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  • 99
    Thermo Fisher mek1 recombinant human protein
    Mek1 Recombinant Human Protein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mek1 2 inhibitor u0126
    SIRT6 promotes NSCLC cell migration and invasion through ERK1/2//MMP9 Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing NSCLC cells and corresponding vector control cells ( A ) Western blotting analysis of p-ERK1/2, ERK1/2 and MMP9 in indicated cells ( B and D ) SIRT6 overexpression increased p-ERK1/2 and MMP9 expression, and treatment with the specific <t>MEK1/2</t> inhibitor <t>U0126</t> abolishes these effects. Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing cells, with or without U0126 ( C ) Wound healing assay results showed that stable SIRT6 overexpression promotes cell migration, which is abolished by concomitant treatment with U0126 ( E ) Migration and invasion assays using a transwell assay system ( F ) SIRT6-overexpressing cells were treated with U0126 or vehicle alone (without U0126). Representative images and quantification of migration and invasion are shown.
    Mek1 2 Inhibitor U0126, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mek1 2
    SIRT6 promotes NSCLC cell migration and invasion through ERK1/2//MMP9 Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing NSCLC cells and corresponding vector control cells ( A ) Western blotting analysis of p-ERK1/2, ERK1/2 and MMP9 in indicated cells ( B and D ) SIRT6 overexpression increased p-ERK1/2 and MMP9 expression, and treatment with the specific <t>MEK1/2</t> inhibitor <t>U0126</t> abolishes these effects. Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing cells, with or without U0126 ( C ) Wound healing assay results showed that stable SIRT6 overexpression promotes cell migration, which is abolished by concomitant treatment with U0126 ( E ) Migration and invasion assays using a transwell assay system ( F ) SIRT6-overexpressing cells were treated with U0126 or vehicle alone (without U0126). Representative images and quantification of migration and invasion are shown.
    Mek1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho mek1 2
    SIRT6 promotes NSCLC cell migration and invasion through ERK1/2//MMP9 Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing NSCLC cells and corresponding vector control cells ( A ) Western blotting analysis of p-ERK1/2, ERK1/2 and MMP9 in indicated cells ( B and D ) SIRT6 overexpression increased p-ERK1/2 and MMP9 expression, and treatment with the specific <t>MEK1/2</t> inhibitor <t>U0126</t> abolishes these effects. Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing cells, with or without U0126 ( C ) Wound healing assay results showed that stable SIRT6 overexpression promotes cell migration, which is abolished by concomitant treatment with U0126 ( E ) Migration and invasion assays using a transwell assay system ( F ) SIRT6-overexpressing cells were treated with U0126 or vehicle alone (without U0126). Representative images and quantification of migration and invasion are shown.
    Phospho Mek1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 809 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti mek1 2
    Multi-cassette induced knockdown of all 4 proteins of the <t>MEK1/2-ERK1/2</t> module. (A) pT2-CAG-puro-tTR + pT2-neo-S2-shMEK2-shMEK1-shERK2-shERK1 (= pT2-neo-S2-MMEE) AMO-1 cells treated with either 1 or 2 μg/ml doxycyclin and harvested for Western blotting 3 and 5 days post-induction (D3/D5). D0 denotes the same cells harvested directly prior to doxycyclin treatment. (B) Similar experiment as in A but conducted with JJN-3 cells. Western blotting shown for cells harvested at day 5 post-induction with 1 or 2 μg/ml doxycyclin. All targets were stained on different blots, but with equal amounts of sample loaded from a single preparation. The respective tubulin loading controls are thus representative for all associated blots.
    Anti Mek1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 611 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc mek1
    <t>MEK1</t> is a direct target of miR‐1271. A, MAP2K1 has one potential miR‐1271 complementary binding site within its 3′UTR (position 131‐137). The 3′UTR (positions 1‐138) of MAP2K1 was cloned into a reporter vector (psiCHECK2‐MAP2K1‐WT or psiCHECK2‐MAP2K1‐Mut) downstream of the Renilla luciferase gene between Pme I and Xho I. A schematic representation of the miR‐1271 seed region in the MAP2K1 3′UTR ( MAP2K1 3′UTR‐WT) and the mutated 3′UTR ( MAP2K1 3′UTR‐Mut) is shown on the right. B, Cotransfection of MKN74 cells with a miR‐1271 mimic and psiCHECK2‐MAP2K1‐WT resulted in a significant decrease in luciferase activity. The graphs represent 3 independent experiments performed in triplicate. Mean ± SD (n = 3). Mann‐Whitney test. ** P
    Mek1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 428 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology mek1
    <t>MEK1</t> activates NFAT through AP-1 signaling. (A) Neonatal cardiomyocytes were infected with AdNFAT-luc to assay for NFAT transcriptional activity, together with an adenovirus encoding constitutively active NFATc3 (ΔNFAT) in the presence or absence
    Mek1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mek1 2 inhibitor u0126
    Effects of proteins kinases inhibitors on the expression of non-phosphorylated CTNNB induced by DPN in the Sertoli cells from 20-day-old rats . Sertoli cells from 20-day-old rats were incubated in the absence (C, control) and presence of DPN (10 nM) for 24 h. The cells were also untreated or pretreated with PP2, an inhibitor of the SRC family of protein tyrosine kinases; <t>U0126,</t> an inhibitor of <t>MEK1/2;</t> Wortmannin, an inhibitor of PI3K and MK-2206, an inhibitor of AKT for 30 min. Afterward, the cells were stimulated with DPN (10 nM) 24h. Immunostaining for CTNNB (green) was detected using an antibody specific for non-phosphorylated CTNNB and Alexa Fluor 488-labeled secondary antibody. Nuclei were stained with DAPI (blue). Negative control was performed using normal rabbit serum at the same dilution of antibody. Scale bar as indicated. The data shown are representative of three independent experiments. Note: Control, C, and DPN are the same results of the Figure 5 . All treatments were performed on the same day (Figures 5 and 6 ).
    Mek1 2 Inhibitor U0126, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 266 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mek1 2 inhibitor
    T3SS1-specific induction of several transcripts requires <t>MEK1/2</t> activity (A) . Immunoblot showing phosphorylated ERK1 and ERK2 (p-ERK1/2) and total ERK1/2 in POR3:T3SS1 + –infected primary human fibroblasts treated with DMSO (vehicle) or U0126 to demonstrate the activity of U0126 as a MEK inhibitor in primary fibroblasts. N=3 independent experiments. (B–E) qRT-PCR showing the expression of RHOB, JUN, FOS, PTGS2, EGR1, KLF2, and ATF3 in primary human fibroblasts that were pretreated with the MEK1/2 inhibitor U0126 or DMSO before being infected with POR3:T3SS1 + or POR4:T3SS1 − compared to uninfected cells (UN). Expression was normalized to the housekeeping gene IPO8 . Data are 2 −ΔΔCq ± SD, N = 3 experiments. * p
    Mek1 2 Inhibitor, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mek1 2
    T3SS1-specific induction of several transcripts requires <t>MEK1/2</t> activity (A) . Immunoblot showing phosphorylated ERK1 and ERK2 (p-ERK1/2) and total ERK1/2 in POR3:T3SS1 + –infected primary human fibroblasts treated with DMSO (vehicle) or U0126 to demonstrate the activity of U0126 as a MEK inhibitor in primary fibroblasts. N=3 independent experiments. (B–E) qRT-PCR showing the expression of RHOB, JUN, FOS, PTGS2, EGR1, KLF2, and ATF3 in primary human fibroblasts that were pretreated with the MEK1/2 inhibitor U0126 or DMSO before being infected with POR3:T3SS1 + or POR4:T3SS1 − compared to uninfected cells (UN). Expression was normalized to the housekeeping gene IPO8 . Data are 2 −ΔΔCq ± SD, N = 3 experiments. * p
    Mek1 2, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phosphorylated mek1 2
    Elevated miR-30d suppresses the MEK/ERK and PI3K/Akt signaling pathways in SK-ES-1 cells. (A) Western blot assay revealed that overexpression of miR-30d reduced the expression levels of <t>p-MEK1/2</t> and p-ERK1/2, but caused no changes in the levels of MEK1/2 and ERK1/2. (B) Ratios of p-MEK/MEK and p-ERK/ERK in miR-30d mimic group were decreased compared with those in the untreated group (**P
    Phosphorylated Mek1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 186 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology mek1 2
    SO 2 derivatives did not inhibit the activation of Erk1/2, <t>MEK1/2</t> and c-Raf molecules directly. ( a ) After transfected with different plasmids as described in the figure, HEK293 cells in experimental groups were treated with Na 2 SO 3 /NaHSO 3 at 15 μ mol/l for 24 h. The phosphorylation of Erk1/2 was assessed by western blotting. * P
    Mek1 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mek1 inhibitor pd98059
    Role ERK1/2 in PA-regulated MMP-2 and TIMP-2 expression in HeLa cells. ( A ) Cells were treated with various concentrations of PA (0 to 30 μM) for 24 h, then harvested and lysed for measurement target proteins by western blotting; ( B ) cells were treated with or without PA in the absence or presence of <t>PD98059</t> (specific <t>MEK1/2</t> inhibitor) for 24 h, followed by measurement of migration and invasion; ( C ) cells were treated as above for 24 h, followed by measurement of relative wound width; ( D , E ) Protein and mRNA expression of MMP-2 and TIMP-2 were measured by western blotting assay and RT-qPCR. Values are means and standard errors of 3 replicates. ** p
    Mek1 Inhibitor Pd98059, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mek 1 2
    Role ERK1/2 in PA-regulated MMP-2 and TIMP-2 expression in HeLa cells. ( A ) Cells were treated with various concentrations of PA (0 to 30 μM) for 24 h, then harvested and lysed for measurement target proteins by western blotting; ( B ) cells were treated with or without PA in the absence or presence of <t>PD98059</t> (specific <t>MEK1/2</t> inhibitor) for 24 h, followed by measurement of migration and invasion; ( C ) cells were treated as above for 24 h, followed by measurement of relative wound width; ( D , E ) Protein and mRNA expression of MMP-2 and TIMP-2 were measured by western blotting assay and RT-qPCR. Values are means and standard errors of 3 replicates. ** p
    Mek 1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mek1  (Abcam)
    94
    Abcam mek1
    Role ERK1/2 in PA-regulated MMP-2 and TIMP-2 expression in HeLa cells. ( A ) Cells were treated with various concentrations of PA (0 to 30 μM) for 24 h, then harvested and lysed for measurement target proteins by western blotting; ( B ) cells were treated with or without PA in the absence or presence of <t>PD98059</t> (specific <t>MEK1/2</t> inhibitor) for 24 h, followed by measurement of migration and invasion; ( C ) cells were treated as above for 24 h, followed by measurement of relative wound width; ( D , E ) Protein and mRNA expression of MMP-2 and TIMP-2 were measured by western blotting assay and RT-qPCR. Values are means and standard errors of 3 replicates. ** p
    Mek1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti mek1 2
    Western blotting of Neuro2a lysates from WT, Lypla1 −/− , Lypla2 −/− , and DKO cells. Relative changes were quantified in triplicate analysis, and representative lanes are shown. A: <t>Phospho-MEK1/2</t> (p-MEK) is significantly increased in DKO cells. B: Phospho-ERK1/2 (p-ERK) is significantly increased in DKO cells. * P
    Rabbit Anti Mek1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology anti mek1 2
    Expression of mutant T210A Plk1 inhibits the PDGF-induced phosphorylation of <t>MEK1/2</t> and ERK1/2, and cell proliferation. a Smooth muscle cells were transfected with plasmids encoding Flag-wild type (WT) or T210A Plk1. Blots of cell extracts were detected with antibodies against Flag and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells transfected with plasmids were normalized to untransfected cells. Values are mean ± SE ( n = 4, * P
    Anti Mek1 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SIRT6 promotes NSCLC cell migration and invasion through ERK1/2//MMP9 Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing NSCLC cells and corresponding vector control cells ( A ) Western blotting analysis of p-ERK1/2, ERK1/2 and MMP9 in indicated cells ( B and D ) SIRT6 overexpression increased p-ERK1/2 and MMP9 expression, and treatment with the specific MEK1/2 inhibitor U0126 abolishes these effects. Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing cells, with or without U0126 ( C ) Wound healing assay results showed that stable SIRT6 overexpression promotes cell migration, which is abolished by concomitant treatment with U0126 ( E ) Migration and invasion assays using a transwell assay system ( F ) SIRT6-overexpressing cells were treated with U0126 or vehicle alone (without U0126). Representative images and quantification of migration and invasion are shown.

    Journal: Oncotarget

    Article Title: Upregulation of SIRT6 predicts poor prognosis and promotes metastasis of non-small cell lung cancer via the ERK1/2/MMP9 pathway

    doi: 10.18632/oncotarget.9750

    Figure Lengend Snippet: SIRT6 promotes NSCLC cell migration and invasion through ERK1/2//MMP9 Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing NSCLC cells and corresponding vector control cells ( A ) Western blotting analysis of p-ERK1/2, ERK1/2 and MMP9 in indicated cells ( B and D ) SIRT6 overexpression increased p-ERK1/2 and MMP9 expression, and treatment with the specific MEK1/2 inhibitor U0126 abolishes these effects. Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing cells, with or without U0126 ( C ) Wound healing assay results showed that stable SIRT6 overexpression promotes cell migration, which is abolished by concomitant treatment with U0126 ( E ) Migration and invasion assays using a transwell assay system ( F ) SIRT6-overexpressing cells were treated with U0126 or vehicle alone (without U0126). Representative images and quantification of migration and invasion are shown.

    Article Snippet: To test the effect of the specific MEK1/2 inhibitor U0126 (Sigma) on the migratory and invasive abilities of cells, A549 and L78 cells were pretreated with 10 μM U0126 for 30 min before migration and invasion assays were performed.

    Techniques: Migration, Activity Assay, Plasmid Preparation, Western Blot, Over Expression, Expressing, Wound Healing Assay, Transwell Assay

    Multi-cassette induced knockdown of all 4 proteins of the MEK1/2-ERK1/2 module. (A) pT2-CAG-puro-tTR + pT2-neo-S2-shMEK2-shMEK1-shERK2-shERK1 (= pT2-neo-S2-MMEE) AMO-1 cells treated with either 1 or 2 μg/ml doxycyclin and harvested for Western blotting 3 and 5 days post-induction (D3/D5). D0 denotes the same cells harvested directly prior to doxycyclin treatment. (B) Similar experiment as in A but conducted with JJN-3 cells. Western blotting shown for cells harvested at day 5 post-induction with 1 or 2 μg/ml doxycyclin. All targets were stained on different blots, but with equal amounts of sample loaded from a single preparation. The respective tubulin loading controls are thus representative for all associated blots.

    Journal: PLoS ONE

    Article Title: A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors

    doi: 10.1371/journal.pone.0205585

    Figure Lengend Snippet: Multi-cassette induced knockdown of all 4 proteins of the MEK1/2-ERK1/2 module. (A) pT2-CAG-puro-tTR + pT2-neo-S2-shMEK2-shMEK1-shERK2-shERK1 (= pT2-neo-S2-MMEE) AMO-1 cells treated with either 1 or 2 μg/ml doxycyclin and harvested for Western blotting 3 and 5 days post-induction (D3/D5). D0 denotes the same cells harvested directly prior to doxycyclin treatment. (B) Similar experiment as in A but conducted with JJN-3 cells. Western blotting shown for cells harvested at day 5 post-induction with 1 or 2 μg/ml doxycyclin. All targets were stained on different blots, but with equal amounts of sample loaded from a single preparation. The respective tubulin loading controls are thus representative for all associated blots.

    Article Snippet: The following antibodies were used: anti-ERK1,2 (Santa Cruz Biotechnology, Heidelberg, Germany; sc-94), anti-phospho-ERK1,2 (Cell Signaling Technology (CST), Frankfurt am Main, Germany; no. 9101), anti-MEK1,2 (CST; no. 9122), anti-phospho-MEK1,2 (CST; no. 9154), anti-HA-tag (Abcam, Cambridge, UK; ab9110), anti-tet-Repressor (Merck, Darmstadt, Germany; AB3541), anti-HSP90β (Enzo Life Sciences, Lörrach, Germany; ADI-SPA-843), anti-α-tubulin (Bio-Rad, München, Germany; MCA78G).

    Techniques: Western Blot, Staining

    Establishment and characterization of vectors for concatenated shRNA expression cassettes. ]). Cells were then harvested for Western blotting at day three post-transfection. Knockdown of both ERK1 and ERK2 from the pSUS double-cassette vectors (lanes 2 and 3) matches the effect obtained with the pSUPER-based single-cassette vectors (lanes 4–6). (B) Left: Establishment of knockdown constructs for MEK2 (three different target sequences tried), based on lowered expression of HA-tagged human MEK2 from a co-transfected expression plasmid (see Methods). The shMEK2-2 sequence was chosen for further experiments (= shMEK2). Right: Knockdown of MEK1 and MEK2 from a pSUS double cassette vector (middle lane) versus combined transfection of the single shRNA expression vectors (right lane). (C) Concentration-dependent effects of the MEK1 2 inhibitor PD0325906 on the levels of phospho-ERK1/2 and phospho-MEK1/2. Cells were treated for 1 h with the drug and harvested for Western blotting. Substantial downregulation of phospho-ERK1/2 results in inceased levels of phospho-MEK1/2. FACS analysis with staining for early (annexin V) and late (PI) apoptotic markers after 3 days of drug treatment does not show increased rates of apoptosis. (D) Western analysis of the efficiency of MEK1+2 and ERK1+2 knockdown from a quadruple cassette shRNA-expression vector (lane 6) in relation to single cassette vectors (ERK1, ERK2, lanes 2 and 3) and double cassette vectors (ERK1+2, lane 4; MEK1+2, lane 5). Cells were harvested at day 3 post-transfection.

    Journal: PLoS ONE

    Article Title: A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors

    doi: 10.1371/journal.pone.0205585

    Figure Lengend Snippet: Establishment and characterization of vectors for concatenated shRNA expression cassettes. ]). Cells were then harvested for Western blotting at day three post-transfection. Knockdown of both ERK1 and ERK2 from the pSUS double-cassette vectors (lanes 2 and 3) matches the effect obtained with the pSUPER-based single-cassette vectors (lanes 4–6). (B) Left: Establishment of knockdown constructs for MEK2 (three different target sequences tried), based on lowered expression of HA-tagged human MEK2 from a co-transfected expression plasmid (see Methods). The shMEK2-2 sequence was chosen for further experiments (= shMEK2). Right: Knockdown of MEK1 and MEK2 from a pSUS double cassette vector (middle lane) versus combined transfection of the single shRNA expression vectors (right lane). (C) Concentration-dependent effects of the MEK1 2 inhibitor PD0325906 on the levels of phospho-ERK1/2 and phospho-MEK1/2. Cells were treated for 1 h with the drug and harvested for Western blotting. Substantial downregulation of phospho-ERK1/2 results in inceased levels of phospho-MEK1/2. FACS analysis with staining for early (annexin V) and late (PI) apoptotic markers after 3 days of drug treatment does not show increased rates of apoptosis. (D) Western analysis of the efficiency of MEK1+2 and ERK1+2 knockdown from a quadruple cassette shRNA-expression vector (lane 6) in relation to single cassette vectors (ERK1, ERK2, lanes 2 and 3) and double cassette vectors (ERK1+2, lane 4; MEK1+2, lane 5). Cells were harvested at day 3 post-transfection.

    Article Snippet: The following antibodies were used: anti-ERK1,2 (Santa Cruz Biotechnology, Heidelberg, Germany; sc-94), anti-phospho-ERK1,2 (Cell Signaling Technology (CST), Frankfurt am Main, Germany; no. 9101), anti-MEK1,2 (CST; no. 9122), anti-phospho-MEK1,2 (CST; no. 9154), anti-HA-tag (Abcam, Cambridge, UK; ab9110), anti-tet-Repressor (Merck, Darmstadt, Germany; AB3541), anti-HSP90β (Enzo Life Sciences, Lörrach, Germany; ADI-SPA-843), anti-α-tubulin (Bio-Rad, München, Germany; MCA78G).

    Techniques: shRNA, Expressing, Western Blot, Transfection, Construct, Plasmid Preparation, Sequencing, Concentration Assay, FACS, Staining

    FMA46C overexpression inhibited cell proliferation. Notes: At 0, 24, 48, and 72 hours after transduction, cell proliferation was evaluated by CCK-8 assay in CAL27 ( A ) and SCC15 ( B ) cells. Data were based on three independent experiments and presented as mean ± SD. The protein expression levels of cleaved caspase 9, cleaved caspase 3, p-ERK1/2, and ERK1/2 in CAL27 ( C ) and SCC15 ( D ) cells after 48 hours of transduction were analyzed by Western blotting. The expression levels of Ras, MEK1/2, and p-MEK1/2 were analyzed by Western blot in CAL27 ( E ) and SCC15 ( F ). Control: original CAL27 or SCC15 cells; vector: CAL27 or SCC15 cells transduced with control lentivirus. ### P

    Journal: OncoTargets and therapy

    Article Title: The potential functions of FAM46C in oral squamous cell carcinoma

    doi: 10.2147/OTT.S185244

    Figure Lengend Snippet: FMA46C overexpression inhibited cell proliferation. Notes: At 0, 24, 48, and 72 hours after transduction, cell proliferation was evaluated by CCK-8 assay in CAL27 ( A ) and SCC15 ( B ) cells. Data were based on three independent experiments and presented as mean ± SD. The protein expression levels of cleaved caspase 9, cleaved caspase 3, p-ERK1/2, and ERK1/2 in CAL27 ( C ) and SCC15 ( D ) cells after 48 hours of transduction were analyzed by Western blotting. The expression levels of Ras, MEK1/2, and p-MEK1/2 were analyzed by Western blot in CAL27 ( E ) and SCC15 ( F ). Control: original CAL27 or SCC15 cells; vector: CAL27 or SCC15 cells transduced with control lentivirus. ### P

    Article Snippet: The membranes were incubated with primary antibodies at room temperature for 2 hours or 4°C overnight (FAM46C, Abcam, Cambridge, UK, Ab169699; p-ERK1/2, CST (Cell Signalling Technology, Danvers, MA, USA), 9101; ERK1/2, CST, 9102; caspase 9, Abcam, Ab202068; caspase 3, Abcam, Ab44976; Ras, Solarbio, Beijing, China, K006385P; MEK1/2, CST, 9122; p-MEK1/2, CST, 4376; GAPDH, CST, 5174).

    Techniques: Over Expression, Transduction, CCK-8 Assay, Expressing, Western Blot, Plasmid Preparation

    MEK1 is a direct target of miR‐1271. A, MAP2K1 has one potential miR‐1271 complementary binding site within its 3′UTR (position 131‐137). The 3′UTR (positions 1‐138) of MAP2K1 was cloned into a reporter vector (psiCHECK2‐MAP2K1‐WT or psiCHECK2‐MAP2K1‐Mut) downstream of the Renilla luciferase gene between Pme I and Xho I. A schematic representation of the miR‐1271 seed region in the MAP2K1 3′UTR ( MAP2K1 3′UTR‐WT) and the mutated 3′UTR ( MAP2K1 3′UTR‐Mut) is shown on the right. B, Cotransfection of MKN74 cells with a miR‐1271 mimic and psiCHECK2‐MAP2K1‐WT resulted in a significant decrease in luciferase activity. The graphs represent 3 independent experiments performed in triplicate. Mean ± SD (n = 3). Mann‐Whitney test. ** P

    Journal: Cancer Medicine

    Article Title: Epigenetic silencing of miR‐1271 enhances MEK1 and TEAD4 expression in gastric cancer, et al. Epigenetic silencing of miR‐1271 enhances MEK1 and TEAD4 expression in gastric cancer

    doi: 10.1002/cam4.1605

    Figure Lengend Snippet: MEK1 is a direct target of miR‐1271. A, MAP2K1 has one potential miR‐1271 complementary binding site within its 3′UTR (position 131‐137). The 3′UTR (positions 1‐138) of MAP2K1 was cloned into a reporter vector (psiCHECK2‐MAP2K1‐WT or psiCHECK2‐MAP2K1‐Mut) downstream of the Renilla luciferase gene between Pme I and Xho I. A schematic representation of the miR‐1271 seed region in the MAP2K1 3′UTR ( MAP2K1 3′UTR‐WT) and the mutated 3′UTR ( MAP2K1 3′UTR‐Mut) is shown on the right. B, Cotransfection of MKN74 cells with a miR‐1271 mimic and psiCHECK2‐MAP2K1‐WT resulted in a significant decrease in luciferase activity. The graphs represent 3 independent experiments performed in triplicate. Mean ± SD (n = 3). Mann‐Whitney test. ** P

    Article Snippet: The membranes were immersed in 5% skim milk or 5% BSA in Tris‐buffered saline containing 0.1% Tween 20 for 1 hour and probed with primary antibodies against TEAD4 (H00007004‐M01, Abnova, 1:1000), MEK1 (#9124, Cell Signaling Technology, 1:1000), phospho‐ERK (#9101, Cell Signaling Technology, 1:1000), ERK (#9102, Cell Signaling Technology, 1:1000), YAP1 (#4912, Cell Signaling Technology, 1:1000), phospho‐YAP (#4911, Cell Signaling Technology 1:1000) or β‐actin (#Ab8227, Abcam, 1:5000) overnight at 4°C.

    Techniques: Binding Assay, Clone Assay, Plasmid Preparation, Luciferase, Cotransfection, Activity Assay, MANN-WHITNEY

    Genes down‐regulated by miR‐1271 are involved in cancer‐associated signaling pathways. A, Overlap between gene sets down‐regulated by the miR‐1271 mimic derived from RNA‐seq analyses of SNU‐601 and MKN74 cells and genes with mirSVR scores less than −0.1 based on conservation (from microRNA.org). Candidate miR‐1271 target genes were narrowed to 40 genes. B, Expression levels of 40 candidate miR‐1271 target genes shown as heatmaps. C, RNA‐seq data were subjected to GSEA analysis (using c6.all.v5.2.symbols.gmt from MSigDB), revealing significant enrichment of the “EGF‐regulated gene set” in a gene set down‐regulated by miR‐1271. D, Expression levels of MAP2K1 upon transfection of a nonsilencing control (Con) or a miR‐1271 mimic (m1271) into MKN74 and SNU‐601 cells. Mean ± SD (n = 3). Mann‐Whitney test. ** P

    Journal: Cancer Medicine

    Article Title: Epigenetic silencing of miR‐1271 enhances MEK1 and TEAD4 expression in gastric cancer, et al. Epigenetic silencing of miR‐1271 enhances MEK1 and TEAD4 expression in gastric cancer

    doi: 10.1002/cam4.1605

    Figure Lengend Snippet: Genes down‐regulated by miR‐1271 are involved in cancer‐associated signaling pathways. A, Overlap between gene sets down‐regulated by the miR‐1271 mimic derived from RNA‐seq analyses of SNU‐601 and MKN74 cells and genes with mirSVR scores less than −0.1 based on conservation (from microRNA.org). Candidate miR‐1271 target genes were narrowed to 40 genes. B, Expression levels of 40 candidate miR‐1271 target genes shown as heatmaps. C, RNA‐seq data were subjected to GSEA analysis (using c6.all.v5.2.symbols.gmt from MSigDB), revealing significant enrichment of the “EGF‐regulated gene set” in a gene set down‐regulated by miR‐1271. D, Expression levels of MAP2K1 upon transfection of a nonsilencing control (Con) or a miR‐1271 mimic (m1271) into MKN74 and SNU‐601 cells. Mean ± SD (n = 3). Mann‐Whitney test. ** P

    Article Snippet: The membranes were immersed in 5% skim milk or 5% BSA in Tris‐buffered saline containing 0.1% Tween 20 for 1 hour and probed with primary antibodies against TEAD4 (H00007004‐M01, Abnova, 1:1000), MEK1 (#9124, Cell Signaling Technology, 1:1000), phospho‐ERK (#9101, Cell Signaling Technology, 1:1000), ERK (#9102, Cell Signaling Technology, 1:1000), YAP1 (#4912, Cell Signaling Technology, 1:1000), phospho‐YAP (#4911, Cell Signaling Technology 1:1000) or β‐actin (#Ab8227, Abcam, 1:5000) overnight at 4°C.

    Techniques: Derivative Assay, RNA Sequencing Assay, Expressing, Transfection, MANN-WHITNEY

    MEK1 activates NFAT through AP-1 signaling. (A) Neonatal cardiomyocytes were infected with AdNFAT-luc to assay for NFAT transcriptional activity, together with an adenovirus encoding constitutively active NFATc3 (ΔNFAT) in the presence or absence

    Journal:

    Article Title: Direct and Indirect Interactions between Calcineurin-NFAT and MEK1-Extracellular Signal-Regulated Kinase 1/2 Signaling Pathways Regulate Cardiac Gene Expression and Cellular Growth

    doi: 10.1128/MCB.25.3.865-878.2005

    Figure Lengend Snippet: MEK1 activates NFAT through AP-1 signaling. (A) Neonatal cardiomyocytes were infected with AdNFAT-luc to assay for NFAT transcriptional activity, together with an adenovirus encoding constitutively active NFATc3 (ΔNFAT) in the presence or absence

    Article Snippet: We first assessed the integrity of the activated MEK1 transgene in the heart by Western blotting for MEK1, phosphorylated ERK1/2, and phosphorylated Elk-1 from cardiac protein extracts (Fig. ).

    Techniques: Infection, Activity Assay

    MEK1 does not affect NFAT nuclear localization. (A) Neonatal cardiomyocytes were infected with AdNFATc1-GFP and the indicated recombinant adenoviruses. After 48 h, cells were fixed and photographed. (B and C) Nuclear localization of NFATc1 was quantified

    Journal:

    Article Title: Direct and Indirect Interactions between Calcineurin-NFAT and MEK1-Extracellular Signal-Regulated Kinase 1/2 Signaling Pathways Regulate Cardiac Gene Expression and Cellular Growth

    doi: 10.1128/MCB.25.3.865-878.2005

    Figure Lengend Snippet: MEK1 does not affect NFAT nuclear localization. (A) Neonatal cardiomyocytes were infected with AdNFATc1-GFP and the indicated recombinant adenoviruses. After 48 h, cells were fixed and photographed. (B and C) Nuclear localization of NFATc1 was quantified

    Article Snippet: We first assessed the integrity of the activated MEK1 transgene in the heart by Western blotting for MEK1, phosphorylated ERK1/2, and phosphorylated Elk-1 from cardiac protein extracts (Fig. ).

    Techniques: Infection, Recombinant

    Calcineurin-NFAT signaling is necessary for MEK1-induced hypertrophy in vitro. (A) Representative immunocytochemical analysis of actin-stained (phalloidin) neonatal cardiomyocytes treated as indicated. (B) Measurement of cell surface area of neonatal

    Journal:

    Article Title: Direct and Indirect Interactions between Calcineurin-NFAT and MEK1-Extracellular Signal-Regulated Kinase 1/2 Signaling Pathways Regulate Cardiac Gene Expression and Cellular Growth

    doi: 10.1128/MCB.25.3.865-878.2005

    Figure Lengend Snippet: Calcineurin-NFAT signaling is necessary for MEK1-induced hypertrophy in vitro. (A) Representative immunocytochemical analysis of actin-stained (phalloidin) neonatal cardiomyocytes treated as indicated. (B) Measurement of cell surface area of neonatal

    Article Snippet: We first assessed the integrity of the activated MEK1 transgene in the heart by Western blotting for MEK1, phosphorylated ERK1/2, and phosphorylated Elk-1 from cardiac protein extracts (Fig. ).

    Techniques: In Vitro, Staining

    MEK1-ERK1/2 signaling is necessary for a full calcineurin-induced hypertrophic response. (A) Representative immunocytochemical analyses of actin-stained (phalloidin) neonatal cardiomyocytes treated as indicated. (B) Measurement of cell surface area of

    Journal:

    Article Title: Direct and Indirect Interactions between Calcineurin-NFAT and MEK1-Extracellular Signal-Regulated Kinase 1/2 Signaling Pathways Regulate Cardiac Gene Expression and Cellular Growth

    doi: 10.1128/MCB.25.3.865-878.2005

    Figure Lengend Snippet: MEK1-ERK1/2 signaling is necessary for a full calcineurin-induced hypertrophic response. (A) Representative immunocytochemical analyses of actin-stained (phalloidin) neonatal cardiomyocytes treated as indicated. (B) Measurement of cell surface area of

    Article Snippet: We first assessed the integrity of the activated MEK1 transgene in the heart by Western blotting for MEK1, phosphorylated ERK1/2, and phosphorylated Elk-1 from cardiac protein extracts (Fig. ).

    Techniques: Staining

    CnA β gene targeting reduces MEK1-induced cardiac growth. (A) Western blot analysis of MEK1-ERK1/2 pathway phosphorylation in the hearts of 2-month-old MEK1 transgenic mice (FVB strain) crossed into the CnA β wild-type (Wt) or null backgrounds.

    Journal:

    Article Title: Direct and Indirect Interactions between Calcineurin-NFAT and MEK1-Extracellular Signal-Regulated Kinase 1/2 Signaling Pathways Regulate Cardiac Gene Expression and Cellular Growth

    doi: 10.1128/MCB.25.3.865-878.2005

    Figure Lengend Snippet: CnA β gene targeting reduces MEK1-induced cardiac growth. (A) Western blot analysis of MEK1-ERK1/2 pathway phosphorylation in the hearts of 2-month-old MEK1 transgenic mice (FVB strain) crossed into the CnA β wild-type (Wt) or null backgrounds.

    Article Snippet: We first assessed the integrity of the activated MEK1 transgene in the heart by Western blotting for MEK1, phosphorylated ERK1/2, and phosphorylated Elk-1 from cardiac protein extracts (Fig. ).

    Techniques: Western Blot, Transgenic Assay, Mouse Assay

    MEK1 induces NFAT transcriptional activity. (A) Rat neonatal ventricular cardiomyocytes were plated in serum-free medium and were infected 48 h after plating with AdNFAT-luc together with AdNFATc3, AdMEK1, or Adβgal adenovirus. Cell extracts were

    Journal:

    Article Title: Direct and Indirect Interactions between Calcineurin-NFAT and MEK1-Extracellular Signal-Regulated Kinase 1/2 Signaling Pathways Regulate Cardiac Gene Expression and Cellular Growth

    doi: 10.1128/MCB.25.3.865-878.2005

    Figure Lengend Snippet: MEK1 induces NFAT transcriptional activity. (A) Rat neonatal ventricular cardiomyocytes were plated in serum-free medium and were infected 48 h after plating with AdNFAT-luc together with AdNFATc3, AdMEK1, or Adβgal adenovirus. Cell extracts were

    Article Snippet: We first assessed the integrity of the activated MEK1 transgene in the heart by Western blotting for MEK1, phosphorylated ERK1/2, and phosphorylated Elk-1 from cardiac protein extracts (Fig. ).

    Techniques: Activity Assay, Infection

    Model of the calcineurin-NFAT and MEK1-ERK1/2 signaling module. While calcineurin-NFAT and MEK1-ERK1/2 receive input from stimuli that either mobilize intracellular calcium or result in Ras activation, the full effectiveness of either pathway likely requires

    Journal:

    Article Title: Direct and Indirect Interactions between Calcineurin-NFAT and MEK1-Extracellular Signal-Regulated Kinase 1/2 Signaling Pathways Regulate Cardiac Gene Expression and Cellular Growth

    doi: 10.1128/MCB.25.3.865-878.2005

    Figure Lengend Snippet: Model of the calcineurin-NFAT and MEK1-ERK1/2 signaling module. While calcineurin-NFAT and MEK1-ERK1/2 receive input from stimuli that either mobilize intracellular calcium or result in Ras activation, the full effectiveness of either pathway likely requires

    Article Snippet: We first assessed the integrity of the activated MEK1 transgene in the heart by Western blotting for MEK1, phosphorylated ERK1/2, and phosphorylated Elk-1 from cardiac protein extracts (Fig. ).

    Techniques: Activation Assay

    MEK1-induced NFAT transcriptional activity is independent of calcineurin. (A) CnB1 -deleted mouse embryonic fibroblasts were obtained as described in Materials and Methods. Cell extracts were analyzed by Western blotting with specific antibodies for CnB1.

    Journal:

    Article Title: Direct and Indirect Interactions between Calcineurin-NFAT and MEK1-Extracellular Signal-Regulated Kinase 1/2 Signaling Pathways Regulate Cardiac Gene Expression and Cellular Growth

    doi: 10.1128/MCB.25.3.865-878.2005

    Figure Lengend Snippet: MEK1-induced NFAT transcriptional activity is independent of calcineurin. (A) CnB1 -deleted mouse embryonic fibroblasts were obtained as described in Materials and Methods. Cell extracts were analyzed by Western blotting with specific antibodies for CnB1.

    Article Snippet: We first assessed the integrity of the activated MEK1 transgene in the heart by Western blotting for MEK1, phosphorylated ERK1/2, and phosphorylated Elk-1 from cardiac protein extracts (Fig. ).

    Techniques: Activity Assay, Western Blot

    MEK1 does not increase NFAT transactivation potential. (A) Schematic representation of NFATc3 and the different Gal4 fusion proteins that were generated. (B) A Gal4-dependent luciferase reporter construct (1.0 μg) was cotransfected into neonatal

    Journal:

    Article Title: Direct and Indirect Interactions between Calcineurin-NFAT and MEK1-Extracellular Signal-Regulated Kinase 1/2 Signaling Pathways Regulate Cardiac Gene Expression and Cellular Growth

    doi: 10.1128/MCB.25.3.865-878.2005

    Figure Lengend Snippet: MEK1 does not increase NFAT transactivation potential. (A) Schematic representation of NFATc3 and the different Gal4 fusion proteins that were generated. (B) A Gal4-dependent luciferase reporter construct (1.0 μg) was cotransfected into neonatal

    Article Snippet: We first assessed the integrity of the activated MEK1 transgene in the heart by Western blotting for MEK1, phosphorylated ERK1/2, and phosphorylated Elk-1 from cardiac protein extracts (Fig. ).

    Techniques: Generated, Luciferase, Construct

    Calcineurin-NFATc3-MEK1-ERK2 form a complex in vivo. (A) Western blots (WB) for ERK2, CnA, or MEK1 following immunoprecipitation (IP) of CnA from the indicated cardiomyocyte cell extracts, generated from cell cultures previously subjected to adenoviral

    Journal:

    Article Title: Direct and Indirect Interactions between Calcineurin-NFAT and MEK1-Extracellular Signal-Regulated Kinase 1/2 Signaling Pathways Regulate Cardiac Gene Expression and Cellular Growth

    doi: 10.1128/MCB.25.3.865-878.2005

    Figure Lengend Snippet: Calcineurin-NFATc3-MEK1-ERK2 form a complex in vivo. (A) Western blots (WB) for ERK2, CnA, or MEK1 following immunoprecipitation (IP) of CnA from the indicated cardiomyocyte cell extracts, generated from cell cultures previously subjected to adenoviral

    Article Snippet: We first assessed the integrity of the activated MEK1 transgene in the heart by Western blotting for MEK1, phosphorylated ERK1/2, and phosphorylated Elk-1 from cardiac protein extracts (Fig. ).

    Techniques: In Vivo, Western Blot, Immunoprecipitation, Generated

    Effects of proteins kinases inhibitors on the expression of non-phosphorylated CTNNB induced by DPN in the Sertoli cells from 20-day-old rats . Sertoli cells from 20-day-old rats were incubated in the absence (C, control) and presence of DPN (10 nM) for 24 h. The cells were also untreated or pretreated with PP2, an inhibitor of the SRC family of protein tyrosine kinases; U0126, an inhibitor of MEK1/2; Wortmannin, an inhibitor of PI3K and MK-2206, an inhibitor of AKT for 30 min. Afterward, the cells were stimulated with DPN (10 nM) 24h. Immunostaining for CTNNB (green) was detected using an antibody specific for non-phosphorylated CTNNB and Alexa Fluor 488-labeled secondary antibody. Nuclei were stained with DAPI (blue). Negative control was performed using normal rabbit serum at the same dilution of antibody. Scale bar as indicated. The data shown are representative of three independent experiments. Note: Control, C, and DPN are the same results of the Figure 5 . All treatments were performed on the same day (Figures 5 and 6 ).

    Journal: Heliyon

    Article Title: The role of estrogen receptors in rat Sertoli cells at different stages of development

    doi: 10.1016/j.heliyon.2020.e05363

    Figure Lengend Snippet: Effects of proteins kinases inhibitors on the expression of non-phosphorylated CTNNB induced by DPN in the Sertoli cells from 20-day-old rats . Sertoli cells from 20-day-old rats were incubated in the absence (C, control) and presence of DPN (10 nM) for 24 h. The cells were also untreated or pretreated with PP2, an inhibitor of the SRC family of protein tyrosine kinases; U0126, an inhibitor of MEK1/2; Wortmannin, an inhibitor of PI3K and MK-2206, an inhibitor of AKT for 30 min. Afterward, the cells were stimulated with DPN (10 nM) 24h. Immunostaining for CTNNB (green) was detected using an antibody specific for non-phosphorylated CTNNB and Alexa Fluor 488-labeled secondary antibody. Nuclei were stained with DAPI (blue). Negative control was performed using normal rabbit serum at the same dilution of antibody. Scale bar as indicated. The data shown are representative of three independent experiments. Note: Control, C, and DPN are the same results of the Figure 5 . All treatments were performed on the same day (Figures 5 and 6 ).

    Article Snippet: The cells were also untreated or pretreated with ESR2-selective antagonist PHTPP (4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo(1,5-a)pyrimidin-3-yl)phenol; 10 nM; Tocris Bioscience), the selective inhibitor of the SRC family of protein tyrosine kinases PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine; 5 nM; Calbiochem, Darmstadt, Germany), MEK1/2 inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2- aminophenylthio] butadiene; 20 μM; Cell Signaling Technology), PI3K inhibitor Wortmannin (11-(acetyloxy)-1S,6bR,7,8,9aS,10,11R,11bR-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3,2-de]indeno[4,5-h]-2- benzopyran-3,6,9-trione; 100 nM; Sigma Chemical Co.) or AKT inhibitor MK-2206 (8-(4-(1-aminocyclobutyl)phenyl)-9-phenyl-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3(2H)-one; 200 nM; Selleckchem, Houston, TX, USA) for 30 min.

    Techniques: Expressing, Incubation, Immunostaining, Labeling, Staining, Negative Control

    T3SS1-specific induction of several transcripts requires MEK1/2 activity (A) . Immunoblot showing phosphorylated ERK1 and ERK2 (p-ERK1/2) and total ERK1/2 in POR3:T3SS1 + –infected primary human fibroblasts treated with DMSO (vehicle) or U0126 to demonstrate the activity of U0126 as a MEK inhibitor in primary fibroblasts. N=3 independent experiments. (B–E) qRT-PCR showing the expression of RHOB, JUN, FOS, PTGS2, EGR1, KLF2, and ATF3 in primary human fibroblasts that were pretreated with the MEK1/2 inhibitor U0126 or DMSO before being infected with POR3:T3SS1 + or POR4:T3SS1 − compared to uninfected cells (UN). Expression was normalized to the housekeeping gene IPO8 . Data are 2 −ΔΔCq ± SD, N = 3 experiments. * p

    Journal: Science signaling

    Article Title: Cytotoxic Vibrio T3SS1 Rewires Host Gene Expression to Subvert Cell Death Signaling and Activate Cell Survival Networks

    doi: 10.1126/scisignal.aal4501

    Figure Lengend Snippet: T3SS1-specific induction of several transcripts requires MEK1/2 activity (A) . Immunoblot showing phosphorylated ERK1 and ERK2 (p-ERK1/2) and total ERK1/2 in POR3:T3SS1 + –infected primary human fibroblasts treated with DMSO (vehicle) or U0126 to demonstrate the activity of U0126 as a MEK inhibitor in primary fibroblasts. N=3 independent experiments. (B–E) qRT-PCR showing the expression of RHOB, JUN, FOS, PTGS2, EGR1, KLF2, and ATF3 in primary human fibroblasts that were pretreated with the MEK1/2 inhibitor U0126 or DMSO before being infected with POR3:T3SS1 + or POR4:T3SS1 − compared to uninfected cells (UN). Expression was normalized to the housekeeping gene IPO8 . Data are 2 −ΔΔCq ± SD, N = 3 experiments. * p

    Article Snippet: For MEK1/2 inhibitor experiments, cells were starved in plain DMEM for 30 minutes and then incubated with 10μM U0126 (Cell Signaling) or 10μM DMSO (vehicle) for one hour prior to infection.

    Techniques: Activity Assay, Infection, Quantitative RT-PCR, Expressing

    Elevated miR-30d suppresses the MEK/ERK and PI3K/Akt signaling pathways in SK-ES-1 cells. (A) Western blot assay revealed that overexpression of miR-30d reduced the expression levels of p-MEK1/2 and p-ERK1/2, but caused no changes in the levels of MEK1/2 and ERK1/2. (B) Ratios of p-MEK/MEK and p-ERK/ERK in miR-30d mimic group were decreased compared with those in the untreated group (**P

    Journal: Oncology Letters

    Article Title: miR-30d inhibits cell biological progression of Ewing's sarcoma by suppressing the MEK/ERK and PI3K/Akt pathways in vitro

    doi: 10.3892/ol.2018.7900

    Figure Lengend Snippet: Elevated miR-30d suppresses the MEK/ERK and PI3K/Akt signaling pathways in SK-ES-1 cells. (A) Western blot assay revealed that overexpression of miR-30d reduced the expression levels of p-MEK1/2 and p-ERK1/2, but caused no changes in the levels of MEK1/2 and ERK1/2. (B) Ratios of p-MEK/MEK and p-ERK/ERK in miR-30d mimic group were decreased compared with those in the untreated group (**P

    Article Snippet: Antibodies against MMP-2 (cat. no. 40994), MMP-9 (cat. no. 13667), Bax (cat. no. 5023), Bcl-2 (cat. no. 4223), caspase-3 (cat. no. 9665), PARP (cat. no. 9532), MEK1/2 (cat. no. 8727), ERK1/2 (cat. no. 4695), phosphorylated MEK1/2 (p-MEK1/2) (cat. no. 9154), phosphorylated ERK1/2 (p-ERK1/2) (cat. no. 4370), phosphoinositide 3-kinase (PI3K) (cat. no. 4249), Akt (cat. no. 4691), phosphorylated Akt (p-Akt) (cat. no. 4060) and β-actin (cat. no. 4970) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Western Blot, Over Expression, Expressing

    SO 2 derivatives did not inhibit the activation of Erk1/2, MEK1/2 and c-Raf molecules directly. ( a ) After transfected with different plasmids as described in the figure, HEK293 cells in experimental groups were treated with Na 2 SO 3 /NaHSO 3 at 15 μ mol/l for 24 h. The phosphorylation of Erk1/2 was assessed by western blotting. * P

    Journal: Cell Death & Disease

    Article Title: Sulfur dioxide inhibits vascular smooth muscle cell proliferation via suppressing the Erk/MAP kinase pathway mediated by cAMP/PKA signaling

    doi: 10.1038/cddis.2014.229

    Figure Lengend Snippet: SO 2 derivatives did not inhibit the activation of Erk1/2, MEK1/2 and c-Raf molecules directly. ( a ) After transfected with different plasmids as described in the figure, HEK293 cells in experimental groups were treated with Na 2 SO 3 /NaHSO 3 at 15 μ mol/l for 24 h. The phosphorylation of Erk1/2 was assessed by western blotting. * P

    Article Snippet: The primary antibodies anti-cyclin D1, anti-c-Raf, anti-phospho-c-Raf (Ser338), anti-phospho-c-Raf (Ser259), phosphor-MEK1/2 (Ser217/221), phosphor-Erk1/2 (Thr202/Tyr204), and anti-PKA and anti-phospho-PKA (Thr197) were all from Cell Signaling Technology; anti-phospho-c-Raf (Ser43) was from Abcam (Cambridge, MA, USA); anti-Erk1/2 and anti-GAPDH were from Kangcheng (Shanghai, China); and MEK1/2 and anti-β -actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Activation Assay, Transfection, Western Blot

    A schematic diagram of the inhibition of proliferation by SO 2 and the pathways. PDGF-BB can promote VSMC proliferation by activating Erk/MAPK pathway after combining with its specific RTKs. The increasing cAMP concentration in VSMCs induces PKA activation, which can disturb Erk/MAPK signaling transduction by inhibiting c-Raf activation. SO 2 derivatives can suppress the activities of Erk1/2, MEK1/2 and c-Raf, which does not result from separate targeting interferences of SO 2 derivatives on the three molecules but from the inhibition of upstream signaling by SO 2 derivatives. SO 2 derivatives might enhance adenylyl cyclase activity to elevate cAMP production from ATP and then activate PKA signal to block c-Raf activation, which finally contributes to the suppression of PDGF-BB-induced VSMC proliferation by SO 2 derivatives

    Journal: Cell Death & Disease

    Article Title: Sulfur dioxide inhibits vascular smooth muscle cell proliferation via suppressing the Erk/MAP kinase pathway mediated by cAMP/PKA signaling

    doi: 10.1038/cddis.2014.229

    Figure Lengend Snippet: A schematic diagram of the inhibition of proliferation by SO 2 and the pathways. PDGF-BB can promote VSMC proliferation by activating Erk/MAPK pathway after combining with its specific RTKs. The increasing cAMP concentration in VSMCs induces PKA activation, which can disturb Erk/MAPK signaling transduction by inhibiting c-Raf activation. SO 2 derivatives can suppress the activities of Erk1/2, MEK1/2 and c-Raf, which does not result from separate targeting interferences of SO 2 derivatives on the three molecules but from the inhibition of upstream signaling by SO 2 derivatives. SO 2 derivatives might enhance adenylyl cyclase activity to elevate cAMP production from ATP and then activate PKA signal to block c-Raf activation, which finally contributes to the suppression of PDGF-BB-induced VSMC proliferation by SO 2 derivatives

    Article Snippet: The primary antibodies anti-cyclin D1, anti-c-Raf, anti-phospho-c-Raf (Ser338), anti-phospho-c-Raf (Ser259), phosphor-MEK1/2 (Ser217/221), phosphor-Erk1/2 (Thr202/Tyr204), and anti-PKA and anti-phospho-PKA (Thr197) were all from Cell Signaling Technology; anti-phospho-c-Raf (Ser43) was from Abcam (Cambridge, MA, USA); anti-Erk1/2 and anti-GAPDH were from Kangcheng (Shanghai, China); and MEK1/2 and anti-β -actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Inhibition, Concentration Assay, Activation Assay, Transduction, Activity Assay, Blocking Assay

    The crosstalk between cAMP/PKA and Erk/MAPK pathways mediated SO 2 -suppressed Erk/MAPK signaling. ( a – c ) Cells, starved for 24 h, were pretreated with or without Na 2 SO 3 /NaHSO 3 at 15 μ mol/l for 30 min and treated with or without PDGF-BB at 20 ng/ml for 30 min. Cellular proteins were extracted for western blotting to determine the phosphorylations of Erk1/2, MEK1/2 and c-Raf on Ser338, Ser259 and Ser43. * P

    Journal: Cell Death & Disease

    Article Title: Sulfur dioxide inhibits vascular smooth muscle cell proliferation via suppressing the Erk/MAP kinase pathway mediated by cAMP/PKA signaling

    doi: 10.1038/cddis.2014.229

    Figure Lengend Snippet: The crosstalk between cAMP/PKA and Erk/MAPK pathways mediated SO 2 -suppressed Erk/MAPK signaling. ( a – c ) Cells, starved for 24 h, were pretreated with or without Na 2 SO 3 /NaHSO 3 at 15 μ mol/l for 30 min and treated with or without PDGF-BB at 20 ng/ml for 30 min. Cellular proteins were extracted for western blotting to determine the phosphorylations of Erk1/2, MEK1/2 and c-Raf on Ser338, Ser259 and Ser43. * P

    Article Snippet: The primary antibodies anti-cyclin D1, anti-c-Raf, anti-phospho-c-Raf (Ser338), anti-phospho-c-Raf (Ser259), phosphor-MEK1/2 (Ser217/221), phosphor-Erk1/2 (Thr202/Tyr204), and anti-PKA and anti-phospho-PKA (Thr197) were all from Cell Signaling Technology; anti-phospho-c-Raf (Ser43) was from Abcam (Cambridge, MA, USA); anti-Erk1/2 and anti-GAPDH were from Kangcheng (Shanghai, China); and MEK1/2 and anti-β -actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Western Blot

    Role ERK1/2 in PA-regulated MMP-2 and TIMP-2 expression in HeLa cells. ( A ) Cells were treated with various concentrations of PA (0 to 30 μM) for 24 h, then harvested and lysed for measurement target proteins by western blotting; ( B ) cells were treated with or without PA in the absence or presence of PD98059 (specific MEK1/2 inhibitor) for 24 h, followed by measurement of migration and invasion; ( C ) cells were treated as above for 24 h, followed by measurement of relative wound width; ( D , E ) Protein and mRNA expression of MMP-2 and TIMP-2 were measured by western blotting assay and RT-qPCR. Values are means and standard errors of 3 replicates. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Praeruptorin A Inhibits Human Cervical Cancer Cell Growth and Invasion by Suppressing MMP-2 Expression and ERK1/2 Signaling

    doi: 10.3390/ijms19010010

    Figure Lengend Snippet: Role ERK1/2 in PA-regulated MMP-2 and TIMP-2 expression in HeLa cells. ( A ) Cells were treated with various concentrations of PA (0 to 30 μM) for 24 h, then harvested and lysed for measurement target proteins by western blotting; ( B ) cells were treated with or without PA in the absence or presence of PD98059 (specific MEK1/2 inhibitor) for 24 h, followed by measurement of migration and invasion; ( C ) cells were treated as above for 24 h, followed by measurement of relative wound width; ( D , E ) Protein and mRNA expression of MMP-2 and TIMP-2 were measured by western blotting assay and RT-qPCR. Values are means and standard errors of 3 replicates. ** p

    Article Snippet: The MEK1/2 inhibitor PD98059 was purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Expressing, Western Blot, Migration, Quantitative RT-PCR

    Western blotting of Neuro2a lysates from WT, Lypla1 −/− , Lypla2 −/− , and DKO cells. Relative changes were quantified in triplicate analysis, and representative lanes are shown. A: Phospho-MEK1/2 (p-MEK) is significantly increased in DKO cells. B: Phospho-ERK1/2 (p-ERK) is significantly increased in DKO cells. * P

    Journal: Journal of Lipid Research

    Article Title: Lysophospholipases cooperate to mediate lipid homeostasis and lysophospholipid signaling

    doi: 10.1194/jlr.M087890

    Figure Lengend Snippet: Western blotting of Neuro2a lysates from WT, Lypla1 −/− , Lypla2 −/− , and DKO cells. Relative changes were quantified in triplicate analysis, and representative lanes are shown. A: Phospho-MEK1/2 (p-MEK) is significantly increased in DKO cells. B: Phospho-ERK1/2 (p-ERK) is significantly increased in DKO cells. * P

    Article Snippet: Marnett.), rabbit anti-ERK1/2 [1:1,000 v/v, Cell Signaling Technologies (CST), Danvers, MA], rabbit anti-phospho-ERK1/2 (1:1,000 v/v, CST), rabbit anti-MEK1/2 (1:1,000 v/v, CST), rabbit anti-phospho-MEK1/2 (1:1,000 v/v, CST), or goat anti-β-actin (1:5,000 v/v, Santa Cruz Biotechnologies, Santa Cruz, CA) overnight at 4°C.

    Techniques: Western Blot

    Expression of mutant T210A Plk1 inhibits the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, and cell proliferation. a Smooth muscle cells were transfected with plasmids encoding Flag-wild type (WT) or T210A Plk1. Blots of cell extracts were detected with antibodies against Flag and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells transfected with plasmids were normalized to untransfected cells. Values are mean ± SE ( n = 4, * P

    Journal: Respiratory Research

    Article Title: Plk1 regulates MEK1/2 and proliferation in airway smooth muscle cells

    doi: 10.1186/s12931-015-0257-8

    Figure Lengend Snippet: Expression of mutant T210A Plk1 inhibits the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, and cell proliferation. a Smooth muscle cells were transfected with plasmids encoding Flag-wild type (WT) or T210A Plk1. Blots of cell extracts were detected with antibodies against Flag and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells transfected with plasmids were normalized to untransfected cells. Values are mean ± SE ( n = 4, * P

    Article Snippet: Antibodies used were anti-phospho-Plk1 (Abcam), anti-Plk1 (EMD Millipore), anti-phospho-MEK1/2 (Santa Cruz Biotech.), anti-MEK1/2 (Santa Cruz Biotech.), anti-phospho-ERK1/2 (Cell signaling), anti-ERK1/2 (Cell signaling), anti-phospho-Raf-1 (Santa Cruz Biotech.), anti-Raf-1 (Santa Cruz Biotech.) and anti-GAPDH (Ambion).

    Techniques: Expressing, Mutagenesis, Transfection

    Stimulation with PDGF induces the phosphorylation of Plk1, MEK1/2 and ERK1/2 in smooth muscle cells. Human airway smooth muscle (HASM) cells were stimulated with 20 ng/ml PDGF for different durations or left unstimulated. The phosphorylation of Plk1 ( a ), MEK1/2 ( b ) and ERK1/2 ( c ) in these cells was evaluated by immunoblot analysis. The phosphorylation levels of the proteins in stimulated cells were normalized to corresponding unstimulated cells. * Significantly higher phosphorylation levels in stimulated cells compared with unstimulated cells ( P

    Journal: Respiratory Research

    Article Title: Plk1 regulates MEK1/2 and proliferation in airway smooth muscle cells

    doi: 10.1186/s12931-015-0257-8

    Figure Lengend Snippet: Stimulation with PDGF induces the phosphorylation of Plk1, MEK1/2 and ERK1/2 in smooth muscle cells. Human airway smooth muscle (HASM) cells were stimulated with 20 ng/ml PDGF for different durations or left unstimulated. The phosphorylation of Plk1 ( a ), MEK1/2 ( b ) and ERK1/2 ( c ) in these cells was evaluated by immunoblot analysis. The phosphorylation levels of the proteins in stimulated cells were normalized to corresponding unstimulated cells. * Significantly higher phosphorylation levels in stimulated cells compared with unstimulated cells ( P

    Article Snippet: Antibodies used were anti-phospho-Plk1 (Abcam), anti-Plk1 (EMD Millipore), anti-phospho-MEK1/2 (Santa Cruz Biotech.), anti-MEK1/2 (Santa Cruz Biotech.), anti-phospho-ERK1/2 (Cell signaling), anti-ERK1/2 (Cell signaling), anti-phospho-Raf-1 (Santa Cruz Biotech.), anti-Raf-1 (Santa Cruz Biotech.) and anti-GAPDH (Ambion).

    Techniques:

    Knockdown (KD) of Plk1 attenuates the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, but not Raf-1 phosphorylation. a Representative immunoblots illustrating the effects of Plk1shRNA on Plk1 expression. Blots of HASM cells infected with lentiviruses encoding control shRNA or Plk1 shRNA were probed with antibodies against Plk1 and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells producing Plk1 shRNA were normalized to ratios obtained from cells producing control shRNA. Values are mean ± SE ( n = 4). *Significantly lower Plk1/GAPDH ratios in cells producing Plk1 shRNA compared with cells producing control shRNA ( P

    Journal: Respiratory Research

    Article Title: Plk1 regulates MEK1/2 and proliferation in airway smooth muscle cells

    doi: 10.1186/s12931-015-0257-8

    Figure Lengend Snippet: Knockdown (KD) of Plk1 attenuates the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, but not Raf-1 phosphorylation. a Representative immunoblots illustrating the effects of Plk1shRNA on Plk1 expression. Blots of HASM cells infected with lentiviruses encoding control shRNA or Plk1 shRNA were probed with antibodies against Plk1 and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells producing Plk1 shRNA were normalized to ratios obtained from cells producing control shRNA. Values are mean ± SE ( n = 4). *Significantly lower Plk1/GAPDH ratios in cells producing Plk1 shRNA compared with cells producing control shRNA ( P

    Article Snippet: Antibodies used were anti-phospho-Plk1 (Abcam), anti-Plk1 (EMD Millipore), anti-phospho-MEK1/2 (Santa Cruz Biotech.), anti-MEK1/2 (Santa Cruz Biotech.), anti-phospho-ERK1/2 (Cell signaling), anti-ERK1/2 (Cell signaling), anti-phospho-Raf-1 (Santa Cruz Biotech.), anti-Raf-1 (Santa Cruz Biotech.) and anti-GAPDH (Ambion).

    Techniques: Western Blot, Expressing, Infection, shRNA