raf-1 Search Results


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  • 96
    Millipore raf 1
    Raf 1, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/raf 1/product/Millipore
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    93
    Santa Cruz Biotechnology raf 1
    Shoc2 and HUWE1 form a complex with M-Ras and <t>RAF-1.</t> (A) Cos1-SR cells were transfected with HA–M-Ras. HA–M-Ras was immunoprecipitated, and precipitates were then analyzed by immunoblotting using anti-HUWE1, anti-RAF-1, and anti-tRFP antibodies.
    Raf 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 843 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology anti raf 1
    Fig. 6. Severe block in BCR-mediated ERK activation in <t>Raf-1/B-Raf</t> double deficient DT40 cells. Populations of DT40MCM/ raf -1 flE3 / B- raf flE6 cells were harvested at 5 days after induction of MCM-mediated deletion (+) and stimulated with M4. Total cellular lysates were subjected to western blot analysis. This result is representative of two experiments with an identical procedure (two independent clones) and at least five other experiments with a similar design. The apparent decrease in the level of ERK2 is caused by sequential detection of phospho-ERK followed by detection with anti-ERK2 antibody on the same western blot membrane.
    Anti Raf 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript raf 1
    Effect of zinc on the in vitro association of the <t>Raf-1/ERK1/2</t> cassette with MOR. (A, B) PAG synaptosomes were incubated with zinc chloride for 4 h at 4°C. (C) The time course for the effect of 3 μ M zinc on MOR association with PKCγ and Raf-1; incubation was conducted at room temperature with and without 100 μ M TPEN. At the end of the procedure, free zinc was removed by three runs of centrifugation washing. Afterward, MOR was immunoprecipitated, and associated proteins were detected with the corresponding antibodies. The study was repeated 3 times on different PAG preparations. Data are expressed relative to their control (no zinc, arbitrary value of 1) as mean±SEM. Equal loading was determined by the MOR signals. *Significantly different from the control group (no zinc), p
    Raf 1, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc raf 1
    Blood endothelial <t>RAF1</t> S259A expression induces lymphatic EC fate specification.
    Raf 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/raf 1/product/Cell Signaling Technology Inc
    Average 92 stars, based on 202 article reviews
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    93
    Horizon Discovery raf1
    <t>Raf1-tr</t> exhibits unique protein binding partners: A ) Outline of experimental procedure for mass spectrometry identification of Raf1-binding partners. B ) Venn diagram of proteins identified to be unique to Raf1-tr (left) and Raf1-fl (right). Proteins that bound both Raf1 species fall in the middle. C ) Volcano plot of proteins that exhibit decreased (left) or increased (right) binding to Raf1-tr relative to Raf1-fl binding. Thresholds of P
    Raf1, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson raf 1
    <t>Raf-1</t> knockdown affects phosphorylation levels of downstream target proteins. (A) Total cell lysates (TCL) from Co- and Eu-hESC cells were analysed for Raf-1, pE/R/M, phospho-paxillin (pPax), E/R/M, pMYPT1, MYPT1 and α-tubulin, 48 hrs after their transfection with either Raf-1 or control siRNA. The Raf-1 and pPax levels were normalized by total α-tubulin. The pE/R/M and pMYPT1 levels were normalized by E/R/M and MYPT1 respectively. Representative blots from biological triplicates (left panel) and graphical representation of Raf-1, pE/R/M, pPax and pMYPT1 (right panel) are shown as protein levels in% (mean value ± S.D.) relative to their normalized levels (mean value set to 100%) in cells transfected with control siRNA, ** P
    Raf 1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 265 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    raf 1  (Abcam)
    95
    Abcam raf 1
    Molecular characterization of <t>RAF1-deficient</t> lesions. ( a ) Immunoblotting of F/F and Δp/np livers collected 30 weeks after DEN treatment. The plots represents a densitometric quantification of the immunoblot performed using ImageJ. The data are expressed as relative band intensity adjusted to TUBA or ACTB, which serve as loading controls (upper plot). Phosphorylation is expressed as the ratio between the phosphospecific antibody signal and the signal obtained with the protein-specific antibody. In both cases, the data are normalized to the F/F non-tumour samples, which were arbitrarily set as 1. ( b ) Immunoblot analysis of signaling pathways in xenograft samples ( n =3, analysed 40 days after transplant). The plots show a quantification of the immunoblots performed as described in ( a ). ( c ) YAP1 expression in the same patient cohort examined in Fig. 1a . Scale bar, 50 μm. Left panel, representative IHC image. Middle panel, comparison of YAP1 expression in matched tumour and non-tumour tissue. Right panel, YAP1 expression in tumours correlates positively with tumour grade and the ratio of RAF1/YAP1 expression in the same tumour negatively correlates with histological grade. ( d ) STAT3 expression in the same cohort. Left panel, representative IHC image. Middle panel, comparison of STAT3 expression in matched tumour and non-tumour tissue. Right panel, RAF1/YAP1 expression in the same tumour negatively correlated with the presence of medium-large clusters of STAT3 nuclear staining. Scale bar 50 μm. In ( a , b ), the data are represented as mean±s.e.m., * P ≤0.05, ** P
    Raf 1, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/raf 1/product/Abcam
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    88
    Cell Signaling Technology Inc p raf1
    Suppression of phosphatidylethanolamine binding protein 1 (PEBP1) degradation eliminates the receptor-interacting protein kinases 4 (RIPK4)-induced activation of the <t>RAF1/MEK/ERK</t> pathway and pancreatic cancer cell migration and invasion. (A) Total and phosphorylated levels of RAF1, MEK1/2 and ERK1/2 in RIPK4-overexpressing and control cells following treatment with MG132 (an inhibitor of the 26S proteasome). (B and C) The effects of MG132-mediated suppression of PEBP1 degradation on RIPK4-overexpressing pancreatic cancer cell (B) migration and (C) invasion. (D) Proposed model of the mechanisms through which RIPK4 promotes pancreatic cancer cell migration and invasion via the PEBP1 degradation-induced activation of the RAF1/MEK/ERK pathway.
    P Raf1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Upstate Biotechnology Inc raf 1
    (A) GILZ sequence alignment with a set of TSC family proteins (upper part of diagram) and alignment of NH 2 -terminal sequence of GILZ to 1vig as proposed by threading server (3DPSSM) (lower part of diagram). PSS indicates the predicted secondary structure for the N-terminal domain. SS indicates the known secondary structure of the library template 1vig . (B) 3D model of human GILZ. α-Helices are represented as light blue cylinders, β-sheets are represented as red ribbons, and proline residues are indicated in red CPK. (C) Molecular modeling of <t>Raf-1-GILZ</t> interaction. Distribution of solutions (i.e., the 100, 50, and 10 top solutions found) of docking experiments of Raf-1 and GILZ; the red balls indicate the center of mass of Raf-1, for each solution, interacting with GILZ.
    Raf 1, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/raf 1/product/Upstate Biotechnology Inc
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    90
    Upstate Biotechnology Inc anti raf 1
    (A) GILZ sequence alignment with a set of TSC family proteins (upper part of diagram) and alignment of NH 2 -terminal sequence of GILZ to 1vig as proposed by threading server (3DPSSM) (lower part of diagram). PSS indicates the predicted secondary structure for the N-terminal domain. SS indicates the known secondary structure of the library template 1vig . (B) 3D model of human GILZ. α-Helices are represented as light blue cylinders, β-sheets are represented as red ribbons, and proline residues are indicated in red CPK. (C) Molecular modeling of <t>Raf-1-GILZ</t> interaction. Distribution of solutions (i.e., the 100, 50, and 10 top solutions found) of docking experiments of Raf-1 and GILZ; the red balls indicate the center of mass of Raf-1, for each solution, interacting with GILZ.
    Anti Raf 1, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 90/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Abnova p raf1
    (A) GILZ sequence alignment with a set of TSC family proteins (upper part of diagram) and alignment of NH 2 -terminal sequence of GILZ to 1vig as proposed by threading server (3DPSSM) (lower part of diagram). PSS indicates the predicted secondary structure for the N-terminal domain. SS indicates the known secondary structure of the library template 1vig . (B) 3D model of human GILZ. α-Helices are represented as light blue cylinders, β-sheets are represented as red ribbons, and proline residues are indicated in red CPK. (C) Molecular modeling of <t>Raf-1-GILZ</t> interaction. Distribution of solutions (i.e., the 100, 50, and 10 top solutions found) of docking experiments of Raf-1 and GILZ; the red balls indicate the center of mass of Raf-1, for each solution, interacting with GILZ.
    P Raf1, supplied by Abnova, used in various techniques. Bioz Stars score: 86/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Shoc2 and HUWE1 form a complex with M-Ras and RAF-1. (A) Cos1-SR cells were transfected with HA–M-Ras. HA–M-Ras was immunoprecipitated, and precipitates were then analyzed by immunoblotting using anti-HUWE1, anti-RAF-1, and anti-tRFP antibodies.

    Journal: Molecular and Cellular Biology

    Article Title: HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold

    doi: 10.1128/MCB.00811-14

    Figure Lengend Snippet: Shoc2 and HUWE1 form a complex with M-Ras and RAF-1. (A) Cos1-SR cells were transfected with HA–M-Ras. HA–M-Ras was immunoprecipitated, and precipitates were then analyzed by immunoblotting using anti-HUWE1, anti-RAF-1, and anti-tRFP antibodies.

    Article Snippet: Similarly, we also observed that overexpression of RAF-1 does not lead to an increased ERK1/2 phosphorylation (see Fig. S6C in the supplemental material), yet hyperactivation of RAF-1 up to a certain threshold was reported to induce the cell cycle and cell proliferation ( , ).

    Techniques: Transfection, Immunoprecipitation

    Diminished Shoc2 ubiquitination alters the ubiquitination of RAF-1 and ERK1/2 signaling. (A) RAF-1 was immunoprecipitated from HeLa cells stably expressing either WT Shoc2-YFP or Shoc2 7KR mutant-YFP. The ubiquitination of RAF-1 was detected with anti-Ub

    Journal: Molecular and Cellular Biology

    Article Title: HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold

    doi: 10.1128/MCB.00811-14

    Figure Lengend Snippet: Diminished Shoc2 ubiquitination alters the ubiquitination of RAF-1 and ERK1/2 signaling. (A) RAF-1 was immunoprecipitated from HeLa cells stably expressing either WT Shoc2-YFP or Shoc2 7KR mutant-YFP. The ubiquitination of RAF-1 was detected with anti-Ub

    Article Snippet: Similarly, we also observed that overexpression of RAF-1 does not lead to an increased ERK1/2 phosphorylation (see Fig. S6C in the supplemental material), yet hyperactivation of RAF-1 up to a certain threshold was reported to induce the cell cycle and cell proliferation ( , ).

    Techniques: Immunoprecipitation, Stable Transfection, Expressing, Mutagenesis

    HUWE1 regulates ubiquitination of Shoc2 and RAF-1. (A) Shoc2 was precipitated from HeLa cells using Shoc2 antibodies. Shoc2 ubiquitination was detected with anti-UB antibodies. Immunoprecipitates and lysates were analyzed by immunoblotting using anti-HUWE1

    Journal: Molecular and Cellular Biology

    Article Title: HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold

    doi: 10.1128/MCB.00811-14

    Figure Lengend Snippet: HUWE1 regulates ubiquitination of Shoc2 and RAF-1. (A) Shoc2 was precipitated from HeLa cells using Shoc2 antibodies. Shoc2 ubiquitination was detected with anti-UB antibodies. Immunoprecipitates and lysates were analyzed by immunoblotting using anti-HUWE1

    Article Snippet: Similarly, we also observed that overexpression of RAF-1 does not lead to an increased ERK1/2 phosphorylation (see Fig. S6C in the supplemental material), yet hyperactivation of RAF-1 up to a certain threshold was reported to induce the cell cycle and cell proliferation ( , ).

    Techniques:

    Model recapitulating the role played by HUWE1 in regulating Shoc2-supported ERK1/2 activity. (A) Shoc2 incorporates the E3 ligase HUWE1 into the Ras and RAF-1 signaling complex. HUWE1-mediated ubiquitination of Shoc2 is necessary for the ubiquitination

    Journal: Molecular and Cellular Biology

    Article Title: HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold

    doi: 10.1128/MCB.00811-14

    Figure Lengend Snippet: Model recapitulating the role played by HUWE1 in regulating Shoc2-supported ERK1/2 activity. (A) Shoc2 incorporates the E3 ligase HUWE1 into the Ras and RAF-1 signaling complex. HUWE1-mediated ubiquitination of Shoc2 is necessary for the ubiquitination

    Article Snippet: Similarly, we also observed that overexpression of RAF-1 does not lead to an increased ERK1/2 phosphorylation (see Fig. S6C in the supplemental material), yet hyperactivation of RAF-1 up to a certain threshold was reported to induce the cell cycle and cell proliferation ( , ).

    Techniques: Activity Assay

    HUWE1 regulates the stability of RAF-1. (A) 293FT, Cos1, or HeLa cells were transiently transfected with HUWE1 siRNA. At 48 h after transfection, cells were harvested for immunoblotting. The expression of Shoc2, HUWE1, B-RAF, A-RAF, RAF-1, EGFR, MEK1/2,

    Journal: Molecular and Cellular Biology

    Article Title: HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold

    doi: 10.1128/MCB.00811-14

    Figure Lengend Snippet: HUWE1 regulates the stability of RAF-1. (A) 293FT, Cos1, or HeLa cells were transiently transfected with HUWE1 siRNA. At 48 h after transfection, cells were harvested for immunoblotting. The expression of Shoc2, HUWE1, B-RAF, A-RAF, RAF-1, EGFR, MEK1/2,

    Article Snippet: Similarly, we also observed that overexpression of RAF-1 does not lead to an increased ERK1/2 phosphorylation (see Fig. S6C in the supplemental material), yet hyperactivation of RAF-1 up to a certain threshold was reported to induce the cell cycle and cell proliferation ( , ).

    Techniques: Transfection, Expressing

    HUWE1 regulates the ubiquitination of RAF-1 in a Shoc2-dependent manner. HeLa-NT, HeLa-LV1, and HeLa-SR cells were transiently transfected with HUWE1 siRNA. At 48 h posttransfection, the cells were treated with MG132. RAF-1 was precipitated using anti-RAF-1

    Journal: Molecular and Cellular Biology

    Article Title: HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold

    doi: 10.1128/MCB.00811-14

    Figure Lengend Snippet: HUWE1 regulates the ubiquitination of RAF-1 in a Shoc2-dependent manner. HeLa-NT, HeLa-LV1, and HeLa-SR cells were transiently transfected with HUWE1 siRNA. At 48 h posttransfection, the cells were treated with MG132. RAF-1 was precipitated using anti-RAF-1

    Article Snippet: Similarly, we also observed that overexpression of RAF-1 does not lead to an increased ERK1/2 phosphorylation (see Fig. S6C in the supplemental material), yet hyperactivation of RAF-1 up to a certain threshold was reported to induce the cell cycle and cell proliferation ( , ).

    Techniques: Transfection

    Molecular characterization of RAF1-deficient lesions. ( a ) Immunoblotting of F/F and Δp/np livers collected 30 weeks after DEN treatment. The plots represents a densitometric quantification of the immunoblot performed using ImageJ. The data are expressed as relative band intensity adjusted to TUBA or ACTB, which serve as loading controls (upper plot). Phosphorylation is expressed as the ratio between the phosphospecific antibody signal and the signal obtained with the protein-specific antibody. In both cases, the data are normalized to the F/F non-tumour samples, which were arbitrarily set as 1. ( b ) Immunoblot analysis of signaling pathways in xenograft samples ( n =3, analysed 40 days after transplant). The plots show a quantification of the immunoblots performed as described in ( a ). ( c ) YAP1 expression in the same patient cohort examined in Fig. 1a . Scale bar, 50 μm. Left panel, representative IHC image. Middle panel, comparison of YAP1 expression in matched tumour and non-tumour tissue. Right panel, YAP1 expression in tumours correlates positively with tumour grade and the ratio of RAF1/YAP1 expression in the same tumour negatively correlates with histological grade. ( d ) STAT3 expression in the same cohort. Left panel, representative IHC image. Middle panel, comparison of STAT3 expression in matched tumour and non-tumour tissue. Right panel, RAF1/YAP1 expression in the same tumour negatively correlated with the presence of medium-large clusters of STAT3 nuclear staining. Scale bar 50 μm. In ( a , b ), the data are represented as mean±s.e.m., * P ≤0.05, ** P

    Journal: Nature Communications

    Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

    doi: 10.1038/ncomms13781

    Figure Lengend Snippet: Molecular characterization of RAF1-deficient lesions. ( a ) Immunoblotting of F/F and Δp/np livers collected 30 weeks after DEN treatment. The plots represents a densitometric quantification of the immunoblot performed using ImageJ. The data are expressed as relative band intensity adjusted to TUBA or ACTB, which serve as loading controls (upper plot). Phosphorylation is expressed as the ratio between the phosphospecific antibody signal and the signal obtained with the protein-specific antibody. In both cases, the data are normalized to the F/F non-tumour samples, which were arbitrarily set as 1. ( b ) Immunoblot analysis of signaling pathways in xenograft samples ( n =3, analysed 40 days after transplant). The plots show a quantification of the immunoblots performed as described in ( a ). ( c ) YAP1 expression in the same patient cohort examined in Fig. 1a . Scale bar, 50 μm. Left panel, representative IHC image. Middle panel, comparison of YAP1 expression in matched tumour and non-tumour tissue. Right panel, YAP1 expression in tumours correlates positively with tumour grade and the ratio of RAF1/YAP1 expression in the same tumour negatively correlates with histological grade. ( d ) STAT3 expression in the same cohort. Left panel, representative IHC image. Middle panel, comparison of STAT3 expression in matched tumour and non-tumour tissue. Right panel, RAF1/YAP1 expression in the same tumour negatively correlated with the presence of medium-large clusters of STAT3 nuclear staining. Scale bar 50 μm. In ( a , b ), the data are represented as mean±s.e.m., * P ≤0.05, ** P

    Article Snippet: The following antibodies were used for immunoblotting (1:1,000 unless otherwise stated): YAP1 (4912, 1:500), pYAP1-S127 (4911), pLATS1-T1079 (8654), LATS1 (3477), pMST1(T183)/MST2(T180) (3681), MST2 (3952), pERK1/2 (9101), ERK1/2 (9102), pSTAT3Y705 (9145), STAT3 (9139), PCNA (2586) all Cell Signaling Technology; GP130 (sc-656), ACTB (sc-1616), pCFL1S3 (sc-12912), ALB (sc-50536), ARAF (sc-408) BRAF (sc-9002), RAF1 (sc-133; all Santa Cruz Biotechnology; RAF1 (610152, 1:500), CD44 (550538), βcatenin (610153) from BD Biosciences; AFP (46799) and pYAP1Y357 (62751) from Abcam; ROKα (04-841, Millipore)and TUBA (T9206, Sigma, 1:10,000).

    Techniques: Western Blot, Expressing, Immunohistochemistry, Staining

    RAF1 ablation increases the number of cancer progenitor cells. ( a ) Quantification of Ki67+ liver cells 8 (top panel) or 12 weeks (bottom panel) after DEN treatment. ( b ) Foci of altered hepatocytes (FAH) in F/F and Δhep or Δp/np livers isolated 12 weeks after DEN injection. Sections were stained with H E or with the indicated antibodies. FAH are delimited by dotted circles ( n =3 per genotype). Scale bars, 50 μm. ( c ) Percentage of cancer progenitor cells (CD44+/CD31−Ter119−CD45−) present in non-aggregate and aggregate fractions of F/F and Δp/np livers, as determined by FACS analysis. Data are represented as mean±s.e.m., * P ≤0.05, ** P

    Journal: Nature Communications

    Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

    doi: 10.1038/ncomms13781

    Figure Lengend Snippet: RAF1 ablation increases the number of cancer progenitor cells. ( a ) Quantification of Ki67+ liver cells 8 (top panel) or 12 weeks (bottom panel) after DEN treatment. ( b ) Foci of altered hepatocytes (FAH) in F/F and Δhep or Δp/np livers isolated 12 weeks after DEN injection. Sections were stained with H E or with the indicated antibodies. FAH are delimited by dotted circles ( n =3 per genotype). Scale bars, 50 μm. ( c ) Percentage of cancer progenitor cells (CD44+/CD31−Ter119−CD45−) present in non-aggregate and aggregate fractions of F/F and Δp/np livers, as determined by FACS analysis. Data are represented as mean±s.e.m., * P ≤0.05, ** P

    Article Snippet: The following antibodies were used for immunoblotting (1:1,000 unless otherwise stated): YAP1 (4912, 1:500), pYAP1-S127 (4911), pLATS1-T1079 (8654), LATS1 (3477), pMST1(T183)/MST2(T180) (3681), MST2 (3952), pERK1/2 (9101), ERK1/2 (9102), pSTAT3Y705 (9145), STAT3 (9139), PCNA (2586) all Cell Signaling Technology; GP130 (sc-656), ACTB (sc-1616), pCFL1S3 (sc-12912), ALB (sc-50536), ARAF (sc-408) BRAF (sc-9002), RAF1 (sc-133; all Santa Cruz Biotechnology; RAF1 (610152, 1:500), CD44 (550538), βcatenin (610153) from BD Biosciences; AFP (46799) and pYAP1Y357 (62751) from Abcam; ROKα (04-841, Millipore)and TUBA (T9206, Sigma, 1:10,000).

    Techniques: Isolation, Injection, Staining, FACS

    Effect of YAP1 silencing, the P6 JAK inhibitor and GP130 silencing on DIH and Hep3B proliferation. ( a ) siRNA-mediated RAF1 silencing in Hep3B cells increases YAP1 and GP130 expression and STAT3 activation without impacting ERK phosphorylation or β-catenin expression/localization. Immunoblot analysis of post-nuclear fraction (PNF; 20 μg, about 8% of total) and nuclear fraction (Nuclei; 20 μg, about 15% of total). ( b ) Silencing of YAP1 in RAF1-proficient and -deficient Hep3B cells (left panel, representative immunoblot analysis) downregulates the expression of the YAP1 target gene CTGF (middle panel, qPCR analysis) and reduces proliferation (right panel). ( c ) Treatment with the JAK inhibitor P6 abrogates STAT3 phosphorylation without impacting ERK phosphorylation or YAP1 expression (left panel, representative immunoblot analysis), decreases BIRC5 expression (middle panel, qPCR analysis) and reduces proliferation in RAF1-deficient Hep3B cells (right panel). ( d,e ) Similar results are obtained by subjecting RAF1-proficient and -deficient DIH to YAP1 silencing ( d ) or P6 treatment ( e ). ( f ) GP130 silencing decreases STAT3 phosphorylation but does not affect YAP1 expression or phosphorylation. Proliferation was assessed 48 h after siRNA transfection (with the exception of c , in which P6 was added 24 h after transfection and proliferation was measured after additional 48 h), gene expression after 24 h, and for immunoblotting cells were lysed after 1 h inhibitor treatment. In ( f ) DIH were treated for 30 min with the indicated concentration of IL6. Experiments were performed in DMEM supplemented with 10% FBS (Hep3B cells) or in DIH medium supplemented with 5% FBS (DIH). The immunoblots are representative of two independent experiments; TUBA was used as loading control. The plots represent the mean±s.e.m. of three independent experiments. * P ≤0.05, ** P

    Journal: Nature Communications

    Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

    doi: 10.1038/ncomms13781

    Figure Lengend Snippet: Effect of YAP1 silencing, the P6 JAK inhibitor and GP130 silencing on DIH and Hep3B proliferation. ( a ) siRNA-mediated RAF1 silencing in Hep3B cells increases YAP1 and GP130 expression and STAT3 activation without impacting ERK phosphorylation or β-catenin expression/localization. Immunoblot analysis of post-nuclear fraction (PNF; 20 μg, about 8% of total) and nuclear fraction (Nuclei; 20 μg, about 15% of total). ( b ) Silencing of YAP1 in RAF1-proficient and -deficient Hep3B cells (left panel, representative immunoblot analysis) downregulates the expression of the YAP1 target gene CTGF (middle panel, qPCR analysis) and reduces proliferation (right panel). ( c ) Treatment with the JAK inhibitor P6 abrogates STAT3 phosphorylation without impacting ERK phosphorylation or YAP1 expression (left panel, representative immunoblot analysis), decreases BIRC5 expression (middle panel, qPCR analysis) and reduces proliferation in RAF1-deficient Hep3B cells (right panel). ( d,e ) Similar results are obtained by subjecting RAF1-proficient and -deficient DIH to YAP1 silencing ( d ) or P6 treatment ( e ). ( f ) GP130 silencing decreases STAT3 phosphorylation but does not affect YAP1 expression or phosphorylation. Proliferation was assessed 48 h after siRNA transfection (with the exception of c , in which P6 was added 24 h after transfection and proliferation was measured after additional 48 h), gene expression after 24 h, and for immunoblotting cells were lysed after 1 h inhibitor treatment. In ( f ) DIH were treated for 30 min with the indicated concentration of IL6. Experiments were performed in DMEM supplemented with 10% FBS (Hep3B cells) or in DIH medium supplemented with 5% FBS (DIH). The immunoblots are representative of two independent experiments; TUBA was used as loading control. The plots represent the mean±s.e.m. of three independent experiments. * P ≤0.05, ** P

    Article Snippet: The following antibodies were used for immunoblotting (1:1,000 unless otherwise stated): YAP1 (4912, 1:500), pYAP1-S127 (4911), pLATS1-T1079 (8654), LATS1 (3477), pMST1(T183)/MST2(T180) (3681), MST2 (3952), pERK1/2 (9101), ERK1/2 (9102), pSTAT3Y705 (9145), STAT3 (9139), PCNA (2586) all Cell Signaling Technology; GP130 (sc-656), ACTB (sc-1616), pCFL1S3 (sc-12912), ALB (sc-50536), ARAF (sc-408) BRAF (sc-9002), RAF1 (sc-133; all Santa Cruz Biotechnology; RAF1 (610152, 1:500), CD44 (550538), βcatenin (610153) from BD Biosciences; AFP (46799) and pYAP1Y357 (62751) from Abcam; ROKα (04-841, Millipore)and TUBA (T9206, Sigma, 1:10,000).

    Techniques: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Transfection, Concentration Assay, Western Blot

    Molecular characterization of RAF1-deficient cells. ( a ) siRNA-mediated RAF1 silencing promotes the proliferation of Hep3B ( n =6), HuH-7 ( n =5) and HepG2 ( n =4) cells and increases the expression of YAP1 and GP130 as well as STAT3 phosphorylation. siRAF1#1 targets the region around nucleotide 721, while siRAF1#2 is a mixture of siRNAs targeting the region from nucleotide 692 to 1,093 in the RAF1 mRNA. ( b ) PCR and immunoblotting analysis of F/F and RAF1Δ/Δ DIH. ( c ) Morphology (x200 magnification) and molecular characterization ( d ) of primary hepatocytes (P-HEPS) compared to DIH (AFP, α-fetoprotein; ALB, albumin; TUBA, loading control). The immunoblot is representative of two independent experiments. ( e ) Proliferation of DIH in decreasing amounts of FBS. ( f ) Molecular defects of RAF1-deficient P-HEPS and DIH treated with the indicated concentrations of IL6 for 30 min. TUBA serves as loading control. Data are presented as mean±s.e.m. * P ≤0.05, ** P

    Journal: Nature Communications

    Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

    doi: 10.1038/ncomms13781

    Figure Lengend Snippet: Molecular characterization of RAF1-deficient cells. ( a ) siRNA-mediated RAF1 silencing promotes the proliferation of Hep3B ( n =6), HuH-7 ( n =5) and HepG2 ( n =4) cells and increases the expression of YAP1 and GP130 as well as STAT3 phosphorylation. siRAF1#1 targets the region around nucleotide 721, while siRAF1#2 is a mixture of siRNAs targeting the region from nucleotide 692 to 1,093 in the RAF1 mRNA. ( b ) PCR and immunoblotting analysis of F/F and RAF1Δ/Δ DIH. ( c ) Morphology (x200 magnification) and molecular characterization ( d ) of primary hepatocytes (P-HEPS) compared to DIH (AFP, α-fetoprotein; ALB, albumin; TUBA, loading control). The immunoblot is representative of two independent experiments. ( e ) Proliferation of DIH in decreasing amounts of FBS. ( f ) Molecular defects of RAF1-deficient P-HEPS and DIH treated with the indicated concentrations of IL6 for 30 min. TUBA serves as loading control. Data are presented as mean±s.e.m. * P ≤0.05, ** P

    Article Snippet: The following antibodies were used for immunoblotting (1:1,000 unless otherwise stated): YAP1 (4912, 1:500), pYAP1-S127 (4911), pLATS1-T1079 (8654), LATS1 (3477), pMST1(T183)/MST2(T180) (3681), MST2 (3952), pERK1/2 (9101), ERK1/2 (9102), pSTAT3Y705 (9145), STAT3 (9139), PCNA (2586) all Cell Signaling Technology; GP130 (sc-656), ACTB (sc-1616), pCFL1S3 (sc-12912), ALB (sc-50536), ARAF (sc-408) BRAF (sc-9002), RAF1 (sc-133; all Santa Cruz Biotechnology; RAF1 (610152, 1:500), CD44 (550538), βcatenin (610153) from BD Biosciences; AFP (46799) and pYAP1Y357 (62751) from Abcam; ROKα (04-841, Millipore)and TUBA (T9206, Sigma, 1:10,000).

    Techniques: Expressing, Polymerase Chain Reaction

    RAF1 ablation correlates with decreased YAP1 and GP130 protein turnover in Hep3B cells, primary hepatocytes (P-HEPS), and DIH. ( a-c ) qPCR analysis showing the expression of the YAP1 and Gp130 genes in Hep3B ( a ), P-HEPS ( b ) and DIH ( c ). qPCR data represent the mean (±s.e.m.) of three independent experiments; according to Student's t test. ( d-f ) Cells were treated with cycloheximide for the indicated amount of time prior to lysis. YAP1 and GP130 expression levels were determined by immunoblotting. A quantification is shown in the right panel; the amount of protein present in each of the untreated samples (normalized to TUBA or ACTB as loading controls) is set as 1.

    Journal: Nature Communications

    Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

    doi: 10.1038/ncomms13781

    Figure Lengend Snippet: RAF1 ablation correlates with decreased YAP1 and GP130 protein turnover in Hep3B cells, primary hepatocytes (P-HEPS), and DIH. ( a-c ) qPCR analysis showing the expression of the YAP1 and Gp130 genes in Hep3B ( a ), P-HEPS ( b ) and DIH ( c ). qPCR data represent the mean (±s.e.m.) of three independent experiments; according to Student's t test. ( d-f ) Cells were treated with cycloheximide for the indicated amount of time prior to lysis. YAP1 and GP130 expression levels were determined by immunoblotting. A quantification is shown in the right panel; the amount of protein present in each of the untreated samples (normalized to TUBA or ACTB as loading controls) is set as 1.

    Article Snippet: The following antibodies were used for immunoblotting (1:1,000 unless otherwise stated): YAP1 (4912, 1:500), pYAP1-S127 (4911), pLATS1-T1079 (8654), LATS1 (3477), pMST1(T183)/MST2(T180) (3681), MST2 (3952), pERK1/2 (9101), ERK1/2 (9102), pSTAT3Y705 (9145), STAT3 (9139), PCNA (2586) all Cell Signaling Technology; GP130 (sc-656), ACTB (sc-1616), pCFL1S3 (sc-12912), ALB (sc-50536), ARAF (sc-408) BRAF (sc-9002), RAF1 (sc-133; all Santa Cruz Biotechnology; RAF1 (610152, 1:500), CD44 (550538), βcatenin (610153) from BD Biosciences; AFP (46799) and pYAP1Y357 (62751) from Abcam; ROKα (04-841, Millipore)and TUBA (T9206, Sigma, 1:10,000).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Lysis

    RAF1 is expressed at low levels in human HCC and suppresses the growth of both HCC xenografts and chemically induced tumours. ( a ) RAF1 expression in a cohort of 31 HCC patients. Left panel, representative IHC image (T, tumour; NT, non-tumour). Scale bar, 50 μm. Middle panel, RAF1 expression in matched tumour and non-tumour tissue (a.u.=arbitrary units). Right panel, RAF1 expression in tumours correlates inversely with tumour grade (ratio: protein expression in tumour/non-tumour tissue). ( b ) Inducible shRNA-mediated RAF1 silencing does not impact A- or BRAF expression (top panel) but increases the proliferation of Hep3B cells in culture (bottom panel; n =6). ( c ) Inducible shRNA-mediated RAF1 silencing strongly promotes the growth of Hep3B xenografts. ( d,e ) Ablation of RAF1 in liver parenchymal cells promotes chemically induced hepatocarcinogenesis. Top panels, experimental protocols. Left bottom panel, macroscopic appearance of F/F and Δhep ( d ) or Δp/np ( e ) tumour-bearing livers 30 weeks (w) after DEN injection; arrows indicate tumours. Scale bars, 0.5 cm. Middle panels, liver:body weight ratio of untreated or DEN/Pb-treated mice. Right panels, tumour numbers and % of tumour-occupied area in control, Δhep ( d ) and Δp/np ( e ) livers. In ( d ), no DEN: F/F n =4, Δhep=6; DEN-treated: F/F n =7, Δhep=8. In ( e ), DEN-treated: F/F n =10, Δp/np n =11. ( f , g ) Quantification of Ki67+ cells: ( f ) and inflammatory cells ( g ; F4/80+ cells, granulocytes and CD3+ cells) in tumour-bearing F/F and Δhep livers. Np=non-parenchymal cells. ( h ) Chemokine levels in the serum of F/F and Δhep mice. ( i ) Chemo-/cytokine levels in tumour-bearing livers. Data are presented as mean ± SEM, * P ≤0.05, ** P

    Journal: Nature Communications

    Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

    doi: 10.1038/ncomms13781

    Figure Lengend Snippet: RAF1 is expressed at low levels in human HCC and suppresses the growth of both HCC xenografts and chemically induced tumours. ( a ) RAF1 expression in a cohort of 31 HCC patients. Left panel, representative IHC image (T, tumour; NT, non-tumour). Scale bar, 50 μm. Middle panel, RAF1 expression in matched tumour and non-tumour tissue (a.u.=arbitrary units). Right panel, RAF1 expression in tumours correlates inversely with tumour grade (ratio: protein expression in tumour/non-tumour tissue). ( b ) Inducible shRNA-mediated RAF1 silencing does not impact A- or BRAF expression (top panel) but increases the proliferation of Hep3B cells in culture (bottom panel; n =6). ( c ) Inducible shRNA-mediated RAF1 silencing strongly promotes the growth of Hep3B xenografts. ( d,e ) Ablation of RAF1 in liver parenchymal cells promotes chemically induced hepatocarcinogenesis. Top panels, experimental protocols. Left bottom panel, macroscopic appearance of F/F and Δhep ( d ) or Δp/np ( e ) tumour-bearing livers 30 weeks (w) after DEN injection; arrows indicate tumours. Scale bars, 0.5 cm. Middle panels, liver:body weight ratio of untreated or DEN/Pb-treated mice. Right panels, tumour numbers and % of tumour-occupied area in control, Δhep ( d ) and Δp/np ( e ) livers. In ( d ), no DEN: F/F n =4, Δhep=6; DEN-treated: F/F n =7, Δhep=8. In ( e ), DEN-treated: F/F n =10, Δp/np n =11. ( f , g ) Quantification of Ki67+ cells: ( f ) and inflammatory cells ( g ; F4/80+ cells, granulocytes and CD3+ cells) in tumour-bearing F/F and Δhep livers. Np=non-parenchymal cells. ( h ) Chemokine levels in the serum of F/F and Δhep mice. ( i ) Chemo-/cytokine levels in tumour-bearing livers. Data are presented as mean ± SEM, * P ≤0.05, ** P

    Article Snippet: The following antibodies were used for immunoblotting (1:1,000 unless otherwise stated): YAP1 (4912, 1:500), pYAP1-S127 (4911), pLATS1-T1079 (8654), LATS1 (3477), pMST1(T183)/MST2(T180) (3681), MST2 (3952), pERK1/2 (9101), ERK1/2 (9102), pSTAT3Y705 (9145), STAT3 (9139), PCNA (2586) all Cell Signaling Technology; GP130 (sc-656), ACTB (sc-1616), pCFL1S3 (sc-12912), ALB (sc-50536), ARAF (sc-408) BRAF (sc-9002), RAF1 (sc-133; all Santa Cruz Biotechnology; RAF1 (610152, 1:500), CD44 (550538), βcatenin (610153) from BD Biosciences; AFP (46799) and pYAP1Y357 (62751) from Abcam; ROKα (04-841, Millipore)and TUBA (T9206, Sigma, 1:10,000).

    Techniques: Expressing, Immunohistochemistry, shRNA, Injection, Mouse Assay

    Fig. 6. MEK* and ΔRaf trigger MAPK activation when phosphatases are inhibited by okadaic acid (OA). ( A ) ERK immunoblot. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and treated with both puromycin and OA, where they resumed meiosis. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lane 1, control GV oocytes; lanes 2, 3 and 4, oocytes matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( B ) MBP kinase assay. Groups of 10 oocytes were subjected to MBP kinase assay. Oocytes, matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, were either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( C ) MEK* triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2), or injected with MEK* (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. ( D ) ΔRaf triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2) or injected with ΔRaf (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Lane 5: control M II-arrested oocytes. Groups of 25 oocytes were immunoblotted with the anti-ERK serum.

    Journal: The EMBO Journal

    Article Title: Mos activates MAP kinase in mouse oocytes through two opposite pathways

    doi: 10.1093/emboj/19.22.6065

    Figure Lengend Snippet: Fig. 6. MEK* and ΔRaf trigger MAPK activation when phosphatases are inhibited by okadaic acid (OA). ( A ) ERK immunoblot. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and treated with both puromycin and OA, where they resumed meiosis. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lane 1, control GV oocytes; lanes 2, 3 and 4, oocytes matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( B ) MBP kinase assay. Groups of 10 oocytes were subjected to MBP kinase assay. Oocytes, matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, were either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( C ) MEK* triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2), or injected with MEK* (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. ( D ) ΔRaf triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2) or injected with ΔRaf (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Lane 5: control M II-arrested oocytes. Groups of 25 oocytes were immunoblotted with the anti-ERK serum.

    Article Snippet: The Raf1, MEK1 and MAP kinases were detected using the anti-Raf1 , anti-MEK1 (a gift of Steven Pelech) and anti-ERK (no. 691, Santa Cruz Biotechnology, Inc.) polyclonal antibodies, respectively.

    Techniques: Activation Assay, Injection, Cell Culture, Kinase Assay

    Fig. 3. Overexpression of ΔRaf does not induce MAPK activation in puromycin-treated oocytes. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and incubated in 10 µg/ml puromycin. Batches of 25 oocytes were immunoblotted with the anti-ERK serum. Lanes 1 and 5, respectively, control GV oocytes and oocytes matured for 1.5 h post-GVBD in M2 medium; lanes 2, 3 and 4, oocytes matured in puromycin-containing medium and collected 1.5 h post-GVBD, either not injected (lane 2) or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4).

    Journal: The EMBO Journal

    Article Title: Mos activates MAP kinase in mouse oocytes through two opposite pathways

    doi: 10.1093/emboj/19.22.6065

    Figure Lengend Snippet: Fig. 3. Overexpression of ΔRaf does not induce MAPK activation in puromycin-treated oocytes. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and incubated in 10 µg/ml puromycin. Batches of 25 oocytes were immunoblotted with the anti-ERK serum. Lanes 1 and 5, respectively, control GV oocytes and oocytes matured for 1.5 h post-GVBD in M2 medium; lanes 2, 3 and 4, oocytes matured in puromycin-containing medium and collected 1.5 h post-GVBD, either not injected (lane 2) or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4).

    Article Snippet: The Raf1, MEK1 and MAP kinases were detected using the anti-Raf1 , anti-MEK1 (a gift of Steven Pelech) and anti-ERK (no. 691, Santa Cruz Biotechnology, Inc.) polyclonal antibodies, respectively.

    Techniques: Over Expression, Activation Assay, Injection, Cell Culture, Incubation

    Fig. 2. ( A ) Overexpression of constitutively active HA-tagged MEK* after microinjection. Fifty immature oocytes were either not injected (lane 1) or injected with mRNAs encoding HA-tagged MEK* cultured overnight in dbcAMP (lane 2) or cultured for 5 (lane 3) or 12 h (lane 4) after GVBD and then collected. Oocytes microinjected with HA-tagged MEK1S218D/S222D were also cultured for 10 h after GVBD and then incubated for 2 h in OA and collected (lane 5). Samples were then subjected to immunoblotting using an anti-HA antibody. ( B ) Overexpression of Raf1 and ΔRaf after microinjection. Fifty immature oocytes were either not injected (lane 1) or injected with mRNAs encoding full-length Raf1 (lane 2) or ΔRaf (lane 3), cultured overnight in dbcAMP and then collected. Control M II oocytes were also collected to show the endogenous Raf (50 M II, lane 4; 100 M II, lane 5). Samples were then subjected to immunoblotting using the anti-Raf1 serum.

    Journal: The EMBO Journal

    Article Title: Mos activates MAP kinase in mouse oocytes through two opposite pathways

    doi: 10.1093/emboj/19.22.6065

    Figure Lengend Snippet: Fig. 2. ( A ) Overexpression of constitutively active HA-tagged MEK* after microinjection. Fifty immature oocytes were either not injected (lane 1) or injected with mRNAs encoding HA-tagged MEK* cultured overnight in dbcAMP (lane 2) or cultured for 5 (lane 3) or 12 h (lane 4) after GVBD and then collected. Oocytes microinjected with HA-tagged MEK1S218D/S222D were also cultured for 10 h after GVBD and then incubated for 2 h in OA and collected (lane 5). Samples were then subjected to immunoblotting using an anti-HA antibody. ( B ) Overexpression of Raf1 and ΔRaf after microinjection. Fifty immature oocytes were either not injected (lane 1) or injected with mRNAs encoding full-length Raf1 (lane 2) or ΔRaf (lane 3), cultured overnight in dbcAMP and then collected. Control M II oocytes were also collected to show the endogenous Raf (50 M II, lane 4; 100 M II, lane 5). Samples were then subjected to immunoblotting using the anti-Raf1 serum.

    Article Snippet: The Raf1, MEK1 and MAP kinases were detected using the anti-Raf1 , anti-MEK1 (a gift of Steven Pelech) and anti-ERK (no. 691, Santa Cruz Biotechnology, Inc.) polyclonal antibodies, respectively.

    Techniques: Over Expression, Injection, Cell Culture, Incubation

    Fig. 4. ( A ) Overexpression of ΔRaf does not induce MAPK activation in mos –/– oocytes while Mos overexpression does. Mos –/– oocytes were injected with mRNAs encoding either full-length Raf1 (as an injection control), ΔRaf or Mos, cultured for 5 h in dbcAMP then removed from dbcAMP and collected at various times after GVBD. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lanes 1 and 2, respectively, Raf1- and ΔRaf-injected mos –/– oocytes collected 3 h after GVBD; lanes 3 and 4, control mos +/– oocytes matured for 2 (lane 3) or 12 h (lane 4) post-GVBD; lanes 5 and 6, respectively, non-injected and Mos-injected mos –/– oocytes collected 3 h after GVBD. ( B ) Mos, but not MEK* or ΔRaf, restores the M II arrest in mos –/– oocytes. Mos –/– oocytes were either not injected (Control) or were injected with RNAs encoding full-length Mos, constitutively active MEK* or ΔRaf in dbcAMP-containing medium. The oocytes were kept for 5 h in this medium and released in M2 medium for overnight culture. We then scored the oocytes with no polar body (MI), with only one polar body (M II) or with two polar bodies (spontaneously activated oocytes, PB2). The numbers in parentheses represent the total number of oocytes injected. These results correspond to at least three independent experiments. ( C ) Mos –/– oocytes injected with Mos and arrested in M II (top) and spontaneously activated control non-injected oocytes with two polar bodies (bottom).

    Journal: The EMBO Journal

    Article Title: Mos activates MAP kinase in mouse oocytes through two opposite pathways

    doi: 10.1093/emboj/19.22.6065

    Figure Lengend Snippet: Fig. 4. ( A ) Overexpression of ΔRaf does not induce MAPK activation in mos –/– oocytes while Mos overexpression does. Mos –/– oocytes were injected with mRNAs encoding either full-length Raf1 (as an injection control), ΔRaf or Mos, cultured for 5 h in dbcAMP then removed from dbcAMP and collected at various times after GVBD. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lanes 1 and 2, respectively, Raf1- and ΔRaf-injected mos –/– oocytes collected 3 h after GVBD; lanes 3 and 4, control mos +/– oocytes matured for 2 (lane 3) or 12 h (lane 4) post-GVBD; lanes 5 and 6, respectively, non-injected and Mos-injected mos –/– oocytes collected 3 h after GVBD. ( B ) Mos, but not MEK* or ΔRaf, restores the M II arrest in mos –/– oocytes. Mos –/– oocytes were either not injected (Control) or were injected with RNAs encoding full-length Mos, constitutively active MEK* or ΔRaf in dbcAMP-containing medium. The oocytes were kept for 5 h in this medium and released in M2 medium for overnight culture. We then scored the oocytes with no polar body (MI), with only one polar body (M II) or with two polar bodies (spontaneously activated oocytes, PB2). The numbers in parentheses represent the total number of oocytes injected. These results correspond to at least three independent experiments. ( C ) Mos –/– oocytes injected with Mos and arrested in M II (top) and spontaneously activated control non-injected oocytes with two polar bodies (bottom).

    Article Snippet: The Raf1, MEK1 and MAP kinases were detected using the anti-Raf1 , anti-MEK1 (a gift of Steven Pelech) and anti-ERK (no. 691, Santa Cruz Biotechnology, Inc.) polyclonal antibodies, respectively.

    Techniques: Over Expression, Activation Assay, Injection, Cell Culture

    Raf-1 S471 is required for the kinase activity of a constitutively active Raf form, Bxb-Raf, both in vivo and in vitro. (A and B) Serum-deprived COS-7 cells expressing HA-ERK (A) or FLAG-MEK (B) together with wild-type GST-Bxb-Raf or a mutant containing

    Journal: Molecular Biology of the Cell

    Article Title: Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

    doi: 10.1091/mbc.E05-02-0090

    Figure Lengend Snippet: Raf-1 S471 is required for the kinase activity of a constitutively active Raf form, Bxb-Raf, both in vivo and in vitro. (A and B) Serum-deprived COS-7 cells expressing HA-ERK (A) or FLAG-MEK (B) together with wild-type GST-Bxb-Raf or a mutant containing

    Article Snippet: Phospho-MEK, phospho-ERK, MEK, and ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA); FLAG antibody from Sigma-Aldrich (St. Louis, MO); glutathione S -transferase (GST) antibody was from Upstate Biotechnology (Lake Placid, NY); HSC-70 and Raf-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); heat-shock protein (HSP)-70 antibody was from Affinity Bioreagents (Golden, CO); and hemagglutinin (HA) and myc epitope tag antibodies were produced from the 12CA5 and 9E10 hybridoma cell lines, respectively.

    Techniques: Activity Assay, In Vivo, In Vitro, Expressing, Mutagenesis

    B-Raf S578, corresponding to Raf-1 S471, is required for B-Raf kinase activation by EGF but not for the constitutive kinase activity induced by the V599E cancer associated mutation. Serum-deprived COS-7 cells expressing HA-ERK alone (lanes 1 and 2) or

    Journal: Molecular Biology of the Cell

    Article Title: Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

    doi: 10.1091/mbc.E05-02-0090

    Figure Lengend Snippet: B-Raf S578, corresponding to Raf-1 S471, is required for B-Raf kinase activation by EGF but not for the constitutive kinase activity induced by the V599E cancer associated mutation. Serum-deprived COS-7 cells expressing HA-ERK alone (lanes 1 and 2) or

    Article Snippet: Phospho-MEK, phospho-ERK, MEK, and ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA); FLAG antibody from Sigma-Aldrich (St. Louis, MO); glutathione S -transferase (GST) antibody was from Upstate Biotechnology (Lake Placid, NY); HSC-70 and Raf-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); heat-shock protein (HSP)-70 antibody was from Affinity Bioreagents (Golden, CO); and hemagglutinin (HA) and myc epitope tag antibodies were produced from the 12CA5 and 9E10 hybridoma cell lines, respectively.

    Techniques: Activation Assay, Activity Assay, Mutagenesis, Expressing

    Dephosphorylation of Raf-1, primarily at EGF-induced sites, results in Raf-1 kinase inactivation. (A and B) Serum-deprived COS-7 cells expressing myc-Raf-1 S259A mutant were metabolically labeled with 32 P for 2 h, followed by stimulation with 100 ng/ml

    Journal: Molecular Biology of the Cell

    Article Title: Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

    doi: 10.1091/mbc.E05-02-0090

    Figure Lengend Snippet: Dephosphorylation of Raf-1, primarily at EGF-induced sites, results in Raf-1 kinase inactivation. (A and B) Serum-deprived COS-7 cells expressing myc-Raf-1 S259A mutant were metabolically labeled with 32 P for 2 h, followed by stimulation with 100 ng/ml

    Article Snippet: Phospho-MEK, phospho-ERK, MEK, and ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA); FLAG antibody from Sigma-Aldrich (St. Louis, MO); glutathione S -transferase (GST) antibody was from Upstate Biotechnology (Lake Placid, NY); HSC-70 and Raf-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); heat-shock protein (HSP)-70 antibody was from Affinity Bioreagents (Golden, CO); and hemagglutinin (HA) and myc epitope tag antibodies were produced from the 12CA5 and 9E10 hybridoma cell lines, respectively.

    Techniques: De-Phosphorylation Assay, Expressing, Mutagenesis, Metabolic Labelling, Labeling

    Raf-1 S471 is critical for Raf-1 function in vivo. Serum-deprived COS-7 cells expressing myc-Raf-1 variants and FLAG-MEK (A) or HA-ERK (B and C), as indicated, were stimulated with 100 ng/ml EGF or vehicle for 15 min. Raf-1 activity in the cells was examined

    Journal: Molecular Biology of the Cell

    Article Title: Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

    doi: 10.1091/mbc.E05-02-0090

    Figure Lengend Snippet: Raf-1 S471 is critical for Raf-1 function in vivo. Serum-deprived COS-7 cells expressing myc-Raf-1 variants and FLAG-MEK (A) or HA-ERK (B and C), as indicated, were stimulated with 100 ng/ml EGF or vehicle for 15 min. Raf-1 activity in the cells was examined

    Article Snippet: Phospho-MEK, phospho-ERK, MEK, and ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA); FLAG antibody from Sigma-Aldrich (St. Louis, MO); glutathione S -transferase (GST) antibody was from Upstate Biotechnology (Lake Placid, NY); HSC-70 and Raf-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); heat-shock protein (HSP)-70 antibody was from Affinity Bioreagents (Golden, CO); and hemagglutinin (HA) and myc epitope tag antibodies were produced from the 12CA5 and 9E10 hybridoma cell lines, respectively.

    Techniques: In Vivo, Expressing, Activity Assay

    Kinase activity comparison of Raf-1 phosphorylation site mutants. The indicated Raf-1 phosphorylation site mutants were expressed in COS-7 cells, and their kinase activity was assayed using the coupled Raf kinase assay. Cells were stimulated with 100

    Journal: Molecular Biology of the Cell

    Article Title: Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

    doi: 10.1091/mbc.E05-02-0090

    Figure Lengend Snippet: Kinase activity comparison of Raf-1 phosphorylation site mutants. The indicated Raf-1 phosphorylation site mutants were expressed in COS-7 cells, and their kinase activity was assayed using the coupled Raf kinase assay. Cells were stimulated with 100

    Article Snippet: Phospho-MEK, phospho-ERK, MEK, and ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA); FLAG antibody from Sigma-Aldrich (St. Louis, MO); glutathione S -transferase (GST) antibody was from Upstate Biotechnology (Lake Placid, NY); HSC-70 and Raf-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); heat-shock protein (HSP)-70 antibody was from Affinity Bioreagents (Golden, CO); and hemagglutinin (HA) and myc epitope tag antibodies were produced from the 12CA5 and 9E10 hybridoma cell lines, respectively.

    Techniques: Activity Assay, Kinase Assay

    Identification of HSP-70 and HSC-70 as Raf-1 associated proteins. (A and B) Serum-deprived COS-7 cells expressing wild-type myc-Raf-1 (lanes 2–4) or control vector (lane 1) were incubated with vehicle (lanes 1–3) or with 20 μM

    Journal: Molecular Biology of the Cell

    Article Title: Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

    doi: 10.1091/mbc.E05-02-0090

    Figure Lengend Snippet: Identification of HSP-70 and HSC-70 as Raf-1 associated proteins. (A and B) Serum-deprived COS-7 cells expressing wild-type myc-Raf-1 (lanes 2–4) or control vector (lane 1) were incubated with vehicle (lanes 1–3) or with 20 μM

    Article Snippet: Phospho-MEK, phospho-ERK, MEK, and ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA); FLAG antibody from Sigma-Aldrich (St. Louis, MO); glutathione S -transferase (GST) antibody was from Upstate Biotechnology (Lake Placid, NY); HSC-70 and Raf-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); heat-shock protein (HSP)-70 antibody was from Affinity Bioreagents (Golden, CO); and hemagglutinin (HA) and myc epitope tag antibodies were produced from the 12CA5 and 9E10 hybridoma cell lines, respectively.

    Techniques: Expressing, Plasmid Preparation, Incubation

    Identification of Raf-1 S29, S289, S296, S301, and S471/T481 as novel Raf-1 phosphorylation sites. (A) Schematic diagram of Raf-1 showing known phosphorylation sites (top) and the newly identified phosphorylation sites (bottom). (B) myc-Raf-1 proteins

    Journal: Molecular Biology of the Cell

    Article Title: Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

    doi: 10.1091/mbc.E05-02-0090

    Figure Lengend Snippet: Identification of Raf-1 S29, S289, S296, S301, and S471/T481 as novel Raf-1 phosphorylation sites. (A) Schematic diagram of Raf-1 showing known phosphorylation sites (top) and the newly identified phosphorylation sites (bottom). (B) myc-Raf-1 proteins

    Article Snippet: Phospho-MEK, phospho-ERK, MEK, and ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA); FLAG antibody from Sigma-Aldrich (St. Louis, MO); glutathione S -transferase (GST) antibody was from Upstate Biotechnology (Lake Placid, NY); HSC-70 and Raf-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); heat-shock protein (HSP)-70 antibody was from Affinity Bioreagents (Golden, CO); and hemagglutinin (HA) and myc epitope tag antibodies were produced from the 12CA5 and 9E10 hybridoma cell lines, respectively.

    Techniques:

    Raf-1 phosphorylation sites. (A) Schematic diagram of Raf-1 showing known phosphorylation sites and potential kinases reported to phosphorylate these sites. The locations of the kinase domain, the Ras binding domain (RBD), the cysteine-rich domain (CRD),

    Journal: Molecular Biology of the Cell

    Article Title: Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

    doi: 10.1091/mbc.E05-02-0090

    Figure Lengend Snippet: Raf-1 phosphorylation sites. (A) Schematic diagram of Raf-1 showing known phosphorylation sites and potential kinases reported to phosphorylate these sites. The locations of the kinase domain, the Ras binding domain (RBD), the cysteine-rich domain (CRD),

    Article Snippet: Phospho-MEK, phospho-ERK, MEK, and ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA); FLAG antibody from Sigma-Aldrich (St. Louis, MO); glutathione S -transferase (GST) antibody was from Upstate Biotechnology (Lake Placid, NY); HSC-70 and Raf-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); heat-shock protein (HSP)-70 antibody was from Affinity Bioreagents (Golden, CO); and hemagglutinin (HA) and myc epitope tag antibodies were produced from the 12CA5 and 9E10 hybridoma cell lines, respectively.

    Techniques: Binding Assay

    Raf-1 S471 is required for Raf-1-MEK binding but not for Raf-1 dimerization or Raf-1 binding to Ras and 14-3-3. (A) COS-7 cells were transfected with FLAG-Ras G12V together with myc-Raf-1forms as indicated. myc-Raf-1 recovery in FLAG-Ras immunoprecipitates

    Journal: Molecular Biology of the Cell

    Article Title: Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

    doi: 10.1091/mbc.E05-02-0090

    Figure Lengend Snippet: Raf-1 S471 is required for Raf-1-MEK binding but not for Raf-1 dimerization or Raf-1 binding to Ras and 14-3-3. (A) COS-7 cells were transfected with FLAG-Ras G12V together with myc-Raf-1forms as indicated. myc-Raf-1 recovery in FLAG-Ras immunoprecipitates

    Article Snippet: Phospho-MEK, phospho-ERK, MEK, and ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA); FLAG antibody from Sigma-Aldrich (St. Louis, MO); glutathione S -transferase (GST) antibody was from Upstate Biotechnology (Lake Placid, NY); HSC-70 and Raf-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); heat-shock protein (HSP)-70 antibody was from Affinity Bioreagents (Golden, CO); and hemagglutinin (HA) and myc epitope tag antibodies were produced from the 12CA5 and 9E10 hybridoma cell lines, respectively.

    Techniques: Binding Assay, Transfection

    Raf-1 S471 is a critical site for Raf-1 kinase activity. Serum-deprived COS-7 cells expressing wild-type myc-Raf-1 or the indicated myc-Raf-1 S471-substituted mutants (A and B), or the indicated myc-Raf-1 T481-substituted mutants (C and D) were stimulated

    Journal: Molecular Biology of the Cell

    Article Title: Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

    doi: 10.1091/mbc.E05-02-0090

    Figure Lengend Snippet: Raf-1 S471 is a critical site for Raf-1 kinase activity. Serum-deprived COS-7 cells expressing wild-type myc-Raf-1 or the indicated myc-Raf-1 S471-substituted mutants (A and B), or the indicated myc-Raf-1 T481-substituted mutants (C and D) were stimulated

    Article Snippet: Phospho-MEK, phospho-ERK, MEK, and ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA); FLAG antibody from Sigma-Aldrich (St. Louis, MO); glutathione S -transferase (GST) antibody was from Upstate Biotechnology (Lake Placid, NY); HSC-70 and Raf-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); heat-shock protein (HSP)-70 antibody was from Affinity Bioreagents (Golden, CO); and hemagglutinin (HA) and myc epitope tag antibodies were produced from the 12CA5 and 9E10 hybridoma cell lines, respectively.

    Techniques: Activity Assay, Expressing

    TNF α induces ERK1/2 activation in a Ras-independent manner and induces Raf-1 kinase activity in a FLIP-L-dependent manner. ( a ) Serum-deprived PC12 cells were treated with 100 ng/ml of TNF α or NGF for 5 min, and activated Ras was pulled down using Raf-RBD conjugated agarose beads. GTP-bound Ras was detected by western blot using an anti-pan-Ras antibody. ( b ) Endogenous Raf-1 was immunoprecipitated from PC12 cells treated with TNF α or NGF and immunoprecipitates were incubated with recombinant MEK and ATP in vitro in order to detect Raf-1 kinase activity. Western blot analysis was performed for Raf-1 and P-MEK1. Inputs were blotted using anti-Raf-1, anti-P-ERK1/2, and anti-ERK1/2 antibody as a loading control. ( c ) Raf-1 kinase activity was assessed as in b , in PC12 cells transfected with Neo or SR-I κ B α , ( d ) or after 3 days of PC12 transduction with scrambled sequence (shRNA Scr) or shRNA against FLIP-L (shRNA FLIP-L) lentiviruses. Black lines indicate that intervening lanes have been spliced. ( e ) Serum-deprived PC12 cells were treated with TNF α for the indicated times prior harvesting and subcellular fractionation. Lysates corresponding to cytosolic (C) and membrane fractions (M) were resolved by SDS-PAGE and Raf-1 subcellular localization was assessed by western blot using an anti-Raf-1 antibody. ERK1/2 phosphorylation was also detected to control MAPK/ERK activation following TNF α stimulation. ( f ) PC12 cells were transfected with siRNA targeting Raf-1 or a scrambled sequence. Three days after transfection, cells were treated with TNF α for the indicated time points and western blot was performed to detect P-ERK1/2, Raf-1 and total ERK1/2 as loading control. ( g ) PC12 cells were transfected with pcDNA3-HA-FLIP-L, pcDNA3-Raf-1 or both plasmids. Cells were harvested 24 h later and FLIP-L was immunoprecipitated using a specific anti-FLIP antibody prior western blot using anti-Raf-1 antibody. Transfection efficiency of both plasmids was checked in the inputs. The asterisk indicates nonspecific bands

    Journal: Cell Death & Disease

    Article Title: TNFα induces survival through the FLIP-L-dependent activation of the MAPK/ERK pathway

    doi: 10.1038/cddis.2013.25

    Figure Lengend Snippet: TNF α induces ERK1/2 activation in a Ras-independent manner and induces Raf-1 kinase activity in a FLIP-L-dependent manner. ( a ) Serum-deprived PC12 cells were treated with 100 ng/ml of TNF α or NGF for 5 min, and activated Ras was pulled down using Raf-RBD conjugated agarose beads. GTP-bound Ras was detected by western blot using an anti-pan-Ras antibody. ( b ) Endogenous Raf-1 was immunoprecipitated from PC12 cells treated with TNF α or NGF and immunoprecipitates were incubated with recombinant MEK and ATP in vitro in order to detect Raf-1 kinase activity. Western blot analysis was performed for Raf-1 and P-MEK1. Inputs were blotted using anti-Raf-1, anti-P-ERK1/2, and anti-ERK1/2 antibody as a loading control. ( c ) Raf-1 kinase activity was assessed as in b , in PC12 cells transfected with Neo or SR-I κ B α , ( d ) or after 3 days of PC12 transduction with scrambled sequence (shRNA Scr) or shRNA against FLIP-L (shRNA FLIP-L) lentiviruses. Black lines indicate that intervening lanes have been spliced. ( e ) Serum-deprived PC12 cells were treated with TNF α for the indicated times prior harvesting and subcellular fractionation. Lysates corresponding to cytosolic (C) and membrane fractions (M) were resolved by SDS-PAGE and Raf-1 subcellular localization was assessed by western blot using an anti-Raf-1 antibody. ERK1/2 phosphorylation was also detected to control MAPK/ERK activation following TNF α stimulation. ( f ) PC12 cells were transfected with siRNA targeting Raf-1 or a scrambled sequence. Three days after transfection, cells were treated with TNF α for the indicated time points and western blot was performed to detect P-ERK1/2, Raf-1 and total ERK1/2 as loading control. ( g ) PC12 cells were transfected with pcDNA3-HA-FLIP-L, pcDNA3-Raf-1 or both plasmids. Cells were harvested 24 h later and FLIP-L was immunoprecipitated using a specific anti-FLIP antibody prior western blot using anti-Raf-1 antibody. Transfection efficiency of both plasmids was checked in the inputs. The asterisk indicates nonspecific bands

    Article Snippet: Raf-1 dependent phosphotransferase activity is measured in a kinase reaction using recombinant MEK1 unactive as a Raf-1 substrate over PC12 lysates where Raf-1 was immunoprecipitated using anti-Raf-1 antibody (Santa Cruz, Biotechnology).

    Techniques: Activation Assay, Activity Assay, Western Blot, Immunoprecipitation, Incubation, Recombinant, In Vitro, Transfection, Transduction, Sequencing, shRNA, Fractionation, SDS Page

    Fig. 6. Severe block in BCR-mediated ERK activation in Raf-1/B-Raf double deficient DT40 cells. Populations of DT40MCM/ raf -1 flE3 / B- raf flE6 cells were harvested at 5 days after induction of MCM-mediated deletion (+) and stimulated with M4. Total cellular lysates were subjected to western blot analysis. This result is representative of two experiments with an identical procedure (two independent clones) and at least five other experiments with a similar design. The apparent decrease in the level of ERK2 is caused by sequential detection of phospho-ERK followed by detection with anti-ERK2 antibody on the same western blot membrane.

    Journal: The EMBO Journal

    Article Title: Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling

    doi: 10.1093/emboj/cdf588

    Figure Lengend Snippet: Fig. 6. Severe block in BCR-mediated ERK activation in Raf-1/B-Raf double deficient DT40 cells. Populations of DT40MCM/ raf -1 flE3 / B- raf flE6 cells were harvested at 5 days after induction of MCM-mediated deletion (+) and stimulated with M4. Total cellular lysates were subjected to western blot analysis. This result is representative of two experiments with an identical procedure (two independent clones) and at least five other experiments with a similar design. The apparent decrease in the level of ERK2 is caused by sequential detection of phospho-ERK followed by detection with anti-ERK2 antibody on the same western blot membrane.

    Article Snippet: The rabbit Abs recognizing the C- (C-19) and N-terminus (H-145) of B-Raf as well as the anti-Raf-1 (C-12), anti-c-Fos (K-25) and anti-EGR-1 (C-19) Abs were purchased from Santa Cruz Biotechnology.

    Techniques: Blocking Assay, Activation Assay, Western Blot

    Fig. 1. The course of the phosphorylation of Raf-1 and B-Raf following BCR engagement is different. ( A ) Western blot analysis of DT40 cells stimulated with anti-IgM Ab (M4). Detection of tyrosine-phosphorylated proteins (pY) indicates successful stimulation. This result is representative of at least five independent experiments. ( B ) DT40 and Ramos B cells were stimulated with anti-IgM Abs as indicated, and Raf proteins were purified using anti-B-Raf H-145 or anti-Raf-1 Abs, respectively. The immunocomplexes were subjected to western blot analysis. Phosphorylation at S445 (B-Raf) and S338 (Raf-1) was detected by the anti-pS338 Ab. This result is representative of at least four (DT40) or two (Ramos) independent experiments.

    Journal: The EMBO Journal

    Article Title: Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling

    doi: 10.1093/emboj/cdf588

    Figure Lengend Snippet: Fig. 1. The course of the phosphorylation of Raf-1 and B-Raf following BCR engagement is different. ( A ) Western blot analysis of DT40 cells stimulated with anti-IgM Ab (M4). Detection of tyrosine-phosphorylated proteins (pY) indicates successful stimulation. This result is representative of at least five independent experiments. ( B ) DT40 and Ramos B cells were stimulated with anti-IgM Abs as indicated, and Raf proteins were purified using anti-B-Raf H-145 or anti-Raf-1 Abs, respectively. The immunocomplexes were subjected to western blot analysis. Phosphorylation at S445 (B-Raf) and S338 (Raf-1) was detected by the anti-pS338 Ab. This result is representative of at least four (DT40) or two (Ramos) independent experiments.

    Article Snippet: The rabbit Abs recognizing the C- (C-19) and N-terminus (H-145) of B-Raf as well as the anti-Raf-1 (C-12), anti-c-Fos (K-25) and anti-EGR-1 (C-19) Abs were purchased from Santa Cruz Biotechnology.

    Techniques: Western Blot, Purification

    Fig. 2. Conditional targeting of the chicken raf- 1 locus. ( A ) Map of the wild-type raf- 1 locus (wt) with exons 2–5 (black boxes). The targeting vector pT raf -1 flE3 contains a lox P site and a lox P-FRT-neo-FRT cassette inserted into the Sna bI and Eco NI sites, respectively. Lox P and FRT sites are indicated by black and white triangles, respectively. ( B ) Map of the modified raf- 1 alleles. The flE3Δneo allele respresents the first targeted allele after excision of the neo R gene by FLP-e expression. The flE3neo allele is the result of the second round of transfection containing the neo R gene. Following 4-HT treatment, MCM excises E3 from the alleles flE3Δneo and flE3neo, thereby generating the ΔE3Δneo and ΔE3neo alleles, respectively. ( C ) MCM-mediated recombination in DT40MCM/ raf -1 flE3 cells was examined by Southern blot analysis. Hin cII-digested genomic DNA derived from two clones, which were either exposed to 4-HT for 24 h (+) or left untreated (–), was detected by the indicated probes. MCM-mediated recombination results in excision of genomic sequences around exon 3 and concomitant loss of a Hin cII site as indicated by the increased fragment size. The polymorphism (by the neo R cassette) of the modified raf -1 loci in these clones was used here to demonstrate the efficient and autonomous recombination of both alleles. ( D ) Western blot analysis of Raf-1 expression in total cellular lysates after induction with 4-HT.

    Journal: The EMBO Journal

    Article Title: Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling

    doi: 10.1093/emboj/cdf588

    Figure Lengend Snippet: Fig. 2. Conditional targeting of the chicken raf- 1 locus. ( A ) Map of the wild-type raf- 1 locus (wt) with exons 2–5 (black boxes). The targeting vector pT raf -1 flE3 contains a lox P site and a lox P-FRT-neo-FRT cassette inserted into the Sna bI and Eco NI sites, respectively. Lox P and FRT sites are indicated by black and white triangles, respectively. ( B ) Map of the modified raf- 1 alleles. The flE3Δneo allele respresents the first targeted allele after excision of the neo R gene by FLP-e expression. The flE3neo allele is the result of the second round of transfection containing the neo R gene. Following 4-HT treatment, MCM excises E3 from the alleles flE3Δneo and flE3neo, thereby generating the ΔE3Δneo and ΔE3neo alleles, respectively. ( C ) MCM-mediated recombination in DT40MCM/ raf -1 flE3 cells was examined by Southern blot analysis. Hin cII-digested genomic DNA derived from two clones, which were either exposed to 4-HT for 24 h (+) or left untreated (–), was detected by the indicated probes. MCM-mediated recombination results in excision of genomic sequences around exon 3 and concomitant loss of a Hin cII site as indicated by the increased fragment size. The polymorphism (by the neo R cassette) of the modified raf -1 loci in these clones was used here to demonstrate the efficient and autonomous recombination of both alleles. ( D ) Western blot analysis of Raf-1 expression in total cellular lysates after induction with 4-HT.

    Article Snippet: The rabbit Abs recognizing the C- (C-19) and N-terminus (H-145) of B-Raf as well as the anti-Raf-1 (C-12), anti-c-Fos (K-25) and anti-EGR-1 (C-19) Abs were purchased from Santa Cruz Biotechnology.

    Techniques: Plasmid Preparation, Modification, Expressing, Transfection, Southern Blot, Derivative Assay, Clone Assay, Genomic Sequencing, Western Blot

    Fig. 4. Loss of Raf-1 does not affect the kinetics of BCR-mediated ERK activation. Populations of DT40MCM/ raf -1 flE3 cells, which were either left untreated (–) or exposed to 4-HT for 24 h (+), were stimulated with M4 at 8 days post-induction. Total cellular lysates were subjected to western blot analysis. This result is representative of at least seven independent, comparable experiments.

    Journal: The EMBO Journal

    Article Title: Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling

    doi: 10.1093/emboj/cdf588

    Figure Lengend Snippet: Fig. 4. Loss of Raf-1 does not affect the kinetics of BCR-mediated ERK activation. Populations of DT40MCM/ raf -1 flE3 cells, which were either left untreated (–) or exposed to 4-HT for 24 h (+), were stimulated with M4 at 8 days post-induction. Total cellular lysates were subjected to western blot analysis. This result is representative of at least seven independent, comparable experiments.

    Article Snippet: The rabbit Abs recognizing the C- (C-19) and N-terminus (H-145) of B-Raf as well as the anti-Raf-1 (C-12), anti-c-Fos (K-25) and anti-EGR-1 (C-19) Abs were purchased from Santa Cruz Biotechnology.

    Techniques: Activation Assay, Western Blot

    Fig. 7. The significance of Ras–Raf interaction for BCR-mediated ERK activation. Western blot analysis of total cellular lysates. ( A ) DT40MCM/ raf -1 flE3 /B- raf flE6 cells were treated with 4-HT to delete both raf -1 and B- raf , and transfected with expression vectors for either wild-type Raf-1 (R), Raf-1 R89L (L) or Raf-1 S259D (S). These cell lines were stimulated with M4 together with parental, Raf-deficient 4-HT-treated DT40MCM/ raf -1 flE3 /B- raf flE6 cells (Δ) and their non-4-HT-treated, Raf-positive sister population (W) as indicated. ( B ) After the deletion of both raf genes, DT40MCM/ raf -1 flE3 /B- raf flE6 cells were transfected with expression vectors for either wild-type HA-tagged B-Raf (R) or HA-B-Raf R188L (L). These cell lines were stimulated with M4 together with parental, Raf-deficient cells (Δ) and their non-4-HT-treated, Raf-positive counterpart (W) as indicated. Detection of BAP37 serves a loading control. ( C ) Detection of S445 phosphorylation in HA-B-Raf and HA-B-Raf R188L by anti-pS338 Ab. DT40MCM/ raf -1 flE3 /B- raf flE6 cells were transfected with expression vectors for HA-B-Raf or HA-B-Raf R188L and then treated with 4-HT. Nine days after induction, the cells were stimulated with M4 for the indicated times and HA-B-Raf proteins were purified as in (B). For each panel, representative results of at least three independent, comparable experiments are shown.

    Journal: The EMBO Journal

    Article Title: Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling

    doi: 10.1093/emboj/cdf588

    Figure Lengend Snippet: Fig. 7. The significance of Ras–Raf interaction for BCR-mediated ERK activation. Western blot analysis of total cellular lysates. ( A ) DT40MCM/ raf -1 flE3 /B- raf flE6 cells were treated with 4-HT to delete both raf -1 and B- raf , and transfected with expression vectors for either wild-type Raf-1 (R), Raf-1 R89L (L) or Raf-1 S259D (S). These cell lines were stimulated with M4 together with parental, Raf-deficient 4-HT-treated DT40MCM/ raf -1 flE3 /B- raf flE6 cells (Δ) and their non-4-HT-treated, Raf-positive sister population (W) as indicated. ( B ) After the deletion of both raf genes, DT40MCM/ raf -1 flE3 /B- raf flE6 cells were transfected with expression vectors for either wild-type HA-tagged B-Raf (R) or HA-B-Raf R188L (L). These cell lines were stimulated with M4 together with parental, Raf-deficient cells (Δ) and their non-4-HT-treated, Raf-positive counterpart (W) as indicated. Detection of BAP37 serves a loading control. ( C ) Detection of S445 phosphorylation in HA-B-Raf and HA-B-Raf R188L by anti-pS338 Ab. DT40MCM/ raf -1 flE3 /B- raf flE6 cells were transfected with expression vectors for HA-B-Raf or HA-B-Raf R188L and then treated with 4-HT. Nine days after induction, the cells were stimulated with M4 for the indicated times and HA-B-Raf proteins were purified as in (B). For each panel, representative results of at least three independent, comparable experiments are shown.

    Article Snippet: The rabbit Abs recognizing the C- (C-19) and N-terminus (H-145) of B-Raf as well as the anti-Raf-1 (C-12), anti-c-Fos (K-25) and anti-EGR-1 (C-19) Abs were purchased from Santa Cruz Biotechnology.

    Techniques: Activation Assay, Western Blot, Transfection, Expressing, Purification

    Fig. 8. BCR-mediated induction of the transcription factors NFAT, c-Fos and Egr-1 is regulated by both Raf kinases. ( A ) DT40 lines allowing inducible deletion of either raf -1, B- raf or both raf genes were exposed to 4-HT for 24 h (white bars) or left untreated (grey bars) and cultivated for an additional 4 days. The cells were then transfected with the NFAT reporter plasmid. Cells were stimulated with 10 µg of M4 for 6 h. ( B ) Raf-1/B-Raf double-deficient DT40 cells were transfected with the NFAT reporter plasmid and 10 µg of the expression vector pFlu/B- raf or the empty vector pFlu as indicated. As wild-type reference, uninduced DT40MCM/ raf -1 flE3 /B- raf flE6 cells were also included. The DT40 lines were treated as described in (A) and stimulated with M4 hybridoma supernatant (a stimulus equivalent to 5–10 µg M4/ml) for 6 h. The mean of the standardized luciferase activity derived from three independent, simultaneously performed transfections is shown. Standard deviation is indicated by an error bar. For each panel, representative results of at least three independent experiments are shown in both (A) and (B). ( C ) Western blot analysis of BCR-mediated synthesis of c-Fos and Egr-1. The DT40 lines were treated as described in (A), stimulated with M4 hybridoma supernatant (a stimulus equivalent to 5–10 µg M4/ml) for 1 h and lysed in RIPA buffer. This result is representative of three independent experiments.

    Journal: The EMBO Journal

    Article Title: Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling

    doi: 10.1093/emboj/cdf588

    Figure Lengend Snippet: Fig. 8. BCR-mediated induction of the transcription factors NFAT, c-Fos and Egr-1 is regulated by both Raf kinases. ( A ) DT40 lines allowing inducible deletion of either raf -1, B- raf or both raf genes were exposed to 4-HT for 24 h (white bars) or left untreated (grey bars) and cultivated for an additional 4 days. The cells were then transfected with the NFAT reporter plasmid. Cells were stimulated with 10 µg of M4 for 6 h. ( B ) Raf-1/B-Raf double-deficient DT40 cells were transfected with the NFAT reporter plasmid and 10 µg of the expression vector pFlu/B- raf or the empty vector pFlu as indicated. As wild-type reference, uninduced DT40MCM/ raf -1 flE3 /B- raf flE6 cells were also included. The DT40 lines were treated as described in (A) and stimulated with M4 hybridoma supernatant (a stimulus equivalent to 5–10 µg M4/ml) for 6 h. The mean of the standardized luciferase activity derived from three independent, simultaneously performed transfections is shown. Standard deviation is indicated by an error bar. For each panel, representative results of at least three independent experiments are shown in both (A) and (B). ( C ) Western blot analysis of BCR-mediated synthesis of c-Fos and Egr-1. The DT40 lines were treated as described in (A), stimulated with M4 hybridoma supernatant (a stimulus equivalent to 5–10 µg M4/ml) for 1 h and lysed in RIPA buffer. This result is representative of three independent experiments.

    Article Snippet: The rabbit Abs recognizing the C- (C-19) and N-terminus (H-145) of B-Raf as well as the anti-Raf-1 (C-12), anti-c-Fos (K-25) and anti-EGR-1 (C-19) Abs were purchased from Santa Cruz Biotechnology.

    Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Derivative Assay, Standard Deviation, Western Blot

    Effect of zinc on the in vitro association of the Raf-1/ERK1/2 cassette with MOR. (A, B) PAG synaptosomes were incubated with zinc chloride for 4 h at 4°C. (C) The time course for the effect of 3 μ M zinc on MOR association with PKCγ and Raf-1; incubation was conducted at room temperature with and without 100 μ M TPEN. At the end of the procedure, free zinc was removed by three runs of centrifugation washing. Afterward, MOR was immunoprecipitated, and associated proteins were detected with the corresponding antibodies. The study was repeated 3 times on different PAG preparations. Data are expressed relative to their control (no zinc, arbitrary value of 1) as mean±SEM. Equal loading was determined by the MOR signals. *Significantly different from the control group (no zinc), p

    Journal: Antioxidants & Redox Signaling

    Article Title: NO-released Zinc Supports the Simultaneous Binding of Raf-1 and PKC? Cysteine-Rich Domains to HINT1 Protein at the Mu-Opioid Receptor

    doi: 10.1089/ars.2010.3511

    Figure Lengend Snippet: Effect of zinc on the in vitro association of the Raf-1/ERK1/2 cassette with MOR. (A, B) PAG synaptosomes were incubated with zinc chloride for 4 h at 4°C. (C) The time course for the effect of 3 μ M zinc on MOR association with PKCγ and Raf-1; incubation was conducted at room temperature with and without 100 μ M TPEN. At the end of the procedure, free zinc was removed by three runs of centrifugation washing. Afterward, MOR was immunoprecipitated, and associated proteins were detected with the corresponding antibodies. The study was repeated 3 times on different PAG preparations. Data are expressed relative to their control (no zinc, arbitrary value of 1) as mean±SEM. Equal loading was determined by the MOR signals. *Significantly different from the control group (no zinc), p

    Article Snippet: High micromolar zinc concentrations reduced the MOR association with PKCγ, Raf-1, and Raf-1–complexed proteins.

    Techniques: In Vitro, Incubation, Centrifugation, Immunoprecipitation

    NO provides the zinc ions required for MOR association with Raf-1 and PKCγ. SNAP and NOR3 were incubated with PAG membranes either for 1 h at RT or for 24 h at 4°C. The NO donors (100 μ M ) were incubated alone or with TPEN (100 μ M ). Subsequently, the influence of these treatments on the MOR association with Raf-1, PKCγ, and nNOS proteins was evaluated. The data in each column are expressed as the mean±SEM of three independent assays. MOR signals served to control loading. *Significantly different from the control group, p

    Journal: Antioxidants & Redox Signaling

    Article Title: NO-released Zinc Supports the Simultaneous Binding of Raf-1 and PKC? Cysteine-Rich Domains to HINT1 Protein at the Mu-Opioid Receptor

    doi: 10.1089/ars.2010.3511

    Figure Lengend Snippet: NO provides the zinc ions required for MOR association with Raf-1 and PKCγ. SNAP and NOR3 were incubated with PAG membranes either for 1 h at RT or for 24 h at 4°C. The NO donors (100 μ M ) were incubated alone or with TPEN (100 μ M ). Subsequently, the influence of these treatments on the MOR association with Raf-1, PKCγ, and nNOS proteins was evaluated. The data in each column are expressed as the mean±SEM of three independent assays. MOR signals served to control loading. *Significantly different from the control group, p

    Article Snippet: High micromolar zinc concentrations reduced the MOR association with PKCγ, Raf-1, and Raf-1–complexed proteins.

    Techniques: Incubation

    Raf-1 and PKCγ CRD bind via zinc to HINT1. Formation of a ternary complex. (A) Interaction of recombinant proteins HINT1, PKCγ, and Raf-1. The HINT1 protomer was used at 200 n M , whereas PKCγ and Raf-1 [whole sequence and catalytic domain (cd)] were used at 100 n M . After TPEN zinc removal, proteins were incubated alone (negative control) or with the GST-tagged protein. The influence of added zinc was evaluated. After incubation, glutathione sepharose (GS) was added to the incubation mixture; the proteins were resolved by SDS-PAGE chromatography and analyzed with Western blotting. A similar study of association was conducted between the recombinant proteins Ras and HINT1. (B) HINT1 mediates the formation of a ternary complex with Raf-1 and PKCγ. After TPEN zinc removal, the whole sequences of Raf-1 and PKCγ showed no interaction with or without added zinc. The binding of Raf-1 to the HINT1 protein was not reduced, whereas the HINT1-PKCγ association increased.

    Journal: Antioxidants & Redox Signaling

    Article Title: NO-released Zinc Supports the Simultaneous Binding of Raf-1 and PKC? Cysteine-Rich Domains to HINT1 Protein at the Mu-Opioid Receptor

    doi: 10.1089/ars.2010.3511

    Figure Lengend Snippet: Raf-1 and PKCγ CRD bind via zinc to HINT1. Formation of a ternary complex. (A) Interaction of recombinant proteins HINT1, PKCγ, and Raf-1. The HINT1 protomer was used at 200 n M , whereas PKCγ and Raf-1 [whole sequence and catalytic domain (cd)] were used at 100 n M . After TPEN zinc removal, proteins were incubated alone (negative control) or with the GST-tagged protein. The influence of added zinc was evaluated. After incubation, glutathione sepharose (GS) was added to the incubation mixture; the proteins were resolved by SDS-PAGE chromatography and analyzed with Western blotting. A similar study of association was conducted between the recombinant proteins Ras and HINT1. (B) HINT1 mediates the formation of a ternary complex with Raf-1 and PKCγ. After TPEN zinc removal, the whole sequences of Raf-1 and PKCγ showed no interaction with or without added zinc. The binding of Raf-1 to the HINT1 protein was not reduced, whereas the HINT1-PKCγ association increased.

    Article Snippet: High micromolar zinc concentrations reduced the MOR association with PKCγ, Raf-1, and Raf-1–complexed proteins.

    Techniques: Recombinant, Sequencing, Incubation, Negative Control, SDS Page, Chromatography, Western Blot, Binding Assay

    Morphine recruits Raf-1and PKCγ to the MOR. (A) The primary structure of Raf-1 and of conventional PKC (α, β, γ). Raf-1: C1–C3 conserved regions; the N-terminal regulatory region contains C1 and C2; the C1 region consists of two subregions: the Ras-binding domain (RBD) and the cysteine-rich domain (CRD); the C-terminal C3 region corresponds to the catalytic domain. PKC: C1–C4 conserved regions; the N-terminus contains the regulatory region with the pseudo substrate (PS); C1 contains tandem C1A and C1B cysteine-rich domains (CRDs) that bind zinc and phorbol esters/diacylglycerol; C2 binds phosphatidylserine and calcium; the C-terminal region contains the ATP-binding site (C3) and the kinase domain (C4) that binds the substrate. In both kinases, the N-terminal regulatory domain regulates (inhibits) the activity of the C-terminal catalytic domain. (B) Morphine (3 nmol and 10 nmol) was injected ICV into mice. Data are the analgesic time course determined by the tail-flick test. Each data point is the mean±SEM, n =8 mice. *Significantly different from the preopioid control interval (0 min), p

    Journal: Antioxidants & Redox Signaling

    Article Title: NO-released Zinc Supports the Simultaneous Binding of Raf-1 and PKC? Cysteine-Rich Domains to HINT1 Protein at the Mu-Opioid Receptor

    doi: 10.1089/ars.2010.3511

    Figure Lengend Snippet: Morphine recruits Raf-1and PKCγ to the MOR. (A) The primary structure of Raf-1 and of conventional PKC (α, β, γ). Raf-1: C1–C3 conserved regions; the N-terminal regulatory region contains C1 and C2; the C1 region consists of two subregions: the Ras-binding domain (RBD) and the cysteine-rich domain (CRD); the C-terminal C3 region corresponds to the catalytic domain. PKC: C1–C4 conserved regions; the N-terminus contains the regulatory region with the pseudo substrate (PS); C1 contains tandem C1A and C1B cysteine-rich domains (CRDs) that bind zinc and phorbol esters/diacylglycerol; C2 binds phosphatidylserine and calcium; the C-terminal region contains the ATP-binding site (C3) and the kinase domain (C4) that binds the substrate. In both kinases, the N-terminal regulatory domain regulates (inhibits) the activity of the C-terminal catalytic domain. (B) Morphine (3 nmol and 10 nmol) was injected ICV into mice. Data are the analgesic time course determined by the tail-flick test. Each data point is the mean±SEM, n =8 mice. *Significantly different from the preopioid control interval (0 min), p

    Article Snippet: High micromolar zinc concentrations reduced the MOR association with PKCγ, Raf-1, and Raf-1–complexed proteins.

    Techniques: Binding Assay, Activity Assay, Injection, Mouse Assay, Tail Flick Test

    Blood endothelial RAF1 S259A expression induces lymphatic EC fate specification.

    Journal: The Journal of Clinical Investigation

    Article Title: Endothelial ERK signaling controls lymphatic fate specification

    doi: 10.1172/JCI63034

    Figure Lengend Snippet: Blood endothelial RAF1 S259A expression induces lymphatic EC fate specification.

    Article Snippet: The following antibodies were used for Western blotting: anti-pERK1/2, ERK1/2, pRAF1 S259, and RAF1 (Cell Signaling Technology); anti–VE-cadherin and CD31 (Santa Cruz Biotechnology); and anti-HA (Covance).

    Techniques: Expressing

    RAF1-AKT crosstalk regulates lymphatic endothelial fate specification by controlling ERK activation.

    Journal: The Journal of Clinical Investigation

    Article Title: Endothelial ERK signaling controls lymphatic fate specification

    doi: 10.1172/JCI63034

    Figure Lengend Snippet: RAF1-AKT crosstalk regulates lymphatic endothelial fate specification by controlling ERK activation.

    Article Snippet: The following antibodies were used for Western blotting: anti-pERK1/2, ERK1/2, pRAF1 S259, and RAF1 (Cell Signaling Technology); anti–VE-cadherin and CD31 (Santa Cruz Biotechnology); and anti-HA (Covance).

    Techniques: Activation Assay

    RAF1 S259A induces Sox18 expression.

    Journal: The Journal of Clinical Investigation

    Article Title: Endothelial ERK signaling controls lymphatic fate specification

    doi: 10.1172/JCI63034

    Figure Lengend Snippet: RAF1 S259A induces Sox18 expression.

    Article Snippet: The following antibodies were used for Western blotting: anti-pERK1/2, ERK1/2, pRAF1 S259, and RAF1 (Cell Signaling Technology); anti–VE-cadherin and CD31 (Santa Cruz Biotechnology); and anti-HA (Covance).

    Techniques: Expressing

    Endothelial-specific expression of RAF1 S259A induces enlarged lymphatic vessels.

    Journal: The Journal of Clinical Investigation

    Article Title: Endothelial ERK signaling controls lymphatic fate specification

    doi: 10.1172/JCI63034

    Figure Lengend Snippet: Endothelial-specific expression of RAF1 S259A induces enlarged lymphatic vessels.

    Article Snippet: The following antibodies were used for Western blotting: anti-pERK1/2, ERK1/2, pRAF1 S259, and RAF1 (Cell Signaling Technology); anti–VE-cadherin and CD31 (Santa Cruz Biotechnology); and anti-HA (Covance).

    Techniques: Expressing

    RAF1 S259A mice develop lymphangiectasia.

    Journal: The Journal of Clinical Investigation

    Article Title: Endothelial ERK signaling controls lymphatic fate specification

    doi: 10.1172/JCI63034

    Figure Lengend Snippet: RAF1 S259A mice develop lymphangiectasia.

    Article Snippet: The following antibodies were used for Western blotting: anti-pERK1/2, ERK1/2, pRAF1 S259, and RAF1 (Cell Signaling Technology); anti–VE-cadherin and CD31 (Santa Cruz Biotechnology); and anti-HA (Covance).

    Techniques: Mouse Assay

    RAF1 S259A induces PROX1 expression in blood ECs.

    Journal: The Journal of Clinical Investigation

    Article Title: Endothelial ERK signaling controls lymphatic fate specification

    doi: 10.1172/JCI63034

    Figure Lengend Snippet: RAF1 S259A induces PROX1 expression in blood ECs.

    Article Snippet: The following antibodies were used for Western blotting: anti-pERK1/2, ERK1/2, pRAF1 S259, and RAF1 (Cell Signaling Technology); anti–VE-cadherin and CD31 (Santa Cruz Biotechnology); and anti-HA (Covance).

    Techniques: Expressing

    SOX18 expression is upregulated in RAF1 S259A embryos.

    Journal: The Journal of Clinical Investigation

    Article Title: Endothelial ERK signaling controls lymphatic fate specification

    doi: 10.1172/JCI63034

    Figure Lengend Snippet: SOX18 expression is upregulated in RAF1 S259A embryos.

    Article Snippet: The following antibodies were used for Western blotting: anti-pERK1/2, ERK1/2, pRAF1 S259, and RAF1 (Cell Signaling Technology); anti–VE-cadherin and CD31 (Santa Cruz Biotechnology); and anti-HA (Covance).

    Techniques: Expressing

    Endothelial-specific expression of RAF1 S259A blocks RAF1-AKT crosstalk and activates ERK.

    Journal: The Journal of Clinical Investigation

    Article Title: Endothelial ERK signaling controls lymphatic fate specification

    doi: 10.1172/JCI63034

    Figure Lengend Snippet: Endothelial-specific expression of RAF1 S259A blocks RAF1-AKT crosstalk and activates ERK.

    Article Snippet: The following antibodies were used for Western blotting: anti-pERK1/2, ERK1/2, pRAF1 S259, and RAF1 (Cell Signaling Technology); anti–VE-cadherin and CD31 (Santa Cruz Biotechnology); and anti-HA (Covance).

    Techniques: Expressing

    Inhibition of ERK signaling rescues the lymphatic phenotype in RAF1 S259A embryos.

    Journal: The Journal of Clinical Investigation

    Article Title: Endothelial ERK signaling controls lymphatic fate specification

    doi: 10.1172/JCI63034

    Figure Lengend Snippet: Inhibition of ERK signaling rescues the lymphatic phenotype in RAF1 S259A embryos.

    Article Snippet: The following antibodies were used for Western blotting: anti-pERK1/2, ERK1/2, pRAF1 S259, and RAF1 (Cell Signaling Technology); anti–VE-cadherin and CD31 (Santa Cruz Biotechnology); and anti-HA (Covance).

    Techniques: Inhibition

    Effects of Raf-1 kinase inhibition on PE-induced MLC 20 ( a ) and MYPT1 ( b ) phosphorylation in rat VSMCs. Left and right panels show concentration- and time-dependent effects of PE, respectively. Cultured cells were pretreated with vehicle or GW5074 (10 μ M ) for 30 min before stimulated with 10 μ M PE. Treated cells were processed as described in Methods. The phosphorylation levels of MLC 20 or MYPT1 were determined by immunoblotting. Top panels show representative blots of phospho-MLC 20 or phospho-MYPT1 and β-actin, bottom panels are the summary of densitometric results. Results were normalized against β-actin and expressed as folds change relative to control. Data represent means of 3–5 independent experiments. * p

    Journal: Journal of Vascular Research

    Article Title: Raf-1 Kinase Regulates Smooth Muscle Contraction in the Rat Mesenteric Arteries

    doi: 10.1159/000277726

    Figure Lengend Snippet: Effects of Raf-1 kinase inhibition on PE-induced MLC 20 ( a ) and MYPT1 ( b ) phosphorylation in rat VSMCs. Left and right panels show concentration- and time-dependent effects of PE, respectively. Cultured cells were pretreated with vehicle or GW5074 (10 μ M ) for 30 min before stimulated with 10 μ M PE. Treated cells were processed as described in Methods. The phosphorylation levels of MLC 20 or MYPT1 were determined by immunoblotting. Top panels show representative blots of phospho-MLC 20 or phospho-MYPT1 and β-actin, bottom panels are the summary of densitometric results. Results were normalized against β-actin and expressed as folds change relative to control. Data represent means of 3–5 independent experiments. * p

    Article Snippet: Membranes were blocked in a PBS solution containing 5% dry milk for 2 h before an overnight incubation in a Tris-buffered saline solution containing 5% milk and probed with phospho-Raf-1 (1:1,000 dilution; Cell Signaling, Danvers, Mass., USA), phospho-MEK (1:2,000 dilution; Cell Signaling), phospho-ERK (1:1,000 dilution; Cell Signaling), phospho-MLC20 (1:4,000 dilution; Sigma), phospho-myosin phosphatase target, MYPT1Thr696 antibody (1:2,000; Upstate, Lake Placid, N.Y., USA).

    Techniques: Inhibition, Concentration Assay, Cell Culture

    Reversal of PE-induced contraction by Raf-1 kinase inhibitors in rat mesenteric arteries (semi-log plot). Submaximal PE contraction was elicited, increasing concentrations of Raf-1 kinase inhibitors, GW5074, L779450 and ZM33637, were added, and then the percentage of relaxation to PE contraction was measured. Data points represent mean ± SEM of measurements in 10–12 mesenteric arterial rings from 5–6 rats.

    Journal: Journal of Vascular Research

    Article Title: Raf-1 Kinase Regulates Smooth Muscle Contraction in the Rat Mesenteric Arteries

    doi: 10.1159/000277726

    Figure Lengend Snippet: Reversal of PE-induced contraction by Raf-1 kinase inhibitors in rat mesenteric arteries (semi-log plot). Submaximal PE contraction was elicited, increasing concentrations of Raf-1 kinase inhibitors, GW5074, L779450 and ZM33637, were added, and then the percentage of relaxation to PE contraction was measured. Data points represent mean ± SEM of measurements in 10–12 mesenteric arterial rings from 5–6 rats.

    Article Snippet: Membranes were blocked in a PBS solution containing 5% dry milk for 2 h before an overnight incubation in a Tris-buffered saline solution containing 5% milk and probed with phospho-Raf-1 (1:1,000 dilution; Cell Signaling, Danvers, Mass., USA), phospho-MEK (1:2,000 dilution; Cell Signaling), phospho-ERK (1:1,000 dilution; Cell Signaling), phospho-MLC20 (1:4,000 dilution; Sigma), phospho-myosin phosphatase target, MYPT1Thr696 antibody (1:2,000; Upstate, Lake Placid, N.Y., USA).

    Techniques:

    a Basal MAP in rats in the absence and presence of GW5074 (250 μg/kg). b Effect of Raf-1 kinase inhibition on PE-induced increase in mean arterial pressure in rats. Dose-response curve for PE evoked increase in blood pressure in the absence and presence of GW5074 (semi-log plot). Rats were pretreated with GW5074 (250 μg/kg; i.v.) or vehicle for 15 min and then increasing concentration of PE was infused (10–300 μg/kg/min). Each dose of PE was infused for a 3-min period and the MAP was determined as the mean value recorded within the last minute of infusion. Hypertensive responses were calculated as percent increase in MAP with respect to the baseline MAP. Values are expressed as mean ± SEM of 5–6 animals.

    Journal: Journal of Vascular Research

    Article Title: Raf-1 Kinase Regulates Smooth Muscle Contraction in the Rat Mesenteric Arteries

    doi: 10.1159/000277726

    Figure Lengend Snippet: a Basal MAP in rats in the absence and presence of GW5074 (250 μg/kg). b Effect of Raf-1 kinase inhibition on PE-induced increase in mean arterial pressure in rats. Dose-response curve for PE evoked increase in blood pressure in the absence and presence of GW5074 (semi-log plot). Rats were pretreated with GW5074 (250 μg/kg; i.v.) or vehicle for 15 min and then increasing concentration of PE was infused (10–300 μg/kg/min). Each dose of PE was infused for a 3-min period and the MAP was determined as the mean value recorded within the last minute of infusion. Hypertensive responses were calculated as percent increase in MAP with respect to the baseline MAP. Values are expressed as mean ± SEM of 5–6 animals.

    Article Snippet: Membranes were blocked in a PBS solution containing 5% dry milk for 2 h before an overnight incubation in a Tris-buffered saline solution containing 5% milk and probed with phospho-Raf-1 (1:1,000 dilution; Cell Signaling, Danvers, Mass., USA), phospho-MEK (1:2,000 dilution; Cell Signaling), phospho-ERK (1:1,000 dilution; Cell Signaling), phospho-MLC20 (1:4,000 dilution; Sigma), phospho-myosin phosphatase target, MYPT1Thr696 antibody (1:2,000; Upstate, Lake Placid, N.Y., USA).

    Techniques: Inhibition, Concentration Assay

    Dose and time course of PE-stimulated phosphorylation of Raf-1 ( a ), MEK ( b ) and ERK ( c ). Left and right panels show concentration- and time-dependent effects of PE, respectively. Cultured VSMCs were treated with varying concentration of PE for 5 min. For time course studies cells pretreated with GW5074 (10 μ M ) or vehicle for 30 min before stimulated with PE (10 μ M ) for indicated time periods. Treated cells were processed as described in Methods. The phosphorylation levels of Raf-1, MEK and ERK were determined by immunoblotting. Top panels show representative blots of respective phosphorylated proteins and β-actin; bottom panels are the summary of densitometric results. Results were normalized against β-actin and expressed as folds change relative to control. Data represent mean of 4–5 independent experiments. * p

    Journal: Journal of Vascular Research

    Article Title: Raf-1 Kinase Regulates Smooth Muscle Contraction in the Rat Mesenteric Arteries

    doi: 10.1159/000277726

    Figure Lengend Snippet: Dose and time course of PE-stimulated phosphorylation of Raf-1 ( a ), MEK ( b ) and ERK ( c ). Left and right panels show concentration- and time-dependent effects of PE, respectively. Cultured VSMCs were treated with varying concentration of PE for 5 min. For time course studies cells pretreated with GW5074 (10 μ M ) or vehicle for 30 min before stimulated with PE (10 μ M ) for indicated time periods. Treated cells were processed as described in Methods. The phosphorylation levels of Raf-1, MEK and ERK were determined by immunoblotting. Top panels show representative blots of respective phosphorylated proteins and β-actin; bottom panels are the summary of densitometric results. Results were normalized against β-actin and expressed as folds change relative to control. Data represent mean of 4–5 independent experiments. * p

    Article Snippet: Membranes were blocked in a PBS solution containing 5% dry milk for 2 h before an overnight incubation in a Tris-buffered saline solution containing 5% milk and probed with phospho-Raf-1 (1:1,000 dilution; Cell Signaling, Danvers, Mass., USA), phospho-MEK (1:2,000 dilution; Cell Signaling), phospho-ERK (1:1,000 dilution; Cell Signaling), phospho-MLC20 (1:4,000 dilution; Sigma), phospho-myosin phosphatase target, MYPT1Thr696 antibody (1:2,000; Upstate, Lake Placid, N.Y., USA).

    Techniques: Concentration Assay, Cell Culture

    Effects of Raf-1 kinase inhibition on PKC-induced Raf-1 ( a ), MLC 20 ( b ), MYPT1 ( c ), MEK ( d ) and ERK ( e ) phosphorylation in rat VSMCs. Cultured cells were pretreated with vehicle or GW5074 (10 μ M ) or U0126 (10 μ M ) or calphostin C (100 n M ) for 30 min before stimulated with 3 μ M PDBu for 15 min. Treated cells were processed as described in Methods. The phosphorylation levels of these proteins were determined by immunoblotting. Top panels show representative blots of the respective phospho-protein and β-actin, bottom panels are the summary of densitometric results. Results were normalized against β-actin and expressed as folds change relative to untreated control. Data represent means of 3 independent experiments. * p

    Journal: Journal of Vascular Research

    Article Title: Raf-1 Kinase Regulates Smooth Muscle Contraction in the Rat Mesenteric Arteries

    doi: 10.1159/000277726

    Figure Lengend Snippet: Effects of Raf-1 kinase inhibition on PKC-induced Raf-1 ( a ), MLC 20 ( b ), MYPT1 ( c ), MEK ( d ) and ERK ( e ) phosphorylation in rat VSMCs. Cultured cells were pretreated with vehicle or GW5074 (10 μ M ) or U0126 (10 μ M ) or calphostin C (100 n M ) for 30 min before stimulated with 3 μ M PDBu for 15 min. Treated cells were processed as described in Methods. The phosphorylation levels of these proteins were determined by immunoblotting. Top panels show representative blots of the respective phospho-protein and β-actin, bottom panels are the summary of densitometric results. Results were normalized against β-actin and expressed as folds change relative to untreated control. Data represent means of 3 independent experiments. * p

    Article Snippet: Membranes were blocked in a PBS solution containing 5% dry milk for 2 h before an overnight incubation in a Tris-buffered saline solution containing 5% milk and probed with phospho-Raf-1 (1:1,000 dilution; Cell Signaling, Danvers, Mass., USA), phospho-MEK (1:2,000 dilution; Cell Signaling), phospho-ERK (1:1,000 dilution; Cell Signaling), phospho-MLC20 (1:4,000 dilution; Sigma), phospho-myosin phosphatase target, MYPT1Thr696 antibody (1:2,000; Upstate, Lake Placid, N.Y., USA).

    Techniques: Inhibition, Cell Culture

    Effect of Raf-1 kinase inhibition on PE-induced increases in [Ca 2+ ] i . Fura-2-loaded cultured VSMCs were pretreated with vehicle ( a ) or GW5074 (10 μ M ) ( b ) for 30 min and then PE-induced increase in [Ca 2+ ] i was recorded in HBSS. c Effect of GW5074 when added at the plateau of PE-induced increase in [Ca 2+ ] i . d Average peak F340/380 values for PE in absence and presence of GW5074. Data are expressed as F340/380 ratio after background correction, and each point represents mean ± SE of 10–15 cells. Tracings are representatives and values are mean ± SE from 3 separate experiments.

    Journal: Journal of Vascular Research

    Article Title: Raf-1 Kinase Regulates Smooth Muscle Contraction in the Rat Mesenteric Arteries

    doi: 10.1159/000277726

    Figure Lengend Snippet: Effect of Raf-1 kinase inhibition on PE-induced increases in [Ca 2+ ] i . Fura-2-loaded cultured VSMCs were pretreated with vehicle ( a ) or GW5074 (10 μ M ) ( b ) for 30 min and then PE-induced increase in [Ca 2+ ] i was recorded in HBSS. c Effect of GW5074 when added at the plateau of PE-induced increase in [Ca 2+ ] i . d Average peak F340/380 values for PE in absence and presence of GW5074. Data are expressed as F340/380 ratio after background correction, and each point represents mean ± SE of 10–15 cells. Tracings are representatives and values are mean ± SE from 3 separate experiments.

    Article Snippet: Membranes were blocked in a PBS solution containing 5% dry milk for 2 h before an overnight incubation in a Tris-buffered saline solution containing 5% milk and probed with phospho-Raf-1 (1:1,000 dilution; Cell Signaling, Danvers, Mass., USA), phospho-MEK (1:2,000 dilution; Cell Signaling), phospho-ERK (1:1,000 dilution; Cell Signaling), phospho-MLC20 (1:4,000 dilution; Sigma), phospho-myosin phosphatase target, MYPT1Thr696 antibody (1:2,000; Upstate, Lake Placid, N.Y., USA).

    Techniques: Inhibition, Cell Culture

    Raf-1 activation via DC-SIGN decreases MV-induced type I IFN expression ( A,C,F,G ) IFN-β, MxA, and ISG15 mRNA expression by DCs 8 h after stimulation with poly(I:C)-LV and/or receptor crosslinking with isotype or DC-SIGN-specific antibodies ( A,C ) or 24 h after infection with rMV KS ( F,G ), in the absence or presence of Raf inhibitor GW5074 ( A,F ) or after Raf-1 silencing ( B,G ), measured by real-time PCR, normalized to GAPDH, and set at 1 in MV- or poly(I:C)-LV-stimulated (control-silenced) cells. Data are presented as mean ± SD. N.s., not statistically significant; *, P

    Journal: Cell host & microbe

    Article Title: Measles virus suppresses RIG-I-like receptor activation in dendritic cells via DC-SIGN-mediated inhibition of PP1 phosphatases

    doi: 10.1016/j.chom.2014.06.008

    Figure Lengend Snippet: Raf-1 activation via DC-SIGN decreases MV-induced type I IFN expression ( A,C,F,G ) IFN-β, MxA, and ISG15 mRNA expression by DCs 8 h after stimulation with poly(I:C)-LV and/or receptor crosslinking with isotype or DC-SIGN-specific antibodies ( A,C ) or 24 h after infection with rMV KS ( F,G ), in the absence or presence of Raf inhibitor GW5074 ( A,F ) or after Raf-1 silencing ( B,G ), measured by real-time PCR, normalized to GAPDH, and set at 1 in MV- or poly(I:C)-LV-stimulated (control-silenced) cells. Data are presented as mean ± SD. N.s., not statistically significant; *, P

    Article Snippet: Raf-1 expression was determined with anti-Raf-1 (9422; Cell Signaling).

    Techniques: Activation Assay, Expressing, Infection, Real-time Polymerase Chain Reaction

    DC-SIGN-Raf-1 signaling inhibits dephosphorylation of RIG-I and Mda5 ( A–D ) RIG-I phosphorylation at Ser8 or Thr170 and Mda5 phosphorylation at Ser88 in DCs left unstimulated; or 3 h after stimulation by crosslinking with isotype or DC-SIGN-specific antibodies ( A,B ), in the absence or presence of Raf inhibitor GW5074 ( B ); or 8 or 16 h after rMV KS infection or 3 h after or rMV IC323 EGFP(1) in the absence or presence of Raf-1 inhibition via GW5074 ( C ) or Raf-1 silencing ( D ), as determined by flow cytometry. Data are representative of at least four ( A,B,C (rMV IC323 , 8 h rMV KS ), two ( C (16 h rMV KS )) or three ( D .

    Journal: Cell host & microbe

    Article Title: Measles virus suppresses RIG-I-like receptor activation in dendritic cells via DC-SIGN-mediated inhibition of PP1 phosphatases

    doi: 10.1016/j.chom.2014.06.008

    Figure Lengend Snippet: DC-SIGN-Raf-1 signaling inhibits dephosphorylation of RIG-I and Mda5 ( A–D ) RIG-I phosphorylation at Ser8 or Thr170 and Mda5 phosphorylation at Ser88 in DCs left unstimulated; or 3 h after stimulation by crosslinking with isotype or DC-SIGN-specific antibodies ( A,B ), in the absence or presence of Raf inhibitor GW5074 ( B ); or 8 or 16 h after rMV KS infection or 3 h after or rMV IC323 EGFP(1) in the absence or presence of Raf-1 inhibition via GW5074 ( C ) or Raf-1 silencing ( D ), as determined by flow cytometry. Data are representative of at least four ( A,B,C (rMV IC323 , 8 h rMV KS ), two ( C (16 h rMV KS )) or three ( D .

    Article Snippet: Raf-1 expression was determined with anti-Raf-1 (9422; Cell Signaling).

    Techniques: De-Phosphorylation Assay, Infection, Inhibition, Flow Cytometry, Cytometry

    DC-SIGN-Raf-1 signaling inhibits RLR activation and type I IFN responses via phosphorylation of GADD34-PP1 inhibitor I-1 ( A,C ) Overall ( A ) or GADD34-specific ( C ) PP1 phosphatase activity in whole cell lysates of DCs 1 h after stimulation with poly(I:C)-LV and/or receptor crosslinking with isotype or DC-SIGN-specific antibodies (left panels) or 24 h after infection with rMV KS or rMV IC323 (right panels), in the absence or presence of Raf inhibitor GW5074. Data are presented as mean ± SD. ( B,E,F ) RIG-I phosphorylation at Ser8 or Thr170 and Mda5 phosphorylation at Ser88 in DCs left unstimulated; or 3 h after stimulation by receptor crosslinking with isotype or DC-SIGN-specific (antibodies ( B,F ); or 8 h after rMV KS infection ( E ), in the absence or presence of GADD34 inhibitor guanabenz (Gb) ( B ) or after GADD34 or I-1 silencing ( E,F ), as determined by flow cytometry. ( D ) I-1 phosphorylation at Ser or Thr residues, and association with PP1α or PP1γ after immunoprecipitation (IP) of I-1 from whole cell lysates of DCs left unstimulated or 3 h after infection with rMV KS , in the absence or presence of GW5074, determined by immunoblotting (IB). ( G,H ) IFN-β, MxA, and ISG15 mRNA expression by DCs 8 h after stimulation with poly(I:C)-LV and/or receptor crosslinking with isotype or DC-SIGN-specific antibodies ( G ) or 24 h after infection with rMV KS ( H ), after GADD34 or I-1 silencing, measured by real-time PCR, normalized to GAPDH, and set at 1 in MV- or poly(I:C)-LV-stimulated control-silenced cells. Data are presented as mean ± SD. *, P

    Journal: Cell host & microbe

    Article Title: Measles virus suppresses RIG-I-like receptor activation in dendritic cells via DC-SIGN-mediated inhibition of PP1 phosphatases

    doi: 10.1016/j.chom.2014.06.008

    Figure Lengend Snippet: DC-SIGN-Raf-1 signaling inhibits RLR activation and type I IFN responses via phosphorylation of GADD34-PP1 inhibitor I-1 ( A,C ) Overall ( A ) or GADD34-specific ( C ) PP1 phosphatase activity in whole cell lysates of DCs 1 h after stimulation with poly(I:C)-LV and/or receptor crosslinking with isotype or DC-SIGN-specific antibodies (left panels) or 24 h after infection with rMV KS or rMV IC323 (right panels), in the absence or presence of Raf inhibitor GW5074. Data are presented as mean ± SD. ( B,E,F ) RIG-I phosphorylation at Ser8 or Thr170 and Mda5 phosphorylation at Ser88 in DCs left unstimulated; or 3 h after stimulation by receptor crosslinking with isotype or DC-SIGN-specific (antibodies ( B,F ); or 8 h after rMV KS infection ( E ), in the absence or presence of GADD34 inhibitor guanabenz (Gb) ( B ) or after GADD34 or I-1 silencing ( E,F ), as determined by flow cytometry. ( D ) I-1 phosphorylation at Ser or Thr residues, and association with PP1α or PP1γ after immunoprecipitation (IP) of I-1 from whole cell lysates of DCs left unstimulated or 3 h after infection with rMV KS , in the absence or presence of GW5074, determined by immunoblotting (IB). ( G,H ) IFN-β, MxA, and ISG15 mRNA expression by DCs 8 h after stimulation with poly(I:C)-LV and/or receptor crosslinking with isotype or DC-SIGN-specific antibodies ( G ) or 24 h after infection with rMV KS ( H ), after GADD34 or I-1 silencing, measured by real-time PCR, normalized to GAPDH, and set at 1 in MV- or poly(I:C)-LV-stimulated control-silenced cells. Data are presented as mean ± SD. *, P

    Article Snippet: Raf-1 expression was determined with anti-Raf-1 (9422; Cell Signaling).

    Techniques: Activation Assay, Activity Assay, Infection, Flow Cytometry, Cytometry, Immunoprecipitation, Expressing, Real-time Polymerase Chain Reaction

    Raf1-tr exhibits unique protein binding partners: A ) Outline of experimental procedure for mass spectrometry identification of Raf1-binding partners. B ) Venn diagram of proteins identified to be unique to Raf1-tr (left) and Raf1-fl (right). Proteins that bound both Raf1 species fall in the middle. C ) Volcano plot of proteins that exhibit decreased (left) or increased (right) binding to Raf1-tr relative to Raf1-fl binding. Thresholds of P

    Journal: The FASEB Journal

    Article Title: Nuclear localized Raf1 isoform alters DNA-dependent protein kinase activity and the DNA damage response

    doi: 10.1096/fj.201800336R

    Figure Lengend Snippet: Raf1-tr exhibits unique protein binding partners: A ) Outline of experimental procedure for mass spectrometry identification of Raf1-binding partners. B ) Venn diagram of proteins identified to be unique to Raf1-tr (left) and Raf1-fl (right). Proteins that bound both Raf1 species fall in the middle. C ) Volcano plot of proteins that exhibit decreased (left) or increased (right) binding to Raf1-tr relative to Raf1-fl binding. Thresholds of P

    Article Snippet: HAP1 parental cells that express Raf1 (C631; Horizon Discovery, Waterbeach, United Kingdom) or HAP1 cells containing a 22 bp deletion in exon 2 (HZGHC002809c001; Horizon Discovery) were cultured in Iscove’s Modified Dulbecco’s Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified 5% CO2 atm.

    Techniques: Protein Binding, Mass Spectrometry, Binding Assay

    Alternative splicing results in a Raf1-tr protein isoform: A ) Exon/intron map of Raf1-fl and the identified splice isoform (Raf1-tr) (top). New exon in Raf1-tr is highlighted in red. Protein product of Raf1-fl (bottom left) showing conserved regions containing a Ras-binding domain and cysteine-rich domain (blue), serine-threonine–rich domain (red), and kinase domain (green). Raf1-tr protein product (bottom right) is truncated and has all conserved regions except for the kinase domain. B ) Measurement of Raf1-tr gene expression in a panel of human tissues. The gene of interest was normalized to GAPDH expression and represented as fold change relative to brain tissue. C ) Western blot of Raf1 in matched normal (N) and adenocarcinoma colon (T) lysate showing that Raf1-tr protein is differentially expressed in normal and colon tumor tissue. D ) Probing for Raf1 with 2 unique N-terminal targeted antibodies demonstrates a 73 kDa Raf1-fl species and a 43 kDa Raf1-tr species in HEK cell lysate. Probing for Raf1 with a C-terminal targeted antibody detects only the 73 kDa Raf1-fl species.

    Journal: The FASEB Journal

    Article Title: Nuclear localized Raf1 isoform alters DNA-dependent protein kinase activity and the DNA damage response

    doi: 10.1096/fj.201800336R

    Figure Lengend Snippet: Alternative splicing results in a Raf1-tr protein isoform: A ) Exon/intron map of Raf1-fl and the identified splice isoform (Raf1-tr) (top). New exon in Raf1-tr is highlighted in red. Protein product of Raf1-fl (bottom left) showing conserved regions containing a Ras-binding domain and cysteine-rich domain (blue), serine-threonine–rich domain (red), and kinase domain (green). Raf1-tr protein product (bottom right) is truncated and has all conserved regions except for the kinase domain. B ) Measurement of Raf1-tr gene expression in a panel of human tissues. The gene of interest was normalized to GAPDH expression and represented as fold change relative to brain tissue. C ) Western blot of Raf1 in matched normal (N) and adenocarcinoma colon (T) lysate showing that Raf1-tr protein is differentially expressed in normal and colon tumor tissue. D ) Probing for Raf1 with 2 unique N-terminal targeted antibodies demonstrates a 73 kDa Raf1-fl species and a 43 kDa Raf1-tr species in HEK cell lysate. Probing for Raf1 with a C-terminal targeted antibody detects only the 73 kDa Raf1-fl species.

    Article Snippet: HAP1 parental cells that express Raf1 (C631; Horizon Discovery, Waterbeach, United Kingdom) or HAP1 cells containing a 22 bp deletion in exon 2 (HZGHC002809c001; Horizon Discovery) were cultured in Iscove’s Modified Dulbecco’s Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified 5% CO2 atm.

    Techniques: Binding Assay, Expressing, Western Blot

    Raf-1 knockdown affects phosphorylation levels of downstream target proteins. (A) Total cell lysates (TCL) from Co- and Eu-hESC cells were analysed for Raf-1, pE/R/M, phospho-paxillin (pPax), E/R/M, pMYPT1, MYPT1 and α-tubulin, 48 hrs after their transfection with either Raf-1 or control siRNA. The Raf-1 and pPax levels were normalized by total α-tubulin. The pE/R/M and pMYPT1 levels were normalized by E/R/M and MYPT1 respectively. Representative blots from biological triplicates (left panel) and graphical representation of Raf-1, pE/R/M, pPax and pMYPT1 (right panel) are shown as protein levels in% (mean value ± S.D.) relative to their normalized levels (mean value set to 100%) in cells transfected with control siRNA, ** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

    doi: 10.1111/j.1582-4934.2011.01520.x

    Figure Lengend Snippet: Raf-1 knockdown affects phosphorylation levels of downstream target proteins. (A) Total cell lysates (TCL) from Co- and Eu-hESC cells were analysed for Raf-1, pE/R/M, phospho-paxillin (pPax), E/R/M, pMYPT1, MYPT1 and α-tubulin, 48 hrs after their transfection with either Raf-1 or control siRNA. The Raf-1 and pPax levels were normalized by total α-tubulin. The pE/R/M and pMYPT1 levels were normalized by E/R/M and MYPT1 respectively. Representative blots from biological triplicates (left panel) and graphical representation of Raf-1, pE/R/M, pPax and pMYPT1 (right panel) are shown as protein levels in% (mean value ± S.D.) relative to their normalized levels (mean value set to 100%) in cells transfected with control siRNA, ** P

    Article Snippet: The down part was incubated with Raf-1 (mouse; BD Transduction Laboratories) to visualize the amount of bound Raf-1.

    Techniques: Transfection

    Raf-1 regulates hESC morphology and migration. (A) Immunofluorescence analysis of cytoskeleton by phalloidin (red), Raf-1 cellular levels (green) and merge (green and red) 48 and 144 hrs after transfection of Eu-hESC cells with siRNA are shown on the left. Average number of cells with contracted phenotype (estimated by two independent investigators) out of three biological triplicates are given on the right as % of cells with contracted phenotype relative to the total cell number (set to 100%, ± S.D.). ** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

    doi: 10.1111/j.1582-4934.2011.01520.x

    Figure Lengend Snippet: Raf-1 regulates hESC morphology and migration. (A) Immunofluorescence analysis of cytoskeleton by phalloidin (red), Raf-1 cellular levels (green) and merge (green and red) 48 and 144 hrs after transfection of Eu-hESC cells with siRNA are shown on the left. Average number of cells with contracted phenotype (estimated by two independent investigators) out of three biological triplicates are given on the right as % of cells with contracted phenotype relative to the total cell number (set to 100%, ± S.D.). ** P

    Article Snippet: The down part was incubated with Raf-1 (mouse; BD Transduction Laboratories) to visualize the amount of bound Raf-1.

    Techniques: Migration, Immunofluorescence, Transfection

    Cellular Raf-1 levels determine hESC motility. (A) Western blot analysis of Raf-1 expression levels in individual hESC cultures (numbered at the top of the immunoblot and corresponding to the sample ID number given in Table S1 (for Ec-hESC; n = 7) and in [ 25 ] for Eu-hESC; n = 7) is shown on the left and the graphical representation of the analysis on the right. The level of the protein is shown as optical density (OD) of the Western blot lanes normalized to the OD of α-tubulin. (B) Box plots of the data obtained using Western blot analysis pE/R/M (left panel; n = 8 per group) and total E/R/M levels (right panel; n = 8 per group) in Co-, Eu- and Ec-hESC are given. The levels of the proteins are shown as OD of the Western blot lanes normalized to the OD of β-actin. The corresponding P -values obtained after anova and Post hoc analysis are additionally inserted on the top of the box plots. (C) The ROCK II activity in Co-, Eu- and Ec-hESC is shown as average values of absorbance (OD 450 nm) determined in six biological replicates (mean ± S.D.) and normalized to the absorbance in Co-hESC. The statistical analysis of the data was performed with anova followed by Post-hoc test, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

    doi: 10.1111/j.1582-4934.2011.01520.x

    Figure Lengend Snippet: Cellular Raf-1 levels determine hESC motility. (A) Western blot analysis of Raf-1 expression levels in individual hESC cultures (numbered at the top of the immunoblot and corresponding to the sample ID number given in Table S1 (for Ec-hESC; n = 7) and in [ 25 ] for Eu-hESC; n = 7) is shown on the left and the graphical representation of the analysis on the right. The level of the protein is shown as optical density (OD) of the Western blot lanes normalized to the OD of α-tubulin. (B) Box plots of the data obtained using Western blot analysis pE/R/M (left panel; n = 8 per group) and total E/R/M levels (right panel; n = 8 per group) in Co-, Eu- and Ec-hESC are given. The levels of the proteins are shown as OD of the Western blot lanes normalized to the OD of β-actin. The corresponding P -values obtained after anova and Post hoc analysis are additionally inserted on the top of the box plots. (C) The ROCK II activity in Co-, Eu- and Ec-hESC is shown as average values of absorbance (OD 450 nm) determined in six biological replicates (mean ± S.D.) and normalized to the absorbance in Co-hESC. The statistical analysis of the data was performed with anova followed by Post-hoc test, * P

    Article Snippet: The down part was incubated with Raf-1 (mouse; BD Transduction Laboratories) to visualize the amount of bound Raf-1.

    Techniques: Western Blot, Expressing, Activity Assay

    Western blot analysis of MYPT1 and E/R/M phosphorylation analysed 24 hrs after treatment of hESC with specific Raf-1 inhibitors (ZM336372 and GW5074 – 1 μM) is shown on the left. Representative blots from three independent experiments are given. Graphical representation of pMYPT1 (right panels) is shown as protein levels in% (mean value ± S.D.) relative to their normalized levels (mean value set to 1) in the non-treated controls, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

    doi: 10.1111/j.1582-4934.2011.01520.x

    Figure Lengend Snippet: Western blot analysis of MYPT1 and E/R/M phosphorylation analysed 24 hrs after treatment of hESC with specific Raf-1 inhibitors (ZM336372 and GW5074 – 1 μM) is shown on the left. Representative blots from three independent experiments are given. Graphical representation of pMYPT1 (right panels) is shown as protein levels in% (mean value ± S.D.) relative to their normalized levels (mean value set to 1) in the non-treated controls, * P

    Article Snippet: The down part was incubated with Raf-1 (mouse; BD Transduction Laboratories) to visualize the amount of bound Raf-1.

    Techniques: Western Blot

    Effects of Raf-1 knockdown on hESC cell morphology. (A) Immunofluorescence analysis of cytoskeleton by vimentin (green), phalloidin (red) and merge (green and red), 48 hrs after transfection of Co-hESC, Eu-hESC and Ec-hESC cells with siRNA are shown, scale bar line = 100 μm. (B) Average number of cells with contracted phenotype (estimated by two independent investigators) out of three biological replicates are given as% of cells with contracted phenotype relative to the total cell number (set to 100%), ** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

    doi: 10.1111/j.1582-4934.2011.01520.x

    Figure Lengend Snippet: Effects of Raf-1 knockdown on hESC cell morphology. (A) Immunofluorescence analysis of cytoskeleton by vimentin (green), phalloidin (red) and merge (green and red), 48 hrs after transfection of Co-hESC, Eu-hESC and Ec-hESC cells with siRNA are shown, scale bar line = 100 μm. (B) Average number of cells with contracted phenotype (estimated by two independent investigators) out of three biological replicates are given as% of cells with contracted phenotype relative to the total cell number (set to 100%), ** P

    Article Snippet: The down part was incubated with Raf-1 (mouse; BD Transduction Laboratories) to visualize the amount of bound Raf-1.

    Techniques: Immunofluorescence, Transfection

    Molecular characterization of RAF1-deficient lesions. ( a ) Immunoblotting of F/F and Δp/np livers collected 30 weeks after DEN treatment. The plots represents a densitometric quantification of the immunoblot performed using ImageJ. The data are expressed as relative band intensity adjusted to TUBA or ACTB, which serve as loading controls (upper plot). Phosphorylation is expressed as the ratio between the phosphospecific antibody signal and the signal obtained with the protein-specific antibody. In both cases, the data are normalized to the F/F non-tumour samples, which were arbitrarily set as 1. ( b ) Immunoblot analysis of signaling pathways in xenograft samples ( n =3, analysed 40 days after transplant). The plots show a quantification of the immunoblots performed as described in ( a ). ( c ) YAP1 expression in the same patient cohort examined in Fig. 1a . Scale bar, 50 μm. Left panel, representative IHC image. Middle panel, comparison of YAP1 expression in matched tumour and non-tumour tissue. Right panel, YAP1 expression in tumours correlates positively with tumour grade and the ratio of RAF1/YAP1 expression in the same tumour negatively correlates with histological grade. ( d ) STAT3 expression in the same cohort. Left panel, representative IHC image. Middle panel, comparison of STAT3 expression in matched tumour and non-tumour tissue. Right panel, RAF1/YAP1 expression in the same tumour negatively correlated with the presence of medium-large clusters of STAT3 nuclear staining. Scale bar 50 μm. In ( a , b ), the data are represented as mean±s.e.m., * P ≤0.05, ** P

    Journal: Nature Communications

    Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

    doi: 10.1038/ncomms13781

    Figure Lengend Snippet: Molecular characterization of RAF1-deficient lesions. ( a ) Immunoblotting of F/F and Δp/np livers collected 30 weeks after DEN treatment. The plots represents a densitometric quantification of the immunoblot performed using ImageJ. The data are expressed as relative band intensity adjusted to TUBA or ACTB, which serve as loading controls (upper plot). Phosphorylation is expressed as the ratio between the phosphospecific antibody signal and the signal obtained with the protein-specific antibody. In both cases, the data are normalized to the F/F non-tumour samples, which were arbitrarily set as 1. ( b ) Immunoblot analysis of signaling pathways in xenograft samples ( n =3, analysed 40 days after transplant). The plots show a quantification of the immunoblots performed as described in ( a ). ( c ) YAP1 expression in the same patient cohort examined in Fig. 1a . Scale bar, 50 μm. Left panel, representative IHC image. Middle panel, comparison of YAP1 expression in matched tumour and non-tumour tissue. Right panel, YAP1 expression in tumours correlates positively with tumour grade and the ratio of RAF1/YAP1 expression in the same tumour negatively correlates with histological grade. ( d ) STAT3 expression in the same cohort. Left panel, representative IHC image. Middle panel, comparison of STAT3 expression in matched tumour and non-tumour tissue. Right panel, RAF1/YAP1 expression in the same tumour negatively correlated with the presence of medium-large clusters of STAT3 nuclear staining. Scale bar 50 μm. In ( a , b ), the data are represented as mean±s.e.m., * P ≤0.05, ** P

    Article Snippet: The following antibodies were used for immunohistochemistry: α-mouse, CD44 (550538, BD Biosciences, 1:50), Ki67 (Novocastra, 1:1,000), YAP1 (4912, Cell Signaling, 1:200), F4/80 (MCA497G, AbD Serotec, 1:50), FSP1 (27957, Abcam, 1:300), CD3 (A0452, DAKO, 1:1,000), βcatenin (32572, Abcam,1:500); α-human YAP1 (12395, Cell Signaling, 1:1,000), RAF1 (154754, Abcam, 1:1,500) and STAT3 (9139, Cell Signaling, 1:800).

    Techniques: Western Blot, Expressing, Immunohistochemistry, Staining

    RAF1 ablation increases the number of cancer progenitor cells. ( a ) Quantification of Ki67+ liver cells 8 (top panel) or 12 weeks (bottom panel) after DEN treatment. ( b ) Foci of altered hepatocytes (FAH) in F/F and Δhep or Δp/np livers isolated 12 weeks after DEN injection. Sections were stained with H E or with the indicated antibodies. FAH are delimited by dotted circles ( n =3 per genotype). Scale bars, 50 μm. ( c ) Percentage of cancer progenitor cells (CD44+/CD31−Ter119−CD45−) present in non-aggregate and aggregate fractions of F/F and Δp/np livers, as determined by FACS analysis. Data are represented as mean±s.e.m., * P ≤0.05, ** P

    Journal: Nature Communications

    Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

    doi: 10.1038/ncomms13781

    Figure Lengend Snippet: RAF1 ablation increases the number of cancer progenitor cells. ( a ) Quantification of Ki67+ liver cells 8 (top panel) or 12 weeks (bottom panel) after DEN treatment. ( b ) Foci of altered hepatocytes (FAH) in F/F and Δhep or Δp/np livers isolated 12 weeks after DEN injection. Sections were stained with H E or with the indicated antibodies. FAH are delimited by dotted circles ( n =3 per genotype). Scale bars, 50 μm. ( c ) Percentage of cancer progenitor cells (CD44+/CD31−Ter119−CD45−) present in non-aggregate and aggregate fractions of F/F and Δp/np livers, as determined by FACS analysis. Data are represented as mean±s.e.m., * P ≤0.05, ** P

    Article Snippet: The following antibodies were used for immunohistochemistry: α-mouse, CD44 (550538, BD Biosciences, 1:50), Ki67 (Novocastra, 1:1,000), YAP1 (4912, Cell Signaling, 1:200), F4/80 (MCA497G, AbD Serotec, 1:50), FSP1 (27957, Abcam, 1:300), CD3 (A0452, DAKO, 1:1,000), βcatenin (32572, Abcam,1:500); α-human YAP1 (12395, Cell Signaling, 1:1,000), RAF1 (154754, Abcam, 1:1,500) and STAT3 (9139, Cell Signaling, 1:800).

    Techniques: Isolation, Injection, Staining, FACS

    Effect of YAP1 silencing, the P6 JAK inhibitor and GP130 silencing on DIH and Hep3B proliferation. ( a ) siRNA-mediated RAF1 silencing in Hep3B cells increases YAP1 and GP130 expression and STAT3 activation without impacting ERK phosphorylation or β-catenin expression/localization. Immunoblot analysis of post-nuclear fraction (PNF; 20 μg, about 8% of total) and nuclear fraction (Nuclei; 20 μg, about 15% of total). ( b ) Silencing of YAP1 in RAF1-proficient and -deficient Hep3B cells (left panel, representative immunoblot analysis) downregulates the expression of the YAP1 target gene CTGF (middle panel, qPCR analysis) and reduces proliferation (right panel). ( c ) Treatment with the JAK inhibitor P6 abrogates STAT3 phosphorylation without impacting ERK phosphorylation or YAP1 expression (left panel, representative immunoblot analysis), decreases BIRC5 expression (middle panel, qPCR analysis) and reduces proliferation in RAF1-deficient Hep3B cells (right panel). ( d,e ) Similar results are obtained by subjecting RAF1-proficient and -deficient DIH to YAP1 silencing ( d ) or P6 treatment ( e ). ( f ) GP130 silencing decreases STAT3 phosphorylation but does not affect YAP1 expression or phosphorylation. Proliferation was assessed 48 h after siRNA transfection (with the exception of c , in which P6 was added 24 h after transfection and proliferation was measured after additional 48 h), gene expression after 24 h, and for immunoblotting cells were lysed after 1 h inhibitor treatment. In ( f ) DIH were treated for 30 min with the indicated concentration of IL6. Experiments were performed in DMEM supplemented with 10% FBS (Hep3B cells) or in DIH medium supplemented with 5% FBS (DIH). The immunoblots are representative of two independent experiments; TUBA was used as loading control. The plots represent the mean±s.e.m. of three independent experiments. * P ≤0.05, ** P

    Journal: Nature Communications

    Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

    doi: 10.1038/ncomms13781

    Figure Lengend Snippet: Effect of YAP1 silencing, the P6 JAK inhibitor and GP130 silencing on DIH and Hep3B proliferation. ( a ) siRNA-mediated RAF1 silencing in Hep3B cells increases YAP1 and GP130 expression and STAT3 activation without impacting ERK phosphorylation or β-catenin expression/localization. Immunoblot analysis of post-nuclear fraction (PNF; 20 μg, about 8% of total) and nuclear fraction (Nuclei; 20 μg, about 15% of total). ( b ) Silencing of YAP1 in RAF1-proficient and -deficient Hep3B cells (left panel, representative immunoblot analysis) downregulates the expression of the YAP1 target gene CTGF (middle panel, qPCR analysis) and reduces proliferation (right panel). ( c ) Treatment with the JAK inhibitor P6 abrogates STAT3 phosphorylation without impacting ERK phosphorylation or YAP1 expression (left panel, representative immunoblot analysis), decreases BIRC5 expression (middle panel, qPCR analysis) and reduces proliferation in RAF1-deficient Hep3B cells (right panel). ( d,e ) Similar results are obtained by subjecting RAF1-proficient and -deficient DIH to YAP1 silencing ( d ) or P6 treatment ( e ). ( f ) GP130 silencing decreases STAT3 phosphorylation but does not affect YAP1 expression or phosphorylation. Proliferation was assessed 48 h after siRNA transfection (with the exception of c , in which P6 was added 24 h after transfection and proliferation was measured after additional 48 h), gene expression after 24 h, and for immunoblotting cells were lysed after 1 h inhibitor treatment. In ( f ) DIH were treated for 30 min with the indicated concentration of IL6. Experiments were performed in DMEM supplemented with 10% FBS (Hep3B cells) or in DIH medium supplemented with 5% FBS (DIH). The immunoblots are representative of two independent experiments; TUBA was used as loading control. The plots represent the mean±s.e.m. of three independent experiments. * P ≤0.05, ** P

    Article Snippet: The following antibodies were used for immunohistochemistry: α-mouse, CD44 (550538, BD Biosciences, 1:50), Ki67 (Novocastra, 1:1,000), YAP1 (4912, Cell Signaling, 1:200), F4/80 (MCA497G, AbD Serotec, 1:50), FSP1 (27957, Abcam, 1:300), CD3 (A0452, DAKO, 1:1,000), βcatenin (32572, Abcam,1:500); α-human YAP1 (12395, Cell Signaling, 1:1,000), RAF1 (154754, Abcam, 1:1,500) and STAT3 (9139, Cell Signaling, 1:800).

    Techniques: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Transfection, Concentration Assay, Western Blot

    Molecular characterization of RAF1-deficient cells. ( a ) siRNA-mediated RAF1 silencing promotes the proliferation of Hep3B ( n =6), HuH-7 ( n =5) and HepG2 ( n =4) cells and increases the expression of YAP1 and GP130 as well as STAT3 phosphorylation. siRAF1#1 targets the region around nucleotide 721, while siRAF1#2 is a mixture of siRNAs targeting the region from nucleotide 692 to 1,093 in the RAF1 mRNA. ( b ) PCR and immunoblotting analysis of F/F and RAF1Δ/Δ DIH. ( c ) Morphology (x200 magnification) and molecular characterization ( d ) of primary hepatocytes (P-HEPS) compared to DIH (AFP, α-fetoprotein; ALB, albumin; TUBA, loading control). The immunoblot is representative of two independent experiments. ( e ) Proliferation of DIH in decreasing amounts of FBS. ( f ) Molecular defects of RAF1-deficient P-HEPS and DIH treated with the indicated concentrations of IL6 for 30 min. TUBA serves as loading control. Data are presented as mean±s.e.m. * P ≤0.05, ** P

    Journal: Nature Communications

    Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

    doi: 10.1038/ncomms13781

    Figure Lengend Snippet: Molecular characterization of RAF1-deficient cells. ( a ) siRNA-mediated RAF1 silencing promotes the proliferation of Hep3B ( n =6), HuH-7 ( n =5) and HepG2 ( n =4) cells and increases the expression of YAP1 and GP130 as well as STAT3 phosphorylation. siRAF1#1 targets the region around nucleotide 721, while siRAF1#2 is a mixture of siRNAs targeting the region from nucleotide 692 to 1,093 in the RAF1 mRNA. ( b ) PCR and immunoblotting analysis of F/F and RAF1Δ/Δ DIH. ( c ) Morphology (x200 magnification) and molecular characterization ( d ) of primary hepatocytes (P-HEPS) compared to DIH (AFP, α-fetoprotein; ALB, albumin; TUBA, loading control). The immunoblot is representative of two independent experiments. ( e ) Proliferation of DIH in decreasing amounts of FBS. ( f ) Molecular defects of RAF1-deficient P-HEPS and DIH treated with the indicated concentrations of IL6 for 30 min. TUBA serves as loading control. Data are presented as mean±s.e.m. * P ≤0.05, ** P

    Article Snippet: The following antibodies were used for immunohistochemistry: α-mouse, CD44 (550538, BD Biosciences, 1:50), Ki67 (Novocastra, 1:1,000), YAP1 (4912, Cell Signaling, 1:200), F4/80 (MCA497G, AbD Serotec, 1:50), FSP1 (27957, Abcam, 1:300), CD3 (A0452, DAKO, 1:1,000), βcatenin (32572, Abcam,1:500); α-human YAP1 (12395, Cell Signaling, 1:1,000), RAF1 (154754, Abcam, 1:1,500) and STAT3 (9139, Cell Signaling, 1:800).

    Techniques: Expressing, Polymerase Chain Reaction

    RAF1 ablation correlates with decreased YAP1 and GP130 protein turnover in Hep3B cells, primary hepatocytes (P-HEPS), and DIH. ( a-c ) qPCR analysis showing the expression of the YAP1 and Gp130 genes in Hep3B ( a ), P-HEPS ( b ) and DIH ( c ). qPCR data represent the mean (±s.e.m.) of three independent experiments; according to Student's t test. ( d-f ) Cells were treated with cycloheximide for the indicated amount of time prior to lysis. YAP1 and GP130 expression levels were determined by immunoblotting. A quantification is shown in the right panel; the amount of protein present in each of the untreated samples (normalized to TUBA or ACTB as loading controls) is set as 1.

    Journal: Nature Communications

    Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

    doi: 10.1038/ncomms13781

    Figure Lengend Snippet: RAF1 ablation correlates with decreased YAP1 and GP130 protein turnover in Hep3B cells, primary hepatocytes (P-HEPS), and DIH. ( a-c ) qPCR analysis showing the expression of the YAP1 and Gp130 genes in Hep3B ( a ), P-HEPS ( b ) and DIH ( c ). qPCR data represent the mean (±s.e.m.) of three independent experiments; according to Student's t test. ( d-f ) Cells were treated with cycloheximide for the indicated amount of time prior to lysis. YAP1 and GP130 expression levels were determined by immunoblotting. A quantification is shown in the right panel; the amount of protein present in each of the untreated samples (normalized to TUBA or ACTB as loading controls) is set as 1.

    Article Snippet: The following antibodies were used for immunohistochemistry: α-mouse, CD44 (550538, BD Biosciences, 1:50), Ki67 (Novocastra, 1:1,000), YAP1 (4912, Cell Signaling, 1:200), F4/80 (MCA497G, AbD Serotec, 1:50), FSP1 (27957, Abcam, 1:300), CD3 (A0452, DAKO, 1:1,000), βcatenin (32572, Abcam,1:500); α-human YAP1 (12395, Cell Signaling, 1:1,000), RAF1 (154754, Abcam, 1:1,500) and STAT3 (9139, Cell Signaling, 1:800).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Lysis

    RAF1 is expressed at low levels in human HCC and suppresses the growth of both HCC xenografts and chemically induced tumours. ( a ) RAF1 expression in a cohort of 31 HCC patients. Left panel, representative IHC image (T, tumour; NT, non-tumour). Scale bar, 50 μm. Middle panel, RAF1 expression in matched tumour and non-tumour tissue (a.u.=arbitrary units). Right panel, RAF1 expression in tumours correlates inversely with tumour grade (ratio: protein expression in tumour/non-tumour tissue). ( b ) Inducible shRNA-mediated RAF1 silencing does not impact A- or BRAF expression (top panel) but increases the proliferation of Hep3B cells in culture (bottom panel; n =6). ( c ) Inducible shRNA-mediated RAF1 silencing strongly promotes the growth of Hep3B xenografts. ( d,e ) Ablation of RAF1 in liver parenchymal cells promotes chemically induced hepatocarcinogenesis. Top panels, experimental protocols. Left bottom panel, macroscopic appearance of F/F and Δhep ( d ) or Δp/np ( e ) tumour-bearing livers 30 weeks (w) after DEN injection; arrows indicate tumours. Scale bars, 0.5 cm. Middle panels, liver:body weight ratio of untreated or DEN/Pb-treated mice. Right panels, tumour numbers and % of tumour-occupied area in control, Δhep ( d ) and Δp/np ( e ) livers. In ( d ), no DEN: F/F n =4, Δhep=6; DEN-treated: F/F n =7, Δhep=8. In ( e ), DEN-treated: F/F n =10, Δp/np n =11. ( f , g ) Quantification of Ki67+ cells: ( f ) and inflammatory cells ( g ; F4/80+ cells, granulocytes and CD3+ cells) in tumour-bearing F/F and Δhep livers. Np=non-parenchymal cells. ( h ) Chemokine levels in the serum of F/F and Δhep mice. ( i ) Chemo-/cytokine levels in tumour-bearing livers. Data are presented as mean ± SEM, * P ≤0.05, ** P

    Journal: Nature Communications

    Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

    doi: 10.1038/ncomms13781

    Figure Lengend Snippet: RAF1 is expressed at low levels in human HCC and suppresses the growth of both HCC xenografts and chemically induced tumours. ( a ) RAF1 expression in a cohort of 31 HCC patients. Left panel, representative IHC image (T, tumour; NT, non-tumour). Scale bar, 50 μm. Middle panel, RAF1 expression in matched tumour and non-tumour tissue (a.u.=arbitrary units). Right panel, RAF1 expression in tumours correlates inversely with tumour grade (ratio: protein expression in tumour/non-tumour tissue). ( b ) Inducible shRNA-mediated RAF1 silencing does not impact A- or BRAF expression (top panel) but increases the proliferation of Hep3B cells in culture (bottom panel; n =6). ( c ) Inducible shRNA-mediated RAF1 silencing strongly promotes the growth of Hep3B xenografts. ( d,e ) Ablation of RAF1 in liver parenchymal cells promotes chemically induced hepatocarcinogenesis. Top panels, experimental protocols. Left bottom panel, macroscopic appearance of F/F and Δhep ( d ) or Δp/np ( e ) tumour-bearing livers 30 weeks (w) after DEN injection; arrows indicate tumours. Scale bars, 0.5 cm. Middle panels, liver:body weight ratio of untreated or DEN/Pb-treated mice. Right panels, tumour numbers and % of tumour-occupied area in control, Δhep ( d ) and Δp/np ( e ) livers. In ( d ), no DEN: F/F n =4, Δhep=6; DEN-treated: F/F n =7, Δhep=8. In ( e ), DEN-treated: F/F n =10, Δp/np n =11. ( f , g ) Quantification of Ki67+ cells: ( f ) and inflammatory cells ( g ; F4/80+ cells, granulocytes and CD3+ cells) in tumour-bearing F/F and Δhep livers. Np=non-parenchymal cells. ( h ) Chemokine levels in the serum of F/F and Δhep mice. ( i ) Chemo-/cytokine levels in tumour-bearing livers. Data are presented as mean ± SEM, * P ≤0.05, ** P

    Article Snippet: The following antibodies were used for immunohistochemistry: α-mouse, CD44 (550538, BD Biosciences, 1:50), Ki67 (Novocastra, 1:1,000), YAP1 (4912, Cell Signaling, 1:200), F4/80 (MCA497G, AbD Serotec, 1:50), FSP1 (27957, Abcam, 1:300), CD3 (A0452, DAKO, 1:1,000), βcatenin (32572, Abcam,1:500); α-human YAP1 (12395, Cell Signaling, 1:1,000), RAF1 (154754, Abcam, 1:1,500) and STAT3 (9139, Cell Signaling, 1:800).

    Techniques: Expressing, Immunohistochemistry, shRNA, Injection, Mouse Assay

    miR-489 decreases migration and invasion by attenuating the PAK5-RAF1-MMP2 axis. Migration and invasion assays were performed with (A) U87 and (B) U251 transfected with either control, miR-489 mimic or miR-489 inh. Representative images of migration and invasion are shown. Magnification ×100. (C) U87 cells were transfected with control, miR-489 mimic or miR-489 inh and western blotting was used to determine the expression of the indicated proteins at 24 h after transfection. U87 cells were transfected with control, (D) miR-489 mimic or (E) miR-489 inh and the mRNA expression levels of MMP2 were measured at 24 h after transfection. **P

    Journal: Oncology Reports

    Article Title: miR-489 promotes apoptosis and inhibits invasiveness of glioma cells by targeting PAK5/RAF1 signaling pathways

    doi: 10.3892/or.2019.7381

    Figure Lengend Snippet: miR-489 decreases migration and invasion by attenuating the PAK5-RAF1-MMP2 axis. Migration and invasion assays were performed with (A) U87 and (B) U251 transfected with either control, miR-489 mimic or miR-489 inh. Representative images of migration and invasion are shown. Magnification ×100. (C) U87 cells were transfected with control, miR-489 mimic or miR-489 inh and western blotting was used to determine the expression of the indicated proteins at 24 h after transfection. U87 cells were transfected with control, (D) miR-489 mimic or (E) miR-489 inh and the mRNA expression levels of MMP2 were measured at 24 h after transfection. **P

    Article Snippet: The membranes were blocked in 5% skimmed milk in TBS-Tween for 1 h at room temperature, and probed with the following primary antibodies: Rabbit polyclonal antibodies PAK5 (cat. no. ab110069; Abcam), p-S338-RAF1 (cat. no. ab51042; Abcam), RAF1 (cat. no. ab137435; Abcam) and matrix metalloproteinase 2 (MMP2; cat. no. ab37150; Abcam); rabbit monoclonal antibodies c-Myc (cat. no. ab32072; Abcam), cyclin D1 (cat. no. ab134175; Abcam), Bcl-2 (cat. no. ab32124; Abcam) and Bax (cat. no. ab32503; Abcam) at 4°C overnight, after which time the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. ab222772 and ab222759; Abcam).

    Techniques: Migration, Transfection, Western Blot, Expressing

    miR-489 suppresses the progression of glioma by targeting PAK5 and inhibiting PAK5/RAF1-mediated signaling pathways. U251 cells were treated with control, miR-489 mimic or a combination of miR-489 mimic and PAK5. (A) MTT assays were performed and the results are presented as the mean ± standard deviation of three individual experiments. **P

    Journal: Oncology Reports

    Article Title: miR-489 promotes apoptosis and inhibits invasiveness of glioma cells by targeting PAK5/RAF1 signaling pathways

    doi: 10.3892/or.2019.7381

    Figure Lengend Snippet: miR-489 suppresses the progression of glioma by targeting PAK5 and inhibiting PAK5/RAF1-mediated signaling pathways. U251 cells were treated with control, miR-489 mimic or a combination of miR-489 mimic and PAK5. (A) MTT assays were performed and the results are presented as the mean ± standard deviation of three individual experiments. **P

    Article Snippet: The membranes were blocked in 5% skimmed milk in TBS-Tween for 1 h at room temperature, and probed with the following primary antibodies: Rabbit polyclonal antibodies PAK5 (cat. no. ab110069; Abcam), p-S338-RAF1 (cat. no. ab51042; Abcam), RAF1 (cat. no. ab137435; Abcam) and matrix metalloproteinase 2 (MMP2; cat. no. ab37150; Abcam); rabbit monoclonal antibodies c-Myc (cat. no. ab32072; Abcam), cyclin D1 (cat. no. ab134175; Abcam), Bcl-2 (cat. no. ab32124; Abcam) and Bax (cat. no. ab32503; Abcam) at 4°C overnight, after which time the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. ab222772 and ab222759; Abcam).

    Techniques: MTT Assay, Standard Deviation

    miR-489 promotes apoptosis in glioma cells by attenuating RAF1-Bax-mediated cell survival pathways. (A and B) U87 and (C and D) U251 cells were transfected with control, miR-489 mimic or miR-489 inh for 24 h, and subsequently stained with Hoechst 33258 and observed under a fluorescence microscope. Flow cytometry analysis was performed to measure the apoptosis in transfected cells after 24 h. (E) U87 cells were transfected with control, miR-489 mimic or miR-489 inh and western blotting was used to determine the expression of the indicated proteins at 24 h post-transfection. U87 cells were transfected with control, (F) miR-489 mimic or (G) miR-489 inh and the mRNA expression levels of the indicated genes were measured at 24 h after transfection. (H) U251 cells were transfected with control, miR-489 mimic or miR-489 inh and western blotting was used to determine the expression of the indicated proteins at 24 h after transfection. U87 cells were transfected with control, (I) miR-489 mimic or (J) miR-489 inh, and the mRNA expression levels of the indicated genes were measured at 24 h after transfection. **P

    Journal: Oncology Reports

    Article Title: miR-489 promotes apoptosis and inhibits invasiveness of glioma cells by targeting PAK5/RAF1 signaling pathways

    doi: 10.3892/or.2019.7381

    Figure Lengend Snippet: miR-489 promotes apoptosis in glioma cells by attenuating RAF1-Bax-mediated cell survival pathways. (A and B) U87 and (C and D) U251 cells were transfected with control, miR-489 mimic or miR-489 inh for 24 h, and subsequently stained with Hoechst 33258 and observed under a fluorescence microscope. Flow cytometry analysis was performed to measure the apoptosis in transfected cells after 24 h. (E) U87 cells were transfected with control, miR-489 mimic or miR-489 inh and western blotting was used to determine the expression of the indicated proteins at 24 h post-transfection. U87 cells were transfected with control, (F) miR-489 mimic or (G) miR-489 inh and the mRNA expression levels of the indicated genes were measured at 24 h after transfection. (H) U251 cells were transfected with control, miR-489 mimic or miR-489 inh and western blotting was used to determine the expression of the indicated proteins at 24 h after transfection. U87 cells were transfected with control, (I) miR-489 mimic or (J) miR-489 inh, and the mRNA expression levels of the indicated genes were measured at 24 h after transfection. **P

    Article Snippet: The membranes were blocked in 5% skimmed milk in TBS-Tween for 1 h at room temperature, and probed with the following primary antibodies: Rabbit polyclonal antibodies PAK5 (cat. no. ab110069; Abcam), p-S338-RAF1 (cat. no. ab51042; Abcam), RAF1 (cat. no. ab137435; Abcam) and matrix metalloproteinase 2 (MMP2; cat. no. ab37150; Abcam); rabbit monoclonal antibodies c-Myc (cat. no. ab32072; Abcam), cyclin D1 (cat. no. ab134175; Abcam), Bcl-2 (cat. no. ab32124; Abcam) and Bax (cat. no. ab32503; Abcam) at 4°C overnight, after which time the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. ab222772 and ab222759; Abcam).

    Techniques: Transfection, Staining, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Western Blot, Expressing

    miR-489 inhibits cell viability by attenuating the PAK5-RAF1-MAPK signaling cascade. (A) U87 and (B) U251 cells were transfected with control, miR-489 mimic or miR-489 inh for the indicated times. MTT assays were performed. **P

    Journal: Oncology Reports

    Article Title: miR-489 promotes apoptosis and inhibits invasiveness of glioma cells by targeting PAK5/RAF1 signaling pathways

    doi: 10.3892/or.2019.7381

    Figure Lengend Snippet: miR-489 inhibits cell viability by attenuating the PAK5-RAF1-MAPK signaling cascade. (A) U87 and (B) U251 cells were transfected with control, miR-489 mimic or miR-489 inh for the indicated times. MTT assays were performed. **P

    Article Snippet: The membranes were blocked in 5% skimmed milk in TBS-Tween for 1 h at room temperature, and probed with the following primary antibodies: Rabbit polyclonal antibodies PAK5 (cat. no. ab110069; Abcam), p-S338-RAF1 (cat. no. ab51042; Abcam), RAF1 (cat. no. ab137435; Abcam) and matrix metalloproteinase 2 (MMP2; cat. no. ab37150; Abcam); rabbit monoclonal antibodies c-Myc (cat. no. ab32072; Abcam), cyclin D1 (cat. no. ab134175; Abcam), Bcl-2 (cat. no. ab32124; Abcam) and Bax (cat. no. ab32503; Abcam) at 4°C overnight, after which time the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. ab222772 and ab222759; Abcam).

    Techniques: Transfection, MTT Assay

    Overexpression of IDO affected the MAPK/ERK pathway. Western blot assay was performed on the protein expression levels of (A) p-Raf-1, Raf-1, p-Mek1/2 Mek1/2 (B) p-Erk1/2 and (C) Erk1/2 in 16HBE cells treated with control, NC, DEX, NC+DEX, and IDO+DEX. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Overexpression of indoleamine 2, 3-dioxygenase contributes to the repair of human airway epithelial cells inhibited by dexamethasone via affecting the MAPK/ERK signaling pathway

    doi: 10.3892/etm.2018.6163

    Figure Lengend Snippet: Overexpression of IDO affected the MAPK/ERK pathway. Western blot assay was performed on the protein expression levels of (A) p-Raf-1, Raf-1, p-Mek1/2 Mek1/2 (B) p-Erk1/2 and (C) Erk1/2 in 16HBE cells treated with control, NC, DEX, NC+DEX, and IDO+DEX. *P

    Article Snippet: After blocking, the membranes were incubated with anti-IDO (dilution, 1:500; ab55305); anti-p-Raf-1 (dilution, 1:500; ab208449); anti-Raf-1 (dilution, 1:1,000; ab50858); anti-p-Mek1/2 (dilution, 1:1,000; ab194754); anti-Mek1/2 (dilution, 1:1,000; ab215263); anti-p-Erk1/2 (dilution, 1:1,000; ab201015); anti-Erk1/2 (dilution, 1:1,000; ab17942); anti-GAPDH (dilution, 1:1,000, ab8245; all from Abcam) antibodies overnight at 4°C.

    Techniques: Over Expression, Western Blot, Expressing

    Altered RAF1 Phosphorylation in HD Models Is Rescued by RRAS Inhibition. (A) Ratio of phospho-S338 to total RAF1 is increased in ST Hdh Q111/Q111 cells due to a reduced level of total RAF1 (n = 3). (B) Enhanced phospho-S338/total RAF1 in transiently transfected HEK293T cells (n = 3). (C) The R6/2 mouse model of Huntington's disease has elevated ratios of phospho-S338 to total RAF1 in regions of the brain affected by the disease (n = 2). **p

    Journal: PLoS Genetics

    Article Title: A Genome-Scale RNA-Interference Screen Identifies RRAS Signaling as a Pathologic Feature of Huntington's Disease

    doi: 10.1371/journal.pgen.1003042

    Figure Lengend Snippet: Altered RAF1 Phosphorylation in HD Models Is Rescued by RRAS Inhibition. (A) Ratio of phospho-S338 to total RAF1 is increased in ST Hdh Q111/Q111 cells due to a reduced level of total RAF1 (n = 3). (B) Enhanced phospho-S338/total RAF1 in transiently transfected HEK293T cells (n = 3). (C) The R6/2 mouse model of Huntington's disease has elevated ratios of phospho-S338 to total RAF1 in regions of the brain affected by the disease (n = 2). **p

    Article Snippet: The gels were transferred to 0.2 µm nitrocellulose, and the membranes then probed with 1∶500 of either anti-phospho-Raf-1 (Ser338) (Upstate, #05-538) to detect p-S338 RAF1, or anti-Raf1 (clone Y198, abcam, #ab32025) to detect total RAF1.

    Techniques: Inhibition, Transfection

    Suppression of phosphatidylethanolamine binding protein 1 (PEBP1) degradation eliminates the receptor-interacting protein kinases 4 (RIPK4)-induced activation of the RAF1/MEK/ERK pathway and pancreatic cancer cell migration and invasion. (A) Total and phosphorylated levels of RAF1, MEK1/2 and ERK1/2 in RIPK4-overexpressing and control cells following treatment with MG132 (an inhibitor of the 26S proteasome). (B and C) The effects of MG132-mediated suppression of PEBP1 degradation on RIPK4-overexpressing pancreatic cancer cell (B) migration and (C) invasion. (D) Proposed model of the mechanisms through which RIPK4 promotes pancreatic cancer cell migration and invasion via the PEBP1 degradation-induced activation of the RAF1/MEK/ERK pathway.

    Journal: International Journal of Oncology

    Article Title: RIPK4/PEBP1 axis promotes pancreatic cancer cell migration and invasion by activating RAF1/MEK/ERK signaling

    doi: 10.3892/ijo.2018.4269

    Figure Lengend Snippet: Suppression of phosphatidylethanolamine binding protein 1 (PEBP1) degradation eliminates the receptor-interacting protein kinases 4 (RIPK4)-induced activation of the RAF1/MEK/ERK pathway and pancreatic cancer cell migration and invasion. (A) Total and phosphorylated levels of RAF1, MEK1/2 and ERK1/2 in RIPK4-overexpressing and control cells following treatment with MG132 (an inhibitor of the 26S proteasome). (B and C) The effects of MG132-mediated suppression of PEBP1 degradation on RIPK4-overexpressing pancreatic cancer cell (B) migration and (C) invasion. (D) Proposed model of the mechanisms through which RIPK4 promotes pancreatic cancer cell migration and invasion via the PEBP1 degradation-induced activation of the RAF1/MEK/ERK pathway.

    Article Snippet: Antibodies against ERK1/2 (cs-9102) and p-RAF1 (cs-9427) were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Binding Assay, Activation Assay, Migration

    Receptor-interacting protein kinases 4 (RIPK4) promotes pancreatic cancer tumor cell metastasis via the RAF1/MEK/ERK pathway. (A) Pathway enrichment analysis of differentially expressed genes using the KEGG pathway database. (B) The levels of total and phosphorylated RAF1, MEK1/2 and ERK1/2 in pancreatic cancer cell lines in which RIPK4 was overexpressed or knocked down. (C) The levels of SMAD2, p-SMAD2, GSK-3β, p-GSK-3β and p-β-catenin in pancreatic cancer cell lines in which RIPK4 was overexpressed were detected by western blot analysis. (D and E) The effects of blocking RAF1/MEK/ERK signaling on pancreatic cancer cell (D) migration and (E) invasion were determined by Transwell assays using RIPK4-overexpressing Capan-1 and SW1990 cell lines. The numbers of migrating or invading cells were calculated, and the quantification of 3 randomly selected fields is shown in the histogram. * P

    Journal: International Journal of Oncology

    Article Title: RIPK4/PEBP1 axis promotes pancreatic cancer cell migration and invasion by activating RAF1/MEK/ERK signaling

    doi: 10.3892/ijo.2018.4269

    Figure Lengend Snippet: Receptor-interacting protein kinases 4 (RIPK4) promotes pancreatic cancer tumor cell metastasis via the RAF1/MEK/ERK pathway. (A) Pathway enrichment analysis of differentially expressed genes using the KEGG pathway database. (B) The levels of total and phosphorylated RAF1, MEK1/2 and ERK1/2 in pancreatic cancer cell lines in which RIPK4 was overexpressed or knocked down. (C) The levels of SMAD2, p-SMAD2, GSK-3β, p-GSK-3β and p-β-catenin in pancreatic cancer cell lines in which RIPK4 was overexpressed were detected by western blot analysis. (D and E) The effects of blocking RAF1/MEK/ERK signaling on pancreatic cancer cell (D) migration and (E) invasion were determined by Transwell assays using RIPK4-overexpressing Capan-1 and SW1990 cell lines. The numbers of migrating or invading cells were calculated, and the quantification of 3 randomly selected fields is shown in the histogram. * P

    Article Snippet: Antibodies against ERK1/2 (cs-9102) and p-RAF1 (cs-9427) were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Western Blot, Blocking Assay, Migration

    Phosphatidylethanolamine binding protein 1 (PEBP1) mediates the interaction between receptor-interacting protein kinases 4 (RIPK4) and RAF1/MEK/ERK signaling and has an inverse relationship with RIPK4. (A) The network analysis between RIPK4 and the RAF1/MEK/ERK signaling pathway. (B and C) PEBP1 mRNA expression in pancreatic cancer cells in which RIPK4 was (B) overexpressed or (C) knocked down. (D and E) Total and phosphorylated PEBP1 protein levels in pancreatic cancer cells in which RIPK4 was (D) overexpressed or (E) knocked down. (F and G) RIPK4 and PEBP1 expression exhibited an inverse correlation in pancreatic cancer patient tissue samples (P=0.0418 with Pearson's χ 2 test). (H) PEBP1 protein levels in RIPK4-overexpressing and control cells following treatment with MG132 (an inhibitor of the 26S proteasome).

    Journal: International Journal of Oncology

    Article Title: RIPK4/PEBP1 axis promotes pancreatic cancer cell migration and invasion by activating RAF1/MEK/ERK signaling

    doi: 10.3892/ijo.2018.4269

    Figure Lengend Snippet: Phosphatidylethanolamine binding protein 1 (PEBP1) mediates the interaction between receptor-interacting protein kinases 4 (RIPK4) and RAF1/MEK/ERK signaling and has an inverse relationship with RIPK4. (A) The network analysis between RIPK4 and the RAF1/MEK/ERK signaling pathway. (B and C) PEBP1 mRNA expression in pancreatic cancer cells in which RIPK4 was (B) overexpressed or (C) knocked down. (D and E) Total and phosphorylated PEBP1 protein levels in pancreatic cancer cells in which RIPK4 was (D) overexpressed or (E) knocked down. (F and G) RIPK4 and PEBP1 expression exhibited an inverse correlation in pancreatic cancer patient tissue samples (P=0.0418 with Pearson's χ 2 test). (H) PEBP1 protein levels in RIPK4-overexpressing and control cells following treatment with MG132 (an inhibitor of the 26S proteasome).

    Article Snippet: Antibodies against ERK1/2 (cs-9102) and p-RAF1 (cs-9427) were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Binding Assay, Expressing

    (A) GILZ sequence alignment with a set of TSC family proteins (upper part of diagram) and alignment of NH 2 -terminal sequence of GILZ to 1vig as proposed by threading server (3DPSSM) (lower part of diagram). PSS indicates the predicted secondary structure for the N-terminal domain. SS indicates the known secondary structure of the library template 1vig . (B) 3D model of human GILZ. α-Helices are represented as light blue cylinders, β-sheets are represented as red ribbons, and proline residues are indicated in red CPK. (C) Molecular modeling of Raf-1-GILZ interaction. Distribution of solutions (i.e., the 100, 50, and 10 top solutions found) of docking experiments of Raf-1 and GILZ; the red balls indicate the center of mass of Raf-1, for each solution, interacting with GILZ.

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: (A) GILZ sequence alignment with a set of TSC family proteins (upper part of diagram) and alignment of NH 2 -terminal sequence of GILZ to 1vig as proposed by threading server (3DPSSM) (lower part of diagram). PSS indicates the predicted secondary structure for the N-terminal domain. SS indicates the known secondary structure of the library template 1vig . (B) 3D model of human GILZ. α-Helices are represented as light blue cylinders, β-sheets are represented as red ribbons, and proline residues are indicated in red CPK. (C) Molecular modeling of Raf-1-GILZ interaction. Distribution of solutions (i.e., the 100, 50, and 10 top solutions found) of docking experiments of Raf-1 and GILZ; the red balls indicate the center of mass of Raf-1, for each solution, interacting with GILZ.

    Article Snippet: Glutathione S -transferase (GST)-GILZ fusion protein was prepared as previously described ( ); GST-Raf fusion protein corresponding to the human Ras-binding domain (RBD; residues 1 to 149) ( ) of Raf-1 (GST-Raf-RBD) was from Upstate Biotechnology.

    Techniques: Sequencing

    GILZ interacts with Raf-1 in COS-7-transfected cells. COS-7 cells were cotransfected with pUSEamp-Raf-1 (2 μg) and myc-GILZ (2 μg) vectors. Immunoprecipitation was performed with anti-myc antibody (3 μg/500 μg of protein), and immunoreactive proteins were visualized with anti-Raf-1 (A) or anti-myc (B) antibodies. Whole-cell lysates were loaded to control GILZ and Raf-1 expression. Serum-starved COS-7 cells, either untransfected or transfected with myc-GILZ, were treated for 15 min with PMA (10 ng/ml). Raf-1 immunoprecipitates were analyzed for kinase activity in the presence of [γ- 32 P]ATP by using GST-MEK (C) or GST-MEK, GST-ERK, and MBP (D) as substrates. P.C., positive control performed with 10 U of purified Raf-1 kinase; N.C., negative control performed with Raf-1 immunocomplex from PMA-stimulated cells in the absence of MEK substrate. (E) MEK phosphorylation was also assayed by Western blot with an antibody specific for phosphorylated MEK (pMEK). myc-GILZ-Raf-cotransfected COS-7 cells were immunostained with anti-myc and anti-Raf antibodies. Single staining and superimposed images are shown. (F) Nuclei were visualized by DAPI staining.

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: GILZ interacts with Raf-1 in COS-7-transfected cells. COS-7 cells were cotransfected with pUSEamp-Raf-1 (2 μg) and myc-GILZ (2 μg) vectors. Immunoprecipitation was performed with anti-myc antibody (3 μg/500 μg of protein), and immunoreactive proteins were visualized with anti-Raf-1 (A) or anti-myc (B) antibodies. Whole-cell lysates were loaded to control GILZ and Raf-1 expression. Serum-starved COS-7 cells, either untransfected or transfected with myc-GILZ, were treated for 15 min with PMA (10 ng/ml). Raf-1 immunoprecipitates were analyzed for kinase activity in the presence of [γ- 32 P]ATP by using GST-MEK (C) or GST-MEK, GST-ERK, and MBP (D) as substrates. P.C., positive control performed with 10 U of purified Raf-1 kinase; N.C., negative control performed with Raf-1 immunocomplex from PMA-stimulated cells in the absence of MEK substrate. (E) MEK phosphorylation was also assayed by Western blot with an antibody specific for phosphorylated MEK (pMEK). myc-GILZ-Raf-cotransfected COS-7 cells were immunostained with anti-myc and anti-Raf antibodies. Single staining and superimposed images are shown. (F) Nuclei were visualized by DAPI staining.

    Article Snippet: Glutathione S -transferase (GST)-GILZ fusion protein was prepared as previously described ( ); GST-Raf fusion protein corresponding to the human Ras-binding domain (RBD; residues 1 to 149) ( ) of Raf-1 (GST-Raf-RBD) was from Upstate Biotechnology.

    Techniques: Transfection, Immunoprecipitation, Expressing, Activity Assay, Positive Control, Purification, Negative Control, Western Blot, Staining

    GILZ interferes with Ras-Raf-1 complex. (A) Activated Ras was expressed in COS-7 cells, and 100 μg of cellular extracts was incubated for 1 h with 2.5 μg of GST-Raf-RBD. The complex was purified by adsorption to glutathione-Sepharose beads, washed, and resuspended in PBS. Purified GILZ was added at the concentrations indicated. After 1 h at 4°C, the GST-Raf-RBD beads were washed and examined for associated proteins by Western blotting with anti-Ras, anti-GILZ, and anti-GST antibodies. N.T., nontransfected cells. Clones transfected with pcDNA3 (PV6) or pcDNA3-GILZ (GIRL-19) were stimulated for 1 h with plastic-bound anti-CD3 MAb. A total of 200 μg of cellular extracts was incubated for 1 h with 2.5 μg of GST-Raf-RBD. (B) Western blotting was performed with anti-Ras antibody. (C) The nitrocellulose membrane was then stripped and reprobed with anti-GILZ antibody.

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: GILZ interferes with Ras-Raf-1 complex. (A) Activated Ras was expressed in COS-7 cells, and 100 μg of cellular extracts was incubated for 1 h with 2.5 μg of GST-Raf-RBD. The complex was purified by adsorption to glutathione-Sepharose beads, washed, and resuspended in PBS. Purified GILZ was added at the concentrations indicated. After 1 h at 4°C, the GST-Raf-RBD beads were washed and examined for associated proteins by Western blotting with anti-Ras, anti-GILZ, and anti-GST antibodies. N.T., nontransfected cells. Clones transfected with pcDNA3 (PV6) or pcDNA3-GILZ (GIRL-19) were stimulated for 1 h with plastic-bound anti-CD3 MAb. A total of 200 μg of cellular extracts was incubated for 1 h with 2.5 μg of GST-Raf-RBD. (B) Western blotting was performed with anti-Ras antibody. (C) The nitrocellulose membrane was then stripped and reprobed with anti-GILZ antibody.

    Article Snippet: Glutathione S -transferase (GST)-GILZ fusion protein was prepared as previously described ( ); GST-Raf fusion protein corresponding to the human Ras-binding domain (RBD; residues 1 to 149) ( ) of Raf-1 (GST-Raf-RBD) was from Upstate Biotechnology.

    Techniques: Incubation, Purification, Adsorption, Western Blot, Clone Assay, Transfection

    DEX inhibits Raf-1, MEK, and ERK-1/2 phosphorylation. Mouse thymocytes left untreated or pretreated for 6 h with DEX (100 nM) were stimulated for 1 h with plastic-bound anti-CD3 MAb. Western blotting was performed with an anti-pERK-1/2 (A), anti-pMEK-1/2 (B), anti-pRaf-1 (C), or anti-GILZ (D) antibody. Western blotting was also performed with an anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibody to verify that no modulation of protein expression occurred or with β-tubulin to verify that equivalent amounts of proteins were loaded in all lanes.

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: DEX inhibits Raf-1, MEK, and ERK-1/2 phosphorylation. Mouse thymocytes left untreated or pretreated for 6 h with DEX (100 nM) were stimulated for 1 h with plastic-bound anti-CD3 MAb. Western blotting was performed with an anti-pERK-1/2 (A), anti-pMEK-1/2 (B), anti-pRaf-1 (C), or anti-GILZ (D) antibody. Western blotting was also performed with an anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibody to verify that no modulation of protein expression occurred or with β-tubulin to verify that equivalent amounts of proteins were loaded in all lanes.

    Article Snippet: Glutathione S -transferase (GST)-GILZ fusion protein was prepared as previously described ( ); GST-Raf fusion protein corresponding to the human Ras-binding domain (RBD; residues 1 to 149) ( ) of Raf-1 (GST-Raf-RBD) was from Upstate Biotechnology.

    Techniques: Western Blot, Expressing

    GILZ overexpression inhibits ERK-1/2, MEK-1/2, and Raf-1 but not JNK phosphorylation. Clones transfected with pcDNA3 (PV6) or pcDNA3-GILZ (GIRL-19) were stimulated for 5 or 60 min with plastic-bound anti-CD3 MAb. Whole-cell lysates were probed with an antibody specific for phosphorylated ERK-1/2 (pERK-1/2) (A), MEK-1/2 (pMEK) (B), or Raf-1 (pRaf-1) (C). Western blots were also performed with anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibodies to verify that no modulation of protein expression occurred or with β-tubulin to verify that an equivalent amount of proteins was loaded in each lane. PV6 or GIRL-19 was stimulated for the times indicated with plastic-bound anti-CD3 MAb. (D) Whole-cell lysates were probed with an antibody recognizing both phosphorylated forms of JNK: p54 and p46 (pSAPK/JNK). C, untreated cells.

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: GILZ overexpression inhibits ERK-1/2, MEK-1/2, and Raf-1 but not JNK phosphorylation. Clones transfected with pcDNA3 (PV6) or pcDNA3-GILZ (GIRL-19) were stimulated for 5 or 60 min with plastic-bound anti-CD3 MAb. Whole-cell lysates were probed with an antibody specific for phosphorylated ERK-1/2 (pERK-1/2) (A), MEK-1/2 (pMEK) (B), or Raf-1 (pRaf-1) (C). Western blots were also performed with anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibodies to verify that no modulation of protein expression occurred or with β-tubulin to verify that an equivalent amount of proteins was loaded in each lane. PV6 or GIRL-19 was stimulated for the times indicated with plastic-bound anti-CD3 MAb. (D) Whole-cell lysates were probed with an antibody recognizing both phosphorylated forms of JNK: p54 and p46 (pSAPK/JNK). C, untreated cells.

    Article Snippet: Glutathione S -transferase (GST)-GILZ fusion protein was prepared as previously described ( ); GST-Raf fusion protein corresponding to the human Ras-binding domain (RBD; residues 1 to 149) ( ) of Raf-1 (GST-Raf-RBD) was from Upstate Biotechnology.

    Techniques: Over Expression, Clone Assay, Transfection, Western Blot, Expressing

    GILZ interacts with endogenous Raf-1 in mouse thymocytes. Mouse thymocytes were treated for 6 h with DEX (100 nM), and cell lysates were incubated with GST or GST-GILZ beads. Binding of Raf-1 (A), MEK-1/2 (B), and ERK-1/2 (C) was visualized by Western blotting. Whole-cell lysates from thymocytes left untreated or treated with DEX were immunoprecipitated with an anti-Raf-1 or control isotype antibody (4 μg/500 μg of protein). (D and E) Nitrocellulose membrane was probed with an anti-GILZ antiserum (D) and then stripped and reprobed with anti-Raf-1 antibody (E).

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: GILZ interacts with endogenous Raf-1 in mouse thymocytes. Mouse thymocytes were treated for 6 h with DEX (100 nM), and cell lysates were incubated with GST or GST-GILZ beads. Binding of Raf-1 (A), MEK-1/2 (B), and ERK-1/2 (C) was visualized by Western blotting. Whole-cell lysates from thymocytes left untreated or treated with DEX were immunoprecipitated with an anti-Raf-1 or control isotype antibody (4 μg/500 μg of protein). (D and E) Nitrocellulose membrane was probed with an anti-GILZ antiserum (D) and then stripped and reprobed with anti-Raf-1 antibody (E).

    Article Snippet: Glutathione S -transferase (GST)-GILZ fusion protein was prepared as previously described ( ); GST-Raf fusion protein corresponding to the human Ras-binding domain (RBD; residues 1 to 149) ( ) of Raf-1 (GST-Raf-RBD) was from Upstate Biotechnology.

    Techniques: Incubation, Binding Assay, Western Blot, Immunoprecipitation

    The GST-Raf-RBD interacts with the GILZ amino-terminal region. (A) GST pulldown was performed with GST-Raf-RBD fusion protein corresponding to the human RBD (residues 1 to 149) of Raf or GST alone, attached to glutathione-Sepharose beads as bait and whole-cell lysates from untreated and DEX-treated thymocytes. The membrane was probed with anti-GILZ antiserum. Total lysates from DEX-treated and untreated thymocytes were loaded to control GILZ expression. (B) GST-Raf-RBD fusion protein was incubated for 18 h with the 35 S-labeled in vitro-transcribed and translated proteins GILZ (lane 3), mutant 6 (lane 6), and mutant 13 (lane 9). Lane 1, GILZ; lane 2, GST plus GILZ; lane 4, mutant 6; lane 5, GST plus mutant 6; lane 7, mutant 13; lane 8, GST plus mutant 13. (C) GST-Raf-RBD fusion protein was incubated for 18 h with the 35 S-labeled in vitro-transcribed and -translated protein GILZ (lane 3) or mutant 2 (lane 6) or mutant 11 7 (line 9). Lane 1, GILZ; lane 2, GST plus GILZ; lane 4, mutant 2; lane 5, GST plus mutant 2; lane 7, mutant 11 7 ; lane 8, GST plus mutant 11 7 . (D) 3DO cells were transfected with the AP-1 luciferase reporter gene, along with GILZ, mutant 2, mutant 6, mutant 11 7 , mutant 13, or GILZ plus activated Raf-1, and then stimulated for 18 h with plastic-bound anti-CD3 MAb. The values are expressed as the increase (n-fold) of luciferase activity compared to that in unstimulated cells. The values of transfected control groups are comparable; only one of them is shown in the figure. Each transfection was performed in triplicate. The standard errors were

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: The GST-Raf-RBD interacts with the GILZ amino-terminal region. (A) GST pulldown was performed with GST-Raf-RBD fusion protein corresponding to the human RBD (residues 1 to 149) of Raf or GST alone, attached to glutathione-Sepharose beads as bait and whole-cell lysates from untreated and DEX-treated thymocytes. The membrane was probed with anti-GILZ antiserum. Total lysates from DEX-treated and untreated thymocytes were loaded to control GILZ expression. (B) GST-Raf-RBD fusion protein was incubated for 18 h with the 35 S-labeled in vitro-transcribed and translated proteins GILZ (lane 3), mutant 6 (lane 6), and mutant 13 (lane 9). Lane 1, GILZ; lane 2, GST plus GILZ; lane 4, mutant 6; lane 5, GST plus mutant 6; lane 7, mutant 13; lane 8, GST plus mutant 13. (C) GST-Raf-RBD fusion protein was incubated for 18 h with the 35 S-labeled in vitro-transcribed and -translated protein GILZ (lane 3) or mutant 2 (lane 6) or mutant 11 7 (line 9). Lane 1, GILZ; lane 2, GST plus GILZ; lane 4, mutant 2; lane 5, GST plus mutant 2; lane 7, mutant 11 7 ; lane 8, GST plus mutant 11 7 . (D) 3DO cells were transfected with the AP-1 luciferase reporter gene, along with GILZ, mutant 2, mutant 6, mutant 11 7 , mutant 13, or GILZ plus activated Raf-1, and then stimulated for 18 h with plastic-bound anti-CD3 MAb. The values are expressed as the increase (n-fold) of luciferase activity compared to that in unstimulated cells. The values of transfected control groups are comparable; only one of them is shown in the figure. Each transfection was performed in triplicate. The standard errors were

    Article Snippet: Glutathione S -transferase (GST)-GILZ fusion protein was prepared as previously described ( ); GST-Raf fusion protein corresponding to the human Ras-binding domain (RBD; residues 1 to 149) ( ) of Raf-1 (GST-Raf-RBD) was from Upstate Biotechnology.

    Techniques: Expressing, Incubation, Labeling, In Vitro, Mutagenesis, Transfection, Luciferase, Activity Assay

    (A) GILZ sequence alignment with a set of TSC family proteins (upper part of diagram) and alignment of NH 2 -terminal sequence of GILZ to 1vig as proposed by threading server (3DPSSM) (lower part of diagram). PSS indicates the predicted secondary structure for the N-terminal domain. SS indicates the known secondary structure of the library template 1vig . (B) 3D model of human GILZ. α-Helices are represented as light blue cylinders, β-sheets are represented as red ribbons, and proline residues are indicated in red CPK. (C) Molecular modeling of Raf-1-GILZ interaction. Distribution of solutions (i.e., the 100, 50, and 10 top solutions found) of docking experiments of Raf-1 and GILZ; the red balls indicate the center of mass of Raf-1, for each solution, interacting with GILZ.

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: (A) GILZ sequence alignment with a set of TSC family proteins (upper part of diagram) and alignment of NH 2 -terminal sequence of GILZ to 1vig as proposed by threading server (3DPSSM) (lower part of diagram). PSS indicates the predicted secondary structure for the N-terminal domain. SS indicates the known secondary structure of the library template 1vig . (B) 3D model of human GILZ. α-Helices are represented as light blue cylinders, β-sheets are represented as red ribbons, and proline residues are indicated in red CPK. (C) Molecular modeling of Raf-1-GILZ interaction. Distribution of solutions (i.e., the 100, 50, and 10 top solutions found) of docking experiments of Raf-1 and GILZ; the red balls indicate the center of mass of Raf-1, for each solution, interacting with GILZ.

    Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

    Techniques: Sequencing

    GILZ interacts with Raf-1 in COS-7-transfected cells. COS-7 cells were cotransfected with pUSEamp-Raf-1 (2 μg) and myc-GILZ (2 μg) vectors. Immunoprecipitation was performed with anti-myc antibody (3 μg/500 μg of protein), and immunoreactive proteins were visualized with anti-Raf-1 (A) or anti-myc (B) antibodies. Whole-cell lysates were loaded to control GILZ and Raf-1 expression. Serum-starved COS-7 cells, either untransfected or transfected with myc-GILZ, were treated for 15 min with PMA (10 ng/ml). Raf-1 immunoprecipitates were analyzed for kinase activity in the presence of [γ- 32 P]ATP by using GST-MEK (C) or GST-MEK, GST-ERK, and MBP (D) as substrates. P.C., positive control performed with 10 U of purified Raf-1 kinase; N.C., negative control performed with Raf-1 immunocomplex from PMA-stimulated cells in the absence of MEK substrate. (E) MEK phosphorylation was also assayed by Western blot with an antibody specific for phosphorylated MEK (pMEK). myc-GILZ-Raf-cotransfected COS-7 cells were immunostained with anti-myc and anti-Raf antibodies. Single staining and superimposed images are shown. (F) Nuclei were visualized by DAPI staining.

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: GILZ interacts with Raf-1 in COS-7-transfected cells. COS-7 cells were cotransfected with pUSEamp-Raf-1 (2 μg) and myc-GILZ (2 μg) vectors. Immunoprecipitation was performed with anti-myc antibody (3 μg/500 μg of protein), and immunoreactive proteins were visualized with anti-Raf-1 (A) or anti-myc (B) antibodies. Whole-cell lysates were loaded to control GILZ and Raf-1 expression. Serum-starved COS-7 cells, either untransfected or transfected with myc-GILZ, were treated for 15 min with PMA (10 ng/ml). Raf-1 immunoprecipitates were analyzed for kinase activity in the presence of [γ- 32 P]ATP by using GST-MEK (C) or GST-MEK, GST-ERK, and MBP (D) as substrates. P.C., positive control performed with 10 U of purified Raf-1 kinase; N.C., negative control performed with Raf-1 immunocomplex from PMA-stimulated cells in the absence of MEK substrate. (E) MEK phosphorylation was also assayed by Western blot with an antibody specific for phosphorylated MEK (pMEK). myc-GILZ-Raf-cotransfected COS-7 cells were immunostained with anti-myc and anti-Raf antibodies. Single staining and superimposed images are shown. (F) Nuclei were visualized by DAPI staining.

    Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

    Techniques: Transfection, Immunoprecipitation, Expressing, Activity Assay, Positive Control, Purification, Negative Control, Western Blot, Staining

    GILZ interferes with Ras-Raf-1 complex. (A) Activated Ras was expressed in COS-7 cells, and 100 μg of cellular extracts was incubated for 1 h with 2.5 μg of GST-Raf-RBD. The complex was purified by adsorption to glutathione-Sepharose beads, washed, and resuspended in PBS. Purified GILZ was added at the concentrations indicated. After 1 h at 4°C, the GST-Raf-RBD beads were washed and examined for associated proteins by Western blotting with anti-Ras, anti-GILZ, and anti-GST antibodies. N.T., nontransfected cells. Clones transfected with pcDNA3 (PV6) or pcDNA3-GILZ (GIRL-19) were stimulated for 1 h with plastic-bound anti-CD3 MAb. A total of 200 μg of cellular extracts was incubated for 1 h with 2.5 μg of GST-Raf-RBD. (B) Western blotting was performed with anti-Ras antibody. (C) The nitrocellulose membrane was then stripped and reprobed with anti-GILZ antibody.

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: GILZ interferes with Ras-Raf-1 complex. (A) Activated Ras was expressed in COS-7 cells, and 100 μg of cellular extracts was incubated for 1 h with 2.5 μg of GST-Raf-RBD. The complex was purified by adsorption to glutathione-Sepharose beads, washed, and resuspended in PBS. Purified GILZ was added at the concentrations indicated. After 1 h at 4°C, the GST-Raf-RBD beads were washed and examined for associated proteins by Western blotting with anti-Ras, anti-GILZ, and anti-GST antibodies. N.T., nontransfected cells. Clones transfected with pcDNA3 (PV6) or pcDNA3-GILZ (GIRL-19) were stimulated for 1 h with plastic-bound anti-CD3 MAb. A total of 200 μg of cellular extracts was incubated for 1 h with 2.5 μg of GST-Raf-RBD. (B) Western blotting was performed with anti-Ras antibody. (C) The nitrocellulose membrane was then stripped and reprobed with anti-GILZ antibody.

    Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

    Techniques: Incubation, Purification, Adsorption, Western Blot, Clone Assay, Transfection

    DEX inhibits Raf-1, MEK, and ERK-1/2 phosphorylation. Mouse thymocytes left untreated or pretreated for 6 h with DEX (100 nM) were stimulated for 1 h with plastic-bound anti-CD3 MAb. Western blotting was performed with an anti-pERK-1/2 (A), anti-pMEK-1/2 (B), anti-pRaf-1 (C), or anti-GILZ (D) antibody. Western blotting was also performed with an anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibody to verify that no modulation of protein expression occurred or with β-tubulin to verify that equivalent amounts of proteins were loaded in all lanes.

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: DEX inhibits Raf-1, MEK, and ERK-1/2 phosphorylation. Mouse thymocytes left untreated or pretreated for 6 h with DEX (100 nM) were stimulated for 1 h with plastic-bound anti-CD3 MAb. Western blotting was performed with an anti-pERK-1/2 (A), anti-pMEK-1/2 (B), anti-pRaf-1 (C), or anti-GILZ (D) antibody. Western blotting was also performed with an anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibody to verify that no modulation of protein expression occurred or with β-tubulin to verify that equivalent amounts of proteins were loaded in all lanes.

    Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

    Techniques: Western Blot, Expressing

    GILZ overexpression inhibits ERK-1/2, MEK-1/2, and Raf-1 but not JNK phosphorylation. Clones transfected with pcDNA3 (PV6) or pcDNA3-GILZ (GIRL-19) were stimulated for 5 or 60 min with plastic-bound anti-CD3 MAb. Whole-cell lysates were probed with an antibody specific for phosphorylated ERK-1/2 (pERK-1/2) (A), MEK-1/2 (pMEK) (B), or Raf-1 (pRaf-1) (C). Western blots were also performed with anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibodies to verify that no modulation of protein expression occurred or with β-tubulin to verify that an equivalent amount of proteins was loaded in each lane. PV6 or GIRL-19 was stimulated for the times indicated with plastic-bound anti-CD3 MAb. (D) Whole-cell lysates were probed with an antibody recognizing both phosphorylated forms of JNK: p54 and p46 (pSAPK/JNK). C, untreated cells.

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: GILZ overexpression inhibits ERK-1/2, MEK-1/2, and Raf-1 but not JNK phosphorylation. Clones transfected with pcDNA3 (PV6) or pcDNA3-GILZ (GIRL-19) were stimulated for 5 or 60 min with plastic-bound anti-CD3 MAb. Whole-cell lysates were probed with an antibody specific for phosphorylated ERK-1/2 (pERK-1/2) (A), MEK-1/2 (pMEK) (B), or Raf-1 (pRaf-1) (C). Western blots were also performed with anti-ERK-1/2, anti-MEK-1/2, or anti-Raf-1 antibodies to verify that no modulation of protein expression occurred or with β-tubulin to verify that an equivalent amount of proteins was loaded in each lane. PV6 or GIRL-19 was stimulated for the times indicated with plastic-bound anti-CD3 MAb. (D) Whole-cell lysates were probed with an antibody recognizing both phosphorylated forms of JNK: p54 and p46 (pSAPK/JNK). C, untreated cells.

    Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

    Techniques: Over Expression, Clone Assay, Transfection, Western Blot, Expressing

    GILZ interacts with endogenous Raf-1 in mouse thymocytes. Mouse thymocytes were treated for 6 h with DEX (100 nM), and cell lysates were incubated with GST or GST-GILZ beads. Binding of Raf-1 (A), MEK-1/2 (B), and ERK-1/2 (C) was visualized by Western blotting. Whole-cell lysates from thymocytes left untreated or treated with DEX were immunoprecipitated with an anti-Raf-1 or control isotype antibody (4 μg/500 μg of protein). (D and E) Nitrocellulose membrane was probed with an anti-GILZ antiserum (D) and then stripped and reprobed with anti-Raf-1 antibody (E).

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: GILZ interacts with endogenous Raf-1 in mouse thymocytes. Mouse thymocytes were treated for 6 h with DEX (100 nM), and cell lysates were incubated with GST or GST-GILZ beads. Binding of Raf-1 (A), MEK-1/2 (B), and ERK-1/2 (C) was visualized by Western blotting. Whole-cell lysates from thymocytes left untreated or treated with DEX were immunoprecipitated with an anti-Raf-1 or control isotype antibody (4 μg/500 μg of protein). (D and E) Nitrocellulose membrane was probed with an anti-GILZ antiserum (D) and then stripped and reprobed with anti-Raf-1 antibody (E).

    Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

    Techniques: Incubation, Binding Assay, Western Blot, Immunoprecipitation

    The GST-Raf-RBD interacts with the GILZ amino-terminal region. (A) GST pulldown was performed with GST-Raf-RBD fusion protein corresponding to the human RBD (residues 1 to 149) of Raf or GST alone, attached to glutathione-Sepharose beads as bait and whole-cell lysates from untreated and DEX-treated thymocytes. The membrane was probed with anti-GILZ antiserum. Total lysates from DEX-treated and untreated thymocytes were loaded to control GILZ expression. (B) GST-Raf-RBD fusion protein was incubated for 18 h with the 35 S-labeled in vitro-transcribed and translated proteins GILZ (lane 3), mutant 6 (lane 6), and mutant 13 (lane 9). Lane 1, GILZ; lane 2, GST plus GILZ; lane 4, mutant 6; lane 5, GST plus mutant 6; lane 7, mutant 13; lane 8, GST plus mutant 13. (C) GST-Raf-RBD fusion protein was incubated for 18 h with the 35 S-labeled in vitro-transcribed and -translated protein GILZ (lane 3) or mutant 2 (lane 6) or mutant 11 7 (line 9). Lane 1, GILZ; lane 2, GST plus GILZ; lane 4, mutant 2; lane 5, GST plus mutant 2; lane 7, mutant 11 7 ; lane 8, GST plus mutant 11 7 . (D) 3DO cells were transfected with the AP-1 luciferase reporter gene, along with GILZ, mutant 2, mutant 6, mutant 11 7 , mutant 13, or GILZ plus activated Raf-1, and then stimulated for 18 h with plastic-bound anti-CD3 MAb. The values are expressed as the increase (n-fold) of luciferase activity compared to that in unstimulated cells. The values of transfected control groups are comparable; only one of them is shown in the figure. Each transfection was performed in triplicate. The standard errors were

    Journal: Molecular and Cellular Biology

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits the Raf-Extracellular Signal-Regulated Kinase Pathway by Binding to Raf-1

    doi: 10.1128/MCB.22.22.7929-7941.2002

    Figure Lengend Snippet: The GST-Raf-RBD interacts with the GILZ amino-terminal region. (A) GST pulldown was performed with GST-Raf-RBD fusion protein corresponding to the human RBD (residues 1 to 149) of Raf or GST alone, attached to glutathione-Sepharose beads as bait and whole-cell lysates from untreated and DEX-treated thymocytes. The membrane was probed with anti-GILZ antiserum. Total lysates from DEX-treated and untreated thymocytes were loaded to control GILZ expression. (B) GST-Raf-RBD fusion protein was incubated for 18 h with the 35 S-labeled in vitro-transcribed and translated proteins GILZ (lane 3), mutant 6 (lane 6), and mutant 13 (lane 9). Lane 1, GILZ; lane 2, GST plus GILZ; lane 4, mutant 6; lane 5, GST plus mutant 6; lane 7, mutant 13; lane 8, GST plus mutant 13. (C) GST-Raf-RBD fusion protein was incubated for 18 h with the 35 S-labeled in vitro-transcribed and -translated protein GILZ (lane 3) or mutant 2 (lane 6) or mutant 11 7 (line 9). Lane 1, GILZ; lane 2, GST plus GILZ; lane 4, mutant 2; lane 5, GST plus mutant 2; lane 7, mutant 11 7 ; lane 8, GST plus mutant 11 7 . (D) 3DO cells were transfected with the AP-1 luciferase reporter gene, along with GILZ, mutant 2, mutant 6, mutant 11 7 , mutant 13, or GILZ plus activated Raf-1, and then stimulated for 18 h with plastic-bound anti-CD3 MAb. The values are expressed as the increase (n-fold) of luciferase activity compared to that in unstimulated cells. The values of transfected control groups are comparable; only one of them is shown in the figure. Each transfection was performed in triplicate. The standard errors were

    Article Snippet: The primary antibodies were a polyclonal rabbit antiserum recognizing GILZ, a polyclonal rabbit anti-mouse phospho-ERK-1/2, and anti-mouse ERK-1/2 (Cell Signaling), anti-Fos, anti-Jun, anti-Raf-1, monoclonal rat anti-mouse phospho-Raf-1 (series 338; Upstate Biotechnology), anti-MEK, and polyclonal rabbit anti-human phospho-MEK (Ser 217/221; Cell Signaling) antibodies.

    Techniques: Expressing, Incubation, Labeling, In Vitro, Mutagenesis, Transfection, Luciferase, Activity Assay