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  • 91
    Cell Signaling Technology Inc p akt akt1
    P Akt Akt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p akt akt1/product/Cell Signaling Technology Inc
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    96
    Cell Signaling Technology Inc p akt
    PUMA induction by anti-EGFR antibodies is mediated by <t>p73</t> (A) DiFi cells transfected with control scrambled or p73 siRNA for 24 hr were re-plated and treated with 10 nM cetuximab (Cmab). Expression of p73 at 8 hr, and PUMA and cleaved (C) caspase-3 at 24 hr after cetuximab treatment was analyzed by western blotting. Cells without siRNA transfection and re-plating were used as the control for analyzing p73 at 8r after treatment. (B) Western blotting of indicated proteins in DiFi cells treated 10 nM cetuximab at the indicated time points. Phospho-p73 (p-p73, Y99); <t>phospho-AKT</t> (p-AKT, S473); phospho-ERK1/2 (p-ERK1/2, T202/Y204). (C) DiFi cells transfected with either a control empty vector or a HA-p73α construct were treated with 10 nM Cmab for the indicated times. Binding of transfected p73α to the PUMA promoter was analyzed by chromatin immunoprecipitation (ChIP) using anti-HA antibody with IgG as control, followed by PCR amplification and analysis of PCR products by agarose gel electrophoresis. (D) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected with siRNA as in (A) and treated with Cmab at the indicated doses for 72 hr. (E) Western blotting of indicated proteins in DiFi cells transfected with control empty vector or constitutively active AKT for 6 hr, and then treated with 10 nM cetuximab for 8 or 24 hr. (F) Crystal violet staining (upper panel) and MTS analysis (lower panel) of DiFi cells transfected as in (E) and treated with Cmab at the indicated doses for 72 hr. (G) A model of PUMA induction by anti-EGFR antibodies.
    P Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 18308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology p akt
    Downregulation of miR-142-5p in CD4 + T cells in NSCLC cell line A549 via the <t>PTEN</t> pathway. PD-L1 and PTEN protein expression using (A) western blotting assay and (B and C) statistical analysis. PI3K and <t>p-Akt</t> protein expression using (D) western blotting and (E and F) statistical analysis. Control, control negative group; anti-miR-142-5p, downregulati on of the miR-142-5p group. ## P
    P Akt, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2427 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Epitomics p akt akt
    Adjudin-pretreated <t>NSCs</t> reduced brain infarct volume and improved neurobehavioral outcome after I/R. Representative sets of cresyl violet staining of brain sections from mice treated with PBS, untreated NSCs, and adjudin-pretreated NSCs 3 days following tMCAO. Dashed line shows the border of the infarct area ( a ). Quantification of infarct volumes ( b ). n = 8 in each group. Adjudin-pretreated NSCs significantly ameliorated neurological deficits 3 days after transplantation when compared to the PBS or NSC group. n = 14 for PBS and untreated NSC group, and n = 19 for adjudin-pretreated NSC group ( c ). Adjudin-pretreated NSCs promoted the phosphorylation of <t>Akt</t> in ipsilateral cortex ( d ) and striatum ( e ) after tMCAO. Representative western blot assay showing that adjudin increased the p-Akt protein level 3 days after tMCAO compared with sham, PBS, and NSC groups. Quantification of densitometric value of the protein bands of cortex and straitum normalized to total Akt ( f , g ). n = 6 in each group. Data are mean ± SEM. * P
    P Akt Akt, supplied by Epitomics, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson p akt
    <t>AKT</t> and Foxo1 link C3aR/C5aR signaling to Foxp3 expression . (A and B) Representative immunoblots of flow-sorted Foxp3-GFP + <t>CD4</t> + nT reg cells lysates 15 min after stimulation with C3a, C5a, both, or control (buffer alone). p-AKT/total AKT (A) and p-Foxo1/total Foxo1 (B) shown with quantification normalized to nonphosphorylated bands (bottom of each panel). (C) Representative immunoblot of flow-sorted Foxp3-GFP + CD4 + nT reg cells lysates 15 min after stimulation with C3a + C5a ± PI-3K inhibitor LY294 for p-AKT, total AKT, p-Foxo1, and total Foxo1. No signal above background was detected for p-AKT or p-Foxo1 in lysates from the LY294-treated cells. Blots are representative of at least independent three experiments. (D) Total number of T conv cells from suppression cultures using WT, Foxo1 −/− , or C3ar1 −/− C5ar1 −/− nT reg cells + C3aR-A/C5aR-A (white) or buffer control (black). *, P
    P Akt, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioworld Antibodies p akt
    Effects of WSZYF on proteins related to insulin signal transduction. (a) P-IRS1 relative expression, (b) <t>P-Akt</t> relative expression, (c) GLUT4 relative expression, and (d) <t>PTP1B</t> relative expression. ∗ P
    P Akt, supplied by Bioworld Antibodies, used in various techniques. Bioz Stars score: 92/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Signalway Antibody p akt
    Expression of <t>HIF-1alpha,</t> <t>p-Akt</t> and Akt by Western blot . The expression of HIF-1alpha, p-Akt and Akt were detected in liver tissues by western blot analysis. The blot shown is representative of three different experiments with similar results (A). Lain 1-5: sham; IPO+I/R; IPO+I/R+L-NAME; I/R; I/R+L-NAME. The expression of the housekeeping gene, glyceraldehydes 3-phosphate dehydrogenase (GAPDH), served as a control. The expression of HIF-1alpha, and p-Akt were significantly higher in the liver tissues with IPO+I/R group than I/R group, and the signals were decreased in liver tissues with L-NAME (16 mg/kg) pre-treatment. HIF-1alpha, p-Akt and Akt proteins were calculated by densitometry relative to GAPDH, and the results were expressed as ratios after normalization at 100% of the control (B). Data are mean ± SD from three separate experiments. * p
    P Akt, supplied by Signalway Antibody, used in various techniques. Bioz Stars score: 92/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Affinity Biosciences p akt
    VS-5584 treatment causes activation of <t>ERK</t> in PDAC cells ( A and B ) BxPC-3 and HPAC cells were treated with vehicle control or variable concentrations of VS-5584 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to vehicle control, are indicated. ( C and D ) BxPC-3 and HPAC cells were treated with vehicle control or 2 μM VS-5584 for 4, 8, 12, 24, or 48 h. Whole cell lysates were subjected to Western blotting and probed with <t>anti-p-AKT(S473),</t> -p-AKT(T308), -AKT, -p-S6, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( E and F ) BxPC-3 and HPAC cells were treated with vehicle control or variable concentrations of VS-5584 for 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-ERK, -ERK, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( G and H ) BxPC-3 and HPAC cells were treated with vehicle control or 2 μM VS-5584 for 4, 8, 12, 24, or 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-ERK, -ERK, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated.
    P Akt, supplied by Affinity Biosciences, used in various techniques. Bioz Stars score: 90/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p akt  (Abcam)
    92
    Abcam p akt
    Effects of juglanin on UVB-induced p38/JNK and <t>PI3K/AKT</t> signaling activity. Western blotting was conducted and relative protein expression levels of (A) p-p38 and p-JNK, and (B) PI3K, p-AKT and p-mTOR were determined. Data are presented as the means ± standard deviation of three independent experiments. ## P
    P Akt, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 2012 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore p akt
    Western blot analysis of <t>c-Raf,</t> p-Erk, Erk, <t>p-Akt,</t> Akt and histone H3 in U937 and MDA-MB-231 cell line after 24h of treatment with compounds ( D3 and D17 ) at 1 µM. Histone H3 was used as a loading control.
    P Akt, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 427 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Servicebio Inc p akt
    Effect of ranitidine and AIG treatment on <t>HSP-70,</t> <t>p-AKT,</t> and PCNA expression in acetic acid-induced gastric tissue of rats. Data are presented as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett's test. ∗ P
    P Akt, supplied by Servicebio Inc, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech p akt
    Icariside II enhanced <t>BDNF/TrkB/CREB</t> signaling in BCCAO-treated rats. The expression of BDNF and TrkB protein, as well as the levels of <t>Akt</t> and CREB phosphorylation were examined by Western blot. (A) The antibody-reactive band of BDNF and TrkB protein together with Akt and CREB phosphorylation. (B) Quantitative analysis of BDNF levels. (C) Quantitative analysis of TrkB levels. (D) Quantitative analysis of Akt phosphorylation. (E) Quantitative analysis of CREB phosphorylation. The relative optical density was normalized to β-actin or Akt or CREB. Data were expressed as mean ± SEM ( n = 3). ∗∗ P
    P Akt, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 90/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime p akt
    Expression levels of <t>ERK,</t> p-ERK, <t>Akt</t> and p-Akt in HGC-27 and AGS cells. ( A ) HGC-27 cells; ( B ) AGS cells; ( C ) relative expression levels of p-ERK/ERK and p-Akt/Akt in HGC-27 cells; ( D ) relative expression levels of p-ERK/ERK and p-Akt/Akt in AGS cells. * p
    P Akt, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher p akt
    HK2 deletion led to enhanced CD4 T cell proliferation without affecting T cell differentiation in vitro. (A-C) Naïve CD4 T cells from WT and HK2 KO mice were CTV labelled and activated for 72 hours. (A) Representative FACS plots and bar graph indicating CTV dilution (as a measure of proliferation) gated on live CD4 T cells. Representative FACS plots and Histogram representing the levels of (B) <t>phosphorylated–AKT</t> and (C) <t>Phosphorylated-S6</t> kinase. (D) Naïve CD4 T cells from WT and HK2 KO mice were cultured in the presence Treg or Th1 or Th17 differentiating conditions for 5 days followed by re-stimulation for 4 hours with PMA/Ionomycin. Histogram showing frequency of Th17, Th1 cells and Treg cells. All the measurements were made on live CD4 T cells. Data represents means ± SEM from two independent experiments (n = 3/group) P≤0.001(***).
    P Akt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 245 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson p akt a488
    HK2 deletion led to enhanced CD4 T cell proliferation without affecting T cell differentiation in vitro. (A-C) Naïve CD4 T cells from WT and HK2 KO mice were CTV labelled and activated for 72 hours. (A) Representative FACS plots and bar graph indicating CTV dilution (as a measure of proliferation) gated on live CD4 T cells. Representative FACS plots and Histogram representing the levels of (B) <t>phosphorylated–AKT</t> and (C) <t>Phosphorylated-S6</t> kinase. (D) Naïve CD4 T cells from WT and HK2 KO mice were cultured in the presence Treg or Th1 or Th17 differentiating conditions for 5 days followed by re-stimulation for 4 hours with PMA/Ionomycin. Histogram showing frequency of Th17, Th1 cells and Treg cells. All the measurements were made on live CD4 T cells. Data represents means ± SEM from two independent experiments (n = 3/group) P≤0.001(***).
    P Akt A488, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Affinity Biosciences p akt ser473
    HK2 deletion led to enhanced CD4 T cell proliferation without affecting T cell differentiation in vitro. (A-C) Naïve CD4 T cells from WT and HK2 KO mice were CTV labelled and activated for 72 hours. (A) Representative FACS plots and bar graph indicating CTV dilution (as a measure of proliferation) gated on live CD4 T cells. Representative FACS plots and Histogram representing the levels of (B) <t>phosphorylated–AKT</t> and (C) <t>Phosphorylated-S6</t> kinase. (D) Naïve CD4 T cells from WT and HK2 KO mice were cultured in the presence Treg or Th1 or Th17 differentiating conditions for 5 days followed by re-stimulation for 4 hours with PMA/Ionomycin. Histogram showing frequency of Th17, Th1 cells and Treg cells. All the measurements were made on live CD4 T cells. Data represents means ± SEM from two independent experiments (n = 3/group) P≤0.001(***).
    P Akt Ser473, supplied by Affinity Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc p akt ser473
    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
    P Akt Ser473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 3655 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


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

    Journal: Oncogene

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

    doi: 10.1038/s41388-018-0289-x

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

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

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

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

    Journal: Oncogene

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

    doi: 10.1038/s41388-018-0289-x

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

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

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

    DLX5 knockdown attenuates AKT signaling pathway by down regulating upstream effectors. (A) AKT signaling is compromised by DLX5 knockdown in IGR-OV1 and OVCAR4 cells. Expression of p-PDK1/PDK1, p-AKT/AKT, p-GSK3, p-p70S6K and p-S6 were analyzed by Western

    Journal: Cancer research

    Article Title: Up Regulation of DLX5 Promotes Ovarian Cancer Cell Proliferation by Enhancing IRS-2-AKT Signaling

    doi: 10.1158/0008-5472.CAN-10-1568

    Figure Lengend Snippet: DLX5 knockdown attenuates AKT signaling pathway by down regulating upstream effectors. (A) AKT signaling is compromised by DLX5 knockdown in IGR-OV1 and OVCAR4 cells. Expression of p-PDK1/PDK1, p-AKT/AKT, p-GSK3, p-p70S6K and p-S6 were analyzed by Western

    Article Snippet: Antibodies against phosphorylated (p)-PDK1, total PDK1, p-AKT/AKT, p-GSK3β/GSK3β, p-p70S6K/p70S6K, p-ERK/ERK, HER2, EGF and p-MET were from Cell Signaling Technology (Danvers, MA).

    Techniques: Expressing, Western Blot

    PI3K and MAPK pathway expression in patients with TCL. ( a ) Gene sets of PI3K and MAPK pathway were analyzed by gene network and pathway analysis on microarray data of TCL according to CHKA expression. The key genes in PI3K and MAPK pathway were indicated on the right side of the heat maps. ( b ) As revealed by immunohistochemistry, increased positivity of p-AKT, p-ERK and MYC expression were observed in tumor samples of TCL patients with high CHKA expression. The tissue samples of reactive hyperplasia were referred as a negative control. ** P

    Journal: Blood Cancer Journal

    Article Title: Dysregulated choline metabolism in T-cell lymphoma: role of choline kinase-α and therapeutic targeting

    doi: 10.1038/bcj.2015.10

    Figure Lengend Snippet: PI3K and MAPK pathway expression in patients with TCL. ( a ) Gene sets of PI3K and MAPK pathway were analyzed by gene network and pathway analysis on microarray data of TCL according to CHKA expression. The key genes in PI3K and MAPK pathway were indicated on the right side of the heat maps. ( b ) As revealed by immunohistochemistry, increased positivity of p-AKT, p-ERK and MYC expression were observed in tumor samples of TCL patients with high CHKA expression. The tissue samples of reactive hyperplasia were referred as a negative control. ** P

    Article Snippet: Antibodies against Ras, phosphorylation of AKT (p-AKT) (Ser473), AKT, phosphorylation of ERK (p-ERK) (Thr202/Tyr204) and RIP3 were from Cell Signaling (Beverly, MA, USA).

    Techniques: Expressing, Microarray, Immunohistochemistry, Negative Control

    In vivo activity of CK37 on murine xenograft T-lymphoma model. ( a ) In the CK37 group, tumors grew more slowly than the untreated group. ( b ) Micro-PET/CT showed decreased standardized uptake value (SUV) intensity after CK37 treatment. ( c ) CK37 restored serum levels of choline metabolites LysoPC (18:0) and LysoPC (16:0). ( d and e ) CK37 inhibited protein levels of p-AKT, p-ERK and MYC ( d ), as well as the activity of Ras-GTP ( e ). ( f ) TUNEL assay showed significantly increased apoptotic cells in the CK37 group. ** P

    Journal: Blood Cancer Journal

    Article Title: Dysregulated choline metabolism in T-cell lymphoma: role of choline kinase-α and therapeutic targeting

    doi: 10.1038/bcj.2015.10

    Figure Lengend Snippet: In vivo activity of CK37 on murine xenograft T-lymphoma model. ( a ) In the CK37 group, tumors grew more slowly than the untreated group. ( b ) Micro-PET/CT showed decreased standardized uptake value (SUV) intensity after CK37 treatment. ( c ) CK37 restored serum levels of choline metabolites LysoPC (18:0) and LysoPC (16:0). ( d and e ) CK37 inhibited protein levels of p-AKT, p-ERK and MYC ( d ), as well as the activity of Ras-GTP ( e ). ( f ) TUNEL assay showed significantly increased apoptotic cells in the CK37 group. ** P

    Article Snippet: Antibodies against Ras, phosphorylation of AKT (p-AKT) (Ser473), AKT, phosphorylation of ERK (p-ERK) (Thr202/Tyr204) and RIP3 were from Cell Signaling (Beverly, MA, USA).

    Techniques: In Vivo, Activity Assay, Micro-PET, TUNEL Assay

    Downregulation of miR-142-5p in CD4 + T cells in NSCLC cell line A549 via the PTEN pathway. PD-L1 and PTEN protein expression using (A) western blotting assay and (B and C) statistical analysis. PI3K and p-Akt protein expression using (D) western blotting and (E and F) statistical analysis. Control, control negative group; anti-miR-142-5p, downregulati on of the miR-142-5p group. ## P

    Journal: Oncology Reports

    Article Title: miR-142-5p regulates CD4+ T cells in human non-small cell lung cancer through PD-L1 expression via the PTEN pathway

    doi: 10.3892/or.2018.6439

    Figure Lengend Snippet: Downregulation of miR-142-5p in CD4 + T cells in NSCLC cell line A549 via the PTEN pathway. PD-L1 and PTEN protein expression using (A) western blotting assay and (B and C) statistical analysis. PI3K and p-Akt protein expression using (D) western blotting and (E and F) statistical analysis. Control, control negative group; anti-miR-142-5p, downregulati on of the miR-142-5p group. ## P

    Article Snippet: The membranes were then blocked at room temperature for 1 h with 5% non-fat milk in TBST and the membranes were incubated at 4°C overnight with the following antibodies: PD-L1 (1:500; cat. no. sc-293425), PTEN (1:500; cat. no. sc-6817-R), PI3K (1:500; cat. no. sc-7175), p-Akt (1:200; cat. no. sc-7985-R), Akt (1:500; cat. no. sc-8312) and GAPDH (1:5,000; cat. no. sc-25778; all from Santa Cruz Biotechnology, Inc., Dallas TX, USA).

    Techniques: Expressing, Western Blot

    Overexpression of miR-142-5p in CD4 + T cells in NSCLC cell line A549 via the PTEN pathway. (A) PD-L1 and PTEN are the potential target genes of miR-142-5p. PD-L1 and PTEN protein expression using (B) western blotting and (C and D) statistical analysis. PI3K and p-Akt protein expression using (E) western blotting assay and (F and G) statistical analysis. Control, control negative group; miR-142-5p, overexpression of the miR-142-5p group. ## P

    Journal: Oncology Reports

    Article Title: miR-142-5p regulates CD4+ T cells in human non-small cell lung cancer through PD-L1 expression via the PTEN pathway

    doi: 10.3892/or.2018.6439

    Figure Lengend Snippet: Overexpression of miR-142-5p in CD4 + T cells in NSCLC cell line A549 via the PTEN pathway. (A) PD-L1 and PTEN are the potential target genes of miR-142-5p. PD-L1 and PTEN protein expression using (B) western blotting and (C and D) statistical analysis. PI3K and p-Akt protein expression using (E) western blotting assay and (F and G) statistical analysis. Control, control negative group; miR-142-5p, overexpression of the miR-142-5p group. ## P

    Article Snippet: The membranes were then blocked at room temperature for 1 h with 5% non-fat milk in TBST and the membranes were incubated at 4°C overnight with the following antibodies: PD-L1 (1:500; cat. no. sc-293425), PTEN (1:500; cat. no. sc-6817-R), PI3K (1:500; cat. no. sc-7175), p-Akt (1:200; cat. no. sc-7985-R), Akt (1:500; cat. no. sc-8312) and GAPDH (1:5,000; cat. no. sc-25778; all from Santa Cruz Biotechnology, Inc., Dallas TX, USA).

    Techniques: Over Expression, Expressing, Western Blot

    Inhibition of PTEN reduces the cancer effects of CD4 + T cells on the PI3K/Akt signaling pathway following miR-142-5p downregulation. PD-L1 and PTEN protein expression using (A) western blotting and (B and C) statistical analysis. PI3K and p-Akt protein expression using (D) western blotting and (E and F) statistical analysis. (G, H and I) The levels of IFN-γ, CCL17, and CCL22, respectively. Control, control negative group; anti-miR-142-5p, downregulation of the miR-142-5p group; PTEN i, PTEN inhibitor (VO-Ohpic trihydrate) and downregulation of the miR-142-5p group. ## P

    Journal: Oncology Reports

    Article Title: miR-142-5p regulates CD4+ T cells in human non-small cell lung cancer through PD-L1 expression via the PTEN pathway

    doi: 10.3892/or.2018.6439

    Figure Lengend Snippet: Inhibition of PTEN reduces the cancer effects of CD4 + T cells on the PI3K/Akt signaling pathway following miR-142-5p downregulation. PD-L1 and PTEN protein expression using (A) western blotting and (B and C) statistical analysis. PI3K and p-Akt protein expression using (D) western blotting and (E and F) statistical analysis. (G, H and I) The levels of IFN-γ, CCL17, and CCL22, respectively. Control, control negative group; anti-miR-142-5p, downregulation of the miR-142-5p group; PTEN i, PTEN inhibitor (VO-Ohpic trihydrate) and downregulation of the miR-142-5p group. ## P

    Article Snippet: The membranes were then blocked at room temperature for 1 h with 5% non-fat milk in TBST and the membranes were incubated at 4°C overnight with the following antibodies: PD-L1 (1:500; cat. no. sc-293425), PTEN (1:500; cat. no. sc-6817-R), PI3K (1:500; cat. no. sc-7175), p-Akt (1:200; cat. no. sc-7985-R), Akt (1:500; cat. no. sc-8312) and GAPDH (1:5,000; cat. no. sc-25778; all from Santa Cruz Biotechnology, Inc., Dallas TX, USA).

    Techniques: Inhibition, Expressing, Western Blot

    AG1478 and NAC attenuate HG-induced EGFR signaling activation, ROS generation, endoplasmic reticulum stress, cell firosis and apoptosis in SV40 cells SV40 cells pretreated with AG1478 (10 μM) or NAC (10 mM) for 1 h were incubated with HG (33 mM) for 1 h. Then cells were lysed and the extracted total proteins were processed for the detection of p-EGFR and p-AKT using Western blot; and statistic figure was shown, data were presented as mean ± SDs. (Six mice in each group were used for above analysis ( A ). ( B ) AG1478 and NAC inhibit high glucose-induced ROS generation. SV40 cells pretreated with AG1478 (10 μM) or NAC (10mM) for 1h were incubated with HG (33 mM) for 2h. DCFH-DA probes were loaded and the ROS positive cells were detected using the fluorescence microscope. Also, after loading with the probes, cells were processed to flow cytometry analysis for O2 level, and mean fluorescence intensity (MFI) value was determined. Data were presented as mean ± SDs. ( n = 3 for each experiment. ** P

    Journal: Oncotarget

    Article Title: EGFR inhibition attenuates diabetic nephropathy through decreasing ROS and endoplasmic reticulum stress

    doi: 10.18632/oncotarget.15948

    Figure Lengend Snippet: AG1478 and NAC attenuate HG-induced EGFR signaling activation, ROS generation, endoplasmic reticulum stress, cell firosis and apoptosis in SV40 cells SV40 cells pretreated with AG1478 (10 μM) or NAC (10 mM) for 1 h were incubated with HG (33 mM) for 1 h. Then cells were lysed and the extracted total proteins were processed for the detection of p-EGFR and p-AKT using Western blot; and statistic figure was shown, data were presented as mean ± SDs. (Six mice in each group were used for above analysis ( A ). ( B ) AG1478 and NAC inhibit high glucose-induced ROS generation. SV40 cells pretreated with AG1478 (10 μM) or NAC (10mM) for 1h were incubated with HG (33 mM) for 2h. DCFH-DA probes were loaded and the ROS positive cells were detected using the fluorescence microscope. Also, after loading with the probes, cells were processed to flow cytometry analysis for O2 level, and mean fluorescence intensity (MFI) value was determined. Data were presented as mean ± SDs. ( n = 3 for each experiment. ** P

    Article Snippet: Antibodies for p-EGFR/EGFR, p-AKT/AKT, p-PERK/PERK, p-eIF2α/ eIF2α, Bax, GAPDH, TGF-β1, collagen IV, CHOP, cleaved-Caspase 3 and were obtained from Santa Cruz Technology (Santa Cruz, CA), and antibodies for ATF4, CHOP were purchased from Abcam (Cambride, MA).

    Techniques: Activation Assay, Incubation, Western Blot, Mouse Assay, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    Scheme for EGFR/AKT/ROS/ER stress signaling pathway in preventing DN Inhibition of EGFR by AG1478 eliminated AKT phosphorylation, sequentially reduced oxidative stress and ER stress, decreased diabetes-induced renal.

    Journal: Oncotarget

    Article Title: EGFR inhibition attenuates diabetic nephropathy through decreasing ROS and endoplasmic reticulum stress

    doi: 10.18632/oncotarget.15948

    Figure Lengend Snippet: Scheme for EGFR/AKT/ROS/ER stress signaling pathway in preventing DN Inhibition of EGFR by AG1478 eliminated AKT phosphorylation, sequentially reduced oxidative stress and ER stress, decreased diabetes-induced renal.

    Article Snippet: Antibodies for p-EGFR/EGFR, p-AKT/AKT, p-PERK/PERK, p-eIF2α/ eIF2α, Bax, GAPDH, TGF-β1, collagen IV, CHOP, cleaved-Caspase 3 and were obtained from Santa Cruz Technology (Santa Cruz, CA), and antibodies for ATF4, CHOP were purchased from Abcam (Cambride, MA).

    Techniques: Inhibition

    AG1478 attenuate diabetes-induced EGFR signaling activation in diabetic kidney ( A ) Representative images for the histochemical staining for p-EGFR and EGFR expression in the formalin-fixed renal tissues (200× magnification). ( B ) Western blot analysis for the expression of p-EGFR in renal tissue. And statistic figure was shown, data were presented as mean ± SDs. ( C ) Representative images for the histochemical staining for p-AKT and AKT expression in the formalin-fixed renal tissues (200× magnification). (Eight mice in each group were used for above analysis. ** P

    Journal: Oncotarget

    Article Title: EGFR inhibition attenuates diabetic nephropathy through decreasing ROS and endoplasmic reticulum stress

    doi: 10.18632/oncotarget.15948

    Figure Lengend Snippet: AG1478 attenuate diabetes-induced EGFR signaling activation in diabetic kidney ( A ) Representative images for the histochemical staining for p-EGFR and EGFR expression in the formalin-fixed renal tissues (200× magnification). ( B ) Western blot analysis for the expression of p-EGFR in renal tissue. And statistic figure was shown, data were presented as mean ± SDs. ( C ) Representative images for the histochemical staining for p-AKT and AKT expression in the formalin-fixed renal tissues (200× magnification). (Eight mice in each group were used for above analysis. ** P

    Article Snippet: Antibodies for p-EGFR/EGFR, p-AKT/AKT, p-PERK/PERK, p-eIF2α/ eIF2α, Bax, GAPDH, TGF-β1, collagen IV, CHOP, cleaved-Caspase 3 and were obtained from Santa Cruz Technology (Santa Cruz, CA), and antibodies for ATF4, CHOP were purchased from Abcam (Cambride, MA).

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

    EGFR and AKT mediate HG-induced ER stress and cell damage in SV40 MES 13 cells ( A ) SV40 cells were transfected with EGFR siRNA or control siRNA for 48 h, the expression of EGFR was detected by Western blot analysis. ( B ) EGFR silencing by siRNA reduced HG-induced ER stress, fibrosis and apoptosis. ( C ) SV40 cells were transfected with AKT siRNA or control siRNA for 48 h, the expression of AKT was detected by Western blot analysis. ( D ) AKT silencing by siRNA reduced HG-induced ER stress, fibrosis and apoptosis. Data were obtained from three independent experiments.

    Journal: Oncotarget

    Article Title: EGFR inhibition attenuates diabetic nephropathy through decreasing ROS and endoplasmic reticulum stress

    doi: 10.18632/oncotarget.15948

    Figure Lengend Snippet: EGFR and AKT mediate HG-induced ER stress and cell damage in SV40 MES 13 cells ( A ) SV40 cells were transfected with EGFR siRNA or control siRNA for 48 h, the expression of EGFR was detected by Western blot analysis. ( B ) EGFR silencing by siRNA reduced HG-induced ER stress, fibrosis and apoptosis. ( C ) SV40 cells were transfected with AKT siRNA or control siRNA for 48 h, the expression of AKT was detected by Western blot analysis. ( D ) AKT silencing by siRNA reduced HG-induced ER stress, fibrosis and apoptosis. Data were obtained from three independent experiments.

    Article Snippet: Antibodies for p-EGFR/EGFR, p-AKT/AKT, p-PERK/PERK, p-eIF2α/ eIF2α, Bax, GAPDH, TGF-β1, collagen IV, CHOP, cleaved-Caspase 3 and were obtained from Santa Cruz Technology (Santa Cruz, CA), and antibodies for ATF4, CHOP were purchased from Abcam (Cambride, MA).

    Techniques: Transfection, Expressing, Western Blot

    Adjudin-pretreated NSCs reduced brain infarct volume and improved neurobehavioral outcome after I/R. Representative sets of cresyl violet staining of brain sections from mice treated with PBS, untreated NSCs, and adjudin-pretreated NSCs 3 days following tMCAO. Dashed line shows the border of the infarct area ( a ). Quantification of infarct volumes ( b ). n = 8 in each group. Adjudin-pretreated NSCs significantly ameliorated neurological deficits 3 days after transplantation when compared to the PBS or NSC group. n = 14 for PBS and untreated NSC group, and n = 19 for adjudin-pretreated NSC group ( c ). Adjudin-pretreated NSCs promoted the phosphorylation of Akt in ipsilateral cortex ( d ) and striatum ( e ) after tMCAO. Representative western blot assay showing that adjudin increased the p-Akt protein level 3 days after tMCAO compared with sham, PBS, and NSC groups. Quantification of densitometric value of the protein bands of cortex and straitum normalized to total Akt ( f , g ). n = 6 in each group. Data are mean ± SEM. * P

    Journal: Stem Cell Research & Therapy

    Article Title: Adjudin-preconditioned neural stem cells enhance neuroprotection after ischemia reperfusion in mice

    doi: 10.1186/s13287-017-0677-0

    Figure Lengend Snippet: Adjudin-pretreated NSCs reduced brain infarct volume and improved neurobehavioral outcome after I/R. Representative sets of cresyl violet staining of brain sections from mice treated with PBS, untreated NSCs, and adjudin-pretreated NSCs 3 days following tMCAO. Dashed line shows the border of the infarct area ( a ). Quantification of infarct volumes ( b ). n = 8 in each group. Adjudin-pretreated NSCs significantly ameliorated neurological deficits 3 days after transplantation when compared to the PBS or NSC group. n = 14 for PBS and untreated NSC group, and n = 19 for adjudin-pretreated NSC group ( c ). Adjudin-pretreated NSCs promoted the phosphorylation of Akt in ipsilateral cortex ( d ) and striatum ( e ) after tMCAO. Representative western blot assay showing that adjudin increased the p-Akt protein level 3 days after tMCAO compared with sham, PBS, and NSC groups. Quantification of densitometric value of the protein bands of cortex and straitum normalized to total Akt ( f , g ). n = 6 in each group. Data are mean ± SEM. * P

    Article Snippet: Compared to the nonpreconditioned NSCs, adjudin pretreatment could dramatically increase the ratio of p-Akt/Akt after H2 O2 stimulation (Fig. ).

    Techniques: Staining, Mouse Assay, Transplantation Assay, Western Blot

    Adjudin pretreatment attenuated cell death and maintained the ATP level of NSCs after H 2 O 2 stimulation. Assays to evaluate whether adjudin pretreatment could attenuate cell death of NSCs after 0.1 mM H 2 O 2 exposure. Cell death and cell survival measured by LDH ( a ) and CCK-8 assay ( b ). ATP level of NSCs after H 2 O 2 stimulation with/without adjudin pretreatment ( c ). Levels of p-Akt and Akt detected by western blot analysis after H 2 O 2 treatment ( d ). Quantification of the densitometric value of protein bands normalized to total Akt ( e ). Bars represent mean ± SEM from three independent experiments. * P

    Journal: Stem Cell Research & Therapy

    Article Title: Adjudin-preconditioned neural stem cells enhance neuroprotection after ischemia reperfusion in mice

    doi: 10.1186/s13287-017-0677-0

    Figure Lengend Snippet: Adjudin pretreatment attenuated cell death and maintained the ATP level of NSCs after H 2 O 2 stimulation. Assays to evaluate whether adjudin pretreatment could attenuate cell death of NSCs after 0.1 mM H 2 O 2 exposure. Cell death and cell survival measured by LDH ( a ) and CCK-8 assay ( b ). ATP level of NSCs after H 2 O 2 stimulation with/without adjudin pretreatment ( c ). Levels of p-Akt and Akt detected by western blot analysis after H 2 O 2 treatment ( d ). Quantification of the densitometric value of protein bands normalized to total Akt ( e ). Bars represent mean ± SEM from three independent experiments. * P

    Article Snippet: Compared to the nonpreconditioned NSCs, adjudin pretreatment could dramatically increase the ratio of p-Akt/Akt after H2 O2 stimulation (Fig. ).

    Techniques: CCK-8 Assay, Western Blot

    Effect of 5-AcTMF on PI3K/AKT/mTOR pathway in CL1-5 cells. Cells were treated with 2.5 μM and 5 μM 5-AcTMF for 24 or 48 h, and then lyzed for protein extraction and subjected to western blot analysis using antibodies

    Journal: Cancer Biology & Therapy

    Article Title: Tangeretin derivative, 5-acetyloxy-6,7,8,4′-tetramethoxyflavone induces G2/M arrest, apoptosis and autophagy in human non-small cell lung cancer cells in vitro and in vivo

    doi: 10.1080/15384047.2015.1108491

    Figure Lengend Snippet: Effect of 5-AcTMF on PI3K/AKT/mTOR pathway in CL1-5 cells. Cells were treated with 2.5 μM and 5 μM 5-AcTMF for 24 or 48 h, and then lyzed for protein extraction and subjected to western blot analysis using antibodies

    Article Snippet: In this study, the primary antibodies were obtained from Cell Signaling Technology Inc. (cleaved caspase-3,8,9, cleaved PARP, XIAP, survivin, Bcl-2, p-ERK, ERK, p-p38, JNK, p-PI3K, p-Akt, p53, p21), from Santa Cruz Biotechnology Inc. (Santa Cruz, CA)(cyclin B1, p-cdc2, cdc25c, β-actin, Bcl-xL, PI3KIII, p-mTOR, mTOR, Bax, p-Akt), from Epitomics (cdc2, survivin, p38, Akt) ,from Millipore (anti-p-JNK) and from abgent (LC3).

    Techniques: Protein Extraction, Western Blot

    AKT and Foxo1 link C3aR/C5aR signaling to Foxp3 expression . (A and B) Representative immunoblots of flow-sorted Foxp3-GFP + CD4 + nT reg cells lysates 15 min after stimulation with C3a, C5a, both, or control (buffer alone). p-AKT/total AKT (A) and p-Foxo1/total Foxo1 (B) shown with quantification normalized to nonphosphorylated bands (bottom of each panel). (C) Representative immunoblot of flow-sorted Foxp3-GFP + CD4 + nT reg cells lysates 15 min after stimulation with C3a + C5a ± PI-3K inhibitor LY294 for p-AKT, total AKT, p-Foxo1, and total Foxo1. No signal above background was detected for p-AKT or p-Foxo1 in lysates from the LY294-treated cells. Blots are representative of at least independent three experiments. (D) Total number of T conv cells from suppression cultures using WT, Foxo1 −/− , or C3ar1 −/− C5ar1 −/− nT reg cells + C3aR-A/C5aR-A (white) or buffer control (black). *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Signaling through C5a receptor and C3a receptor diminishes function of murine natural regulatory T cells

    doi: 10.1084/jem.20121525

    Figure Lengend Snippet: AKT and Foxo1 link C3aR/C5aR signaling to Foxp3 expression . (A and B) Representative immunoblots of flow-sorted Foxp3-GFP + CD4 + nT reg cells lysates 15 min after stimulation with C3a, C5a, both, or control (buffer alone). p-AKT/total AKT (A) and p-Foxo1/total Foxo1 (B) shown with quantification normalized to nonphosphorylated bands (bottom of each panel). (C) Representative immunoblot of flow-sorted Foxp3-GFP + CD4 + nT reg cells lysates 15 min after stimulation with C3a + C5a ± PI-3K inhibitor LY294 for p-AKT, total AKT, p-Foxo1, and total Foxo1. No signal above background was detected for p-AKT or p-Foxo1 in lysates from the LY294-treated cells. Blots are representative of at least independent three experiments. (D) Total number of T conv cells from suppression cultures using WT, Foxo1 −/− , or C3ar1 −/− C5ar1 −/− nT reg cells + C3aR-A/C5aR-A (white) or buffer control (black). *, P

    Article Snippet: Antibodies against CD4, Foxp3, GR1, CTLA4 (eBioscience), p-AKT (Ser473; BD), CD88, CD45.1 (BioLegend) were used for flow cytometry.

    Techniques: Expressing, Western Blot, Flow Cytometry

    Effects of WSZYF on proteins related to insulin signal transduction. (a) P-IRS1 relative expression, (b) P-Akt relative expression, (c) GLUT4 relative expression, and (d) PTP1B relative expression. ∗ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Wushenziye Formula Improves Skeletal Muscle Insulin Resistance in Type 2 Diabetes Mellitus via PTP1B-IRS1-Akt-GLUT4 Signaling Pathway

    doi: 10.1155/2017/4393529

    Figure Lengend Snippet: Effects of WSZYF on proteins related to insulin signal transduction. (a) P-IRS1 relative expression, (b) P-Akt relative expression, (c) GLUT4 relative expression, and (d) PTP1B relative expression. ∗ P

    Article Snippet: The primary antibodies against PTP1B, p-Akt (phospho S473), GLUT4, and GAPDH were from Bioworld Technology (USA) and antibody against p-IRS1 (phospho Y612) was from Abcam (USA).

    Techniques: Transduction, Expressing

    Expression of HIF-1alpha, p-Akt and Akt by Western blot . The expression of HIF-1alpha, p-Akt and Akt were detected in liver tissues by western blot analysis. The blot shown is representative of three different experiments with similar results (A). Lain 1-5: sham; IPO+I/R; IPO+I/R+L-NAME; I/R; I/R+L-NAME. The expression of the housekeeping gene, glyceraldehydes 3-phosphate dehydrogenase (GAPDH), served as a control. The expression of HIF-1alpha, and p-Akt were significantly higher in the liver tissues with IPO+I/R group than I/R group, and the signals were decreased in liver tissues with L-NAME (16 mg/kg) pre-treatment. HIF-1alpha, p-Akt and Akt proteins were calculated by densitometry relative to GAPDH, and the results were expressed as ratios after normalization at 100% of the control (B). Data are mean ± SD from three separate experiments. * p

    Journal: Journal of Biomedical Science

    Article Title: Ischemic postconditioning attenuates liver warm ischemia-reperfusion injury through Akt-eNOS-NO-HIF pathway

    doi: 10.1186/1423-0127-18-79

    Figure Lengend Snippet: Expression of HIF-1alpha, p-Akt and Akt by Western blot . The expression of HIF-1alpha, p-Akt and Akt were detected in liver tissues by western blot analysis. The blot shown is representative of three different experiments with similar results (A). Lain 1-5: sham; IPO+I/R; IPO+I/R+L-NAME; I/R; I/R+L-NAME. The expression of the housekeeping gene, glyceraldehydes 3-phosphate dehydrogenase (GAPDH), served as a control. The expression of HIF-1alpha, and p-Akt were significantly higher in the liver tissues with IPO+I/R group than I/R group, and the signals were decreased in liver tissues with L-NAME (16 mg/kg) pre-treatment. HIF-1alpha, p-Akt and Akt proteins were calculated by densitometry relative to GAPDH, and the results were expressed as ratios after normalization at 100% of the control (B). Data are mean ± SD from three separate experiments. * p

    Article Snippet: After overnight blocking at 4°C, the membranes were incubated and shaken for 2 h at 37 °C with a mouse monoclonal antibody against HIF-1α (diluted 1:500, AbCam, Canbridge, UK); p-Akt (diluted 1:500, Signalway Antibody); rabbit polyclonal antibody against Akt (diluted 1:500, Signalway Antibody); followed by a secondary antibodies (diluted 1:2000, Santa Cruz, CA).

    Techniques: Expressing, Western Blot

    VS-5584 treatment causes activation of ERK in PDAC cells ( A and B ) BxPC-3 and HPAC cells were treated with vehicle control or variable concentrations of VS-5584 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to vehicle control, are indicated. ( C and D ) BxPC-3 and HPAC cells were treated with vehicle control or 2 μM VS-5584 for 4, 8, 12, 24, or 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-AKT(S473), -p-AKT(T308), -AKT, -p-S6, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( E and F ) BxPC-3 and HPAC cells were treated with vehicle control or variable concentrations of VS-5584 for 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-ERK, -ERK, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( G and H ) BxPC-3 and HPAC cells were treated with vehicle control or 2 μM VS-5584 for 4, 8, 12, 24, or 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-ERK, -ERK, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated.

    Journal: Oncotarget

    Article Title: Targeting ERK enhances the cytotoxic effect of the novel PI3K and mTOR dual inhibitor VS-5584 in preclinical models of pancreatic cancer

    doi: 10.18632/oncotarget.17869

    Figure Lengend Snippet: VS-5584 treatment causes activation of ERK in PDAC cells ( A and B ) BxPC-3 and HPAC cells were treated with vehicle control or variable concentrations of VS-5584 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to vehicle control, are indicated. ( C and D ) BxPC-3 and HPAC cells were treated with vehicle control or 2 μM VS-5584 for 4, 8, 12, 24, or 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-AKT(S473), -p-AKT(T308), -AKT, -p-S6, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( E and F ) BxPC-3 and HPAC cells were treated with vehicle control or variable concentrations of VS-5584 for 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-ERK, -ERK, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( G and H ) BxPC-3 and HPAC cells were treated with vehicle control or 2 μM VS-5584 for 4, 8, 12, 24, or 48 h. Whole cell lysates were subjected to Western blotting and probed with anti-p-ERK, -ERK, or -β-actin antibody. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated.

    Article Snippet: Whole cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Inc., Rockford, IL, USA) and immunoblotted with anti-PARP, -Mcl-1, -Bcl-2, -Bcl-xL, -Bax, -actin, -Bad, -ERK (Proteintech, Chicago, IL, USA), -p-AKT (T308), -p-AKT (S473) (Affinity Biosciences, Changzhou, Jiangsu Province, China), -Bim, -pS6 (Cell Signaling Technologies, Danvers, MA, USA), -p-ERK, or -AKT (Abcam, Cambridge, MA, USA), as previously described [ , , , ].

    Techniques: Activation Assay, Western Blot

    Effects of juglanin on UVB-induced p38/JNK and PI3K/AKT signaling activity. Western blotting was conducted and relative protein expression levels of (A) p-p38 and p-JNK, and (B) PI3K, p-AKT and p-mTOR were determined. Data are presented as the means ± standard deviation of three independent experiments. ## P

    Journal: International Journal of Molecular Medicine

    Article Title: Juglanin ameliorates UVB-induced skin carcinogenesis via anti-inflammatory and proapoptotic effects in vivo and in vitro

    doi: 10.3892/ijmm.2018.3601

    Figure Lengend Snippet: Effects of juglanin on UVB-induced p38/JNK and PI3K/AKT signaling activity. Western blotting was conducted and relative protein expression levels of (A) p-p38 and p-JNK, and (B) PI3K, p-AKT and p-mTOR were determined. Data are presented as the means ± standard deviation of three independent experiments. ## P

    Article Snippet: The primary polyclonal antibodies used were as follows: rabbit anti-GAPDH (1:500; cat. no. sc-293335; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p38 (1:1,000; cat. no. 8690; Cell Signaling Technology, Inc., Danvers, MA, USA), p-p38 (1:1,000; cat. no. 4511, Cell Signaling Technology, Inc.), p-JNK (1:1,000; cat. no. 4668, Cell Signaling Technology, Inc.), JNK (1:1,000; cat. no. 9252, Cell Signaling Technology, Inc.), TNF-α (1:1,000; cat. no. ab1793; Abcam), IL-18 (1:1,000; cat. no. ab71495; Abcam), IL-1β (1:1,000; cat. no. ab9722; Abcam), p-NF-κB (1:1,000; cat. no. ab86299; Abcam), NF-κB (1:1,000; cat. no. ab207297; Abcam), PI3K (1:1,000; cat. no. ab86714; Abcam), p-AKT (1:1,000; cat. no. 81283; Abcam), AKT (1:1,000; cat. no. ab8805; Abcam), p-mammalian target of rapamycin (mTOR; 1:1,000; cat. no. ab109268; Abcam), mTOR (1:1,000; cat. no. ab2732; Abcam), poly (ADP-ribose) polymerase (PARP; 1:1,000; cat. no. ab13907; Abcam), cyclin-dependent kinase (CDK1; 1:1,000; cat. no. ab18; Abcam), cyclin D (1:1,000; cat. no. ab134175; Abcam), p53 (1:1,000; cat. no. ab26; Abcam), p21 (1:1,000; cat. no. ab109199; Abcam), p27 (1:1,000; cat. no. ab62364; Abcam), caspase-8 (1:1,000; cat. no. ab25901; Abcam), caspase-3 (1:1,000; cat. no. ab52293; Abcam) and PCNA (1:1,000; cat. no. ab29; Abcam).

    Techniques: Activity Assay, Western Blot, Expressing, Standard Deviation

    Lnc-Myd88 induces an upregulation of Myd88 by enhanced acetylation of the promoter of Myd88 and then activates NF- κ B and PI3K/AKT signal pathways. ( a ) Enrichment prediction of H3K4m3, H3K27Ac and H3K27m3 in the promoter region of Myd88 through ENCODE database. ( b ) The enrichment of H3K4m3, H3K27Ac and H3K4m3 in the promoter region of Myd88 in HCC patients. ( c ) The alteration of the enrichment of H3K27Ac in the promoter region of Myd88 in HCC cells treated with Lnc-Myd88. ( d and e ) The alteration of mRNA and protein level of Myd88 in HCC cells induced by Lnc-Myd88. GAPDH was used as a loading control. ( f and g ) The levels of NF- κ B, p-NF- κ B, AKT, p-AKT and GAPDH were examined by western blotting in SMMC-7721 and Huh7 cells treated with Lnc-Myd88. The experiments were performed in triplicate; the data are expressed as the mean±S.E.M. (* P

    Journal: Cell Death & Disease

    Article Title: Long non-coding RNA Myd88 promotes growth and metastasis in hepatocellular carcinoma via regulating Myd88 expression through H3K27 modification

    doi: 10.1038/cddis.2017.519

    Figure Lengend Snippet: Lnc-Myd88 induces an upregulation of Myd88 by enhanced acetylation of the promoter of Myd88 and then activates NF- κ B and PI3K/AKT signal pathways. ( a ) Enrichment prediction of H3K4m3, H3K27Ac and H3K27m3 in the promoter region of Myd88 through ENCODE database. ( b ) The enrichment of H3K4m3, H3K27Ac and H3K4m3 in the promoter region of Myd88 in HCC patients. ( c ) The alteration of the enrichment of H3K27Ac in the promoter region of Myd88 in HCC cells treated with Lnc-Myd88. ( d and e ) The alteration of mRNA and protein level of Myd88 in HCC cells induced by Lnc-Myd88. GAPDH was used as a loading control. ( f and g ) The levels of NF- κ B, p-NF- κ B, AKT, p-AKT and GAPDH were examined by western blotting in SMMC-7721 and Huh7 cells treated with Lnc-Myd88. The experiments were performed in triplicate; the data are expressed as the mean±S.E.M. (* P

    Article Snippet: And then the membrane was incubated at 4 °C overnight with human-specific antibody of Myd88 (Abcam, London, UK), p-NF-κ B and NF-κ B (CST, Boston, MA, USA), p-AKT/AKT (Abcam, London, UK), GAPDH (CST).

    Techniques: Western Blot

    Western blot analysis of c-Raf, p-Erk, Erk, p-Akt, Akt and histone H3 in U937 and MDA-MB-231 cell line after 24h of treatment with compounds ( D3 and D17 ) at 1 µM. Histone H3 was used as a loading control.

    Journal: ChemMedChem

    Article Title: Novel Histone Deacetylase Inhibitors with Enhanced Enzymatic Inhibition Effects and Potent in vitro and in vivo Anti-tumor Activities

    doi: 10.1002/cmdc.201300297

    Figure Lengend Snippet: Western blot analysis of c-Raf, p-Erk, Erk, p-Akt, Akt and histone H3 in U937 and MDA-MB-231 cell line after 24h of treatment with compounds ( D3 and D17 ) at 1 µM. Histone H3 was used as a loading control.

    Article Snippet: Membrane was blocked with 5% milk in TBS-T for 1 h at room temperature, then incubated with a 1:1000 or 1:2000 dilution of primary antibody overnight at 4 °C including tubulin (Sigma), acetylated tubulin (Sigma), histone H3 (Sigma), acetylated histone H3 (Sigma), acetylated histone H4 (Sigma), p21 (Cell Signaling), c-Raf (Sigma), p-Erk (Sigma), Erk (Sigma), p-Akt (Sigma) and Akt (Sigma).

    Techniques: Western Blot, Multiple Displacement Amplification

    Effect of ranitidine and AIG treatment on HSP-70, p-AKT, and PCNA expression in acetic acid-induced gastric tissue of rats. Data are presented as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett's test. ∗ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Mechanisms of Antiulcer Effect of an Active Ingredient Group of Modified Xiao Chaihu Decoction

    doi: 10.1155/2018/5498698

    Figure Lengend Snippet: Effect of ranitidine and AIG treatment on HSP-70, p-AKT, and PCNA expression in acetic acid-induced gastric tissue of rats. Data are presented as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett's test. ∗ P

    Article Snippet: Sections were then incubated with primary antibodies against PCNA (Servicebio, China) (1 : 500), HSP-70 (Ruiying Biological, China) (1 : 100), or p-AKT (Servicebio, China) (1 : 200).

    Techniques: Expressing

    Icariside II enhanced BDNF/TrkB/CREB signaling in BCCAO-treated rats. The expression of BDNF and TrkB protein, as well as the levels of Akt and CREB phosphorylation were examined by Western blot. (A) The antibody-reactive band of BDNF and TrkB protein together with Akt and CREB phosphorylation. (B) Quantitative analysis of BDNF levels. (C) Quantitative analysis of TrkB levels. (D) Quantitative analysis of Akt phosphorylation. (E) Quantitative analysis of CREB phosphorylation. The relative optical density was normalized to β-actin or Akt or CREB. Data were expressed as mean ± SEM ( n = 3). ∗∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Icariside II Ameliorates Cognitive Impairments Induced by Chronic Cerebral Hypoperfusion by Inhibiting the Amyloidogenic Pathway: Involvement of BDNF/TrkB/CREB Signaling and Up-Regulation of PPARα and PPARγ in Rats

    doi: 10.3389/fphar.2018.01211

    Figure Lengend Snippet: Icariside II enhanced BDNF/TrkB/CREB signaling in BCCAO-treated rats. The expression of BDNF and TrkB protein, as well as the levels of Akt and CREB phosphorylation were examined by Western blot. (A) The antibody-reactive band of BDNF and TrkB protein together with Akt and CREB phosphorylation. (B) Quantitative analysis of BDNF levels. (C) Quantitative analysis of TrkB levels. (D) Quantitative analysis of Akt phosphorylation. (E) Quantitative analysis of CREB phosphorylation. The relative optical density was normalized to β-actin or Akt or CREB. Data were expressed as mean ± SEM ( n = 3). ∗∗ P

    Article Snippet: Immunoblots were probed using primary antibodies against: Aβ1-42 (1:1000, Abcam, United States), Aβ1-40 (1:1000, Abcam, United States), IDE (1:1000, Abcam, United States), BACE1 (1:1000, Abcam, United States), ADAM10 (1:1000, Abcam, United States), APP (1:1000, Sangon Biotech, China), PPARα (1:1000, Abcam, United States), PPARβ (1:1000, Abcam, United States), PPARγ (1:1000, Abcam, United States), BDNF (1:500, Abcam, United States), TrkB (1:500, Abcam, United States), Akt (1:1000, Sangon Biotech, China), p-Akt (1:1000, Sangon Biotech, China), CREB (1:500, Cell Signaling Technology, United States), p-CREB (1:500, Cell Signaling Technology, United States), β-actin (1:5000, Beyotime, China), and GAPDH (1:5000, Beyotime, China).

    Techniques: Expressing, Western Blot

    Expression levels of ERK, p-ERK, Akt and p-Akt in HGC-27 and AGS cells. ( A ) HGC-27 cells; ( B ) AGS cells; ( C ) relative expression levels of p-ERK/ERK and p-Akt/Akt in HGC-27 cells; ( D ) relative expression levels of p-ERK/ERK and p-Akt/Akt in AGS cells. * p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Atractylenolide II Inhibits Proliferation, Motility and Induces Apoptosis in Human Gastric Carcinoma Cell Lines HGC-27 and AGS

    doi: 10.3390/molecules22111886

    Figure Lengend Snippet: Expression levels of ERK, p-ERK, Akt and p-Akt in HGC-27 and AGS cells. ( A ) HGC-27 cells; ( B ) AGS cells; ( C ) relative expression levels of p-ERK/ERK and p-Akt/Akt in HGC-27 cells; ( D ) relative expression levels of p-ERK/ERK and p-Akt/Akt in AGS cells. * p

    Article Snippet: CCK-8 Kit and human Bcl-2, Bax, Akt, p-Akt, ERK, p-ERK and β-actin polyclonal antibodies were purchased from Beyotime (Shanghai, China).

    Techniques: Expressing

    HK2 deletion led to enhanced CD4 T cell proliferation without affecting T cell differentiation in vitro. (A-C) Naïve CD4 T cells from WT and HK2 KO mice were CTV labelled and activated for 72 hours. (A) Representative FACS plots and bar graph indicating CTV dilution (as a measure of proliferation) gated on live CD4 T cells. Representative FACS plots and Histogram representing the levels of (B) phosphorylated–AKT and (C) Phosphorylated-S6 kinase. (D) Naïve CD4 T cells from WT and HK2 KO mice were cultured in the presence Treg or Th1 or Th17 differentiating conditions for 5 days followed by re-stimulation for 4 hours with PMA/Ionomycin. Histogram showing frequency of Th17, Th1 cells and Treg cells. All the measurements were made on live CD4 T cells. Data represents means ± SEM from two independent experiments (n = 3/group) P≤0.001(***).

    Journal: PLoS ONE

    Article Title: Hexokinase II may be dispensable for CD4 T cell responses against a virus infection

    doi: 10.1371/journal.pone.0191533

    Figure Lengend Snippet: HK2 deletion led to enhanced CD4 T cell proliferation without affecting T cell differentiation in vitro. (A-C) Naïve CD4 T cells from WT and HK2 KO mice were CTV labelled and activated for 72 hours. (A) Representative FACS plots and bar graph indicating CTV dilution (as a measure of proliferation) gated on live CD4 T cells. Representative FACS plots and Histogram representing the levels of (B) phosphorylated–AKT and (C) Phosphorylated-S6 kinase. (D) Naïve CD4 T cells from WT and HK2 KO mice were cultured in the presence Treg or Th1 or Th17 differentiating conditions for 5 days followed by re-stimulation for 4 hours with PMA/Ionomycin. Histogram showing frequency of Th17, Th1 cells and Treg cells. All the measurements were made on live CD4 T cells. Data represents means ± SEM from two independent experiments (n = 3/group) P≤0.001(***).

    Article Snippet: P-AKT (S473-SDRNR) from ebiosciences and P-S6 (S235/236-D57.2.2E) from cell signaling.

    Techniques: Cell Differentiation, In Vitro, Mouse Assay, FACS, Cell Culture

    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