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  • 99
    Cell Signaling Technology Inc akt
    <t>ARF6-GTP</t> is sufficient for PI3K and <t>AKT</t> activation. (a) Catalytic activity of endogenous PI3K in mouse tumor cell lines, one–way ANOVA with Tukey’s multiple comparisons test (a). (b) Mouse melanoma cells Braf CA ; Cdkn2a f/f control (5588), Braf CA ; Cdkn2a f/f + Arf6 Q67L (6431 and 6455). (c) Adenoviral delivery of MYC-tagged ARF6 Q67L to serum-starved NIH3T3 cells. (b-c) Graphs show individual data points normalized to control along with geometric means, 95% C.I., ratio paired t test, two tailed. Phosphorylated AKT S473 detected by immunohistochemistry in (d) primary tumors from Arf6 Q67L mice but not Braf CA ;Cdkn2a f/f controls. Scale bars = 20μm, 400x magnification. (e) pAKT stains tended to highlight tumor cells at the invasive front (arrows). Mouse tumors 8845, 6807 and 6808 scale bars = 20 μm, 400x magnification. In 6807 pAKT highlights tumor cells invading edematous subcutaneous tissue (top right of image). In 6808, pAKT highlights tumor cells invading adipose (left portion of image). Mouse 7760 shows pAKT highlighting tumor invading skeletal muscle, scale bar = 50 μm, 100x magnification. (f) Heterogeneous pAKT staining of metastatic tumors. Pulmonary metastasis (met), scale bars = 20 μm, 400x magnification. Lymph node met scale bars = 50 μm, 200x magnification. Braf CA ;Cdkn2a f/f mice are known to develop benign pulmonary adenomas (unrelated to melanoma) and these fail to stain with pAKT, providing a negative internal control (right panel, scale bar 100μm, 100x magnification).
    Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 55577 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc anti akt
    The involvement of the <t>FASN/ERα/Akt</t> pathway in hypoxia regulation of IGFBP-2
    Anti Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 17768 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 3687 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.
    Anti P Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti akt
    Loss of FlnB induces Cdk1 activity changes through β1 <t>integrin-Pi3k/Akt</t> pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p
    Rabbit Anti Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 3541 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho akt s473
    Arsenic growth inhibition correlates with decreased temsirolimus-induced <t>phospho-AKT.</t> MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10 -6 N NaOH and 2.5 x 10 -5 % EtOH in PBS), 1-2 µM ATO, 0.5-5 ng/ml temsirolimus and the combinations for 24 hours. Whole cell extracts were used in immunoblotting experiments for <t>phospho-S473-AKT,</t> phospho-T308-AKT, AKT, and β-actin. Immunoblots shown are representative of experiments performed at least 3 times.
    Phospho Akt S473, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1721 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti phospho akt
    Immunofluorescent detection of Ki-67 ( panels a, b ), <t>phospho-PDGFR-β</t> ( panels c, d ), and <t>phospho-Akt</t> ( e,f ) in control ( a,c,e ) and injured carotid arteries ( b,d,f ) of smLrp1 −/− mice 14 days after endothelial denudation. The elastic
    Rabbit Anti Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1932 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ARF6-GTP is sufficient for PI3K and AKT activation. (a) Catalytic activity of endogenous PI3K in mouse tumor cell lines, one–way ANOVA with Tukey’s multiple comparisons test (a). (b) Mouse melanoma cells Braf CA ; Cdkn2a f/f control (5588), Braf CA ; Cdkn2a f/f + Arf6 Q67L (6431 and 6455). (c) Adenoviral delivery of MYC-tagged ARF6 Q67L to serum-starved NIH3T3 cells. (b-c) Graphs show individual data points normalized to control along with geometric means, 95% C.I., ratio paired t test, two tailed. Phosphorylated AKT S473 detected by immunohistochemistry in (d) primary tumors from Arf6 Q67L mice but not Braf CA ;Cdkn2a f/f controls. Scale bars = 20μm, 400x magnification. (e) pAKT stains tended to highlight tumor cells at the invasive front (arrows). Mouse tumors 8845, 6807 and 6808 scale bars = 20 μm, 400x magnification. In 6807 pAKT highlights tumor cells invading edematous subcutaneous tissue (top right of image). In 6808, pAKT highlights tumor cells invading adipose (left portion of image). Mouse 7760 shows pAKT highlighting tumor invading skeletal muscle, scale bar = 50 μm, 100x magnification. (f) Heterogeneous pAKT staining of metastatic tumors. Pulmonary metastasis (met), scale bars = 20 μm, 400x magnification. Lymph node met scale bars = 50 μm, 200x magnification. Braf CA ;Cdkn2a f/f mice are known to develop benign pulmonary adenomas (unrelated to melanoma) and these fail to stain with pAKT, providing a negative internal control (right panel, scale bar 100μm, 100x magnification).

    Journal: Cancer research

    Article Title: The small GTPase ARF6 activates PI3K in melanoma to induce a pro-metastatic state

    doi: 10.1158/0008-5472.CAN-18-3026

    Figure Lengend Snippet: ARF6-GTP is sufficient for PI3K and AKT activation. (a) Catalytic activity of endogenous PI3K in mouse tumor cell lines, one–way ANOVA with Tukey’s multiple comparisons test (a). (b) Mouse melanoma cells Braf CA ; Cdkn2a f/f control (5588), Braf CA ; Cdkn2a f/f + Arf6 Q67L (6431 and 6455). (c) Adenoviral delivery of MYC-tagged ARF6 Q67L to serum-starved NIH3T3 cells. (b-c) Graphs show individual data points normalized to control along with geometric means, 95% C.I., ratio paired t test, two tailed. Phosphorylated AKT S473 detected by immunohistochemistry in (d) primary tumors from Arf6 Q67L mice but not Braf CA ;Cdkn2a f/f controls. Scale bars = 20μm, 400x magnification. (e) pAKT stains tended to highlight tumor cells at the invasive front (arrows). Mouse tumors 8845, 6807 and 6808 scale bars = 20 μm, 400x magnification. In 6807 pAKT highlights tumor cells invading edematous subcutaneous tissue (top right of image). In 6808, pAKT highlights tumor cells invading adipose (left portion of image). Mouse 7760 shows pAKT highlighting tumor invading skeletal muscle, scale bar = 50 μm, 100x magnification. (f) Heterogeneous pAKT staining of metastatic tumors. Pulmonary metastasis (met), scale bars = 20 μm, 400x magnification. Lymph node met scale bars = 50 μm, 200x magnification. Braf CA ;Cdkn2a f/f mice are known to develop benign pulmonary adenomas (unrelated to melanoma) and these fail to stain with pAKT, providing a negative internal control (right panel, scale bar 100μm, 100x magnification).

    Article Snippet: Antibodies dilutions for western blots include ARF6 (CST-5740) 1:1000, HA tag (CST-3724) 1:1000, MYC tag (CST-2276) 1:5000, AKT (CST-9272) 1:1000, pAKT S473 (CST-4060) 1:2000, p44/42 MAPK (Erk1/2) (CST-9107) 1:1000, phospho-p44/42 MAPK (Erk1/2 Thr202/Tyr204)(CST-4377) 1:2000.

    Techniques: Activation Assay, Activity Assay, Two Tailed Test, Immunohistochemistry, Mouse Assay, Staining

    ARF6 is necessary for PI3K and AKT activation. (a-c) Adenoviral delivery of MYC-tagged ARF6 T27N , a GDP-bound, inactive form of ARF6. (a) Catalytic activity of endogenous PI3K in A2058 human melanoma cells. Unpaired t-test, two tailed. (b) 15 melanoma and one carcinoma (HEY-T30) cell lines were screened for AKT activation. 11/16 (68%) cell lines show reduced pAKT S473 with ARF6 T27N expression. (c) Reproducible reduction in pAKT by ARF6 T27N expression in A375 cells (n=4), A2058 cells (n=4), and HEY-T30 cells (n=5). (d) Reduced pAKT with siRNA knockdown of ARF6 for quantitative data.

    Journal: Cancer research

    Article Title: The small GTPase ARF6 activates PI3K in melanoma to induce a pro-metastatic state

    doi: 10.1158/0008-5472.CAN-18-3026

    Figure Lengend Snippet: ARF6 is necessary for PI3K and AKT activation. (a-c) Adenoviral delivery of MYC-tagged ARF6 T27N , a GDP-bound, inactive form of ARF6. (a) Catalytic activity of endogenous PI3K in A2058 human melanoma cells. Unpaired t-test, two tailed. (b) 15 melanoma and one carcinoma (HEY-T30) cell lines were screened for AKT activation. 11/16 (68%) cell lines show reduced pAKT S473 with ARF6 T27N expression. (c) Reproducible reduction in pAKT by ARF6 T27N expression in A375 cells (n=4), A2058 cells (n=4), and HEY-T30 cells (n=5). (d) Reduced pAKT with siRNA knockdown of ARF6 for quantitative data.

    Article Snippet: Antibodies dilutions for western blots include ARF6 (CST-5740) 1:1000, HA tag (CST-3724) 1:1000, MYC tag (CST-2276) 1:5000, AKT (CST-9272) 1:1000, pAKT S473 (CST-4060) 1:2000, p44/42 MAPK (Erk1/2) (CST-9107) 1:1000, phospho-p44/42 MAPK (Erk1/2 Thr202/Tyr204)(CST-4377) 1:2000.

    Techniques: Activation Assay, Activity Assay, Two Tailed Test, Expressing

    ARF6-GTP upregulates PI3K expression and AKT activation. (a) RNA sequencing of mouse tumors reveals differential expression between melanomas from control (n=4) and Arf6 Q67L mice (n=6). Left panel = heat map of relative expression shown, range of 2 to −2 (log2). Right panel = Upregulated gene biosets in ARF6 Q67L tumors. * = gene sets that include Pik3r1 . (b) ARF6 Q67L induces 1.4503-fold increase in Pik3r1 expression in tumors. Unpaired t-test, two-tailed. (c) In mouse tumor cell lines derived from a distinct group of mice from those sequenced, ARF6 Q67L increases both p85 and p110 PI3K protein levels. Ratio paired t-test, two-tailed. HA immunoblot detects ectopic HA-tagged ARF6 Q67L . (d) Heatmap representing differences in signaling protein levels detected with reverse phase protein array. Columns represent individual mouse tumors. Unpaired t-test, two-tailed.

    Journal: Cancer research

    Article Title: The small GTPase ARF6 activates PI3K in melanoma to induce a pro-metastatic state

    doi: 10.1158/0008-5472.CAN-18-3026

    Figure Lengend Snippet: ARF6-GTP upregulates PI3K expression and AKT activation. (a) RNA sequencing of mouse tumors reveals differential expression between melanomas from control (n=4) and Arf6 Q67L mice (n=6). Left panel = heat map of relative expression shown, range of 2 to −2 (log2). Right panel = Upregulated gene biosets in ARF6 Q67L tumors. * = gene sets that include Pik3r1 . (b) ARF6 Q67L induces 1.4503-fold increase in Pik3r1 expression in tumors. Unpaired t-test, two-tailed. (c) In mouse tumor cell lines derived from a distinct group of mice from those sequenced, ARF6 Q67L increases both p85 and p110 PI3K protein levels. Ratio paired t-test, two-tailed. HA immunoblot detects ectopic HA-tagged ARF6 Q67L . (d) Heatmap representing differences in signaling protein levels detected with reverse phase protein array. Columns represent individual mouse tumors. Unpaired t-test, two-tailed.

    Article Snippet: Antibodies dilutions for western blots include ARF6 (CST-5740) 1:1000, HA tag (CST-3724) 1:1000, MYC tag (CST-2276) 1:5000, AKT (CST-9272) 1:1000, pAKT S473 (CST-4060) 1:2000, p44/42 MAPK (Erk1/2) (CST-9107) 1:1000, phospho-p44/42 MAPK (Erk1/2 Thr202/Tyr204)(CST-4377) 1:2000.

    Techniques: Expressing, Activation Assay, RNA Sequencing Assay, Mouse Assay, Two Tailed Test, Derivative Assay, Protein Array

    PI3K and AKT are necessary for ARF6-GTP-dependent melanoma invasion. (a,c) Matrigel invasion of A2058 cells is increased by ectopic expression of Myc-tagged ARF6 Q67L (a-d). (a) The AKT inhibitor MK2206 (1μM) or (c) the PI3K inhibitor GDC0941 (1μM) eliminates ARF6 Q67L -induced invasion. Error bars = S.D., ****p

    Journal: Cancer research

    Article Title: The small GTPase ARF6 activates PI3K in melanoma to induce a pro-metastatic state

    doi: 10.1158/0008-5472.CAN-18-3026

    Figure Lengend Snippet: PI3K and AKT are necessary for ARF6-GTP-dependent melanoma invasion. (a,c) Matrigel invasion of A2058 cells is increased by ectopic expression of Myc-tagged ARF6 Q67L (a-d). (a) The AKT inhibitor MK2206 (1μM) or (c) the PI3K inhibitor GDC0941 (1μM) eliminates ARF6 Q67L -induced invasion. Error bars = S.D., ****p

    Article Snippet: Antibodies dilutions for western blots include ARF6 (CST-5740) 1:1000, HA tag (CST-3724) 1:1000, MYC tag (CST-2276) 1:5000, AKT (CST-9272) 1:1000, pAKT S473 (CST-4060) 1:2000, p44/42 MAPK (Erk1/2) (CST-9107) 1:1000, phospho-p44/42 MAPK (Erk1/2 Thr202/Tyr204)(CST-4377) 1:2000.

    Techniques: Expressing

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.22081

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

    Article Snippet: The tissue sections were incubated with primary antibodies, rabbit anti-Ki-67 (ab16667, Abcam, Cambridge, MA, USA), phospho-EGFR (Tyr1068) (CST3777, Cell Signaling technology, Boston, MA, USA), phospho-Akt (p-Akt, Ser473) (CST4060, Cell Signaling technology, Boston, MA, USA), β-catenin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), cyclin D1(ab134175, Abcam, Cambridge, MA, USA) and rabbit anti-MUC2 (Santa Cruz Biotechnology, Inc), overnight at 4°C in a humidity-controlled chamber.

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

    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

    A subset of amino terminal mutants of p110a is associated with increased AKT phosphorylation on Ser473 ( A ). Total protein from stably transfected, serum starved U2OS cells expressing vector, wild-type FLAG-p110α, or mutant Flag-p110α were analyzed by western blotting. Constructs expressing the known activating mutants, p110α-H1047R and p110α-E365K, served as positive controls for AKT phosphorylation ( B ) FLAG- p110α and p-AKT Ser473 bands were normalized to Actin. The ratio of normalized p-AKT Ser473 to normalized FLAG- p110α relative to wild-type p110α is shown.

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

    Article Title: A unique spectrum of somatic PIK3CA (p110?) mutations within primary endometrial carcinomas

    doi: 10.1158/1078-0432.CCR-10-0540

    Figure Lengend Snippet: A subset of amino terminal mutants of p110a is associated with increased AKT phosphorylation on Ser473 ( A ). Total protein from stably transfected, serum starved U2OS cells expressing vector, wild-type FLAG-p110α, or mutant Flag-p110α were analyzed by western blotting. Constructs expressing the known activating mutants, p110α-H1047R and p110α-E365K, served as positive controls for AKT phosphorylation ( B ) FLAG- p110α and p-AKT Ser473 bands were normalized to Actin. The ratio of normalized p-AKT Ser473 to normalized FLAG- p110α relative to wild-type p110α is shown.

    Article Snippet: Normalized band intensities for FLAG-, phospho-AKTSer473 , and total AKT were determined by comparison to respective β-Actin band intensities.

    Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Construct

    Insulin-stimulated cell signaling in skeletal muscle. Phosphorylation of insulin receptor substrate 1 (IRS1) on Tyr632 ( A ), Akt on Thr308 ( B ), and Ser473 ( C ), and PAS160 ( D ) and relative total protein levels of Akt1, Akt2, and AS160 ( E ) were determined

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: Low intrinsic running capacity is associated with reduced skeletal muscle substrate oxidation and lower mitochondrial content in white skeletal muscle

    doi: 10.1152/ajpregu.00659.2010

    Figure Lengend Snippet: Insulin-stimulated cell signaling in skeletal muscle. Phosphorylation of insulin receptor substrate 1 (IRS1) on Tyr632 ( A ), Akt on Thr308 ( B ), and Ser473 ( C ), and PAS160 ( D ) and relative total protein levels of Akt1, Akt2, and AS160 ( E ) were determined

    Article Snippet: The membranes were then blocked (5% nonfat dry milk), and incubated overnight at 4°C with primary antibodies specific for either phospho-Akt Ser473, phospho-Akt Thr308, Akt1, Akt2, phospho-Akt substrate (1:1,000; Cell Signaling, Beverly, MA), phospho-insulin receptor substrate 1 (IRS1) Tyr632, CPTI (1:200–500; Santa Cruz Biotechnology, Santa Cruz, CA), or AS160 (TBCD14), which was produced as previously described , using a region of human AS160 from amino acids 621–766 fused with glutathione S -transferase (1:1,000, a gift from Prof. David James, Garvan Institute, Sydney, Australia).

    Techniques:

    NCOA5 upregulates Cyclin D1 and MMP9 as well as downreuglates P27 in CRC cells via the PI3K/AKT pathway (A) Western blot analysis of NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27 and MMP9. The lysates derived from SW620-shNCOA5 2 # , SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells were immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27, MMP9 and GAPDH (a loading control), respectively. The representative figures of Western blot assay were shown. (B) Western blot analysis after PI3K inhibition. The SW480-LV-NCOA5 CRC cells were pretreated with different concentrations (0, 5, 10 and 20 μM) of PI3K inhibitor LY294002. The lysates of LY294002-treated and -untreated SW480-LV-NCOA5 CRC cells were harvested and then immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, Cyclin D1, P27, MMP9 and GAPDH (a loading control). The representative figures of Western blot assay were shown. (C) ELISA analysis of MMP9 secretion.SW620-shNCOA5 2 # and SW620-shNCOA5 3 # compared with SW620-shNTC group: ** , P

    Journal: Oncotarget

    Article Title: NCOA5 promotes proliferation, migration and invasion of colorectal cancer cells via activation of PI3K/AKT pathway

    doi: 10.18632/oncotarget.22429

    Figure Lengend Snippet: NCOA5 upregulates Cyclin D1 and MMP9 as well as downreuglates P27 in CRC cells via the PI3K/AKT pathway (A) Western blot analysis of NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27 and MMP9. The lysates derived from SW620-shNCOA5 2 # , SW620-shNCOA5 3 # and SW620-shNTC; SW480-LV-NCOA5 and SW480-LV CRC cells were immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, p-ERK1/2, ERK1/2, Cyclin D1, P27, MMP9 and GAPDH (a loading control), respectively. The representative figures of Western blot assay were shown. (B) Western blot analysis after PI3K inhibition. The SW480-LV-NCOA5 CRC cells were pretreated with different concentrations (0, 5, 10 and 20 μM) of PI3K inhibitor LY294002. The lysates of LY294002-treated and -untreated SW480-LV-NCOA5 CRC cells were harvested and then immunoblotted with a panel of antibodies specific for NCOA5, p-AKT, AKT, Cyclin D1, P27, MMP9 and GAPDH (a loading control). The representative figures of Western blot assay were shown. (C) ELISA analysis of MMP9 secretion.SW620-shNCOA5 2 # and SW620-shNCOA5 3 # compared with SW620-shNTC group: ** , P

    Article Snippet: The membranes transferred by cell lysates were incubated with primary antibodies including anti-NCOA5 (1:1000; Cat. No. A300-789A, Bethyl, Montgomery, TX, USA), anti-phospho-AKT (anti-p-AKT) (Ser473) (clone No. D9E) (1:1000; Cat. No. 4060S, Cell Signaling Technology, Danvers, MA, USA), anti-AKT (1:1000; Cat. No. AP52689-100, ABGENT, San Diego, CA, USA), anti-phospho-extracellular signal-regulated kinase 1/2 (anti-p-ERK1/2) (Thr202/Tyr204) (clone No. 20G11) (1:1000; Cat. No. 4376S, Cell Signaling Technology, Danvers, MA, USA), anti-ERK1/2 (clone No. 137F5) (1:1000; Cat. No. 4695S, Cell Signaling Technology, Danvers, MA, USA), anti-Cyclin D1 (clone No. 92G2) (1:1000; Cat. No. 2978S, Cell Signaling Technology, Danvers, MA, USA), anti-P27 (clone No. D69C12) (1:1000; Cat. No. 3686S, Cell Signaling Technology, Danvers, MA, USA), anti-MMP9 (clone No. 5G3) (1:1000; Cat. No. GTX60482, GeneTex, Irvine, CA, USA) and anti-GAPDH (1:3000; Cat. No. AP0063, Bioworld, St. Louis Park, MN, USA) (a loading control), respectively.

    Techniques: Western Blot, Derivative Assay, Inhibition, Enzyme-linked Immunosorbent Assay

    Activation of mTOR in jejunum. (Up) Representative western blots of total 4EBP1(T-4EBP1), phosphorylated 4EBP1 (Ser65) (P-4EBP1), total Akt (T-Akt), phosphorylated Akt (Ser473) (P-Akt), total mTOR (T- mTOR), phosphorylated mTOR (Ser2448) (P- mTOR) in jejunum of piglets fed the various dietary treatments. β-actin was the loading control. (Down) Quantification by image analysis of 4EBP1, Akt and mTOR phosphorylation. Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4 mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% (g/g) glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% (g/g) glutamic acid supplementation. n = 7 for treatments.

    Journal: PLoS ONE

    Article Title: Therapeutic Effects of Glutamic Acid in Piglets Challenged with Deoxynivalenol

    doi: 10.1371/journal.pone.0100591

    Figure Lengend Snippet: Activation of mTOR in jejunum. (Up) Representative western blots of total 4EBP1(T-4EBP1), phosphorylated 4EBP1 (Ser65) (P-4EBP1), total Akt (T-Akt), phosphorylated Akt (Ser473) (P-Akt), total mTOR (T- mTOR), phosphorylated mTOR (Ser2448) (P- mTOR) in jejunum of piglets fed the various dietary treatments. β-actin was the loading control. (Down) Quantification by image analysis of 4EBP1, Akt and mTOR phosphorylation. Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4 mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% (g/g) glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% (g/g) glutamic acid supplementation. n = 7 for treatments.

    Article Snippet: The following primary antibodies were incubated overnight at 4°C with gentle rocking: β-actin (Santa Vruz, 1∶400), total 4EBP1 (T-4EBP1, Cell Signaling, 1∶1000), phosphorylated 4EBP1 (Ser 65) (Cell Signaling, 1∶1000), total Akt (T-Akt, Cell Signaling, 1∶1000), phosphorylated Akt (Ser 473) (Cell Signaling, 1∶1000), total mTOR (T-mTOR, Cell Signaling, 1∶1000) or phosphorylated mTOR (Ser 2448) (Cell Signaling, 1∶1000).

    Techniques: Activation Assay, Western Blot

    Activation of mTOR in ileum. (Up) Representative western blots of total 4EBP1(T-4EBP1), phosphorylated 4EBP1 (Ser65) (P-4EBP1), total Akt (T-Akt), phosphorylated Akt (Ser473) (P-Akt), total mTOR (T- mTOR), phosphorylated mTOR (Ser2448) (P- mTOR) in ileal of piglets fed the various dietary treatments. β-actin was the loading control. (Down) Quantification by image analysis of 4EBP1, Akt and mTOR phosphorylation. Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4 mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% (g/g) glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% (g/g) glutamic acid supplementation. n = 7 for treatments.

    Journal: PLoS ONE

    Article Title: Therapeutic Effects of Glutamic Acid in Piglets Challenged with Deoxynivalenol

    doi: 10.1371/journal.pone.0100591

    Figure Lengend Snippet: Activation of mTOR in ileum. (Up) Representative western blots of total 4EBP1(T-4EBP1), phosphorylated 4EBP1 (Ser65) (P-4EBP1), total Akt (T-Akt), phosphorylated Akt (Ser473) (P-Akt), total mTOR (T- mTOR), phosphorylated mTOR (Ser2448) (P- mTOR) in ileal of piglets fed the various dietary treatments. β-actin was the loading control. (Down) Quantification by image analysis of 4EBP1, Akt and mTOR phosphorylation. Dietary treatments were NC, an uncontaminated basal diet, DON, the basal contaminated with 4 mg/kg deoxynivalenol, GLU, uncontaminated basal diet with 2% (g/g) glutamic acid supplementation, and DG, deoxynivalenol-contaminated (4 mg/kg) basal diet with 2% (g/g) glutamic acid supplementation. n = 7 for treatments.

    Article Snippet: The following primary antibodies were incubated overnight at 4°C with gentle rocking: β-actin (Santa Vruz, 1∶400), total 4EBP1 (T-4EBP1, Cell Signaling, 1∶1000), phosphorylated 4EBP1 (Ser 65) (Cell Signaling, 1∶1000), total Akt (T-Akt, Cell Signaling, 1∶1000), phosphorylated Akt (Ser 473) (Cell Signaling, 1∶1000), total mTOR (T-mTOR, Cell Signaling, 1∶1000) or phosphorylated mTOR (Ser 2448) (Cell Signaling, 1∶1000).

    Techniques: Activation Assay, Western Blot

    TRIP6 regulates growth factor-induced AKT activation and T157 phosphorylation of p27 KIP1 . (A) Knockdown of TRIP6 specifically eliminates serum-induced T157 phosphorylation of p27 KIP1 . Confluent U373-MG cells, as indicated, were starved overnight. After pretreatment with MG-132 for 1 h, cells were stimulated with serum for 15 min. Immunoblotting was performed to detect phosphorylated p27 KIP1 (pT157, pT198, and pS10), total p27 KIP1 , or TRIP6 in the lysates. (B to D) Knockdown of TRIP6 eliminates growth factor-induced T157 phosphorylation; however, this effect can be reversed by reconstitution with TRIP6. Confluent U373-MG cells (B) or SKOV-3 cells (C and D), as indicated, were starved overnight, followed by stimulation with the indicated growth factors for 15 min (B) or 5 min (C and D). Immunoblotting was performed to detect pT157-p27 KIP1 or total p27 KIP1 . The expression of EGFP-TRIP6 and TRIP6 in the whole-cell lysates was detected by using anti-TRIP6 antibody. (E) TRIP6 promotes cytosolic mislocalization of p27 KIP1 through the regulation of its phosphorylation at T157. WT or T157D EGFP-p27 KIP1 was expressed in SKOV-3 cells (siScr and siTRIP6). Subcellular fractionation was performed to determine the expression level of EGFP-p27 KIP1 or EGFP-T157D p27 KIP1 in the nucleus (N) or cytosol (C). The intensity of EGFP-p27 KIP1 or EGFP-T157D p27 KIP1 was quantified by NIH IMAGE J software to determine the relative levels of nuclear versus cytosolic p27 KIP1 . Histone H3 and GAPDH served as nuclear and cytosolic markers, respectively. (F and G) Knockdown of TRIP6 attenuates EGF-induced AKT activation; however, this effect can be reversed by TRIP6 overexpression. U373-MG cells (F) or SKOV-3 cells (G), as indicated, were starved overnight, followed by stimulation with EGF for 15 min (F or G) or 30 min (F). Immunoblotting was performed to detect the levels of pT308-AKT, AKT1, pS9–GSK-3β, GSK-3β, and TRIP6. (H) TRIP6 enhances AKT-mediated in vitro phosphorylation of p27 KIP1 at T157 but not at T198. An in vitro kinase assay was performed by incubating purified recombinant p27 KIP1 with active AKT1 in the presence or absence of TRIP6 at 30°C for 30 min. Immunoblotting (IB) was performed to detect pT157-p27 KIP1 , pT198-p27 KIP1 , pT308-AKT1, p27 KIP1 , or TRIP6. The relative intensity of pT157-p27 KIP1 or pT198-p27 KIP1 was quantified by using NIH IMAGE J software.

    Journal: Molecular and Cellular Biology

    Article Title: TRIP6 Regulates p27KIP1 To Promote Tumorigenesis

    doi: 10.1128/MCB.01149-12

    Figure Lengend Snippet: TRIP6 regulates growth factor-induced AKT activation and T157 phosphorylation of p27 KIP1 . (A) Knockdown of TRIP6 specifically eliminates serum-induced T157 phosphorylation of p27 KIP1 . Confluent U373-MG cells, as indicated, were starved overnight. After pretreatment with MG-132 for 1 h, cells were stimulated with serum for 15 min. Immunoblotting was performed to detect phosphorylated p27 KIP1 (pT157, pT198, and pS10), total p27 KIP1 , or TRIP6 in the lysates. (B to D) Knockdown of TRIP6 eliminates growth factor-induced T157 phosphorylation; however, this effect can be reversed by reconstitution with TRIP6. Confluent U373-MG cells (B) or SKOV-3 cells (C and D), as indicated, were starved overnight, followed by stimulation with the indicated growth factors for 15 min (B) or 5 min (C and D). Immunoblotting was performed to detect pT157-p27 KIP1 or total p27 KIP1 . The expression of EGFP-TRIP6 and TRIP6 in the whole-cell lysates was detected by using anti-TRIP6 antibody. (E) TRIP6 promotes cytosolic mislocalization of p27 KIP1 through the regulation of its phosphorylation at T157. WT or T157D EGFP-p27 KIP1 was expressed in SKOV-3 cells (siScr and siTRIP6). Subcellular fractionation was performed to determine the expression level of EGFP-p27 KIP1 or EGFP-T157D p27 KIP1 in the nucleus (N) or cytosol (C). The intensity of EGFP-p27 KIP1 or EGFP-T157D p27 KIP1 was quantified by NIH IMAGE J software to determine the relative levels of nuclear versus cytosolic p27 KIP1 . Histone H3 and GAPDH served as nuclear and cytosolic markers, respectively. (F and G) Knockdown of TRIP6 attenuates EGF-induced AKT activation; however, this effect can be reversed by TRIP6 overexpression. U373-MG cells (F) or SKOV-3 cells (G), as indicated, were starved overnight, followed by stimulation with EGF for 15 min (F or G) or 30 min (F). Immunoblotting was performed to detect the levels of pT308-AKT, AKT1, pS9–GSK-3β, GSK-3β, and TRIP6. (H) TRIP6 enhances AKT-mediated in vitro phosphorylation of p27 KIP1 at T157 but not at T198. An in vitro kinase assay was performed by incubating purified recombinant p27 KIP1 with active AKT1 in the presence or absence of TRIP6 at 30°C for 30 min. Immunoblotting (IB) was performed to detect pT157-p27 KIP1 , pT198-p27 KIP1 , pT308-AKT1, p27 KIP1 , or TRIP6. The relative intensity of pT157-p27 KIP1 or pT198-p27 KIP1 was quantified by using NIH IMAGE J software.

    Article Snippet: We found that although p27KIP1 bound to AKT1 weakly, this association could be greatly improved by overexpression of TRIP6 ( ).

    Techniques: Activation Assay, Expressing, Fractionation, Software, Over Expression, In Vitro, Kinase Assay, Purification, Recombinant

    The LIM domain and pre-LIM region of TRIP6 regulate its function in AKT-mediated T157 phosphorylation of p27 KIP1 and ovarian tumor sphere proliferation. (A) The effect of TRIP6 knockdown on the inhibition of AKT activity and T157 phosphorylation of p27 KIP1 is rescued by overexpression of wild-type TRIP6 but not a TRIP6 mutant that lacks the LIM1 domain. EGFP, EGFP-TRIP6, or EGFP-ΔLIM1-TRIP6 was overexpressed in SKOV-3 cells stably expressing TRIP6 shRNA (SKOV-3–siTRIP6). After starvation overnight, cells were stimulated with EGF for 10 min. Immunoblotting was performed to detect pT308-AKT, pT157-p27 KIP1 , AKT1, p27 KIP1 , EGFP, EGFP-TRIP6, or EGFP-ΔLIM1-TRIP6 in the lysates. (B) Overexpression of a TRIP6 mutant that contains LIM1 and the pre-LIM region of TRIP6 attenuates EGF-induced AKT activation and T157 phosphorylation of p27 KIP1 . The EGFP-1-350 mutant, which contains LIM1 and the pre-LIM region of TRIP6, was overexpressed in SKOV-3–siScr cells. After starvation overnight, cells were stimulated with EGF for 10 min. Immunoblotting was performed to detect pT308-AKT, pT157-p27 KIP1 , AKT1, p27 KIP1 , or EGFP-1-350-TRIP6 in the lysates. (C and D) Deletion of LIM1 eliminates the function of TRIP6 in promoting ovarian tumor sphere proliferation, whereas overexpression of LIM1 and the pre-LIM region of TRIP6 attenuates this effect. SKOV-3–siTRIP6 cells stably expressing EGFP-TRIP6 or EGFP-ΔLIM1-TRIP6 or not (C) or SKOV-3–siScr cells stably expressing EGFP-1-350-TRIP6 or not (D) were seeded onto Matrigel-coated chamber slides in medium containing 2% horse serum with EGF, insulin, hydrocortisone, cholera toxin, and 2% Matrigel. The diameter of tumor spheres formed in 3D cultures was measured on days 3, 6, 9, and 12. The left panel shows the images of spheres on day 12. The right panel shows the average diameter of the spheres. Data shown are the means ± standard errors of the means for 40 spheres. ∗, P

    Journal: Molecular and Cellular Biology

    Article Title: TRIP6 Regulates p27KIP1 To Promote Tumorigenesis

    doi: 10.1128/MCB.01149-12

    Figure Lengend Snippet: The LIM domain and pre-LIM region of TRIP6 regulate its function in AKT-mediated T157 phosphorylation of p27 KIP1 and ovarian tumor sphere proliferation. (A) The effect of TRIP6 knockdown on the inhibition of AKT activity and T157 phosphorylation of p27 KIP1 is rescued by overexpression of wild-type TRIP6 but not a TRIP6 mutant that lacks the LIM1 domain. EGFP, EGFP-TRIP6, or EGFP-ΔLIM1-TRIP6 was overexpressed in SKOV-3 cells stably expressing TRIP6 shRNA (SKOV-3–siTRIP6). After starvation overnight, cells were stimulated with EGF for 10 min. Immunoblotting was performed to detect pT308-AKT, pT157-p27 KIP1 , AKT1, p27 KIP1 , EGFP, EGFP-TRIP6, or EGFP-ΔLIM1-TRIP6 in the lysates. (B) Overexpression of a TRIP6 mutant that contains LIM1 and the pre-LIM region of TRIP6 attenuates EGF-induced AKT activation and T157 phosphorylation of p27 KIP1 . The EGFP-1-350 mutant, which contains LIM1 and the pre-LIM region of TRIP6, was overexpressed in SKOV-3–siScr cells. After starvation overnight, cells were stimulated with EGF for 10 min. Immunoblotting was performed to detect pT308-AKT, pT157-p27 KIP1 , AKT1, p27 KIP1 , or EGFP-1-350-TRIP6 in the lysates. (C and D) Deletion of LIM1 eliminates the function of TRIP6 in promoting ovarian tumor sphere proliferation, whereas overexpression of LIM1 and the pre-LIM region of TRIP6 attenuates this effect. SKOV-3–siTRIP6 cells stably expressing EGFP-TRIP6 or EGFP-ΔLIM1-TRIP6 or not (C) or SKOV-3–siScr cells stably expressing EGFP-1-350-TRIP6 or not (D) were seeded onto Matrigel-coated chamber slides in medium containing 2% horse serum with EGF, insulin, hydrocortisone, cholera toxin, and 2% Matrigel. The diameter of tumor spheres formed in 3D cultures was measured on days 3, 6, 9, and 12. The left panel shows the images of spheres on day 12. The right panel shows the average diameter of the spheres. Data shown are the means ± standard errors of the means for 40 spheres. ∗, P

    Article Snippet: We found that although p27KIP1 bound to AKT1 weakly, this association could be greatly improved by overexpression of TRIP6 ( ).

    Techniques: Inhibition, Activity Assay, Over Expression, Mutagenesis, Stable Transfection, Expressing, shRNA, Activation Assay

    TRIP6 binds to both p27 KIP1 and AKT in vitro and in cells. (A) The pre-LIM region and LIM1 domain of TRIP6 are both required for its binding to p27 KIP1 . Purified recombinant p27 KIP1 was incubated with GST, GST-TRIP6, or a deletion mutant of GST-TRIP6 at 4°C for 3 h. p27 KIP1 pulled down by GST-TRIP6 was detected by immunoblotting using anti-p27 KIP1 antibody. Coomassie blue staining shows the expression of GST proteins used in this experiment. The right panel shows the schematic structure of TRIP6 constructs and their ability to bind p27 KIP1 . (B) The pre-LIM region and LIM1 domain of TRIP6 associate with p27 KIP1 in HEK 293T cells. HA-p27 KIP1 was coexpressed with EGFP-TRIP6 or the EGFP-1-350 mutant, which contains the pre-LIM region and LIM1 domain of TRIP6, in HEK 293T cells. After MG-132 treatment for 1 h, HA-p27 KIP1 was immunoprecipitated (IP) with anti-HA mouse monoclonal antibody-conjugated agarose beads, and coimmunoprecipitated EGFP-TRIP6 or EGFP-1-350-TRIP6 was detected by immunoblotting using anti-GFP antibody. (C) The internal region of p27 KIP1 is required for its binding to TRIP6. HEK 293T cells expressing FLAG-TRIP6 with either EGFP or one of the EGFP-p27 KIP1 constructs (WT, aa 1 to 109 or aa 1 to159) were treated with MG-132 for 1 h. FLAG-TRIP6 was immunoprecipitated with anti-FLAG M2 antibody-conjugated agarose beads, and the immunoblot was probed with anti-GFP or anti-FLAG rabbit antibody. The bottom panel shows the expression of EGFP constructs in the lysates. (D) TRIP6 binds to AKT1 directly through its LIM domains. Purified recombinant AKT1 was incubated with GST, GST-TRIP6, or a GST-TRIP6 mutant containing residues 279 to 476, 340 to 476, or 398 to 476 at 4°C for 3 h. AKT1 pulled down by GST-TRIP6 or the GST-TRIP6 mutant was detected by immunoblotting using anti-AKT1 antibody. The bottom panel shows Ponceau S staining of the GST proteins used in this experiment. (E) Deletion of the LIM1 domain eliminates the ability of TRIP6 to bind to AKT1. HA-AKT1 was coexpressed with EGFP-TRIP6, EGFP-LIM1, or EGFP-ΔLIM1-TRIP6 in HEK 293T cells. HA-AKT1 was immunoprecipitated with anti-HA mouse monoclonal antibody-conjugated agarose beads, and immunoblotting was performed to detect coimmunoprecipitated EGFP-TRIP6 or EGFP-LIM1. (F) LIM1 is the minimal domain required for TRIP6 binding to AKT1. FLAG-TRIP6 or a FLAG-TRIP6 mutant that contains residues 1 to 278, 1 to 350, or 1 to 398 of TRIP6 was expressed in HEK 293T cells. FLAG proteins were immunoprecipitated with anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads, followed by immunoblotting to detect coimmunoprecipitated endogenous AKT1. The bottom panel shows the expression of AKT1 in the lysates. (G) Deletion of the PH domain eliminates AKT1 binding to TRIP6. HEK 293T cells were transfected with the expression vector of FLAG-TRIP6 or an HA-tagged myristoylated AKT1 mutant that lacks the PH domain (Myr-ΔPH-AKT1). FLAG-TRIP6 was immunoprecipitated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads, followed by immunoblotting using anti-HA or anti-FLAG antibody. (H) LPP and TRIP6, but not zyxin, bind to p27 KIP1 and AKT1 directly in vitro . Purified recombinant p27 KIP1 or AKT1 was incubated with GST, GST-LPP, GST-zyxin, or GST-TRIP6 at 4°C for 3 h. p27 KIP1 or AKT1 pulled down by the glutathione beads was detected by immunoblotting using an antibody specific to p27 KIP1 or AKT1. The bottom panel shows Coomassie blue staining of the GST proteins used in this experiment. (I) Knockdown of LPP attenuates EGF-induced AKT activation and T157 phosphorylation of p27 KIP1 in SKOV-3 cells. Confluent SKOV-3 cells stably expressing a scrambled shRNA (siScr) or LPP shRNA (siLPP) were starved overnight, followed by stimulation with EGF for 10 min. Immunoblotting was performed to detect pT308-AKT, pS473-AKT, pT157-p27 KIP1 , AKT1, p27 KIP1 , or LPP in the lysates.

    Journal: Molecular and Cellular Biology

    Article Title: TRIP6 Regulates p27KIP1 To Promote Tumorigenesis

    doi: 10.1128/MCB.01149-12

    Figure Lengend Snippet: TRIP6 binds to both p27 KIP1 and AKT in vitro and in cells. (A) The pre-LIM region and LIM1 domain of TRIP6 are both required for its binding to p27 KIP1 . Purified recombinant p27 KIP1 was incubated with GST, GST-TRIP6, or a deletion mutant of GST-TRIP6 at 4°C for 3 h. p27 KIP1 pulled down by GST-TRIP6 was detected by immunoblotting using anti-p27 KIP1 antibody. Coomassie blue staining shows the expression of GST proteins used in this experiment. The right panel shows the schematic structure of TRIP6 constructs and their ability to bind p27 KIP1 . (B) The pre-LIM region and LIM1 domain of TRIP6 associate with p27 KIP1 in HEK 293T cells. HA-p27 KIP1 was coexpressed with EGFP-TRIP6 or the EGFP-1-350 mutant, which contains the pre-LIM region and LIM1 domain of TRIP6, in HEK 293T cells. After MG-132 treatment for 1 h, HA-p27 KIP1 was immunoprecipitated (IP) with anti-HA mouse monoclonal antibody-conjugated agarose beads, and coimmunoprecipitated EGFP-TRIP6 or EGFP-1-350-TRIP6 was detected by immunoblotting using anti-GFP antibody. (C) The internal region of p27 KIP1 is required for its binding to TRIP6. HEK 293T cells expressing FLAG-TRIP6 with either EGFP or one of the EGFP-p27 KIP1 constructs (WT, aa 1 to 109 or aa 1 to159) were treated with MG-132 for 1 h. FLAG-TRIP6 was immunoprecipitated with anti-FLAG M2 antibody-conjugated agarose beads, and the immunoblot was probed with anti-GFP or anti-FLAG rabbit antibody. The bottom panel shows the expression of EGFP constructs in the lysates. (D) TRIP6 binds to AKT1 directly through its LIM domains. Purified recombinant AKT1 was incubated with GST, GST-TRIP6, or a GST-TRIP6 mutant containing residues 279 to 476, 340 to 476, or 398 to 476 at 4°C for 3 h. AKT1 pulled down by GST-TRIP6 or the GST-TRIP6 mutant was detected by immunoblotting using anti-AKT1 antibody. The bottom panel shows Ponceau S staining of the GST proteins used in this experiment. (E) Deletion of the LIM1 domain eliminates the ability of TRIP6 to bind to AKT1. HA-AKT1 was coexpressed with EGFP-TRIP6, EGFP-LIM1, or EGFP-ΔLIM1-TRIP6 in HEK 293T cells. HA-AKT1 was immunoprecipitated with anti-HA mouse monoclonal antibody-conjugated agarose beads, and immunoblotting was performed to detect coimmunoprecipitated EGFP-TRIP6 or EGFP-LIM1. (F) LIM1 is the minimal domain required for TRIP6 binding to AKT1. FLAG-TRIP6 or a FLAG-TRIP6 mutant that contains residues 1 to 278, 1 to 350, or 1 to 398 of TRIP6 was expressed in HEK 293T cells. FLAG proteins were immunoprecipitated with anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads, followed by immunoblotting to detect coimmunoprecipitated endogenous AKT1. The bottom panel shows the expression of AKT1 in the lysates. (G) Deletion of the PH domain eliminates AKT1 binding to TRIP6. HEK 293T cells were transfected with the expression vector of FLAG-TRIP6 or an HA-tagged myristoylated AKT1 mutant that lacks the PH domain (Myr-ΔPH-AKT1). FLAG-TRIP6 was immunoprecipitated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads, followed by immunoblotting using anti-HA or anti-FLAG antibody. (H) LPP and TRIP6, but not zyxin, bind to p27 KIP1 and AKT1 directly in vitro . Purified recombinant p27 KIP1 or AKT1 was incubated with GST, GST-LPP, GST-zyxin, or GST-TRIP6 at 4°C for 3 h. p27 KIP1 or AKT1 pulled down by the glutathione beads was detected by immunoblotting using an antibody specific to p27 KIP1 or AKT1. The bottom panel shows Coomassie blue staining of the GST proteins used in this experiment. (I) Knockdown of LPP attenuates EGF-induced AKT activation and T157 phosphorylation of p27 KIP1 in SKOV-3 cells. Confluent SKOV-3 cells stably expressing a scrambled shRNA (siScr) or LPP shRNA (siLPP) were starved overnight, followed by stimulation with EGF for 10 min. Immunoblotting was performed to detect pT308-AKT, pS473-AKT, pT157-p27 KIP1 , AKT1, p27 KIP1 , or LPP in the lysates.

    Article Snippet: We found that although p27KIP1 bound to AKT1 weakly, this association could be greatly improved by overexpression of TRIP6 ( ).

    Techniques: In Vitro, Binding Assay, Purification, Recombinant, Incubation, Mutagenesis, Staining, Expressing, Construct, Immunoprecipitation, Transfection, Plasmid Preparation, Activation Assay, Stable Transfection, shRNA

    TRIP6 forms a ternary complex with p27 KIP1 and AKT in the cytosol and promotes the association of p27 KIP1 with AKT1 as well as membrane translocation of AKT1. (A) TRIP6 binds to p27 KIP1 at late G 1 to S phase of the cell cycle. U373-MG cells were synchronized at the late-G 1 /S-phase border by using the double-thymidine-block procedure. After serum stimulation for various times, p27 KIP1 from the whole-cell lysates was immunoprecipitated with anti-p27 KIP1 mouse antibody or control mouse IgG, and immunoblotting was performed to detect coimmunoprecipitated TRIP6. The induction of cyclin D1 served as an indicator of S-phase entry of the cell cycle. (B) TRIP6 associates with AKT1 upon serum stimulation. Starved U373-MG cells (siScr and siTRIP6) were treated with serum for 15 or 30 min. Endogenous AKT1 was immunoprecipitated with anti-AKT1 mouse antibody or control mouse IgG, followed by immunoblotting to detect coimmunoprecipitated TRIP6. The bottom panels show the expression of TRIP6, pT308-AKT, pS473-AKT, AKT1, pT157-p27 KIP1 , or p27 KIP1 in the lysates. (C) TRIP6 forms a ternary complex with p27 KIP1 and AKT1 in the cytosol. HEK 293T cells overexpressing FLAG-TRIP6, p27 KIP1 , or both were pretreated with MG-132 for 2 h under serum-free conditions, followed by stimulation with serum for 15 min. After in-cell cross-linking with DSP, subcellular fractionation was performed to separate nuclei and the cytosol. The FLAG-TRIP6-containing complex was first immunoprecipitated by using anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads. After competitive elution with excess free FLAG peptide, the eluted complex was subsequently immunoprecipitated with anti-p27 KIP1 mouse antibody, followed by immunoblotting to detect endogenous AKT1 and transfected FLAG-TRIP6 or p27 KIP1 in the complex. The bottom panels show the expression of p27 KIP1 , FLAG-TRIP6, endogenous TRIP6, or AKT1 in each fraction. GAPDH and histone H3 served as cytosolic and nuclear markers, respectively. (D) TRIP6 promotes the recruitment of p27 KIP1 to AKT1. HEK 293T cells expressing FLAG-TRIP6 or FLAG-p27 KIP1 with or without HA-TRIP6 were stimulated with serum for 15 min. FLAG-TRIP6 or FLAG-p27 KIP1 was immunoprecipitated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads, and immunoblotting was performed to detect coimmunoprecipitated endogenous AKT1. The bottom two panels show the expression of endogenous AKT1, TRIP6, or transfected TRIP6 in the lysates. (E) Knockdown of TRIP6 reduces the association of p27 KIP1 with AKT1, whereas reconstitution with TRIP6 reverses this effect. U373-MG stable cell lines (siScr, siTRIP6, or siTRIP6 with reconstituted TRIP6) were starved overnight. After pretreatment with MG-132 for 2 h, cells were stimulated with serum for 15 min. Endogenous p27 KIP1 was immunoprecipitated with anti-p27 KIP1 mouse monoclonal antibody or control mouse IgG, followed by immunoblotting to detect coimmunoprecipitated AKT1 and TRIP6. (F) Knockdown of TRIP6 impairs the membrane translocation of AKT1. Starved U373-MG cells, as indicated, were stimulated with EGF for 15 min. Differential centrifugation was performed to separate plasma membrane from the cytosol. Immunoblotting was performed to detect the expression of AKT1 or TRIP6 in each fraction. LPP and GAPDH served as membrane and cytosolic markers, respectively.

    Journal: Molecular and Cellular Biology

    Article Title: TRIP6 Regulates p27KIP1 To Promote Tumorigenesis

    doi: 10.1128/MCB.01149-12

    Figure Lengend Snippet: TRIP6 forms a ternary complex with p27 KIP1 and AKT in the cytosol and promotes the association of p27 KIP1 with AKT1 as well as membrane translocation of AKT1. (A) TRIP6 binds to p27 KIP1 at late G 1 to S phase of the cell cycle. U373-MG cells were synchronized at the late-G 1 /S-phase border by using the double-thymidine-block procedure. After serum stimulation for various times, p27 KIP1 from the whole-cell lysates was immunoprecipitated with anti-p27 KIP1 mouse antibody or control mouse IgG, and immunoblotting was performed to detect coimmunoprecipitated TRIP6. The induction of cyclin D1 served as an indicator of S-phase entry of the cell cycle. (B) TRIP6 associates with AKT1 upon serum stimulation. Starved U373-MG cells (siScr and siTRIP6) were treated with serum for 15 or 30 min. Endogenous AKT1 was immunoprecipitated with anti-AKT1 mouse antibody or control mouse IgG, followed by immunoblotting to detect coimmunoprecipitated TRIP6. The bottom panels show the expression of TRIP6, pT308-AKT, pS473-AKT, AKT1, pT157-p27 KIP1 , or p27 KIP1 in the lysates. (C) TRIP6 forms a ternary complex with p27 KIP1 and AKT1 in the cytosol. HEK 293T cells overexpressing FLAG-TRIP6, p27 KIP1 , or both were pretreated with MG-132 for 2 h under serum-free conditions, followed by stimulation with serum for 15 min. After in-cell cross-linking with DSP, subcellular fractionation was performed to separate nuclei and the cytosol. The FLAG-TRIP6-containing complex was first immunoprecipitated by using anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads. After competitive elution with excess free FLAG peptide, the eluted complex was subsequently immunoprecipitated with anti-p27 KIP1 mouse antibody, followed by immunoblotting to detect endogenous AKT1 and transfected FLAG-TRIP6 or p27 KIP1 in the complex. The bottom panels show the expression of p27 KIP1 , FLAG-TRIP6, endogenous TRIP6, or AKT1 in each fraction. GAPDH and histone H3 served as cytosolic and nuclear markers, respectively. (D) TRIP6 promotes the recruitment of p27 KIP1 to AKT1. HEK 293T cells expressing FLAG-TRIP6 or FLAG-p27 KIP1 with or without HA-TRIP6 were stimulated with serum for 15 min. FLAG-TRIP6 or FLAG-p27 KIP1 was immunoprecipitated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads, and immunoblotting was performed to detect coimmunoprecipitated endogenous AKT1. The bottom two panels show the expression of endogenous AKT1, TRIP6, or transfected TRIP6 in the lysates. (E) Knockdown of TRIP6 reduces the association of p27 KIP1 with AKT1, whereas reconstitution with TRIP6 reverses this effect. U373-MG stable cell lines (siScr, siTRIP6, or siTRIP6 with reconstituted TRIP6) were starved overnight. After pretreatment with MG-132 for 2 h, cells were stimulated with serum for 15 min. Endogenous p27 KIP1 was immunoprecipitated with anti-p27 KIP1 mouse monoclonal antibody or control mouse IgG, followed by immunoblotting to detect coimmunoprecipitated AKT1 and TRIP6. (F) Knockdown of TRIP6 impairs the membrane translocation of AKT1. Starved U373-MG cells, as indicated, were stimulated with EGF for 15 min. Differential centrifugation was performed to separate plasma membrane from the cytosol. Immunoblotting was performed to detect the expression of AKT1 or TRIP6 in each fraction. LPP and GAPDH served as membrane and cytosolic markers, respectively.

    Article Snippet: We found that although p27KIP1 bound to AKT1 weakly, this association could be greatly improved by overexpression of TRIP6 ( ).

    Techniques: Translocation Assay, Blocking Assay, Immunoprecipitation, Expressing, Fractionation, Transfection, Stable Transfection, Centrifugation

    Intra-venous delivery of Rictor si-NPs inhibits tumor mTORC2 activity and cooperates with lapatinib to support tumor regression A. MDA-MB-361 tumors grown in nude mice were treated with 1 mg/kg si-NPs (i.v. delivered on treatment days 0, 2, 4, and 7) and 100 mg/kg lapatinib (orally, once daily for 21 days). B-C. Tumors were harvested on treatment day 8, 24 h after final si-NP treatment, and 1 hour after lapatinib treatment. B. Whole tumor RNA was used to assess RICTOR gene expression levels by RT-qPCR. RICTOR Ct values were corrected for housekeeping gene 36B4, and are shown as the average values (± S.E.) relative to the average value measured in control tumors treated with vehicle and Scramble si-NP. N = 5. C. IHC for Rictor, P-Akt S473, and P-HER-2. Representative images are shown. N = 3. D-F. Tumor volume was measured throughout the 21 day treatment period. Values shown are the average (± S.E.), N = 7. G-I. Histological analysis of tumors collected on treatment day 21 was performed on H E stained sections at low power (20X, G ) and high power (400X, H ), demonstrating smaller size and decreased cellularity. I. The number of nuclei per mm 2 was determined using CellSense software. N = 3 randomly chosen fields per sample, N = 5.

    Journal: Cancer research

    Article Title: Selective mTORC2 inhibitor therapeutically blocks breast cancer cell growth and survival

    doi: 10.1158/0008-5472.CAN-17-2388

    Figure Lengend Snippet: Intra-venous delivery of Rictor si-NPs inhibits tumor mTORC2 activity and cooperates with lapatinib to support tumor regression A. MDA-MB-361 tumors grown in nude mice were treated with 1 mg/kg si-NPs (i.v. delivered on treatment days 0, 2, 4, and 7) and 100 mg/kg lapatinib (orally, once daily for 21 days). B-C. Tumors were harvested on treatment day 8, 24 h after final si-NP treatment, and 1 hour after lapatinib treatment. B. Whole tumor RNA was used to assess RICTOR gene expression levels by RT-qPCR. RICTOR Ct values were corrected for housekeeping gene 36B4, and are shown as the average values (± S.E.) relative to the average value measured in control tumors treated with vehicle and Scramble si-NP. N = 5. C. IHC for Rictor, P-Akt S473, and P-HER-2. Representative images are shown. N = 3. D-F. Tumor volume was measured throughout the 21 day treatment period. Values shown are the average (± S.E.), N = 7. G-I. Histological analysis of tumors collected on treatment day 21 was performed on H E stained sections at low power (20X, G ) and high power (400X, H ), demonstrating smaller size and decreased cellularity. I. The number of nuclei per mm 2 was determined using CellSense software. N = 3 randomly chosen fields per sample, N = 5.

    Article Snippet: IHC on paraffin-embedded sections was performed as described previously ( ) using: Rictor (Santa Cruz Biotechnologies), Ki67 (Santa Cruz Biotechnologies) and P-Akt S473 (Cell Signaling Technologies) antibodies.

    Techniques: Activity Assay, Multiple Displacement Amplification, Mouse Assay, Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Software

    Intra-tumoral delivery of nanoparticles provides therapeutic mTORC2 inhibition in vivo A. MDA-MB-231-Luciferase xenografts grown in contralateral mammary fatpads were treated by intra-tumoral injection with Luciferase si-NPs (siLuc) or Scarmble si-NPs (siScr). Intravital imaging of tumor luminescence was measured 24h after treatment with si-NPs using IVIS software. Left panel shows representative images of a single mouse imaged at 0h and again at 24h after intra-tumoral si-NP delivery. Panel at right shows luciferase activity at 24h post-treatment, as a percentage of values measured at 0h. Average values (±S.E.) shown, N = 3. B. Timeline schematic illustrates the dosing schedule for MDA-MB-231 and MDA-MB-361 tumors treated with si-NPs. Three doses were given at 48h intervals. Tumors were measured immediately prior to dosing. C. Western analysis of whole tumor extracts harvested on day 5, 24 h after the final si-NP treatment. Each lane represents a tumor collected from different mice. Antibodies are shown at left of each panel. D. Rictor and P-Akt S473 were detected by IHC in tumors harvested on treatment day 5. Upper panels show representative images taken at 200 X. Lower panels show quantitative analysis of staining using Scion Image software, expressed as pixels/field. Each point represents the average of 5 randomly chosen fields per sample. Midlines are the average (± S.E.). N = 3 (Rictor IHC) and 5 (P-Akt S473 IHC). E. Tumor volumes were measured on treatments days 0, 2, and 4. N = 5. F. TUNEL analysis. Left panels show representative images. Right panel shows quantitation of TUNEL staining, expressed as the percentage of nuclei that are TUNEL+. N = 6, each assessed in 5 randomly chosen 400X fields.

    Journal: Cancer research

    Article Title: Selective mTORC2 inhibitor therapeutically blocks breast cancer cell growth and survival

    doi: 10.1158/0008-5472.CAN-17-2388

    Figure Lengend Snippet: Intra-tumoral delivery of nanoparticles provides therapeutic mTORC2 inhibition in vivo A. MDA-MB-231-Luciferase xenografts grown in contralateral mammary fatpads were treated by intra-tumoral injection with Luciferase si-NPs (siLuc) or Scarmble si-NPs (siScr). Intravital imaging of tumor luminescence was measured 24h after treatment with si-NPs using IVIS software. Left panel shows representative images of a single mouse imaged at 0h and again at 24h after intra-tumoral si-NP delivery. Panel at right shows luciferase activity at 24h post-treatment, as a percentage of values measured at 0h. Average values (±S.E.) shown, N = 3. B. Timeline schematic illustrates the dosing schedule for MDA-MB-231 and MDA-MB-361 tumors treated with si-NPs. Three doses were given at 48h intervals. Tumors were measured immediately prior to dosing. C. Western analysis of whole tumor extracts harvested on day 5, 24 h after the final si-NP treatment. Each lane represents a tumor collected from different mice. Antibodies are shown at left of each panel. D. Rictor and P-Akt S473 were detected by IHC in tumors harvested on treatment day 5. Upper panels show representative images taken at 200 X. Lower panels show quantitative analysis of staining using Scion Image software, expressed as pixels/field. Each point represents the average of 5 randomly chosen fields per sample. Midlines are the average (± S.E.). N = 3 (Rictor IHC) and 5 (P-Akt S473 IHC). E. Tumor volumes were measured on treatments days 0, 2, and 4. N = 5. F. TUNEL analysis. Left panels show representative images. Right panel shows quantitation of TUNEL staining, expressed as the percentage of nuclei that are TUNEL+. N = 6, each assessed in 5 randomly chosen 400X fields.

    Article Snippet: IHC on paraffin-embedded sections was performed as described previously ( ) using: Rictor (Santa Cruz Biotechnologies), Ki67 (Santa Cruz Biotechnologies) and P-Akt S473 (Cell Signaling Technologies) antibodies.

    Techniques: Inhibition, In Vivo, Multiple Displacement Amplification, Luciferase, Injection, Imaging, Software, Activity Assay, Western Blot, Mouse Assay, Immunohistochemistry, Staining, TUNEL Assay, Quantitation Assay

    Increased serum phosphorylates retinoblastoma, Akt Ser473 , and AKT Thr308 and decreases p27 KIP1 in confluent PASMC, but not in confluent PAEC. A and B) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and 24 hours later, cell lysates were harvested for protein analysis. Representative Western blots are shown. C) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were infected for 2 hours at a multiple of infectivity of 200 with a replication-deficient adenovirus serotype 5 containing either a human p27 KIP1 ( Ad p27) or alkaline phosphatase ( Ad C) cDNA driven by a CMV promoter. Cells were then exposed to 5% serum and cell lysates harvested 24 hours later. A representative Western blot is shown. [pRB: hypophosphorylated retinoblastoma; ppRB: hyperphosphorylated retinoblastoma]; D) BrdU incorporation 24 hours after exposure to 5% serum in p27 KIP1 -infected human PAEC and PASMC.

    Journal: PLoS ONE

    Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

    doi: 10.1371/journal.pone.0071490

    Figure Lengend Snippet: Increased serum phosphorylates retinoblastoma, Akt Ser473 , and AKT Thr308 and decreases p27 KIP1 in confluent PASMC, but not in confluent PAEC. A and B) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and 24 hours later, cell lysates were harvested for protein analysis. Representative Western blots are shown. C) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were infected for 2 hours at a multiple of infectivity of 200 with a replication-deficient adenovirus serotype 5 containing either a human p27 KIP1 ( Ad p27) or alkaline phosphatase ( Ad C) cDNA driven by a CMV promoter. Cells were then exposed to 5% serum and cell lysates harvested 24 hours later. A representative Western blot is shown. [pRB: hypophosphorylated retinoblastoma; ppRB: hyperphosphorylated retinoblastoma]; D) BrdU incorporation 24 hours after exposure to 5% serum in p27 KIP1 -infected human PAEC and PASMC.

    Article Snippet: AKT (9272), phospho-AKT (Ser473) (4058), and phospho-AKT (Thr308) (4056) all were from Cell Signaling.

    Techniques: Western Blot, Infection, BrdU Incorporation Assay

    Insulin-like growth factor 2 mRNA-binding protein 1 enhances pancreatic cancer cell proliferation via the AKT signaling pathway. A and B: Western blot analysis of AKT and p-AKT (Ser473) expression in the indicated cells and their corresponding control cells; C: Panc-1 sh-insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) or control cells were subcutaneously injected into BALB/c nude mice for xenograft assays; D-E: The average weights and tumor growth curves of each group; F: Immunohistochemistry staining of the xenograft tumors. Scale bar = 25 μm (red line). P

    Journal: World Journal of Gastroenterology

    Article Title: Insulin-like growth factor 2 mRNA-binding protein 1 promotes cell proliferation via activation of AKT and is directly targeted by microRNA-494 in pancreatic cancer

    doi: 10.3748/wjg.v25.i40.6063

    Figure Lengend Snippet: Insulin-like growth factor 2 mRNA-binding protein 1 enhances pancreatic cancer cell proliferation via the AKT signaling pathway. A and B: Western blot analysis of AKT and p-AKT (Ser473) expression in the indicated cells and their corresponding control cells; C: Panc-1 sh-insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) or control cells were subcutaneously injected into BALB/c nude mice for xenograft assays; D-E: The average weights and tumor growth curves of each group; F: Immunohistochemistry staining of the xenograft tumors. Scale bar = 25 μm (red line). P

    Article Snippet: The primary antibodies included IGF2BP1 (ab124930, Abcam), pan-AKT (C67E7, CST), p-AKT (D9E, CST), and GAPDH (Boster, Wuhan, China).

    Techniques: Binding Assay, Western Blot, Expressing, Injection, Mouse Assay, Immunohistochemistry, Staining

    MiR-494 reexpression partly abrogates the oncogenic effect of insulin-like growth factor 2 mRNA-binding protein 1 in pancreatic cancer. A: Enhanced cell viability of Mia PaCa-2 cells due to insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) overexpression was partly reduced following the reexpression of miR-494, which was detected by CCK-8 assays; B: Cell apoptosis was analyzed by flow cytometry when IGF2BP1-overexpressing Mia PaCa-2 cells were cotransfected with miR-494 mimics or their corresponding controls; C: The cell cycle was analyzed by flow cytometry when IGF2BP1-overexpressing Mia PaCa-2 cells were co-transfected with miR-494 mimics or control; D: Western blot analysis of AKT and p-AKT (Ser473) expression in the indicated cells and their corresponding control group; E: Correlations between the expression of miR-494 and IGF2BP1 in 30 pancreatic cancer specimens were determined by Pearson's correlation analysis; F: Kaplan-Meier analysis indicated that low miR-494 expression predicts a poorer overall survival rate than high miR-494 expression. All data are expressed as the mean ± standard deviation from three independent experiments at least and P

    Journal: World Journal of Gastroenterology

    Article Title: Insulin-like growth factor 2 mRNA-binding protein 1 promotes cell proliferation via activation of AKT and is directly targeted by microRNA-494 in pancreatic cancer

    doi: 10.3748/wjg.v25.i40.6063

    Figure Lengend Snippet: MiR-494 reexpression partly abrogates the oncogenic effect of insulin-like growth factor 2 mRNA-binding protein 1 in pancreatic cancer. A: Enhanced cell viability of Mia PaCa-2 cells due to insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) overexpression was partly reduced following the reexpression of miR-494, which was detected by CCK-8 assays; B: Cell apoptosis was analyzed by flow cytometry when IGF2BP1-overexpressing Mia PaCa-2 cells were cotransfected with miR-494 mimics or their corresponding controls; C: The cell cycle was analyzed by flow cytometry when IGF2BP1-overexpressing Mia PaCa-2 cells were co-transfected with miR-494 mimics or control; D: Western blot analysis of AKT and p-AKT (Ser473) expression in the indicated cells and their corresponding control group; E: Correlations between the expression of miR-494 and IGF2BP1 in 30 pancreatic cancer specimens were determined by Pearson's correlation analysis; F: Kaplan-Meier analysis indicated that low miR-494 expression predicts a poorer overall survival rate than high miR-494 expression. All data are expressed as the mean ± standard deviation from three independent experiments at least and P

    Article Snippet: The primary antibodies included IGF2BP1 (ab124930, Abcam), pan-AKT (C67E7, CST), p-AKT (D9E, CST), and GAPDH (Boster, Wuhan, China).

    Techniques: Binding Assay, Over Expression, CCK-8 Assay, Flow Cytometry, Cytometry, Transfection, Western Blot, Expressing, Standard Deviation

    DCAC up-regulated the AKT/mTOR and MAPK signaling in KCs. KCs were pre-treated with DCAC at concentrations of 0, 1, 2.5 or 5 mM for 24 h. (A) Representative images of the cellular levels of phos-AKT S473, phos-AKT T308, total AKT, phos-ERK and ERK, as determined by Western blotting, (B) Relative phos-ERK level, as determined by Western blotting, (C) Relative protein levels of phos-AKT(Ser-473) and phos-AKT(Thr-308), as determined by Western blotting, and (D) Representative images and relative phos-mTOR, as determined by Western blotting. n = 3 in each group. *p

    Journal: PLoS ONE

    Article Title: Trichloroethene metabolite dichloroacetyl chloride induces apoptosis and compromises phagocytosis in Kupffer Cells: Activation of inflammasome and MAPKs

    doi: 10.1371/journal.pone.0210200

    Figure Lengend Snippet: DCAC up-regulated the AKT/mTOR and MAPK signaling in KCs. KCs were pre-treated with DCAC at concentrations of 0, 1, 2.5 or 5 mM for 24 h. (A) Representative images of the cellular levels of phos-AKT S473, phos-AKT T308, total AKT, phos-ERK and ERK, as determined by Western blotting, (B) Relative phos-ERK level, as determined by Western blotting, (C) Relative protein levels of phos-AKT(Ser-473) and phos-AKT(Thr-308), as determined by Western blotting, and (D) Representative images and relative phos-mTOR, as determined by Western blotting. n = 3 in each group. *p

    Article Snippet: Primary antibodies against AKT, phosphor-AKT (S473), phosphor-AKT (T308), phosphor-ERK, phosphor-mTOR (Ser2448), ERK and NLRP3 were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Western Blot

    The plausible mechanism of DCAC-induced autoimmune response. DCAC can induce inflammasome NLRP3/caspase1 and ROS-related ERK/AKT/mTOR activation in Kupffer cells, leading to increased apoptosis and impaired phagocytosis. Ultimately, delayed clearance of apoptotic bodies due to compromised Kupffer cell function will result in the breakdown of self-tolerance, autoimmunity, and eventually AIH. “------”, established with parent compound TCE.

    Journal: PLoS ONE

    Article Title: Trichloroethene metabolite dichloroacetyl chloride induces apoptosis and compromises phagocytosis in Kupffer Cells: Activation of inflammasome and MAPKs

    doi: 10.1371/journal.pone.0210200

    Figure Lengend Snippet: The plausible mechanism of DCAC-induced autoimmune response. DCAC can induce inflammasome NLRP3/caspase1 and ROS-related ERK/AKT/mTOR activation in Kupffer cells, leading to increased apoptosis and impaired phagocytosis. Ultimately, delayed clearance of apoptotic bodies due to compromised Kupffer cell function will result in the breakdown of self-tolerance, autoimmunity, and eventually AIH. “------”, established with parent compound TCE.

    Article Snippet: Primary antibodies against AKT, phosphor-AKT (S473), phosphor-AKT (T308), phosphor-ERK, phosphor-mTOR (Ser2448), ERK and NLRP3 were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Activation Assay, Cell Function Assay

    Draxin treatment of cortical neurons inhibits Akt activity and activates GSK-3β. Immunoblot analyses of cortical neurons from newborn wild-type ( A ) or MAP1B-/- ( B ) mice cultured for 60 h, treated with draxin for the indicated times, lysed and probed using the indicated antibodies. The GSK-3β doublets represent the GSK-3β1 and GSK-3β2 isoforms [ 33 ].The relative levels of GSK-3β phosphorylated at Ser9 (P-GSK-3β) and Akt phosphorylated on Ser473 (P-Akt) were determined by normalizing the signals for the phosphorylated proteins to the corresponding signals for the total proteins in 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Repulsive Axon Guidance by Draxin Is Mediated by Protein Kinase B (Akt), Glycogen Synthase Kinase-3β (GSK-3β) and Microtubule-Associated Protein 1B

    doi: 10.1371/journal.pone.0119524

    Figure Lengend Snippet: Draxin treatment of cortical neurons inhibits Akt activity and activates GSK-3β. Immunoblot analyses of cortical neurons from newborn wild-type ( A ) or MAP1B-/- ( B ) mice cultured for 60 h, treated with draxin for the indicated times, lysed and probed using the indicated antibodies. The GSK-3β doublets represent the GSK-3β1 and GSK-3β2 isoforms [ 33 ].The relative levels of GSK-3β phosphorylated at Ser9 (P-GSK-3β) and Akt phosphorylated on Ser473 (P-Akt) were determined by normalizing the signals for the phosphorylated proteins to the corresponding signals for the total proteins in 3 independent experiments.

    Article Snippet: Primary antibodies: mouse monoclonal antibodies against phospho-MAP1B (SMI31; 1:1000; Covance) and neurofilament H (1:1000; Cell Signaling); rabbit polyclonal antibodies against total-MAP1B raised against peptides ATVVVEATEPEPSGC and ETVTEEHLRRAIGN [ ], phospho Ser473 Akt (1:1000; Cell Signaling), total Akt (1:1000; Cell Signaling) and GAPDH (1:3000; Sigma-Aldrich); rabbit monoclonal antibodies against phospho Ser9-GSK-3β (1:1000; Cell Signaling) and total GSK-3β (1:1000; Cell Signaling).

    Techniques: Activity Assay, Mouse Assay, Cell Culture

    AKT activation by metastatic site. A , Average expression levels of P-AKT-Ser473 (blue), P-AKT-Thr308 (red), P-GSK3α/β (yellow), and PTEN (green) in melanoma metastases resected from the brain, liver, and lung, as determined by RPPA. *,

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

    Article Title: Integrated Molecular and Clinical Analysis of AKT Activation in Metastatic Melanoma

    doi: 10.1158/1078-0432.CCR-09-1985

    Figure Lengend Snippet: AKT activation by metastatic site. A , Average expression levels of P-AKT-Ser473 (blue), P-AKT-Thr308 (red), P-GSK3α/β (yellow), and PTEN (green) in melanoma metastases resected from the brain, liver, and lung, as determined by RPPA. *,

    Article Snippet: Levels of PTEN, a negative regulator of AKT activation, negatively correlated with levels of P-AKT-Ser473 ( r =−.574; P < .001) and P-AKT-Thr308 ( r =−.634; P < .001).

    Techniques: Activation Assay, Expressing

    AKT activation and clinical outcomes. A , Unsupervised hierarchical clustering of 56 regional(lymph node, soft tissue) metastases based on the expression levels of P-AKT-Ser473, P-AKT-Thr308, and PTEN. For comparative analyses, tumors in the upper cluster

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

    Article Title: Integrated Molecular and Clinical Analysis of AKT Activation in Metastatic Melanoma

    doi: 10.1158/1078-0432.CCR-09-1985

    Figure Lengend Snippet: AKT activation and clinical outcomes. A , Unsupervised hierarchical clustering of 56 regional(lymph node, soft tissue) metastases based on the expression levels of P-AKT-Ser473, P-AKT-Thr308, and PTEN. For comparative analyses, tumors in the upper cluster

    Article Snippet: Levels of PTEN, a negative regulator of AKT activation, negatively correlated with levels of P-AKT-Ser473 ( r =−.574; P < .001) and P-AKT-Thr308 ( r =−.634; P < .001).

    Techniques: Activation Assay, Expressing

    RPPA analysis of AKT activation in melanoma clinical specimens and correlation with activating mutations. A , Average expression levels of P-AKT-Ser473 (blue), P-AKT-Thr308 (red), P-GSK3α/β (yellow), and PTEN (green) in BRAF-mutant or NRAS-mutant

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

    Article Title: Integrated Molecular and Clinical Analysis of AKT Activation in Metastatic Melanoma

    doi: 10.1158/1078-0432.CCR-09-1985

    Figure Lengend Snippet: RPPA analysis of AKT activation in melanoma clinical specimens and correlation with activating mutations. A , Average expression levels of P-AKT-Ser473 (blue), P-AKT-Thr308 (red), P-GSK3α/β (yellow), and PTEN (green) in BRAF-mutant or NRAS-mutant

    Article Snippet: Levels of PTEN, a negative regulator of AKT activation, negatively correlated with levels of P-AKT-Ser473 ( r =−.574; P < .001) and P-AKT-Thr308 ( r =−.634; P < .001).

    Techniques: Activation Assay, Expressing, Mutagenesis

    The involvement of the FASN/ERα/Akt pathway in hypoxia regulation of IGFBP-2

    Journal: Oncotarget

    Article Title: Hypoxia negates hyperglycaemia-induced chemo-resistance in breast cancer cells: the role of insulin-like growth factor binding protein 2

    doi: 10.18632/oncotarget.20287

    Figure Lengend Snippet: The involvement of the FASN/ERα/Akt pathway in hypoxia regulation of IGFBP-2

    Article Snippet: The membranes were probed with anti-FASN (mouse, 1:2000) and anti-HIF-Iα (mouse, 1:500), purchased from BD Biosciences Oxford, UK, anti-ERα (mouse, 1:750) and anti-IGFBP-2 (goat, 1:1000) from Santa Cruz Heidelberg, Germany, anti-Akt (rabbit, 1:1000) and anti-p-Akt (rabbit, 1:1000) purchased from Cell Signaling Hertfordshire, UK, and anti-α-tubulin (mouse, 1:5000) purchased from Merck Millipore Hertfordshire, UK.

    Techniques:

    HCA2 receptor agonists increase AKT, AMPKα and ERK1/2 phosphorylation in bovine neutrophils. Neutrophils were treated for 3 min with ( A ) BHB, ( B ) MK-1903, ( C ) nicotinic acid and ( D ) nicotinamide. Total protein was analyzed by SDS/PAGE and immunoblotting using antibodies against the phosphorylated forms of AKT, AMPKα and ERK1/2. Unphosphorylated AKT, ERK 1/2 and β-actin were also analyzed, and the ratio was calculated by densitometric analysis. Each dot represents an independent experiment and the line shows the mean. N = 3 different heifers. * P

    Journal: Scientific Reports

    Article Title: β-hydroxybutyrate and hydroxycarboxylic acid receptor 2 agonists activate the AKT, ERK and AMPK pathways, which are involved in bovine neutrophil chemotaxis

    doi: 10.1038/s41598-020-69500-2

    Figure Lengend Snippet: HCA2 receptor agonists increase AKT, AMPKα and ERK1/2 phosphorylation in bovine neutrophils. Neutrophils were treated for 3 min with ( A ) BHB, ( B ) MK-1903, ( C ) nicotinic acid and ( D ) nicotinamide. Total protein was analyzed by SDS/PAGE and immunoblotting using antibodies against the phosphorylated forms of AKT, AMPKα and ERK1/2. Unphosphorylated AKT, ERK 1/2 and β-actin were also analyzed, and the ratio was calculated by densitometric analysis. Each dot represents an independent experiment and the line shows the mean. N = 3 different heifers. * P

    Article Snippet: The membrane was then incubated with a polyclonal ERK 1 or AKT antibody (Cell Signaling, USA) at a dilution of 1:2000, and β-actin HRP (Santa Cruz Biotechnology, USA) was also used.

    Techniques: SDS Page

    Effect of co-stimulation with ATP and prostaglandin (PG)E 2 on Syk, extracellular signal-regulated kinase (ERK)1/2, and Akt phosphorylation in bone marrow-derived mast cells (BMMCs). ( A ) BMMCs were stimulated with ATP (100 μM) with or without PGE 2 (1 μM, upper) or 2,4-dinitrophenyl human serum albumin (DNP-HAS,10 ng/mL, lower) for 1 min. Cell lysates were subjected to western blot analysis for phospho-Syk and total-Syk. ( B ) BMMCs were stimulated with ATP (100 μM) with or without PGE 2 (1 μM) for 1 (left) or 3 (right) min. Cell lysates were subjected to western blot analysis for phospho-Akt and total Akt (upper) or phospho-ERK 1/2 and total-ERK 1/2 (lower). The numbers below each image indicate normalized relative phosphorylated protein intensity; the results for no stimulation are set to one. Blots are representative of three independent experiments.

    Journal: International Journal of Molecular Sciences

    Article Title: Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells

    doi: 10.3390/ijms20205157

    Figure Lengend Snippet: Effect of co-stimulation with ATP and prostaglandin (PG)E 2 on Syk, extracellular signal-regulated kinase (ERK)1/2, and Akt phosphorylation in bone marrow-derived mast cells (BMMCs). ( A ) BMMCs were stimulated with ATP (100 μM) with or without PGE 2 (1 μM, upper) or 2,4-dinitrophenyl human serum albumin (DNP-HAS,10 ng/mL, lower) for 1 min. Cell lysates were subjected to western blot analysis for phospho-Syk and total-Syk. ( B ) BMMCs were stimulated with ATP (100 μM) with or without PGE 2 (1 μM) for 1 (left) or 3 (right) min. Cell lysates were subjected to western blot analysis for phospho-Akt and total Akt (upper) or phospho-ERK 1/2 and total-ERK 1/2 (lower). The numbers below each image indicate normalized relative phosphorylated protein intensity; the results for no stimulation are set to one. Blots are representative of three independent experiments.

    Article Snippet: Anti-phospho-Syk, anti-Syk, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-Akt, and anti-Akt antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Derivative Assay, Western Blot

    Effect of tyrosine kinase and phosphoinositide 3-kinase (PI3K)/Akt signaling pathway inhibitors on mast cell (MC) degranulation induced by co-stimulation with ATP and prostaglandin (PG)E 2 . ( A ) Bone marrow-derived MCs (BMMCs) were preincubated with a vehicle or the Src tyrosine kinase inhibitor PP2 (1 μM) for 5 min and then stimulated with ATP (100 μM) with or without PGE 2 (1 μM) or 2,4-dinitrophenyl human serum albumin (DNP-HSA, 10 ng/mL). ( B ) BMMCs were preincubated with a vehicle or the Syk inhibitor R406 (2 μM) for 5 min and then stimulated with ATP (100 μM) with or without PGE 2 (1 μM) ( n = 3). ( C ) BMMCs were preincubated with a vehicle, the PI3K inhibitor LY294002 (10 μM), or the control compound LY303511 (10 μM) for 5 min and then stimulated with ATP (0.1 mM) with or without PGE 2 (1 μM) ( n = 3). ( D ) BMMCs were preincubated with a vehicle or the PI3Kγ inhibitor AS605240 (1 μM) for 5 min and then stimulated with ATP (100 μM) with or without PGE 2 (1 μM) ( n = 3). ( E ) BMMCs were preincubated with a vehicle or the Akt inhibitor triciribin (10 μM) for 5 min, and then stimulated with ATP (100 μM) with or without PGE 2 (1 μM) ( n = 3). Data are shown as the mean ± SEM. N.S. no significant difference, ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells

    doi: 10.3390/ijms20205157

    Figure Lengend Snippet: Effect of tyrosine kinase and phosphoinositide 3-kinase (PI3K)/Akt signaling pathway inhibitors on mast cell (MC) degranulation induced by co-stimulation with ATP and prostaglandin (PG)E 2 . ( A ) Bone marrow-derived MCs (BMMCs) were preincubated with a vehicle or the Src tyrosine kinase inhibitor PP2 (1 μM) for 5 min and then stimulated with ATP (100 μM) with or without PGE 2 (1 μM) or 2,4-dinitrophenyl human serum albumin (DNP-HSA, 10 ng/mL). ( B ) BMMCs were preincubated with a vehicle or the Syk inhibitor R406 (2 μM) for 5 min and then stimulated with ATP (100 μM) with or without PGE 2 (1 μM) ( n = 3). ( C ) BMMCs were preincubated with a vehicle, the PI3K inhibitor LY294002 (10 μM), or the control compound LY303511 (10 μM) for 5 min and then stimulated with ATP (0.1 mM) with or without PGE 2 (1 μM) ( n = 3). ( D ) BMMCs were preincubated with a vehicle or the PI3Kγ inhibitor AS605240 (1 μM) for 5 min and then stimulated with ATP (100 μM) with or without PGE 2 (1 μM) ( n = 3). ( E ) BMMCs were preincubated with a vehicle or the Akt inhibitor triciribin (10 μM) for 5 min, and then stimulated with ATP (100 μM) with or without PGE 2 (1 μM) ( n = 3). Data are shown as the mean ± SEM. N.S. no significant difference, ** p

    Article Snippet: Anti-phospho-Syk, anti-Syk, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-Akt, and anti-Akt antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Derivative Assay

    Targeting IGF-1R affects cancer stem cell properties and Akt activation of breast cancer stem cells . (A) CD44 + cells of AS-B244 cells were treated with picropodophyllin (PPP) for 12 hours and cell migration within 18 hours was determined (upper panel). E-cadherin expression was determined by immunofluorescence stain (middle panel, 10× objective lens). Cell morphology was observed under microscope (bright field, 20× objective lens). (B) The expression of epithelial-mesenchymal transition-related molecules after PPP treatment was determined by western blot. (C) Cells were sorted as described in Figure 1A and the phosphorylation of Akt or mammalian target of rapamycin (mTOR) was determined by western blot. GAPDH and actin were used as internal control for BC0145 and for BC0244, respectively. (D) AS-B145 or AS-B244 cells were transfected with 100 nM negative control siRNA (ctrl) or insulin-like growth factor 1 receptor (IGF-1R) specific siRNA (KD) for 48 hours and the expression of IGF-1R, Akt or p-Akt was determined by western blot. (E) ALDH + AS-B145 or ALDH + AS-B244 cells were treated with PPP for 48 hours. pAkt ser473 was determined by western blot. All experiments were repeated at least twice and results shown were from a representative experiment. ALDH, aldehyde dehydrogenase; DMSO, dimethylsulfoxide.

    Journal: Breast Cancer Research : BCR

    Article Title: The expression and significance of insulin-like growth factor-1 receptor and its pathway on breast cancer stem/progenitors

    doi: 10.1186/bcr3423

    Figure Lengend Snippet: Targeting IGF-1R affects cancer stem cell properties and Akt activation of breast cancer stem cells . (A) CD44 + cells of AS-B244 cells were treated with picropodophyllin (PPP) for 12 hours and cell migration within 18 hours was determined (upper panel). E-cadherin expression was determined by immunofluorescence stain (middle panel, 10× objective lens). Cell morphology was observed under microscope (bright field, 20× objective lens). (B) The expression of epithelial-mesenchymal transition-related molecules after PPP treatment was determined by western blot. (C) Cells were sorted as described in Figure 1A and the phosphorylation of Akt or mammalian target of rapamycin (mTOR) was determined by western blot. GAPDH and actin were used as internal control for BC0145 and for BC0244, respectively. (D) AS-B145 or AS-B244 cells were transfected with 100 nM negative control siRNA (ctrl) or insulin-like growth factor 1 receptor (IGF-1R) specific siRNA (KD) for 48 hours and the expression of IGF-1R, Akt or p-Akt was determined by western blot. (E) ALDH + AS-B145 or ALDH + AS-B244 cells were treated with PPP for 48 hours. pAkt ser473 was determined by western blot. All experiments were repeated at least twice and results shown were from a representative experiment. ALDH, aldehyde dehydrogenase; DMSO, dimethylsulfoxide.

    Article Snippet: The membrane was then incubated with antibodies against Akt, phosphor-Akt (Ser473), mTOR, phosphor-mTOR (Ser2448), GAPDH (Cell Signaling Technology, Danvers, MA, USA), phospho-insulin receptor (Tyr972), insulin receptor (IR; GeneTex Inc., Irvine, CA, USA) phospho-IGF-1R (Tyr1165/1166; Santa Cruz Biotechnology), β-actin (Sigma-Aldrich, St. Louis, MO, USA), and the IGF-1R (R & D Systems, Minneapolis, MN, USA).

    Techniques: Activation Assay, Migration, Expressing, Immunofluorescence, Staining, Microscopy, Western Blot, Transfection, Negative Control

    Targeting the PI3K/Akt/mTOR pathway inhibits the cancer stem cell population of breast cancer cells . (A) AS-B145 cells or (B) AS-B244 cells were treated with small molecule inhibitors of phosphoinositide-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) (PI-103), Akt (CB-124005 or FPA-124) or mTOR (rapamycin) for 48 hours and the aldehyde dehydrogenase (ALDH) + cell population within 7-aminoactinomycin D (7-AAD)-negative cells was determined with Aldefluor assay and displayed as the relative percentage of dimethylsulfoxide (DMSO) control. * P

    Journal: Breast Cancer Research : BCR

    Article Title: The expression and significance of insulin-like growth factor-1 receptor and its pathway on breast cancer stem/progenitors

    doi: 10.1186/bcr3423

    Figure Lengend Snippet: Targeting the PI3K/Akt/mTOR pathway inhibits the cancer stem cell population of breast cancer cells . (A) AS-B145 cells or (B) AS-B244 cells were treated with small molecule inhibitors of phosphoinositide-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) (PI-103), Akt (CB-124005 or FPA-124) or mTOR (rapamycin) for 48 hours and the aldehyde dehydrogenase (ALDH) + cell population within 7-aminoactinomycin D (7-AAD)-negative cells was determined with Aldefluor assay and displayed as the relative percentage of dimethylsulfoxide (DMSO) control. * P

    Article Snippet: The membrane was then incubated with antibodies against Akt, phosphor-Akt (Ser473), mTOR, phosphor-mTOR (Ser2448), GAPDH (Cell Signaling Technology, Danvers, MA, USA), phospho-insulin receptor (Tyr972), insulin receptor (IR; GeneTex Inc., Irvine, CA, USA) phospho-IGF-1R (Tyr1165/1166; Santa Cruz Biotechnology), β-actin (Sigma-Aldrich, St. Louis, MO, USA), and the IGF-1R (R & D Systems, Minneapolis, MN, USA).

    Techniques:

    Effect on vascular phospho-eNOS (p-eNOS) (A), total eNOS (t-eNOS) (B), phospho-Akt (p-Akt) (C), and total Akt (t-Akt) (D) of salt-loaded SHRSP. Abbreviations used are the same as in Fig. 1. The upper panels in (A), (B), (C), and (D) indicate representative western blots in each group. Each value represents the mean ± SEM (n = 4 in each group).

    Journal: PLoS ONE

    Article Title: Calcium Channel Blockers, More than Diuretics, Enhance Vascular Protective Effects of Angiotensin Receptor Blockers in Salt-Loaded Hypertensive Rats

    doi: 10.1371/journal.pone.0039162

    Figure Lengend Snippet: Effect on vascular phospho-eNOS (p-eNOS) (A), total eNOS (t-eNOS) (B), phospho-Akt (p-Akt) (C), and total Akt (t-Akt) (D) of salt-loaded SHRSP. Abbreviations used are the same as in Fig. 1. The upper panels in (A), (B), (C), and (D) indicate representative western blots in each group. Each value represents the mean ± SEM (n = 4 in each group).

    Article Snippet: Antibodies used were as follows: anti-phospho eNOS (Ser1177) (×2000, BD Transduction Laboratories, Tokyo, Japan), anti-total eNOS (×5000, BD Transduction Laboratories, Tokyo, Japan), anti-total Akt (×2000, Cell signaling Technology Inc. Tokyo, Japan), anti-phospho Akt (×2000, Cell signaling Technology Inc. Tokyo, Japan), anti-α-tubulin (×5000, Oncogene, Tokyo, Japan), anti-phospho-ERK (×2500, Cell signaling Technology Inc. Tokyo, Japan).

    Techniques: Western Blot

    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

    Total and phosphorylated Akt in WT and Akt3 −/− platelets. (A,D) Washed Akt3 −/− and WT mouse platelets were stimulated with thrombin (0.018 U/mL) for 1, 3, and 5 minutes, solubilized, and immunoblotted with antibodies directed against: (A) Akt3, total Akt (Akt1, Akt2, and Akt3), phosphorylated Ser473 of Akt, and GSK-3β (loading control), and (D) phosphorylated Thr 308 of Akt, total Akt and α-tubulin (loading control). (B,C) Western blot results from each of 3 experiments as shown in panel A were scanned and quantitated using NIH ImageJ for uncalibrated optical density. The relative quantity of total Akt (B) and phosphorylated Ser473 of Akt (C) in wild-type and Akt3 −/− platelets are shown (mean ± SE). (E) Washed Akt3 −/− and WT mouse platelets were solubilized and immunoblotted with antibodies directed against Akt1, Akt2, and Akt3 and α-tubulin (loading control).

    Journal: Blood

    Article Title: An important role for Akt3 in platelet activation and thrombosis

    doi: 10.1182/blood-2010-12-323204

    Figure Lengend Snippet: Total and phosphorylated Akt in WT and Akt3 −/− platelets. (A,D) Washed Akt3 −/− and WT mouse platelets were stimulated with thrombin (0.018 U/mL) for 1, 3, and 5 minutes, solubilized, and immunoblotted with antibodies directed against: (A) Akt3, total Akt (Akt1, Akt2, and Akt3), phosphorylated Ser473 of Akt, and GSK-3β (loading control), and (D) phosphorylated Thr 308 of Akt, total Akt and α-tubulin (loading control). (B,C) Western blot results from each of 3 experiments as shown in panel A were scanned and quantitated using NIH ImageJ for uncalibrated optical density. The relative quantity of total Akt (B) and phosphorylated Ser473 of Akt (C) in wild-type and Akt3 −/− platelets are shown (mean ± SE). (E) Washed Akt3 −/− and WT mouse platelets were solubilized and immunoblotted with antibodies directed against Akt1, Akt2, and Akt3 and α-tubulin (loading control).

    Article Snippet: The membranes were immunoblotted with an anti-Akt3 rabbit monoclonal antibody, anti–GSK-3β, anti–phosphoGSK-3β, anti-phosphoAkt Thr308 , anti-phospho Akt Ser473, anti-Akt1, anti-Akt2 (Cell Signaling Technology), and total Akt (recognizing Akt1, Akt2 and Akt3; Santa Cruz Biotechnology Inc).

    Techniques: Western Blot

    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

    Loss of FlnB induces Cdk1 activity changes through β1 integrin-Pi3k/Akt pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p

    Journal: PLoS ONE

    Article Title: Filamin B Regulates Chondrocyte Proliferation and Differentiation through Cdk1 Signaling

    doi: 10.1371/journal.pone.0089352

    Figure Lengend Snippet: Loss of FlnB induces Cdk1 activity changes through β1 integrin-Pi3k/Akt pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p

    Article Snippet: The primary antibodies (for immunostaining and some also for western blotting) were: rabbit anti-FlnA monoclonal antibody (1∶300, Cat.# 2242, Epitomics, Burlingame, CA, USA); rabbit anti-FlnB polyclonal antibody (Gifted by Dr. Kao, CWRU); mouse anti-Col2a1 (Cat.# Ab3092, ABCAM, USA); rabbit anti-Col10a1 (kindly gifted by Dr. Horton and Dr. Lunstrum, Shriners Hospital for Children, Portland, OR, USA; ); rabbit anti-Pthr1 (Cat.# Ab75150, ABCAM, USA);rabbit anti-Ihh (Cat.# sc-13088, Santa Cruz); rabbit anti-Runx2 (Cat.# sc-10758, Santa Cruz); rabbit anti-Sox9 pab (1∶300, AB5535, Millipore); rabbit anti-Sox9 pab (O9-1, gift of Professor Dr. Michael Wegner, Institute of Biochemistry, Friedrich-Alexander-University, Erlangen-Nurnberg, Germany); rat anti-BrdU (1∶150, Cat.# MCA2060, AbD Serotec, Raleigh, NC, USA); rabbit anti-Ki-67 mab (1∶200, Cat.# 4203, Epitomics); rabbit anti-PH3 pab (1∶250, Cat:# 06-570, Millipore, Billerica, MA, USA); mouse and rabbit anti-Wee1 (1∶50, Cat.# sc-5285 and sc-325, Santa Cruz); rabbit anti-Pkmyt1 (1∶100, Cat.# 3303, Epitomics); anti-pan 14-3-3 (Santa Cruz, sc-629); mouse and rabbit anti-cyclin B1 (1∶100, Cat.# sc-245 and sc-752, Santa Cruz); mouse anti-Cdc20 (1∶100, Cat.# sc-13162, Santa Cruz); mouse and rabbit anti-cdc25c (1∶100, Cat.# sc-55513 and sc-327, Santa Cruz); rabbit-anti-Cdk1 (Cat.# PC25,Calbiochem, San Diego, CA, USA); mouse anti-Cdk1(pY15) (Cat.# BD612306, BD, Franklin Lakes, NJ USA); rabbit anti-Pi3k (p85 subunit alpha, Cat#: 1675, Epitomics); rabbit anti-Akt (phospho-S473, Cat# 4060, Cell Signaling); rabbit anti-Erk1/2 (phospho-T202/Y204, Cat.# 4370, Cell Signaling); rat anti-β1 integrin (Cat.# mab1997, Millipore); rabbit anti- β1 integrin(pS785) (Cat.# OPA1-03177, Affinity BioReagents).

    Techniques: Activity Assay, Immunostaining, Knock-Out, Western Blot, Activation Assay, Incubation, Laser Capture Microdissection

    Arsenic growth inhibition correlates with decreased temsirolimus-induced phospho-AKT. MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10 -6 N NaOH and 2.5 x 10 -5 % EtOH in PBS), 1-2 µM ATO, 0.5-5 ng/ml temsirolimus and the combinations for 24 hours. Whole cell extracts were used in immunoblotting experiments for phospho-S473-AKT, phospho-T308-AKT, AKT, and β-actin. Immunoblots shown are representative of experiments performed at least 3 times.

    Journal: PLoS ONE

    Article Title: Arsenic Trioxide Overcomes Rapamycin-Induced Feedback Activation of AKT and ERK Signaling to Enhance the Anti-Tumor Effects in Breast Cancer

    doi: 10.1371/journal.pone.0085995

    Figure Lengend Snippet: Arsenic growth inhibition correlates with decreased temsirolimus-induced phospho-AKT. MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10 -6 N NaOH and 2.5 x 10 -5 % EtOH in PBS), 1-2 µM ATO, 0.5-5 ng/ml temsirolimus and the combinations for 24 hours. Whole cell extracts were used in immunoblotting experiments for phospho-S473-AKT, phospho-T308-AKT, AKT, and β-actin. Immunoblots shown are representative of experiments performed at least 3 times.

    Article Snippet: Membranes were incubated overnight with primary antibodies: phospho-AKT (S473) (1:500, Cell Signaling), phospho-AKT (T308) (1:750, Cell Signaling), AKT (1:750 Cell Signaling), phospho-p44/42 MAPK (T202/Y204) (1:1000, Cell Signaling), ERK2 (1:2000, SantaCruz), phospho-S6 (1:1500, Cell Signaling), S6 (1:1000, Cell Signaling), and β–actin (1:5000, Sigma).

    Techniques: Inhibition, Multiple Displacement Amplification, Western Blot

    Addition of ATO decreases rapamycin-induced AKT activation in vivo . Tumor samples were stained with antibody against phospho-S473-AKT. Representative pictures of each treatment are shown (A). Quantification of staining intensity was performed using algorithms provided with the ImageScope software (B). Individual animals are represented with mean and standard error bars (n=4-5 mice). (C-D) Tumors at the end of the experiment were analyzed in immunoblotting experiments with antibodies against phospho-S473-AKT (C), phospho-T308-AKT (D), AKT, and β-actin. Band densitometry was performed and the relative intensity of phospho-AKT/AKT/β-actin was calculated. Individual animals are represented with mean and standard error bars (n= 8-9 mice).

    Journal: PLoS ONE

    Article Title: Arsenic Trioxide Overcomes Rapamycin-Induced Feedback Activation of AKT and ERK Signaling to Enhance the Anti-Tumor Effects in Breast Cancer

    doi: 10.1371/journal.pone.0085995

    Figure Lengend Snippet: Addition of ATO decreases rapamycin-induced AKT activation in vivo . Tumor samples were stained with antibody against phospho-S473-AKT. Representative pictures of each treatment are shown (A). Quantification of staining intensity was performed using algorithms provided with the ImageScope software (B). Individual animals are represented with mean and standard error bars (n=4-5 mice). (C-D) Tumors at the end of the experiment were analyzed in immunoblotting experiments with antibodies against phospho-S473-AKT (C), phospho-T308-AKT (D), AKT, and β-actin. Band densitometry was performed and the relative intensity of phospho-AKT/AKT/β-actin was calculated. Individual animals are represented with mean and standard error bars (n= 8-9 mice).

    Article Snippet: Membranes were incubated overnight with primary antibodies: phospho-AKT (S473) (1:500, Cell Signaling), phospho-AKT (T308) (1:750, Cell Signaling), AKT (1:750 Cell Signaling), phospho-p44/42 MAPK (T202/Y204) (1:1000, Cell Signaling), ERK2 (1:2000, SantaCruz), phospho-S6 (1:1500, Cell Signaling), S6 (1:1000, Cell Signaling), and β–actin (1:5000, Sigma).

    Techniques: Activation Assay, In Vivo, Staining, Software, Mouse Assay

    Immunofluorescent detection of Ki-67 ( panels a, b ), phospho-PDGFR-β ( panels c, d ), and phospho-Akt ( e,f ) in control ( a,c,e ) and injured carotid arteries ( b,d,f ) of smLrp1 −/− mice 14 days after endothelial denudation. The elastic

    Journal:

    Article Title: Smooth Muscle LDL Receptor-related Protein-1 Inactivation Reduces Vascular Reactivity and Promotes Injury-induced Neointima Formation

    doi: 10.1161/ATVBAHA.109.194357

    Figure Lengend Snippet: Immunofluorescent detection of Ki-67 ( panels a, b ), phospho-PDGFR-β ( panels c, d ), and phospho-Akt ( e,f ) in control ( a,c,e ) and injured carotid arteries ( b,d,f ) of smLrp1 −/− mice 14 days after endothelial denudation. The elastic

    Article Snippet: Cell proliferation and PDGFR signaling were assessed by incubating deparaffinized tissue sections overnight at 4°C with rabbit anti-Ki67 (Vector Laboratories), goat anti-phospho-PDGFR (Santa Cruz Biotechnology), or rabbit anti-phospho-Akt (Cell Signaling) at a 1:1000 dilution in Antibody Diluent (Zymed).

    Techniques: Mouse Assay