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  • 99
    Name:
    Akt Antibody
    Description:
    Akt also referred to as PKB or Rac plays a critical role in controlling survival and apoptosis 1 3 This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin sensitive pathway involving PI3 kinase 2 3 Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 4 and by phosphorylation within the carboxy terminus at Ser473 The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin mTOR in a rapamycin insensitive complex with rictor and Sin1 5 6 Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets including Bad 7 forkhead transcription factors 8 c Raf 9 and caspase 9 PTEN phosphatase is a major negative regulator of the PI3 kinase Akt signaling pathway 10 LY294002 is a specific PI3 kinase inhibitor 11 Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK 3α and β 12 13 Akt may also play a role in insulin stimulation of glucose transport 12 In addition to its role in survival and glycogen synthesis Akt is involved in cell cycle regulation by preventing GSK 3β mediated phosphorylation and degradation of cyclin D1 14 and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 15 and p21 Waf1 Cip1 16 Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin sensitive complex containing raptor 17 More importantly Akt phosphorylates and inactivates tuberin TSC2 an inhibitor of mTOR within the mTOR raptor complex 18 19
    Catalog Number:
    9272
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation, Immunofluorescence, Flow Cytometry
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the carboxy-terminal sequence of mouse Akt. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Hamster Monkey Chicken D melanogaster Bovine Dog Pig Guinea Pig
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc anti p akt
    Effects of clofarabine and resveratrol on <t>Akt</t> and Erk1/2 phosphorylation. (A) MSTO-211H cells were treated with clofarabine (40 nM) and resveratrol (15 μM) for the indicated times. (B) Cells were pretreated with or without Ly294002 (20 μM), PD98059 (50 nM), and NAC (5 mM) for 1 h prior to combined treatment for 24 h. Cell lysates were analyzed by immunoblotting using <t>anti-p-Akt,</t> anti-p-Erk, anti-Nrf2, and anti-HO-1 antibodies. (C) Cells were treated with or without NAC (5 mM) for 1 h prior to the combination treatment for 24 h. The percentage of viable cells was then determined by the trypan blue exclusion assay. *P < 0.05 compared with respective controls.
    Akt also referred to as PKB or Rac plays a critical role in controlling survival and apoptosis 1 3 This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin sensitive pathway involving PI3 kinase 2 3 Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 4 and by phosphorylation within the carboxy terminus at Ser473 The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin mTOR in a rapamycin insensitive complex with rictor and Sin1 5 6 Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets including Bad 7 forkhead transcription factors 8 c Raf 9 and caspase 9 PTEN phosphatase is a major negative regulator of the PI3 kinase Akt signaling pathway 10 LY294002 is a specific PI3 kinase inhibitor 11 Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK 3α and β 12 13 Akt may also play a role in insulin stimulation of glucose transport 12 In addition to its role in survival and glycogen synthesis Akt is involved in cell cycle regulation by preventing GSK 3β mediated phosphorylation and degradation of cyclin D1 14 and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 15 and p21 Waf1 Cip1 16 Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin sensitive complex containing raptor 17 More importantly Akt phosphorylates and inactivates tuberin TSC2 an inhibitor of mTOR within the mTOR raptor complex 18 19
    https://www.bioz.com/result/anti p akt/product/Cell Signaling Technology Inc
    Average 99 stars, based on 112 article reviews
    Price from $9.99 to $1999.99
    anti p akt - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Synergistic inhibition of mesothelioma cell growth by the combination of clofarabine and resveratrol involves Nrf2 downregulation"

    Article Title: Synergistic inhibition of mesothelioma cell growth by the combination of clofarabine and resveratrol involves Nrf2 downregulation

    Journal: BMB Reports

    doi: 10.5483/BMBRep.2012.45.11.111

    Effects of clofarabine and resveratrol on Akt and Erk1/2 phosphorylation. (A) MSTO-211H cells were treated with clofarabine (40 nM) and resveratrol (15 μM) for the indicated times. (B) Cells were pretreated with or without Ly294002 (20 μM), PD98059 (50 nM), and NAC (5 mM) for 1 h prior to combined treatment for 24 h. Cell lysates were analyzed by immunoblotting using anti-p-Akt, anti-p-Erk, anti-Nrf2, and anti-HO-1 antibodies. (C) Cells were treated with or without NAC (5 mM) for 1 h prior to the combination treatment for 24 h. The percentage of viable cells was then determined by the trypan blue exclusion assay. *P < 0.05 compared with respective controls.
    Figure Legend Snippet: Effects of clofarabine and resveratrol on Akt and Erk1/2 phosphorylation. (A) MSTO-211H cells were treated with clofarabine (40 nM) and resveratrol (15 μM) for the indicated times. (B) Cells were pretreated with or without Ly294002 (20 μM), PD98059 (50 nM), and NAC (5 mM) for 1 h prior to combined treatment for 24 h. Cell lysates were analyzed by immunoblotting using anti-p-Akt, anti-p-Erk, anti-Nrf2, and anti-HO-1 antibodies. (C) Cells were treated with or without NAC (5 mM) for 1 h prior to the combination treatment for 24 h. The percentage of viable cells was then determined by the trypan blue exclusion assay. *P < 0.05 compared with respective controls.

    Techniques Used: Trypan Blue Exclusion Assay

    2) Product Images from "Luteolin induces apoptosis in vitro through suppressing the MAPK and PI3K signaling pathways in gastric cancer"

    Article Title: Luteolin induces apoptosis in vitro through suppressing the MAPK and PI3K signaling pathways in gastric cancer

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.6380

    Luteolin inhibits the activation of the PI3K and MAPK signaling pathways in BGC-823 cells. Cells were seeded in 60-mm plates and cultured to 80–90% confluence. The cells were then treated with various doses of luteolin (20, 40 and 60 µM) for 48 h. Cells treated with dimethyl sulfoxide alone were used as the control. (A) Luteolin inhibited the PI3K signaling pathway in BGC-823 cells. Cells were treated as described above, and the cell extracts were subjected to immunoblot analysis using anti-p-PI3K, anti-p-AKT, anti-p-mTOR and anti-β-actin antibodies. (B) Effect of luteolin on ERK1/2, p38 and JNK pathways. Cells were treated as described above, and the cell extracts were subjected to immunoblotting using anti-p-ERK, anti-p-p38, anti-p-JNK and anti-β-actin antibodies. (C) Effects of luteolin on the Bcl-2 and Bax in BGC-823 cells. Cells were treated as described above, and the cell extracts were subjected to immunoblotting using anti-p-ERK, anti-Bcl-2, anti-Bax and anti-β-actin antibodies. (D) The relative intensity of Bax/Bcl-2. Values are the mean ± standard deviation. The data were analyzed by one-way analysis of variance. **The control group vs. all the other groups (P
    Figure Legend Snippet: Luteolin inhibits the activation of the PI3K and MAPK signaling pathways in BGC-823 cells. Cells were seeded in 60-mm plates and cultured to 80–90% confluence. The cells were then treated with various doses of luteolin (20, 40 and 60 µM) for 48 h. Cells treated with dimethyl sulfoxide alone were used as the control. (A) Luteolin inhibited the PI3K signaling pathway in BGC-823 cells. Cells were treated as described above, and the cell extracts were subjected to immunoblot analysis using anti-p-PI3K, anti-p-AKT, anti-p-mTOR and anti-β-actin antibodies. (B) Effect of luteolin on ERK1/2, p38 and JNK pathways. Cells were treated as described above, and the cell extracts were subjected to immunoblotting using anti-p-ERK, anti-p-p38, anti-p-JNK and anti-β-actin antibodies. (C) Effects of luteolin on the Bcl-2 and Bax in BGC-823 cells. Cells were treated as described above, and the cell extracts were subjected to immunoblotting using anti-p-ERK, anti-Bcl-2, anti-Bax and anti-β-actin antibodies. (D) The relative intensity of Bax/Bcl-2. Values are the mean ± standard deviation. The data were analyzed by one-way analysis of variance. **The control group vs. all the other groups (P

    Techniques Used: Activation Assay, Cell Culture, Standard Deviation

    3) Product Images from "p53 and PI3K/AKT Signalings Are Up-Regulated in Flies with Defects in the THO Complex"

    Article Title: p53 and PI3K/AKT Signalings Are Up-Regulated in Flies with Defects in the THO Complex

    Journal: Molecules and Cells

    doi: 10.1007/s10059-013-0009-x

    Increased level of p-AKT in the THO mutant testes. (A) Antibody staining of testes with anti-p-AKT antibody in wild-type (WT), thoc5 1 /thoc5 e00906 ( thoc5 ), thoc6 e00298 ( thoc6 ) and thoc7 d05792 ( thoc7 ). In wild-type, p-AKT was highly accumulated in the tip region where cells at the early-stages of germline development reside, and gradually disappeared as spermatogenesis proceeded. In thoc5 , however, the level of p-AKT was not only elevated, but also sustained over spermatogenesis. Similar, but much milder, defects were seen in the thoc7 mutant testis. No significant differences from wild-type and thoc6 . were observed. (B) Western blot analysis of testis extracts from wildtype, thoc5 1 /thoc5 e00906 , thoc6 e00298 and thoc7 d05792 . The level of p-AKT was increased in thoc5 and thoc7 , but not in thoc6 . The result was well matched with that of antibody staining (C) FLP/FRT-mediated clonal analysis of thoc5 1 mutant cells in the testis. In the thoc5 1 mutant cells indicated by the absence of GFP signals (green), the immunoreactivities against p-AKT (Red) were highly elevated compared to their neighboring wild-type cells which showed only faint signals of p-AKT. Hoechst 33258 was used to visualize DNA (blue). Genotype: y w P{hs-FLP}; P{FRT42D} thoc5 1 /P{FRT42D} P{Ubi-GFP} . Scale bars, 20 μm (A) and 10 μm (C).
    Figure Legend Snippet: Increased level of p-AKT in the THO mutant testes. (A) Antibody staining of testes with anti-p-AKT antibody in wild-type (WT), thoc5 1 /thoc5 e00906 ( thoc5 ), thoc6 e00298 ( thoc6 ) and thoc7 d05792 ( thoc7 ). In wild-type, p-AKT was highly accumulated in the tip region where cells at the early-stages of germline development reside, and gradually disappeared as spermatogenesis proceeded. In thoc5 , however, the level of p-AKT was not only elevated, but also sustained over spermatogenesis. Similar, but much milder, defects were seen in the thoc7 mutant testis. No significant differences from wild-type and thoc6 . were observed. (B) Western blot analysis of testis extracts from wildtype, thoc5 1 /thoc5 e00906 , thoc6 e00298 and thoc7 d05792 . The level of p-AKT was increased in thoc5 and thoc7 , but not in thoc6 . The result was well matched with that of antibody staining (C) FLP/FRT-mediated clonal analysis of thoc5 1 mutant cells in the testis. In the thoc5 1 mutant cells indicated by the absence of GFP signals (green), the immunoreactivities against p-AKT (Red) were highly elevated compared to their neighboring wild-type cells which showed only faint signals of p-AKT. Hoechst 33258 was used to visualize DNA (blue). Genotype: y w P{hs-FLP}; P{FRT42D} thoc5 1 /P{FRT42D} P{Ubi-GFP} . Scale bars, 20 μm (A) and 10 μm (C).

    Techniques Used: Mutagenesis, Staining, Western Blot

    4) Product Images from "The Cytokine Midkine and its Receptor RPTP? Regulate B Cell Survival in a Pathway Induced by CD74"

    Article Title: The Cytokine Midkine and its Receptor RPTP? Regulate B Cell Survival in a Pathway Induced by CD74

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

    doi: 10.4049/jimmunol.1101468

    MK induced cascade in B cells B cells derived from C57BL/6 mice were incubated in the presence or absence of MK (100 ng/ml) for various periods. Immediately after stimulation, cells were washed and fast frozen in liquid N 2 . The cells were lysed as described in Materials and Methods (A) Lysates were separated on 10% (w/v) SDS-PAGE, and proteins were blotted with anti-p-Akt or anti-Tubulin antibodies. The results presented are representative of at least four different experiments . (B) An aliquot from the lysates was reserved for total Syk analysis. Phosphorylated proteins from the remaining lysate were immunoprecipitated (IP) with an anti-Tyr(P) antibody. Immunoprecipitates and total lysate proteins were separated on 10% (w/v) SDS-PAGE and blotted with an anti-Syk antibody. The results presented are representative of at least three different experiments. (C) RNA was purified from control B cells after stimulation with PBS or MK (100ng/ml) for 6h. Quantitative Real time PCR was performed using primers for Bcl-2 and β-actin. β-actin levels were used to normalize samples for calculation of the relative expression levels of Bcl-2. Results are expressed as a fold change in MK expression in stimulated cells compared to non-stimulated cells, which was defined as 1. Results shown represent an average of at least eight separate experiments. (D) B220+ B cells derived from C57BL/6 mice were incubated in the presence or absence of MK (100ng/ml) for 6h. Cells were lysed, and levels of Bcl-2 and Tubulin were analyzed by western blot analysis. The results presented are representative of at least four different experiments.
    Figure Legend Snippet: MK induced cascade in B cells B cells derived from C57BL/6 mice were incubated in the presence or absence of MK (100 ng/ml) for various periods. Immediately after stimulation, cells were washed and fast frozen in liquid N 2 . The cells were lysed as described in Materials and Methods (A) Lysates were separated on 10% (w/v) SDS-PAGE, and proteins were blotted with anti-p-Akt or anti-Tubulin antibodies. The results presented are representative of at least four different experiments . (B) An aliquot from the lysates was reserved for total Syk analysis. Phosphorylated proteins from the remaining lysate were immunoprecipitated (IP) with an anti-Tyr(P) antibody. Immunoprecipitates and total lysate proteins were separated on 10% (w/v) SDS-PAGE and blotted with an anti-Syk antibody. The results presented are representative of at least three different experiments. (C) RNA was purified from control B cells after stimulation with PBS or MK (100ng/ml) for 6h. Quantitative Real time PCR was performed using primers for Bcl-2 and β-actin. β-actin levels were used to normalize samples for calculation of the relative expression levels of Bcl-2. Results are expressed as a fold change in MK expression in stimulated cells compared to non-stimulated cells, which was defined as 1. Results shown represent an average of at least eight separate experiments. (D) B220+ B cells derived from C57BL/6 mice were incubated in the presence or absence of MK (100ng/ml) for 6h. Cells were lysed, and levels of Bcl-2 and Tubulin were analyzed by western blot analysis. The results presented are representative of at least four different experiments.

    Techniques Used: Derivative Assay, Mouse Assay, Incubation, SDS Page, Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction, Expressing, Western Blot

    5) Product Images from "Sur8/Shoc2 promotes cell motility and metastasis through activation of Ras-PI3K signaling"

    Article Title: Sur8/Shoc2 promotes cell motility and metastasis through activation of Ras-PI3K signaling

    Journal: Oncotarget

    doi:

    Effect of Sur8 on Rac and Akt activation A–C. The shCon-GFP and shSur8-GFP NIH3T3 cells were treated with EGF for 10 minutes (A), HEK293 cells were transfected with the indicated plasmids or siRNAs (siGFP or siSur8 #1 and #2) (B, C). For GTP-Rac measurement, WCLs were incubated with GST-PAK-CD and analyzed by immunoblotting with an anti-Rac1 antibody. For all other measurements, WCLs were immunoblotted against the indicated proteins. D. NIH3T3 cells were transfected with either Con-GFP or Sur8-GFP plasmids, and immunocytochemical analysis was performed using anti-p-ERK or -p-Akt antibody. Cell nuclei were counterstained with DAPI. Relative intensities of the markers stained were quantified for at least 15 different cells using NIS-Elements AR 3.1. Scale bars, 50 μm. The values are mean ± s.e.m. of three independent experiments.
    Figure Legend Snippet: Effect of Sur8 on Rac and Akt activation A–C. The shCon-GFP and shSur8-GFP NIH3T3 cells were treated with EGF for 10 minutes (A), HEK293 cells were transfected with the indicated plasmids or siRNAs (siGFP or siSur8 #1 and #2) (B, C). For GTP-Rac measurement, WCLs were incubated with GST-PAK-CD and analyzed by immunoblotting with an anti-Rac1 antibody. For all other measurements, WCLs were immunoblotted against the indicated proteins. D. NIH3T3 cells were transfected with either Con-GFP or Sur8-GFP plasmids, and immunocytochemical analysis was performed using anti-p-ERK or -p-Akt antibody. Cell nuclei were counterstained with DAPI. Relative intensities of the markers stained were quantified for at least 15 different cells using NIS-Elements AR 3.1. Scale bars, 50 μm. The values are mean ± s.e.m. of three independent experiments.

    Techniques Used: Activation Assay, Transfection, Incubation, Staining

    6) Product Images from "ΔNp63 promotes IGF1 signalling through IRS1 in squamous cell carcinoma"

    Article Title: ΔNp63 promotes IGF1 signalling through IRS1 in squamous cell carcinoma

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101725

    D epletion of p63 reduces the responsiveness of HNSCC cells to ligand stimulation. ( A ) Fadu cells were transfected with siScr or different p63 (sip63#1, sip63#2, siΔNp63) siRNAs. Forty-eight h after transfection, cells were serum starved for 4 h, and then stimulated with 5 nM IGF1 (upper panel) or 500 ng/ml insulin (lower panel) for 10 min. Protein amounts of p63, IRS1 and p-IRS1 were detected by western blot analysis. β-actin served as loading control. Blots are representative of three individual experiments. ( B ) Fadu cells were transfected with siScr, sip63#1 and sip63#2, serum starved for 4 h and then stimulated with 5 nM IGF1 for 10 min. Cellular extracts were analysed with the following antibodies: anti-IRS1, anti-p-IRS1, anti-p-AKT, anti-AKT, anti-p-S6 Ribosomal Protein, anti-S6, anti-p44/42 MAPK (p-ERK1/2), anti-ERK1/2, p63 and β-actin as loading control. Blots are representative of three individual experiments. ( C ) Fadu cells were transfected with siScr or siIRS1 (upper panel) and with sip63#1, ΔNp63, or siScr (lower panel). Forty-eight h after transfection, cells were seeded in 6-cm plates at 500,000/plate and growth was followed until day 6.
    Figure Legend Snippet: D epletion of p63 reduces the responsiveness of HNSCC cells to ligand stimulation. ( A ) Fadu cells were transfected with siScr or different p63 (sip63#1, sip63#2, siΔNp63) siRNAs. Forty-eight h after transfection, cells were serum starved for 4 h, and then stimulated with 5 nM IGF1 (upper panel) or 500 ng/ml insulin (lower panel) for 10 min. Protein amounts of p63, IRS1 and p-IRS1 were detected by western blot analysis. β-actin served as loading control. Blots are representative of three individual experiments. ( B ) Fadu cells were transfected with siScr, sip63#1 and sip63#2, serum starved for 4 h and then stimulated with 5 nM IGF1 for 10 min. Cellular extracts were analysed with the following antibodies: anti-IRS1, anti-p-IRS1, anti-p-AKT, anti-AKT, anti-p-S6 Ribosomal Protein, anti-S6, anti-p44/42 MAPK (p-ERK1/2), anti-ERK1/2, p63 and β-actin as loading control. Blots are representative of three individual experiments. ( C ) Fadu cells were transfected with siScr or siIRS1 (upper panel) and with sip63#1, ΔNp63, or siScr (lower panel). Forty-eight h after transfection, cells were seeded in 6-cm plates at 500,000/plate and growth was followed until day 6.

    Techniques Used: Transfection, Western Blot

    7) Product Images from "Agonist antibody that induces human malignant cells to kill one another"

    Article Title: Agonist antibody that induces human malignant cells to kill one another

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1519079112

    Activation of TPOR signal transduction by antibody in AML cells. ( A ) After treatment with various doses of antibody or 10 ng/mL of TPO for 1 h, the phosphorylation of STAT-3, AKT, and ERK was analyzed by Western blotting using anti–p-STAT-3, anti–p-AKT,
    Figure Legend Snippet: Activation of TPOR signal transduction by antibody in AML cells. ( A ) After treatment with various doses of antibody or 10 ng/mL of TPO for 1 h, the phosphorylation of STAT-3, AKT, and ERK was analyzed by Western blotting using anti–p-STAT-3, anti–p-AKT,

    Techniques Used: Activation Assay, Transduction, Western Blot

    8) Product Images from "CD84 regulates PD-1/PD-L1 expression and function in chronic lymphocytic leukemia"

    Article Title: CD84 regulates PD-1/PD-L1 expression and function in chronic lymphocytic leukemia

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI96610

    Activation of cell-surface CD84 elevates PD-L1 levels through the p-AKT/mTOR pathway. ( A and B ) M210B4 cells (1 × 10 5 ) were stimulated with anti-CD84 (4 μg/ml) for 15 minutes, followed by anti-FAB cross-linking for 5 minutes. ( A ) Cells were lysed, and lysates were separated on 12% (wt/vol) SDS-PAGE and blotted with anti–p-S6, anti–p-AKT, anti–p-ERK, or actin. Blots are representative of 3 independent experiments. ( B ) Cells were fixed, permeabilized, and subsequently stained with anti–p-AKT and anti–p-S6 antibodies followed by a secondary anti-rabbit allophycocyanin-conjugated (APC-conjugated) antibody. Histograms are representative of 2 independent experiments. ( C – E ) Primary CLL cells were stimulated with control (IgG) or anti-CD84–activating (5 μg/ml) antibodies for 5 minutes, followed by anti-FAB cross-linking for an additional 5 minutes. Then, the cells were fixed, and p-ERK, p-AKT, and p-S6 levels were analyzed by flow cytometry ( n = 3; * P
    Figure Legend Snippet: Activation of cell-surface CD84 elevates PD-L1 levels through the p-AKT/mTOR pathway. ( A and B ) M210B4 cells (1 × 10 5 ) were stimulated with anti-CD84 (4 μg/ml) for 15 minutes, followed by anti-FAB cross-linking for 5 minutes. ( A ) Cells were lysed, and lysates were separated on 12% (wt/vol) SDS-PAGE and blotted with anti–p-S6, anti–p-AKT, anti–p-ERK, or actin. Blots are representative of 3 independent experiments. ( B ) Cells were fixed, permeabilized, and subsequently stained with anti–p-AKT and anti–p-S6 antibodies followed by a secondary anti-rabbit allophycocyanin-conjugated (APC-conjugated) antibody. Histograms are representative of 2 independent experiments. ( C – E ) Primary CLL cells were stimulated with control (IgG) or anti-CD84–activating (5 μg/ml) antibodies for 5 minutes, followed by anti-FAB cross-linking for an additional 5 minutes. Then, the cells were fixed, and p-ERK, p-AKT, and p-S6 levels were analyzed by flow cytometry ( n = 3; * P

    Techniques Used: Activation Assay, SDS Page, Staining, Flow Cytometry, Cytometry

    9) Product Images from "Autophagy induction by the pathogen receptor NECTIN4 and sustained autophagy contribute to peste des petits ruminants virus infectivity"

    Article Title: Autophagy induction by the pathogen receptor NECTIN4 and sustained autophagy contribute to peste des petits ruminants virus infectivity

    Journal: Autophagy

    doi: 10.1080/15548627.2019.1643184

    PPRV-H and NECTIN4 are sufficient to induce the first wave of autophagy via AKT and MTOR dephosphorylation. (A) His-tagged V, H and N were expressed in E. coli BL-21 and purified using Ni-NTA columns. The purified products were separated using SDS-PAGE and analyzed by immunoblotting with an anti-His antibody. (B) EECs were transfected with GFP-LC3 for 24 h and incubated with 100 ng/mL His-GST, His-V, His-H, or His-N for 1.5 h. Autophagy was monitored by evaluating the number of GFP + -LC3 vesicles per cell profile by confocal immunofluorescence microscopy. Scale bars, 20 μm. (C) Corresponding graph representing the numbers of GFP + -LC3 vesicles per cell profile in EECs pre-treated with His-GST, His-V, His-H, or His-N. (D) EECs were cultured in DMEM/F12 supplemented with 2% fetal bovine serum containing His-GST, His-V, His-H or His-N for 1.5 h and analyzed by immunoblotting with anti-LC3, anti-SQSTM1, anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT, and anti-ACTB (loading control) antibodies. The relative target protein levels were determined by densitometry in His-GST, His-V, His-H and His-N pre-treated cells. (E) EECs were treated with si-control or si- NECTIN4 and infected with PPRV (MOI = 3) for 1.5 h. The cell samples were analyzed by immunoblotting with anti-LC3, anti-SQSTM1, anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT, anti-NECTIN4 and anti-ACTB (loading control) antibodies. The relative target protein levels in the siRNA-transfected cells were determined by densitometry. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; ** P
    Figure Legend Snippet: PPRV-H and NECTIN4 are sufficient to induce the first wave of autophagy via AKT and MTOR dephosphorylation. (A) His-tagged V, H and N were expressed in E. coli BL-21 and purified using Ni-NTA columns. The purified products were separated using SDS-PAGE and analyzed by immunoblotting with an anti-His antibody. (B) EECs were transfected with GFP-LC3 for 24 h and incubated with 100 ng/mL His-GST, His-V, His-H, or His-N for 1.5 h. Autophagy was monitored by evaluating the number of GFP + -LC3 vesicles per cell profile by confocal immunofluorescence microscopy. Scale bars, 20 μm. (C) Corresponding graph representing the numbers of GFP + -LC3 vesicles per cell profile in EECs pre-treated with His-GST, His-V, His-H, or His-N. (D) EECs were cultured in DMEM/F12 supplemented with 2% fetal bovine serum containing His-GST, His-V, His-H or His-N for 1.5 h and analyzed by immunoblotting with anti-LC3, anti-SQSTM1, anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT, and anti-ACTB (loading control) antibodies. The relative target protein levels were determined by densitometry in His-GST, His-V, His-H and His-N pre-treated cells. (E) EECs were treated with si-control or si- NECTIN4 and infected with PPRV (MOI = 3) for 1.5 h. The cell samples were analyzed by immunoblotting with anti-LC3, anti-SQSTM1, anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT, anti-NECTIN4 and anti-ACTB (loading control) antibodies. The relative target protein levels in the siRNA-transfected cells were determined by densitometry. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; ** P

    Techniques Used: De-Phosphorylation Assay, Purification, SDS Page, Transfection, Incubation, Immunofluorescence, Microscopy, Cell Culture, Infection

    The role of the AKT-MTOR pathway in PPRV-induced autophagy in host cells. (A and B) EECs were mock-infected or infected with PPRV (MOI = 3). The cells were harvested at 1.5, 3, 6, 9, 12 and 24 h and then immunoblotted with anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT, and anti-ACTB antibodies. (C) The p-MTOR levels relative to the MTOR levels were determined by densitometry. (D) The p-AKT levels relative to the AKT levels were determined by densitometry. (E and F) EECs were pre-treated with INS (1 μM) for 6 h prior to viral infection. Then, the cells were infected with PPRV or UV-irradiated PPRV (MOI = 3) for 1.5 or 12 h. The cell samples were analyzed by immunoblotting with anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT, anti-LC3, anti-PPRV-N and anti-ACTB (loading control) antibodies. (G) The relative target protein levels in the INS-pre-treated cells at 1.5 hpi were determined by densitometry. (H) The relative target protein levels in the INS-pre-treated cells at 12 hpi were determined by densitometry. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; ** P
    Figure Legend Snippet: The role of the AKT-MTOR pathway in PPRV-induced autophagy in host cells. (A and B) EECs were mock-infected or infected with PPRV (MOI = 3). The cells were harvested at 1.5, 3, 6, 9, 12 and 24 h and then immunoblotted with anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT, and anti-ACTB antibodies. (C) The p-MTOR levels relative to the MTOR levels were determined by densitometry. (D) The p-AKT levels relative to the AKT levels were determined by densitometry. (E and F) EECs were pre-treated with INS (1 μM) for 6 h prior to viral infection. Then, the cells were infected with PPRV or UV-irradiated PPRV (MOI = 3) for 1.5 or 12 h. The cell samples were analyzed by immunoblotting with anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT, anti-LC3, anti-PPRV-N and anti-ACTB (loading control) antibodies. (G) The relative target protein levels in the INS-pre-treated cells at 1.5 hpi were determined by densitometry. (H) The relative target protein levels in the INS-pre-treated cells at 12 hpi were determined by densitometry. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; ** P

    Techniques Used: Infection, Irradiation

    PPRV-H binding to NECTIN4 triggers autophagy via AKT-MTOR dephosphorylation. (A) Co-IP assay results demonstrating that endogenous NECTIN4 binds to H-HA in transfected cells. EECs were transfected with pCDNA3.1-H-HA for 48 h and harvested. Cell lysates from transfected cells and untransfected control cells were immunoprecipitated with an antibody against HA and then subjected to immunoblotting. (B) Exogenous NECTIN4-Flag and H-HA coexpression in EECs. Cell lysates from cells co-transfected with NECTIN4-Flag and H-HA or transfected with NECTIN4-Flag alone were immunoprecipitated with an antibody against HA and then subjected to immunoblotting. (C) Results of reciprocal co-IP experiments showing that the anti-Flag antibody precipitated H-HA. (D) EECs were transfected with pCDNA3.1-NECTIN4-Flag for 48 h and then infected with PPRV (MOI = 3) for 1.5 or 3 h. Cell lysates from the transfected cells were immunoprecipitated with an antibody against Flag and then subjected to immunoblotting. (E) EECs were transfected with pCDNA3.1-NECTIN4-Flag for 48 h and then incubated with 100 ng/mL His-GST, His-V, His-H, or His-N for 1.5 h. Cell lysates from the transfected cells were immunoprecipitated with an antibody against Flag and then subjected to immunoblotting. (F) Immunoblotting was performed using anti-LC3 and anti-SQSTM1 antibodies on lysates from EECs cultured in uncoated plates or in plates coated with anti-NECTIN4 or an irrelevant isotype control IgG for 4 h at 37°C. The target protein levels relative to the ACTB levels in cells pre-treated with anti-NECTIN4 or an irrelevant isotype control IgG were determined by densitometry. (G) EECs were transfected with GFP-LC3 for 24 h and cultured in plates coated with the irrelevant isotype control IgG or anti-NECTIN4 for 4 h. Autophagy was monitored by evaluating the number of GFP + -LC3 vesicles per cell profile by confocal immunofluorescence microscopy. Scale bars, 20 μm. The corresponding graph shows the number of GFP + -LC3 vesicles per cell profile among the EECs pre-treated with anti-NECTIN4 or the irrelevant isotype control IgG. (H) EECs were incubated with His-GST, a mixture of His-GST and NECTIN4 (His-GST:NECTIN4 = 1:3), a mixture of His-H and NECTIN4 (His-H:NECTIN4 = 1:3), or His-H for 1.5 h and analyzed by immunoblotting with anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT, anti-LC3, and anti-ACTB (loading control) antibodies. The relative target protein levels in the pre-treated cells were determined by densitometry. (I) EECs were incubated with His-GST (100 ng/mL) or His-H (100 ng/mL) and PPRV for 1.5 h at 37°C or 4°C. The cells were analyzed by immunoblotting with anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT, anti-LC3, and anti-ACTB (loading control) antibodies. The relative target protein levels in the pre-treated cells were determined by densitometry. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; ** P
    Figure Legend Snippet: PPRV-H binding to NECTIN4 triggers autophagy via AKT-MTOR dephosphorylation. (A) Co-IP assay results demonstrating that endogenous NECTIN4 binds to H-HA in transfected cells. EECs were transfected with pCDNA3.1-H-HA for 48 h and harvested. Cell lysates from transfected cells and untransfected control cells were immunoprecipitated with an antibody against HA and then subjected to immunoblotting. (B) Exogenous NECTIN4-Flag and H-HA coexpression in EECs. Cell lysates from cells co-transfected with NECTIN4-Flag and H-HA or transfected with NECTIN4-Flag alone were immunoprecipitated with an antibody against HA and then subjected to immunoblotting. (C) Results of reciprocal co-IP experiments showing that the anti-Flag antibody precipitated H-HA. (D) EECs were transfected with pCDNA3.1-NECTIN4-Flag for 48 h and then infected with PPRV (MOI = 3) for 1.5 or 3 h. Cell lysates from the transfected cells were immunoprecipitated with an antibody against Flag and then subjected to immunoblotting. (E) EECs were transfected with pCDNA3.1-NECTIN4-Flag for 48 h and then incubated with 100 ng/mL His-GST, His-V, His-H, or His-N for 1.5 h. Cell lysates from the transfected cells were immunoprecipitated with an antibody against Flag and then subjected to immunoblotting. (F) Immunoblotting was performed using anti-LC3 and anti-SQSTM1 antibodies on lysates from EECs cultured in uncoated plates or in plates coated with anti-NECTIN4 or an irrelevant isotype control IgG for 4 h at 37°C. The target protein levels relative to the ACTB levels in cells pre-treated with anti-NECTIN4 or an irrelevant isotype control IgG were determined by densitometry. (G) EECs were transfected with GFP-LC3 for 24 h and cultured in plates coated with the irrelevant isotype control IgG or anti-NECTIN4 for 4 h. Autophagy was monitored by evaluating the number of GFP + -LC3 vesicles per cell profile by confocal immunofluorescence microscopy. Scale bars, 20 μm. The corresponding graph shows the number of GFP + -LC3 vesicles per cell profile among the EECs pre-treated with anti-NECTIN4 or the irrelevant isotype control IgG. (H) EECs were incubated with His-GST, a mixture of His-GST and NECTIN4 (His-GST:NECTIN4 = 1:3), a mixture of His-H and NECTIN4 (His-H:NECTIN4 = 1:3), or His-H for 1.5 h and analyzed by immunoblotting with anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT, anti-LC3, and anti-ACTB (loading control) antibodies. The relative target protein levels in the pre-treated cells were determined by densitometry. (I) EECs were incubated with His-GST (100 ng/mL) or His-H (100 ng/mL) and PPRV for 1.5 h at 37°C or 4°C. The cells were analyzed by immunoblotting with anti-p-MTOR, anti-MTOR, anti-p-AKT, anti-AKT, anti-LC3, and anti-ACTB (loading control) antibodies. The relative target protein levels in the pre-treated cells were determined by densitometry. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; ** P

    Techniques Used: Binding Assay, De-Phosphorylation Assay, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Infection, Incubation, Cell Culture, Immunofluorescence, Microscopy

    10) Product Images from "Inhibition of pancreatic oxidative damage by stilbene derivative dihydro-resveratrol: implication for treatment of acute pancreatitis"

    Article Title: Inhibition of pancreatic oxidative damage by stilbene derivative dihydro-resveratrol: implication for treatment of acute pancreatitis

    Journal: Scientific Reports

    doi: 10.1038/srep22859

    Dihydro-resveratrol decreased IκB degradation and AKT phosphorylation. ( a ) IκB- α degradation and AKT phosphorylation in rat pancreatic tissues were visualized on immunoblots probed with anti-IκB- α , anti-p-AKT and anti-total AKT antibodies. Tubulin was served as a loading control. ( b ) Isolated pancreatic acini were pre-incubated with H 2 O 2 for 30 minutes, and treated with dihydro-resveratrol, and/or LY294002 for 1 hour. The phosphorylation of AKT in isolated pancreatic acini was examined by means of Western blotting. Tubulin was served as a reference control of the cytoplasmic fraction. ( c ) Acinar cells were treated with a series concentration of dihydro-resveratrol or trans -resveratrol (0 to 1000 μM) for 24 hours. The cytotoxicity of dihydro-resveratrol in isolated acini was assessed using MTT cell viability assay.
    Figure Legend Snippet: Dihydro-resveratrol decreased IκB degradation and AKT phosphorylation. ( a ) IκB- α degradation and AKT phosphorylation in rat pancreatic tissues were visualized on immunoblots probed with anti-IκB- α , anti-p-AKT and anti-total AKT antibodies. Tubulin was served as a loading control. ( b ) Isolated pancreatic acini were pre-incubated with H 2 O 2 for 30 minutes, and treated with dihydro-resveratrol, and/or LY294002 for 1 hour. The phosphorylation of AKT in isolated pancreatic acini was examined by means of Western blotting. Tubulin was served as a reference control of the cytoplasmic fraction. ( c ) Acinar cells were treated with a series concentration of dihydro-resveratrol or trans -resveratrol (0 to 1000 μM) for 24 hours. The cytotoxicity of dihydro-resveratrol in isolated acini was assessed using MTT cell viability assay.

    Techniques Used: Western Blot, Isolation, Incubation, Concentration Assay, MTT Assay, Viability Assay

    11) Product Images from "Anti-metastatic potential of somatostatin analog SOM230: Indirect pharmacological targeting of pancreatic cancer-associated fibroblasts"

    Article Title: Anti-metastatic potential of somatostatin analog SOM230: Indirect pharmacological targeting of pancreatic cancer-associated fibroblasts

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9296

    A-D. Immunoblotting using an anti-P-Akt, or Akt (loading control) antibody of protein extracts from Panc-1 cells A, C-D. or BxPC-3 cells B. treated or not for 15 min with SOM230 (10 −7 M), or with recombinant IL-6 (rIL-6), or with IL-6 Ab, or with CM from untreated or SOM230-treated CAFs in the presence or not of the IL-6 neutralizing antibody (IL-6 Ab) (representative of n=3). E-J. Panc-1 E-G. and BxPC-3 H-J. cell viability E, H. was assessed by MTT, and cell migration F, I. or invasion G, J. using modified Boyden chambers. Cells were pre-treated or not for 30 min with LY294002, and then incubated in the presence of recombinant IL-6 (rIL-6), or CM from untreated or SOM230-treated CAFs. Results are presented as a percentage of the survival, migration or invasion observed in the condition where cancer cells were not exposed to rIL-6 or CAF CM (=100%) (n=3). *: effect of treatment vs. untreated cells; #: effect of LY294002.
    Figure Legend Snippet: A-D. Immunoblotting using an anti-P-Akt, or Akt (loading control) antibody of protein extracts from Panc-1 cells A, C-D. or BxPC-3 cells B. treated or not for 15 min with SOM230 (10 −7 M), or with recombinant IL-6 (rIL-6), or with IL-6 Ab, or with CM from untreated or SOM230-treated CAFs in the presence or not of the IL-6 neutralizing antibody (IL-6 Ab) (representative of n=3). E-J. Panc-1 E-G. and BxPC-3 H-J. cell viability E, H. was assessed by MTT, and cell migration F, I. or invasion G, J. using modified Boyden chambers. Cells were pre-treated or not for 30 min with LY294002, and then incubated in the presence of recombinant IL-6 (rIL-6), or CM from untreated or SOM230-treated CAFs. Results are presented as a percentage of the survival, migration or invasion observed in the condition where cancer cells were not exposed to rIL-6 or CAF CM (=100%) (n=3). *: effect of treatment vs. untreated cells; #: effect of LY294002.

    Techniques Used: Recombinant, MTT Assay, Migration, Modification, Incubation

    12) Product Images from "Ras signaling regulates osteoprogenitor cell proliferation and bone formation"

    Article Title: Ras signaling regulates osteoprogenitor cell proliferation and bone formation

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.314

    Activation of Kras oncogene in Col2 cells at perinatal age increases stromal cell numbers (see also Supplementary figure S3 ). ( a - b ) Hematoxylin/eosin-stained paraffin sections showing the stromal cells between the trabeculae in P28 mice (secondary spongiosa) control ( a ) and Kras G12D ( b ) after Kras activation at E18.5. × 40 magnification. ( c - f ) In situ hybridization for collagen 1 (Col1 ISH) ( c , d ) and osteocalcin (Oc ISH) ( e , f ) in the secondary spongiosa of P28-old mice control ( c , e ) or Kras G12D ( d , f ) after tamoxifen injection at E18.5. Stromal cells are not stained for these markers. Black arrows, osteoblasts; red asterisks, stromal cells. ( g - j ) Immunohistochemistry for p-ERK at P1.5 ( g , h ) and P10 ( i , j ) in the tibia secondary spongiosa of control ( g , i ) and Kras G12D ( h , j ) after tamoxifen injection at E18.5. Arrows show representative cells stained with anti- p-ERK. ( k - l ) Immunohistochemistry for p-Akt at P10 in the tibia secondary spongiosa of control ( k ) or Kras G12D ( l ) after tamoxifen injection at E18.5. Arrows show representative cells stained with anti- p-Akt antibody. ( m - p ) BrdU (Bromodeoxyuridine) labeling of proliferating cells in the metaphysis of mice (tibia) control ( m , o ) and Kras G12D ( n , p ) at P10 after Kras activation at E18.5. BrdU-positive cells are stained brown. ( q ) The BrdU index, calculated as the percentage of BrdU-labeled stromal cells, was significantly increased upon Kras G12D activation. Stromal cells were defined as the non-hematopoietic cells that were not attached to the bone matrix. Data are represented as mean±S.E.M.; n =3, * P =0.002
    Figure Legend Snippet: Activation of Kras oncogene in Col2 cells at perinatal age increases stromal cell numbers (see also Supplementary figure S3 ). ( a - b ) Hematoxylin/eosin-stained paraffin sections showing the stromal cells between the trabeculae in P28 mice (secondary spongiosa) control ( a ) and Kras G12D ( b ) after Kras activation at E18.5. × 40 magnification. ( c - f ) In situ hybridization for collagen 1 (Col1 ISH) ( c , d ) and osteocalcin (Oc ISH) ( e , f ) in the secondary spongiosa of P28-old mice control ( c , e ) or Kras G12D ( d , f ) after tamoxifen injection at E18.5. Stromal cells are not stained for these markers. Black arrows, osteoblasts; red asterisks, stromal cells. ( g - j ) Immunohistochemistry for p-ERK at P1.5 ( g , h ) and P10 ( i , j ) in the tibia secondary spongiosa of control ( g , i ) and Kras G12D ( h , j ) after tamoxifen injection at E18.5. Arrows show representative cells stained with anti- p-ERK. ( k - l ) Immunohistochemistry for p-Akt at P10 in the tibia secondary spongiosa of control ( k ) or Kras G12D ( l ) after tamoxifen injection at E18.5. Arrows show representative cells stained with anti- p-Akt antibody. ( m - p ) BrdU (Bromodeoxyuridine) labeling of proliferating cells in the metaphysis of mice (tibia) control ( m , o ) and Kras G12D ( n , p ) at P10 after Kras activation at E18.5. BrdU-positive cells are stained brown. ( q ) The BrdU index, calculated as the percentage of BrdU-labeled stromal cells, was significantly increased upon Kras G12D activation. Stromal cells were defined as the non-hematopoietic cells that were not attached to the bone matrix. Data are represented as mean±S.E.M.; n =3, * P =0.002

    Techniques Used: Activation Assay, Staining, Mouse Assay, In Situ Hybridization, Injection, Immunohistochemistry, Labeling

    13) Product Images from "Chaperone-E3 Ligase Complex HSP70-CHIP Mediates Ubiquitination of Ribosomal Protein S3"

    Article Title: Chaperone-E3 Ligase Complex HSP70-CHIP Mediates Ubiquitination of Ribosomal Protein S3

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092723

    Ribosomal protein S3 (RPS3) is highly upregulated in the hippocampus and cerebral cortex after starvation. C57BL/6 mice were subjected to nutrient starvation, with only water supplied, for 0, 24, 72, and 120 h and were euthanized to isolate the hippocampal area and cortical area of the brains. Endogenous protein levels of RPS3 or phospho (p)-RPS3 in the ( A ) hippocampus or ( C ) cortex were measured by immunoblotting with anti-RPS3 and anti-p-RPS3 antibodies, and neuronal apoptosis was evaluated with an anti-poly-ADP-ribose polymerase (PARP) antibody. ( B , D ) RPS3/p-RPS3 (left), AKT/p-AKT (right) protein levels were determined by densitometric analysis in ( B ) the hippocampus or ( D ) cortex. Densities of immunoblotting were measured with Image J and its normalized with control mice (0 h) brain lysates. Values in this figure represent the mean ± SEM from three independent experiments, and the images shown are representative of at least three independent experiments. Statistical significance was calculated using a one-way ANOVA test followed by Turkey’s post-test (* p
    Figure Legend Snippet: Ribosomal protein S3 (RPS3) is highly upregulated in the hippocampus and cerebral cortex after starvation. C57BL/6 mice were subjected to nutrient starvation, with only water supplied, for 0, 24, 72, and 120 h and were euthanized to isolate the hippocampal area and cortical area of the brains. Endogenous protein levels of RPS3 or phospho (p)-RPS3 in the ( A ) hippocampus or ( C ) cortex were measured by immunoblotting with anti-RPS3 and anti-p-RPS3 antibodies, and neuronal apoptosis was evaluated with an anti-poly-ADP-ribose polymerase (PARP) antibody. ( B , D ) RPS3/p-RPS3 (left), AKT/p-AKT (right) protein levels were determined by densitometric analysis in ( B ) the hippocampus or ( D ) cortex. Densities of immunoblotting were measured with Image J and its normalized with control mice (0 h) brain lysates. Values in this figure represent the mean ± SEM from three independent experiments, and the images shown are representative of at least three independent experiments. Statistical significance was calculated using a one-way ANOVA test followed by Turkey’s post-test (* p

    Techniques Used: Mouse Assay

    14) Product Images from "Astrocyte-Specific Overexpression of Insulin-Like Growth Factor-1 Protects Hippocampal Neurons and Reduces Behavioral Deficits following Traumatic Brain Injury in Mice"

    Article Title: Astrocyte-Specific Overexpression of Insulin-Like Growth Factor-1 Protects Hippocampal Neurons and Reduces Behavioral Deficits following Traumatic Brain Injury in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067204

    Akt phosphorylation is augmented in IGF-1Tg mice. A) Representative western blot images for ipsilateral hippocampal samples probed with anti-p-Akt and anti-actin antibodies. Blots illustrate expression at 72 h after severe (1.0 mm) controlled cortical impact (CCI) brain injury or sham injury (Sh). Relative expression of hippocampal p-Akt at 24 h ( B,C ) and 72 h ( D,E ) following either 0.5 mm ( B,D ) or 1.0 mm ( C,E ) CCI in wildtype (WT, open bars) and IGF-1 transgenic (IGF-1Tg, closed bars) mice. Optical density from each band was normalised to its respective actin band and then group means were normalised to the mean of WT sham group. Data are represented as mean+SEM. # p
    Figure Legend Snippet: Akt phosphorylation is augmented in IGF-1Tg mice. A) Representative western blot images for ipsilateral hippocampal samples probed with anti-p-Akt and anti-actin antibodies. Blots illustrate expression at 72 h after severe (1.0 mm) controlled cortical impact (CCI) brain injury or sham injury (Sh). Relative expression of hippocampal p-Akt at 24 h ( B,C ) and 72 h ( D,E ) following either 0.5 mm ( B,D ) or 1.0 mm ( C,E ) CCI in wildtype (WT, open bars) and IGF-1 transgenic (IGF-1Tg, closed bars) mice. Optical density from each band was normalised to its respective actin band and then group means were normalised to the mean of WT sham group. Data are represented as mean+SEM. # p

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

    15) Product Images from "Co-expression modules of NF1, PTEN and sprouty enable distinction of adult diffuse gliomas according to pathway activities of receptor tyrosine kinases"

    Article Title: Co-expression modules of NF1, PTEN and sprouty enable distinction of adult diffuse gliomas according to pathway activities of receptor tyrosine kinases

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10359

    Activation of RTK signaling in vessel endothelial cells and TAMs in RMPA high gliomas Sections of the classified RMPA high or RMPA low gliomas were co-stained with mAbs against CD31 or CD68 in combination with mAbs towards p-AKT or p-ERK. The staining of anti-CD31 or anti-CD68 was detected with Alexa Fluor ® 555 conjugated secondary antibody, and anti-p-AKT or anti-p-ERK with Alexa Fluor ® 647 conjugated secondary antibody. Sections were further stainined with DAPI and evaluated using a confocal microscope (ZEISS LSM 700). Images of representative RMPA high A. or RMPA low . B. gliomas are shown. The control stainings were performed in the absence of the primary antibody. Staining results of other RMPA classified or non-classified samples are summarized in Supplementary Figure S11 .
    Figure Legend Snippet: Activation of RTK signaling in vessel endothelial cells and TAMs in RMPA high gliomas Sections of the classified RMPA high or RMPA low gliomas were co-stained with mAbs against CD31 or CD68 in combination with mAbs towards p-AKT or p-ERK. The staining of anti-CD31 or anti-CD68 was detected with Alexa Fluor ® 555 conjugated secondary antibody, and anti-p-AKT or anti-p-ERK with Alexa Fluor ® 647 conjugated secondary antibody. Sections were further stainined with DAPI and evaluated using a confocal microscope (ZEISS LSM 700). Images of representative RMPA high A. or RMPA low . B. gliomas are shown. The control stainings were performed in the absence of the primary antibody. Staining results of other RMPA classified or non-classified samples are summarized in Supplementary Figure S11 .

    Techniques Used: Activation Assay, Staining, Microscopy

    16) Product Images from "TROP2 is epigenetically inactivated and modulates IGF-1R signalling in lung adenocarcinoma"

    Article Title: TROP2 is epigenetically inactivated and modulates IGF-1R signalling in lung adenocarcinoma

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201200222

    TROP2 expression modulates cell proliferation and colony formation Trop-2 was overexpressed in H1299 cells and knocked down in H322M cells, and the activities of AKT and ERK were determined by Western blotting with anti-pAKT and anti-p-ERK antibodies, respectively. Forced expression of Trop-2 in H1299 cells inhibited cell proliferation compared to the vector control, as determined by cell counting. Conversely, Trop-2 knockdown by either lenti-shTROP2A or lenti-shTROP2C enhanced cell proliferation. The tumour growth ability of lung CL was measured by the formation of colonies in soft agar. The results are presented as means ± SD, data were compared between groups using the t -test, and p
    Figure Legend Snippet: TROP2 expression modulates cell proliferation and colony formation Trop-2 was overexpressed in H1299 cells and knocked down in H322M cells, and the activities of AKT and ERK were determined by Western blotting with anti-pAKT and anti-p-ERK antibodies, respectively. Forced expression of Trop-2 in H1299 cells inhibited cell proliferation compared to the vector control, as determined by cell counting. Conversely, Trop-2 knockdown by either lenti-shTROP2A or lenti-shTROP2C enhanced cell proliferation. The tumour growth ability of lung CL was measured by the formation of colonies in soft agar. The results are presented as means ± SD, data were compared between groups using the t -test, and p

    Techniques Used: Expressing, Western Blot, Plasmid Preparation, Cell Counting

    17) Product Images from "p53 and PI3K/AKT Signalings Are Up-Regulated in Flies with Defects in the THO Complex"

    Article Title: p53 and PI3K/AKT Signalings Are Up-Regulated in Flies with Defects in the THO Complex

    Journal: Molecules and Cells

    doi: 10.1007/s10059-013-0009-x

    Increased level of p-AKT in the THO mutant testes. (A) Antibody staining of testes with anti-p-AKT antibody in wild-type (WT), thoc5 1 /thoc5 e00906 ( thoc5 ), thoc6 e00298 ( thoc6 ) and thoc7 d05792 ( thoc7 ). In wild-type, p-AKT was highly accumulated in the tip region where cells at the early-stages of germline development reside, and gradually disappeared as spermatogenesis proceeded. In thoc5 , however, the level of p-AKT was not only elevated, but also sustained over spermatogenesis. Similar, but much milder, defects were seen in the thoc7 mutant testis. No significant differences from wild-type and thoc6 . were observed. (B) Western blot analysis of testis extracts from wildtype, thoc5 1 /thoc5 e00906 , thoc6 e00298 and thoc7 d05792 . The level of p-AKT was increased in thoc5 and thoc7 , but not in thoc6 . The result was well matched with that of antibody staining (C) FLP/FRT-mediated clonal analysis of thoc5 1 mutant cells in the testis. In the thoc5 1 mutant cells indicated by the absence of GFP signals (green), the immunoreactivities against p-AKT (Red) were highly elevated compared to their neighboring wild-type cells which showed only faint signals of p-AKT. Hoechst 33258 was used to visualize DNA (blue). Genotype: y w P{hs-FLP}; P{FRT42D} thoc5 1 /P{FRT42D} P{Ubi-GFP} . Scale bars, 20 μm (A) and 10 μm (C).
    Figure Legend Snippet: Increased level of p-AKT in the THO mutant testes. (A) Antibody staining of testes with anti-p-AKT antibody in wild-type (WT), thoc5 1 /thoc5 e00906 ( thoc5 ), thoc6 e00298 ( thoc6 ) and thoc7 d05792 ( thoc7 ). In wild-type, p-AKT was highly accumulated in the tip region where cells at the early-stages of germline development reside, and gradually disappeared as spermatogenesis proceeded. In thoc5 , however, the level of p-AKT was not only elevated, but also sustained over spermatogenesis. Similar, but much milder, defects were seen in the thoc7 mutant testis. No significant differences from wild-type and thoc6 . were observed. (B) Western blot analysis of testis extracts from wildtype, thoc5 1 /thoc5 e00906 , thoc6 e00298 and thoc7 d05792 . The level of p-AKT was increased in thoc5 and thoc7 , but not in thoc6 . The result was well matched with that of antibody staining (C) FLP/FRT-mediated clonal analysis of thoc5 1 mutant cells in the testis. In the thoc5 1 mutant cells indicated by the absence of GFP signals (green), the immunoreactivities against p-AKT (Red) were highly elevated compared to their neighboring wild-type cells which showed only faint signals of p-AKT. Hoechst 33258 was used to visualize DNA (blue). Genotype: y w P{hs-FLP}; P{FRT42D} thoc5 1 /P{FRT42D} P{Ubi-GFP} . Scale bars, 20 μm (A) and 10 μm (C).

    Techniques Used: Mutagenesis, Staining, Western Blot

    18) Product Images from "Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity"

    Article Title: Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2012.80

    Biochemical pathways induced by MT2 in PC12 cells. ( a ) TrkA phosphorylation. Serum-starved PC12 cells were stimulated with 10 μ M MT2, or 4 nM hrNGF as positive control, for 15 min. Cells lysates were blotted with antibodies to P-Y490, P-Y674/675, P-Y785 and with anti-actin as loading control. Membranes were stripped and stained with anti-total TrkA IgG. The relative histograms represent the data of densitometric analysis and are expressed as the ratio between phosphoprotein (P) and total protein (T) of five different experiments (mean±S.E.). Statistical analysis was performed by paired Student's t -test. ( b ) Kinases phosphorylation. Serum-starved PC12 cells were stimulated with 10 μ M MT2, or 4 nM hrNGF, for 30 min. Cell lysates were blotted with rabbit anti-P-ERK, anti-P-Akt, anti-P-p38 MAPK, anti-P-JNK. Then, membranes were stripped and stained with antibodies to the respective total protein and with anti-actin antibodies as loading control. The relative histograms represent the data of densitometric analysis and are expressed as the ratio between phosphoprotein (P) and total protein (T) of five different experiments (mean±S.E.). Statistical analysis was performed by paired Student's t -test. ( c ) Phosphatase. Serum-starved PC12 cells were stimulated with 10 μ M MT2, or 4 nM hrNGF as positive control, for 30 min. Cell lysates were blotted with rabbit anti-MKP1 IgG and anti-actin antibodies as loading control. The relative histogram represents the data of densitometric analysis and is expressed as the ratio between MKP1 expression and actin of five different experiments (mean±S.E.). Statistical analysis was performed by paired Student's t -test
    Figure Legend Snippet: Biochemical pathways induced by MT2 in PC12 cells. ( a ) TrkA phosphorylation. Serum-starved PC12 cells were stimulated with 10 μ M MT2, or 4 nM hrNGF as positive control, for 15 min. Cells lysates were blotted with antibodies to P-Y490, P-Y674/675, P-Y785 and with anti-actin as loading control. Membranes were stripped and stained with anti-total TrkA IgG. The relative histograms represent the data of densitometric analysis and are expressed as the ratio between phosphoprotein (P) and total protein (T) of five different experiments (mean±S.E.). Statistical analysis was performed by paired Student's t -test. ( b ) Kinases phosphorylation. Serum-starved PC12 cells were stimulated with 10 μ M MT2, or 4 nM hrNGF, for 30 min. Cell lysates were blotted with rabbit anti-P-ERK, anti-P-Akt, anti-P-p38 MAPK, anti-P-JNK. Then, membranes were stripped and stained with antibodies to the respective total protein and with anti-actin antibodies as loading control. The relative histograms represent the data of densitometric analysis and are expressed as the ratio between phosphoprotein (P) and total protein (T) of five different experiments (mean±S.E.). Statistical analysis was performed by paired Student's t -test. ( c ) Phosphatase. Serum-starved PC12 cells were stimulated with 10 μ M MT2, or 4 nM hrNGF as positive control, for 30 min. Cell lysates were blotted with rabbit anti-MKP1 IgG and anti-actin antibodies as loading control. The relative histogram represents the data of densitometric analysis and is expressed as the ratio between MKP1 expression and actin of five different experiments (mean±S.E.). Statistical analysis was performed by paired Student's t -test

    Techniques Used: Positive Control, Staining, Expressing

    19) Product Images from "Chaperone-E3 Ligase Complex HSP70-CHIP Mediates Ubiquitination of Ribosomal Protein S3"

    Article Title: Chaperone-E3 Ligase Complex HSP70-CHIP Mediates Ubiquitination of Ribosomal Protein S3

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092723

    Ribosomal protein S3 (RPS3) is highly upregulated in the hippocampus and cerebral cortex after starvation. C57BL/6 mice were subjected to nutrient starvation, with only water supplied, for 0, 24, 72, and 120 h and were euthanized to isolate the hippocampal area and cortical area of the brains. Endogenous protein levels of RPS3 or phospho (p)-RPS3 in the ( A ) hippocampus or ( C ) cortex were measured by immunoblotting with anti-RPS3 and anti-p-RPS3 antibodies, and neuronal apoptosis was evaluated with an anti-poly-ADP-ribose polymerase (PARP) antibody. ( B , D ) RPS3/p-RPS3 (left), AKT/p-AKT (right) protein levels were determined by densitometric analysis in ( B ) the hippocampus or ( D ) cortex. Densities of immunoblotting were measured with Image J and its normalized with control mice (0 h) brain lysates. Values in this figure represent the mean ± SEM from three independent experiments, and the images shown are representative of at least three independent experiments. Statistical significance was calculated using a one-way ANOVA test followed by Turkey’s post-test (* p
    Figure Legend Snippet: Ribosomal protein S3 (RPS3) is highly upregulated in the hippocampus and cerebral cortex after starvation. C57BL/6 mice were subjected to nutrient starvation, with only water supplied, for 0, 24, 72, and 120 h and were euthanized to isolate the hippocampal area and cortical area of the brains. Endogenous protein levels of RPS3 or phospho (p)-RPS3 in the ( A ) hippocampus or ( C ) cortex were measured by immunoblotting with anti-RPS3 and anti-p-RPS3 antibodies, and neuronal apoptosis was evaluated with an anti-poly-ADP-ribose polymerase (PARP) antibody. ( B , D ) RPS3/p-RPS3 (left), AKT/p-AKT (right) protein levels were determined by densitometric analysis in ( B ) the hippocampus or ( D ) cortex. Densities of immunoblotting were measured with Image J and its normalized with control mice (0 h) brain lysates. Values in this figure represent the mean ± SEM from three independent experiments, and the images shown are representative of at least three independent experiments. Statistical significance was calculated using a one-way ANOVA test followed by Turkey’s post-test (* p

    Techniques Used: Mouse Assay

    20) Product Images from "ΔNp63 promotes IGF1 signalling through IRS1 in squamous cell carcinoma"

    Article Title: ΔNp63 promotes IGF1 signalling through IRS1 in squamous cell carcinoma

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101725

    D epletion of p63 reduces the responsiveness of HNSCC cells to ligand stimulation. ( A ) Fadu cells were transfected with siScr or different p63 (sip63#1, sip63#2, siΔNp63) siRNAs. Forty-eight h after transfection, cells were serum starved for 4 h, and then stimulated with 5 nM IGF1 (upper panel) or 500 ng/ml insulin (lower panel) for 10 min. Protein amounts of p63, IRS1 and p-IRS1 were detected by western blot analysis. β-actin served as loading control. Blots are representative of three individual experiments. ( B ) Fadu cells were transfected with siScr, sip63#1 and sip63#2, serum starved for 4 h and then stimulated with 5 nM IGF1 for 10 min. Cellular extracts were analysed with the following antibodies: anti-IRS1, anti-p-IRS1, anti-p-AKT, anti-AKT, anti-p-S6 Ribosomal Protein, anti-S6, anti-p44/42 MAPK (p-ERK1/2), anti-ERK1/2, p63 and β-actin as loading control. Blots are representative of three individual experiments. ( C ) Fadu cells were transfected with siScr or siIRS1 (upper panel) and with sip63#1, ΔNp63, or siScr (lower panel). Forty-eight h after transfection, cells were seeded in 6-cm plates at 500,000/plate and growth was followed until day 6.
    Figure Legend Snippet: D epletion of p63 reduces the responsiveness of HNSCC cells to ligand stimulation. ( A ) Fadu cells were transfected with siScr or different p63 (sip63#1, sip63#2, siΔNp63) siRNAs. Forty-eight h after transfection, cells were serum starved for 4 h, and then stimulated with 5 nM IGF1 (upper panel) or 500 ng/ml insulin (lower panel) for 10 min. Protein amounts of p63, IRS1 and p-IRS1 were detected by western blot analysis. β-actin served as loading control. Blots are representative of three individual experiments. ( B ) Fadu cells were transfected with siScr, sip63#1 and sip63#2, serum starved for 4 h and then stimulated with 5 nM IGF1 for 10 min. Cellular extracts were analysed with the following antibodies: anti-IRS1, anti-p-IRS1, anti-p-AKT, anti-AKT, anti-p-S6 Ribosomal Protein, anti-S6, anti-p44/42 MAPK (p-ERK1/2), anti-ERK1/2, p63 and β-actin as loading control. Blots are representative of three individual experiments. ( C ) Fadu cells were transfected with siScr or siIRS1 (upper panel) and with sip63#1, ΔNp63, or siScr (lower panel). Forty-eight h after transfection, cells were seeded in 6-cm plates at 500,000/plate and growth was followed until day 6.

    Techniques Used: Transfection, Western Blot

    21) Product Images from "Aurora-A overexpression enhances cell-aggregation of Ha-ras transformants through the MEK/ERK signaling pathway"

    Article Title: Aurora-A overexpression enhances cell-aggregation of Ha-ras transformants through the MEK/ERK signaling pathway

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-9-435

    Wild-type Aurora-A increases phosphorylation of MEK/ERK, AKT and the activity of RalA of Ras V12 transformants . (A) The phosphorylation of MEK was detected by anti-p-MEK antibody. The expression levels of AKT and p-AKT were detected using anti-AKT and anti-p-AKT specific antibodies.Ral A activity was detected by Ral pull-down assay. The expression level of each protein in three cells was shown graphically at the bottom panel. (B) After Aurora-A siRNA (5 μg) was transfected into WT cells for 6 h, IPTG was added and incubated for another 48 h. The total cell lysates (100 μg) were separated by 12% SDS-PAGE. Ral A, Ras, Aurora-A and β-actin were detected using Western blotting. The level of each protein was quantified by a densitometer. The expression level of each protein in Vector cells without IPTG induction was set as 1.0 fold.
    Figure Legend Snippet: Wild-type Aurora-A increases phosphorylation of MEK/ERK, AKT and the activity of RalA of Ras V12 transformants . (A) The phosphorylation of MEK was detected by anti-p-MEK antibody. The expression levels of AKT and p-AKT were detected using anti-AKT and anti-p-AKT specific antibodies.Ral A activity was detected by Ral pull-down assay. The expression level of each protein in three cells was shown graphically at the bottom panel. (B) After Aurora-A siRNA (5 μg) was transfected into WT cells for 6 h, IPTG was added and incubated for another 48 h. The total cell lysates (100 μg) were separated by 12% SDS-PAGE. Ral A, Ras, Aurora-A and β-actin were detected using Western blotting. The level of each protein was quantified by a densitometer. The expression level of each protein in Vector cells without IPTG induction was set as 1.0 fold. " * ": p ≦ 0.01

    Techniques Used: Activity Assay, Expressing, Pull Down Assay, Transfection, Incubation, SDS Page, Western Blot, Plasmid Preparation

    22) Product Images from "Reactive Oxygen Species Signaling Facilitates FOXO-3a/FBXO-Dependent Vascular BK Channel ?1 Subunit Degradation in Diabetic Mice"

    Article Title: Reactive Oxygen Species Signaling Facilitates FOXO-3a/FBXO-Dependent Vascular BK Channel ?1 Subunit Degradation in Diabetic Mice

    Journal: Diabetes

    doi: 10.2337/db11-1658

    Role of Akt signaling in the FOXO-3a/atrogin-1-dependent downregulation of BK-β 1 expression. A and B : Immunoblots against anti-Akt and anti- p -Akt(S473) antibodies showed reduced p -Akt(S473) level but no change in the total Akt expression in diabetic aortas ( A ) and in human coronary SMCs with HG culture ( B ). C : A 24-h incubation with 7 μmol/L LY294002 (LY) mimicked the effects of HG on the levels of p -Akt(S473), p -FOXO-3a(T32), atrogin-1, and BK-β 1 expression in human coronary SMCs cultured in NG.
    Figure Legend Snippet: Role of Akt signaling in the FOXO-3a/atrogin-1-dependent downregulation of BK-β 1 expression. A and B : Immunoblots against anti-Akt and anti- p -Akt(S473) antibodies showed reduced p -Akt(S473) level but no change in the total Akt expression in diabetic aortas ( A ) and in human coronary SMCs with HG culture ( B ). C : A 24-h incubation with 7 μmol/L LY294002 (LY) mimicked the effects of HG on the levels of p -Akt(S473), p -FOXO-3a(T32), atrogin-1, and BK-β 1 expression in human coronary SMCs cultured in NG.

    Techniques Used: Expressing, Western Blot, Incubation, Cell Culture

    23) Product Images from "CCR7 ligands control basal T cell motility within lymph node slices in a phosphoinositide 3-kinase- independent manner"

    Article Title: CCR7 ligands control basal T cell motility within lymph node slices in a phosphoinositide 3-kinase- independent manner

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20062079

    Absence of a Rac- and class Ia PI3K–dependent PIP3 amplification system in T lymphocytes. (A) Human PBT cells transfected with EGFP or EGFP-p85α were left unstimulated or stimulated with either soluble 10 μg/ml anti-CD3 for 15 min or 100 ng/ml CCL19 for 2 min, fixed, stained with anti–P-AKT, and analyzed by flow cytometry. (B) PBT cells were preincubated in the presence of medium alone, 100 nM WMN for 30 min, or IC87114 for 30 min, and were stimulated with either anti-CD3 or CCL19, stained, and analyzed as in A. Results are representative of three independent experiments. The y axis corresponds to the number of T cells. (C) PBT cells were transfected with vector alone or vector encoding a constitutively active (CA) form of Rac. P-AKT was detected by flow cytometry as in Fig. 1 B . The y axis corresponds to the number of T cells. (D) NIH-3T3 fibroblasts were transfected with vector alone or vector encoding a constitutively active (CA) form of Rac. P-AKT was detected on adherent cells by immunofluorescence.
    Figure Legend Snippet: Absence of a Rac- and class Ia PI3K–dependent PIP3 amplification system in T lymphocytes. (A) Human PBT cells transfected with EGFP or EGFP-p85α were left unstimulated or stimulated with either soluble 10 μg/ml anti-CD3 for 15 min or 100 ng/ml CCL19 for 2 min, fixed, stained with anti–P-AKT, and analyzed by flow cytometry. (B) PBT cells were preincubated in the presence of medium alone, 100 nM WMN for 30 min, or IC87114 for 30 min, and were stimulated with either anti-CD3 or CCL19, stained, and analyzed as in A. Results are representative of three independent experiments. The y axis corresponds to the number of T cells. (C) PBT cells were transfected with vector alone or vector encoding a constitutively active (CA) form of Rac. P-AKT was detected by flow cytometry as in Fig. 1 B . The y axis corresponds to the number of T cells. (D) NIH-3T3 fibroblasts were transfected with vector alone or vector encoding a constitutively active (CA) form of Rac. P-AKT was detected on adherent cells by immunofluorescence.

    Techniques Used: IA, Amplification, Transfection, Staining, Flow Cytometry, Cytometry, Plasmid Preparation, Immunofluorescence

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    Article Snippet: Antibodies The following antibodies were used for immunoblotting: PLCδ 1, PLCδ 3 (lab-made); β -actin, Tubulin (SIGMA); PKCα (BD Biosciences, San Jose, CA, USA); PKCβ 1 , PKCθ , Caveolin1, Bax (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); and PKCɛ , Thr538 p-PKCθ , Bad, Bcl-2, Akt, Ser473 p-Akt, ERK, p-ERK, cleaved Caspase 9, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology Inc., Danver, MA, USA).

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    Article Title: Autocrine IGF-I/insulin receptor axis compensates for inhibition of AKT in ER-positive breast cancer cells with resistance to estrogen deprivation
    Article Snippet: .. Immunoblot analysis and RTK arrays Lysates from cells treated with AZD5363 [ ], IGF-I, IGF-II, IGFBP-3 (R & D Systems, Minneapolis, MN, USA), AEW541 [ ] or BKM120 [ ] (Selleck Chemicals, Houston, TX, USA) were subjected to SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblot analysis [ ] using antibodies against P-AKTS473 , P-AKTT308 , AKT, P-PRAS40, P-GSK-3α/β, P-S6S240/244 , S6, P-IGF-IRβY1131 /P-InsRβY1146 , P-HER3Y1197 , P-HER2Y1248 , P-SrcY416 , P-FRS2-αY436 , EGFR (Cell Signaling, Danvers, MA, USA), InsRβ, IGF-IRβ, ERα (F-10), HER3, HER4, FGFR2 (Santa Cruz Biotechnology, Dallas, TX, USA), HER2 (NeoMarkers, Fremont, CA, USA), PR (Dako, Carpinteria, CA, USA), IRS-1 (EMD Millipore, Billerica, MA, USA), and actin (Sigma-Aldrich, St. Louis, MO, USA). ..

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    Effects of clofarabine and resveratrol on <t>Akt</t> and Erk1/2 phosphorylation. (A) MSTO-211H cells were treated with clofarabine (40 nM) and resveratrol (15 μM) for the indicated times. (B) Cells were pretreated with or without Ly294002 (20 μM), PD98059 (50 nM), and NAC (5 mM) for 1 h prior to combined treatment for 24 h. Cell lysates were analyzed by immunoblotting using <t>anti-p-Akt,</t> anti-p-Erk, anti-Nrf2, and anti-HO-1 antibodies. (C) Cells were treated with or without NAC (5 mM) for 1 h prior to the combination treatment for 24 h. The percentage of viable cells was then determined by the trypan blue exclusion assay. *P < 0.05 compared with respective controls.
    Anti P Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Westerns blots of frontal cortex of Fmr1 KO and control mice on vehicle and rapamycin treatment. (A) Representative Western blot images. (B) p-mTOR levels did not differ among the groups. (C) mTOR levels did not differ among the groups. (D) p-mTOR/Total mTOR did not differ among the groups. (E) p-p70S6k did not differ among the groups. (F) p70S6k did not differ among the groups. (G) p-p70S6k/Total p70S6k did not differ among the groups. (H) The genotype × treatment interaction for pS6 235/236 was statistically significant. Post hoc t -tests revealed that vehicle-treated Fmr1 KO animals had significantly higher p-S6 235/236 ( p = 0.002) compared to vehicle-treated controls. This was significantly reduced by rapamycin treatment ( p = 0.002). (I) S6 levels did not differ among groups. (J) The genotype × treatment interaction for p-S6 (235/236)/Total S6 approached statistical significance. We looked at individual differences by means of post hoc t -tests and found that the difference between vehicle-treated controls and vehicle-treated Fmr1 KO mice was statistically significant ( p = 0.004). (K) The genotype × treatment interaction for p-S6 240/244 was statistically significant. ( Post hoc t -tests revealed that vehicle-treated Fmr1 KO animals had significantly higher p-S6 240/244 levels compared to vehicle-treated controls ( p = 0.010). This was significantly reduced with rapamycin treatment ( p = 0.006). (L) Total S6 levels did not differ among the groups. (M) The genotype × treatment interaction for p-S6 (240/244)/Total S6 approached statistical significance. We looked at individual differences by means of post hoc t -tests and found that the difference between vehicle-treated controls and vehicle-treated Fmr1 KO mice was statistically significant ( p = 0.016). (N) The genotype × treatment interaction for <t>p-AKT</t> <t>Ser473</t> was statistically significant. Post hoc t -tests revealed that vehicle-treated Fmr1 KO animals had higher p-AKT compared to vehicle-treated controls ( p = 0.020). p-AKT Ser473 levels were reduced in Fmr1 KO animals after rapamycin treatment ( p = 0.013). (O) Total AKT levels did not differ among the groups. (P) p-Akt (473)/Akt did not differ among the groups. (Q) The main effect of treatment for p-ERK levels was statistically significant indicating that regardless of genotype, rapamycin reduced p-ERK. (R) The main effect of treatment for ERK levels was statistically significant indicating that regardless of genotype, rapamycin reduced ERK. (S) p-ERK/ERK did not differ among the groups. (B–S) Levels were normalized to total protein in the blot. Values presented are relative to the mean of vehicle-treated control values. Bars represent mean ± SEM. ∼0.05
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    An endogenous <t>AKT</t> protein resides in lipid raft fractions of SQ20B cells and confers resistance to targeted and nontargeted effects. ( A ) SQ20B cells treated without (untreated) or with MBCD were incubated with Alexa-488-conjugated cholera toxin B (CTxB) following incubation with p-AKT <t>S473.</t> ( B ) SQ20B cells were pretreated in the presence or absence of MBCD, and total cell lysates (upper panel) or fractions isolated from non-raft and raft domains (bottom panel) were subjected to SDS-PAGE followed by western blotting for p-AKT S473. ( C ) SQ20B cells were preincubated or not with the phosphatidylinositol-3-kinase (PI3K) inhibitor (0.5 µM wortmannin) for 60 min followed by irradiation (donor cells) or CM from SQ20B donor cells (recipient cells), and clonogenic survival was determined. The results are the mean ± SD of three experiments performed in triplicate. * p
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    Transcriptional and post-transcriptional modifications of the <t>PI3K/AKT/FOXO</t> pathway in human skeletal muscle by CR (A) Transcriptional down-regulation of the PI3K/AKT/FOXO signaling pathway by CR. GH: growth hormone; IGF: insulin-like growth factor; PI3K: phosphatidylinositol 3-kinase; AKT: protein kinase B (PKB); FOXO-3A: forkhead box O3; FOXO-4: forkhead box O4; mTOR: mammalian target of rapamycin; LC3: microtubule-associated protein 1 light chain 3; AMPK: adenosine monophosphate-activated protein kinase; NFκB: nuclear factor-kappaB; SOD2: superoxide dismutase 2; DDB1: damage-specific DNA binding protein 1; FASL: fas ligand. Western blot of human skeletal muscle from individuals on a Western diet (WD) or caloric restricted (CR) diet. Immunoblot (B) and quantification (C) of western blots in panel a for <t>p-T308</t> AKT and p-S473 AKT was performed using NIH ImageJ, and normalized to total AKT expression. (*p
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    Effects of clofarabine and resveratrol on Akt and Erk1/2 phosphorylation. (A) MSTO-211H cells were treated with clofarabine (40 nM) and resveratrol (15 μM) for the indicated times. (B) Cells were pretreated with or without Ly294002 (20 μM), PD98059 (50 nM), and NAC (5 mM) for 1 h prior to combined treatment for 24 h. Cell lysates were analyzed by immunoblotting using anti-p-Akt, anti-p-Erk, anti-Nrf2, and anti-HO-1 antibodies. (C) Cells were treated with or without NAC (5 mM) for 1 h prior to the combination treatment for 24 h. The percentage of viable cells was then determined by the trypan blue exclusion assay. *P < 0.05 compared with respective controls.

    Journal: BMB Reports

    Article Title: Synergistic inhibition of mesothelioma cell growth by the combination of clofarabine and resveratrol involves Nrf2 downregulation

    doi: 10.5483/BMBRep.2012.45.11.111

    Figure Lengend Snippet: Effects of clofarabine and resveratrol on Akt and Erk1/2 phosphorylation. (A) MSTO-211H cells were treated with clofarabine (40 nM) and resveratrol (15 μM) for the indicated times. (B) Cells were pretreated with or without Ly294002 (20 μM), PD98059 (50 nM), and NAC (5 mM) for 1 h prior to combined treatment for 24 h. Cell lysates were analyzed by immunoblotting using anti-p-Akt, anti-p-Erk, anti-Nrf2, and anti-HO-1 antibodies. (C) Cells were treated with or without NAC (5 mM) for 1 h prior to the combination treatment for 24 h. The percentage of viable cells was then determined by the trypan blue exclusion assay. *P < 0.05 compared with respective controls.

    Article Snippet: The membranes were incubated for 2 h at room temperature with a 1:500 dilution of anti-Nrf2 (Santa Cruz Biotechnology), anti-HO-1 (Stressgen Biotechnologies), anti-p-Akt, and anti-p-Erk antibodies (Cell Signaling Technologies).

    Techniques: Trypan Blue Exclusion Assay

    Westerns blots of frontal cortex of Fmr1 KO and control mice on vehicle and rapamycin treatment. (A) Representative Western blot images. (B) p-mTOR levels did not differ among the groups. (C) mTOR levels did not differ among the groups. (D) p-mTOR/Total mTOR did not differ among the groups. (E) p-p70S6k did not differ among the groups. (F) p70S6k did not differ among the groups. (G) p-p70S6k/Total p70S6k did not differ among the groups. (H) The genotype × treatment interaction for pS6 235/236 was statistically significant. Post hoc t -tests revealed that vehicle-treated Fmr1 KO animals had significantly higher p-S6 235/236 ( p = 0.002) compared to vehicle-treated controls. This was significantly reduced by rapamycin treatment ( p = 0.002). (I) S6 levels did not differ among groups. (J) The genotype × treatment interaction for p-S6 (235/236)/Total S6 approached statistical significance. We looked at individual differences by means of post hoc t -tests and found that the difference between vehicle-treated controls and vehicle-treated Fmr1 KO mice was statistically significant ( p = 0.004). (K) The genotype × treatment interaction for p-S6 240/244 was statistically significant. ( Post hoc t -tests revealed that vehicle-treated Fmr1 KO animals had significantly higher p-S6 240/244 levels compared to vehicle-treated controls ( p = 0.010). This was significantly reduced with rapamycin treatment ( p = 0.006). (L) Total S6 levels did not differ among the groups. (M) The genotype × treatment interaction for p-S6 (240/244)/Total S6 approached statistical significance. We looked at individual differences by means of post hoc t -tests and found that the difference between vehicle-treated controls and vehicle-treated Fmr1 KO mice was statistically significant ( p = 0.016). (N) The genotype × treatment interaction for p-AKT Ser473 was statistically significant. Post hoc t -tests revealed that vehicle-treated Fmr1 KO animals had higher p-AKT compared to vehicle-treated controls ( p = 0.020). p-AKT Ser473 levels were reduced in Fmr1 KO animals after rapamycin treatment ( p = 0.013). (O) Total AKT levels did not differ among the groups. (P) p-Akt (473)/Akt did not differ among the groups. (Q) The main effect of treatment for p-ERK levels was statistically significant indicating that regardless of genotype, rapamycin reduced p-ERK. (R) The main effect of treatment for ERK levels was statistically significant indicating that regardless of genotype, rapamycin reduced ERK. (S) p-ERK/ERK did not differ among the groups. (B–S) Levels were normalized to total protein in the blot. Values presented are relative to the mean of vehicle-treated control values. Bars represent mean ± SEM. ∼0.05

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Negative Effects of Chronic Rapamycin Treatment on Behavior in a Mouse Model of Fragile X Syndrome

    doi: 10.3389/fnmol.2017.00452

    Figure Lengend Snippet: Westerns blots of frontal cortex of Fmr1 KO and control mice on vehicle and rapamycin treatment. (A) Representative Western blot images. (B) p-mTOR levels did not differ among the groups. (C) mTOR levels did not differ among the groups. (D) p-mTOR/Total mTOR did not differ among the groups. (E) p-p70S6k did not differ among the groups. (F) p70S6k did not differ among the groups. (G) p-p70S6k/Total p70S6k did not differ among the groups. (H) The genotype × treatment interaction for pS6 235/236 was statistically significant. Post hoc t -tests revealed that vehicle-treated Fmr1 KO animals had significantly higher p-S6 235/236 ( p = 0.002) compared to vehicle-treated controls. This was significantly reduced by rapamycin treatment ( p = 0.002). (I) S6 levels did not differ among groups. (J) The genotype × treatment interaction for p-S6 (235/236)/Total S6 approached statistical significance. We looked at individual differences by means of post hoc t -tests and found that the difference between vehicle-treated controls and vehicle-treated Fmr1 KO mice was statistically significant ( p = 0.004). (K) The genotype × treatment interaction for p-S6 240/244 was statistically significant. ( Post hoc t -tests revealed that vehicle-treated Fmr1 KO animals had significantly higher p-S6 240/244 levels compared to vehicle-treated controls ( p = 0.010). This was significantly reduced with rapamycin treatment ( p = 0.006). (L) Total S6 levels did not differ among the groups. (M) The genotype × treatment interaction for p-S6 (240/244)/Total S6 approached statistical significance. We looked at individual differences by means of post hoc t -tests and found that the difference between vehicle-treated controls and vehicle-treated Fmr1 KO mice was statistically significant ( p = 0.016). (N) The genotype × treatment interaction for p-AKT Ser473 was statistically significant. Post hoc t -tests revealed that vehicle-treated Fmr1 KO animals had higher p-AKT compared to vehicle-treated controls ( p = 0.020). p-AKT Ser473 levels were reduced in Fmr1 KO animals after rapamycin treatment ( p = 0.013). (O) Total AKT levels did not differ among the groups. (P) p-Akt (473)/Akt did not differ among the groups. (Q) The main effect of treatment for p-ERK levels was statistically significant indicating that regardless of genotype, rapamycin reduced p-ERK. (R) The main effect of treatment for ERK levels was statistically significant indicating that regardless of genotype, rapamycin reduced ERK. (S) p-ERK/ERK did not differ among the groups. (B–S) Levels were normalized to total protein in the blot. Values presented are relative to the mean of vehicle-treated control values. Bars represent mean ± SEM. ∼0.05

    Article Snippet: Primary antibodies were used at a 1:1000 dilution and were as follows: FMRP (Abcam 27455), p-mTOR (Cell Signaling 5536), mTOR (Cell Signaling 2983), p-p70S6K (Cell Signaling 9234), p70S6K (Cell Signaling 2708), p-S6 235/236 (Cell Signaling 2211), p-S6 240/244 (Cell Signaling 2215), S6 (Cell Signaling 2217), p-ERK (Cell Signaling 4370), ERK (Cell Signaling 7124), p-AKT Ser473 (Cell Signaling 4060), and AKT (Cell Signaling 9272).

    Techniques: Mouse Assay, Western Blot

    An endogenous AKT protein resides in lipid raft fractions of SQ20B cells and confers resistance to targeted and nontargeted effects. ( A ) SQ20B cells treated without (untreated) or with MBCD were incubated with Alexa-488-conjugated cholera toxin B (CTxB) following incubation with p-AKT S473. ( B ) SQ20B cells were pretreated in the presence or absence of MBCD, and total cell lysates (upper panel) or fractions isolated from non-raft and raft domains (bottom panel) were subjected to SDS-PAGE followed by western blotting for p-AKT S473. ( C ) SQ20B cells were preincubated or not with the phosphatidylinositol-3-kinase (PI3K) inhibitor (0.5 µM wortmannin) for 60 min followed by irradiation (donor cells) or CM from SQ20B donor cells (recipient cells), and clonogenic survival was determined. The results are the mean ± SD of three experiments performed in triplicate. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Ceramide-Enriched Membrane Domains Contribute to Targeted and Nontargeted Effects of Radiation through Modulation of PI3K/AKT Signaling in HNSCC Cells

    doi: 10.3390/ijms21197200

    Figure Lengend Snippet: An endogenous AKT protein resides in lipid raft fractions of SQ20B cells and confers resistance to targeted and nontargeted effects. ( A ) SQ20B cells treated without (untreated) or with MBCD were incubated with Alexa-488-conjugated cholera toxin B (CTxB) following incubation with p-AKT S473. ( B ) SQ20B cells were pretreated in the presence or absence of MBCD, and total cell lysates (upper panel) or fractions isolated from non-raft and raft domains (bottom panel) were subjected to SDS-PAGE followed by western blotting for p-AKT S473. ( C ) SQ20B cells were preincubated or not with the phosphatidylinositol-3-kinase (PI3K) inhibitor (0.5 µM wortmannin) for 60 min followed by irradiation (donor cells) or CM from SQ20B donor cells (recipient cells), and clonogenic survival was determined. The results are the mean ± SD of three experiments performed in triplicate. * p

    Article Snippet: Membranes were then incubated with anti-p-AKT (S473), AKT-pan (Cell Signaling Technology, Danvers, MA, USA), anti-flotillin-1, and anti-caveolin-1 (Santa Cruz Biotechnology, Inc., Heidelberg, Germany) primary antibodies at dilution 1:1000.

    Techniques: Incubation, Isolation, SDS Page, Western Blot, Irradiation

    Transcriptional and post-transcriptional modifications of the PI3K/AKT/FOXO pathway in human skeletal muscle by CR (A) Transcriptional down-regulation of the PI3K/AKT/FOXO signaling pathway by CR. GH: growth hormone; IGF: insulin-like growth factor; PI3K: phosphatidylinositol 3-kinase; AKT: protein kinase B (PKB); FOXO-3A: forkhead box O3; FOXO-4: forkhead box O4; mTOR: mammalian target of rapamycin; LC3: microtubule-associated protein 1 light chain 3; AMPK: adenosine monophosphate-activated protein kinase; NFκB: nuclear factor-kappaB; SOD2: superoxide dismutase 2; DDB1: damage-specific DNA binding protein 1; FASL: fas ligand. Western blot of human skeletal muscle from individuals on a Western diet (WD) or caloric restricted (CR) diet. Immunoblot (B) and quantification (C) of western blots in panel a for p-T308 AKT and p-S473 AKT was performed using NIH ImageJ, and normalized to total AKT expression. (*p

    Journal: Aging cell

    Article Title: Calorie restriction in humans inhibits the PI3K/AKT pathway and induces a younger transcription profile

    doi: 10.1111/acel.12088

    Figure Lengend Snippet: Transcriptional and post-transcriptional modifications of the PI3K/AKT/FOXO pathway in human skeletal muscle by CR (A) Transcriptional down-regulation of the PI3K/AKT/FOXO signaling pathway by CR. GH: growth hormone; IGF: insulin-like growth factor; PI3K: phosphatidylinositol 3-kinase; AKT: protein kinase B (PKB); FOXO-3A: forkhead box O3; FOXO-4: forkhead box O4; mTOR: mammalian target of rapamycin; LC3: microtubule-associated protein 1 light chain 3; AMPK: adenosine monophosphate-activated protein kinase; NFκB: nuclear factor-kappaB; SOD2: superoxide dismutase 2; DDB1: damage-specific DNA binding protein 1; FASL: fas ligand. Western blot of human skeletal muscle from individuals on a Western diet (WD) or caloric restricted (CR) diet. Immunoblot (B) and quantification (C) of western blots in panel a for p-T308 AKT and p-S473 AKT was performed using NIH ImageJ, and normalized to total AKT expression. (*p

    Article Snippet: Antibodies to p-T308 AKT (CST 2965), p-S473 AKT (CST 4060), and total AKT (CST 4691) were from Cell Signaling Technology.

    Techniques: Binding Assay, Western Blot, Expressing