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  • 99
    Millipore mitogen activated protein kinase extracellular signal regulated kinase kinase mek inhibitor pd98059
    IGF-1 activates the <t>PI3K/Akt</t> and <t>MEK/ERK</t> signaling pathways. A, IGF-1 increases the phosphorylation of Akt at 30 min and (B) ERK at 10 min. IGF-1 treatment preferentially induced phosphorylation of ERK2 (p42), while phospho-ERK1 (p44) signal was barely
    Mitogen Activated Protein Kinase Extracellular Signal Regulated Kinase Kinase Mek Inhibitor Pd98059, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Pfizer Inc mitogen activated protein kinase extracellular signal regulated kinase kinase 1
    EREG expression is EGFR dependent. A, EREG mRNA was detected by semiquantitative PCR analysis of RNA prepared from a panel of NSCLC cell lines in which EGFR is mutant (HCC827, H3255, and HCC2279) or wild-type (H1299, H460, A549, H322, and H596). Actin was used as the control for RNA integrity. B, kinase-specific regulation of EREG expression. HCC827 cells were treated with inhibitors of EGFR (gefitinib), mitogen-activated protein kinase/extracellular signal-regulated kinase <t>kinase</t> 1 (CI-1040), p38 (SB202190), mammalian target of rapamycin (CCI-779), c-jun NH 2 -terminal kinase (SP600125), or AKT (A838450.3). Reverse transcription-PCR analysis of EREG and actin ( top ). EREG bands were quantified by densitometric analysis, normalized for loading differences based on actin, and expressed relative to that of untreated cells, which was set at 1.0. To evaluate dose-dependent target inhibition by the inhibitors, p38 kinase assays ( KA ) and Western blot analyses were done ( bottom ) of phosphorylated Y1068-EGFR ( p-EGFR ), T202/204-extracellular signal–regulated kinase ( p-ERK ), S235/236-S6 ( p-S6 ), S21/9-glycogen synthase kinase 3 ( p-GSK ), S63-c-Jun ( p-cJun ), and corresponding total proteins.
    Mitogen Activated Protein Kinase Extracellular Signal Regulated Kinase Kinase 1, supplied by Pfizer Inc, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Cell Signaling Technology Inc mitogen activated protein kinase extracellular signal regulated kinase kinase 1 2
    EREG expression is EGFR dependent. A, EREG mRNA was detected by semiquantitative PCR analysis of RNA prepared from a panel of NSCLC cell lines in which EGFR is mutant (HCC827, H3255, and HCC2279) or wild-type (H1299, H460, A549, H322, and H596). Actin was used as the control for RNA integrity. B, kinase-specific regulation of EREG expression. HCC827 cells were treated with inhibitors of EGFR (gefitinib), mitogen-activated protein kinase/extracellular signal-regulated kinase <t>kinase</t> 1 (CI-1040), p38 (SB202190), mammalian target of rapamycin (CCI-779), c-jun NH 2 -terminal kinase (SP600125), or AKT (A838450.3). Reverse transcription-PCR analysis of EREG and actin ( top ). EREG bands were quantified by densitometric analysis, normalized for loading differences based on actin, and expressed relative to that of untreated cells, which was set at 1.0. To evaluate dose-dependent target inhibition by the inhibitors, p38 kinase assays ( KA ) and Western blot analyses were done ( bottom ) of phosphorylated Y1068-EGFR ( p-EGFR ), T202/204-extracellular signal–regulated kinase ( p-ERK ), S235/236-S6 ( p-S6 ), S21/9-glycogen synthase kinase 3 ( p-GSK ), S63-c-Jun ( p-cJun ), and corresponding total proteins.
    Mitogen Activated Protein Kinase Extracellular Signal Regulated Kinase Kinase 1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore mek
    Sustained infusion of an <t>MEK</t> inhibitor reduces CFA-induced inflammatory pain. The MEK inhibitor <t>U0126</t> delivered by osmotic pump (0.5 μg · μl −1 · hr −1 ) before CFA injection reduces thermal hyperalgesia ( a ) and mechanical allodynia ( b ) 24 and 48 hr after CFA injection. These were measured by paw-withdrawal latency and paw-withdrawal threshold, respectively, and expressed as percentage of pre-CFA baseline measurements of vehicle control (50% DMSO). * p
    Mek, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    SignalChem mek 2
    Sustained infusion of an <t>MEK</t> inhibitor reduces CFA-induced inflammatory pain. The MEK inhibitor <t>U0126</t> delivered by osmotic pump (0.5 μg · μl −1 · hr −1 ) before CFA injection reduces thermal hyperalgesia ( a ) and mechanical allodynia ( b ) 24 and 48 hr after CFA injection. These were measured by paw-withdrawal latency and paw-withdrawal threshold, respectively, and expressed as percentage of pre-CFA baseline measurements of vehicle control (50% DMSO). * p
    Mek 2, supplied by SignalChem, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cayman Chemical mek inhibitor
    The effect of ERK1/2 signaling activity on proliferation of XY PGCs cultured with high FGF9. A ) Five thousand XY PGCs isolated from 12.5 dpc male gonads were incubated with high FGF9 for 2 hr to promote phosphorylation of ERK1/2. When the cells were incubated with both high FGF9 and <t>U0126</t> <t>MEK</t> inhibitor (10 μM) for 30 min or 1 hr, ERK1/2 activity induced by high FGF9 was completely suppressed. The levels of protein bands were quantified by densitometry and normalized to the β-ACTIN levels. The relative optical densities of phosphorylated protein levels were calculated based on the level in the high FGF9 treatment group set as 1.00. B) To determine the relationship between the ERK1/2 signaling pathway and PGC proliferation when stimulated by high FGF9 treatment, XY PGCs isolated at 12.5 dpc were cultured with high FGF9 and U0126 inhibitor (1, 10 μM) for 2 days and then subjected to the EdU cell proliferation assay. Cells treated with high FGF9 alone significantly increased cell proliferation ( P
    Mek Inhibitor, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 91/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific mek inhibitor
    The effect of ERK1/2 signaling activity on proliferation of XY PGCs cultured with high FGF9. A ) Five thousand XY PGCs isolated from 12.5 dpc male gonads were incubated with high FGF9 for 2 hr to promote phosphorylation of ERK1/2. When the cells were incubated with both high FGF9 and <t>U0126</t> <t>MEK</t> inhibitor (10 μM) for 30 min or 1 hr, ERK1/2 activity induced by high FGF9 was completely suppressed. The levels of protein bands were quantified by densitometry and normalized to the β-ACTIN levels. The relative optical densities of phosphorylated protein levels were calculated based on the level in the high FGF9 treatment group set as 1.00. B) To determine the relationship between the ERK1/2 signaling pathway and PGC proliferation when stimulated by high FGF9 treatment, XY PGCs isolated at 12.5 dpc were cultured with high FGF9 and U0126 inhibitor (1, 10 μM) for 2 days and then subjected to the EdU cell proliferation assay. Cells treated with high FGF9 alone significantly increased cell proliferation ( P
    Mek Inhibitor, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore mek inhibitor
    DAZAP1 regulates cell proliferation (a) Stable over-expression of DAZAP1 in non-small cell lung carcinoma NCI-H157 cells as detected by western blots. Control cells are stably transfected with vector only. (b) The expression of DAZAP1 inhibited cell proliferation as judged by colony formation assay with H157 cells. A representative picture from the triplicate experiments, the means and s.d. were plotted. (c) DAZAP1 inhibited the anchorage independent cell growth of H157 as judged by soft agar assays. A representative picture from the triplicates and the quantification were shown. The box plot was drawn using 25 and 75 quartiles in <t>Sigma</t> Plot with whiskers indicating max and min value and dots indicating outliers. P values were determined using paired t-test, *=p ≤ 5.0×10 −5 . (d) DAZAP1 inhibited cell migration as judged by wound healing assay. The picture of a representative plate (left panel) and the quantification of percent of gap closure were shown (right panel). Data from three independent assays were performed error bar indicate s.d. (e) The quantification of cell cycle stages (G1, S and G2/M phases) in DAZAP1 over-expressed or control cells. The cell cycle stages were analyzed by flow-cytometry measurement of DNA content. Data from two independent flow analysis, error bar indicate s.d. (f) Expression of endogenous DAZAP1 protein during cell cycle as detected by western blots. Tubulin and canonical cell cycle markers (cyclin A1 and B1) were also probed as controls. (g) An integrated model for DAZAP1 function and the regulation of its activity through the <t>MEK/Erk</t> pathway.
    Mek Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 411 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stemgent mek inhibitor
    Susceptibility to signaling inputs was highly dynamic around the exit from pluripotency. a – c Fractions of cells classified as XEN-like, ectoderm-like, double positive and double negative after 96 h, based on CD24 and PDGFRA expression. Expression of the two markers was measured by antibody staining and flow cytometry. a Cells were pulsed with 0.25 µM RA for x h (pulse) and subsequently differentiated in basal medium (N2B27) complemented with an RA receptor antagonist. b Cells were incubated with 0.25 µM RA for x h (pulse) after which 0.5 µM PD0325901 <t>(MEK</t> inhibitor) was added for the remainder of the time course. c Cells were first incubated with basal medium (N2B27) for x h (delay) and then exposed to 0.25 µM RA for the remainder of the time course. d Schematic representation of a minimal gene regulatory network that can model a lineage decision 32 . Pointy arrows indicate (auto-)activation; blunted arrows indicate repression. E and X represent expression of ectoderm-like and XEN-like transcriptional programs, respectively. P stands for the pluripotency network. RA increases the auto-activation of the XEN program. e Results of the stochastic simulations of the network shown in d . The relative frequency of XEN-like cells after 96 h is shown vs. the length of an RA pulse (solid lines) or the length of the delay before RA exposure is started (dashed lines). In all cases the pluripotency network was turned off after 12 h. Simulations were run with different amounts of gene expression noise (D: noise power / time, see Methods). See Supplementary Fig. 8b for exemplary trajectories
    Mek Inhibitor, supplied by Stemgent, used in various techniques. Bioz Stars score: 92/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc p mek
    S1P‐induced TIMP ‐3 expression is mediated through the <t>c‐Src/</t> MAPK signaling axis in human chondrosarcoma cells. (A) Starved JJ 012 cells were incubated with 10 μ m of S1P for 10, 15, 30, 60, and 120 min, and the phosphorylation of c‐Src, <t>MEK</t> , and ERK was examined by immunoblotting using anti‐phospho c‐Src, MEK , or ERK antibodies, followed by reprobing against total c‐Src, MEK , ERK , and β‐actin to show the equal loading amounts. (B) The JJ 012 cells were transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h, and the protein expressions of c‐Src, MEK , and ERK were then determined to show the knockdown efficiency. Starved JJ 012 (open bar) and SW 1353 (closed bar) cells were pretreated with 10 μ m of PP 2, PD 98059, and U0126 for 1 h or transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h, followed by treatment with 10 μ m of S1P for another 24 h, and expressions of miR‐101 (C), TIMP ‐3 (E), and MMP ‐2 (F) mRNA , as well as the protein expressions of TIMP ‐3 and MMP ‐2 (G), and migratory activity (H), were then determined by real‐time PCR , immunoblotting, and transwell analyses, respectively. (D) JJ 012 and SW 1353 cells were transfected with plasmids harboring with WT ‐ TIMP ‐3 3′ UTR , followed by treatment with 10 μ m of PP 2, PD 98059, and U0126 for 1 h or transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h prior to incubation with 10 μ m of S1P for another 24 h. The promoter activity of TIMP ‐3 was then determined by luciferase activity assay. (I) Starved JJ 012 cells were pretreated with 10 μ m of PP 2 and PD 98059 for 1 h, followed by treatment with 10 μ m of S1P for another 10 min, and the phosphorylation of Src, MEK , and ERK was then determined by immunoblotting analysis. Cells without treatment were used as the untreated control (set to 1 or 100), and data were shown as multiples of that. The results are expressed as mean ± SEM . * P
    P Mek, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 725 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Cell Signaling Technology Inc antiphospho mek
    S1P‐induced TIMP ‐3 expression is mediated through the <t>c‐Src/</t> MAPK signaling axis in human chondrosarcoma cells. (A) Starved JJ 012 cells were incubated with 10 μ m of S1P for 10, 15, 30, 60, and 120 min, and the phosphorylation of c‐Src, <t>MEK</t> , and ERK was examined by immunoblotting using anti‐phospho c‐Src, MEK , or ERK antibodies, followed by reprobing against total c‐Src, MEK , ERK , and β‐actin to show the equal loading amounts. (B) The JJ 012 cells were transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h, and the protein expressions of c‐Src, MEK , and ERK were then determined to show the knockdown efficiency. Starved JJ 012 (open bar) and SW 1353 (closed bar) cells were pretreated with 10 μ m of PP 2, PD 98059, and U0126 for 1 h or transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h, followed by treatment with 10 μ m of S1P for another 24 h, and expressions of miR‐101 (C), TIMP ‐3 (E), and MMP ‐2 (F) mRNA , as well as the protein expressions of TIMP ‐3 and MMP ‐2 (G), and migratory activity (H), were then determined by real‐time PCR , immunoblotting, and transwell analyses, respectively. (D) JJ 012 and SW 1353 cells were transfected with plasmids harboring with WT ‐ TIMP ‐3 3′ UTR , followed by treatment with 10 μ m of PP 2, PD 98059, and U0126 for 1 h or transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h prior to incubation with 10 μ m of S1P for another 24 h. The promoter activity of TIMP ‐3 was then determined by luciferase activity assay. (I) Starved JJ 012 cells were pretreated with 10 μ m of PP 2 and PD 98059 for 1 h, followed by treatment with 10 μ m of S1P for another 10 min, and the phosphorylation of Src, MEK , and ERK was then determined by immunoblotting analysis. Cells without treatment were used as the untreated control (set to 1 or 100), and data were shown as multiples of that. The results are expressed as mean ± SEM . * P
    Antiphospho Mek, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Genentech mek inhibitor
    S1P‐induced TIMP ‐3 expression is mediated through the <t>c‐Src/</t> MAPK signaling axis in human chondrosarcoma cells. (A) Starved JJ 012 cells were incubated with 10 μ m of S1P for 10, 15, 30, 60, and 120 min, and the phosphorylation of c‐Src, <t>MEK</t> , and ERK was examined by immunoblotting using anti‐phospho c‐Src, MEK , or ERK antibodies, followed by reprobing against total c‐Src, MEK , ERK , and β‐actin to show the equal loading amounts. (B) The JJ 012 cells were transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h, and the protein expressions of c‐Src, MEK , and ERK were then determined to show the knockdown efficiency. Starved JJ 012 (open bar) and SW 1353 (closed bar) cells were pretreated with 10 μ m of PP 2, PD 98059, and U0126 for 1 h or transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h, followed by treatment with 10 μ m of S1P for another 24 h, and expressions of miR‐101 (C), TIMP ‐3 (E), and MMP ‐2 (F) mRNA , as well as the protein expressions of TIMP ‐3 and MMP ‐2 (G), and migratory activity (H), were then determined by real‐time PCR , immunoblotting, and transwell analyses, respectively. (D) JJ 012 and SW 1353 cells were transfected with plasmids harboring with WT ‐ TIMP ‐3 3′ UTR , followed by treatment with 10 μ m of PP 2, PD 98059, and U0126 for 1 h or transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h prior to incubation with 10 μ m of S1P for another 24 h. The promoter activity of TIMP ‐3 was then determined by luciferase activity assay. (I) Starved JJ 012 cells were pretreated with 10 μ m of PP 2 and PD 98059 for 1 h, followed by treatment with 10 μ m of S1P for another 10 min, and the phosphorylation of Src, MEK , and ERK was then determined by immunoblotting analysis. Cells without treatment were used as the untreated control (set to 1 or 100), and data were shown as multiples of that. The results are expressed as mean ± SEM . * P
    Mek Inhibitor, supplied by Genentech, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega mek inhibitor
    <t>MEK</t> inhibitor inhibits CVB3-mediated RasGAP cleavage. HeLa cells were pretreated for 30 min with various concentrations of <t>U0126</t> and then were infected by CVB3 for 1 h. At 9 h after addition of CVB3, HeLa cells were harvested and Western blot analysis was performed using an antibody that recognizes RasGAP. Results were quantitated by densitometric analysis using National Institutes of Health Image, version 1.61, and normalized to the control level (sham-infected cells without U0126), which was arbitrarily set to 1.0. Values are means ± standard errors ( n = 3).
    Mek Inhibitor, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc t mek
    <t>RAGE/Raf/MEK/ERK</t> pathway is involved in AGE-BSA induced autophagy. A , Western blotting showing RAGE protein levels in hFOB 1.19 cells treated with AGE-BSA at various concentrations for 24 h. B , quantitative analysis of RAGE protein expression. Each bar
    T Mek, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    InvivoGen mek inhibitor
    <t>RAGE/Raf/MEK/ERK</t> pathway is involved in AGE-BSA induced autophagy. A , Western blotting showing RAGE protein levels in hFOB 1.19 cells treated with AGE-BSA at various concentrations for 24 h. B , quantitative analysis of RAGE protein expression. Each bar
    Mek Inhibitor, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega mek signaling
    <t>RAGE/Raf/MEK/ERK</t> pathway is involved in AGE-BSA induced autophagy. A , Western blotting showing RAGE protein levels in hFOB 1.19 cells treated with AGE-BSA at various concentrations for 24 h. B , quantitative analysis of RAGE protein expression. Each bar
    Mek Signaling, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Epitomics p mek
    Migration and proliferation ability of HSC is related to CCN3-ERK signaling. There was no significant difference in OS and CRR between patients with or without cirrhosis ( a ). Recombinant CCN3 could significantly enhance the migration and proliferation ( c ) of LX2, which could be reversed by S (sorafenib) or U (U0126) ( b ). The diminished number of HSC and subcutaneous tumor weight were found in mice injected with MHCC97H-CCN3-sh cells in nude mouse models ( d ). The increased number of HSC and the enhanced subcutaneous tumor growth were found in the MHCC97H-CCN3 group ( e ). CCN3 could activate ERK signaling pathways with upregulation of <t>RAF,</t> p-RAF, <t>p-MEK</t> and p-ERK, and sorafenib or U0126 showed significant inhibition on ERK signaling ( f )
    P Mek, supplied by Epitomics, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc pp mek
    Localisation of phosphorylated <t>MEK</t> and ERK. (A) Nuclear GFP-ΔBRAF has kinase activity towards the MEK-ERK pathway. NIH3T3 cells were transfected with vectors expressing GFP or GFP-ΔBRAF. Whole cell lysates were prepared from the GFP-transfected cells while nuclear/cytoplasmic fractions were prepared from the GFP-ΔBRAF transfected cells. GFP expression levels and purity of fractions are indicated by the western blots on the bottom. Samples were subjected to immunoprecipitation for GFP and kinase cascade assays were performed. Data shows mean ± SD of three independent experiments. (B) Nuclear GFP-ΔBRAF binds to phosphorylated and non-phosphorylated MEK and ERK. NIH3T3 cells were transfected with vectors expressing GFP or GFP-ΔBRAF, nuclear/cytoplasmic fractions were prepared and analysed for GFP, α-Tubulin or PARP as well as components of the <t>MAPK</t> pathway. A portion of the fractions was subjected to GFP-TRAP immunoprecipitation and immunoprecipitates were subjected to western blot analysis with the antibodies indicated. (C) Phosphorylated MEK and ERK accumulate in the nucleus of cells expressing GFP-ΔBRAF. NIH3T3 cells were transfected with the vectors indicated. Nuclear and cytoplasmic fractions were prepared and analysed by western blot with the indicated antibodies. (D) Quantitation of western blot data from (C). Image J analysis was used to determine density signals of each band on western blots which were adjusted for loading using signals for α-Tubulin (cytoplasmic loading) or PARP (nuclear loading). In the upper bar chart, PP-MEK value were adjusted for total MEK values while in the lower bar chart PP-ERK values were adjusted for ERK2 values. The data are presented as Arbitrary Units (AU) and represent mean ± SEM (n = 3 for each condition).
    Pp Mek, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DuPont de Nemours mek inhibitor
    Localisation of phosphorylated <t>MEK</t> and ERK. (A) Nuclear GFP-ΔBRAF has kinase activity towards the MEK-ERK pathway. NIH3T3 cells were transfected with vectors expressing GFP or GFP-ΔBRAF. Whole cell lysates were prepared from the GFP-transfected cells while nuclear/cytoplasmic fractions were prepared from the GFP-ΔBRAF transfected cells. GFP expression levels and purity of fractions are indicated by the western blots on the bottom. Samples were subjected to immunoprecipitation for GFP and kinase cascade assays were performed. Data shows mean ± SD of three independent experiments. (B) Nuclear GFP-ΔBRAF binds to phosphorylated and non-phosphorylated MEK and ERK. NIH3T3 cells were transfected with vectors expressing GFP or GFP-ΔBRAF, nuclear/cytoplasmic fractions were prepared and analysed for GFP, α-Tubulin or PARP as well as components of the <t>MAPK</t> pathway. A portion of the fractions was subjected to GFP-TRAP immunoprecipitation and immunoprecipitates were subjected to western blot analysis with the antibodies indicated. (C) Phosphorylated MEK and ERK accumulate in the nucleus of cells expressing GFP-ΔBRAF. NIH3T3 cells were transfected with the vectors indicated. Nuclear and cytoplasmic fractions were prepared and analysed by western blot with the indicated antibodies. (D) Quantitation of western blot data from (C). Image J analysis was used to determine density signals of each band on western blots which were adjusted for loading using signals for α-Tubulin (cytoplasmic loading) or PARP (nuclear loading). In the upper bar chart, PP-MEK value were adjusted for total MEK values while in the lower bar chart PP-ERK values were adjusted for ERK2 values. The data are presented as Arbitrary Units (AU) and represent mean ± SEM (n = 3 for each condition).
    Mek Inhibitor, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pfizer Inc mek inhibitor
    Rationale for Targeting Both the <t>Ras/Raf/MEK/ERK</t> and Ras/PI3K/PTEN/Akt/mTOR Pathways for Suppressing Cancer Growth A: The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways are both activated by upstream receptor ligation and frequently co-regulate many downstream targets in parallel. Thus for effective elimination of many cancers or prevention of aging, it may be necessary to target both signaling pathways. Activation of these pathways could also result in increased transcription of many genes that promote cellular growth and malignant transformation. B. Inhibition of mTOR can result in the induction of autophagy, which is a very important mechanism of cell death, especially in solid tumors. C. As described previously, both the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways regulate the activity of apoptotic proteins by post-translational mechanisms. Targeting this pathway may also contribute to the induction of apoptosis. Signaling molecules promoting phosphorylation events are indicated in green. Stimulatory signaling events are indicted in green lines with a green arrow before the target of the phophorylation. Small molecule inhibitors are indicated in red. Inhibitory phosphorylation events are indicated in red lines with a block on the end before the target of the inhibition. Inhibitory signaling or proapoptotic molecules or inactivated molecules are indicated in yellow. A growth factor and a growth factor receptor are indicated in purple. Active transcription factors are indicated in purple diamonds. Inactivated transcription factors are indicated in yellow diamonds.
    Mek Inhibitor, supplied by Pfizer Inc, used in various techniques. Bioz Stars score: 92/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p mek  (Abcam)
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    RAS-MAPK pathway is activated in keratinocytes during squamatization. ( a ) Immunohistochemical staining demonstrates that basaloid keratinocytic cells typical of BCC, such as those contained in the dashed box, have minimal expression of p-ERK, whereas squamatized keratinocytes and adjacent basaloid cells, such as those contained in the solid box, show high expression of p-ERK. Left panel, H E staining original magnification ×20. Right panel, p-ERK staining, original magnification ×20. Bar = 50 μm. ( b ) Representativepicturesofthe H E, DAPI, nuclear <t>Gli1</t> (as readoutfor Hh pathwayactivation) and <t>p-MEK</t> (as readout for Ras/MAPK pathway activation) immunostainings in human BSC samples. Higher magnifications of the basal and squamous areas of the tumor are shown in the top left and top midline framed pictures, respectively, with nuclei highlighted by white dashed lines. Basaloid keratinocytic cells typical of BCC have a higher expression of nuclear Gli1 and low p-MEK, whereas squamatized keratinocytes have high p-MEK and low Gli1. BCC, basal cell carcinoma; BSC, basosquamous carcinoma; H E, hematoxylin and eosin; Hh, Hedgehog; MAPK, mitogen-activated protein kinase; SCC, squamous cell carcinoma. Bar = 25 μm.
    P Mek, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RAS-MAPK pathway is activated in keratinocytes during squamatization. ( a ) Immunohistochemical staining demonstrates that basaloid keratinocytic cells typical of BCC, such as those contained in the dashed box, have minimal expression of p-ERK, whereas squamatized keratinocytes and adjacent basaloid cells, such as those contained in the solid box, show high expression of p-ERK. Left panel, H E staining original magnification ×20. Right panel, p-ERK staining, original magnification ×20. Bar = 50 μm. ( b ) Representativepicturesofthe H E, DAPI, nuclear <t>Gli1</t> (as readoutfor Hh pathwayactivation) and <t>p-MEK</t> (as readout for Ras/MAPK pathway activation) immunostainings in human BSC samples. Higher magnifications of the basal and squamous areas of the tumor are shown in the top left and top midline framed pictures, respectively, with nuclei highlighted by white dashed lines. Basaloid keratinocytic cells typical of BCC have a higher expression of nuclear Gli1 and low p-MEK, whereas squamatized keratinocytes have high p-MEK and low Gli1. BCC, basal cell carcinoma; BSC, basosquamous carcinoma; H E, hematoxylin and eosin; Hh, Hedgehog; MAPK, mitogen-activated protein kinase; SCC, squamous cell carcinoma. Bar = 25 μm.
    P Mek, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RAS-MAPK pathway is activated in keratinocytes during squamatization. ( a ) Immunohistochemical staining demonstrates that basaloid keratinocytic cells typical of BCC, such as those contained in the dashed box, have minimal expression of p-ERK, whereas squamatized keratinocytes and adjacent basaloid cells, such as those contained in the solid box, show high expression of p-ERK. Left panel, H E staining original magnification ×20. Right panel, p-ERK staining, original magnification ×20. Bar = 50 μm. ( b ) Representativepicturesofthe H E, DAPI, nuclear <t>Gli1</t> (as readoutfor Hh pathwayactivation) and <t>p-MEK</t> (as readout for Ras/MAPK pathway activation) immunostainings in human BSC samples. Higher magnifications of the basal and squamous areas of the tumor are shown in the top left and top midline framed pictures, respectively, with nuclei highlighted by white dashed lines. Basaloid keratinocytic cells typical of BCC have a higher expression of nuclear Gli1 and low p-MEK, whereas squamatized keratinocytes have high p-MEK and low Gli1. BCC, basal cell carcinoma; BSC, basosquamous carcinoma; H E, hematoxylin and eosin; Hh, Hedgehog; MAPK, mitogen-activated protein kinase; SCC, squamous cell carcinoma. Bar = 25 μm.
    Mek 1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    IGF-1 activates the PI3K/Akt and MEK/ERK signaling pathways. A, IGF-1 increases the phosphorylation of Akt at 30 min and (B) ERK at 10 min. IGF-1 treatment preferentially induced phosphorylation of ERK2 (p42), while phospho-ERK1 (p44) signal was barely

    Journal: The Journal of Neuroscience

    Article Title: IGF-1 promotes G1/S cell cycle progression through bidirectional regulation of cyclins and CDK inhibitors via the PI3K/Akt pathway in developing rat cerebral cortex

    doi: 10.1523/JNEUROSCI.1700-08.2009

    Figure Lengend Snippet: IGF-1 activates the PI3K/Akt and MEK/ERK signaling pathways. A, IGF-1 increases the phosphorylation of Akt at 30 min and (B) ERK at 10 min. IGF-1 treatment preferentially induced phosphorylation of ERK2 (p42), while phospho-ERK1 (p44) signal was barely

    Article Snippet: Transferrin, PI3K inhibitor (LY294002; 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and MEK inhibitors (PD98059, 2-Amino-3-methoxyflavone; U0126, 1,4-Diamino-2,3-dicyano-1,4- bis (2-aminophenylthio)butadiene) were obtained from Calbiochem (La Jolla, CA).

    Techniques:

    Effects of IGF-1 ICV injections on the PI3K/Akt and MEK/ERK pathways. A, IGF-1 injections into the lateral ventricles of E16.5 rat embryos (30 ng per embryo) resulted in a 4 fold increase in the phosphorylation of Akt by 30 min, revealed by Western blotting.

    Journal: The Journal of Neuroscience

    Article Title: IGF-1 promotes G1/S cell cycle progression through bidirectional regulation of cyclins and CDK inhibitors via the PI3K/Akt pathway in developing rat cerebral cortex

    doi: 10.1523/JNEUROSCI.1700-08.2009

    Figure Lengend Snippet: Effects of IGF-1 ICV injections on the PI3K/Akt and MEK/ERK pathways. A, IGF-1 injections into the lateral ventricles of E16.5 rat embryos (30 ng per embryo) resulted in a 4 fold increase in the phosphorylation of Akt by 30 min, revealed by Western blotting.

    Article Snippet: Transferrin, PI3K inhibitor (LY294002; 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and MEK inhibitors (PD98059, 2-Amino-3-methoxyflavone; U0126, 1,4-Diamino-2,3-dicyano-1,4- bis (2-aminophenylthio)butadiene) were obtained from Calbiochem (La Jolla, CA).

    Techniques: Western Blot

    Blockade of PI3K/Akt signaling, but not the MEK/ERK pathway, inhibits IGF-1 stimulatory effects on DNA synthesis of cortical precursors. A, Cells were pre-incubated with PI3K inhibitor LY294002 (LY) for 30 min before IGF-1 addition, and DNA synthesis

    Journal: The Journal of Neuroscience

    Article Title: IGF-1 promotes G1/S cell cycle progression through bidirectional regulation of cyclins and CDK inhibitors via the PI3K/Akt pathway in developing rat cerebral cortex

    doi: 10.1523/JNEUROSCI.1700-08.2009

    Figure Lengend Snippet: Blockade of PI3K/Akt signaling, but not the MEK/ERK pathway, inhibits IGF-1 stimulatory effects on DNA synthesis of cortical precursors. A, Cells were pre-incubated with PI3K inhibitor LY294002 (LY) for 30 min before IGF-1 addition, and DNA synthesis

    Article Snippet: Transferrin, PI3K inhibitor (LY294002; 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and MEK inhibitors (PD98059, 2-Amino-3-methoxyflavone; U0126, 1,4-Diamino-2,3-dicyano-1,4- bis (2-aminophenylthio)butadiene) were obtained from Calbiochem (La Jolla, CA).

    Techniques: DNA Synthesis, Incubation

    EREG expression is EGFR dependent. A, EREG mRNA was detected by semiquantitative PCR analysis of RNA prepared from a panel of NSCLC cell lines in which EGFR is mutant (HCC827, H3255, and HCC2279) or wild-type (H1299, H460, A549, H322, and H596). Actin was used as the control for RNA integrity. B, kinase-specific regulation of EREG expression. HCC827 cells were treated with inhibitors of EGFR (gefitinib), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (CI-1040), p38 (SB202190), mammalian target of rapamycin (CCI-779), c-jun NH 2 -terminal kinase (SP600125), or AKT (A838450.3). Reverse transcription-PCR analysis of EREG and actin ( top ). EREG bands were quantified by densitometric analysis, normalized for loading differences based on actin, and expressed relative to that of untreated cells, which was set at 1.0. To evaluate dose-dependent target inhibition by the inhibitors, p38 kinase assays ( KA ) and Western blot analyses were done ( bottom ) of phosphorylated Y1068-EGFR ( p-EGFR ), T202/204-extracellular signal–regulated kinase ( p-ERK ), S235/236-S6 ( p-S6 ), S21/9-glycogen synthase kinase 3 ( p-GSK ), S63-c-Jun ( p-cJun ), and corresponding total proteins.

    Journal: Cancer Prevention Research (Philadelphia, Pa.)

    Article Title: Intratumoral Epiregulin Is a Marker of Advanced Disease in Non-Small Cell Lung Cancer Patients and Confers Invasive Properties on EGFR-Mutant Cells

    doi: 10.1158/1940-6207.CAPR-08-0014

    Figure Lengend Snippet: EREG expression is EGFR dependent. A, EREG mRNA was detected by semiquantitative PCR analysis of RNA prepared from a panel of NSCLC cell lines in which EGFR is mutant (HCC827, H3255, and HCC2279) or wild-type (H1299, H460, A549, H322, and H596). Actin was used as the control for RNA integrity. B, kinase-specific regulation of EREG expression. HCC827 cells were treated with inhibitors of EGFR (gefitinib), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (CI-1040), p38 (SB202190), mammalian target of rapamycin (CCI-779), c-jun NH 2 -terminal kinase (SP600125), or AKT (A838450.3). Reverse transcription-PCR analysis of EREG and actin ( top ). EREG bands were quantified by densitometric analysis, normalized for loading differences based on actin, and expressed relative to that of untreated cells, which was set at 1.0. To evaluate dose-dependent target inhibition by the inhibitors, p38 kinase assays ( KA ) and Western blot analyses were done ( bottom ) of phosphorylated Y1068-EGFR ( p-EGFR ), T202/204-extracellular signal–regulated kinase ( p-ERK ), S235/236-S6 ( p-S6 ), S21/9-glycogen synthase kinase 3 ( p-GSK ), S63-c-Jun ( p-cJun ), and corresponding total proteins.

    Article Snippet: We obtained small-molecule inhibitors of EGFR (gefitinib, AstraZeneca), mammalian target of rapamycin (CCI-779, Wyeth-Ayerst), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (CI-1040, Pfizer Pharmaceuticals), c-jun NH2 -terminal kinase (SP600125, Celgene Pharmaceuticals), and AKT (A838450.3, Abbott Pharmaceutical Products).

    Techniques: Expressing, Polymerase Chain Reaction, Mutagenesis, Inhibition, Western Blot

    Sustained infusion of an MEK inhibitor reduces CFA-induced inflammatory pain. The MEK inhibitor U0126 delivered by osmotic pump (0.5 μg · μl −1 · hr −1 ) before CFA injection reduces thermal hyperalgesia ( a ) and mechanical allodynia ( b ) 24 and 48 hr after CFA injection. These were measured by paw-withdrawal latency and paw-withdrawal threshold, respectively, and expressed as percentage of pre-CFA baseline measurements of vehicle control (50% DMSO). * p

    Journal: The Journal of Neuroscience

    Article Title: ERK MAP Kinase Activation in Superficial Spinal Cord Neurons Induces Prodynorphin and NK-1 Upregulation and Contributes to Persistent Inflammatory Pain Hypersensitivity

    doi: 10.1523/JNEUROSCI.22-02-00478.2002

    Figure Lengend Snippet: Sustained infusion of an MEK inhibitor reduces CFA-induced inflammatory pain. The MEK inhibitor U0126 delivered by osmotic pump (0.5 μg · μl −1 · hr −1 ) before CFA injection reduces thermal hyperalgesia ( a ) and mechanical allodynia ( b ) 24 and 48 hr after CFA injection. These were measured by paw-withdrawal latency and paw-withdrawal threshold, respectively, and expressed as percentage of pre-CFA baseline measurements of vehicle control (50% DMSO). * p

    Article Snippet: For intrathecal drug delivery, a polyethylene-10 catheter was implanted into the intrathecal space of the spinal cord at the lumbar enlargement, and 10 μl of the MEK (MAP kinase kinase) inhibitor U0126 (1 μg; dissolved in 10% DMSO; Calbiochem, La Jolla, CA) was administered.

    Techniques: Injection

    Post-treatment with an MEK inhibitor has a delayed effect on inflammatory pain. U0126 (1 μg) or vehicle (10% DMSO) was intrathecally administered 24 hr after CFA injection. Heat hyperalgesia ( a ) and mechanical allodynia ( b ) were tested 30 min, 6 hr, and 24 hr after the administration of the U0126. * p

    Journal: The Journal of Neuroscience

    Article Title: ERK MAP Kinase Activation in Superficial Spinal Cord Neurons Induces Prodynorphin and NK-1 Upregulation and Contributes to Persistent Inflammatory Pain Hypersensitivity

    doi: 10.1523/JNEUROSCI.22-02-00478.2002

    Figure Lengend Snippet: Post-treatment with an MEK inhibitor has a delayed effect on inflammatory pain. U0126 (1 μg) or vehicle (10% DMSO) was intrathecally administered 24 hr after CFA injection. Heat hyperalgesia ( a ) and mechanical allodynia ( b ) were tested 30 min, 6 hr, and 24 hr after the administration of the U0126. * p

    Article Snippet: For intrathecal drug delivery, a polyethylene-10 catheter was implanted into the intrathecal space of the spinal cord at the lumbar enlargement, and 10 μl of the MEK (MAP kinase kinase) inhibitor U0126 (1 μg; dissolved in 10% DMSO; Calbiochem, La Jolla, CA) was administered.

    Techniques: Injection

    The effect of ERK1/2 signaling activity on proliferation of XY PGCs cultured with high FGF9. A ) Five thousand XY PGCs isolated from 12.5 dpc male gonads were incubated with high FGF9 for 2 hr to promote phosphorylation of ERK1/2. When the cells were incubated with both high FGF9 and U0126 MEK inhibitor (10 μM) for 30 min or 1 hr, ERK1/2 activity induced by high FGF9 was completely suppressed. The levels of protein bands were quantified by densitometry and normalized to the β-ACTIN levels. The relative optical densities of phosphorylated protein levels were calculated based on the level in the high FGF9 treatment group set as 1.00. B) To determine the relationship between the ERK1/2 signaling pathway and PGC proliferation when stimulated by high FGF9 treatment, XY PGCs isolated at 12.5 dpc were cultured with high FGF9 and U0126 inhibitor (1, 10 μM) for 2 days and then subjected to the EdU cell proliferation assay. Cells treated with high FGF9 alone significantly increased cell proliferation ( P

    Journal: Biology of Reproduction

    Article Title: Dose-dependent functions of fibroblast growth factor 9 regulate the fate of murine XY primordial germ cells

    doi: 10.1095/biolreprod.116.143941

    Figure Lengend Snippet: The effect of ERK1/2 signaling activity on proliferation of XY PGCs cultured with high FGF9. A ) Five thousand XY PGCs isolated from 12.5 dpc male gonads were incubated with high FGF9 for 2 hr to promote phosphorylation of ERK1/2. When the cells were incubated with both high FGF9 and U0126 MEK inhibitor (10 μM) for 30 min or 1 hr, ERK1/2 activity induced by high FGF9 was completely suppressed. The levels of protein bands were quantified by densitometry and normalized to the β-ACTIN levels. The relative optical densities of phosphorylated protein levels were calculated based on the level in the high FGF9 treatment group set as 1.00. B) To determine the relationship between the ERK1/2 signaling pathway and PGC proliferation when stimulated by high FGF9 treatment, XY PGCs isolated at 12.5 dpc were cultured with high FGF9 and U0126 inhibitor (1, 10 μM) for 2 days and then subjected to the EdU cell proliferation assay. Cells treated with high FGF9 alone significantly increased cell proliferation ( P

    Article Snippet: In some experiments, PGCs were cultured with p38 inhibitors (BIRB796; Cayman Chemical Company, Ann Arbor, MI, SB202190; Sigma-Aldrich) at 10 μM or MEK inhibitor (U0126; Cayman Chemical Company) at 1 or 10 μM.

    Techniques: Activity Assay, Cell Culture, Isolation, Incubation, Pyrolysis Gas Chromatography, Proliferation Assay

    DAZAP1 regulates cell proliferation (a) Stable over-expression of DAZAP1 in non-small cell lung carcinoma NCI-H157 cells as detected by western blots. Control cells are stably transfected with vector only. (b) The expression of DAZAP1 inhibited cell proliferation as judged by colony formation assay with H157 cells. A representative picture from the triplicate experiments, the means and s.d. were plotted. (c) DAZAP1 inhibited the anchorage independent cell growth of H157 as judged by soft agar assays. A representative picture from the triplicates and the quantification were shown. The box plot was drawn using 25 and 75 quartiles in Sigma Plot with whiskers indicating max and min value and dots indicating outliers. P values were determined using paired t-test, *=p ≤ 5.0×10 −5 . (d) DAZAP1 inhibited cell migration as judged by wound healing assay. The picture of a representative plate (left panel) and the quantification of percent of gap closure were shown (right panel). Data from three independent assays were performed error bar indicate s.d. (e) The quantification of cell cycle stages (G1, S and G2/M phases) in DAZAP1 over-expressed or control cells. The cell cycle stages were analyzed by flow-cytometry measurement of DNA content. Data from two independent flow analysis, error bar indicate s.d. (f) Expression of endogenous DAZAP1 protein during cell cycle as detected by western blots. Tubulin and canonical cell cycle markers (cyclin A1 and B1) were also probed as controls. (g) An integrated model for DAZAP1 function and the regulation of its activity through the MEK/Erk pathway.

    Journal: Nature communications

    Article Title: The splicing activator DAZAP1 integrates splicing control into MEK/Erk regulated cell proliferation and migration

    doi: 10.1038/ncomms4078

    Figure Lengend Snippet: DAZAP1 regulates cell proliferation (a) Stable over-expression of DAZAP1 in non-small cell lung carcinoma NCI-H157 cells as detected by western blots. Control cells are stably transfected with vector only. (b) The expression of DAZAP1 inhibited cell proliferation as judged by colony formation assay with H157 cells. A representative picture from the triplicate experiments, the means and s.d. were plotted. (c) DAZAP1 inhibited the anchorage independent cell growth of H157 as judged by soft agar assays. A representative picture from the triplicates and the quantification were shown. The box plot was drawn using 25 and 75 quartiles in Sigma Plot with whiskers indicating max and min value and dots indicating outliers. P values were determined using paired t-test, *=p ≤ 5.0×10 −5 . (d) DAZAP1 inhibited cell migration as judged by wound healing assay. The picture of a representative plate (left panel) and the quantification of percent of gap closure were shown (right panel). Data from three independent assays were performed error bar indicate s.d. (e) The quantification of cell cycle stages (G1, S and G2/M phases) in DAZAP1 over-expressed or control cells. The cell cycle stages were analyzed by flow-cytometry measurement of DNA content. Data from two independent flow analysis, error bar indicate s.d. (f) Expression of endogenous DAZAP1 protein during cell cycle as detected by western blots. Tubulin and canonical cell cycle markers (cyclin A1 and B1) were also probed as controls. (g) An integrated model for DAZAP1 function and the regulation of its activity through the MEK/Erk pathway.

    Article Snippet: Addition of MEK inhibitor (U0126, Sigma) was followed 2 h post transfection and cells were collected after 16–18h-post treatment for protein and splicing analysis.

    Techniques: Over Expression, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Colony Assay, Migration, Wound Healing Assay, Flow Cytometry, Cytometry, Activity Assay

    DAZAP1 is regulated by Erk1/2 catalyzed threonine phosphorylation in the CTD ( a ). A series of truncated CTDs of DAZAP1 were tethered to the exon of a splicing reporter to test which regions are critical for its activity. The full length DAZAP1 was used as positive control, and the two Erk1/2 phosphorylation sites (Thr269 and Thr315) are marked by red triangle. The experimental conditions were identical to Fig. 3b . The mean PSI values and s.d. are shown below a representative gel. P values were determined with paired t-test by comparing to vector-only control. In all panels, #: p ≤0.05; *= p ≤0.01; **= p ≤0.002 ; *** = p ≤0.0008; ns= not significant. ( b ) A schematic model showing that DAZAP1 activity is mediated by MEK/Erk pathway (left panel). The tethering experiments similar to panel a were conducted in cells treated with MEK/Erk activator (PMA) or inhibitor (U0126). ( c ). Phosphorylation of DAZAP1 was inhibited by two specific MEK inhibitors as judged by western blot with Phos-tag labeling. The phosphorylated forms of DAZAP1 were indicated with arrows. (d) Mutation of the two DAZAP1 threonine sites phosphorylated by Erk1/2 abolished DAZAP1 activity as judged by tethering experiments. The experiments were conducted in triplicates and the mean and s.d. of PSI values are plotted below a representative gel. The western blot was shown on the right to confirm equal expression between wild type and mutated DAZAP1. The band indicated by the asterisk is likely a degradation product of MS2-DAZAP1. (e) Inhibition of MEK/Erk by U0126 affected the splicing of endogenous DAZAP1 targets FIP1L1 and MELK. A representative gel from duplicated experiments and the relative changes of PSI were plotted with ranges as error bars. ( f ) Inhibition of MEK/Erk pathway affected nuclear/cytoplasmic translocation of DAZAP1. The cells were transfected with tagged DAZAP1 and hnRNP A1 and detected by corresponding antibodies. Bar, 5 μm. The percents of cells with DAZAP1 localized in nucleus (N), cytoplasm (C) or both compartments (N+C) were counted and plotted in the right panel (n=80 for each sample in two independent experiments). ( g ) Inhibition of MEK/Erk pathway affected nuclear/cytoplasmic translocation of endogenous DAZAP1. Scale bar, 2.5 μm.

    Journal: Nature communications

    Article Title: The splicing activator DAZAP1 integrates splicing control into MEK/Erk regulated cell proliferation and migration

    doi: 10.1038/ncomms4078

    Figure Lengend Snippet: DAZAP1 is regulated by Erk1/2 catalyzed threonine phosphorylation in the CTD ( a ). A series of truncated CTDs of DAZAP1 were tethered to the exon of a splicing reporter to test which regions are critical for its activity. The full length DAZAP1 was used as positive control, and the two Erk1/2 phosphorylation sites (Thr269 and Thr315) are marked by red triangle. The experimental conditions were identical to Fig. 3b . The mean PSI values and s.d. are shown below a representative gel. P values were determined with paired t-test by comparing to vector-only control. In all panels, #: p ≤0.05; *= p ≤0.01; **= p ≤0.002 ; *** = p ≤0.0008; ns= not significant. ( b ) A schematic model showing that DAZAP1 activity is mediated by MEK/Erk pathway (left panel). The tethering experiments similar to panel a were conducted in cells treated with MEK/Erk activator (PMA) or inhibitor (U0126). ( c ). Phosphorylation of DAZAP1 was inhibited by two specific MEK inhibitors as judged by western blot with Phos-tag labeling. The phosphorylated forms of DAZAP1 were indicated with arrows. (d) Mutation of the two DAZAP1 threonine sites phosphorylated by Erk1/2 abolished DAZAP1 activity as judged by tethering experiments. The experiments were conducted in triplicates and the mean and s.d. of PSI values are plotted below a representative gel. The western blot was shown on the right to confirm equal expression between wild type and mutated DAZAP1. The band indicated by the asterisk is likely a degradation product of MS2-DAZAP1. (e) Inhibition of MEK/Erk by U0126 affected the splicing of endogenous DAZAP1 targets FIP1L1 and MELK. A representative gel from duplicated experiments and the relative changes of PSI were plotted with ranges as error bars. ( f ) Inhibition of MEK/Erk pathway affected nuclear/cytoplasmic translocation of DAZAP1. The cells were transfected with tagged DAZAP1 and hnRNP A1 and detected by corresponding antibodies. Bar, 5 μm. The percents of cells with DAZAP1 localized in nucleus (N), cytoplasm (C) or both compartments (N+C) were counted and plotted in the right panel (n=80 for each sample in two independent experiments). ( g ) Inhibition of MEK/Erk pathway affected nuclear/cytoplasmic translocation of endogenous DAZAP1. Scale bar, 2.5 μm.

    Article Snippet: Addition of MEK inhibitor (U0126, Sigma) was followed 2 h post transfection and cells were collected after 16–18h-post treatment for protein and splicing analysis.

    Techniques: Activity Assay, Positive Control, Plasmid Preparation, Western Blot, Labeling, Mutagenesis, Expressing, Inhibition, Translocation Assay, Transfection

    Susceptibility to signaling inputs was highly dynamic around the exit from pluripotency. a – c Fractions of cells classified as XEN-like, ectoderm-like, double positive and double negative after 96 h, based on CD24 and PDGFRA expression. Expression of the two markers was measured by antibody staining and flow cytometry. a Cells were pulsed with 0.25 µM RA for x h (pulse) and subsequently differentiated in basal medium (N2B27) complemented with an RA receptor antagonist. b Cells were incubated with 0.25 µM RA for x h (pulse) after which 0.5 µM PD0325901 (MEK inhibitor) was added for the remainder of the time course. c Cells were first incubated with basal medium (N2B27) for x h (delay) and then exposed to 0.25 µM RA for the remainder of the time course. d Schematic representation of a minimal gene regulatory network that can model a lineage decision 32 . Pointy arrows indicate (auto-)activation; blunted arrows indicate repression. E and X represent expression of ectoderm-like and XEN-like transcriptional programs, respectively. P stands for the pluripotency network. RA increases the auto-activation of the XEN program. e Results of the stochastic simulations of the network shown in d . The relative frequency of XEN-like cells after 96 h is shown vs. the length of an RA pulse (solid lines) or the length of the delay before RA exposure is started (dashed lines). In all cases the pluripotency network was turned off after 12 h. Simulations were run with different amounts of gene expression noise (D: noise power / time, see Methods). See Supplementary Fig. 8b for exemplary trajectories

    Journal: Nature Communications

    Article Title: Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells

    doi: 10.1038/s41467-017-01076-4

    Figure Lengend Snippet: Susceptibility to signaling inputs was highly dynamic around the exit from pluripotency. a – c Fractions of cells classified as XEN-like, ectoderm-like, double positive and double negative after 96 h, based on CD24 and PDGFRA expression. Expression of the two markers was measured by antibody staining and flow cytometry. a Cells were pulsed with 0.25 µM RA for x h (pulse) and subsequently differentiated in basal medium (N2B27) complemented with an RA receptor antagonist. b Cells were incubated with 0.25 µM RA for x h (pulse) after which 0.5 µM PD0325901 (MEK inhibitor) was added for the remainder of the time course. c Cells were first incubated with basal medium (N2B27) for x h (delay) and then exposed to 0.25 µM RA for the remainder of the time course. d Schematic representation of a minimal gene regulatory network that can model a lineage decision 32 . Pointy arrows indicate (auto-)activation; blunted arrows indicate repression. E and X represent expression of ectoderm-like and XEN-like transcriptional programs, respectively. P stands for the pluripotency network. RA increases the auto-activation of the XEN program. e Results of the stochastic simulations of the network shown in d . The relative frequency of XEN-like cells after 96 h is shown vs. the length of an RA pulse (solid lines) or the length of the delay before RA exposure is started (dashed lines). In all cases the pluripotency network was turned off after 12 h. Simulations were run with different amounts of gene expression noise (D: noise power / time, see Methods). See Supplementary Fig. 8b for exemplary trajectories

    Article Snippet: Cell culture All cell lines were grown routinely in modified 2i medium plus LIF (2i/L): DMEM/F12 (Life technologies) supplemented with 0.5x N2 supplement, 0.5x B27 supplement, 0.5mM L -glutamine (Gibco), 20 µg/ml human insulin (Sigma-Aldrich), 1 × 100U/ml penicillin/streptomycin (Gibco), 0.5x MEM Non-Essential Amino Acids (Gibco), 0.1 mM 2-Mercaptoethanol (Sigma-Aldrich), 1 µM MEK inhibitor (PD0325901, Stemgent), 3 µM GSK3 inhibitor (CHIR99021, Stemgent), 1000 U/ml mouse LIF (ESGRO).

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry, Incubation, Activation Assay

    S1P‐induced TIMP ‐3 expression is mediated through the c‐Src/ MAPK signaling axis in human chondrosarcoma cells. (A) Starved JJ 012 cells were incubated with 10 μ m of S1P for 10, 15, 30, 60, and 120 min, and the phosphorylation of c‐Src, MEK , and ERK was examined by immunoblotting using anti‐phospho c‐Src, MEK , or ERK antibodies, followed by reprobing against total c‐Src, MEK , ERK , and β‐actin to show the equal loading amounts. (B) The JJ 012 cells were transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h, and the protein expressions of c‐Src, MEK , and ERK were then determined to show the knockdown efficiency. Starved JJ 012 (open bar) and SW 1353 (closed bar) cells were pretreated with 10 μ m of PP 2, PD 98059, and U0126 for 1 h or transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h, followed by treatment with 10 μ m of S1P for another 24 h, and expressions of miR‐101 (C), TIMP ‐3 (E), and MMP ‐2 (F) mRNA , as well as the protein expressions of TIMP ‐3 and MMP ‐2 (G), and migratory activity (H), were then determined by real‐time PCR , immunoblotting, and transwell analyses, respectively. (D) JJ 012 and SW 1353 cells were transfected with plasmids harboring with WT ‐ TIMP ‐3 3′ UTR , followed by treatment with 10 μ m of PP 2, PD 98059, and U0126 for 1 h or transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h prior to incubation with 10 μ m of S1P for another 24 h. The promoter activity of TIMP ‐3 was then determined by luciferase activity assay. (I) Starved JJ 012 cells were pretreated with 10 μ m of PP 2 and PD 98059 for 1 h, followed by treatment with 10 μ m of S1P for another 10 min, and the phosphorylation of Src, MEK , and ERK was then determined by immunoblotting analysis. Cells without treatment were used as the untreated control (set to 1 or 100), and data were shown as multiples of that. The results are expressed as mean ± SEM . * P

    Journal: Molecular Oncology

    Article Title: Sphingosine‐1‐phosphate suppresses chondrosarcoma metastasis by upregulation of tissue inhibitor of metalloproteinase 3 through suppressing miR‐101 expression

    doi: 10.1002/1878-0261.12106

    Figure Lengend Snippet: S1P‐induced TIMP ‐3 expression is mediated through the c‐Src/ MAPK signaling axis in human chondrosarcoma cells. (A) Starved JJ 012 cells were incubated with 10 μ m of S1P for 10, 15, 30, 60, and 120 min, and the phosphorylation of c‐Src, MEK , and ERK was examined by immunoblotting using anti‐phospho c‐Src, MEK , or ERK antibodies, followed by reprobing against total c‐Src, MEK , ERK , and β‐actin to show the equal loading amounts. (B) The JJ 012 cells were transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h, and the protein expressions of c‐Src, MEK , and ERK were then determined to show the knockdown efficiency. Starved JJ 012 (open bar) and SW 1353 (closed bar) cells were pretreated with 10 μ m of PP 2, PD 98059, and U0126 for 1 h or transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h, followed by treatment with 10 μ m of S1P for another 24 h, and expressions of miR‐101 (C), TIMP ‐3 (E), and MMP ‐2 (F) mRNA , as well as the protein expressions of TIMP ‐3 and MMP ‐2 (G), and migratory activity (H), were then determined by real‐time PCR , immunoblotting, and transwell analyses, respectively. (D) JJ 012 and SW 1353 cells were transfected with plasmids harboring with WT ‐ TIMP ‐3 3′ UTR , followed by treatment with 10 μ m of PP 2, PD 98059, and U0126 for 1 h or transfected with si RNA of scramble, c‐Src, MEK , and ERK for 24 h prior to incubation with 10 μ m of S1P for another 24 h. The promoter activity of TIMP ‐3 was then determined by luciferase activity assay. (I) Starved JJ 012 cells were pretreated with 10 μ m of PP 2 and PD 98059 for 1 h, followed by treatment with 10 μ m of S1P for another 10 min, and the phosphorylation of Src, MEK , and ERK was then determined by immunoblotting analysis. Cells without treatment were used as the untreated control (set to 1 or 100), and data were shown as multiples of that. The results are expressed as mean ± SEM . * P

    Article Snippet: The anti‐rabbit sphingosine kinase 1, a tissue inhibitor of metalloproteinase 3, was obtained from Genetex (Irvine, CA, USA); anti‐rabbit p‐c‐Src and p‐MEK were obtained from Cell Signaling Technology (Danvers, MA, USA); anti‐rabbit p‐ERK, ERK, MEK, MMP‐2, and c‐Src were obtained from Santa Cruz (Santa Cruz, CA, USA).

    Techniques: Expressing, Incubation, Transfection, Activity Assay, Real-time Polymerase Chain Reaction, Luciferase

    N. caninum infection induces ERK-MEK pathway activation of PMϕ. (A) PMϕ were infected with freshly purified N. caninum -1 tachyzoites (MOI = 10) or treated with LPS (50 ng/ml) for different times at 37°C. For the last lane, cell monolayers were treated with tachyzoites or LPS in the presence of PD98059 (20 μM) for 1 h. After treatments, cells were collected for determination of MEK1/2 phosphorylation at Ser217/221 and total MEK by Western blot. (B) After incubated with PD98059 (5–20 μM) or DMSO for 1 h, PMϕ were infected with tachyzoites at a MOI of 10 for 1 h at 37°C, followed by determination of ERK1/2 phosphorylation at Thr202/Tyr204 and total ERK. The intensities of phospho-ERK or MEK were calculated using ImageJ (NIH) and normalized against total ERK or MEK. Data represent the mean of 3 independent experiments ± SEM. ∗ P

    Journal: Frontiers in Microbiology

    Article Title: Activation of ERK Signaling via TLR11 Induces IL-12p40 Production in Peritoneal Macrophages Challenged by Neospora caninum

    doi: 10.3389/fmicb.2017.01393

    Figure Lengend Snippet: N. caninum infection induces ERK-MEK pathway activation of PMϕ. (A) PMϕ were infected with freshly purified N. caninum -1 tachyzoites (MOI = 10) or treated with LPS (50 ng/ml) for different times at 37°C. For the last lane, cell monolayers were treated with tachyzoites or LPS in the presence of PD98059 (20 μM) for 1 h. After treatments, cells were collected for determination of MEK1/2 phosphorylation at Ser217/221 and total MEK by Western blot. (B) After incubated with PD98059 (5–20 μM) or DMSO for 1 h, PMϕ were infected with tachyzoites at a MOI of 10 for 1 h at 37°C, followed by determination of ERK1/2 phosphorylation at Thr202/Tyr204 and total ERK. The intensities of phospho-ERK or MEK were calculated using ImageJ (NIH) and normalized against total ERK or MEK. Data represent the mean of 3 independent experiments ± SEM. ∗ P

    Article Snippet: Membranes were incubated with the specific Phospho-ERK (Thr202/Tyr204, 197G2, 44/42 kDa), Phospho-MEK (Ser217/221, 41G9, 43/44 kDa), ERK (137F5, 44/42 kDa), MEK (D1A5, 43/44 kDa) (1/1,000; all from Cell Signaling) and TLR11 (ab47097, 95 kDa, 1/500; Abcam) rabbit mAb overnight at 4°C followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1/2,000; Proteintech, United States) for 1 h at 37°C in TBST.

    Techniques: Infection, Activation Assay, Purification, Western Blot, Incubation

    MEK inhibitor inhibits CVB3-mediated RasGAP cleavage. HeLa cells were pretreated for 30 min with various concentrations of U0126 and then were infected by CVB3 for 1 h. At 9 h after addition of CVB3, HeLa cells were harvested and Western blot analysis was performed using an antibody that recognizes RasGAP. Results were quantitated by densitometric analysis using National Institutes of Health Image, version 1.61, and normalized to the control level (sham-infected cells without U0126), which was arbitrarily set to 1.0. Values are means ± standard errors ( n = 3).

    Journal: Journal of Virology

    Article Title: Coxsackievirus B3 Replication Is Reduced by Inhibition of the Extracellular Signal-Regulated Kinase (ERK) Signaling Pathway

    doi: 10.1128/JVI.76.7.3365-3373.2002

    Figure Lengend Snippet: MEK inhibitor inhibits CVB3-mediated RasGAP cleavage. HeLa cells were pretreated for 30 min with various concentrations of U0126 and then were infected by CVB3 for 1 h. At 9 h after addition of CVB3, HeLa cells were harvested and Western blot analysis was performed using an antibody that recognizes RasGAP. Results were quantitated by densitometric analysis using National Institutes of Health Image, version 1.61, and normalized to the control level (sham-infected cells without U0126), which was arbitrarily set to 1.0. Values are means ± standard errors ( n = 3).

    Article Snippet: U0126, the MEK inhibitor, was purchased from Promega.

    Techniques: Infection, Western Blot

    RAGE/Raf/MEK/ERK pathway is involved in AGE-BSA induced autophagy. A , Western blotting showing RAGE protein levels in hFOB 1.19 cells treated with AGE-BSA at various concentrations for 24 h. B , quantitative analysis of RAGE protein expression. Each bar

    Journal: The Journal of Biological Chemistry

    Article Title: Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the Receptor of Advanced Glycation End Products/Raf Protein/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (RAGE/Raf/MEK/ERK) Pathway *

    doi: 10.1074/jbc.M115.669499

    Figure Lengend Snippet: RAGE/Raf/MEK/ERK pathway is involved in AGE-BSA induced autophagy. A , Western blotting showing RAGE protein levels in hFOB 1.19 cells treated with AGE-BSA at various concentrations for 24 h. B , quantitative analysis of RAGE protein expression. Each bar

    Article Snippet: Primary antibodies for LC3, phospho-c-Raf (p-c-Raf), total ERK1/2 (t-ERK1/2), p-ERK1/2, p-MEK1/2, and t-MEK were purchased from Cell Signaling Technology, and antibodies for beclin-1, p62/SQSTM1, OPG, OCN, RANKL, and RAGE were purchased from Abcam.

    Techniques: Western Blot, Expressing

    Migration and proliferation ability of HSC is related to CCN3-ERK signaling. There was no significant difference in OS and CRR between patients with or without cirrhosis ( a ). Recombinant CCN3 could significantly enhance the migration and proliferation ( c ) of LX2, which could be reversed by S (sorafenib) or U (U0126) ( b ). The diminished number of HSC and subcutaneous tumor weight were found in mice injected with MHCC97H-CCN3-sh cells in nude mouse models ( d ). The increased number of HSC and the enhanced subcutaneous tumor growth were found in the MHCC97H-CCN3 group ( e ). CCN3 could activate ERK signaling pathways with upregulation of RAF, p-RAF, p-MEK and p-ERK, and sorafenib or U0126 showed significant inhibition on ERK signaling ( f )

    Journal: BMC Cancer

    Article Title: Remodeling of hepatic stellate cells orchestrated the stroma-derived oxaliplatin-resistance through CCN3 paracrine in hepatocellular carcinoma

    doi: 10.1186/s12885-019-6362-1

    Figure Lengend Snippet: Migration and proliferation ability of HSC is related to CCN3-ERK signaling. There was no significant difference in OS and CRR between patients with or without cirrhosis ( a ). Recombinant CCN3 could significantly enhance the migration and proliferation ( c ) of LX2, which could be reversed by S (sorafenib) or U (U0126) ( b ). The diminished number of HSC and subcutaneous tumor weight were found in mice injected with MHCC97H-CCN3-sh cells in nude mouse models ( d ). The increased number of HSC and the enhanced subcutaneous tumor growth were found in the MHCC97H-CCN3 group ( e ). CCN3 could activate ERK signaling pathways with upregulation of RAF, p-RAF, p-MEK and p-ERK, and sorafenib or U0126 showed significant inhibition on ERK signaling ( f )

    Article Snippet: Primary antibodies used for immunofluorescence, immunoblotting and/or, immunohistochemistry were as follows: CCN3, α-SMA, p-C-RAF, ERK1/2, NFκB, TIMP-2, TGF-β, and p-ERK1/2 (Abcam, Cambridge, MA, USA), RANTES and C-RAF (Cell Signaling, Beverly, MA, USA), p-MEK (Epitomics, Burlingame, CA, USA), Actin (Jackson Labs, Bar Harbor, ME, USA).

    Techniques: Migration, Recombinant, Mouse Assay, Injection, Inhibition

    Localisation of phosphorylated MEK and ERK. (A) Nuclear GFP-ΔBRAF has kinase activity towards the MEK-ERK pathway. NIH3T3 cells were transfected with vectors expressing GFP or GFP-ΔBRAF. Whole cell lysates were prepared from the GFP-transfected cells while nuclear/cytoplasmic fractions were prepared from the GFP-ΔBRAF transfected cells. GFP expression levels and purity of fractions are indicated by the western blots on the bottom. Samples were subjected to immunoprecipitation for GFP and kinase cascade assays were performed. Data shows mean ± SD of three independent experiments. (B) Nuclear GFP-ΔBRAF binds to phosphorylated and non-phosphorylated MEK and ERK. NIH3T3 cells were transfected with vectors expressing GFP or GFP-ΔBRAF, nuclear/cytoplasmic fractions were prepared and analysed for GFP, α-Tubulin or PARP as well as components of the MAPK pathway. A portion of the fractions was subjected to GFP-TRAP immunoprecipitation and immunoprecipitates were subjected to western blot analysis with the antibodies indicated. (C) Phosphorylated MEK and ERK accumulate in the nucleus of cells expressing GFP-ΔBRAF. NIH3T3 cells were transfected with the vectors indicated. Nuclear and cytoplasmic fractions were prepared and analysed by western blot with the indicated antibodies. (D) Quantitation of western blot data from (C). Image J analysis was used to determine density signals of each band on western blots which were adjusted for loading using signals for α-Tubulin (cytoplasmic loading) or PARP (nuclear loading). In the upper bar chart, PP-MEK value were adjusted for total MEK values while in the lower bar chart PP-ERK values were adjusted for ERK2 values. The data are presented as Arbitrary Units (AU) and represent mean ± SEM (n = 3 for each condition).

    Journal: Heliyon

    Article Title: Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK

    doi: 10.1016/j.heliyon.2018.e01065

    Figure Lengend Snippet: Localisation of phosphorylated MEK and ERK. (A) Nuclear GFP-ΔBRAF has kinase activity towards the MEK-ERK pathway. NIH3T3 cells were transfected with vectors expressing GFP or GFP-ΔBRAF. Whole cell lysates were prepared from the GFP-transfected cells while nuclear/cytoplasmic fractions were prepared from the GFP-ΔBRAF transfected cells. GFP expression levels and purity of fractions are indicated by the western blots on the bottom. Samples were subjected to immunoprecipitation for GFP and kinase cascade assays were performed. Data shows mean ± SD of three independent experiments. (B) Nuclear GFP-ΔBRAF binds to phosphorylated and non-phosphorylated MEK and ERK. NIH3T3 cells were transfected with vectors expressing GFP or GFP-ΔBRAF, nuclear/cytoplasmic fractions were prepared and analysed for GFP, α-Tubulin or PARP as well as components of the MAPK pathway. A portion of the fractions was subjected to GFP-TRAP immunoprecipitation and immunoprecipitates were subjected to western blot analysis with the antibodies indicated. (C) Phosphorylated MEK and ERK accumulate in the nucleus of cells expressing GFP-ΔBRAF. NIH3T3 cells were transfected with the vectors indicated. Nuclear and cytoplasmic fractions were prepared and analysed by western blot with the indicated antibodies. (D) Quantitation of western blot data from (C). Image J analysis was used to determine density signals of each band on western blots which were adjusted for loading using signals for α-Tubulin (cytoplasmic loading) or PARP (nuclear loading). In the upper bar chart, PP-MEK value were adjusted for total MEK values while in the lower bar chart PP-ERK values were adjusted for ERK2 values. The data are presented as Arbitrary Units (AU) and represent mean ± SEM (n = 3 for each condition).

    Article Snippet: Cells were immunostained with a primary antibody for PP-ERK1/2 (1:200 clone MAPK-YT, Sigma M9692) or PP-MEK (1:200, Cell Signaling, 9154) using AlexaFluor® 546-conjugated goat anti-mouse IgG (H + L) (1:200, Thermo Fisher, A-11003) or AlexaFluor® 546-conjugated goat anti-rabbit IgG (Fab) antibody (1:200, Thermo Fisher, A11018) and counterstained with DAPI as previously reported [ , ].

    Techniques: Activity Assay, Transfection, Expressing, Western Blot, Immunoprecipitation, Quantitation Assay

    Rationale for Targeting Both the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR Pathways for Suppressing Cancer Growth A: The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways are both activated by upstream receptor ligation and frequently co-regulate many downstream targets in parallel. Thus for effective elimination of many cancers or prevention of aging, it may be necessary to target both signaling pathways. Activation of these pathways could also result in increased transcription of many genes that promote cellular growth and malignant transformation. B. Inhibition of mTOR can result in the induction of autophagy, which is a very important mechanism of cell death, especially in solid tumors. C. As described previously, both the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways regulate the activity of apoptotic proteins by post-translational mechanisms. Targeting this pathway may also contribute to the induction of apoptosis. Signaling molecules promoting phosphorylation events are indicated in green. Stimulatory signaling events are indicted in green lines with a green arrow before the target of the phophorylation. Small molecule inhibitors are indicated in red. Inhibitory phosphorylation events are indicated in red lines with a block on the end before the target of the inhibition. Inhibitory signaling or proapoptotic molecules or inactivated molecules are indicated in yellow. A growth factor and a growth factor receptor are indicated in purple. Active transcription factors are indicated in purple diamonds. Inactivated transcription factors are indicated in yellow diamonds.

    Journal: Oncotarget

    Article Title: Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Inhibitors: Rationale and Importance to Inhibiting These Pathways in Human Health

    doi:

    Figure Lengend Snippet: Rationale for Targeting Both the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR Pathways for Suppressing Cancer Growth A: The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways are both activated by upstream receptor ligation and frequently co-regulate many downstream targets in parallel. Thus for effective elimination of many cancers or prevention of aging, it may be necessary to target both signaling pathways. Activation of these pathways could also result in increased transcription of many genes that promote cellular growth and malignant transformation. B. Inhibition of mTOR can result in the induction of autophagy, which is a very important mechanism of cell death, especially in solid tumors. C. As described previously, both the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways regulate the activity of apoptotic proteins by post-translational mechanisms. Targeting this pathway may also contribute to the induction of apoptosis. Signaling molecules promoting phosphorylation events are indicated in green. Stimulatory signaling events are indicted in green lines with a green arrow before the target of the phophorylation. Small molecule inhibitors are indicated in red. Inhibitory phosphorylation events are indicated in red lines with a block on the end before the target of the inhibition. Inhibitory signaling or proapoptotic molecules or inactivated molecules are indicated in yellow. A growth factor and a growth factor receptor are indicated in purple. Active transcription factors are indicated in purple diamonds. Inactivated transcription factors are indicated in yellow diamonds.

    Article Snippet: Sorafenib may target the VEGFR and other membrane receptors expressed on the particular cancer cells, whereas the MEK inhibitor would specifically suppress the Raf/MEK/ERK cascade which is abnormally activated by the BRAF oncogene or other mutant upstream signaling molecules.

    Techniques: Ligation, Activation Assay, Transformation Assay, Inhibition, Activity Assay, Blocking Assay

    Dysregulated Expression of Upstream Receptors and Kinases Can Result in Activation of the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR Pathway Sometimes dysregulated expression of growth factor receptors occurs by increased expression, genetic translocations or genomic amplifications which can lead to activation of the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways. Alternatively chromosomal translocations can occur in non-receptor kinases and other genes which result in activation of these pathways. Genes in the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways that have activating mutations detected in human cancer and proliferative diseases are indicated in blue ovals. Genes overexpressed in certain cancers are indicated in purple ovals. Tumor suppressor genes mutated in human cancer are indicated in red rectangles. Other key genes are indicated in green ovals. Genes inactivated by the Ras/PI3K/PTEN/Akt/mTOR pathway are indicated in orange ovals. Green arrows indicate activating events in pathways. Blocked red arrows indicating inactivating events in pathway.

    Journal: Oncotarget

    Article Title: Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Inhibitors: Rationale and Importance to Inhibiting These Pathways in Human Health

    doi:

    Figure Lengend Snippet: Dysregulated Expression of Upstream Receptors and Kinases Can Result in Activation of the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR Pathway Sometimes dysregulated expression of growth factor receptors occurs by increased expression, genetic translocations or genomic amplifications which can lead to activation of the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways. Alternatively chromosomal translocations can occur in non-receptor kinases and other genes which result in activation of these pathways. Genes in the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways that have activating mutations detected in human cancer and proliferative diseases are indicated in blue ovals. Genes overexpressed in certain cancers are indicated in purple ovals. Tumor suppressor genes mutated in human cancer are indicated in red rectangles. Other key genes are indicated in green ovals. Genes inactivated by the Ras/PI3K/PTEN/Akt/mTOR pathway are indicated in orange ovals. Green arrows indicate activating events in pathways. Blocked red arrows indicating inactivating events in pathway.

    Article Snippet: Sorafenib may target the VEGFR and other membrane receptors expressed on the particular cancer cells, whereas the MEK inhibitor would specifically suppress the Raf/MEK/ERK cascade which is abnormally activated by the BRAF oncogene or other mutant upstream signaling molecules.

    Techniques: Expressing, Activation Assay

    Conceptual Overview of Targeting the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR Pathways to Suppress Malignant Growth The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways can interact at many different levels. In this diagram, we have focused on how they interact to regulate mTOR, p70S6K and protein synthesis and autophagy. Targeting both of these pathways may be an effective means to regulate cell growth. Signaling molecules promoting phosphorylation events are indicated in green. Stimulatory signaling events are indicted in green lines with a green arrow before the target of the phosphorylation. Small molecule inhibitors are indicated in red. Inhibitory phosphorylation events are indicated in red lines with a block on the end before the target of the inhibition. More tentative inhibitory phosphorylation events are indicated in dotted red lines with a block on the end before the target of the inhibition. Inhibitory signaling or proapoptotic molecules or inactivated molecules are indicated in yellow. A growth factor and a growth factor receptor are indicated in purple. Active transcription factors are indicated in purple diamonds. Inactivated transcription factors are indicated in yellow diamonds.

    Journal: Oncotarget

    Article Title: Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Inhibitors: Rationale and Importance to Inhibiting These Pathways in Human Health

    doi:

    Figure Lengend Snippet: Conceptual Overview of Targeting the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR Pathways to Suppress Malignant Growth The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways can interact at many different levels. In this diagram, we have focused on how they interact to regulate mTOR, p70S6K and protein synthesis and autophagy. Targeting both of these pathways may be an effective means to regulate cell growth. Signaling molecules promoting phosphorylation events are indicated in green. Stimulatory signaling events are indicted in green lines with a green arrow before the target of the phosphorylation. Small molecule inhibitors are indicated in red. Inhibitory phosphorylation events are indicated in red lines with a block on the end before the target of the inhibition. More tentative inhibitory phosphorylation events are indicated in dotted red lines with a block on the end before the target of the inhibition. Inhibitory signaling or proapoptotic molecules or inactivated molecules are indicated in yellow. A growth factor and a growth factor receptor are indicated in purple. Active transcription factors are indicated in purple diamonds. Inactivated transcription factors are indicated in yellow diamonds.

    Article Snippet: Sorafenib may target the VEGFR and other membrane receptors expressed on the particular cancer cells, whereas the MEK inhibitor would specifically suppress the Raf/MEK/ERK cascade which is abnormally activated by the BRAF oncogene or other mutant upstream signaling molecules.

    Techniques: Blocking Assay, Inhibition

    Targeting Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR Pathways May Prevent Drug Resistance and Reemergence of Cancer Initiating Cells Chemotherapeutic drugs such as Doxorubicin and Docetaxel may also induce the Raf/MEK/ERK pathway which may contribute to emergence of drug resistant clones. The Raf/MEK/ERK pathway may regulate downstream transcription factors such as GATA-1 which control the transcription of genes such as XRCC1 and ERCC1 which are involved in DNA repair and their aberrant expression may contribute to drug resistance. Treatment of drug resistant cells with MEK inhibitors, or combined treatments consisting of a chemotherapeutic drug and a MEK inhibitor, may be an effective approach to prevent drug resistance. Treatment of certain cancer initiating cells with Akt or mTOR inhibitors may prevent their reemergence. Various components of the Ras/PI3K/PTEN/Akt/mTOR pathway are implicated in drug resistance. Changes in Akt expression may occur to mutations at PI3K or PTEN. Furthermore, altered expression of microRNAs may be involved in decreasing PTEN expression which results in drug resistance. The roles of these various genetic changes in cancer initiating cells are beginning to become apparent. Chemotherapeutic drugs are indicated in irregular black elipses. Treatment of certain cancer initiating cells with Akt or mTOR inhibitors may prevent their reemergence. Signaling molecules promoting phosphorylation events are indicated in green. Stimulatory signaling events are indicted in green lines with a green arrow before the target of the phosphorylation. Small molecule inhibitors are indicated in red. Inhibitory phosphorylation events are indicated in red lines with a block on the end before the target of the inhibition. More tentative inhibitory phosphorylation events are indicated in dotted red lines with a block on the end before the target of the inhibition. Inhibitory signaling or proapoptotic molecules or inactivated molecules are indicated in yellow. A growth factor and a growth factor receptor are indicated in purple. Active transcription factors are indicated in purple diamonds. Inactivated transcription factors are indicated in yellow diamonds.

    Journal: Oncotarget

    Article Title: Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Inhibitors: Rationale and Importance to Inhibiting These Pathways in Human Health

    doi:

    Figure Lengend Snippet: Targeting Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR Pathways May Prevent Drug Resistance and Reemergence of Cancer Initiating Cells Chemotherapeutic drugs such as Doxorubicin and Docetaxel may also induce the Raf/MEK/ERK pathway which may contribute to emergence of drug resistant clones. The Raf/MEK/ERK pathway may regulate downstream transcription factors such as GATA-1 which control the transcription of genes such as XRCC1 and ERCC1 which are involved in DNA repair and their aberrant expression may contribute to drug resistance. Treatment of drug resistant cells with MEK inhibitors, or combined treatments consisting of a chemotherapeutic drug and a MEK inhibitor, may be an effective approach to prevent drug resistance. Treatment of certain cancer initiating cells with Akt or mTOR inhibitors may prevent their reemergence. Various components of the Ras/PI3K/PTEN/Akt/mTOR pathway are implicated in drug resistance. Changes in Akt expression may occur to mutations at PI3K or PTEN. Furthermore, altered expression of microRNAs may be involved in decreasing PTEN expression which results in drug resistance. The roles of these various genetic changes in cancer initiating cells are beginning to become apparent. Chemotherapeutic drugs are indicated in irregular black elipses. Treatment of certain cancer initiating cells with Akt or mTOR inhibitors may prevent their reemergence. Signaling molecules promoting phosphorylation events are indicated in green. Stimulatory signaling events are indicted in green lines with a green arrow before the target of the phosphorylation. Small molecule inhibitors are indicated in red. Inhibitory phosphorylation events are indicated in red lines with a block on the end before the target of the inhibition. More tentative inhibitory phosphorylation events are indicated in dotted red lines with a block on the end before the target of the inhibition. Inhibitory signaling or proapoptotic molecules or inactivated molecules are indicated in yellow. A growth factor and a growth factor receptor are indicated in purple. Active transcription factors are indicated in purple diamonds. Inactivated transcription factors are indicated in yellow diamonds.

    Article Snippet: Sorafenib may target the VEGFR and other membrane receptors expressed on the particular cancer cells, whereas the MEK inhibitor would specifically suppress the Raf/MEK/ERK cascade which is abnormally activated by the BRAF oncogene or other mutant upstream signaling molecules.

    Techniques: Clone Assay, Expressing, Blocking Assay, Inhibition

    RAS-MAPK pathway is activated in keratinocytes during squamatization. ( a ) Immunohistochemical staining demonstrates that basaloid keratinocytic cells typical of BCC, such as those contained in the dashed box, have minimal expression of p-ERK, whereas squamatized keratinocytes and adjacent basaloid cells, such as those contained in the solid box, show high expression of p-ERK. Left panel, H E staining original magnification ×20. Right panel, p-ERK staining, original magnification ×20. Bar = 50 μm. ( b ) Representativepicturesofthe H E, DAPI, nuclear Gli1 (as readoutfor Hh pathwayactivation) and p-MEK (as readout for Ras/MAPK pathway activation) immunostainings in human BSC samples. Higher magnifications of the basal and squamous areas of the tumor are shown in the top left and top midline framed pictures, respectively, with nuclei highlighted by white dashed lines. Basaloid keratinocytic cells typical of BCC have a higher expression of nuclear Gli1 and low p-MEK, whereas squamatized keratinocytes have high p-MEK and low Gli1. BCC, basal cell carcinoma; BSC, basosquamous carcinoma; H E, hematoxylin and eosin; Hh, Hedgehog; MAPK, mitogen-activated protein kinase; SCC, squamous cell carcinoma. Bar = 25 μm.

    Journal: The Journal of investigative dermatology

    Article Title: Genetic Mutations Underlying Phenotypic Plasticity in Basosquamous Carcinoma

    doi: 10.1016/j.jid.2019.03.1163

    Figure Lengend Snippet: RAS-MAPK pathway is activated in keratinocytes during squamatization. ( a ) Immunohistochemical staining demonstrates that basaloid keratinocytic cells typical of BCC, such as those contained in the dashed box, have minimal expression of p-ERK, whereas squamatized keratinocytes and adjacent basaloid cells, such as those contained in the solid box, show high expression of p-ERK. Left panel, H E staining original magnification ×20. Right panel, p-ERK staining, original magnification ×20. Bar = 50 μm. ( b ) Representativepicturesofthe H E, DAPI, nuclear Gli1 (as readoutfor Hh pathwayactivation) and p-MEK (as readout for Ras/MAPK pathway activation) immunostainings in human BSC samples. Higher magnifications of the basal and squamous areas of the tumor are shown in the top left and top midline framed pictures, respectively, with nuclei highlighted by white dashed lines. Basaloid keratinocytic cells typical of BCC have a higher expression of nuclear Gli1 and low p-MEK, whereas squamatized keratinocytes have high p-MEK and low Gli1. BCC, basal cell carcinoma; BSC, basosquamous carcinoma; H E, hematoxylin and eosin; Hh, Hedgehog; MAPK, mitogen-activated protein kinase; SCC, squamous cell carcinoma. Bar = 25 μm.

    Article Snippet: Gli1 (NBP1–78259, 1:200, Novus Biologicals, Littleton, CO), p-MEK (EPR3338, 1:250, Abcam, Cambridge, MA), and nuclei (Hoechst 33342, 1:2000, Invitrogen, Waltham, MA) were stained using a standard immunofluorescence protocol for formalin-fixed paraffin-embedded tissues.

    Techniques: Immunohistochemistry, Staining, Expressing, Activation Assay