Structured Review

Santa Cruz Biotechnology raf 1
Effect of MnTBAP on glucose-induced activation of H-Ras. Endothelial cells were incubated in 5 mM glucose or 20 mM glucose medium for 96 h in the presence or absence of 200 μM MnTBAP for 96 h. Activation of H-Ras was estimated by ( A ) Western blot technique and <t>Raf-1</t> ( B ) binding assay. Each experiment was repeated with at least three separate cell preparations. The histogram represents the ratio of the densities of H-Ras and β-actin in the same lane as quantified using Un-Scan-It gel software. The values obtained from the cells incubated in 5 mM glucose conditions are considered 100%. Asterisk (*) marks p
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Images

1) Product Images from "Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina"

Article Title: Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina

Journal: Molecular Vision

doi:

Effect of MnTBAP on glucose-induced activation of H-Ras. Endothelial cells were incubated in 5 mM glucose or 20 mM glucose medium for 96 h in the presence or absence of 200 μM MnTBAP for 96 h. Activation of H-Ras was estimated by ( A ) Western blot technique and Raf-1 ( B ) binding assay. Each experiment was repeated with at least three separate cell preparations. The histogram represents the ratio of the densities of H-Ras and β-actin in the same lane as quantified using Un-Scan-It gel software. The values obtained from the cells incubated in 5 mM glucose conditions are considered 100%. Asterisk (*) marks p
Figure Legend Snippet: Effect of MnTBAP on glucose-induced activation of H-Ras. Endothelial cells were incubated in 5 mM glucose or 20 mM glucose medium for 96 h in the presence or absence of 200 μM MnTBAP for 96 h. Activation of H-Ras was estimated by ( A ) Western blot technique and Raf-1 ( B ) binding assay. Each experiment was repeated with at least three separate cell preparations. The histogram represents the ratio of the densities of H-Ras and β-actin in the same lane as quantified using Un-Scan-It gel software. The values obtained from the cells incubated in 5 mM glucose conditions are considered 100%. Asterisk (*) marks p

Techniques Used: Activation Assay, Incubation, Western Blot, Binding Assay, Software

Effect of MnTBAP on glucose-induced activation of Raf-1 and phosphorylation of p38 MAP kinase. Activation of Raf-1 and phosphorylation of p38 MAP kinase were determined by Western blot using β-actin as a loading standard. Each sample was run in duplicate, and the experiment was repeated with three or more cell preparations. The histogram represents the density of Raf-1 ( A ), or p-p38 ( B ) band that has been adjusted to the density of β-actin band in the same lane. The ratio obtained from 5 mM glucose is considered as 100%. Asterisk (*) represent p
Figure Legend Snippet: Effect of MnTBAP on glucose-induced activation of Raf-1 and phosphorylation of p38 MAP kinase. Activation of Raf-1 and phosphorylation of p38 MAP kinase were determined by Western blot using β-actin as a loading standard. Each sample was run in duplicate, and the experiment was repeated with three or more cell preparations. The histogram represents the density of Raf-1 ( A ), or p-p38 ( B ) band that has been adjusted to the density of β-actin band in the same lane. The ratio obtained from 5 mM glucose is considered as 100%. Asterisk (*) represent p

Techniques Used: Activation Assay, Western Blot

Effect of hydrogen peroxide on the activation of H-Ras and its signaling pathway. Bovine retinal endothelial cells were incubated with 250 μM H 2 O 2 for 1 h. At the end of the incubation, the cells were rinsed with DMEM, and incubated in 5 mM glucose and 20 mM glucose media for 96 h. The activation of H-Ras was determined by Raf-1 binding assay, Raf-1 by measuring the expression of Raf-1 by Western blot, and that of MAP kinase by quantifying the expression of phospho-p38 of MAP kinase. Each sample was analyzed in duplicate, and the Western blots presented are representative of at least 3-4 experiments.
Figure Legend Snippet: Effect of hydrogen peroxide on the activation of H-Ras and its signaling pathway. Bovine retinal endothelial cells were incubated with 250 μM H 2 O 2 for 1 h. At the end of the incubation, the cells were rinsed with DMEM, and incubated in 5 mM glucose and 20 mM glucose media for 96 h. The activation of H-Ras was determined by Raf-1 binding assay, Raf-1 by measuring the expression of Raf-1 by Western blot, and that of MAP kinase by quantifying the expression of phospho-p38 of MAP kinase. Each sample was analyzed in duplicate, and the Western blots presented are representative of at least 3-4 experiments.

Techniques Used: Activation Assay, Incubation, Binding Assay, Expressing, Western Blot

2) Product Images from "Azaspirene, a fungal product, inhibits angiogenesis by blocking Raf-1 activation"

Article Title: Azaspirene, a fungal product, inhibits angiogenesis by blocking Raf-1 activation

Journal: Cancer science

doi: 10.1111/j.1349-7006.2008.00890.x

Effect of azaspirene on the autophosphorylation of KDR/Flk-1, phosphorylation of Raf-1 and disruption of Raf-1 complexes.
Figure Legend Snippet: Effect of azaspirene on the autophosphorylation of KDR/Flk-1, phosphorylation of Raf-1 and disruption of Raf-1 complexes.

Techniques Used:

3) Product Images from "cAMP-Dependent Cytosolic Mislocalization of p27kip-Cyclin D1 During Quinol-Thioether-Induced Tuberous Sclerosis Renal Cell Carcinoma"

Article Title: cAMP-Dependent Cytosolic Mislocalization of p27kip-Cyclin D1 During Quinol-Thioether-Induced Tuberous Sclerosis Renal Cell Carcinoma

Journal: Toxicological Sciences

doi: 10.1093/toxsci/kfr118

Effect of sorafenib and Raf siRNA on p27 expression in QTRRE cells. (A) QTRRE cells were treated with sorafenib (50μM) for 30, 60, and 90 min. QTRRE cells were transfected with (B) 100nM B-Raf or (C) Raf-1 ON_TARGETplus SMARTpool siRNA or siCONTROL
Figure Legend Snippet: Effect of sorafenib and Raf siRNA on p27 expression in QTRRE cells. (A) QTRRE cells were treated with sorafenib (50μM) for 30, 60, and 90 min. QTRRE cells were transfected with (B) 100nM B-Raf or (C) Raf-1 ON_TARGETplus SMARTpool siRNA or siCONTROL

Techniques Used: Expressing, Transfection

4) Product Images from "Spatial control of Shoc2-scaffold-mediated ERK1/2 signaling requires remodeling activity of the ATPase PSMC5"

Article Title: Spatial control of Shoc2-scaffold-mediated ERK1/2 signaling requires remodeling activity of the ATPase PSMC5

Journal: Journal of Cell Science

doi: 10.1242/jcs.177543

PSMC5 controls levels of ubiquitylation of Shoc2 and RAF-1. (A–C) Shoc2 was immunoprecipitated (IP) from cells transfected with CFP–PSMC5 (A), PSMC5 siRNA (siPSMC5; B), or full-length GST-PSMC5 (WT) or the GST–PSMC5 mutants (ΔCC
Figure Legend Snippet: PSMC5 controls levels of ubiquitylation of Shoc2 and RAF-1. (A–C) Shoc2 was immunoprecipitated (IP) from cells transfected with CFP–PSMC5 (A), PSMC5 siRNA (siPSMC5; B), or full-length GST-PSMC5 (WT) or the GST–PSMC5 mutants (ΔCC

Techniques Used: Immunoprecipitation, Transfection

5) Product Images from "Choice of Biological Source Material Supersedes Oxidative Stress in Its Influence on DJ-1 in Vivo Interactions with Hsp90"

Article Title: Choice of Biological Source Material Supersedes Oxidative Stress in Its Influence on DJ-1 in Vivo Interactions with Hsp90

Journal: Journal of proteome research

doi: 10.1021/pr200225c

DJ-1 influences the Hsp90-dependent stabilization of Raf-1 and Akt. (A) DJ-1-deficient, DJ-1 heterozygote, and wild-type ES cells were grown in the presence of carrier solvent or 1 μ M of the Hsp90 inhibitor geldanamycin (GA) for 16 h and subsequently
Figure Legend Snippet: DJ-1 influences the Hsp90-dependent stabilization of Raf-1 and Akt. (A) DJ-1-deficient, DJ-1 heterozygote, and wild-type ES cells were grown in the presence of carrier solvent or 1 μ M of the Hsp90 inhibitor geldanamycin (GA) for 16 h and subsequently

Techniques Used:

6) Product Images from "Mutant N-Ras protects colorectal cancer cells from stress-induced apoptosis and contributes to cancer development and progression"

Article Title: Mutant N-Ras protects colorectal cancer cells from stress-induced apoptosis and contributes to cancer development and progression

Journal: Cancer discovery

doi: 10.1158/2159-8290.CD-12-0198

Mutant N-Ras signals through RAF-1 to confer resistance to apoptosis induced by sodium butyrate. A. Apoptotic phenotypes of colon cancer cell lines with mutant forms of Ras. Cells expressing wild-type Ras or H-Ras G12V were sensitive to induction of apoptosis
Figure Legend Snippet: Mutant N-Ras signals through RAF-1 to confer resistance to apoptosis induced by sodium butyrate. A. Apoptotic phenotypes of colon cancer cell lines with mutant forms of Ras. Cells expressing wild-type Ras or H-Ras G12V were sensitive to induction of apoptosis

Techniques Used: Mutagenesis, Expressing

7) Product Images from "Translocation of H-Ras and its implications in the development of diabetic retinopathy"

Article Title: Translocation of H-Ras and its implications in the development of diabetic retinopathy

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2009.07.038

Diabetes translocates retinal H-Ras and Raf-1 from cytosol to the membrane
Figure Legend Snippet: Diabetes translocates retinal H-Ras and Raf-1 from cytosol to the membrane

Techniques Used:

8) Product Images from "Translocation of H-Ras and its implications in the development of diabetic retinopathy"

Article Title: Translocation of H-Ras and its implications in the development of diabetic retinopathy

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2009.07.038

Diabetes translocates retinal H-Ras and Raf-1 from cytosol to the membrane
Figure Legend Snippet: Diabetes translocates retinal H-Ras and Raf-1 from cytosol to the membrane

Techniques Used:

9) Product Images from "Translocation of H-Ras and its implications in the development of diabetic retinopathy"

Article Title: Translocation of H-Ras and its implications in the development of diabetic retinopathy

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2009.07.038

Diabetes translocates retinal H-Ras and Raf-1 from cytosol to the membrane
Figure Legend Snippet: Diabetes translocates retinal H-Ras and Raf-1 from cytosol to the membrane

Techniques Used:

10) Product Images from "Thrombopoietin-Mediated Sustained Activation of Extracellular Signal-Regulated Kinase in UT7-Mpl Cells Requires Both Ras-Raf-1- and Rap1-B-Raf-Dependent Pathways"

Article Title: Thrombopoietin-Mediated Sustained Activation of Extracellular Signal-Regulated Kinase in UT7-Mpl Cells Requires Both Ras-Raf-1- and Rap1-B-Raf-Dependent Pathways

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.8.2659-2670.2001

In UT7-Mpl cells, Raf-1 but not B-Raf mediates the Ras-induced activation of Elk1. UT7-Mpl cells were transiently transfected with pFA-Elk1 and pFR-luc plasmids along with 10 μg of empty pcDNA or pcDNA-RasV12, alone or together with pcDNA encoding wild-type or dominant negative forms of Raf-1 or B-Raf, as indicated. Luciferase activity was measured 30 h after transfection in cells incubated in the absence of cytokine. In the same experiment, the functionality of dominant negative mutants of Raf-1 and B-Raf (Nter-Raf-1 and Nter-B-Raf, respectively) was tested by measuring luciferase activity in cells stimulated with TPO during the last 24 h of incubation. (A) Results from a representative experiment. Luciferase activity is expressed as fold induction over the values obtained in cells transfected with empty pcDNA alone, in the absence of TPO. (B) Effects of wild-type and dominant negative forms of Raf-1 and B-Raf on RasV12-induced Elk1 activation, summarized from four independent experiments. Luciferase activity is expressed as a percentage of the response induced upon expression of RasV12 alone and is presented as average values with error bars indicating SEs.
Figure Legend Snippet: In UT7-Mpl cells, Raf-1 but not B-Raf mediates the Ras-induced activation of Elk1. UT7-Mpl cells were transiently transfected with pFA-Elk1 and pFR-luc plasmids along with 10 μg of empty pcDNA or pcDNA-RasV12, alone or together with pcDNA encoding wild-type or dominant negative forms of Raf-1 or B-Raf, as indicated. Luciferase activity was measured 30 h after transfection in cells incubated in the absence of cytokine. In the same experiment, the functionality of dominant negative mutants of Raf-1 and B-Raf (Nter-Raf-1 and Nter-B-Raf, respectively) was tested by measuring luciferase activity in cells stimulated with TPO during the last 24 h of incubation. (A) Results from a representative experiment. Luciferase activity is expressed as fold induction over the values obtained in cells transfected with empty pcDNA alone, in the absence of TPO. (B) Effects of wild-type and dominant negative forms of Raf-1 and B-Raf on RasV12-induced Elk1 activation, summarized from four independent experiments. Luciferase activity is expressed as a percentage of the response induced upon expression of RasV12 alone and is presented as average values with error bars indicating SEs.

Techniques Used: Activation Assay, Transfection, Dominant Negative Mutation, Luciferase, Activity Assay, Incubation, Expressing

TPO-mediated Ras and ERK activation in UT7-MplWt and UT7-MplΔ3 cells. UT7 cells expressing MplWt (WT) or MplΔ3 (Δ3) receptors were stimulated for various times with 10 nM TPO mimetic peptide. At the indicated times, cells were pelleted and lysed either directly in Laemmli sample buffer for ERK activation (A) or in NP-40 lysis buffer for Ras activation (B). The activity of ERKs was assessed by detecting their phosphorylation upon Western blotting (WB) with a polyclonal anti-phospho-ERK antibody. Total ERK was determined by reprobing the membranes with an antibody recognizing ERK1 and ERK2 in both resting and activated forms. The GTP-bound form of Ras was trapped by pull-down assay using the Raf-1 RBD-GST fusion protein and detected by Western blotting with anti-Ras monoclonal antibody.
Figure Legend Snippet: TPO-mediated Ras and ERK activation in UT7-MplWt and UT7-MplΔ3 cells. UT7 cells expressing MplWt (WT) or MplΔ3 (Δ3) receptors were stimulated for various times with 10 nM TPO mimetic peptide. At the indicated times, cells were pelleted and lysed either directly in Laemmli sample buffer for ERK activation (A) or in NP-40 lysis buffer for Ras activation (B). The activity of ERKs was assessed by detecting their phosphorylation upon Western blotting (WB) with a polyclonal anti-phospho-ERK antibody. Total ERK was determined by reprobing the membranes with an antibody recognizing ERK1 and ERK2 in both resting and activated forms. The GTP-bound form of Ras was trapped by pull-down assay using the Raf-1 RBD-GST fusion protein and detected by Western blotting with anti-Ras monoclonal antibody.

Techniques Used: Activation Assay, Expressing, Lysis, Activity Assay, Western Blot, Pull Down Assay

Kinetics of activation of endogenous Raf-1 and B-Raf in UT7 cells expressing MplWt or MplΔ3. UT7-MplWt and UT7-MplΔ3 cells were stimulated for the various times indicated and lysed. Raf-1 (A) or B-Raf (B) was immunoprecipitated and subjected to in vitro kinase assay, using recombinant inactive MEK as a substrate. Radioactivity incorporated into MEK was revealed by autoradiography. Immunoprecipitated Raf-1 or B-Raf levels were verified by Western blotting (WB) using specific antisera, as indicated. Two sets of kinetics (short and long terms) are shown for UT7-MplWt cells.
Figure Legend Snippet: Kinetics of activation of endogenous Raf-1 and B-Raf in UT7 cells expressing MplWt or MplΔ3. UT7-MplWt and UT7-MplΔ3 cells were stimulated for the various times indicated and lysed. Raf-1 (A) or B-Raf (B) was immunoprecipitated and subjected to in vitro kinase assay, using recombinant inactive MEK as a substrate. Radioactivity incorporated into MEK was revealed by autoradiography. Immunoprecipitated Raf-1 or B-Raf levels were verified by Western blotting (WB) using specific antisera, as indicated. Two sets of kinetics (short and long terms) are shown for UT7-MplWt cells.

Techniques Used: Activation Assay, Expressing, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Radioactivity, Autoradiography, Western Blot

11) Product Images from "HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold"

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00811-14

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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used:

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
Figure Legend 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

Techniques Used: 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,
Figure Legend 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,

Techniques Used: 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
Figure Legend 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

Techniques Used: Transfection

12) Product Images from "HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold"

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00811-14

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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used:

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
Figure Legend 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

Techniques Used: 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,
Figure Legend 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,

Techniques Used: 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
Figure Legend 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

Techniques Used: Transfection

13) Product Images from "HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold"

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00811-14

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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used:

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
Figure Legend 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

Techniques Used: 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,
Figure Legend 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,

Techniques Used: 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
Figure Legend 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

Techniques Used: Transfection

14) Product Images from "HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold"

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00811-14

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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used:

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
Figure Legend 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

Techniques Used: 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,
Figure Legend 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,

Techniques Used: 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
Figure Legend 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

Techniques Used: Transfection

15) Product Images from "HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold"

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00811-14

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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used:

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
Figure Legend 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

Techniques Used: 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,
Figure Legend 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,

Techniques Used: 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
Figure Legend 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

Techniques Used: Transfection

16) Product Images from "HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold"

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00811-14

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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used:

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
Figure Legend 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

Techniques Used: 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,
Figure Legend 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,

Techniques Used: 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
Figure Legend 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

Techniques Used: Transfection

17) Product Images from "Inhibition of Hsp90 Down-regulates Mutant Epidermal Growth Factor Receptor (EGFR) Expression and Sensitizes EGFR Mutant Tumors to Paclitaxel"

Article Title: Inhibition of Hsp90 Down-regulates Mutant Epidermal Growth Factor Receptor (EGFR) Expression and Sensitizes EGFR Mutant Tumors to Paclitaxel

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-07-1570

17-AAG induced the degradation of the L858R, T790M, EGFR mutant in H1975 lung adenocarcinoma cells. A , immunoblots showing that 17-AAG causes degradation of mutant EGFR and down-regulation of phospho-MAPK and phospho-Akt expression in H1975 cells. B , in Calu-1 cells (wild-type EGFR), EGFR expression is only minimally affected by 17-AAG. However, other Hsp90 clients, such as Raf-1, are degraded in Calu-1. Cells were treated with 17-AAG for 24 h at the doses indicated. C , both EGFR mutant and EGFR wild-type lung cancer cell lines were sensitive to 17-AAG in vitro . Cellular proliferation was assayed at 72 h by Alamar Blue and graphed as a percentage of DMSO-treated cells.
Figure Legend Snippet: 17-AAG induced the degradation of the L858R, T790M, EGFR mutant in H1975 lung adenocarcinoma cells. A , immunoblots showing that 17-AAG causes degradation of mutant EGFR and down-regulation of phospho-MAPK and phospho-Akt expression in H1975 cells. B , in Calu-1 cells (wild-type EGFR), EGFR expression is only minimally affected by 17-AAG. However, other Hsp90 clients, such as Raf-1, are degraded in Calu-1. Cells were treated with 17-AAG for 24 h at the doses indicated. C , both EGFR mutant and EGFR wild-type lung cancer cell lines were sensitive to 17-AAG in vitro . Cellular proliferation was assayed at 72 h by Alamar Blue and graphed as a percentage of DMSO-treated cells.

Techniques Used: Mutagenesis, Western Blot, Expressing, In Vitro

17-AAG, at nontoxic doses, down-regulates mutant EGFR expression in xenograft tumors. For dose-response studies, mice were treated with either 50 or 75 mg/kg of 17-AAG and sacrificed 6 h later. In time course experiments mice were treated with a single dose of 75 mg/kg of 17-AAG and sacrificed at the time points indicated (0–48 h). A , immunoblots of EGFR, Raf-1, Akt, and cyclin D1 in H1650 xenografts treated with 17-AAG. 17-AAG treatment resulted in a > 95% reduction in mutant EGFR expression and inhibition of AKT and MAPK activity in H1650 xenografts. Raf-1 was less sensitive than mutant EGFR to 17-AAG treatment, and Akt expression was unaffected by 17-AAG. B , quantitation of the effects of 17-AAG on the expression of EGFR, Raf-1, and Akt in H1650 xenografts. MAPK, a protein unaffected by Hsp90 inhibition, is included as a loading control. C , similar results were observed in H3255 and H1975 xenografts ( C and data not shown). D , 17-AAG does not induce the degradation of wild-type EGFR but does down-regulate the expression of HER2 in A549 xenografts.
Figure Legend Snippet: 17-AAG, at nontoxic doses, down-regulates mutant EGFR expression in xenograft tumors. For dose-response studies, mice were treated with either 50 or 75 mg/kg of 17-AAG and sacrificed 6 h later. In time course experiments mice were treated with a single dose of 75 mg/kg of 17-AAG and sacrificed at the time points indicated (0–48 h). A , immunoblots of EGFR, Raf-1, Akt, and cyclin D1 in H1650 xenografts treated with 17-AAG. 17-AAG treatment resulted in a > 95% reduction in mutant EGFR expression and inhibition of AKT and MAPK activity in H1650 xenografts. Raf-1 was less sensitive than mutant EGFR to 17-AAG treatment, and Akt expression was unaffected by 17-AAG. B , quantitation of the effects of 17-AAG on the expression of EGFR, Raf-1, and Akt in H1650 xenografts. MAPK, a protein unaffected by Hsp90 inhibition, is included as a loading control. C , similar results were observed in H3255 and H1975 xenografts ( C and data not shown). D , 17-AAG does not induce the degradation of wild-type EGFR but does down-regulate the expression of HER2 in A549 xenografts.

Techniques Used: Mutagenesis, Expressing, Mouse Assay, Western Blot, Inhibition, Activity Assay, Quantitation Assay

Mutant forms of EGFR are more sensitive than wild-type EGFR to 17-AAG–induced degradation. A , NIH-3T3 cells were stably transfected with wild-type and mutant forms of EGFR (L858R and ΔL747_E749, A750P). Immunoblots of EGFR, Raf-1, and p85 phosphatidylinositol 3-kinase (PI3K) show that mutant forms of EGFR were degraded at earlier time points than wild-type EGFR after treatment with 0.1 μmol/L of 17-AAG. p85 PI3K, a protein unaffected by Hsp90 inhibition, was included as a loading control. B , immunoblots of EGFR, Raf-1, and p85 PI3K, showing that lower concentrations of 17-AAG were required to down-regulate mutant forms of EGFR (L858R and ΔL747_E749, A750P) compared with wild-type EGFR. Cells were harvested 24 h after treatment with 17-AAG.
Figure Legend Snippet: Mutant forms of EGFR are more sensitive than wild-type EGFR to 17-AAG–induced degradation. A , NIH-3T3 cells were stably transfected with wild-type and mutant forms of EGFR (L858R and ΔL747_E749, A750P). Immunoblots of EGFR, Raf-1, and p85 phosphatidylinositol 3-kinase (PI3K) show that mutant forms of EGFR were degraded at earlier time points than wild-type EGFR after treatment with 0.1 μmol/L of 17-AAG. p85 PI3K, a protein unaffected by Hsp90 inhibition, was included as a loading control. B , immunoblots of EGFR, Raf-1, and p85 PI3K, showing that lower concentrations of 17-AAG were required to down-regulate mutant forms of EGFR (L858R and ΔL747_E749, A750P) compared with wild-type EGFR. Cells were harvested 24 h after treatment with 17-AAG.

Techniques Used: Mutagenesis, Stable Transfection, Transfection, Western Blot, Inhibition

18) Product Images from "14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity"

Article Title: 14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity

Journal: Molecular and Cellular Biology

doi:

Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.
Figure Legend Snippet: Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.

Techniques Used: Binding Assay, Activity Assay, Activation Assay

Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).
Figure Legend Snippet: Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).

Techniques Used: Purification, Labeling, In Vitro, High Performance Liquid Chromatography, Radioactivity, SDS Page, Chromatography, Staining

S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.
Figure Legend Snippet: S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.

Techniques Used: Activity Assay, Labeling, Infection, Recombinant, Construct, Cell Culture, Lysis, SDS Page, Expressing, In Vitro

19) Product Images from "14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity"

Article Title: 14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity

Journal: Molecular and Cellular Biology

doi:

Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.
Figure Legend Snippet: Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.

Techniques Used: Binding Assay, Activity Assay, Activation Assay

Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).
Figure Legend Snippet: Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).

Techniques Used: Purification, Labeling, In Vitro, High Performance Liquid Chromatography, Radioactivity, SDS Page, Chromatography, Staining

S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.
Figure Legend Snippet: S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.

Techniques Used: Activity Assay, Labeling, Infection, Recombinant, Construct, Cell Culture, Lysis, SDS Page, Expressing, In Vitro

20) Product Images from "14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity"

Article Title: 14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity

Journal: Molecular and Cellular Biology

doi:

Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.
Figure Legend Snippet: Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.

Techniques Used: Binding Assay, Activity Assay, Activation Assay

Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).
Figure Legend Snippet: Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).

Techniques Used: Purification, Labeling, In Vitro, High Performance Liquid Chromatography, Radioactivity, SDS Page, Chromatography, Staining

S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.
Figure Legend Snippet: S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.

Techniques Used: Activity Assay, Labeling, Infection, Recombinant, Construct, Cell Culture, Lysis, SDS Page, Expressing, In Vitro

21) Product Images from "14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity"

Article Title: 14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity

Journal: Molecular and Cellular Biology

doi:

Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.
Figure Legend Snippet: Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.

Techniques Used: Binding Assay, Activity Assay, Activation Assay

Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).
Figure Legend Snippet: Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).

Techniques Used: Purification, Labeling, In Vitro, High Performance Liquid Chromatography, Radioactivity, SDS Page, Chromatography, Staining

S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.
Figure Legend Snippet: S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.

Techniques Used: Activity Assay, Labeling, Infection, Recombinant, Construct, Cell Culture, Lysis, SDS Page, Expressing, In Vitro

22) Product Images from "The Thrombopoietin Receptor Can Mediate Proliferation without Activation of the Jak-STAT Pathway "

Article Title: The Thrombopoietin Receptor Can Mediate Proliferation without Activation of the Jak-STAT Pathway

Journal: The Journal of Experimental Medicine

doi:

Effect of TPO stimulation on Shc, Vav, the receptor itself, Raf-1, and MAPK. Growth factor-deprived BAF-mplwt and BAF-mplΔ7 cells were either left untreated or stimulated with TPO for the indicated times and cell extracts were prepared. Immunoprecipitations were performed with antibodies to Shc ( a ), Vav ( b ), and myc ( c ) and the immunoprecipitates were blotted with antiphosphotyrosine antibodies ( a–c ). To confirm equal loading of protein, membranes were stripped and reprobed with the antibodies used for immunoprecipitations (lower panel of a–c ). In ( c ) a higher amount of c-mplΔ7 protein was immunoprecipitated. ( d ) Antiphosphotyrosine immunoblot of total cell lysates. ( e ) Cell lysates were immunoblotted with an antibody to Raf-1. The lower mobility of Raf-1 seen after stimulation with TPO in BAF-mplwt and BAF-mplΔ7 reflects the increased phosphorylation of Raf-1 on serine. ( f ) Cell lysates were immunoblotted with anti-active MAPK antibodies which recognize the active forms of Erk-1 and Erk-2 (different exposures of the same membrane are shown in the upper and middle panel). Membranes were stripped and reprobed with anti-Erk2 antibodies to confirm equal protein loading.
Figure Legend Snippet: Effect of TPO stimulation on Shc, Vav, the receptor itself, Raf-1, and MAPK. Growth factor-deprived BAF-mplwt and BAF-mplΔ7 cells were either left untreated or stimulated with TPO for the indicated times and cell extracts were prepared. Immunoprecipitations were performed with antibodies to Shc ( a ), Vav ( b ), and myc ( c ) and the immunoprecipitates were blotted with antiphosphotyrosine antibodies ( a–c ). To confirm equal loading of protein, membranes were stripped and reprobed with the antibodies used for immunoprecipitations (lower panel of a–c ). In ( c ) a higher amount of c-mplΔ7 protein was immunoprecipitated. ( d ) Antiphosphotyrosine immunoblot of total cell lysates. ( e ) Cell lysates were immunoblotted with an antibody to Raf-1. The lower mobility of Raf-1 seen after stimulation with TPO in BAF-mplwt and BAF-mplΔ7 reflects the increased phosphorylation of Raf-1 on serine. ( f ) Cell lysates were immunoblotted with anti-active MAPK antibodies which recognize the active forms of Erk-1 and Erk-2 (different exposures of the same membrane are shown in the upper and middle panel). Membranes were stripped and reprobed with anti-Erk2 antibodies to confirm equal protein loading.

Techniques Used: Immunoprecipitation

23) Product Images from "Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells"

Article Title: Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells

Journal: BMC Cancer

doi: 10.1186/s12885-015-1568-3

Extracellular vesicle mediated changes in cellular protein expression. EVs were isolated from malignant HCT116 cells. EVs were resuspended in PBS and co-cultured with non-malignant 1459 cells and malignant HCT116 cells. Upon completion of the 7-day co-culture, whole cell lysates were prepared for Western blot analysis, as reported. Western blot analysis results show and increased expression of Raf-1, pRKIP, STAT1, Prohibitin and 14-3-3 zeta/delta in 1459 + HCT116 EV co-cultures, as compared to 1459 control. A slight decrease expression of RKIP is also shown in this comparison. We examined these proteins based on LC-MS data (Table 1 )
Figure Legend Snippet: Extracellular vesicle mediated changes in cellular protein expression. EVs were isolated from malignant HCT116 cells. EVs were resuspended in PBS and co-cultured with non-malignant 1459 cells and malignant HCT116 cells. Upon completion of the 7-day co-culture, whole cell lysates were prepared for Western blot analysis, as reported. Western blot analysis results show and increased expression of Raf-1, pRKIP, STAT1, Prohibitin and 14-3-3 zeta/delta in 1459 + HCT116 EV co-cultures, as compared to 1459 control. A slight decrease expression of RKIP is also shown in this comparison. We examined these proteins based on LC-MS data (Table 1 )

Techniques Used: Expressing, Isolation, Cell Culture, Co-Culture Assay, Western Blot, Liquid Chromatography with Mass Spectroscopy

24) Product Images from "Activated Ras Induces Cytoplasmic Vacuolation and Non-Apoptotic Death in Glioblastoma Cells via Novel Effector Pathways"

Article Title: Activated Ras Induces Cytoplasmic Vacuolation and Non-Apoptotic Death in Glioblastoma Cells via Novel Effector Pathways

Journal: Cellular signalling

doi: 10.1016/j.cellsig.2006.11.010

Overexpression of constitutively active Raf-1 does not mimic the effect of activated H-Ras. U251 cells were nucleofected with empty vector or with expression vectors encoding myc-Raf-C aax or myc-H-Ras(G12V). The cells were processed for western blot analysis or immunofluorescence 24 h later. (a) Immunoblot analysis with an antibody against Raf confirms that myc-Raf-C aax (upper band) is overexpressed relative to endogenous Raf. (b) Phospho-ERK and total ERK were determined by immunoblot analysis (representative blot shown). The ratio of phospho-ERK to total ERK was determined by quantifying the immunoblot signals with a Kodak Image station. The results shown in the bar graph are the means (±SD) of separate determinations performed on three cultures. (c) Cells expressing myc-Raf-C aax or myc-H- Ras(G12V) were identified by immunofluorescence using antibody against the myc epitope. The same cells were examined under phase contrast to assess the presence of vacuoles. The nucleofection efficiencies in both sets of cultures were approximately 50%. There were no vacuolated myc-positive cells in the cultures expressing myc-Raf-C aax .
Figure Legend Snippet: Overexpression of constitutively active Raf-1 does not mimic the effect of activated H-Ras. U251 cells were nucleofected with empty vector or with expression vectors encoding myc-Raf-C aax or myc-H-Ras(G12V). The cells were processed for western blot analysis or immunofluorescence 24 h later. (a) Immunoblot analysis with an antibody against Raf confirms that myc-Raf-C aax (upper band) is overexpressed relative to endogenous Raf. (b) Phospho-ERK and total ERK were determined by immunoblot analysis (representative blot shown). The ratio of phospho-ERK to total ERK was determined by quantifying the immunoblot signals with a Kodak Image station. The results shown in the bar graph are the means (±SD) of separate determinations performed on three cultures. (c) Cells expressing myc-Raf-C aax or myc-H- Ras(G12V) were identified by immunofluorescence using antibody against the myc epitope. The same cells were examined under phase contrast to assess the presence of vacuoles. The nucleofection efficiencies in both sets of cultures were approximately 50%. There were no vacuolated myc-positive cells in the cultures expressing myc-Raf-C aax .

Techniques Used: Over Expression, Plasmid Preparation, Expressing, Western Blot, Immunofluorescence

25) Product Images from "Raf-1 activation disrupts its binding to keratins during cell stress"

Article Title: Raf-1 activation disrupts its binding to keratins during cell stress

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200402051

Keratin association with Raf-1 kinase. (A) HT29 cells (lanes 1 and 2) were cultured in the presence or absence of OA (1 μg/ml, 2 h), then solubilized with 1% NP-40. Alternatively, transgenic mice (lanes 3–6) that overexpress human K18 ( Ku et al., 2002 ) were injected with MLR (30 μg/kg) in saline (+) or with saline alone (−). After 2 h, the livers were homogenized with 1% NP-40. K8/18 immunoprecipitates were obtained from HT29 and liver NP-40 lysates then separated by SDS-PAGE. Duplicate gels were stained with Coomassie blue or transferred then blotted with Ab to Raf or 14-3-3. Note heat shock protein 70 (hsp70) association with K8/18. (B) BHK cells were transfected with vector alone, Raf, K8/18, K8/19, Raf+K8/18, or Raf+K8/19 constructs. After 3 d, transfected cells were solubilized followed by precipitation of K8/18 or K8/19 using K18- or K19-specific mAb. Immunoprecipitates (i.p.) were analyzed as in A. Arrow positioned between K18 and K19 highlights a nonspecific band, and arrowheads indicate previously characterized K18 fragments ( Ku et al., 1997 ). (C) A total cell lysate and K8/18 precipitates were prepared from HT29 cells. After separation by SDS-PAGE, duplicate gels were stained with Coomassie blue (lanes 1 and 2) or transferred to a membrane for overlay assay (lanes 1′ and 2′). The membrane was incubated with recombinant Raf kinase, washed, and blotted with anti-Raf Ab (lanes 1′ and 2′). (D) K8/18 precipitates were prepared from HT29 cells then incubated (30 min, 22°C) in the absence or presence of Mg-ATP, followed by washing then blotting with Ab to Raf or hsp70. (E) HT29 cells were incubated in the presence or absence of H 2 O 2 for 1 h followed by processing 80% of the cells for K8/18 precipitation and the remaining to prepare a total cell lysate. Precipitates were analyzed by blotting with the indicated Ab, including phospho-Erk1/2 as an indicator of Raf kinase activity.
Figure Legend Snippet: Keratin association with Raf-1 kinase. (A) HT29 cells (lanes 1 and 2) were cultured in the presence or absence of OA (1 μg/ml, 2 h), then solubilized with 1% NP-40. Alternatively, transgenic mice (lanes 3–6) that overexpress human K18 ( Ku et al., 2002 ) were injected with MLR (30 μg/kg) in saline (+) or with saline alone (−). After 2 h, the livers were homogenized with 1% NP-40. K8/18 immunoprecipitates were obtained from HT29 and liver NP-40 lysates then separated by SDS-PAGE. Duplicate gels were stained with Coomassie blue or transferred then blotted with Ab to Raf or 14-3-3. Note heat shock protein 70 (hsp70) association with K8/18. (B) BHK cells were transfected with vector alone, Raf, K8/18, K8/19, Raf+K8/18, or Raf+K8/19 constructs. After 3 d, transfected cells were solubilized followed by precipitation of K8/18 or K8/19 using K18- or K19-specific mAb. Immunoprecipitates (i.p.) were analyzed as in A. Arrow positioned between K18 and K19 highlights a nonspecific band, and arrowheads indicate previously characterized K18 fragments ( Ku et al., 1997 ). (C) A total cell lysate and K8/18 precipitates were prepared from HT29 cells. After separation by SDS-PAGE, duplicate gels were stained with Coomassie blue (lanes 1 and 2) or transferred to a membrane for overlay assay (lanes 1′ and 2′). The membrane was incubated with recombinant Raf kinase, washed, and blotted with anti-Raf Ab (lanes 1′ and 2′). (D) K8/18 precipitates were prepared from HT29 cells then incubated (30 min, 22°C) in the absence or presence of Mg-ATP, followed by washing then blotting with Ab to Raf or hsp70. (E) HT29 cells were incubated in the presence or absence of H 2 O 2 for 1 h followed by processing 80% of the cells for K8/18 precipitation and the remaining to prepare a total cell lysate. Precipitates were analyzed by blotting with the indicated Ab, including phospho-Erk1/2 as an indicator of Raf kinase activity.

Techniques Used: Cell Culture, Transgenic Assay, Mouse Assay, Injection, SDS Page, Staining, Transfection, Plasmid Preparation, Construct, Overlay Assay, Incubation, Recombinant, Activity Assay

26) Product Images from "Stimulation of β-adrenergic receptors plays a protective role via increased expression of RAF-1 and PDX-1 in hyperglycemic rat pancreatic islet (RIN-m5F) cells"

Article Title: Stimulation of β-adrenergic receptors plays a protective role via increased expression of RAF-1 and PDX-1 in hyperglycemic rat pancreatic islet (RIN-m5F) cells

Journal: Archives of Medical Science : AMS

doi: 10.5114/aoms.2016.64131

RIN-m5F cells were treated with glucose alone, glucose + isoproterenol (Iso) (β-adrenergic receptors antagonist) and glucose (Glu) + isoproterenol (Iso) + propranolol (Pro) (β-adrenergic receptor antagonist). Betaadrenergic receptor stimulation by isoproterenol caused increased mRNA level of RAF-1 ( A and B ) and PDX-1 ( C and D ) as compared to control and glucose treated cells Data are presented as mean ± standard deviation and compare glucose treated vs. glucose + isoproterenol treated vs. glucose + isoproterenol + propranolol treated (*p
Figure Legend Snippet: RIN-m5F cells were treated with glucose alone, glucose + isoproterenol (Iso) (β-adrenergic receptors antagonist) and glucose (Glu) + isoproterenol (Iso) + propranolol (Pro) (β-adrenergic receptor antagonist). Betaadrenergic receptor stimulation by isoproterenol caused increased mRNA level of RAF-1 ( A and B ) and PDX-1 ( C and D ) as compared to control and glucose treated cells Data are presented as mean ± standard deviation and compare glucose treated vs. glucose + isoproterenol treated vs. glucose + isoproterenol + propranolol treated (*p

Techniques Used: Standard Deviation

Stimulation of β-adrenergic receptors with isoproterenol induced the expression of RAF-1 in hyperglycemic RIN-m5F cells ( A and B ). Isoproterenol treated cells were then subjected to propranolol. Increase in the expression of RAF-1 was dependent on concentration but independent of time periods. The isoproterenol generated effect on RAF-1 protein was reversed when the cells were treated with isoproterenol + propranolol ( C and D ) Data are presented as mean ± standard deviation and compare glucose treated vs. glucose + isoproterenol treated vs. glucose + isoproterenol + propranolol treated (*p
Figure Legend Snippet: Stimulation of β-adrenergic receptors with isoproterenol induced the expression of RAF-1 in hyperglycemic RIN-m5F cells ( A and B ). Isoproterenol treated cells were then subjected to propranolol. Increase in the expression of RAF-1 was dependent on concentration but independent of time periods. The isoproterenol generated effect on RAF-1 protein was reversed when the cells were treated with isoproterenol + propranolol ( C and D ) Data are presented as mean ± standard deviation and compare glucose treated vs. glucose + isoproterenol treated vs. glucose + isoproterenol + propranolol treated (*p

Techniques Used: Expressing, Concentration Assay, Generated, Standard Deviation

27) Product Images from "Inhibition of Hsp90 Down-regulates Mutant Epidermal Growth Factor Receptor (EGFR) Expression and Sensitizes EGFR Mutant Tumors to Paclitaxel"

Article Title: Inhibition of Hsp90 Down-regulates Mutant Epidermal Growth Factor Receptor (EGFR) Expression and Sensitizes EGFR Mutant Tumors to Paclitaxel

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-07-1570

17-AAG induced the degradation of the L858R, T790M, EGFR mutant in H1975 lung adenocarcinoma cells. A , immunoblots showing that 17-AAG causes degradation of mutant EGFR and down-regulation of phospho-MAPK and phospho-Akt expression in H1975 cells. B , in Calu-1 cells (wild-type EGFR), EGFR expression is only minimally affected by 17-AAG. However, other Hsp90 clients, such as Raf-1, are degraded in Calu-1. Cells were treated with 17-AAG for 24 h at the doses indicated. C , both EGFR mutant and EGFR wild-type lung cancer cell lines were sensitive to 17-AAG in vitro . Cellular proliferation was assayed at 72 h by Alamar Blue and graphed as a percentage of DMSO-treated cells.
Figure Legend Snippet: 17-AAG induced the degradation of the L858R, T790M, EGFR mutant in H1975 lung adenocarcinoma cells. A , immunoblots showing that 17-AAG causes degradation of mutant EGFR and down-regulation of phospho-MAPK and phospho-Akt expression in H1975 cells. B , in Calu-1 cells (wild-type EGFR), EGFR expression is only minimally affected by 17-AAG. However, other Hsp90 clients, such as Raf-1, are degraded in Calu-1. Cells were treated with 17-AAG for 24 h at the doses indicated. C , both EGFR mutant and EGFR wild-type lung cancer cell lines were sensitive to 17-AAG in vitro . Cellular proliferation was assayed at 72 h by Alamar Blue and graphed as a percentage of DMSO-treated cells.

Techniques Used: Mutagenesis, Western Blot, Expressing, In Vitro

17-AAG, at nontoxic doses, down-regulates mutant EGFR expression in xenograft tumors. For dose-response studies, mice were treated with either 50 or 75 mg/kg of 17-AAG and sacrificed 6 h later. In time course experiments mice were treated with a single dose of 75 mg/kg of 17-AAG and sacrificed at the time points indicated (0–48 h). A , immunoblots of EGFR, Raf-1, Akt, and cyclin D1 in H1650 xenografts treated with 17-AAG. 17-AAG treatment resulted in a > 95% reduction in mutant EGFR expression and inhibition of AKT and MAPK activity in H1650 xenografts. Raf-1 was less sensitive than mutant EGFR to 17-AAG treatment, and Akt expression was unaffected by 17-AAG. B , quantitation of the effects of 17-AAG on the expression of EGFR, Raf-1, and Akt in H1650 xenografts. MAPK, a protein unaffected by Hsp90 inhibition, is included as a loading control. C , similar results were observed in H3255 and H1975 xenografts ( C and data not shown). D , 17-AAG does not induce the degradation of wild-type EGFR but does down-regulate the expression of HER2 in A549 xenografts.
Figure Legend Snippet: 17-AAG, at nontoxic doses, down-regulates mutant EGFR expression in xenograft tumors. For dose-response studies, mice were treated with either 50 or 75 mg/kg of 17-AAG and sacrificed 6 h later. In time course experiments mice were treated with a single dose of 75 mg/kg of 17-AAG and sacrificed at the time points indicated (0–48 h). A , immunoblots of EGFR, Raf-1, Akt, and cyclin D1 in H1650 xenografts treated with 17-AAG. 17-AAG treatment resulted in a > 95% reduction in mutant EGFR expression and inhibition of AKT and MAPK activity in H1650 xenografts. Raf-1 was less sensitive than mutant EGFR to 17-AAG treatment, and Akt expression was unaffected by 17-AAG. B , quantitation of the effects of 17-AAG on the expression of EGFR, Raf-1, and Akt in H1650 xenografts. MAPK, a protein unaffected by Hsp90 inhibition, is included as a loading control. C , similar results were observed in H3255 and H1975 xenografts ( C and data not shown). D , 17-AAG does not induce the degradation of wild-type EGFR but does down-regulate the expression of HER2 in A549 xenografts.

Techniques Used: Mutagenesis, Expressing, Mouse Assay, Western Blot, Inhibition, Activity Assay, Quantitation Assay

Mutant forms of EGFR are more sensitive than wild-type EGFR to 17-AAG–induced degradation. A , NIH-3T3 cells were stably transfected with wild-type and mutant forms of EGFR (L858R and ΔL747_E749, A750P). Immunoblots of EGFR, Raf-1, and p85 phosphatidylinositol 3-kinase (PI3K) show that mutant forms of EGFR were degraded at earlier time points than wild-type EGFR after treatment with 0.1 μmol/L of 17-AAG. p85 PI3K, a protein unaffected by Hsp90 inhibition, was included as a loading control. B , immunoblots of EGFR, Raf-1, and p85 PI3K, showing that lower concentrations of 17-AAG were required to down-regulate mutant forms of EGFR (L858R and ΔL747_E749, A750P) compared with wild-type EGFR. Cells were harvested 24 h after treatment with 17-AAG.
Figure Legend Snippet: Mutant forms of EGFR are more sensitive than wild-type EGFR to 17-AAG–induced degradation. A , NIH-3T3 cells were stably transfected with wild-type and mutant forms of EGFR (L858R and ΔL747_E749, A750P). Immunoblots of EGFR, Raf-1, and p85 phosphatidylinositol 3-kinase (PI3K) show that mutant forms of EGFR were degraded at earlier time points than wild-type EGFR after treatment with 0.1 μmol/L of 17-AAG. p85 PI3K, a protein unaffected by Hsp90 inhibition, was included as a loading control. B , immunoblots of EGFR, Raf-1, and p85 PI3K, showing that lower concentrations of 17-AAG were required to down-regulate mutant forms of EGFR (L858R and ΔL747_E749, A750P) compared with wild-type EGFR. Cells were harvested 24 h after treatment with 17-AAG.

Techniques Used: Mutagenesis, Stable Transfection, Transfection, Western Blot, Inhibition

28) Product Images from "Activated Ras Induces Cytoplasmic Vacuolation and Non-Apoptotic Death in Glioblastoma Cells via Novel Effector Pathways"

Article Title: Activated Ras Induces Cytoplasmic Vacuolation and Non-Apoptotic Death in Glioblastoma Cells via Novel Effector Pathways

Journal: Cellular signalling

doi: 10.1016/j.cellsig.2006.11.010

Overexpression of constitutively active Raf-1 does not mimic the effect of activated H-Ras. U251 cells were nucleofected with empty vector or with expression vectors encoding myc-Raf-C aax or myc-H-Ras(G12V). The cells were processed for western blot analysis or immunofluorescence 24 h later. (a) Immunoblot analysis with an antibody against Raf confirms that myc-Raf-C aax (upper band) is overexpressed relative to endogenous Raf. (b) Phospho-ERK and total ERK were determined by immunoblot analysis (representative blot shown). The ratio of phospho-ERK to total ERK was determined by quantifying the immunoblot signals with a Kodak Image station. The results shown in the bar graph are the means (±SD) of separate determinations performed on three cultures. (c) Cells expressing myc-Raf-C aax or myc-H- Ras(G12V) were identified by immunofluorescence using antibody against the myc epitope. The same cells were examined under phase contrast to assess the presence of vacuoles. The nucleofection efficiencies in both sets of cultures were approximately 50%. There were no vacuolated myc-positive cells in the cultures expressing myc-Raf-C aax .
Figure Legend Snippet: Overexpression of constitutively active Raf-1 does not mimic the effect of activated H-Ras. U251 cells were nucleofected with empty vector or with expression vectors encoding myc-Raf-C aax or myc-H-Ras(G12V). The cells were processed for western blot analysis or immunofluorescence 24 h later. (a) Immunoblot analysis with an antibody against Raf confirms that myc-Raf-C aax (upper band) is overexpressed relative to endogenous Raf. (b) Phospho-ERK and total ERK were determined by immunoblot analysis (representative blot shown). The ratio of phospho-ERK to total ERK was determined by quantifying the immunoblot signals with a Kodak Image station. The results shown in the bar graph are the means (±SD) of separate determinations performed on three cultures. (c) Cells expressing myc-Raf-C aax or myc-H- Ras(G12V) were identified by immunofluorescence using antibody against the myc epitope. The same cells were examined under phase contrast to assess the presence of vacuoles. The nucleofection efficiencies in both sets of cultures were approximately 50%. There were no vacuolated myc-positive cells in the cultures expressing myc-Raf-C aax .

Techniques Used: Over Expression, Plasmid Preparation, Expressing, Western Blot, Immunofluorescence

29) Product Images from "HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold"

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00811-14

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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used:

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
Figure Legend 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

Techniques Used: 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,
Figure Legend 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,

Techniques Used: 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
Figure Legend 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

Techniques Used: Transfection

30) Product Images from "ERK-dependent T-cell receptor threshold calibration in rheumatoid arthritis"

Article Title: ERK-dependent T-cell receptor threshold calibration in rheumatoid arthritis

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.0901784

Sustained Ras-Raf complex formation in RA T cells. T cells were stimulated, fixed, and stained with antibodies for signaling molecules. Representative cells stained for Raf-1 and K-Ras (A) or N-Ras (B) are shown. In C, D, F, and G, fluorescence intensities in membrane-gated regions were quantified; results are expressed as fold-difference compared to unstimulated control cells and are shown as box plots of 75 cells from 5 patients (shaded boxes) and 5 controls (open boxes). In Figures 5E and H, co-localization of green and red fluorescence (indicated by yellow color in A, B) was examined. Fluorescence for each pixel was correlated, and the data are shown as the correlation coefficients. (I) T cells were stimulated by CD3 cross-linking; cell lysates were obtained at indicated time points and analyzed by Western blotting for p-c-Raf. Results are representative of 3 experiments with 9 RA patients and 9 controls. * p
Figure Legend Snippet: Sustained Ras-Raf complex formation in RA T cells. T cells were stimulated, fixed, and stained with antibodies for signaling molecules. Representative cells stained for Raf-1 and K-Ras (A) or N-Ras (B) are shown. In C, D, F, and G, fluorescence intensities in membrane-gated regions were quantified; results are expressed as fold-difference compared to unstimulated control cells and are shown as box plots of 75 cells from 5 patients (shaded boxes) and 5 controls (open boxes). In Figures 5E and H, co-localization of green and red fluorescence (indicated by yellow color in A, B) was examined. Fluorescence for each pixel was correlated, and the data are shown as the correlation coefficients. (I) T cells were stimulated by CD3 cross-linking; cell lysates were obtained at indicated time points and analyzed by Western blotting for p-c-Raf. Results are representative of 3 experiments with 9 RA patients and 9 controls. * p

Techniques Used: Staining, Fluorescence, Western Blot

31) Product Images from "ERK-dependent T-cell receptor threshold calibration in rheumatoid arthritis"

Article Title: ERK-dependent T-cell receptor threshold calibration in rheumatoid arthritis

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.0901784

Sustained Ras-Raf complex formation in RA T cells. T cells were stimulated, fixed, and stained with antibodies for signaling molecules. Representative cells stained for Raf-1 and K-Ras (A) or N-Ras (B) are shown. In C, D, F, and G, fluorescence intensities in membrane-gated regions were quantified; results are expressed as fold-difference compared to unstimulated control cells and are shown as box plots of 75 cells from 5 patients (shaded boxes) and 5 controls (open boxes). In Figures 5E and H, co-localization of green and red fluorescence (indicated by yellow color in A, B) was examined. Fluorescence for each pixel was correlated, and the data are shown as the correlation coefficients. (I) T cells were stimulated by CD3 cross-linking; cell lysates were obtained at indicated time points and analyzed by Western blotting for p-c-Raf. Results are representative of 3 experiments with 9 RA patients and 9 controls. * p
Figure Legend Snippet: Sustained Ras-Raf complex formation in RA T cells. T cells were stimulated, fixed, and stained with antibodies for signaling molecules. Representative cells stained for Raf-1 and K-Ras (A) or N-Ras (B) are shown. In C, D, F, and G, fluorescence intensities in membrane-gated regions were quantified; results are expressed as fold-difference compared to unstimulated control cells and are shown as box plots of 75 cells from 5 patients (shaded boxes) and 5 controls (open boxes). In Figures 5E and H, co-localization of green and red fluorescence (indicated by yellow color in A, B) was examined. Fluorescence for each pixel was correlated, and the data are shown as the correlation coefficients. (I) T cells were stimulated by CD3 cross-linking; cell lysates were obtained at indicated time points and analyzed by Western blotting for p-c-Raf. Results are representative of 3 experiments with 9 RA patients and 9 controls. * p

Techniques Used: Staining, Fluorescence, Western Blot

32) Product Images from "14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity"

Article Title: 14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity

Journal: Molecular and Cellular Biology

doi:

Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.
Figure Legend Snippet: Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.

Techniques Used: Binding Assay, Activity Assay, Activation Assay

Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).
Figure Legend Snippet: Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).

Techniques Used: Purification, Labeling, In Vitro, High Performance Liquid Chromatography, Radioactivity, SDS Page, Chromatography, Staining

S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.
Figure Legend Snippet: S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.

Techniques Used: Activity Assay, Labeling, Infection, Recombinant, Construct, Cell Culture, Lysis, SDS Page, Expressing, In Vitro

33) Product Images from "HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold"

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00811-14

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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used:

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
Figure Legend 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

Techniques Used: 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,
Figure Legend 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,

Techniques Used: 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
Figure Legend 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

Techniques Used: Transfection

34) Product Images from "Inhibition of Hsp90 Down-regulates Mutant Epidermal Growth Factor Receptor (EGFR) Expression and Sensitizes EGFR Mutant Tumors to Paclitaxel"

Article Title: Inhibition of Hsp90 Down-regulates Mutant Epidermal Growth Factor Receptor (EGFR) Expression and Sensitizes EGFR Mutant Tumors to Paclitaxel

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-07-1570

17-AAG induced the degradation of the L858R, T790M, EGFR mutant in H1975 lung adenocarcinoma cells. A , immunoblots showing that 17-AAG causes degradation of mutant EGFR and down-regulation of phospho-MAPK and phospho-Akt expression in H1975 cells. B , in Calu-1 cells (wild-type EGFR), EGFR expression is only minimally affected by 17-AAG. However, other Hsp90 clients, such as Raf-1, are degraded in Calu-1. Cells were treated with 17-AAG for 24 h at the doses indicated. C , both EGFR mutant and EGFR wild-type lung cancer cell lines were sensitive to 17-AAG in vitro . Cellular proliferation was assayed at 72 h by Alamar Blue and graphed as a percentage of DMSO-treated cells.
Figure Legend Snippet: 17-AAG induced the degradation of the L858R, T790M, EGFR mutant in H1975 lung adenocarcinoma cells. A , immunoblots showing that 17-AAG causes degradation of mutant EGFR and down-regulation of phospho-MAPK and phospho-Akt expression in H1975 cells. B , in Calu-1 cells (wild-type EGFR), EGFR expression is only minimally affected by 17-AAG. However, other Hsp90 clients, such as Raf-1, are degraded in Calu-1. Cells were treated with 17-AAG for 24 h at the doses indicated. C , both EGFR mutant and EGFR wild-type lung cancer cell lines were sensitive to 17-AAG in vitro . Cellular proliferation was assayed at 72 h by Alamar Blue and graphed as a percentage of DMSO-treated cells.

Techniques Used: Mutagenesis, Western Blot, Expressing, In Vitro

17-AAG, at nontoxic doses, down-regulates mutant EGFR expression in xenograft tumors. For dose-response studies, mice were treated with either 50 or 75 mg/kg of 17-AAG and sacrificed 6 h later. In time course experiments mice were treated with a single dose of 75 mg/kg of 17-AAG and sacrificed at the time points indicated (0–48 h). A , immunoblots of EGFR, Raf-1, Akt, and cyclin D1 in H1650 xenografts treated with 17-AAG. 17-AAG treatment resulted in a > 95% reduction in mutant EGFR expression and inhibition of AKT and MAPK activity in H1650 xenografts. Raf-1 was less sensitive than mutant EGFR to 17-AAG treatment, and Akt expression was unaffected by 17-AAG. B , quantitation of the effects of 17-AAG on the expression of EGFR, Raf-1, and Akt in H1650 xenografts. MAPK, a protein unaffected by Hsp90 inhibition, is included as a loading control. C , similar results were observed in H3255 and H1975 xenografts ( C and data not shown). D , 17-AAG does not induce the degradation of wild-type EGFR but does down-regulate the expression of HER2 in A549 xenografts.
Figure Legend Snippet: 17-AAG, at nontoxic doses, down-regulates mutant EGFR expression in xenograft tumors. For dose-response studies, mice were treated with either 50 or 75 mg/kg of 17-AAG and sacrificed 6 h later. In time course experiments mice were treated with a single dose of 75 mg/kg of 17-AAG and sacrificed at the time points indicated (0–48 h). A , immunoblots of EGFR, Raf-1, Akt, and cyclin D1 in H1650 xenografts treated with 17-AAG. 17-AAG treatment resulted in a > 95% reduction in mutant EGFR expression and inhibition of AKT and MAPK activity in H1650 xenografts. Raf-1 was less sensitive than mutant EGFR to 17-AAG treatment, and Akt expression was unaffected by 17-AAG. B , quantitation of the effects of 17-AAG on the expression of EGFR, Raf-1, and Akt in H1650 xenografts. MAPK, a protein unaffected by Hsp90 inhibition, is included as a loading control. C , similar results were observed in H3255 and H1975 xenografts ( C and data not shown). D , 17-AAG does not induce the degradation of wild-type EGFR but does down-regulate the expression of HER2 in A549 xenografts.

Techniques Used: Mutagenesis, Expressing, Mouse Assay, Western Blot, Inhibition, Activity Assay, Quantitation Assay

Mutant forms of EGFR are more sensitive than wild-type EGFR to 17-AAG–induced degradation. A , NIH-3T3 cells were stably transfected with wild-type and mutant forms of EGFR (L858R and ΔL747_E749, A750P). Immunoblots of EGFR, Raf-1, and p85 phosphatidylinositol 3-kinase (PI3K) show that mutant forms of EGFR were degraded at earlier time points than wild-type EGFR after treatment with 0.1 μmol/L of 17-AAG. p85 PI3K, a protein unaffected by Hsp90 inhibition, was included as a loading control. B , immunoblots of EGFR, Raf-1, and p85 PI3K, showing that lower concentrations of 17-AAG were required to down-regulate mutant forms of EGFR (L858R and ΔL747_E749, A750P) compared with wild-type EGFR. Cells were harvested 24 h after treatment with 17-AAG.
Figure Legend Snippet: Mutant forms of EGFR are more sensitive than wild-type EGFR to 17-AAG–induced degradation. A , NIH-3T3 cells were stably transfected with wild-type and mutant forms of EGFR (L858R and ΔL747_E749, A750P). Immunoblots of EGFR, Raf-1, and p85 phosphatidylinositol 3-kinase (PI3K) show that mutant forms of EGFR were degraded at earlier time points than wild-type EGFR after treatment with 0.1 μmol/L of 17-AAG. p85 PI3K, a protein unaffected by Hsp90 inhibition, was included as a loading control. B , immunoblots of EGFR, Raf-1, and p85 PI3K, showing that lower concentrations of 17-AAG were required to down-regulate mutant forms of EGFR (L858R and ΔL747_E749, A750P) compared with wild-type EGFR. Cells were harvested 24 h after treatment with 17-AAG.

Techniques Used: Mutagenesis, Stable Transfection, Transfection, Western Blot, Inhibition

35) Product Images from "Protein Farnesylation-Dependent Raf/Extracellular Signal-Related Kinase Signaling Links to Cytoskeletal Remodeling to Facilitate Glucose-Induced Insulin Secretion in Pancreatic ?-Cells"

Article Title: Protein Farnesylation-Dependent Raf/Extracellular Signal-Related Kinase Signaling Links to Cytoskeletal Remodeling to Facilitate Glucose-Induced Insulin Secretion in Pancreatic ?-Cells

Journal: Diabetes

doi: 10.2337/db09-1334

Raf-1 kinase activation is necessary for glucose-induced ERK1/2 phosphorylation and insulin secretion in INS 832/13 β-cells. A : INS 832/13 cells were treated with either diluent alone or GW-5074 (10 μmol/l) as indicated in the figure, and cultured overnight in low-glucose media. The cells were further incubated in the presence of either low (2.5 mmol/l) or high (20 mmol/l) glucose for 30 min at 37°C in the continuous presence of either GW-5074 or diluent. Cell lysates were then separated by SDS-PAGE followed by transfer onto a nitrocellulose membrane. The membranes were then blocked and incubated with anti–p-ERK1/2 followed by incubation with HRP-conjugated secondary antibody. The same blots were then stripped and reprobed with ERK1/2 antibody. A representative blot from three experiments is shown here. B : The relative intensities of p-ERK1/2:total ERK1/2 ratio quantitated by densitometry. Data are expressed as fold increase and are means ± SEM from three experiments. * P
Figure Legend Snippet: Raf-1 kinase activation is necessary for glucose-induced ERK1/2 phosphorylation and insulin secretion in INS 832/13 β-cells. A : INS 832/13 cells were treated with either diluent alone or GW-5074 (10 μmol/l) as indicated in the figure, and cultured overnight in low-glucose media. The cells were further incubated in the presence of either low (2.5 mmol/l) or high (20 mmol/l) glucose for 30 min at 37°C in the continuous presence of either GW-5074 or diluent. Cell lysates were then separated by SDS-PAGE followed by transfer onto a nitrocellulose membrane. The membranes were then blocked and incubated with anti–p-ERK1/2 followed by incubation with HRP-conjugated secondary antibody. The same blots were then stripped and reprobed with ERK1/2 antibody. A representative blot from three experiments is shown here. B : The relative intensities of p-ERK1/2:total ERK1/2 ratio quantitated by densitometry. Data are expressed as fold increase and are means ± SEM from three experiments. * P

Techniques Used: Activation Assay, Cell Culture, Incubation, SDS Page

Inhibition of Raf-1 kinase or FTase markedly attenuates glucose-induced activation of Rac1 in INS 832/13 cells. INS 832/13 cells were cultured overnight in low-serum, low-glucose media with either diluent alone or GW-5074 (10 μmol/l) as indicated in the figure. The cells were then incubated further (30 min) in the presence of either low (5 mmol/l) or high (20 mmol/l) glucose in the continuous presence of GW-5074 or diluent. The degree of Rac1 activation was determined by PAK/PBD pull-down assay. A : A representative blot from four independent experiments yielding similar results. B : Densitometric analysis of the ratio of total Rac1 and Rac1.GTP. Data are mean ± SEM from four determinations in each case. * P
Figure Legend Snippet: Inhibition of Raf-1 kinase or FTase markedly attenuates glucose-induced activation of Rac1 in INS 832/13 cells. INS 832/13 cells were cultured overnight in low-serum, low-glucose media with either diluent alone or GW-5074 (10 μmol/l) as indicated in the figure. The cells were then incubated further (30 min) in the presence of either low (5 mmol/l) or high (20 mmol/l) glucose in the continuous presence of GW-5074 or diluent. The degree of Rac1 activation was determined by PAK/PBD pull-down assay. A : A representative blot from four independent experiments yielding similar results. B : Densitometric analysis of the ratio of total Rac1 and Rac1.GTP. Data are mean ± SEM from four determinations in each case. * P

Techniques Used: Inhibition, Activation Assay, Cell Culture, Incubation, Pull Down Assay

36) Product Images from "IL-6 Increases MMP-13 Expression and Motility in Human Chondrosarcoma Cells *"

Article Title: IL-6 Increases MMP-13 Expression and Motility in Human Chondrosarcoma Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.204081

Involvement of Ras and Raf-1 signaling pathways in response to IL-6 in chondrosarcoma cells. A , cells were pretreated with manumycin A (3 μ m ) and GW5074 (3 μ m ) for 30 min followed by stimulation with IL-6 for 24 h, and in vitro migration
Figure Legend Snippet: Involvement of Ras and Raf-1 signaling pathways in response to IL-6 in chondrosarcoma cells. A , cells were pretreated with manumycin A (3 μ m ) and GW5074 (3 μ m ) for 30 min followed by stimulation with IL-6 for 24 h, and in vitro migration

Techniques Used: In Vitro, Migration

37) Product Images from "Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina"

Article Title: Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina

Journal: Molecular Vision

doi:

Effect of MnTBAP on glucose-induced activation of H-Ras. Endothelial cells were incubated in 5 mM glucose or 20 mM glucose medium for 96 h in the presence or absence of 200 μM MnTBAP for 96 h. Activation of H-Ras was estimated by ( A ) Western blot technique and Raf-1 ( B ) binding assay. Each experiment was repeated with at least three separate cell preparations. The histogram represents the ratio of the densities of H-Ras and β-actin in the same lane as quantified using Un-Scan-It gel software. The values obtained from the cells incubated in 5 mM glucose conditions are considered 100%. Asterisk (*) marks p
Figure Legend Snippet: Effect of MnTBAP on glucose-induced activation of H-Ras. Endothelial cells were incubated in 5 mM glucose or 20 mM glucose medium for 96 h in the presence or absence of 200 μM MnTBAP for 96 h. Activation of H-Ras was estimated by ( A ) Western blot technique and Raf-1 ( B ) binding assay. Each experiment was repeated with at least three separate cell preparations. The histogram represents the ratio of the densities of H-Ras and β-actin in the same lane as quantified using Un-Scan-It gel software. The values obtained from the cells incubated in 5 mM glucose conditions are considered 100%. Asterisk (*) marks p

Techniques Used: Activation Assay, Incubation, Western Blot, Binding Assay, Software

Effect of MnTBAP on glucose-induced activation of Raf-1 and phosphorylation of p38 MAP kinase. Activation of Raf-1 and phosphorylation of p38 MAP kinase were determined by Western blot using β-actin as a loading standard. Each sample was run in duplicate, and the experiment was repeated with three or more cell preparations. The histogram represents the density of Raf-1 ( A ), or p-p38 ( B ) band that has been adjusted to the density of β-actin band in the same lane. The ratio obtained from 5 mM glucose is considered as 100%. Asterisk (*) represent p
Figure Legend Snippet: Effect of MnTBAP on glucose-induced activation of Raf-1 and phosphorylation of p38 MAP kinase. Activation of Raf-1 and phosphorylation of p38 MAP kinase were determined by Western blot using β-actin as a loading standard. Each sample was run in duplicate, and the experiment was repeated with three or more cell preparations. The histogram represents the density of Raf-1 ( A ), or p-p38 ( B ) band that has been adjusted to the density of β-actin band in the same lane. The ratio obtained from 5 mM glucose is considered as 100%. Asterisk (*) represent p

Techniques Used: Activation Assay, Western Blot

Effect of hydrogen peroxide on the activation of H-Ras and its signaling pathway. Bovine retinal endothelial cells were incubated with 250 μM H 2 O 2 for 1 h. At the end of the incubation, the cells were rinsed with DMEM, and incubated in 5 mM glucose and 20 mM glucose media for 96 h. The activation of H-Ras was determined by Raf-1 binding assay, Raf-1 by measuring the expression of Raf-1 by Western blot, and that of MAP kinase by quantifying the expression of phospho-p38 of MAP kinase. Each sample was analyzed in duplicate, and the Western blots presented are representative of at least 3-4 experiments.
Figure Legend Snippet: Effect of hydrogen peroxide on the activation of H-Ras and its signaling pathway. Bovine retinal endothelial cells were incubated with 250 μM H 2 O 2 for 1 h. At the end of the incubation, the cells were rinsed with DMEM, and incubated in 5 mM glucose and 20 mM glucose media for 96 h. The activation of H-Ras was determined by Raf-1 binding assay, Raf-1 by measuring the expression of Raf-1 by Western blot, and that of MAP kinase by quantifying the expression of phospho-p38 of MAP kinase. Each sample was analyzed in duplicate, and the Western blots presented are representative of at least 3-4 experiments.

Techniques Used: Activation Assay, Incubation, Binding Assay, Expressing, Western Blot

38) Product Images from "Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina"

Article Title: Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina

Journal: Molecular Vision

doi:

Effect of MnTBAP on glucose-induced activation of H-Ras. Endothelial cells were incubated in 5 mM glucose or 20 mM glucose medium for 96 h in the presence or absence of 200 μM MnTBAP for 96 h. Activation of H-Ras was estimated by ( A ) Western blot technique and Raf-1 ( B ) binding assay. Each experiment was repeated with at least three separate cell preparations. The histogram represents the ratio of the densities of H-Ras and β-actin in the same lane as quantified using Un-Scan-It gel software. The values obtained from the cells incubated in 5 mM glucose conditions are considered 100%. Asterisk (*) marks p
Figure Legend Snippet: Effect of MnTBAP on glucose-induced activation of H-Ras. Endothelial cells were incubated in 5 mM glucose or 20 mM glucose medium for 96 h in the presence or absence of 200 μM MnTBAP for 96 h. Activation of H-Ras was estimated by ( A ) Western blot technique and Raf-1 ( B ) binding assay. Each experiment was repeated with at least three separate cell preparations. The histogram represents the ratio of the densities of H-Ras and β-actin in the same lane as quantified using Un-Scan-It gel software. The values obtained from the cells incubated in 5 mM glucose conditions are considered 100%. Asterisk (*) marks p

Techniques Used: Activation Assay, Incubation, Western Blot, Binding Assay, Software

Effect of MnTBAP on glucose-induced activation of Raf-1 and phosphorylation of p38 MAP kinase. Activation of Raf-1 and phosphorylation of p38 MAP kinase were determined by Western blot using β-actin as a loading standard. Each sample was run in duplicate, and the experiment was repeated with three or more cell preparations. The histogram represents the density of Raf-1 ( A ), or p-p38 ( B ) band that has been adjusted to the density of β-actin band in the same lane. The ratio obtained from 5 mM glucose is considered as 100%. Asterisk (*) represent p
Figure Legend Snippet: Effect of MnTBAP on glucose-induced activation of Raf-1 and phosphorylation of p38 MAP kinase. Activation of Raf-1 and phosphorylation of p38 MAP kinase were determined by Western blot using β-actin as a loading standard. Each sample was run in duplicate, and the experiment was repeated with three or more cell preparations. The histogram represents the density of Raf-1 ( A ), or p-p38 ( B ) band that has been adjusted to the density of β-actin band in the same lane. The ratio obtained from 5 mM glucose is considered as 100%. Asterisk (*) represent p

Techniques Used: Activation Assay, Western Blot

Effect of hydrogen peroxide on the activation of H-Ras and its signaling pathway. Bovine retinal endothelial cells were incubated with 250 μM H 2 O 2 for 1 h. At the end of the incubation, the cells were rinsed with DMEM, and incubated in 5 mM glucose and 20 mM glucose media for 96 h. The activation of H-Ras was determined by Raf-1 binding assay, Raf-1 by measuring the expression of Raf-1 by Western blot, and that of MAP kinase by quantifying the expression of phospho-p38 of MAP kinase. Each sample was analyzed in duplicate, and the Western blots presented are representative of at least 3-4 experiments.
Figure Legend Snippet: Effect of hydrogen peroxide on the activation of H-Ras and its signaling pathway. Bovine retinal endothelial cells were incubated with 250 μM H 2 O 2 for 1 h. At the end of the incubation, the cells were rinsed with DMEM, and incubated in 5 mM glucose and 20 mM glucose media for 96 h. The activation of H-Ras was determined by Raf-1 binding assay, Raf-1 by measuring the expression of Raf-1 by Western blot, and that of MAP kinase by quantifying the expression of phospho-p38 of MAP kinase. Each sample was analyzed in duplicate, and the Western blots presented are representative of at least 3-4 experiments.

Techniques Used: Activation Assay, Incubation, Binding Assay, Expressing, Western Blot

39) Product Images from "14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity"

Article Title: 14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity

Journal: Molecular and Cellular Biology

doi:

Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.
Figure Legend Snippet: Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.

Techniques Used: Binding Assay, Activity Assay, Activation Assay

Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).
Figure Legend Snippet: Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).

Techniques Used: Purification, Labeling, In Vitro, High Performance Liquid Chromatography, Radioactivity, SDS Page, Chromatography, Staining

S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.
Figure Legend Snippet: S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.

Techniques Used: Activity Assay, Labeling, Infection, Recombinant, Construct, Cell Culture, Lysis, SDS Page, Expressing, In Vitro

40) Product Images from "14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity"

Article Title: 14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity

Journal: Molecular and Cellular Biology

doi:

Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.
Figure Legend Snippet: Potential role for 14-3-3 in Raf-1 maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.

Techniques Used: Binding Assay, Activity Assay, Activation Assay

Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).
Figure Legend Snippet: Specificity of the anti-Raf phosphoserine 621 antibody. (A) Tryptic peptide analysis of CT-Raf phosphorylated by C-TAK1. CT-Raf was expressed in bacteria as a GST fusion protein. After purification, it was labeled in vitro with purified C-TAK1 and [γ- 32 P]ATP and then digested with trypsin. Peptides were resolved by HPLC. Radioactivity associated with each fraction was measured by scintillation counting. (B) Manual Edman degradation of tryptic fraction 51. Fraction 51 from panel A was subjected to manual Edman degradation. Bars represent radioactivity released from the membrane. The starting radioactivity associated with the membrane was 376 cpm. (C) Anti-phospho-S621 immunoblotting. CT-Raf or histidine-tagged Cdc25C was either expressed alone (lanes 1 and 3) or coexpressed with C-TAK1 (lanes 2 and 4) in bacteria. Lanes 1 and 2, cell extracts were analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to Raf-1 (top panel) or with anti-p621 (lower panel). Lanes 3 and 4, His-tagged Cdc25C was purified by nickel chromatography and Coomassie blue stained (upper panel) or blotted with anti-p621 (lower panel).

Techniques Used: Purification, Labeling, In Vitro, High Performance Liquid Chromatography, Radioactivity, SDS Page, Chromatography, Staining

S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.
Figure Legend Snippet: S621 phosphorylation and kinase activity of wild-type or mutated CT-Raf proteins. (A) Phosphate labeling of mutated CT-Raf proteins. Sf9 cells infected with recombinant baculoviruses encoding the indicated CT-Raf constructs or uninfected cells (control) were cultured in the presence of [ 32 P]orthophosphoric acid for 12 h. Cells were lysed in NP-40 lysis buffer, and Raf-1 immunoprecipitates were prepared. After resolution by SDS-PAGE, the labeled proteins were visualized by phosphorimaging. Equivalent expression of each construct was verified separately by immunoblot analysis (data not shown). The data shown are representative of three separate experiments. (B) In vitro kinase activity of wild-type or mutated CT-Raf proteins. Sf9 cells were infected with a recombinant baculovirus encoding MEK-1 alone (control) or coinfected with the MEK-1 virus and a second recombinant baculovirus encoding the indicated CT-Raf construct. At 48 h postinfection, cell lysates were prepared. Anti-MEK-1 immunoprecipitations from each lysate were analyzed for MEK-1 kinase activity by using recombinant, kinase-deficient (KD) MAPK as a substrate. Lane 1, cells infected with MEK-1 alone. Lanes 2 to 7, cells infected with MEK-1 and the following CT-Raf constructs: lane 2, wild type; lane 3, K375M (kinase dead); lane 4, S621A; lane 5, +2 Gly (P623G); lane 6, +2 Leu (P623L); lane 7, −2 Lys (S619K). Data shown are representative of four separate experiments. (C) Expression levels of CT-Raf constructs used for panel B. Equal aliquots of lysates used for panel B were resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an anti-Raf antibody. Lane contents are identical to those in panel B.

Techniques Used: Activity Assay, Labeling, Infection, Recombinant, Construct, Cell Culture, Lysis, SDS Page, Expressing, In Vitro

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Activation Assay:

Article Title: Mutant N-Ras protects colorectal cancer cells from stress-induced apoptosis and contributes to cancer development and progression
Article Snippet: .. Elad-Sfadia G, Haklai R, Ballan E, Gabius HJ, Kloog Y. Galectin-1 augments Ras activation and diverts Ras signals to Raf-1 at the expense of phosphoinositide 3-kinase. ..

Article Title: Choice of Biological Source Material Supersedes Oxidative Stress in Its Influence on DJ-1 in Vivo Interactions with Hsp90
Article Snippet: .. It is involved in the maturation, stabilization, and/or activation of a select but structurally broad clientele of proteins, including many kinases such as Raf-1 and PKB/Akt. ..

other:

Article Title: Spatial control of Shoc2-scaffold-mediated ERK1/2 signaling requires remodeling activity of the ATPase PSMC5
Article Snippet: Antibodies against the following proteins were used: GST, RAF-1, HA, PSMC5, GAPDH, phosphorylated ERK1/2 (pERK1/2) and EGFR (Santa Cruz Biotechnology); Shoc2 (Proteintech); EEA1 and phosphorylated RAF-1 (pRAF-1; Cell Signaling); Rab5 (BD Biosciences); RFP and Na+ /K+ ATPase (Thermo Scientific); FLAG (SydLabs); HUWE1 (Bethyl); ubiquitin (Covance); PP1c (Millipore); Rpt1 (Enzo); LAMP1 (DSHB); TSG101 (GeneTex); anti-Cyclin B1 antibody was provided by Tianyan Gao (University of Kentucky, Lexington, KY).

Article Title: Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina
Article Snippet: Raf-1 is a key downstream effector protein of Ras function that in its inactive state is localized in the cytosol.

Expressing:

Article Title: Translocation of H-Ras and its implications in the development of diabetic retinopathy
Article Snippet: .. In the same diabetic rats the gene expression of H-Ras in the retinal membranes was 1.7 fold higher compared to normal rats , and H-Ras was activated by about 40 %, as determined by its binding to Raf-1 ( ). .. As shown in , the expression of Raf-1 was significantly increased in the membrane fraction in diabetic rats compared to normal rats; the ratio expression of Raf-1 expression in membrane to cytosol was 0.7 in normal rat retina and 1.2 in diabetic rat retina.

Binding Assay:

Article Title: Translocation of H-Ras and its implications in the development of diabetic retinopathy
Article Snippet: .. In the same diabetic rats the gene expression of H-Ras in the retinal membranes was 1.7 fold higher compared to normal rats , and H-Ras was activated by about 40 %, as determined by its binding to Raf-1 ( ). .. As shown in , the expression of Raf-1 was significantly increased in the membrane fraction in diabetic rats compared to normal rats; the ratio expression of Raf-1 expression in membrane to cytosol was 0.7 in normal rat retina and 1.2 in diabetic rat retina.

Translocation Assay:

Article Title: Translocation of H-Ras and its implications in the development of diabetic retinopathy
Article Snippet: .. Transfection of BRECs with H-Ras-siRNA abrogated glucose-induced membrane translocation of H-Ras and Raf-1. .. The gene expression of H-Ras in the membrane fraction of cells incubated in high glucose was significantly lower in the H-Ras-siRNA transfected cells compared to the mock cells, and the values were similar to those obtained from cells incubated in normal glucose ( ).

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    Santa Cruz Biotechnology raf 1
    Effect of MnTBAP on glucose-induced activation of H-Ras. Endothelial cells were incubated in 5 mM glucose or 20 mM glucose medium for 96 h in the presence or absence of 200 μM MnTBAP for 96 h. Activation of H-Ras was estimated by ( A ) Western blot technique and <t>Raf-1</t> ( B ) binding assay. Each experiment was repeated with at least three separate cell preparations. The histogram represents the ratio of the densities of H-Ras and β-actin in the same lane as quantified using Un-Scan-It gel software. The values obtained from the cells incubated in 5 mM glucose conditions are considered 100%. Asterisk (*) marks p
    Raf 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/raf 1/product/Santa Cruz Biotechnology
    Average 93 stars, based on 97 article reviews
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    85
    Santa Cruz Biotechnology c anti raf 1 antisera
    Inhibition of <t>raf-1</t> kinase activity by p38. ATDC5 cells were stimulated with IGF-I (100 ng/ml) for 15 min; cell lysates immunoprecipitated for endogenous raf-1 protein were incubated with or without recombinant (His) 6 -p38 protein for 30 min, then kinase
    C Anti Raf 1 Antisera, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c anti raf 1 antisera/product/Santa Cruz Biotechnology
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    c anti raf 1 antisera - by Bioz Stars, 2020-07
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    Effect of MnTBAP on glucose-induced activation of H-Ras. Endothelial cells were incubated in 5 mM glucose or 20 mM glucose medium for 96 h in the presence or absence of 200 μM MnTBAP for 96 h. Activation of H-Ras was estimated by ( A ) Western blot technique and Raf-1 ( B ) binding assay. Each experiment was repeated with at least three separate cell preparations. The histogram represents the ratio of the densities of H-Ras and β-actin in the same lane as quantified using Un-Scan-It gel software. The values obtained from the cells incubated in 5 mM glucose conditions are considered 100%. Asterisk (*) marks p

    Journal: Molecular Vision

    Article Title: Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina

    doi:

    Figure Lengend Snippet: Effect of MnTBAP on glucose-induced activation of H-Ras. Endothelial cells were incubated in 5 mM glucose or 20 mM glucose medium for 96 h in the presence or absence of 200 μM MnTBAP for 96 h. Activation of H-Ras was estimated by ( A ) Western blot technique and Raf-1 ( B ) binding assay. Each experiment was repeated with at least three separate cell preparations. The histogram represents the ratio of the densities of H-Ras and β-actin in the same lane as quantified using Un-Scan-It gel software. The values obtained from the cells incubated in 5 mM glucose conditions are considered 100%. Asterisk (*) marks p

    Article Snippet: Raf-1 is a key downstream effector protein of Ras function that in its inactive state is localized in the cytosol.

    Techniques: Activation Assay, Incubation, Western Blot, Binding Assay, Software

    Effect of MnTBAP on glucose-induced activation of Raf-1 and phosphorylation of p38 MAP kinase. Activation of Raf-1 and phosphorylation of p38 MAP kinase were determined by Western blot using β-actin as a loading standard. Each sample was run in duplicate, and the experiment was repeated with three or more cell preparations. The histogram represents the density of Raf-1 ( A ), or p-p38 ( B ) band that has been adjusted to the density of β-actin band in the same lane. The ratio obtained from 5 mM glucose is considered as 100%. Asterisk (*) represent p

    Journal: Molecular Vision

    Article Title: Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina

    doi:

    Figure Lengend Snippet: Effect of MnTBAP on glucose-induced activation of Raf-1 and phosphorylation of p38 MAP kinase. Activation of Raf-1 and phosphorylation of p38 MAP kinase were determined by Western blot using β-actin as a loading standard. Each sample was run in duplicate, and the experiment was repeated with three or more cell preparations. The histogram represents the density of Raf-1 ( A ), or p-p38 ( B ) band that has been adjusted to the density of β-actin band in the same lane. The ratio obtained from 5 mM glucose is considered as 100%. Asterisk (*) represent p

    Article Snippet: Raf-1 is a key downstream effector protein of Ras function that in its inactive state is localized in the cytosol.

    Techniques: Activation Assay, Western Blot

    Effect of hydrogen peroxide on the activation of H-Ras and its signaling pathway. Bovine retinal endothelial cells were incubated with 250 μM H 2 O 2 for 1 h. At the end of the incubation, the cells were rinsed with DMEM, and incubated in 5 mM glucose and 20 mM glucose media for 96 h. The activation of H-Ras was determined by Raf-1 binding assay, Raf-1 by measuring the expression of Raf-1 by Western blot, and that of MAP kinase by quantifying the expression of phospho-p38 of MAP kinase. Each sample was analyzed in duplicate, and the Western blots presented are representative of at least 3-4 experiments.

    Journal: Molecular Vision

    Article Title: Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina

    doi:

    Figure Lengend Snippet: Effect of hydrogen peroxide on the activation of H-Ras and its signaling pathway. Bovine retinal endothelial cells were incubated with 250 μM H 2 O 2 for 1 h. At the end of the incubation, the cells were rinsed with DMEM, and incubated in 5 mM glucose and 20 mM glucose media for 96 h. The activation of H-Ras was determined by Raf-1 binding assay, Raf-1 by measuring the expression of Raf-1 by Western blot, and that of MAP kinase by quantifying the expression of phospho-p38 of MAP kinase. Each sample was analyzed in duplicate, and the Western blots presented are representative of at least 3-4 experiments.

    Article Snippet: Raf-1 is a key downstream effector protein of Ras function that in its inactive state is localized in the cytosol.

    Techniques: Activation Assay, Incubation, Binding Assay, Expressing, Western Blot

    Effect of azaspirene on the autophosphorylation of KDR/Flk-1, phosphorylation of Raf-1 and disruption of Raf-1 complexes.

    Journal: Cancer science

    Article Title: Azaspirene, a fungal product, inhibits angiogenesis by blocking Raf-1 activation

    doi: 10.1111/j.1349-7006.2008.00890.x

    Figure Lengend Snippet: Effect of azaspirene on the autophosphorylation of KDR/Flk-1, phosphorylation of Raf-1 and disruption of Raf-1 complexes.

    Article Snippet: Rabbit polyclonal antibodies; KDR/Flk-1, Raf-1, ERK1/2 and MEK1/2 were purchased from Santa Cruz Biotechnology, Zymed Laboratories (South San Francisco, CA) and Cell Signaling Technology, respectively.

    Techniques:

    Effect of sorafenib and Raf siRNA on p27 expression in QTRRE cells. (A) QTRRE cells were treated with sorafenib (50μM) for 30, 60, and 90 min. QTRRE cells were transfected with (B) 100nM B-Raf or (C) Raf-1 ON_TARGETplus SMARTpool siRNA or siCONTROL

    Journal: Toxicological Sciences

    Article Title: cAMP-Dependent Cytosolic Mislocalization of p27kip-Cyclin D1 During Quinol-Thioether-Induced Tuberous Sclerosis Renal Cell Carcinoma

    doi: 10.1093/toxsci/kfr118

    Figure Lengend Snippet: Effect of sorafenib and Raf siRNA on p27 expression in QTRRE cells. (A) QTRRE cells were treated with sorafenib (50μM) for 30, 60, and 90 min. QTRRE cells were transfected with (B) 100nM B-Raf or (C) Raf-1 ON_TARGETplus SMARTpool siRNA or siCONTROL

    Article Snippet: Primary antibodies used were cyclin D1 (A-12), B-Raf (H-145), Raf-1 (C-20), SP1 (E3), p27 (F-8), Rap1B (Santa Cruz Biotechnologies); p42/44, phospho-p42/44 (T202/Y204) (20G11) (Cell Signaling Technologies); and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Ambion, Austin, TX).

    Techniques: Expressing, Transfection

    Inhibition of raf-1 kinase activity by p38. ATDC5 cells were stimulated with IGF-I (100 ng/ml) for 15 min; cell lysates immunoprecipitated for endogenous raf-1 protein were incubated with or without recombinant (His) 6 -p38 protein for 30 min, then kinase

    Journal: Molecular Endocrinology

    Article Title: BDNF Alters ERK/p38 MAPK Activity Ratios to Promote Differentiation in Growth Plate Chondrocytes

    doi: 10.1210/me.2012-1063

    Figure Lengend Snippet: Inhibition of raf-1 kinase activity by p38. ATDC5 cells were stimulated with IGF-I (100 ng/ml) for 15 min; cell lysates immunoprecipitated for endogenous raf-1 protein were incubated with or without recombinant (His) 6 -p38 protein for 30 min, then kinase

    Article Snippet: Lysates normalized for total protein content were precleared with 30 μl of protein-A agarose (Invitrogen) for 30 min at 4 C. Anti-raf-1 antisera (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added at 1:500 plus 30 μl of protein-A agarose, and lysates incubated for 2 h with rocking at 4 C. Pellets were washed three times with lysis buffer and then washed three times with kinase buffer (50 m m Tris 7.4, 50 m m NaCl, 5 m m MnCl2 , 2 m m dithiothreitol, and 1 m m phenylmethylsulfonylfluoride) plus phosphatase inhibitors as above.

    Techniques: Inhibition, Activity Assay, Immunoprecipitation, Incubation, Recombinant

    Involvement of p38 in the regulation of raf-1 activity by BDNF and CNP. Isolated bovine reserve/proliferative chondrocytes were treated as in ; whole-cell lysates normalized for total protein content were immunoprecipitated with antisera to raf-1.

    Journal: Molecular Endocrinology

    Article Title: BDNF Alters ERK/p38 MAPK Activity Ratios to Promote Differentiation in Growth Plate Chondrocytes

    doi: 10.1210/me.2012-1063

    Figure Lengend Snippet: Involvement of p38 in the regulation of raf-1 activity by BDNF and CNP. Isolated bovine reserve/proliferative chondrocytes were treated as in ; whole-cell lysates normalized for total protein content were immunoprecipitated with antisera to raf-1.

    Article Snippet: Lysates normalized for total protein content were precleared with 30 μl of protein-A agarose (Invitrogen) for 30 min at 4 C. Anti-raf-1 antisera (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added at 1:500 plus 30 μl of protein-A agarose, and lysates incubated for 2 h with rocking at 4 C. Pellets were washed three times with lysis buffer and then washed three times with kinase buffer (50 m m Tris 7.4, 50 m m NaCl, 5 m m MnCl2 , 2 m m dithiothreitol, and 1 m m phenylmethylsulfonylfluoride) plus phosphatase inhibitors as above.

    Techniques: Activity Assay, Isolation, Immunoprecipitation