. There is significantly increased TUNEL staining in HCD group representing apoptosis. The markers for apoptosis are significantly altered in the myocardial tissue. p-Akt is significantly decreased, which may be involved in apoptosis and leads to up-regulation of p38MAPK leading to pathological remodeling. (*p-value
    Figure Legend Snippet: Apoptosis in the myocardium. There is significantly increased TUNEL staining in HCD group representing apoptosis. The markers for apoptosis are significantly altered in the myocardial tissue. p-Akt is significantly decreased, which may be involved in apoptosis and leads to up-regulation of p38MAPK leading to pathological remodeling. (*p-value

    Techniques Used: TUNEL Assay, Staining

    6) Product Images from "The stem cell adjuvant with Exendin-4 repairs the heart after myocardial infarction via STAT3 activation"

    Article Title: The stem cell adjuvant with Exendin-4 repairs the heart after myocardial infarction via STAT3 activation

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12272

    Adipose-derived stem cells (ADSCs) adjuvant with Exendin-4 activated the Akt, ERK1/2 and STAT3 in ischaemic myocardium. ( A ) Representative western blotting of p-Akt, Akt, p-ERK, ERK, p-STAT3, STAT3 and GAPDH in ischaemic myocardium. ( B – D ) The relative optical density ratio of p-Akt, p-ERK, p-STAT3 ( n = 6). * P
    Figure Legend Snippet: Adipose-derived stem cells (ADSCs) adjuvant with Exendin-4 activated the Akt, ERK1/2 and STAT3 in ischaemic myocardium. ( A ) Representative western blotting of p-Akt, Akt, p-ERK, ERK, p-STAT3, STAT3 and GAPDH in ischaemic myocardium. ( B – D ) The relative optical density ratio of p-Akt, p-ERK, p-STAT3 ( n = 6). * P

    Techniques Used: Derivative Assay, Western Blot

    Exendin-4 prevented H 2 O 2 /SD- induced apoptosis of adipose-derived stem cells (ADSCs) and activated Akt, ERK and STAT3. ( A ) In vitro BLI determined that Exendin-4 at 5 nM promoted the attenuated viability of ADSCs exposed to H 2 O 2 /SD ( n = 6). ( B ) Apoptosis was evaluated with flow cytometry analysis. ( C ) The quantitative analysis of apoptotic index (apoptotic cells/total cells) in ADSCs ( n = 6). ( D ) Caspase-3 activity determined by using Caspase-3 ELISA kit ( n = 6). ( E ) Representative blots of p-Akt, Akt, p-ERK, ERK, p-STAT3, STAT3 in ADSCs exposed to H 2 O 2 /SD and quantitative analysis of p-Akt, p-ERK, p-STAT3. β-actin is used as internal parameter ( n = 6). ( F ) Representative blots of Bcl-2 and Bax in ADSCs exposed to H 2 O 2 /SD and quantitative analysis of Bcl-2 and Bax ( n = 6); * P
    Figure Legend Snippet: Exendin-4 prevented H 2 O 2 /SD- induced apoptosis of adipose-derived stem cells (ADSCs) and activated Akt, ERK and STAT3. ( A ) In vitro BLI determined that Exendin-4 at 5 nM promoted the attenuated viability of ADSCs exposed to H 2 O 2 /SD ( n = 6). ( B ) Apoptosis was evaluated with flow cytometry analysis. ( C ) The quantitative analysis of apoptotic index (apoptotic cells/total cells) in ADSCs ( n = 6). ( D ) Caspase-3 activity determined by using Caspase-3 ELISA kit ( n = 6). ( E ) Representative blots of p-Akt, Akt, p-ERK, ERK, p-STAT3, STAT3 in ADSCs exposed to H 2 O 2 /SD and quantitative analysis of p-Akt, p-ERK, p-STAT3. β-actin is used as internal parameter ( n = 6). ( F ) Representative blots of Bcl-2 and Bax in ADSCs exposed to H 2 O 2 /SD and quantitative analysis of Bcl-2 and Bax ( n = 6); * P

    Techniques Used: Derivative Assay, In Vitro, Flow Cytometry, Cytometry, Activity Assay, Enzyme-linked Immunosorbent Assay

    7) Product Images from "Dietary feeding of freeze-dried whole cranberry inhibits intestinal tumor development in Apcmin/+ mice"

    Article Title: Dietary feeding of freeze-dried whole cranberry inhibits intestinal tumor development in Apcmin/+ mice

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22081

    Cranberry supplementation down-regulated EGFR signaling pathway in intestinal tumors ( A – B ) p-EGFR and p-Akt from the diatal small intestine of both groups were shown by immunohistochemical staining (400×). Scale bars, 50 μm. Data were semiquantified as mean percentage of positive cells at five randomly selected fields. ( C ) Protein lysates from tumors were analyzed by Western blot analysis using anti-total EGFR and p-EGFR antibodies, and anti-total Akt and p-Akt antibodies, which anti-β-actin antibody was used as an internal control for total protein. The fold changes were calculated by comparing the relative densities of total EGFR, p-EGFR and p-Akt bands to corresponding β-actin bands from the same mouse. Proteins were quantified by densitometry using Image J to calculate the average ratio, and the average ratio in control was set as 100%. Columns, means from at least six mice in each group; bars, standard deviation. * p
    Figure Legend Snippet: Cranberry supplementation down-regulated EGFR signaling pathway in intestinal tumors ( A – B ) p-EGFR and p-Akt from the diatal small intestine of both groups were shown by immunohistochemical staining (400×). Scale bars, 50 μm. Data were semiquantified as mean percentage of positive cells at five randomly selected fields. ( C ) Protein lysates from tumors were analyzed by Western blot analysis using anti-total EGFR and p-EGFR antibodies, and anti-total Akt and p-Akt antibodies, which anti-β-actin antibody was used as an internal control for total protein. The fold changes were calculated by comparing the relative densities of total EGFR, p-EGFR and p-Akt bands to corresponding β-actin bands from the same mouse. Proteins were quantified by densitometry using Image J to calculate the average ratio, and the average ratio in control was set as 100%. Columns, means from at least six mice in each group; bars, standard deviation. * p

    Techniques Used: Immunohistochemistry, Staining, Western Blot, Mouse Assay, Standard Deviation

    8) Product Images from "Combination of galectin inhibitor GCS-100 and BH3 mimetics eliminates both p53 wild type and p53 null AML cells"

    Article Title: Combination of galectin inhibitor GCS-100 and BH3 mimetics eliminates both p53 wild type and p53 null AML cells

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamcr.2015.12.008

    GCS-100 kills AML cells and inhibits ERK and AKT activation and MCL-1 expression. A. OCI-AML3 and THP-1 cells were treated with vehicle (10% PBS) or varying doses of GCS-100 for 72 h and cell viability assessed by trypan blue staining. * represents p
    Figure Legend Snippet: GCS-100 kills AML cells and inhibits ERK and AKT activation and MCL-1 expression. A. OCI-AML3 and THP-1 cells were treated with vehicle (10% PBS) or varying doses of GCS-100 for 72 h and cell viability assessed by trypan blue staining. * represents p

    Techniques Used: Activation Assay, Expressing, Staining

    9) Product Images from "Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells"

    Article Title: Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23855

    Autophagy, ER stress, and p-AKT change after CQ (Q), Rapa (R) and TMZ (T) in different combination treatment GBM8401 cells and U87MG cells were treated with CQ, Rapa and TMZ in different combinations, harvested at 24 hours and immunoblotted for LC3-I, LC3-II and ( A ) p62, ( B ) p-AKT, ( C ) p-mTOR and p-S6K, ( D ) GRP78 and CHOP.
    Figure Legend Snippet: Autophagy, ER stress, and p-AKT change after CQ (Q), Rapa (R) and TMZ (T) in different combination treatment GBM8401 cells and U87MG cells were treated with CQ, Rapa and TMZ in different combinations, harvested at 24 hours and immunoblotted for LC3-I, LC3-II and ( A ) p62, ( B ) p-AKT, ( C ) p-mTOR and p-S6K, ( D ) GRP78 and CHOP.

    Techniques Used:

    10) Product Images from "3, 3?-Diindolylmethane Exhibits Antileukemic Activity In Vitro and In Vivo through a Akt-Dependent Process"

    Article Title: 3, 3?-Diindolylmethane Exhibits Antileukemic Activity In Vitro and In Vivo through a Akt-Dependent Process

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031783

    DIM induces apoptosis, caspase activation, downregulation of Mcl-1, upregulation of p21, inactivation of Akt, and activation of JNK in U937 human leukemia cells in dose- and time-dependent manners. U937 cells were treated with various concentrations of DIM as indicated for 12 h and 24 h or treated with 80 µM DIM for 1, 3, 6, 9, 12, and 24 h. (a) Cells were washed twice with PBS and stained with Annexin V/propidium iodide (PI), and apoptosis was determined using flow cytometry. Both early apoptotic (Annexin V-positive, PI-negative) and late apoptotic (Annexin V-positive and PI-positive) cells were included in cell death determinations. The values obtained from annexin V/PI assays represent the mean ± SD for three separate experiments. (b–d) Total cellular extracts were prepared and subjected to Western blot assay using antibodies as indicated.
    Figure Legend Snippet: DIM induces apoptosis, caspase activation, downregulation of Mcl-1, upregulation of p21, inactivation of Akt, and activation of JNK in U937 human leukemia cells in dose- and time-dependent manners. U937 cells were treated with various concentrations of DIM as indicated for 12 h and 24 h or treated with 80 µM DIM for 1, 3, 6, 9, 12, and 24 h. (a) Cells were washed twice with PBS and stained with Annexin V/propidium iodide (PI), and apoptosis was determined using flow cytometry. Both early apoptotic (Annexin V-positive, PI-negative) and late apoptotic (Annexin V-positive and PI-positive) cells were included in cell death determinations. The values obtained from annexin V/PI assays represent the mean ± SD for three separate experiments. (b–d) Total cellular extracts were prepared and subjected to Western blot assay using antibodies as indicated.

    Techniques Used: Activation Assay, Staining, Flow Cytometry, Cytometry, Western Blot

    Effects of inhibition of caspases by Z-VAD-FMK on apoptosis, expression of Mcl-1 and p21, and phosphorylation of Akt and JNK. U937 cells were pretreated with the caspase inhibitor Z-VAD-FMK (20 µM) for 1 h, followed by treatment with 80 µM DIM for 12 h and 24 h. (a) Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. **Values for cells treated with DIM and Z-VAD-FMK were significantly reduced compared to values obtained for DIM alone by Student's t-test, p
    Figure Legend Snippet: Effects of inhibition of caspases by Z-VAD-FMK on apoptosis, expression of Mcl-1 and p21, and phosphorylation of Akt and JNK. U937 cells were pretreated with the caspase inhibitor Z-VAD-FMK (20 µM) for 1 h, followed by treatment with 80 µM DIM for 12 h and 24 h. (a) Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. **Values for cells treated with DIM and Z-VAD-FMK were significantly reduced compared to values obtained for DIM alone by Student's t-test, p

    Techniques Used: Inhibition, Expressing, Staining, Flow Cytometry, Cytometry

    11) Product Images from "Disruption of the EGFR E884-R958 ion pair conserved in the human kinome differentially alters signaling and inhibitor sensitivity"

    Article Title: Disruption of the EGFR E884-R958 ion pair conserved in the human kinome differentially alters signaling and inhibitor sensitivity

    Journal: Oncogene

    doi: 10.1038/onc.2008.411

    Effects of mutational disruption of the Glu(E)884-Arg(R)958 ion pair in EGFR signaling (A) Stable COS-7 transfectant cells expressing the various EGFR variants were cultured in 0.5% BSA-containing serum-free media, followed by EGF stimulation (100 ng/ml, 10 min). Whole cell lysates were prepared for SDS-PAGE and immunoblotted using the antibodies against: phosphotyrosine (pY-EGFR), EGFR, p-CBL[Y774], CBL, p-AKT [S473], AKT, p-ERK1/2 [T202/Y204], ERK1/2, p-STAT3 [Y705], STAT3, and β-actin. E884K, either alone or in- cis with L858R, modulated differential activation of downstream mutant EGFR signaling. (B) The EGFR double mutations L858R+E884K conferred a significantly higher cell proliferation rate than L858R alone in the COS-7 cells stably expressing the transduced mutant EGFR. Cellular viability assay was performed with the cells growing in regular growth media (10% FBS) up to 5 days as described in Materials and Methods. The MTS viability assay was performed in triplicate. Error bar, S.D. *, P =0.0013.
    Figure Legend Snippet: Effects of mutational disruption of the Glu(E)884-Arg(R)958 ion pair in EGFR signaling (A) Stable COS-7 transfectant cells expressing the various EGFR variants were cultured in 0.5% BSA-containing serum-free media, followed by EGF stimulation (100 ng/ml, 10 min). Whole cell lysates were prepared for SDS-PAGE and immunoblotted using the antibodies against: phosphotyrosine (pY-EGFR), EGFR, p-CBL[Y774], CBL, p-AKT [S473], AKT, p-ERK1/2 [T202/Y204], ERK1/2, p-STAT3 [Y705], STAT3, and β-actin. E884K, either alone or in- cis with L858R, modulated differential activation of downstream mutant EGFR signaling. (B) The EGFR double mutations L858R+E884K conferred a significantly higher cell proliferation rate than L858R alone in the COS-7 cells stably expressing the transduced mutant EGFR. Cellular viability assay was performed with the cells growing in regular growth media (10% FBS) up to 5 days as described in Materials and Methods. The MTS viability assay was performed in triplicate. Error bar, S.D. *, P =0.0013.

    Techniques Used: Transfection, Expressing, Cell Culture, SDS Page, Activation Assay, Mutagenesis, Stable Transfection, Cell Viability Assay, Viability Assay

    Mutational disruption of the conserved E1271-R1345 ion pair in MET kinase salt bridge causes inhibitor specific modulation of sensitivity to SU11274 (unchanged) and PHA665752 (more sensitive) (A) MET kinase domain crystal structure (PDB accession code: 2RFS) ( Bellon et al. , 2008 ) highlighting the salt bridge between E1271 and R1345. Crystal structure was solved in complex with SU11274, shown. The conserved Glu-Arg ion pair is shown in stick format, with oxygen atoms colored red, nitrogens colored blue and carbon colored yellow. Figure was prepared using the program PYMOL ( www.pymol.org ). (B) Stable COS-7 transfects expressing E1271K mutant MET were cultured in 0.5% BSA-containing serum-free media for 16 hours, then incubated with increasing concentrations of the MET inhibitors SU11274 (top) and PHA665752 (bottom) as indicated, in the presence of HGF stimulation (50ng/ml). Whole cell lysates were extracted for immunoblotting using antibodies against p-MET [Y1234/Y1235], MET, p-AKT, AKT, p-ERK1/2, ERK1/2 and β-actin. Wild-type MET expressing COS-7 tranfectant cells were included as control. E1271K mutation of MET increased the sensitivity of MET kinase phosphorylation inhibition by PHA665752.
    Figure Legend Snippet: Mutational disruption of the conserved E1271-R1345 ion pair in MET kinase salt bridge causes inhibitor specific modulation of sensitivity to SU11274 (unchanged) and PHA665752 (more sensitive) (A) MET kinase domain crystal structure (PDB accession code: 2RFS) ( Bellon et al. , 2008 ) highlighting the salt bridge between E1271 and R1345. Crystal structure was solved in complex with SU11274, shown. The conserved Glu-Arg ion pair is shown in stick format, with oxygen atoms colored red, nitrogens colored blue and carbon colored yellow. Figure was prepared using the program PYMOL ( www.pymol.org ). (B) Stable COS-7 transfects expressing E1271K mutant MET were cultured in 0.5% BSA-containing serum-free media for 16 hours, then incubated with increasing concentrations of the MET inhibitors SU11274 (top) and PHA665752 (bottom) as indicated, in the presence of HGF stimulation (50ng/ml). Whole cell lysates were extracted for immunoblotting using antibodies against p-MET [Y1234/Y1235], MET, p-AKT, AKT, p-ERK1/2, ERK1/2 and β-actin. Wild-type MET expressing COS-7 tranfectant cells were included as control. E1271K mutation of MET increased the sensitivity of MET kinase phosphorylation inhibition by PHA665752.

    Techniques Used: Expressing, Mutagenesis, Cell Culture, Incubation, Inhibition

    E884K mutation of EGFR worked in concert with L858R to differentially alter sensitivity to EGFR kinase inhibitors erlotinib and gefitinib (A) Stable COS-7 transfects expressing the L858R and double mutant L858R+E884K variants of EGFR were used in the experiment. The endogenous wild type EGFR expression of parental COS-7 cells is negligible (data not shown). Cells were cultured in 0.5% BSA-containing serum-free media for 16 hours and then incubated with increasing concentrations of either erlotinib or gefitinib in the presence of EGF (100 ng/ml). Whole cell lysates were extracted for SDS-PAGE and immunoblotting using antibodies against: p-EGFR [Y1068], phosphotyrosine (p-Tyr), EGFR, p-AKT [S473], AKT, p-STAT3 [Y705], STAT3, c-PARP [cleaved-PARP(Asp214)], and β-actin. The experiment was performed in duplicate with reproducible results. The E884K mutation negatively modulated the effect of L858R to erlotinib inhibition in a dominant fashion but enhanced sensitivity of the mutant receptor to gefitinib inhibition. (B) Densitometric quantitation of the p-EGFR [Y1068] levels showing differential alteration of sensitivity to erlotinib (more resistant) and gefitinib (more sensitive) by the E884K mutation when in- cis with L858R. The densitometric scanning of the p-EGFR immunoblot bands was performed digitally using the NIH ImageJ software program, and was normalized to the total EGFR expression levels. (C) Relative expression of the apoptotic marker, cleaved-PARP(Asp214) in L858R and L858R+E884K EGFR variants treated with increasing concentrations of erlotinib (left) and gefitinib (right). The immunoblot using anti-cleaved-PARP(Asp214) antibody is shown here (above) together with the densitometric quantitation (below) adjusted to β-actin loading control using the NIH ImageJ software program. (D) COS-7 cells with stable transduced expression of L858R or L858R+E884K mutant EGFR were tested in cellular cytotoxicity assay in vitro under drug treatment with either erlotinib or gefitinib at indicated concentrations. Results are shown in percent change of cell viability of L858R+E884K EGFR-COS-7 compared to the control L858R EGFR-COS-7 cells at each concentration of TKI tested. E884K mutation, when in- cis with L858R, significantly decreased the sensitivity of cell viability inhibition by erlotinib compared with L858R alone; however, it significantly increased the sensitivity of cell viability inhibition by gefitinib compared with L858R alone. In the case of erlotinib, E884K was desensitizing to L858R, leading to lower cytotoxicity (56.3 ±2.68% increased viable cells after inhibition at 5 μM, P =0.0004) compared with L858R alone. Conversely, in gefitinib inhibition, E884K further sensitized L858R in- cis , leading to significantly higher cytotoxicity (63.5 ±6.86% decreased viable cells after inhibition at 5 μM, P =0.0013) compared with L858R alone. Error bar, S.D. (N=3). *, P
    Figure Legend Snippet: E884K mutation of EGFR worked in concert with L858R to differentially alter sensitivity to EGFR kinase inhibitors erlotinib and gefitinib (A) Stable COS-7 transfects expressing the L858R and double mutant L858R+E884K variants of EGFR were used in the experiment. The endogenous wild type EGFR expression of parental COS-7 cells is negligible (data not shown). Cells were cultured in 0.5% BSA-containing serum-free media for 16 hours and then incubated with increasing concentrations of either erlotinib or gefitinib in the presence of EGF (100 ng/ml). Whole cell lysates were extracted for SDS-PAGE and immunoblotting using antibodies against: p-EGFR [Y1068], phosphotyrosine (p-Tyr), EGFR, p-AKT [S473], AKT, p-STAT3 [Y705], STAT3, c-PARP [cleaved-PARP(Asp214)], and β-actin. The experiment was performed in duplicate with reproducible results. The E884K mutation negatively modulated the effect of L858R to erlotinib inhibition in a dominant fashion but enhanced sensitivity of the mutant receptor to gefitinib inhibition. (B) Densitometric quantitation of the p-EGFR [Y1068] levels showing differential alteration of sensitivity to erlotinib (more resistant) and gefitinib (more sensitive) by the E884K mutation when in- cis with L858R. The densitometric scanning of the p-EGFR immunoblot bands was performed digitally using the NIH ImageJ software program, and was normalized to the total EGFR expression levels. (C) Relative expression of the apoptotic marker, cleaved-PARP(Asp214) in L858R and L858R+E884K EGFR variants treated with increasing concentrations of erlotinib (left) and gefitinib (right). The immunoblot using anti-cleaved-PARP(Asp214) antibody is shown here (above) together with the densitometric quantitation (below) adjusted to β-actin loading control using the NIH ImageJ software program. (D) COS-7 cells with stable transduced expression of L858R or L858R+E884K mutant EGFR were tested in cellular cytotoxicity assay in vitro under drug treatment with either erlotinib or gefitinib at indicated concentrations. Results are shown in percent change of cell viability of L858R+E884K EGFR-COS-7 compared to the control L858R EGFR-COS-7 cells at each concentration of TKI tested. E884K mutation, when in- cis with L858R, significantly decreased the sensitivity of cell viability inhibition by erlotinib compared with L858R alone; however, it significantly increased the sensitivity of cell viability inhibition by gefitinib compared with L858R alone. In the case of erlotinib, E884K was desensitizing to L858R, leading to lower cytotoxicity (56.3 ±2.68% increased viable cells after inhibition at 5 μM, P =0.0004) compared with L858R alone. Conversely, in gefitinib inhibition, E884K further sensitized L858R in- cis , leading to significantly higher cytotoxicity (63.5 ±6.86% decreased viable cells after inhibition at 5 μM, P =0.0013) compared with L858R alone. Error bar, S.D. (N=3). *, P

    Techniques Used: Mutagenesis, Expressing, Cell Culture, Incubation, SDS Page, Inhibition, Quantitation Assay, Software, Marker, Cytotoxicity Assay, In Vitro, Concentration Assay

    12) Product Images from "Oligonol, a Low-Molecular Weight Polyphenol Derived from Lychee, Alleviates Muscle Loss in Diabetes by Suppressing Atrogin-1 and MuRF1"

    Article Title: Oligonol, a Low-Molecular Weight Polyphenol Derived from Lychee, Alleviates Muscle Loss in Diabetes by Suppressing Atrogin-1 and MuRF1

    Journal: Nutrients

    doi: 10.3390/nu9091040

    Representative blots of nuclear FoxO3a ( A ), SIRT1 ( B ), phospho-Akt (Thr473), Akt ( C ), phospho-FoxO3a (Ser253), and total FoxO3a ( D ) in m/m and db/db mice with or without oligonol (OLG) supplement are shown ( n = 5–6/group). Significance ( p
    Figure Legend Snippet: Representative blots of nuclear FoxO3a ( A ), SIRT1 ( B ), phospho-Akt (Thr473), Akt ( C ), phospho-FoxO3a (Ser253), and total FoxO3a ( D ) in m/m and db/db mice with or without oligonol (OLG) supplement are shown ( n = 5–6/group). Significance ( p

    Techniques Used: Mouse Assay

    13) Product Images from "A Novel Network Pharmacology Strategy to Decode Mechanism of Lang Chuang Wan in Treating Systemic Lupus Erythematosus"

    Article Title: A Novel Network Pharmacology Strategy to Decode Mechanism of Lang Chuang Wan in Treating Systemic Lupus Erythematosus

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2020.512877

    Effects of ferulic acid (A) and liquiritin (B) on resiquimod-induced RAW 264.7 cells IL-6 cytokines expression and the protein expression of p-ERK1/2, p-AKT, and p-PI3K of resiquimod induced RAW264.7 cells (C–F) . ## p
    Figure Legend Snippet: Effects of ferulic acid (A) and liquiritin (B) on resiquimod-induced RAW 264.7 cells IL-6 cytokines expression and the protein expression of p-ERK1/2, p-AKT, and p-PI3K of resiquimod induced RAW264.7 cells (C–F) . ## p

    Techniques Used: Expressing

    14) Product Images from "TLR2 ligand induces protection against cerebral ischemia/reperfusion injury: via activation of PI3K/Akt signaling"

    Article Title: TLR2 ligand induces protection against cerebral ischemia/reperfusion injury: via activation of PI3K/Akt signaling

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1003428

    Pam3CSK4 administration increases the levels of Akt and GSK-3β phosphorylation and association of TLR2 with PI3K in the brain tissues following cerebral I/R. Mice were treated with or without Pam3CSK4 one hr prior to cerebral ischemia (45 min)
    Figure Legend Snippet: Pam3CSK4 administration increases the levels of Akt and GSK-3β phosphorylation and association of TLR2 with PI3K in the brain tissues following cerebral I/R. Mice were treated with or without Pam3CSK4 one hr prior to cerebral ischemia (45 min)

    Techniques Used: Mouse Assay

    15) Product Images from "Age-dependent Accumulation of Soluble Amyloid ? (A?) Oligomers Reverses the Neuroprotective Effect of Soluble Amyloid Precursor Protein-? (sAPP?) by Modulating Phosphatidylinositol 3-Kinase (PI3K)/Akt-GSK-3? Pathway in Alzheimer Mouse Model *"

    Article Title: Age-dependent Accumulation of Soluble Amyloid ? (A?) Oligomers Reverses the Neuroprotective Effect of Soluble Amyloid Precursor Protein-? (sAPP?) by Modulating Phosphatidylinositol 3-Kinase (PI3K)/Akt-GSK-3? Pathway in Alzheimer Mouse Model *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.209718

    Soluble fractions, derived from 6- or 18-month-old PS1×APP mice, produced the stimulation or inhibition of Akt-GSK-3β phosphorylation. A , N2a cultures were serum-deprived for 12 h and treated with 50 μg of soluble proteins derived
    Figure Legend Snippet: Soluble fractions, derived from 6- or 18-month-old PS1×APP mice, produced the stimulation or inhibition of Akt-GSK-3β phosphorylation. A , N2a cultures were serum-deprived for 12 h and treated with 50 μg of soluble proteins derived

    Techniques Used: Derivative Assay, Mouse Assay, Produced, Inhibition

    Synthetic sAPPα and Aβ oligomers (ADDLs) stimulated or inhibited the Akt-GSK-3β phosphorylation in N2a cultures. A , sAPPα activated the IGF-1R/IR Akt GSK-3β pathway. Serum-deprived N2a cultures were treated with
    Figure Legend Snippet: Synthetic sAPPα and Aβ oligomers (ADDLs) stimulated or inhibited the Akt-GSK-3β phosphorylation in N2a cultures. A , sAPPα activated the IGF-1R/IR Akt GSK-3β pathway. Serum-deprived N2a cultures were treated with

    Techniques Used:

    16) Product Images from "Initial Testing (Stage 1) of the Phosphatidylinositol 3’ Kinase Inhibitor, SAR245408 (XL147) by the Pediatric Preclinical Testing Program"

    Article Title: Initial Testing (Stage 1) of the Phosphatidylinositol 3’ Kinase Inhibitor, SAR245408 (XL147) by the Pediatric Preclinical Testing Program

    Journal: Pediatric blood & cancer

    doi: 10.1002/pbc.24301

    ); B. Protein levels for PTEN, Akt and phospho-Akt in solid tumor
    Figure Legend Snippet: ); B. Protein levels for PTEN, Akt and phospho-Akt in solid tumor

    Techniques Used:

    17) Product Images from "Induction of autophagy contributes to crizotinib resistance in ALK-positive lung cancer"

    Article Title: Induction of autophagy contributes to crizotinib resistance in ALK-positive lung cancer

    Journal: Cancer Biology & Therapy

    doi: 10.4161/cbt.28162

    Figure 3. Akt/mTOR pathway is involved in autophagy activation in crizotinib-resistant lung cancer cells. H3122 and H3122CR-1 cells were treated with the indicated concentrations of crizonitib or vehicle for 2 h. The levels of phospho-ALK, phospho-Akt,
    Figure Legend Snippet: Figure 3. Akt/mTOR pathway is involved in autophagy activation in crizotinib-resistant lung cancer cells. H3122 and H3122CR-1 cells were treated with the indicated concentrations of crizonitib or vehicle for 2 h. The levels of phospho-ALK, phospho-Akt,

    Techniques Used: Activation Assay

    18) Product Images from "IKBKE is required during KRAS-induced pancreatic tumorigenesis"

    Article Title: IKBKE is required during KRAS-induced pancreatic tumorigenesis

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-15-1684

    IKBKE mediates AKT reactivation post-mTOR inhibition (A–B) Western blot showing phosphorylation of Akt and other mTOR substrates, S6K and 4EBP1, in control Panc-1 cells (A) and Panc-1 cells with IKBKE knockdown (B) in response to treatment with Torin-1 up to 24 hours. (C) Relative cell viability of Panc-1 cells measured by MTT assay 5 days after treatment with increasing dosage of the mTOR inhibitor Torin-1 in the presence and absence of IKBKE knockdown. (D) Cleaved-Caspase 3 staining in Panc-1 cells indicates that while mTOR inhibition alone using Torin-1 does not lead to a significant increase in apoptosis, combined inhibition of IKBKE and mTOR leads to a significant increase in apoptosis. (E) Soft agar colony formation assay in Panc-1 cells indicates that while mTOR inhibition alone does not affect tumorigenicity of the cells, combined inhibition of mTOR and IKBKE leads to significant decrease in tumorigenicity. (F) Tumor volume measurement indicates significant decrease in growth of MiaPaca-2 cell line derived xenograft tumors (n = 6) in response to inducible IKBKE knockdown, and synergistic effect with combined IKBKE and mTOR knockdown while mTOR knockdown has no effect. (G) Representative images of xenograft tumors 4 weeks after induction of shRNA indicate significant reduction in tumor volume in response to IKBKE knockdown and synergistic reduction with combined IKBKE / mTOR knockdown. (H–K) Representative IHC images showing phosphorylation of AKT at Serine-473 in xenograft tumors derived from Control (H), shmTOR (I), shIKBKE (J), and shIKBKE/shmTOR (K) MiaPaca-2 cell lines. Error bars represent Standard Deviation. Statistical significance was determined using a Student’s two-tailed t-test. * P
    Figure Legend Snippet: IKBKE mediates AKT reactivation post-mTOR inhibition (A–B) Western blot showing phosphorylation of Akt and other mTOR substrates, S6K and 4EBP1, in control Panc-1 cells (A) and Panc-1 cells with IKBKE knockdown (B) in response to treatment with Torin-1 up to 24 hours. (C) Relative cell viability of Panc-1 cells measured by MTT assay 5 days after treatment with increasing dosage of the mTOR inhibitor Torin-1 in the presence and absence of IKBKE knockdown. (D) Cleaved-Caspase 3 staining in Panc-1 cells indicates that while mTOR inhibition alone using Torin-1 does not lead to a significant increase in apoptosis, combined inhibition of IKBKE and mTOR leads to a significant increase in apoptosis. (E) Soft agar colony formation assay in Panc-1 cells indicates that while mTOR inhibition alone does not affect tumorigenicity of the cells, combined inhibition of mTOR and IKBKE leads to significant decrease in tumorigenicity. (F) Tumor volume measurement indicates significant decrease in growth of MiaPaca-2 cell line derived xenograft tumors (n = 6) in response to inducible IKBKE knockdown, and synergistic effect with combined IKBKE and mTOR knockdown while mTOR knockdown has no effect. (G) Representative images of xenograft tumors 4 weeks after induction of shRNA indicate significant reduction in tumor volume in response to IKBKE knockdown and synergistic reduction with combined IKBKE / mTOR knockdown. (H–K) Representative IHC images showing phosphorylation of AKT at Serine-473 in xenograft tumors derived from Control (H), shmTOR (I), shIKBKE (J), and shIKBKE/shmTOR (K) MiaPaca-2 cell lines. Error bars represent Standard Deviation. Statistical significance was determined using a Student’s two-tailed t-test. * P

    Techniques Used: Inhibition, Western Blot, MTT Assay, Staining, Soft Agar Assay, Derivative Assay, shRNA, Immunohistochemistry, Standard Deviation, Two Tailed Test

    IKBKE regulates AKT activation in PDAC cells in vivo and in vitro (A–B) Immunohistochemistry for AKT phosphorylation at Serine-473 in stage matched PanIN lesions of P48Cre;Kras G12D (A), and P48Cre;Kras G12D ; IKBKE −/− (B) mouse pancreas. P48Cre;Kras G12D ;IKBKE −/− PanIN lesions have significantly reduced AKT phosphorylation compared to P48Cre;Kras G12D lesions. (C) Correlation of IKBKE protein levels and AKT Serine-473 phosphorylation levels in human PDAC tissue microarray. Representative images of immunohistochemistry for IKBKE (i–iii) and phospho-AKT Serine-473 (iv–vi) in matched human PDAC tissue samples. (D) Western blot showing phosphorylation of AKT and ERK in serum starved Panc-1 cells in response to shRNA mediated IKBKE knockdown. IKBKE knockdown leads to decrease in phosphorylation of AKT at Serine-473, but not ERK phosphorylation. (E) Western blot of Panc-1 cells with or without inhibition of mTOR using Torin-1, and shRNA mediated knockdown of IKBKE . Inhibition of IKBKE and mTOR leads to decrease in phosphorylation of AKT at Serine-473 and Threonine-308.
    Figure Legend Snippet: IKBKE regulates AKT activation in PDAC cells in vivo and in vitro (A–B) Immunohistochemistry for AKT phosphorylation at Serine-473 in stage matched PanIN lesions of P48Cre;Kras G12D (A), and P48Cre;Kras G12D ; IKBKE −/− (B) mouse pancreas. P48Cre;Kras G12D ;IKBKE −/− PanIN lesions have significantly reduced AKT phosphorylation compared to P48Cre;Kras G12D lesions. (C) Correlation of IKBKE protein levels and AKT Serine-473 phosphorylation levels in human PDAC tissue microarray. Representative images of immunohistochemistry for IKBKE (i–iii) and phospho-AKT Serine-473 (iv–vi) in matched human PDAC tissue samples. (D) Western blot showing phosphorylation of AKT and ERK in serum starved Panc-1 cells in response to shRNA mediated IKBKE knockdown. IKBKE knockdown leads to decrease in phosphorylation of AKT at Serine-473, but not ERK phosphorylation. (E) Western blot of Panc-1 cells with or without inhibition of mTOR using Torin-1, and shRNA mediated knockdown of IKBKE . Inhibition of IKBKE and mTOR leads to decrease in phosphorylation of AKT at Serine-473 and Threonine-308.

    Techniques Used: Activation Assay, In Vivo, In Vitro, Immunohistochemistry, Microarray, Western Blot, shRNA, Inhibition

    19) Product Images from "Memory enhancement and protective effects of crocin against D-galactose aging model in the hippocampus of Wistar rats"

    Article Title: Memory enhancement and protective effects of crocin against D-galactose aging model in the hippocampus of Wistar rats

    Journal: Iranian Journal of Basic Medical Sciences

    doi: 10.22038/IJBMS.2017.9541

    Proposed Schematic mechanism of crocin against D-gal neurotoxicity. This diagram shows possible mechanisms of crocin on synaptic plasticity, which prevented memory impairment, through MAPK and Akt pathways activation in D-gal induced aging rat model. In addition, crocin decreases ROS induction, AGEs formation and neuroinflammation
    Figure Legend Snippet: Proposed Schematic mechanism of crocin against D-gal neurotoxicity. This diagram shows possible mechanisms of crocin on synaptic plasticity, which prevented memory impairment, through MAPK and Akt pathways activation in D-gal induced aging rat model. In addition, crocin decreases ROS induction, AGEs formation and neuroinflammation

    Techniques Used: Activation Assay

    20) Product Images from "Effect of Targeted Therapy With Pazopanib on Expression Levels of Transcription, Growth Factors and Components of AKT/m-TOR Signaling Pathway in Patients with Renal Cell Carcinoma"

    Article Title: Effect of Targeted Therapy With Pazopanib on Expression Levels of Transcription, Growth Factors and Components of AKT/m-TOR Signaling Pathway in Patients with Renal Cell Carcinoma

    Journal: Asian Pacific Journal of Cancer Prevention : APJCP

    doi: 10.22034/APJCP.2017.18.11.2977

    Immunoblots of AKT/m-TOR Components in Kidney Cancer Tissues. a – AKT, phospho-PDK1, phospho-c-Raf, phospho-GSK-3beta and phospho-PTEN level in non transformed (1) and cancer (2) tissues; b – m-TOR, phospho-m-TOR, 70S 6 kinase and 4E-BP1 levels in non transformed (1) and cancer (2) tissues. Note: The results were standardized using the beta-actin expression in a sample and were expressed in percentages to the protein content in non-transformed tissues. The level of protein in normal non-altered tissue was indicated as 100%. It is found the activation of AKT/m-TOR signaling pathway in kidney cancers by increase of AKT, its phosphorylated form, proteinkinase m-TOR, glycogen regulator GSK-3-ß and transcription inhibitor 4E-BP1.
    Figure Legend Snippet: Immunoblots of AKT/m-TOR Components in Kidney Cancer Tissues. a – AKT, phospho-PDK1, phospho-c-Raf, phospho-GSK-3beta and phospho-PTEN level in non transformed (1) and cancer (2) tissues; b – m-TOR, phospho-m-TOR, 70S 6 kinase and 4E-BP1 levels in non transformed (1) and cancer (2) tissues. Note: The results were standardized using the beta-actin expression in a sample and were expressed in percentages to the protein content in non-transformed tissues. The level of protein in normal non-altered tissue was indicated as 100%. It is found the activation of AKT/m-TOR signaling pathway in kidney cancers by increase of AKT, its phosphorylated form, proteinkinase m-TOR, glycogen regulator GSK-3-ß and transcription inhibitor 4E-BP1.

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

    21) Product Images from "TGFβ-Induced Deptor Suppression Recruits mTORC1 and Not mTORC2 to Enhance Collagen I (α2) Gene Expression"

    Article Title: TGFβ-Induced Deptor Suppression Recruits mTORC1 and Not mTORC2 to Enhance Collagen I (α2) Gene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0109608

    TGFβ-induced suppression of deptor regulates collagen expression in proximal tubular epithelial cells. (A–D) TGFβ decreases deptor resulting in increased mTORC1 and mTORC2 activity. Human proximal tubular epithelial cells were incubated with 2 ng/ml TGFβ for indicated period of time. The cell lysates were immunoblotted with deptor, actin (panel A), phospho-S6 kinase (Thr-389), S6 kinase (panel B), phospho-4EBP-1 (Thr-37/46), 4EBP-1 (panel C) and phospho-Akt (Ser-473), phospho-Akt (Thr-308) and Akt (panel D) antibodies as indicated. (E–G and I) Expression of deptor inhibits mTORC1 and mTORC2 activities to block collagen expression. Human proximal tubular epithelial cells were transfected with FLAG-tagged Deptor expression plasmid or vector. Transfected cells were incubated with 2 ng/ml TGFβ for 24 hours. The cell lysates were immunoblotted with phospho-S6 kinase, S6 kinase (panel E), phospho-4EBP-1, 4EBP-1 (panel F), phospho-Akt, Akt (panel G), collagen I (α2), actin (panel I) as indicated. The same lysates were used to immunoblot with FLAG antibody to demonstrate deptor expression. Quantifications of panels A–G are shown in Fig. S2A – S2G . (H) Expression of deptor inhibits mTORC1 and mTORC2 activities to block collagen mRNA expression. Human proximal tubular epithelial cells were transfected with FLAG-tagged Deptor expression plasmid or vector. Transfected cells were incubated with 2 ng/ml TGFβ for 24 hours. Total RNAs were prepared and used for real time RT-PCR to detect collagen mRNA as described in the Materials and Methods . Mean ± SE of triplicate measurements is shown. *p
    Figure Legend Snippet: TGFβ-induced suppression of deptor regulates collagen expression in proximal tubular epithelial cells. (A–D) TGFβ decreases deptor resulting in increased mTORC1 and mTORC2 activity. Human proximal tubular epithelial cells were incubated with 2 ng/ml TGFβ for indicated period of time. The cell lysates were immunoblotted with deptor, actin (panel A), phospho-S6 kinase (Thr-389), S6 kinase (panel B), phospho-4EBP-1 (Thr-37/46), 4EBP-1 (panel C) and phospho-Akt (Ser-473), phospho-Akt (Thr-308) and Akt (panel D) antibodies as indicated. (E–G and I) Expression of deptor inhibits mTORC1 and mTORC2 activities to block collagen expression. Human proximal tubular epithelial cells were transfected with FLAG-tagged Deptor expression plasmid or vector. Transfected cells were incubated with 2 ng/ml TGFβ for 24 hours. The cell lysates were immunoblotted with phospho-S6 kinase, S6 kinase (panel E), phospho-4EBP-1, 4EBP-1 (panel F), phospho-Akt, Akt (panel G), collagen I (α2), actin (panel I) as indicated. The same lysates were used to immunoblot with FLAG antibody to demonstrate deptor expression. Quantifications of panels A–G are shown in Fig. S2A – S2G . (H) Expression of deptor inhibits mTORC1 and mTORC2 activities to block collagen mRNA expression. Human proximal tubular epithelial cells were transfected with FLAG-tagged Deptor expression plasmid or vector. Transfected cells were incubated with 2 ng/ml TGFβ for 24 hours. Total RNAs were prepared and used for real time RT-PCR to detect collagen mRNA as described in the Materials and Methods . Mean ± SE of triplicate measurements is shown. *p

    Techniques Used: Expressing, Activity Assay, Incubation, Blocking Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR

    22) Product Images from "A positive feedback loop involving Erk5 and Akt turns on mesangial cell proliferation in response to PDGF"

    Article Title: A positive feedback loop involving Erk5 and Akt turns on mesangial cell proliferation in response to PDGF

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00387.2013

    Akt kinase regulates PDGF-induced phosphorylation of Erk5. Mesangial cells were treated with MK2206 before incubation with PDGF for 5 min ( A and B ). Mesangial cells were transfected with dominant negative Akt K179M ( C and D ) or Myr Akt ( E – G ).
    Figure Legend Snippet: Akt kinase regulates PDGF-induced phosphorylation of Erk5. Mesangial cells were treated with MK2206 before incubation with PDGF for 5 min ( A and B ). Mesangial cells were transfected with dominant negative Akt K179M ( C and D ) or Myr Akt ( E – G ).

    Techniques Used: Incubation, Transfection, Dominant Negative Mutation

    Erk5 regulates PDGF-stimulated Akt activity. A and B : mesangial cells were treated with XMD8-92 before incubation with PDGF for 5 min. C – K : mesangial cells were transfected with dominant negative Erk5 ( C and D ) or siErk5A ( E and F ) or siErk5B
    Figure Legend Snippet: Erk5 regulates PDGF-stimulated Akt activity. A and B : mesangial cells were treated with XMD8-92 before incubation with PDGF for 5 min. C – K : mesangial cells were transfected with dominant negative Erk5 ( C and D ) or siErk5A ( E and F ) or siErk5B

    Techniques Used: Activity Assay, Incubation, Transfection, Dominant Negative Mutation

    23) Product Images from "Molecular analysis of the dual targeting of the epidermal growth factor receptor and the O6-methylguanine-DNA methyltransferase with a double arm hybrid molecule"

    Article Title: Molecular analysis of the dual targeting of the epidermal growth factor receptor and the O6-methylguanine-DNA methyltransferase with a double arm hybrid molecule

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25120

    Schematic representation of the dual mechanism of action of MR30 Through its EGFR targeting arm MR30 can block RAF-MAPK-ERK1/2 and the PI3K-AKT pathways. Through its MGMT targeting arm, it can induce S-benzylation and subsequent down-regulation of MGMT and potentiate TMZ in tumour cells co-expressing EGFR and MGMT.
    Figure Legend Snippet: Schematic representation of the dual mechanism of action of MR30 Through its EGFR targeting arm MR30 can block RAF-MAPK-ERK1/2 and the PI3K-AKT pathways. Through its MGMT targeting arm, it can induce S-benzylation and subsequent down-regulation of MGMT and potentiate TMZ in tumour cells co-expressing EGFR and MGMT.

    Techniques Used: Blocking Assay, Expressing

    Effects of MR30 on cell signaling (A) Inhibition of phosphorylation of EGFR and downstream signaling proteins, p-ERK1/2 and p-AKT by MR30 was studied at the 0.1, 1, 2 and 10x IC 50 concentrations on A549 (wild-type EGFR), A375 (wild-type EGFR) and H1650 (EGFR d746-750) by western blot. MR30 strongly inhibited EGFR autophosphorylation at submicromolar concentrations, which has led to inhibition of downstream signaling by inhibiting p-ERK1/2 and p-AKT. (B) MR30 and gefitinib were tested in in vitro EGFR kinase assay. MR30 as well as gefitinib showed dose dependent inhibition of EGFR phosphorylation. Gefitinib induced stronger inhibition of EGFR phosphorylation than MR30.
    Figure Legend Snippet: Effects of MR30 on cell signaling (A) Inhibition of phosphorylation of EGFR and downstream signaling proteins, p-ERK1/2 and p-AKT by MR30 was studied at the 0.1, 1, 2 and 10x IC 50 concentrations on A549 (wild-type EGFR), A375 (wild-type EGFR) and H1650 (EGFR d746-750) by western blot. MR30 strongly inhibited EGFR autophosphorylation at submicromolar concentrations, which has led to inhibition of downstream signaling by inhibiting p-ERK1/2 and p-AKT. (B) MR30 and gefitinib were tested in in vitro EGFR kinase assay. MR30 as well as gefitinib showed dose dependent inhibition of EGFR phosphorylation. Gefitinib induced stronger inhibition of EGFR phosphorylation than MR30.

    Techniques Used: Inhibition, Western Blot, In Vitro, Kinase Assay

    24) Product Images from "Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity"

    Article Title: Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI35721

    Inhibition of phospho-CRKL by imatinib in CML stem and progenitor cells. ( A ) Phospho-CRKL levels in normal CD34 + cells were compared with CML CD34 + cells treated or not with imatinib. Total CRKL and actin are shown as loading controls. ( B ) Phospho-CRKL and ( C ) phospho-STAT5 immunoblots with lysate from sorted CD34 + CD38 + and CD34 + CD38 – CML cells treated 4 hours with or without 5 μM imatinib demonstrated inhibition of BCR-ABL by imatinib in both populations. Total CRKL and total STAT5 are shown as respective loading controls. ( D ) Phospho-CRKL, phospho-STAT5, and phospho-AKT immunocytochemical stains of sorted CD34 + CD38 – and CD34 + CD38 + CML cells treated 4 hours with or without 5 μM imatinib demonstrated BCR-ABL inhibition in all cells within the population. Representative cells are shown. Identical data were obtained for a second sample (not shown).
    Figure Legend Snippet: Inhibition of phospho-CRKL by imatinib in CML stem and progenitor cells. ( A ) Phospho-CRKL levels in normal CD34 + cells were compared with CML CD34 + cells treated or not with imatinib. Total CRKL and actin are shown as loading controls. ( B ) Phospho-CRKL and ( C ) phospho-STAT5 immunoblots with lysate from sorted CD34 + CD38 + and CD34 + CD38 – CML cells treated 4 hours with or without 5 μM imatinib demonstrated inhibition of BCR-ABL by imatinib in both populations. Total CRKL and total STAT5 are shown as respective loading controls. ( D ) Phospho-CRKL, phospho-STAT5, and phospho-AKT immunocytochemical stains of sorted CD34 + CD38 – and CD34 + CD38 + CML cells treated 4 hours with or without 5 μM imatinib demonstrated BCR-ABL inhibition in all cells within the population. Representative cells are shown. Identical data were obtained for a second sample (not shown).

    Techniques Used: Inhibition, Western Blot

    25) Product Images from "Antrocin Sensitizes Prostate Cancer Cells to Radiotherapy through Inhibiting PI3K/AKT and MAPK Signaling Pathways"

    Article Title: Antrocin Sensitizes Prostate Cancer Cells to Radiotherapy through Inhibiting PI3K/AKT and MAPK Signaling Pathways

    Journal: Cancers

    doi: 10.3390/cancers11010034

    Proposed mechanism of action for antrocin-induced sensitization to radiotherapy in radioresistant PCa cells. IGF-1 binds to IGF-1R and induces IRS/Shc phosphorylation, which leads to the activation of PI3K/AKT and MAPK signaling. Co-treatment of cells with antrocin and radiation inhibits IGF1-induced activation of the PI3K/AKT and MAPK pathways, effectively sensitizing PCa cells to radiotherapy. IRS: insulin receptor substrate; Shc: src homology/collagen; LEF: lymphoid enhancer factor; TCF: transcription factor; Ub: ubiquitin.
    Figure Legend Snippet: Proposed mechanism of action for antrocin-induced sensitization to radiotherapy in radioresistant PCa cells. IGF-1 binds to IGF-1R and induces IRS/Shc phosphorylation, which leads to the activation of PI3K/AKT and MAPK signaling. Co-treatment of cells with antrocin and radiation inhibits IGF1-induced activation of the PI3K/AKT and MAPK pathways, effectively sensitizing PCa cells to radiotherapy. IRS: insulin receptor substrate; Shc: src homology/collagen; LEF: lymphoid enhancer factor; TCF: transcription factor; Ub: ubiquitin.

    Techniques Used: Activation Assay

    Antrocin inhibits the PI3K/AKT and MAPK signaling pathways and phosphorylation of IGF-1R. PC3-KD cells were treated with IR (2 Gy), antrocin (100 μM), or IR plus antrocin, followed by incubation for 48 h. ( A ) The represented gene network was identified by ingenuity pathway analysis. The color of each line indicates the regulation of gene expression. ( B ) Cell lysates were harvested and subjected to Western blot analysis using antibodies against p85, p110, p-AKT, AKT, GSK3-β, p-GSK3-β, p-β-catenin, and β-catenin, and ( C ) antibodies against IGF-1R, p38, JNK, Erk1/2, and their respective phosphorylated forms. The data represent one of three independent experiments. β-actin was used as a loading control. The expression level of each protein was quantified by the signal intensity and normalized with each vehicle untreated group. The relative level of each protein expression was indicated at the bottom of each lane.
    Figure Legend Snippet: Antrocin inhibits the PI3K/AKT and MAPK signaling pathways and phosphorylation of IGF-1R. PC3-KD cells were treated with IR (2 Gy), antrocin (100 μM), or IR plus antrocin, followed by incubation for 48 h. ( A ) The represented gene network was identified by ingenuity pathway analysis. The color of each line indicates the regulation of gene expression. ( B ) Cell lysates were harvested and subjected to Western blot analysis using antibodies against p85, p110, p-AKT, AKT, GSK3-β, p-GSK3-β, p-β-catenin, and β-catenin, and ( C ) antibodies against IGF-1R, p38, JNK, Erk1/2, and their respective phosphorylated forms. The data represent one of three independent experiments. β-actin was used as a loading control. The expression level of each protein was quantified by the signal intensity and normalized with each vehicle untreated group. The relative level of each protein expression was indicated at the bottom of each lane.

    Techniques Used: Incubation, Expressing, Western Blot

    Representative immunohistochemical (IHC) staining patterns in xenograft PCa tissue. IHC analysis of paraffin sections show staining with specific antibodies against PARP, cleaved-caspase 9, phospho-AKT, phospho-β-catenin, and cyclin D1, respectively. Images were photographed at 200× magnification.
    Figure Legend Snippet: Representative immunohistochemical (IHC) staining patterns in xenograft PCa tissue. IHC analysis of paraffin sections show staining with specific antibodies against PARP, cleaved-caspase 9, phospho-AKT, phospho-β-catenin, and cyclin D1, respectively. Images were photographed at 200× magnification.

    Techniques Used: Immunohistochemistry, Staining

    26) Product Images from "LonP1 Differently Modulates Mitochondrial Function and Bioenergetics of Primary Versus Metastatic Colon Cancer Cells"

    Article Title: LonP1 Differently Modulates Mitochondrial Function and Bioenergetics of Primary Versus Metastatic Colon Cancer Cells

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2018.00254

    LonP1 modulates β-ctn by regulating Akt/GSK3β signaling. (A) Representative Western blot analysis and relative protein levels of phosphorylated Akt (Ser473), Akt, phosphorylated GSK-3β (Ser9), and GSK-3β in SW480 cells overexpressing LonP1. (B) Representative Western blot analysis and relative protein levels of phosphorylated Akt (Ser473), Akt, phosphorylated GSK-3β (Ser9), and GSK-3β in SW620 downregulating LonP1. β-actin was used as loading control. Densitometries are reported in histograms, and data are reported as mean ± SD ( n = 3). * P
    Figure Legend Snippet: LonP1 modulates β-ctn by regulating Akt/GSK3β signaling. (A) Representative Western blot analysis and relative protein levels of phosphorylated Akt (Ser473), Akt, phosphorylated GSK-3β (Ser9), and GSK-3β in SW480 cells overexpressing LonP1. (B) Representative Western blot analysis and relative protein levels of phosphorylated Akt (Ser473), Akt, phosphorylated GSK-3β (Ser9), and GSK-3β in SW620 downregulating LonP1. β-actin was used as loading control. Densitometries are reported in histograms, and data are reported as mean ± SD ( n = 3). * P

    Techniques Used: Western Blot

    27) Product Images from "(-)-EPIGALLOCATECHIN-3-GALLATE IS A NOVEL HSP90 INHIBITOR †"

    Article Title: (-)-EPIGALLOCATECHIN-3-GALLATE IS A NOVEL HSP90 INHIBITOR †

    Journal: Biochemistry

    doi: 10.1021/bi801637q

    EGCG modifies the interaction of hsp90 with cochaperones, and leads to the degradation of the hsp90 client proteins ErbB2, Raf-1 and phospho-AKT. A. Hepa cytosol was incubated with DMSO or 200μM EGCG (E) for 1h at RT and immunoprecipitated with
    Figure Legend Snippet: EGCG modifies the interaction of hsp90 with cochaperones, and leads to the degradation of the hsp90 client proteins ErbB2, Raf-1 and phospho-AKT. A. Hepa cytosol was incubated with DMSO or 200μM EGCG (E) for 1h at RT and immunoprecipitated with

    Techniques Used: Incubation, Immunoprecipitation

    28) Product Images from "Germline KRAS Mutations Cause Aberrant Biochemical and Physical Properties Leading to Developmental Disorders"

    Article Title: Germline KRAS Mutations Cause Aberrant Biochemical and Physical Properties Leading to Developmental Disorders

    Journal: Human mutation

    doi: 10.1002/humu.21377

    Increased downstream signaling activity of the germline RAS mutants under serum-free conditions. KRAS mutants, transiently transfected in COS-7 cells were analyzed for the phosphorylation level of MEK1/2 (pMEK1/2), ERK1/2 (pERK1/2), and AKT (pAKT) under serum-free culture conditions. The amounts of total RAS, MEK1/2, ERK1/2, and AKT in the cleared cell lysates as well as RAS wt , RAS G12V , and RAS F28L were included as controls. Results of experiments in the presence of serum are shown in Supp. Fig. S4.
    Figure Legend Snippet: Increased downstream signaling activity of the germline RAS mutants under serum-free conditions. KRAS mutants, transiently transfected in COS-7 cells were analyzed for the phosphorylation level of MEK1/2 (pMEK1/2), ERK1/2 (pERK1/2), and AKT (pAKT) under serum-free culture conditions. The amounts of total RAS, MEK1/2, ERK1/2, and AKT in the cleared cell lysates as well as RAS wt , RAS G12V , and RAS F28L were included as controls. Results of experiments in the presence of serum are shown in Supp. Fig. S4.

    Techniques Used: Activity Assay, Transfection

    29) Product Images from "SERPINA3 promotes endometrial cancer cells growth by regulating G2/M cell cycle checkpoint and apoptosis"

    Article Title: SERPINA3 promotes endometrial cancer cells growth by regulating G2/M cell cycle checkpoint and apoptosis

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

    Knockdown of SERPINA3 inhibits activation of ERK1/2 and AKT. Analysis of activation of ERK1/2, AKT, Src, EGFR and FAK in SERPINA3 knockdown HEC-1A and KLE cells, using GAPDH as a loading control.
    Figure Legend Snippet: Knockdown of SERPINA3 inhibits activation of ERK1/2 and AKT. Analysis of activation of ERK1/2, AKT, Src, EGFR and FAK in SERPINA3 knockdown HEC-1A and KLE cells, using GAPDH as a loading control.

    Techniques Used: Activation Assay

    30) Product Images from "DiSCoVERing innovative therapies for rare tumors: combining genetically accurate disease models with in silico analysis to identify novel therapeutic targets"

    Article Title: DiSCoVERing innovative therapies for rare tumors: combining genetically accurate disease models with in silico analysis to identify novel therapeutic targets

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-15-3011

    A) Stem cells transduced with MYC have increased proliferation compared to untransduced neural stem cells and those immortalized with SV40. B) Cerebellar neural stem cells transduced with DN-TP53 hT AKT and MYC form tumors that kill mice in 117 days. Cells transduced with SV40 alone do not form tumors. C) These tumors express the introduced oncogenes. Images show tumor adjacent to normal brain. D) Tumors formed from cerebellar neural stem cells transduced with all four oncogenes form aggressive, anaplastic tumors (i) that spread to the leptominenges (ii) and metastasize to the spine (iii). E) The tumors are positive for MAP2 and NESTIN expression and negative for GFAP expression.
    Figure Legend Snippet: A) Stem cells transduced with MYC have increased proliferation compared to untransduced neural stem cells and those immortalized with SV40. B) Cerebellar neural stem cells transduced with DN-TP53 hT AKT and MYC form tumors that kill mice in 117 days. Cells transduced with SV40 alone do not form tumors. C) These tumors express the introduced oncogenes. Images show tumor adjacent to normal brain. D) Tumors formed from cerebellar neural stem cells transduced with all four oncogenes form aggressive, anaplastic tumors (i) that spread to the leptominenges (ii) and metastasize to the spine (iii). E) The tumors are positive for MAP2 and NESTIN expression and negative for GFAP expression.

    Techniques Used: Transduction, Mouse Assay, Expressing

    A) Diagram illustrating the creation of novel cancer models using human stem cells and oncogenic elements of interest. B) Human cerebellar neurosphere. C) Western blot indicating the expression of stem cell markers in the human cerebellar neural stem and progenitor cells. D) 65-sample medulloblastoma tissue microarray (TMA) reveals that Group 3 samples have the highest MYC expression. Lower panel: an example of MYC staining in a Group 3 sample. E) Staining the TMA reveals that Group 3 samples also have the highest expression of TP53, indicating inactivation of this pathway. Lower Panel: an example of TP53 staining in a Group 3 sample. F) Phospho-AKT, which indicates activation of AKT, is expressed in all three subgroups present on the array. Lower panel: an example of Phospho-AKT staining in a Group 3 sample. Magnification of all TMA images 400x.
    Figure Legend Snippet: A) Diagram illustrating the creation of novel cancer models using human stem cells and oncogenic elements of interest. B) Human cerebellar neurosphere. C) Western blot indicating the expression of stem cell markers in the human cerebellar neural stem and progenitor cells. D) 65-sample medulloblastoma tissue microarray (TMA) reveals that Group 3 samples have the highest MYC expression. Lower panel: an example of MYC staining in a Group 3 sample. E) Staining the TMA reveals that Group 3 samples also have the highest expression of TP53, indicating inactivation of this pathway. Lower Panel: an example of TP53 staining in a Group 3 sample. F) Phospho-AKT, which indicates activation of AKT, is expressed in all three subgroups present on the array. Lower panel: an example of Phospho-AKT staining in a Group 3 sample. Magnification of all TMA images 400x.

    Techniques Used: Western Blot, Expressing, Microarray, Staining, Activation Assay

    31) Product Images from "Inhibition of epidermal growth factor signaling by the cardiac glycoside ouabain in medulloblastoma"

    Article Title: Inhibition of epidermal growth factor signaling by the cardiac glycoside ouabain in medulloblastoma

    Journal: Cancer Medicine

    doi: 10.1002/cam4.314

    Activation of EGFR and HER2/ErbB2 signaling in DAOY cells. DAOY cells in serum-free medium were treated with 10 ng/mL EGF for the indicated times. Activation of EGFR (A) and HER2/ErbB2 (B) was determined by immunoblotting using antiphospho-EGFR (Tyr1173) and antiphospho-HER2 antibodies, respectively, which recognize only the activated form of the receptor. Immunoblots for total EGFR (A) and HER2 (B) confirm equal loading. Erk1/2 and Akt activation as determined by immunoblotting with phosphorylation-specific antibodies against Erk1/2 (T202/Y204) (C) and Akt (S473) (D), respectively. Equal loading was confirmed with antibodies for total Erk1/2 and Akt. (E) Phalloidin staining for filamentous actin in EGF-treated DAOY cells. Bar, 20 μ m. EGF, epidermal growth factor.
    Figure Legend Snippet: Activation of EGFR and HER2/ErbB2 signaling in DAOY cells. DAOY cells in serum-free medium were treated with 10 ng/mL EGF for the indicated times. Activation of EGFR (A) and HER2/ErbB2 (B) was determined by immunoblotting using antiphospho-EGFR (Tyr1173) and antiphospho-HER2 antibodies, respectively, which recognize only the activated form of the receptor. Immunoblots for total EGFR (A) and HER2 (B) confirm equal loading. Erk1/2 and Akt activation as determined by immunoblotting with phosphorylation-specific antibodies against Erk1/2 (T202/Y204) (C) and Akt (S473) (D), respectively. Equal loading was confirmed with antibodies for total Erk1/2 and Akt. (E) Phalloidin staining for filamentous actin in EGF-treated DAOY cells. Bar, 20 μ m. EGF, epidermal growth factor.

    Techniques Used: Activation Assay, Western Blot, Staining

    Ouabain inhibits EGF-induced Erk1/2 and Akt signaling. DAOY cells were incubated for 15 min with EGF in the presence or absence of 50 μ mol/L ouabain. (A) Activation of the MAPK signaling cascade was monitored by immunoblotting with phospho-specific anti-Erk1/2 and anti-Raf antibodies. Total Erk1/2 and actin levels confirm equal loading, respectively. (B) Activation of the Akt signaling cascade was determined with anti-phospho-Akt and anti-phospho-GSK antibodies that recognize only the activated forms of these proteins. Equal loading of cell lysate was confirmed with total Akt and actin immunoblots, respectively. EGF, epidermal growth factor.
    Figure Legend Snippet: Ouabain inhibits EGF-induced Erk1/2 and Akt signaling. DAOY cells were incubated for 15 min with EGF in the presence or absence of 50 μ mol/L ouabain. (A) Activation of the MAPK signaling cascade was monitored by immunoblotting with phospho-specific anti-Erk1/2 and anti-Raf antibodies. Total Erk1/2 and actin levels confirm equal loading, respectively. (B) Activation of the Akt signaling cascade was determined with anti-phospho-Akt and anti-phospho-GSK antibodies that recognize only the activated forms of these proteins. Equal loading of cell lysate was confirmed with total Akt and actin immunoblots, respectively. EGF, epidermal growth factor.

    Techniques Used: Incubation, Activation Assay, Western Blot

    32) Product Images from "Effects of Rosa laevigata Michx. extract on reactive oxygen species production and mitochondrial membrane potential in lens epithelial cells cultured under high glucose"

    Article Title: Effects of Rosa laevigata Michx. extract on reactive oxygen species production and mitochondrial membrane potential in lens epithelial cells cultured under high glucose

    Journal: International Journal of Clinical and Experimental Medicine

    doi:

    RLM induced HO-1 expression through the PI3K/Akt and Nrf2/ARE pathways in SRA01/04 LECs. A, B. The PI3/Akt pathway was involved in the function of RLM in LECs cultured under high glucose condition. A. RLM significantly elevated the levels of phosphorylated Akt (p-Akt) in SRA01/04 cells under high glucose. B. PI3K inhibitor down-regulated the expression levels of HO-1 in SRA01/04 cells under high glucose. C-E. The Nrf2/ARE pathway was involved in the function of RLM in LECs cultured under high glucose condition. C. RLM stimulated the translocation of Nrf2 from cytoplasm into nucleus in SRA01/04 cells. D. RNA silencing of Nrf2. Cells were treated with 30 μmol/L RLM, 30 μmol/L RLM + control siRNA, and 30 μmol/L RLM + Nrf2 siRNA, respectively. E. RNS silencing of Hrf2 canceled the inducing effects of RLM on HO-1 expression in SRA01/04 cells. RLM extract was administrated at the concentration of 30 μmol/L.
    Figure Legend Snippet: RLM induced HO-1 expression through the PI3K/Akt and Nrf2/ARE pathways in SRA01/04 LECs. A, B. The PI3/Akt pathway was involved in the function of RLM in LECs cultured under high glucose condition. A. RLM significantly elevated the levels of phosphorylated Akt (p-Akt) in SRA01/04 cells under high glucose. B. PI3K inhibitor down-regulated the expression levels of HO-1 in SRA01/04 cells under high glucose. C-E. The Nrf2/ARE pathway was involved in the function of RLM in LECs cultured under high glucose condition. C. RLM stimulated the translocation of Nrf2 from cytoplasm into nucleus in SRA01/04 cells. D. RNA silencing of Nrf2. Cells were treated with 30 μmol/L RLM, 30 μmol/L RLM + control siRNA, and 30 μmol/L RLM + Nrf2 siRNA, respectively. E. RNS silencing of Hrf2 canceled the inducing effects of RLM on HO-1 expression in SRA01/04 cells. RLM extract was administrated at the concentration of 30 μmol/L.

    Techniques Used: Expressing, Cell Culture, Translocation Assay, Concentration Assay

    33) Product Images from "Scopolamine rapidly increases mTORC1 signaling, synaptogenesis, and antidepressant behavioral responses"

    Article Title: Scopolamine rapidly increases mTORC1 signaling, synaptogenesis, and antidepressant behavioral responses

    Journal: Biological psychiatry

    doi: 10.1016/j.biopsych.2013.04.025

    Muscarinic receptor antagonist administration increases mTORC1 signaling in rat frontal cortex Rats were administered scopolamine (25 µg/kg, i.p.) or telenzapine (3 mg/kg, s.c.) and levels of mTORC1 signaling were determined 1 or 2 hr as indicated. Levels of the phosphorylated and activated forms of mTORC1, ERK, Akt, S6K were determined by western blot analysis. Levels of total mTORC1 and GAPDH were determined to control for loading differences. (a) Representative western blot images for each signaling protein are shown. (b) Results were quantified and are the mean ± SEM, percent of control (n = 6 animals; *P
    Figure Legend Snippet: Muscarinic receptor antagonist administration increases mTORC1 signaling in rat frontal cortex Rats were administered scopolamine (25 µg/kg, i.p.) or telenzapine (3 mg/kg, s.c.) and levels of mTORC1 signaling were determined 1 or 2 hr as indicated. Levels of the phosphorylated and activated forms of mTORC1, ERK, Akt, S6K were determined by western blot analysis. Levels of total mTORC1 and GAPDH were determined to control for loading differences. (a) Representative western blot images for each signaling protein are shown. (b) Results were quantified and are the mean ± SEM, percent of control (n = 6 animals; *P

    Techniques Used: Western Blot

    34) Product Images from "Targeting the glucagon receptor improves cardiac function and enhances insulin sensitivity following a myocardial infarction"

    Article Title: Targeting the glucagon receptor improves cardiac function and enhances insulin sensitivity following a myocardial infarction

    Journal: Cardiovascular Diabetology

    doi: 10.1186/s12933-019-0806-4

    Schematic illustration of the proposed cardioprotective mechanism of mAb A treatment in hearts following a myocardial infarction. Glucagon receptor antagonism increases the insulin signalling pathway and the BCAA catabolic pathway, while inhibiting the mTOR pathway. IRS - 1 insulin receptor substrate-1, Akt protein kinase B, GSK - 3β glycogen synthase kinase-3 beta, mTOR mammalian target of rapamycin, P70S6K ribosomal protein S6 kinase, AS160 Akt substrate 160, GLUT4 glucose transporter 4, PDH pyruvate dehydrogenase, TAK1 transforming growth factor beta-activated kinase 1, p38 MAPK p38 mitogen-activated protein kinase, KLF15 Krüppel-like factor 15, BCATm mitochondrial branched chain aminotransferase, BCAA branched chain amino acids, TCA tricarboxylic acid, MPC mitochondrial pyruvate carrier. Red arrows indicate the cellular changes following mAb A treatment in post-MI heart
    Figure Legend Snippet: Schematic illustration of the proposed cardioprotective mechanism of mAb A treatment in hearts following a myocardial infarction. Glucagon receptor antagonism increases the insulin signalling pathway and the BCAA catabolic pathway, while inhibiting the mTOR pathway. IRS - 1 insulin receptor substrate-1, Akt protein kinase B, GSK - 3β glycogen synthase kinase-3 beta, mTOR mammalian target of rapamycin, P70S6K ribosomal protein S6 kinase, AS160 Akt substrate 160, GLUT4 glucose transporter 4, PDH pyruvate dehydrogenase, TAK1 transforming growth factor beta-activated kinase 1, p38 MAPK p38 mitogen-activated protein kinase, KLF15 Krüppel-like factor 15, BCATm mitochondrial branched chain aminotransferase, BCAA branched chain amino acids, TCA tricarboxylic acid, MPC mitochondrial pyruvate carrier. Red arrows indicate the cellular changes following mAb A treatment in post-MI heart

    Techniques Used:

    mAb A treatment enhances insulin signalling in the heart post-MI. a Representative blots for insulin signalling kinases. Densiometric analysis for b phosphorylated insulin receptor substaret-1 (p-IRS-1 Tyr628 )/IRS-1, c phosphorylated protein kinase B (p-Akt Ser473 )/Akt, d phosphorylated glycogen synthase kinase-beta (p-GSK-3β Ser9 )/GSK-3β, e phosphorylated Akt substrate 160 (AS160 Thr642 )/AS160 f glucose transporter type-4 (GLUT4)/α-tubulin, and g phosphorylated pyruvate dehydrogenase (p-PDH-E1α Ser293 )/PDH. Protein bands were normalised for either to their correspondent total protein band or to α-tubulin bands for intra-experiment variation. *p
    Figure Legend Snippet: mAb A treatment enhances insulin signalling in the heart post-MI. a Representative blots for insulin signalling kinases. Densiometric analysis for b phosphorylated insulin receptor substaret-1 (p-IRS-1 Tyr628 )/IRS-1, c phosphorylated protein kinase B (p-Akt Ser473 )/Akt, d phosphorylated glycogen synthase kinase-beta (p-GSK-3β Ser9 )/GSK-3β, e phosphorylated Akt substrate 160 (AS160 Thr642 )/AS160 f glucose transporter type-4 (GLUT4)/α-tubulin, and g phosphorylated pyruvate dehydrogenase (p-PDH-E1α Ser293 )/PDH. Protein bands were normalised for either to their correspondent total protein band or to α-tubulin bands for intra-experiment variation. *p

    Techniques Used:

    35) Product Images from "Deoxycholic acid activates epidermal growth factor receptor and promotes intestinal carcinogenesis by ADAM17‐dependent ligand release, et al. Deoxycholic acid activates epidermal growth factor receptor and promotes intestinal carcinogenesis by ADAM17‐dependent ligand release"

    Article Title: Deoxycholic acid activates epidermal growth factor receptor and promotes intestinal carcinogenesis by ADAM17‐dependent ligand release, et al. Deoxycholic acid activates epidermal growth factor receptor and promotes intestinal carcinogenesis by ADAM17‐dependent ligand release

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13709

    ADAM ‐17/ EGFR signalling axis was activated in intestinal tumours of deoxycholic acid–treated Apc min/+ mice. A, Immunohistochemistry showed that DCA treatment increased the percentage of positive cells of ADAM ‐17 in intestinal tumour in Apc min/+ mice. B, DCA treatment increased the percentage of positive cells of amphiregulin in intestinal tumour in Apc min/+ mice. C,D, Phosphorylation of epidermal growth factor receptor ( EGFR ) and Akt in intestinal tumours was up‐regulated after DCA treatment. E, Western blot analysis showed that DCA increased the phosphorylation of EGFR and Akt in Apc min/+ mice. DCA , deoxycholic acid. Scale bar: 20 μm. ** P
    Figure Legend Snippet: ADAM ‐17/ EGFR signalling axis was activated in intestinal tumours of deoxycholic acid–treated Apc min/+ mice. A, Immunohistochemistry showed that DCA treatment increased the percentage of positive cells of ADAM ‐17 in intestinal tumour in Apc min/+ mice. B, DCA treatment increased the percentage of positive cells of amphiregulin in intestinal tumour in Apc min/+ mice. C,D, Phosphorylation of epidermal growth factor receptor ( EGFR ) and Akt in intestinal tumours was up‐regulated after DCA treatment. E, Western blot analysis showed that DCA increased the phosphorylation of EGFR and Akt in Apc min/+ mice. DCA , deoxycholic acid. Scale bar: 20 μm. ** P

    Techniques Used: Mouse Assay, Immunohistochemistry, Western Blot

    Deoxycholic acid activated epidermal growth factor receptor ( EGFR ) in intestinal tumour cells. A,C, Protein levels of phosphorylation and total levels of EGFR and Akt expressions in Immorto‐Min colonic epithelial cell line or human colorectal cancer cell line cells were analysed by Western blot along with time variation, using β‐actin as internal control. B,D, Proteins were quantified by densitometry using an Imaging processor program (ImageJ). DCA , deoxycholic acid. *, P
    Figure Legend Snippet: Deoxycholic acid activated epidermal growth factor receptor ( EGFR ) in intestinal tumour cells. A,C, Protein levels of phosphorylation and total levels of EGFR and Akt expressions in Immorto‐Min colonic epithelial cell line or human colorectal cancer cell line cells were analysed by Western blot along with time variation, using β‐actin as internal control. B,D, Proteins were quantified by densitometry using an Imaging processor program (ImageJ). DCA , deoxycholic acid. *, P

    Techniques Used: Western Blot, Imaging

    Model of ADAM ‐17/ EGFR signalling axis activation induced by deoxycholic acid in intestinal carcinogenesis. DCA stimulates ADAM ‐17 activation and AREG release, which is required for EGF receptor activation, EGFR /Akt signalling pathway activation and apoptosis resistance in intestinal tumour cells. ADAM ‐17, a disintegrin and metalloprotease domain‐containing protein 17; AREG , amphiregulin; DCA , deoxycholic acid; EGFR , epidermal growth factor receptor [Colour figure can be viewed at http://wileyonlinelibrary.com ]
    Figure Legend Snippet: Model of ADAM ‐17/ EGFR signalling axis activation induced by deoxycholic acid in intestinal carcinogenesis. DCA stimulates ADAM ‐17 activation and AREG release, which is required for EGF receptor activation, EGFR /Akt signalling pathway activation and apoptosis resistance in intestinal tumour cells. ADAM ‐17, a disintegrin and metalloprotease domain‐containing protein 17; AREG , amphiregulin; DCA , deoxycholic acid; EGFR , epidermal growth factor receptor [Colour figure can be viewed at http://wileyonlinelibrary.com ]

    Techniques Used: Activation Assay

    36) Product Images from "TRPV4 channel activation selectively inhibits tumor endothelial cell proliferation"

    Article Title: TRPV4 channel activation selectively inhibits tumor endothelial cell proliferation

    Journal: Scientific Reports

    doi: 10.1038/srep14257

    Pharmacological activation of TRPV4 decreases TEC proliferation via modulation of ERK1/2 but not AKT pathway. ( A ) Representative Western blots showing ERK1/2 phosphorylation in control and GSK treated NEC and TEC. ( B ) Densitometric analysis of the Western blots showing a decrease in ERK1/2 phosphorylation in TEC treated with GSK (100 nM). ERK1/2 phosphorylation was measured by normalizing phospho-ERK1/2 to total-ERK1/2 and was expressed as a fold change relative to NEC. ( C ) Representative Western blots depicting ERK1/2 phosphorylation TEC transfected with TRPV4-EGFP. ( D ) Densitometric analysis of the Western blots for ERK1/2 phosphorylation. ERK1/2 phosphorylation was measured by normalizing phospho-ERK1/2 to total-ERK1/2 and was expressed as a fold change relative to TEC. All the data shown is mean ± SEM from at least three independent experiments.
    Figure Legend Snippet: Pharmacological activation of TRPV4 decreases TEC proliferation via modulation of ERK1/2 but not AKT pathway. ( A ) Representative Western blots showing ERK1/2 phosphorylation in control and GSK treated NEC and TEC. ( B ) Densitometric analysis of the Western blots showing a decrease in ERK1/2 phosphorylation in TEC treated with GSK (100 nM). ERK1/2 phosphorylation was measured by normalizing phospho-ERK1/2 to total-ERK1/2 and was expressed as a fold change relative to NEC. ( C ) Representative Western blots depicting ERK1/2 phosphorylation TEC transfected with TRPV4-EGFP. ( D ) Densitometric analysis of the Western blots for ERK1/2 phosphorylation. ERK1/2 phosphorylation was measured by normalizing phospho-ERK1/2 to total-ERK1/2 and was expressed as a fold change relative to TEC. All the data shown is mean ± SEM from at least three independent experiments.

    Techniques Used: Activation Assay, Western Blot, Transfection

    37) Product Images from "Rewiring cellular metabolism via the AKT/mTOR pathway contributes to host defence against Mycobacterium tuberculosis in human and murine cells"

    Article Title: Rewiring cellular metabolism via the AKT/mTOR pathway contributes to host defence against Mycobacterium tuberculosis in human and murine cells

    Journal: European Journal of Immunology

    doi: 10.1002/eji.201546259

    Induction of glycolysis in human PBMCs is mediated by the AKT‐mTOR pathway. (A–D) PBMCs were stimulated with RPMI or Mtb in a time‐dependent manner in the presence or absence of DMSO (vehicle control), wortmannin (PI3K/AKT inhibitor), or rapamycin (mTOR inhibitor). (C) CD14 + and CD3 + T cells were separated from PBMCs stimulated for 2 h with Mtb. Where indicated, GM‐CSF stimulation was included as a positive control. (A, B) AKT, (C, D) p70‐S6K and 4E‐BP1 phosphorylation and actin levels were determined by Western blot using specific antibodies. (A, B) Cell lysates were harvested at 15, 30, 60, and 120 min poststimulation. (C, D) Cell lysates were harvested at 30, 60, 120, and 240 min poststimulation. Representative blots from two of four donors are shown. (E–G) PBMCs were preincubated with 10 nM rapamycin, 100 nM torin, or 100 nM wortmannin for 1 h prior to stimulation with Mtb lysate. Data are shown as means ± SEM of n = 9, pooled from three independent experiments. Means were compared using the Wilcoxon signed‐rank test (* p
    Figure Legend Snippet: Induction of glycolysis in human PBMCs is mediated by the AKT‐mTOR pathway. (A–D) PBMCs were stimulated with RPMI or Mtb in a time‐dependent manner in the presence or absence of DMSO (vehicle control), wortmannin (PI3K/AKT inhibitor), or rapamycin (mTOR inhibitor). (C) CD14 + and CD3 + T cells were separated from PBMCs stimulated for 2 h with Mtb. Where indicated, GM‐CSF stimulation was included as a positive control. (A, B) AKT, (C, D) p70‐S6K and 4E‐BP1 phosphorylation and actin levels were determined by Western blot using specific antibodies. (A, B) Cell lysates were harvested at 15, 30, 60, and 120 min poststimulation. (C, D) Cell lysates were harvested at 30, 60, 120, and 240 min poststimulation. Representative blots from two of four donors are shown. (E–G) PBMCs were preincubated with 10 nM rapamycin, 100 nM torin, or 100 nM wortmannin for 1 h prior to stimulation with Mtb lysate. Data are shown as means ± SEM of n = 9, pooled from three independent experiments. Means were compared using the Wilcoxon signed‐rank test (* p

    Techniques Used: Positive Control, Western Blot

    38) Product Images from "Neuroprotection by Curcumin in Ischemic Brain Injury Involves the Akt/Nrf2 Pathway"

    Article Title: Neuroprotection by Curcumin in Ischemic Brain Injury Involves the Akt/Nrf2 Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059843

    Western blot analysis of Akt, phospho-Akt, Nrf2 and NQO1 in the rat cortex after focal cerebral ischemia. A, Representative Western blots and semiquantitative analyses of phospho-Akt and Akt levels in MCA cortical tissues at 24 h. B, Representative Western blots and semiquantitative analyses of Nrf2 levels in MCA cortical tissues at 24 h. C, Representative Western blots and semiquantitative analyses of NQO1 levels in MCA cortical tissues at 24 h. Bars represent mean±SEM (n = 6–8 for each group). * p
    Figure Legend Snippet: Western blot analysis of Akt, phospho-Akt, Nrf2 and NQO1 in the rat cortex after focal cerebral ischemia. A, Representative Western blots and semiquantitative analyses of phospho-Akt and Akt levels in MCA cortical tissues at 24 h. B, Representative Western blots and semiquantitative analyses of Nrf2 levels in MCA cortical tissues at 24 h. C, Representative Western blots and semiquantitative analyses of NQO1 levels in MCA cortical tissues at 24 h. Bars represent mean±SEM (n = 6–8 for each group). * p

    Techniques Used: Western Blot

    Effects of PI3K/Akt activation on curcumin-induced NQO1 expression. A–D: Effects of inhibitors of PI3K and MAPKs on curcumin-induced NQO1 protein expression. Cortical neurons were post-treated with various concentrations of (A) LY294002, (B) SB203580, (C) SP600125, or (D) PD98059 and were incubated with 5 µM curcumin for 24 h. Data were normalized by vehicle group as 100%. Bars represent the means±SE (n = 4–6), *p
    Figure Legend Snippet: Effects of PI3K/Akt activation on curcumin-induced NQO1 expression. A–D: Effects of inhibitors of PI3K and MAPKs on curcumin-induced NQO1 protein expression. Cortical neurons were post-treated with various concentrations of (A) LY294002, (B) SB203580, (C) SP600125, or (D) PD98059 and were incubated with 5 µM curcumin for 24 h. Data were normalized by vehicle group as 100%. Bars represent the means±SE (n = 4–6), *p

    Techniques Used: Activation Assay, Expressing, Incubation

    39) Product Images from "BAFF promotes proliferation of human mesangial cells through interaction with BAFF-R"

    Article Title: BAFF promotes proliferation of human mesangial cells through interaction with BAFF-R

    Journal: BMC Nephrology

    doi: 10.1186/s12882-015-0064-y

    BAFF signal transduction in human mesangial cells. Human mesangial cells were subjected to serum starvation for 4 h, and then BAFF treatment (Veh, 5, 20, 100 ng/mL, indicated with the symbol ( ) for 10 min. The harvested cells were lysed for western blot, with antibodies used as indicated ( a ). b This experiment was repeated independently at least three times and expression ratios of target proteins were normalized to GAPDH or the unphosphorylated form of the protein. Specifically, NF-κBp100: NF-κBp100/GAPDH; p-NF-κBp65: p-NF-κBp65/NF-κBp65; p-Akt: p-Akt/Akt; p-Erk: p-Erk/Erk; p-p38: p-p38/p38. Statistical significance is indicated as * p
    Figure Legend Snippet: BAFF signal transduction in human mesangial cells. Human mesangial cells were subjected to serum starvation for 4 h, and then BAFF treatment (Veh, 5, 20, 100 ng/mL, indicated with the symbol ( ) for 10 min. The harvested cells were lysed for western blot, with antibodies used as indicated ( a ). b This experiment was repeated independently at least three times and expression ratios of target proteins were normalized to GAPDH or the unphosphorylated form of the protein. Specifically, NF-κBp100: NF-κBp100/GAPDH; p-NF-κBp65: p-NF-κBp65/NF-κBp65; p-Akt: p-Akt/Akt; p-Erk: p-Erk/Erk; p-p38: p-p38/p38. Statistical significance is indicated as * p

    Techniques Used: Transduction, Western Blot, Expressing

    40) Product Images from "Elevation of CD109 promotes metastasis and drug resistance in lung cancer via activation of EGFR‐AKT‐mTOR signaling, et al. Elevation of CD109 promotes metastasis and drug resistance in lung cancer via activation of EGFR‐AKT‐mTOR signaling"

    Article Title: Elevation of CD109 promotes metastasis and drug resistance in lung cancer via activation of EGFR‐AKT‐mTOR signaling, et al. Elevation of CD109 promotes metastasis and drug resistance in lung cancer via activation of EGFR‐AKT‐mTOR signaling

    Journal: Cancer Science

    doi: 10.1111/cas.14373

    Expression of CD109 is associated with AKT/mammalian target of rapamycin (mTOR) signaling. A, Blot images from a Human Phospho‐Kinase array performed in A549/shcon and A549/shCD109 cells (left panel). Densitometry analyses were normalized to multiples relative to the sh‐control group (right). B, Western blot analysis of AKT signaling molecules in control and CD109‐knockdown lung cancer cell lines. C, Gene set enrichment analysis (GSEA) of CD109‐associated oncogenic signatures in lung cancer datasets (left panel). Intersecting results are depicted in the right panel, and mTOR signaling is shown in red. D, Representative GSEA showing a correlation of CD109 with the mTOR signature. NES, net enrichment score
    Figure Legend Snippet: Expression of CD109 is associated with AKT/mammalian target of rapamycin (mTOR) signaling. A, Blot images from a Human Phospho‐Kinase array performed in A549/shcon and A549/shCD109 cells (left panel). Densitometry analyses were normalized to multiples relative to the sh‐control group (right). B, Western blot analysis of AKT signaling molecules in control and CD109‐knockdown lung cancer cell lines. C, Gene set enrichment analysis (GSEA) of CD109‐associated oncogenic signatures in lung cancer datasets (left panel). Intersecting results are depicted in the right panel, and mTOR signaling is shown in red. D, Representative GSEA showing a correlation of CD109 with the mTOR signature. NES, net enrichment score

    Techniques Used: Expressing, Western Blot

    CD109 is associated with EGFR and confers resistance to EGFR‐tyrosine kinase inhibitor. A, Coimmunoprecipitation analysis of the association between CD109 and the EGFR in A549 cells. B, Western blot analysis of phosphorylated EGFR in control and CD109‐silenced A549 cells. C, Suppression of CD109 blocked the EGFR signaling cascade. A549/shcon and A549/shCD109 cells were treated with the EGF (25 ng/mL) for different time periods, and phosphorylation of the EGFR and AKT was analyzed by Western blotting. D, A549/shcon and A549/shCD109 cells were treated with different concentrations of gefitinib (0, 5, and 20 µmol/L), and phosphorylation of AKT and mTOR was analyzed by Western blotting. E, A549 cells were treated with the EGF (25 ng/mL) in the presence of gefitinib (20 µmol/L). Cell lysates were subjected to Western blotting using specific antibodies. F, Silencing of CD109 increased the sensitivity of A549 and CL1‐5 cells to gefitinib. A549 and CL1‐5 cells were treated with different concentrations of gefitinib for 48 h, and cell viability was determined by trypan blue exclusion assay. G, Western blot analysis of the EGFR signaling cascade in PC9/shcon and PC9/shCD109 cells in response to gefitinib for 2 h. H, PC9 cells were treated with different concentrations of gefitinib for 48 h, and cell viability was determined by trypan blue exclusion assay. Data are presented as the percentage relative to the control. * P
    Figure Legend Snippet: CD109 is associated with EGFR and confers resistance to EGFR‐tyrosine kinase inhibitor. A, Coimmunoprecipitation analysis of the association between CD109 and the EGFR in A549 cells. B, Western blot analysis of phosphorylated EGFR in control and CD109‐silenced A549 cells. C, Suppression of CD109 blocked the EGFR signaling cascade. A549/shcon and A549/shCD109 cells were treated with the EGF (25 ng/mL) for different time periods, and phosphorylation of the EGFR and AKT was analyzed by Western blotting. D, A549/shcon and A549/shCD109 cells were treated with different concentrations of gefitinib (0, 5, and 20 µmol/L), and phosphorylation of AKT and mTOR was analyzed by Western blotting. E, A549 cells were treated with the EGF (25 ng/mL) in the presence of gefitinib (20 µmol/L). Cell lysates were subjected to Western blotting using specific antibodies. F, Silencing of CD109 increased the sensitivity of A549 and CL1‐5 cells to gefitinib. A549 and CL1‐5 cells were treated with different concentrations of gefitinib for 48 h, and cell viability was determined by trypan blue exclusion assay. G, Western blot analysis of the EGFR signaling cascade in PC9/shcon and PC9/shCD109 cells in response to gefitinib for 2 h. H, PC9 cells were treated with different concentrations of gefitinib for 48 h, and cell viability was determined by trypan blue exclusion assay. Data are presented as the percentage relative to the control. * P

    Techniques Used: Western Blot, Trypan Blue Exclusion Assay

    Related Articles

    Incubation:

    Article Title: Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells
    Article Snippet: .. The membrane was then incubated with antibodies against β-actin (Sigma, 1:10000), GAPDH (Sigma, 1:10000), LC3 (Novus Biologicals Inc., Littleton, CO, 1:10000), SQSTM1/p62 (MBL international, 1:1000), PARP (Cell Signaling Technologies, 1:1000), phospho-mTOR, phospho-Akt (Ser 473) (Cell Signaling Technologies, 1:1000), phospho-p70S6K (Cell Signaling Technologies, 1:1000), ABCA1 (Abcam, 1:1000), LDLR (Abcam, 1:5000), SREBP1 (BD, 1:1000), SREBP2 (BD, 1:1000), CHOP (Cell Signaling Technologies, 1:1000) and GRP78 (Cell Signaling Technologies, 1:1000) overnight at 4°C in PBS containing 0.05% Tween 20 and 5% nonfat milk, followed by incubation for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in the same buffer. .. Blots were developed using a chemiluminescent detection system (ECL; GE Life Science, Buckinghamshire, UK).

    Article Title: Overexpression of 14-3-3? in cancer cells activates PI3K via binding the p85 regulatory subunit
    Article Snippet: .. The slides were then incubated with 14-3-3ζ polyclonal antibody (C-16; Santa Cruz, Santa Cruz, CA, USA) or phospho-Akt Ser473 (Cell Signaling, Danvers, MA, USA) at 4°C overnight. .. Immunodetection was performed using streptavidin-biotin detection with the LSAB2 system (DAKO).

    Article Title: Effects of trabecular calcium phosphate scaffolds on stress-signaling in osteoblast precursor cells
    Article Snippet: .. Primary rabbit antibodies against phospho-p44/42 (ERK1/2, Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), phospho-SAPK/JNK (Thr183/Tyr185), and phospho-AKT (Ser 473, Cell Signaling Technology, Danvers, MA) were added in 5% FCS-PBS-T and incubated overnight at 4 °C with 20 rpm rocking. ..

    Article Title: A Novel Approach Based on Metabolomics Coupled With Intestinal Flora Analysis and Network Pharmacology to Explain the Mechanisms of Action of Bekhogainsam Decoction in the Improvement of Symptoms of Streptozotocin-Induced Diabetic Nephropathy in Mice
    Article Snippet: .. The membranes were incubated in 5% skimmed milk, dissolved in Tris-buffered saline with 1% Tween-20 (TBST), pH 7.5, for 30 min at RT for blocking, and then incubated with anti-PKCα, I-κB, NF-κB (p 65), α-SMA, TGF-β1, COX-2 (Santa Cruz Biotechnology, Santa Cruz, Dallas, TX, USA), iNOS (BD Biosciences, San Jose, CA, USA), total-AKT (Ser473), phospho-AKT (Ser473), total-IRS (Ser 307), phospho-IRS (Ser 307), PI3K (p85) (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Sigma-Aldrich, Inc., St. Louis, MO, USA) antibodies at 1:1,000 overnight at 4°C and incubated with HRP-conjugated mouse or rabbit secondary antibodies for 3 h at RT. .. Blots were scanned and analysed with ChemiDocTM MP imaging system.

    other:

    Article Title: Hyperglycemia- and hyperinsulinemia-induced insulin resistance causes alterations in cellular bioenergetics and activation of inflammatory signaling in lymphatic muscle
    Article Snippet: Phospho-MLC20 (Ser19 ), phospho-p38 MAPK, p-38MAPK, phospho-ERK1/2, ERK1/2, phospho-Akt (Thr308 ), phospho-Akt (Ser473 ), Akt, β-actin, phospho-glycogen synthase kinase (GSK)-3β (Ser9 ), phospho-IRS1 (Ser1101 ), IRS1, phospho-IκB, phosphor JNK, JNK, and PI3Kp85, were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Article Title: AMPK ?1 Activation Is Required for Stimulation of Glucose Uptake by Twitch Contraction, but Not by H2O2, in Mouse Skeletal Muscle
    Article Snippet: Lysates were subjected to standard immunoblotting techniques , using the following phospho-specific antibodies: AMPK Thr172 (Cell Signaling Technology, MA), ACCβ Ser221 (Upstate Biotechnologies, MA), Akt Ser473 (Cell Signaling Technology), and phospho-Akt substrate motif RX RXX S/T (PAS) (Cell Signaling Technology) recognizing phospho-AS160 .

    Blocking Assay:

    Article Title: A Novel Approach Based on Metabolomics Coupled With Intestinal Flora Analysis and Network Pharmacology to Explain the Mechanisms of Action of Bekhogainsam Decoction in the Improvement of Symptoms of Streptozotocin-Induced Diabetic Nephropathy in Mice
    Article Snippet: .. The membranes were incubated in 5% skimmed milk, dissolved in Tris-buffered saline with 1% Tween-20 (TBST), pH 7.5, for 30 min at RT for blocking, and then incubated with anti-PKCα, I-κB, NF-κB (p 65), α-SMA, TGF-β1, COX-2 (Santa Cruz Biotechnology, Santa Cruz, Dallas, TX, USA), iNOS (BD Biosciences, San Jose, CA, USA), total-AKT (Ser473), phospho-AKT (Ser473), total-IRS (Ser 307), phospho-IRS (Ser 307), PI3K (p85) (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Sigma-Aldrich, Inc., St. Louis, MO, USA) antibodies at 1:1,000 overnight at 4°C and incubated with HRP-conjugated mouse or rabbit secondary antibodies for 3 h at RT. .. Blots were scanned and analysed with ChemiDocTM MP imaging system.

    Mouse Assay:

    Article Title: ?-Mangostin extracted from the pericarp of the mangosteen (Garcinia mangostana Linn) reduces tumor growth and lymph node metastasis in an immunocompetent xenograft model of metastatic mammary cancer carrying a p53 mutation
    Article Snippet: .. There were wide variations among mice, but, overall, phospho-Akt-Ser473 tended to marginally decrease with α-mangostin treatment (Figure ). .. In vivo Compared to expression of phospho-Akt-Thr308 in control mammary carcinomas (Figure ), the number of positive cells and their staining intensity was markedly lower in mammary carcinomas of mice treated with 20 mg/kg/day α-mangostin (Figure ).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    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: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phosphot akt ser473/product/Cell Signaling Technology Inc
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    anti phosphot akt ser473 - by Bioz Stars, 2020-11
    85/100 stars
      Buy from Supplier

    95
    Cell Signaling Technology Inc phospho akt
    Profilin induces cellular responses and activates classical signaling cascades in rat VSMCs. ( A ) DNA-synthesis as assessed by measurement of BrdU incorporation. ( B ) Chemotaxis was evaluated utilizing modified Boyden chemotaxis chambers. ( C ) Quiescent VSMCs were stimulated with recombinant profilin-1 (1 µM) for various time points as indicated. The cells were lysed, and equal amounts of protein were subjected to SDS-PAGE and Western blot analyses, using phospho-specific antibodies recognizing phosphorylated p70 S6K , <t>Akt,</t> and Erk1/2. <t>RasGAP</t> served as a lysate control. ( D ) Cells were stimulated with recombinant profilin-1 (1 µM) for 15 min in the presence of pharmacological inhibitors as indicated. Data in C and D were quantified by densitometry and are expressed as fold increase compared to buffer-treated control cells. ( E ) DNA synthesis was assessed by measurement of BrdU incorporation, and recombinant profilin was added in the presence of pharmacological inhibitors as indicated. Data are expressed as the percentage of the maximal profilin-response. ( F ) Chemotaxis was evaluated utilizing modified Boyden chemotaxis chambers in the presence of pharmacological inhibitors. Data are expressed as fold increase compared to buffer-treated control cells. Pharmacological inhibitors: PI3K inhibitors wortmannin (W; 100 nM) and LY294002 (LY; 20 µM), MEK inhibitor PD98059 (PD; 30 µM), rapamycin (Rap; 10 nM), tyrosine kinase inhibitor genestein (Gen; 50 µM), Src inhibitors SU6656 (SU; 2.5 µM) or PP2 (50 nM), PLCγ inhibitor U73122 (U; 10 µM). All data represent means ± SEM from at least three independent experiments. * P
    Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 2368 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho akt/product/Cell Signaling Technology Inc
    Average 95 stars, based on 2368 article reviews
    Price from $9.99 to $1999.99
    phospho akt - by Bioz Stars, 2020-11
    95/100 stars
      Buy from Supplier

    Image Search Results


    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

    Profilin induces cellular responses and activates classical signaling cascades in rat VSMCs. ( A ) DNA-synthesis as assessed by measurement of BrdU incorporation. ( B ) Chemotaxis was evaluated utilizing modified Boyden chemotaxis chambers. ( C ) Quiescent VSMCs were stimulated with recombinant profilin-1 (1 µM) for various time points as indicated. The cells were lysed, and equal amounts of protein were subjected to SDS-PAGE and Western blot analyses, using phospho-specific antibodies recognizing phosphorylated p70 S6K , Akt, and Erk1/2. RasGAP served as a lysate control. ( D ) Cells were stimulated with recombinant profilin-1 (1 µM) for 15 min in the presence of pharmacological inhibitors as indicated. Data in C and D were quantified by densitometry and are expressed as fold increase compared to buffer-treated control cells. ( E ) DNA synthesis was assessed by measurement of BrdU incorporation, and recombinant profilin was added in the presence of pharmacological inhibitors as indicated. Data are expressed as the percentage of the maximal profilin-response. ( F ) Chemotaxis was evaluated utilizing modified Boyden chemotaxis chambers in the presence of pharmacological inhibitors. Data are expressed as fold increase compared to buffer-treated control cells. Pharmacological inhibitors: PI3K inhibitors wortmannin (W; 100 nM) and LY294002 (LY; 20 µM), MEK inhibitor PD98059 (PD; 30 µM), rapamycin (Rap; 10 nM), tyrosine kinase inhibitor genestein (Gen; 50 µM), Src inhibitors SU6656 (SU; 2.5 µM) or PP2 (50 nM), PLCγ inhibitor U73122 (U; 10 µM). All data represent means ± SEM from at least three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Profilin-1 Is Expressed in Human Atherosclerotic Plaques and Induces Atherogenic Effects on Vascular Smooth Muscle Cells

    doi: 10.1371/journal.pone.0013608

    Figure Lengend Snippet: Profilin induces cellular responses and activates classical signaling cascades in rat VSMCs. ( A ) DNA-synthesis as assessed by measurement of BrdU incorporation. ( B ) Chemotaxis was evaluated utilizing modified Boyden chemotaxis chambers. ( C ) Quiescent VSMCs were stimulated with recombinant profilin-1 (1 µM) for various time points as indicated. The cells were lysed, and equal amounts of protein were subjected to SDS-PAGE and Western blot analyses, using phospho-specific antibodies recognizing phosphorylated p70 S6K , Akt, and Erk1/2. RasGAP served as a lysate control. ( D ) Cells were stimulated with recombinant profilin-1 (1 µM) for 15 min in the presence of pharmacological inhibitors as indicated. Data in C and D were quantified by densitometry and are expressed as fold increase compared to buffer-treated control cells. ( E ) DNA synthesis was assessed by measurement of BrdU incorporation, and recombinant profilin was added in the presence of pharmacological inhibitors as indicated. Data are expressed as the percentage of the maximal profilin-response. ( F ) Chemotaxis was evaluated utilizing modified Boyden chemotaxis chambers in the presence of pharmacological inhibitors. Data are expressed as fold increase compared to buffer-treated control cells. Pharmacological inhibitors: PI3K inhibitors wortmannin (W; 100 nM) and LY294002 (LY; 20 µM), MEK inhibitor PD98059 (PD; 30 µM), rapamycin (Rap; 10 nM), tyrosine kinase inhibitor genestein (Gen; 50 µM), Src inhibitors SU6656 (SU; 2.5 µM) or PP2 (50 nM), PLCγ inhibitor U73122 (U; 10 µM). All data represent means ± SEM from at least three independent experiments. * P

    Article Snippet: The cells were harvested, the lysates were resolved by SDS-PAGE, and subjected to Western blotting as described , , using antisera against RasGAP (lysate control), phospho-Erk1/2 (thr202/tyr204), phospho-Akt (ser473), or phospho-p70S6K (Cell Signaling).

    Techniques: DNA Synthesis, BrdU Incorporation Assay, Chemotaxis Assay, Modification, Recombinant, SDS Page, Western Blot

    Effect of sialostatins on the signalling pathways activated by LTA in dendritic cells. Dendritic cells were seeded in 24-well plate. Next day DCs were incubated 2 h with tick cystatins (both 3 μM) prior to the addition of LTA (2 μg/ml) and further incubated for indicated times. Afterwards, cells were lysed and obtained protein extract was further analysed by immunoblotting using antibodies recognizing phosphorylated form of tested kinases. Membranes were stripped and reprobed with antibodies against total kinase protein, which served as a control ( a ). Proteins were visualized by enhanced chemiluminescence. Bands were quantified using scanning densitometry and phosphorylation/activities of Erk1/2 ( b ), Akt ( c ), and NF-κB ( d ) kinases were normalized by total kinase protein level (relative activity = phospho kinase/total kinase). Three independent experiments were performed and representative blots are shown. Graphs represent the average ± SD from 2-3 experiments, the relative phosphorylation of kinase achieved at 60 min upon LTA stimulation was set up to 1 to allow pooling of data

    Journal: Parasites & Vectors

    Article Title: Tick sialostatins L and L2 differentially influence dendritic cell responses to Borrelia spirochetes

    doi: 10.1186/s13071-015-0887-1

    Figure Lengend Snippet: Effect of sialostatins on the signalling pathways activated by LTA in dendritic cells. Dendritic cells were seeded in 24-well plate. Next day DCs were incubated 2 h with tick cystatins (both 3 μM) prior to the addition of LTA (2 μg/ml) and further incubated for indicated times. Afterwards, cells were lysed and obtained protein extract was further analysed by immunoblotting using antibodies recognizing phosphorylated form of tested kinases. Membranes were stripped and reprobed with antibodies against total kinase protein, which served as a control ( a ). Proteins were visualized by enhanced chemiluminescence. Bands were quantified using scanning densitometry and phosphorylation/activities of Erk1/2 ( b ), Akt ( c ), and NF-κB ( d ) kinases were normalized by total kinase protein level (relative activity = phospho kinase/total kinase). Three independent experiments were performed and representative blots are shown. Graphs represent the average ± SD from 2-3 experiments, the relative phosphorylation of kinase achieved at 60 min upon LTA stimulation was set up to 1 to allow pooling of data

    Article Snippet: The blots were incubated overnight at 4 °C with the antibody recognizing phospho-NF-κB p65 (Ser536 ), phospho-p44/42 MAPK (Erk1/2) (Thr202 /Tyr204 ), phospho-p38 MAPK (Thr180 /Tyr182 ), phospho-Akt (Ser473 ), total NF-κB p65, p44/42 MAPK (Erk1/2), p38 MAPK, Akt, and β-actin (all from Cell Signalling) followed by incubation with secondary antibody conjungated with horse radish peroxidase.

    Techniques: Incubation, Activity Assay

    Western blot analysis showing effects of asaronic acid on induction of CARKL, GLUT1, phospho-Akt, phospho-mTOR, and phospho-AMPKα ( A – D ). J774A.1 macrophages were exposed to 40 ng/mL IL-4 in the absence and presence of 1-20 μM asaronic acid for 48 h. Cell lysates were subject to 8–12% SDS-PAGE and Western blot analysis with a primary antibody against CARKL, GLUT1, Akt, phospho-Akt, mTOR, phospho-mTOR, AMPKα, and phospho-AMPKα. β-Actin antibody was used as an internal control. The bar graphs (mean ± SEM, n = 3) in the panels represent quantitative results of the blot bands obtained from a densitometer. Mean values in respective bar graphs not sharing a same lower-case alphabet letter are significantly different at p

    Journal: Nutrients

    Article Title: Asaronic Acid Inhibited Glucose-Triggered M2-Phenotype Shift Through Disrupting the Formation of Coordinated Signaling of IL-4Rα-Tyk2-STAT6 and GLUT1-Akt-mTOR-AMPK

    doi: 10.3390/nu12072006

    Figure Lengend Snippet: Western blot analysis showing effects of asaronic acid on induction of CARKL, GLUT1, phospho-Akt, phospho-mTOR, and phospho-AMPKα ( A – D ). J774A.1 macrophages were exposed to 40 ng/mL IL-4 in the absence and presence of 1-20 μM asaronic acid for 48 h. Cell lysates were subject to 8–12% SDS-PAGE and Western blot analysis with a primary antibody against CARKL, GLUT1, Akt, phospho-Akt, mTOR, phospho-mTOR, AMPKα, and phospho-AMPKα. β-Actin antibody was used as an internal control. The bar graphs (mean ± SEM, n = 3) in the panels represent quantitative results of the blot bands obtained from a densitometer. Mean values in respective bar graphs not sharing a same lower-case alphabet letter are significantly different at p

    Article Snippet: Antibodies of arginas-1, PPARγ, phospho-tyrosine kinase 2 (Tyk2), TGF-β, glucose transporter 1 (GLUT1), Akt, phospho-Akt, mammalian target of rapamycin complex (mTOR), phospho-mTOR, 5’-adenosine monophosphate-activated protein kinase (AMPK), and phospho-AMPK were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Western Blot, SDS Page

    Inhibition of induction of GLUT1, phospho-Akt, phospho-mTOR, and phospho-AMPKα by asaronic acid ( A – C ), and blockade of ROS production by 20 μM asaronic acid ( D ). J774A.1 macrophages were exposed to 33 mM glucose in the absence and presence of 1-20 μM asaronic acid for 48 h. Cell lysates were subject to 8–12% SDS-PAGE and Western blot analysis with a primary antibody against GLUT1, Akt, phospho-Akt, phospho-mTOR, and phospho-AMPKα. β-Actin antibody was used as an internal control. The bar graphs (mean ± SEM, n = 3) in the panels represent quantitative results of the upper blot bands obtained from a densitometer. Mean values in respective bar graphs not sharing a same lower-case alphabet letter are significantly different at p

    Journal: Nutrients

    Article Title: Asaronic Acid Inhibited Glucose-Triggered M2-Phenotype Shift Through Disrupting the Formation of Coordinated Signaling of IL-4Rα-Tyk2-STAT6 and GLUT1-Akt-mTOR-AMPK

    doi: 10.3390/nu12072006

    Figure Lengend Snippet: Inhibition of induction of GLUT1, phospho-Akt, phospho-mTOR, and phospho-AMPKα by asaronic acid ( A – C ), and blockade of ROS production by 20 μM asaronic acid ( D ). J774A.1 macrophages were exposed to 33 mM glucose in the absence and presence of 1-20 μM asaronic acid for 48 h. Cell lysates were subject to 8–12% SDS-PAGE and Western blot analysis with a primary antibody against GLUT1, Akt, phospho-Akt, phospho-mTOR, and phospho-AMPKα. β-Actin antibody was used as an internal control. The bar graphs (mean ± SEM, n = 3) in the panels represent quantitative results of the upper blot bands obtained from a densitometer. Mean values in respective bar graphs not sharing a same lower-case alphabet letter are significantly different at p

    Article Snippet: Antibodies of arginas-1, PPARγ, phospho-tyrosine kinase 2 (Tyk2), TGF-β, glucose transporter 1 (GLUT1), Akt, phospho-Akt, mammalian target of rapamycin complex (mTOR), phospho-mTOR, 5’-adenosine monophosphate-activated protein kinase (AMPK), and phospho-AMPK were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Inhibition, SDS Page, Western Blot