raf 1 activity  (Millipore)


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    RAF1
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    Structured Review

    Millipore raf 1 activity
    PM-targeted active Ras efficiently activates Raf. (A) NIH 3T3 cells were cotransfected to express <t>CFP-Raf-1,</t> together with either YFP-tagged H-Ras(61L) or PM-Ras(61L). Thirty-six hours after transfection, cells positive for both YFP and CFP were analyzed

    https://www.bioz.com/result/raf 1 activity/product/Millipore
    Average 96 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    raf 1 activity - by Bioz Stars, 2020-07
    96/100 stars

    Images

    1) Product Images from "Compartmentalized Ras Proteins Transform NIH 3T3 Cells with Different Efficiencies ▿Compartmentalized Ras Proteins Transform NIH 3T3 Cells with Different Efficiencies ▿ †"

    Article Title: Compartmentalized Ras Proteins Transform NIH 3T3 Cells with Different Efficiencies ▿Compartmentalized Ras Proteins Transform NIH 3T3 Cells with Different Efficiencies ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00137-10

    PM-targeted active Ras efficiently activates Raf. (A) NIH 3T3 cells were cotransfected to express CFP-Raf-1, together with either YFP-tagged H-Ras(61L) or PM-Ras(61L). Thirty-six hours after transfection, cells positive for both YFP and CFP were analyzed
    Figure Legend Snippet: PM-targeted active Ras efficiently activates Raf. (A) NIH 3T3 cells were cotransfected to express CFP-Raf-1, together with either YFP-tagged H-Ras(61L) or PM-Ras(61L). Thirty-six hours after transfection, cells positive for both YFP and CFP were analyzed

    Techniques Used: Transfection

    2) Product Images from "TNFα induces survival through the FLIP-L-dependent activation of the MAPK/ERK pathway"

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

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.25

    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
    Figure Legend 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

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

    Related Articles

    Nucleic Acid Electrophoresis:

    Article Title: Cyclic AMP-Mediated Inhibition of Cell Growth Requires the Small G Protein Rap1
    Article Snippet: .. For detection of Raf-1, Myc–Raf-1, ERK2, Myc-ERK2, HA, Flag, Ras, Rap1, pAKT, and pERK1/2, equal protein amounts of cell lysate per treatment condition were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted onto polyrinylidene difluoride (PVDF) (Millipore Corporation, Bedford, Mass.) membranes, and probed with the corresponding antibodies according to the manufacturer's guidelines. .. Seventy to eighty percent confluent Hek293 or NIH 3T3 cells were cotransfected with the indicated cDNAs by using a Lipofectamine kit (Gibco BRL) according to the manufacturer's instructions.

    Transfection:

    Article Title: p21 Activated Kinase 5 Activates Raf-1 and Targets it to Mitochondria
    Article Snippet: .. To test for co-localization of Raf-1 and PAK5, HEK293 cells were plated on glass cover slips pre-coated with 25 μg/ml fibronectin (Sigma), and then transfected with expression vectors for Flag-tagged wt Raf-1 and Myc-tagged wt PAK5, alone or together. ..

    Positron Emission Tomography:

    Article Title: KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1
    Article Snippet: .. Hexahistidine (His)-tagged and FLAG-tagged constructs of murine wild-type KSR1, murine kinase-inactive KSR1 (D683A/D700A), His-tagged wild-type Raf-1, and His-tagged kinase-inactive Raf-1 (Raf-1 K375M) were subcloned into the pET DUET (Novagen, Darmstadt, Germany) bacterial expression vector. .. Murine KSR1 harboring an N-terminal truncation of 521 amino acids (KSR1ΔN521), and murine KSR1 harboring a deletion of the CA1 domain (KSR1ΔCA1) were generated by PCR based mutagenesis and subcloned into the pET DUET bacterial expression vector.

    In Vitro:

    Article Title: KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1
    Article Snippet: .. For some assays, recombinant constitutively active truncated Raf-1 (rCA-Raf-1) (Millipore) was used as a positive control for in vitro MAPK cascade activation. .. MBP phosphorylation assays were performed using the MBP Kinase Flex Assay Kit (Millipore) with rKSR1, rD683/D700A, rKSR1ΔN521, rRaf-1, or rRaf-1 K375M proteins following manufacturer’s protocol.

    Positive Control:

    Article Title: KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1
    Article Snippet: .. For some assays, recombinant constitutively active truncated Raf-1 (rCA-Raf-1) (Millipore) was used as a positive control for in vitro MAPK cascade activation. .. MBP phosphorylation assays were performed using the MBP Kinase Flex Assay Kit (Millipore) with rKSR1, rD683/D700A, rKSR1ΔN521, rRaf-1, or rRaf-1 K375M proteins following manufacturer’s protocol.

    Construct:

    Article Title: KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1
    Article Snippet: .. Hexahistidine (His)-tagged and FLAG-tagged constructs of murine wild-type KSR1, murine kinase-inactive KSR1 (D683A/D700A), His-tagged wild-type Raf-1, and His-tagged kinase-inactive Raf-1 (Raf-1 K375M) were subcloned into the pET DUET (Novagen, Darmstadt, Germany) bacterial expression vector. .. Murine KSR1 harboring an N-terminal truncation of 521 amino acids (KSR1ΔN521), and murine KSR1 harboring a deletion of the CA1 domain (KSR1ΔCA1) were generated by PCR based mutagenesis and subcloned into the pET DUET bacterial expression vector.

    Activation Assay:

    Article Title: KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1
    Article Snippet: .. For some assays, recombinant constitutively active truncated Raf-1 (rCA-Raf-1) (Millipore) was used as a positive control for in vitro MAPK cascade activation. .. MBP phosphorylation assays were performed using the MBP Kinase Flex Assay Kit (Millipore) with rKSR1, rD683/D700A, rKSR1ΔN521, rRaf-1, or rRaf-1 K375M proteins following manufacturer’s protocol.

    Article Title: Peroxynitrite activates ERK via Raf-1 and MEK, independently from EGF receptor and p21Ras in H9C2 cardiomyocytes
    Article Snippet: .. To address the role of the various signaling intermediates possibly involved in ERK activation by ONOO− , cells were treated with the following inhibitors before ONOO− stimulation: AG1478, a specific inhibitor of EGFR tyrosine kinase (100 nM, Calbiochem, San Diego, CA), BAY 43–9006, a Raf-1 kinase inhibitor (5 μM, Calbiochem) [ ], and PD98059, a specific MEK 1 inhibitor (50 μM, Calbiochem). ..

    Incubation:

    Article Title: In vitro silencing of the insulin receptor attenuates cellular accumulation of fibronectin in renal mesangial cells
    Article Snippet: .. Normalized cell lysates were incubated with Raf-1 RBD preconjugated agarose (Millipore) for 2 hours at 4°C. ..

    other:

    Article Title: Propofol induces MAPK/ERK cascade dependant expression of cFos and Egr-1 in rat hippocampal slices
    Article Snippet: FTI-277 (RAS Inhibitor-Cat # 344555), RAF1 Kinase Inhibitor I (Cat # 553008), U0126 (MEK Inhibitor-Cat # 662005), PD98059 (ERK Inhibitor-Cat # 513000), and SB203580 (p38-MAPK Inhibitor-Cat # 559389), were purchased from Calbiochem (San Diego, CA).

    Activity Assay:

    Article Title: Sorafenib and its derivative SC-49 sensitize hepatocellular carcinoma cells to CS-1008, a humanized anti-TNFRSF10B (DR5) antibody
    Article Snippet: .. The Raf-1 kinase cascade assay kit (Upstate-Millipore, Billerica, MA) was used to examine Raf-1 kinase activity. .. The VEGFR1 kinase activity kit was purchased from Reaction Biology Corp. (Malvern, PA, USA).

    Expressing:

    Article Title: KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1
    Article Snippet: .. Hexahistidine (His)-tagged and FLAG-tagged constructs of murine wild-type KSR1, murine kinase-inactive KSR1 (D683A/D700A), His-tagged wild-type Raf-1, and His-tagged kinase-inactive Raf-1 (Raf-1 K375M) were subcloned into the pET DUET (Novagen, Darmstadt, Germany) bacterial expression vector. .. Murine KSR1 harboring an N-terminal truncation of 521 amino acids (KSR1ΔN521), and murine KSR1 harboring a deletion of the CA1 domain (KSR1ΔCA1) were generated by PCR based mutagenesis and subcloned into the pET DUET bacterial expression vector.

    Article Title: p21 Activated Kinase 5 Activates Raf-1 and Targets it to Mitochondria
    Article Snippet: .. To test for co-localization of Raf-1 and PAK5, HEK293 cells were plated on glass cover slips pre-coated with 25 μg/ml fibronectin (Sigma), and then transfected with expression vectors for Flag-tagged wt Raf-1 and Myc-tagged wt PAK5, alone or together. ..

    Recombinant:

    Article Title: KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1
    Article Snippet: .. For some assays, recombinant constitutively active truncated Raf-1 (rCA-Raf-1) (Millipore) was used as a positive control for in vitro MAPK cascade activation. .. MBP phosphorylation assays were performed using the MBP Kinase Flex Assay Kit (Millipore) with rKSR1, rD683/D700A, rKSR1ΔN521, rRaf-1, or rRaf-1 K375M proteins following manufacturer’s protocol.

    SDS Page:

    Article Title: Cyclic AMP-Mediated Inhibition of Cell Growth Requires the Small G Protein Rap1
    Article Snippet: .. For detection of Raf-1, Myc–Raf-1, ERK2, Myc-ERK2, HA, Flag, Ras, Rap1, pAKT, and pERK1/2, equal protein amounts of cell lysate per treatment condition were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted onto polyrinylidene difluoride (PVDF) (Millipore Corporation, Bedford, Mass.) membranes, and probed with the corresponding antibodies according to the manufacturer's guidelines. .. Seventy to eighty percent confluent Hek293 or NIH 3T3 cells were cotransfected with the indicated cDNAs by using a Lipofectamine kit (Gibco BRL) according to the manufacturer's instructions.

    Plasmid Preparation:

    Article Title: KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1
    Article Snippet: .. Hexahistidine (His)-tagged and FLAG-tagged constructs of murine wild-type KSR1, murine kinase-inactive KSR1 (D683A/D700A), His-tagged wild-type Raf-1, and His-tagged kinase-inactive Raf-1 (Raf-1 K375M) were subcloned into the pET DUET (Novagen, Darmstadt, Germany) bacterial expression vector. .. Murine KSR1 harboring an N-terminal truncation of 521 amino acids (KSR1ΔN521), and murine KSR1 harboring a deletion of the CA1 domain (KSR1ΔCA1) were generated by PCR based mutagenesis and subcloned into the pET DUET bacterial expression vector.

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    Millipore raf 1 kinase activity
    . β -Actin was used as the control. (B) Both OPRM1 and <t>Raf-1</t> are required for AC superactivation. MEF-Raf-1 WT cells were transfected with OPRM1 (△ Raf-1 WT + OPRM1) or Raf-1 (◇ Raf-1 WT + Raf-1) cDNA. Raf-1 KO cells were transfected with OPRM1 (■ Raf-1 KO + OPRM1), Raf-1 (● Raf-1 KO + Raf-1), or both (▼ Raf-1 KO + OPRM1 + Raf-1) cDNA. After 48 hours, the cells were treated with morphine (1 μ M, 4 hours), and cAMP assays were performed in the presence of various concentrations of naloxone. Forskolin-induced cAMP in cells without drug treatment is used as 100% control. (C) Bar graph representation of the results from B when 10 −4 M naloxone was added in the assay. ** P
    Raf 1 Kinase Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/raf 1 kinase activity/product/Millipore
    Average 96 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    raf 1 kinase activity - by Bioz Stars, 2020-07
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    . β -Actin was used as the control. (B) Both OPRM1 and Raf-1 are required for AC superactivation. MEF-Raf-1 WT cells were transfected with OPRM1 (△ Raf-1 WT + OPRM1) or Raf-1 (◇ Raf-1 WT + Raf-1) cDNA. Raf-1 KO cells were transfected with OPRM1 (■ Raf-1 KO + OPRM1), Raf-1 (● Raf-1 KO + Raf-1), or both (▼ Raf-1 KO + OPRM1 + Raf-1) cDNA. After 48 hours, the cells were treated with morphine (1 μ M, 4 hours), and cAMP assays were performed in the presence of various concentrations of naloxone. Forskolin-induced cAMP in cells without drug treatment is used as 100% control. (C) Bar graph representation of the results from B when 10 −4 M naloxone was added in the assay. ** P

    Journal: Molecular Pharmacology

    Article Title: A Novel Noncanonical Signaling Pathway for the µ

    doi: 10.1124/mol.113.088278

    Figure Lengend Snippet: . β -Actin was used as the control. (B) Both OPRM1 and Raf-1 are required for AC superactivation. MEF-Raf-1 WT cells were transfected with OPRM1 (△ Raf-1 WT + OPRM1) or Raf-1 (◇ Raf-1 WT + Raf-1) cDNA. Raf-1 KO cells were transfected with OPRM1 (■ Raf-1 KO + OPRM1), Raf-1 (● Raf-1 KO + Raf-1), or both (▼ Raf-1 KO + OPRM1 + Raf-1) cDNA. After 48 hours, the cells were treated with morphine (1 μ M, 4 hours), and cAMP assays were performed in the presence of various concentrations of naloxone. Forskolin-induced cAMP in cells without drug treatment is used as 100% control. (C) Bar graph representation of the results from B when 10 −4 M naloxone was added in the assay. ** P

    Article Snippet: Raf-1 kinase activity was determined with the assay kit supplied by Millipore.

    Techniques: Transfection

    Raf-1 activation is not observed in cells expressing Tyr mutant OPRM1. (A) Raf-1 activation. HEKMT or HEKMT-Y336F cells were treated with or without 1 μ M morphine (Mor) for 4 hours followed by 0 or 10 μ . The blots were probed with anti-pMEK1, anti–Raf-1, and anti-G β . (B) Bar graph summary of Western blots as shown in A. The ratio of the density of pMEK1 to the density of Raf-1 from untreated cells was used as 100% control. * P

    Journal: Molecular Pharmacology

    Article Title: A Novel Noncanonical Signaling Pathway for the µ

    doi: 10.1124/mol.113.088278

    Figure Lengend Snippet: Raf-1 activation is not observed in cells expressing Tyr mutant OPRM1. (A) Raf-1 activation. HEKMT or HEKMT-Y336F cells were treated with or without 1 μ M morphine (Mor) for 4 hours followed by 0 or 10 μ . The blots were probed with anti-pMEK1, anti–Raf-1, and anti-G β . (B) Bar graph summary of Western blots as shown in A. The ratio of the density of pMEK1 to the density of Raf-1 from untreated cells was used as 100% control. * P

    Article Snippet: Raf-1 kinase activity was determined with the assay kit supplied by Millipore.

    Techniques: Activation Assay, Expressing, Mutagenesis, Western Blot

    Schematic summary of OPRM1 differential signal pathways upon acute and chronic morphine administration. During acute morphine treatment, OPRM1 interacts with G α i2 ) and causes inhibition of adenylyl cyclase activity and a decrease of the intracellular cAMP level. Under prolonged treatment, Src kinase is recruited by the OPRM1 signal complex, where it is activated. The activated Src phosphorylates OPRM1 at Tyr 336 . The phosphorylated Tyr 336 serves as a docking site to recruit Grb/SOS/Ras and Raf-1, which converts the OPRM1 from a classic G i /G o -coupled receptor into an RTK-like entity. The activated Raf-1 eventually phosphorylates and activates the AC isozymes, most likely AC5/6, resulting in AC superactivation. By doing so, OPRM1 can lead to an alternative signaling pathway that is initiated by the same agonist but is different depending on the duration of agonist exposure.

    Journal: Molecular Pharmacology

    Article Title: A Novel Noncanonical Signaling Pathway for the µ

    doi: 10.1124/mol.113.088278

    Figure Lengend Snippet: Schematic summary of OPRM1 differential signal pathways upon acute and chronic morphine administration. During acute morphine treatment, OPRM1 interacts with G α i2 ) and causes inhibition of adenylyl cyclase activity and a decrease of the intracellular cAMP level. Under prolonged treatment, Src kinase is recruited by the OPRM1 signal complex, where it is activated. The activated Src phosphorylates OPRM1 at Tyr 336 . The phosphorylated Tyr 336 serves as a docking site to recruit Grb/SOS/Ras and Raf-1, which converts the OPRM1 from a classic G i /G o -coupled receptor into an RTK-like entity. The activated Raf-1 eventually phosphorylates and activates the AC isozymes, most likely AC5/6, resulting in AC superactivation. By doing so, OPRM1 can lead to an alternative signaling pathway that is initiated by the same agonist but is different depending on the duration of agonist exposure.

    Article Snippet: Raf-1 kinase activity was determined with the assay kit supplied by Millipore.

    Techniques: Inhibition, Activity Assay

    Raf-1 is activated during chronic morphine treatment. (A) Raf-1 is activated within the OPRM1 signal complex in a chronic morphine– and Src kinase–dependent manner. The cells were treated with or without morphine (Mor; 1 μ M, 4 hours) and naloxone (Nal, 10 μ M, 15 minutes), as indicated. Src inhibitor (PP2; 2 μ M) or Raf-1 inhibitor GW5074 (GW; 10 μ . (B) Bar graph summary of Western blots as shown in A. The ratio of the density of pRaf1, pSrc, or AC5/6 to the density of OPRM1 from untreated cells was used as 100% control. * P

    Journal: Molecular Pharmacology

    Article Title: A Novel Noncanonical Signaling Pathway for the µ

    doi: 10.1124/mol.113.088278

    Figure Lengend Snippet: Raf-1 is activated during chronic morphine treatment. (A) Raf-1 is activated within the OPRM1 signal complex in a chronic morphine– and Src kinase–dependent manner. The cells were treated with or without morphine (Mor; 1 μ M, 4 hours) and naloxone (Nal, 10 μ M, 15 minutes), as indicated. Src inhibitor (PP2; 2 μ M) or Raf-1 inhibitor GW5074 (GW; 10 μ . (B) Bar graph summary of Western blots as shown in A. The ratio of the density of pRaf1, pSrc, or AC5/6 to the density of OPRM1 from untreated cells was used as 100% control. * P

    Article Snippet: Raf-1 kinase activity was determined with the assay kit supplied by Millipore.

    Techniques: Western Blot

    . β -Actin was used as control. (B) AC activity in the presence of naloxone after 4 hours of morphine treatment. MEF-Raf-1 KO cells were transfected with OPRM1 and WT Raf-1 (■ OPRM1 + Raf-1), or Raf-1 Y340A mutant (● OPRM1 + Y340A), or Raf-1 Y341A mutant (□ OPRM1 + Y341A), or double Tyr mutant Y340/341AA (▼ OPRM1+Y340/341AA), or constitutive active mutant Y340/341DD (◇ OPRM1+Y340/341DD) cDNA. After 48 hours, the cells were treated with 1 μ M morphine for 4 hours, then cAMP assays were performed in the presence of the indicated naloxone concentrations. Forskolin-induced cAMP in cells without drug treatment is used as 100% control. (C) Bar graph representation of the results from B in the presence of 10 −4 M naloxone. ** P

    Journal: Molecular Pharmacology

    Article Title: A Novel Noncanonical Signaling Pathway for the µ

    doi: 10.1124/mol.113.088278

    Figure Lengend Snippet: . β -Actin was used as control. (B) AC activity in the presence of naloxone after 4 hours of morphine treatment. MEF-Raf-1 KO cells were transfected with OPRM1 and WT Raf-1 (■ OPRM1 + Raf-1), or Raf-1 Y340A mutant (● OPRM1 + Y340A), or Raf-1 Y341A mutant (□ OPRM1 + Y341A), or double Tyr mutant Y340/341AA (▼ OPRM1+Y340/341AA), or constitutive active mutant Y340/341DD (◇ OPRM1+Y340/341DD) cDNA. After 48 hours, the cells were treated with 1 μ M morphine for 4 hours, then cAMP assays were performed in the presence of the indicated naloxone concentrations. Forskolin-induced cAMP in cells without drug treatment is used as 100% control. (C) Bar graph representation of the results from B in the presence of 10 −4 M naloxone. ** P

    Article Snippet: Raf-1 kinase activity was determined with the assay kit supplied by Millipore.

    Techniques: Activity Assay, Transfection, Mutagenesis

    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: Determination of Raf-1 kinase activity In order to determine Raf-1 activity, we used a Raf-1 kinase assay kit with chemiluminescence detection (Millipore).

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

    PM-targeted active Ras efficiently activates Raf. (A) NIH 3T3 cells were cotransfected to express CFP-Raf-1, together with either YFP-tagged H-Ras(61L) or PM-Ras(61L). Thirty-six hours after transfection, cells positive for both YFP and CFP were analyzed

    Journal: Molecular and Cellular Biology

    Article Title: Compartmentalized Ras Proteins Transform NIH 3T3 Cells with Different Efficiencies ▿Compartmentalized Ras Proteins Transform NIH 3T3 Cells with Different Efficiencies ▿ †

    doi: 10.1128/MCB.00137-10

    Figure Lengend Snippet: PM-targeted active Ras efficiently activates Raf. (A) NIH 3T3 cells were cotransfected to express CFP-Raf-1, together with either YFP-tagged H-Ras(61L) or PM-Ras(61L). Thirty-six hours after transfection, cells positive for both YFP and CFP were analyzed

    Article Snippet: Ten micrograms of protein from the P100 fraction was taken to measure Raf-1 activity using a MEK1/Erk2-coupled in vitro kinase assay kit from Millipore (Billerica, MA).

    Techniques: Transfection

    Inhibition of Raf/MEK/ERK activation by MCP compounds. ( A ) HEK293 cells were cotransfected with expression vectors for Flag-tagged Raf-1 and H-Ras (V12). Cells were serum-starved in the presence of DMSO (−) or 20 μM MCP110. Raf-1 assay was performed by immunoprecipitating Flag-tagged Raf-1. Mean ± SEM ( n = 3). ( B ) HT1080 cells were cultured for 20 h in the presence of DMSO (−) or MCP compounds as indicated. Endogenous Raf-1 activity was measured. Mean ± SEM ( n = 4). ( C ) HT1080 cells were cultured in the presence of DMSO (−), 20 μM MCP compounds or PD98059, or 10 μM U0126. Endogenous MEK-1 activity was measured. Mean ± SEM ( n = 3 or 4). ( D ) HT1080 cells were serum-starved for 20 h in the presence of DMSO (−), 10 μM U0126, or 20 μM MCP compounds. Total as well as phosphorylated ERK1/2 level was examined by immunoblotting. ( E ) A549 cells were serum-starved and stimulated with 100 ng/ml EGF for 5 min. Two hours before EGF stimulation, cells were incubated with DMSO (−) or 20 μM MCP. Endogenous Raf-1 activity was measured. Mean ± SEM ( n = 6).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells

    doi: 10.1073/pnas.222222699

    Figure Lengend Snippet: Inhibition of Raf/MEK/ERK activation by MCP compounds. ( A ) HEK293 cells were cotransfected with expression vectors for Flag-tagged Raf-1 and H-Ras (V12). Cells were serum-starved in the presence of DMSO (−) or 20 μM MCP110. Raf-1 assay was performed by immunoprecipitating Flag-tagged Raf-1. Mean ± SEM ( n = 3). ( B ) HT1080 cells were cultured for 20 h in the presence of DMSO (−) or MCP compounds as indicated. Endogenous Raf-1 activity was measured. Mean ± SEM ( n = 4). ( C ) HT1080 cells were cultured in the presence of DMSO (−), 20 μM MCP compounds or PD98059, or 10 μM U0126. Endogenous MEK-1 activity was measured. Mean ± SEM ( n = 3 or 4). ( D ) HT1080 cells were serum-starved for 20 h in the presence of DMSO (−), 10 μM U0126, or 20 μM MCP compounds. Total as well as phosphorylated ERK1/2 level was examined by immunoblotting. ( E ) A549 cells were serum-starved and stimulated with 100 ng/ml EGF for 5 min. Two hours before EGF stimulation, cells were incubated with DMSO (−) or 20 μM MCP. Endogenous Raf-1 activity was measured. Mean ± SEM ( n = 6).

    Article Snippet: In contrast, MCP110 does not inhibit Raf-1 catalytic activity, as examined by the incubation of active MCP compounds versus Raf kinase inhibitor (Calbiochem) with purified preactivated Raf-1 kinase (data not shown).

    Techniques: Inhibition, Activation Assay, Expressing, Cell Culture, Activity Assay, Incubation

    Identification of compounds that inhibit Ras/Raf interaction by subtractive two-hybrid screening. ( A ) Improved sensitivity of the modified two-hybrid strains to drugs. SKY191 (wild-type two-hybrid) and SKY48 (wild-type dual bait), modified SKY197 (permeability-enhanced two-hybrid), and SKY54 (permeability-enhanced dual bait) strains were grown, and sensitivity to cycloheximide (CYH), 4-nitroquinoline N -oxide (NQO), sulfomethuron methyl (SMM), and zeocin (Zeo) was analyzed by spotting chemicals onto the lawn of yeast after serial 4-fold dilution. ( B ) Screening of compounds that inhibit Ras/Raf interaction. SKY54 expressing cI-DBD-H-Ras and AD-Raf-1 ( Upper ) and SKY54 expressing LexA-DBD-hsRPB7 and AD-hsRPB4 ( Lower ) were plated. Compounds were spotted and the appearance of white zones due to decreased expression of β-gal was examined. A specific compound (MCP307) identified in this way and studied further herein is shown. ( C ) Inhibition of β-gal activity by 30 μM MCP307 and MCP1 was examined.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells

    doi: 10.1073/pnas.222222699

    Figure Lengend Snippet: Identification of compounds that inhibit Ras/Raf interaction by subtractive two-hybrid screening. ( A ) Improved sensitivity of the modified two-hybrid strains to drugs. SKY191 (wild-type two-hybrid) and SKY48 (wild-type dual bait), modified SKY197 (permeability-enhanced two-hybrid), and SKY54 (permeability-enhanced dual bait) strains were grown, and sensitivity to cycloheximide (CYH), 4-nitroquinoline N -oxide (NQO), sulfomethuron methyl (SMM), and zeocin (Zeo) was analyzed by spotting chemicals onto the lawn of yeast after serial 4-fold dilution. ( B ) Screening of compounds that inhibit Ras/Raf interaction. SKY54 expressing cI-DBD-H-Ras and AD-Raf-1 ( Upper ) and SKY54 expressing LexA-DBD-hsRPB7 and AD-hsRPB4 ( Lower ) were plated. Compounds were spotted and the appearance of white zones due to decreased expression of β-gal was examined. A specific compound (MCP307) identified in this way and studied further herein is shown. ( C ) Inhibition of β-gal activity by 30 μM MCP307 and MCP1 was examined.

    Article Snippet: In contrast, MCP110 does not inhibit Raf-1 catalytic activity, as examined by the incubation of active MCP compounds versus Raf kinase inhibitor (Calbiochem) with purified preactivated Raf-1 kinase (data not shown).

    Techniques: Two Hybrid Screening, Modification, Permeability, Expressing, Inhibition, Activity Assay

    Inhibition of transcriptional activation by MCP compounds. ( A ) CHO cells were transfected with pSRE-luc and serum-starved. Cells were preincubated with DMSO (−), MCP307 or 1 as indicated and stimulated with 10% FBS for 5 h. Lucifererase assay was performed. ( B ) HEK293 cells were cotransfected with pAP-1-luc and pcDNA3-H-Ras (V12). Cells were serum-starved in the presence of compounds as indicated and luciferase assay was performed. Mean ± SD ( n = 3). ( C ) HT1080 cells were incubated with DMSO (−), 40 μM MCPs, or 20 μM radicicol for 40 h, and the level of Raf-1 was examined by immunoblotting.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells

    doi: 10.1073/pnas.222222699

    Figure Lengend Snippet: Inhibition of transcriptional activation by MCP compounds. ( A ) CHO cells were transfected with pSRE-luc and serum-starved. Cells were preincubated with DMSO (−), MCP307 or 1 as indicated and stimulated with 10% FBS for 5 h. Lucifererase assay was performed. ( B ) HEK293 cells were cotransfected with pAP-1-luc and pcDNA3-H-Ras (V12). Cells were serum-starved in the presence of compounds as indicated and luciferase assay was performed. Mean ± SD ( n = 3). ( C ) HT1080 cells were incubated with DMSO (−), 40 μM MCPs, or 20 μM radicicol for 40 h, and the level of Raf-1 was examined by immunoblotting.

    Article Snippet: In contrast, MCP110 does not inhibit Raf-1 catalytic activity, as examined by the incubation of active MCP compounds versus Raf kinase inhibitor (Calbiochem) with purified preactivated Raf-1 kinase (data not shown).

    Techniques: Inhibition, Activation Assay, Transfection, Luciferase, Incubation