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  • 94
    Name:
    A Raf Antibody
    Description:
    A Raf B Raf and c Raf Raf 1 are the main effectors recruited by GTP bound Ras to activate the MEK MAP kinase pathway 1 Activation of c Raf is the best understood and involves phosphorylation at multiple activating sites including Ser338 Tyr341 Thr491 Ser494 Ser497 and Ser499 2 p21 activated protein kinase PAK has been shown to phosphorylate c Raf at Ser338 and the Src family phosphorylates Tyr341 to induce c Raf activity 3 4 Ser338 of c Raf corresponds to similar sites in A Raf Ser299 and B Raf Ser445 although this site is constitutively phosphorylated in B Raf 5 Inhibitory 14 3 3 binding sites on c Raf Ser259 and Ser621 can be phosphorylated by Akt and AMPK respectively 6 7 While A Raf B Raf and c Raf are similar in sequence and function differential regulation has been observed 8 Of particular interest B Raf contains three consensus Akt phosphorylation sites Ser364 Ser428 and Thr439 and lacks a site equivalent to Tyr341 of c Raf 8 9 Research studies have shown that the B Raf mutation V600E results in elevated kinase activity and is commonly found in malignant melanoma 10 Six residues of c Raf Ser29 Ser43 Ser289 Ser296 Ser301 and Ser642 become hyperphosphorylated in a manner consistent with c Raf inactivation The hyperphosphorylation of these six sites is dependent on downstream MEK signaling and renders c Raf unresponsive to subsequent activation events 11
    Catalog Number:
    4432
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues close to the linker domain of human A-Raf. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc a raf
    Endogenous <t>RanBPM</t> and <t>c-Raf</t> interaction in HeLa cells using PLA. Duolink II proximity ligation assay (PLA) was performed in: ( A ) control shRNA HeLa cells, without the addition of primary antibodies (negative control); ( B ) control shRNA HeLa cells, with Hsp90 and c-Raf primary antibodies (positive control); ( C ) control shRNA HeLa cells, using c-Raf and RanBPM primary antibodies; ( D ). HeLa RanBPM shRNA cells, with c-Raf and RanBPM primary antibodies (negative control); ( E ) control shRNA HeLa cells, using MAEA (Macrophage erythroblast attacher) and RanBPM primary antibodies (positive control). ( F ) Control shRNA HeLa cells, using c-Raf and MAEA primary antibodies. The DAPI filter was used to visualize the nuclei, while the Cyanine 3 (Cy3) filter was used to visualize the PLA dots representing protein–protein interactions. Representative images from one of three independent experiments are shown. Scale bars, 10 μm.
    A Raf B Raf and c Raf Raf 1 are the main effectors recruited by GTP bound Ras to activate the MEK MAP kinase pathway 1 Activation of c Raf is the best understood and involves phosphorylation at multiple activating sites including Ser338 Tyr341 Thr491 Ser494 Ser497 and Ser499 2 p21 activated protein kinase PAK has been shown to phosphorylate c Raf at Ser338 and the Src family phosphorylates Tyr341 to induce c Raf activity 3 4 Ser338 of c Raf corresponds to similar sites in A Raf Ser299 and B Raf Ser445 although this site is constitutively phosphorylated in B Raf 5 Inhibitory 14 3 3 binding sites on c Raf Ser259 and Ser621 can be phosphorylated by Akt and AMPK respectively 6 7 While A Raf B Raf and c Raf are similar in sequence and function differential regulation has been observed 8 Of particular interest B Raf contains three consensus Akt phosphorylation sites Ser364 Ser428 and Thr439 and lacks a site equivalent to Tyr341 of c Raf 8 9 Research studies have shown that the B Raf mutation V600E results in elevated kinase activity and is commonly found in malignant melanoma 10 Six residues of c Raf Ser29 Ser43 Ser289 Ser296 Ser301 and Ser642 become hyperphosphorylated in a manner consistent with c Raf inactivation The hyperphosphorylation of these six sites is dependent on downstream MEK signaling and renders c Raf unresponsive to subsequent activation events 11
    https://www.bioz.com/result/a raf/product/Cell Signaling Technology Inc
    Average 94 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    a raf - by Bioz Stars, 2020-07
    94/100 stars

    Images

    1) Product Images from "Regulation of c-Raf Stability through the CTLH Complex"

    Article Title: Regulation of c-Raf Stability through the CTLH Complex

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20040934

    Endogenous RanBPM and c-Raf interaction in HeLa cells using PLA. Duolink II proximity ligation assay (PLA) was performed in: ( A ) control shRNA HeLa cells, without the addition of primary antibodies (negative control); ( B ) control shRNA HeLa cells, with Hsp90 and c-Raf primary antibodies (positive control); ( C ) control shRNA HeLa cells, using c-Raf and RanBPM primary antibodies; ( D ). HeLa RanBPM shRNA cells, with c-Raf and RanBPM primary antibodies (negative control); ( E ) control shRNA HeLa cells, using MAEA (Macrophage erythroblast attacher) and RanBPM primary antibodies (positive control). ( F ) Control shRNA HeLa cells, using c-Raf and MAEA primary antibodies. The DAPI filter was used to visualize the nuclei, while the Cyanine 3 (Cy3) filter was used to visualize the PLA dots representing protein–protein interactions. Representative images from one of three independent experiments are shown. Scale bars, 10 μm.
    Figure Legend Snippet: Endogenous RanBPM and c-Raf interaction in HeLa cells using PLA. Duolink II proximity ligation assay (PLA) was performed in: ( A ) control shRNA HeLa cells, without the addition of primary antibodies (negative control); ( B ) control shRNA HeLa cells, with Hsp90 and c-Raf primary antibodies (positive control); ( C ) control shRNA HeLa cells, using c-Raf and RanBPM primary antibodies; ( D ). HeLa RanBPM shRNA cells, with c-Raf and RanBPM primary antibodies (negative control); ( E ) control shRNA HeLa cells, using MAEA (Macrophage erythroblast attacher) and RanBPM primary antibodies (positive control). ( F ) Control shRNA HeLa cells, using c-Raf and MAEA primary antibodies. The DAPI filter was used to visualize the nuclei, while the Cyanine 3 (Cy3) filter was used to visualize the PLA dots representing protein–protein interactions. Representative images from one of three independent experiments are shown. Scale bars, 10 μm.

    Techniques Used: Proximity Ligation Assay, shRNA, Negative Control, Positive Control

    Downregulation of RanBPM promotes tumour formation in NOD/SCID/gamma mice. ( A ) re-expression of RanBPM in HEK293 cells via Tet-off pBIG expression vector. HEK293 pool of cells stably expressing RanBPM shRNA were transfected with pBIG-RanBPM WT (si-mt) and maintained in media with 2 μg/mL Tetracycline and 250 μg/mL hygromycin to select for integration of the pBIG vector. Following selection, cells were either maintained (+) in Tetracyclin-containing media, or cultured in absence of Tetracyclin (−) for 24 h to allow induction of RanBPM. Tetracyclin removal leads to re-expression of RanBPM (lane 4); ( B ) ERK pathway activation in RanBPM shRNA cells. Samples shown in ( A ) were analyzed for ERK phosphorylation and A-Raf and B-Raf expression. The Western blot was analyzed with the indicated antibodies; ( C ) injections with HEK293 control and RanBPM shRNA pools were injected subcutaneously in the flank of 6–8 weeks old NOD/SCID/gamma. Tumour measurements were taken twice per week and a digital caliper was used to measure Length × Width × Depth of the tumour upon excision in order to calculate volume. n = 7, error bars represent SEM; ( D ) injections with HEK293 RanBPM shRNA pool of cells stably re-expressing RanBPM via pBIG Tet-off expression system (see C , lanes 3,4). Mice were fed chow containing Dox (purple line) or regular chow (green line). n = 6, error bars represent SEM. p
    Figure Legend Snippet: Downregulation of RanBPM promotes tumour formation in NOD/SCID/gamma mice. ( A ) re-expression of RanBPM in HEK293 cells via Tet-off pBIG expression vector. HEK293 pool of cells stably expressing RanBPM shRNA were transfected with pBIG-RanBPM WT (si-mt) and maintained in media with 2 μg/mL Tetracycline and 250 μg/mL hygromycin to select for integration of the pBIG vector. Following selection, cells were either maintained (+) in Tetracyclin-containing media, or cultured in absence of Tetracyclin (−) for 24 h to allow induction of RanBPM. Tetracyclin removal leads to re-expression of RanBPM (lane 4); ( B ) ERK pathway activation in RanBPM shRNA cells. Samples shown in ( A ) were analyzed for ERK phosphorylation and A-Raf and B-Raf expression. The Western blot was analyzed with the indicated antibodies; ( C ) injections with HEK293 control and RanBPM shRNA pools were injected subcutaneously in the flank of 6–8 weeks old NOD/SCID/gamma. Tumour measurements were taken twice per week and a digital caliper was used to measure Length × Width × Depth of the tumour upon excision in order to calculate volume. n = 7, error bars represent SEM; ( D ) injections with HEK293 RanBPM shRNA pool of cells stably re-expressing RanBPM via pBIG Tet-off expression system (see C , lanes 3,4). Mice were fed chow containing Dox (purple line) or regular chow (green line). n = 6, error bars represent SEM. p

    Techniques Used: Mouse Assay, Expressing, Plasmid Preparation, Stable Transfection, shRNA, Transfection, Selection, Cell Culture, Activation Assay, Western Blot, Injection

    RanBPM C-terminal CRA domain directly interacts with ΔN-c-Raf and is necessary for C-Raf regulation. ( A ) diagram of WT RanBPM, N2 domain and C1 domain cloned into the bacterial expression vector pGEX-4T-1; ( B ) Left, Western blot analysis of GST pull-down assays for N c-Raf performed using GST, GST-WT-RanBPM, GST-N2-domain and GST-C1-domain E. coli extracts. A representative image is shown. Right, pull down assays experiments were quantified by normalizing ΔN-c-Raf levels to pulled-down GST, GST-WT-RanBPM, GST-N2 or GST-C1 and statistical analyses were performed ( n = 6, error bar indicates SEM) with different letters indicating statistical difference ( p
    Figure Legend Snippet: RanBPM C-terminal CRA domain directly interacts with ΔN-c-Raf and is necessary for C-Raf regulation. ( A ) diagram of WT RanBPM, N2 domain and C1 domain cloned into the bacterial expression vector pGEX-4T-1; ( B ) Left, Western blot analysis of GST pull-down assays for N c-Raf performed using GST, GST-WT-RanBPM, GST-N2-domain and GST-C1-domain E. coli extracts. A representative image is shown. Right, pull down assays experiments were quantified by normalizing ΔN-c-Raf levels to pulled-down GST, GST-WT-RanBPM, GST-N2 or GST-C1 and statistical analyses were performed ( n = 6, error bar indicates SEM) with different letters indicating statistical difference ( p

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Western Blot

    Analysis of RanBPM domains that control C-Raf stability. ( A ) schematic representation of RanBPM mutants. ( B ) and ( C ) Western blot analyses of HeLa RanBPM shRNA cells transfected with pEBG-GST-ΔN-c-Raf and either pCMV-HA (empty vector), pCMV-HA-WT-RanBPM or pCMV-HA RanBPM mutant constructs as indicated. c-Raf and HA antibodies were used to detect the levels of ΔN-c-Raf and RanBPM, respectively. β-actin was used as a loading control. A representative Western blot is shown (top) and quantifications of c-Raf levels are shown (bottom graph) with error bars indicating SEM ( n = 5). Deletion of RanBPM C-terminal domain (ΔC1) impairs RanBPM interaction with GST-ΔN-c-Raf. ( D ) and ( E ) GST-Pull-down assays. HeLa RanBPM shRNA cells were transfected with pEBG-ΔN-c-Raf and either pCMV-HA (empty vector), pCMV-HA-WT-RanBPM or pCMV-HA RanBPM mutant constructs. ΔN-c-Raf was pulled down through binding to glutathione-sepharose beads and interaction of RanBPM WT and mutants with GST-ΔN-c-Raf assessed by Western blot with an HA antibody. Below: Quantifications were performed by normalizing RanBPM mutant levels to pulled-down GST or GST-ΔN-c-Raf and statistical analyses were performed ( n = 4–7, SEM shown). Different letters are statistically different ( p
    Figure Legend Snippet: Analysis of RanBPM domains that control C-Raf stability. ( A ) schematic representation of RanBPM mutants. ( B ) and ( C ) Western blot analyses of HeLa RanBPM shRNA cells transfected with pEBG-GST-ΔN-c-Raf and either pCMV-HA (empty vector), pCMV-HA-WT-RanBPM or pCMV-HA RanBPM mutant constructs as indicated. c-Raf and HA antibodies were used to detect the levels of ΔN-c-Raf and RanBPM, respectively. β-actin was used as a loading control. A representative Western blot is shown (top) and quantifications of c-Raf levels are shown (bottom graph) with error bars indicating SEM ( n = 5). Deletion of RanBPM C-terminal domain (ΔC1) impairs RanBPM interaction with GST-ΔN-c-Raf. ( D ) and ( E ) GST-Pull-down assays. HeLa RanBPM shRNA cells were transfected with pEBG-ΔN-c-Raf and either pCMV-HA (empty vector), pCMV-HA-WT-RanBPM or pCMV-HA RanBPM mutant constructs. ΔN-c-Raf was pulled down through binding to glutathione-sepharose beads and interaction of RanBPM WT and mutants with GST-ΔN-c-Raf assessed by Western blot with an HA antibody. Below: Quantifications were performed by normalizing RanBPM mutant levels to pulled-down GST or GST-ΔN-c-Raf and statistical analyses were performed ( n = 4–7, SEM shown). Different letters are statistically different ( p

    Techniques Used: Western Blot, shRNA, Transfection, Plasmid Preparation, Mutagenesis, Construct, Binding Assay

    C-Raf is regulated by the proteasome through the CTLH complex. Non-targeting shRNA control and shRNA RanBPM cells were treated with 10 μM MG132 or DMSO, as vehicle, for 24 h. RIPA buffered whole cell extracts of HeLa ( A ), and HCT116 ( B ) were analyzed by Western blot with RanBPM, c-Raf and β-actin antibodies to detect RanBPM, c-Raf and β-actin proteins, respectively. c-Raf protein levels were normalized to β-actin levels. Quantifications of relative c-Raf protein levels are shown with error bars indicating SD ( n = 4). * p
    Figure Legend Snippet: C-Raf is regulated by the proteasome through the CTLH complex. Non-targeting shRNA control and shRNA RanBPM cells were treated with 10 μM MG132 or DMSO, as vehicle, for 24 h. RIPA buffered whole cell extracts of HeLa ( A ), and HCT116 ( B ) were analyzed by Western blot with RanBPM, c-Raf and β-actin antibodies to detect RanBPM, c-Raf and β-actin proteins, respectively. c-Raf protein levels were normalized to β-actin levels. Quantifications of relative c-Raf protein levels are shown with error bars indicating SD ( n = 4). * p

    Techniques Used: shRNA, Western Blot

    Model of regulation of c-Raf by the RanBPM/CTLH complex. Three RanBPM regions, N-terminal (1–102), LisH/CTLH (360–460) and C-terminal CRA (615–729) are necessary to regulate c-Raf expression/stability, but only the CRA domain is able to directly interact with c-Raf in vitro. Our data suggest that RanBPM interacts with c-Raf through the CRA domain and recruits c-Raf to the CTLH complex to which RanBPM is associated through its LisH/CTLH domain. The CTLH complex promotes c-Raf ubiquitination and degradation. The role of RanBPM N-terminal domain is unclear, but it may be involved in RanBPM stability and folding and potentially stabilizes c-Raf interaction (dashed line). The minimum region of c-Raf defined so far as necessary for interaction with RanBPM is ∆N-c-Raf, which is comprised of conserved region CR3 and short flanking sequences. The position of the CR1, CR2 and CR3 conserved regions are shown. The location of the c-Raf catalytic domain (KD, kinase domain) is indicated. The thick double-head arrow indicates interaction. The dashed arrow indicates a regulation of c-Raf by the RanBPM N-terminal domain. Ubiquitination of c-Raf by the CTLH complex is indicated by the pink arrow. The bracket indicates that the LiSH/CTLH domain mediates interaction with CTLH complex members.
    Figure Legend Snippet: Model of regulation of c-Raf by the RanBPM/CTLH complex. Three RanBPM regions, N-terminal (1–102), LisH/CTLH (360–460) and C-terminal CRA (615–729) are necessary to regulate c-Raf expression/stability, but only the CRA domain is able to directly interact with c-Raf in vitro. Our data suggest that RanBPM interacts with c-Raf through the CRA domain and recruits c-Raf to the CTLH complex to which RanBPM is associated through its LisH/CTLH domain. The CTLH complex promotes c-Raf ubiquitination and degradation. The role of RanBPM N-terminal domain is unclear, but it may be involved in RanBPM stability and folding and potentially stabilizes c-Raf interaction (dashed line). The minimum region of c-Raf defined so far as necessary for interaction with RanBPM is ∆N-c-Raf, which is comprised of conserved region CR3 and short flanking sequences. The position of the CR1, CR2 and CR3 conserved regions are shown. The location of the c-Raf catalytic domain (KD, kinase domain) is indicated. The thick double-head arrow indicates interaction. The dashed arrow indicates a regulation of c-Raf by the RanBPM N-terminal domain. Ubiquitination of c-Raf by the CTLH complex is indicated by the pink arrow. The bracket indicates that the LiSH/CTLH domain mediates interaction with CTLH complex members.

    Techniques Used: Expressing, In Vitro

    CTLH complex members RMND5A and RanBPM regulate c-Raf levels and cell proliferation. ( A ) RMND5A regulates endogenous c-Raf protein levels. Whole cell extracts from wild-type (WT) HEK293 cells and CRISPR KO RMND5A HEK293 cells untransfected (−) or transfected with pCGN-HA-RMND5A (+) were prepared and analyzed by Western blot. The top shows a representative analysis using c-Raf, HA (hemagglutinin), RMND5A, and β-actin antibodies to detect endogenous c-Raf, exogenous HA-RMND5A, endogenous RMND5A, and β-actin, respectively. Below, relative endogenous c-Raf protein levels were quantified by normalizing c-Raf to β-actin, and comparing values to wild-type HEK293 when set to a value of 1. Quantifications are shown with error bars indicating SD. p
    Figure Legend Snippet: CTLH complex members RMND5A and RanBPM regulate c-Raf levels and cell proliferation. ( A ) RMND5A regulates endogenous c-Raf protein levels. Whole cell extracts from wild-type (WT) HEK293 cells and CRISPR KO RMND5A HEK293 cells untransfected (−) or transfected with pCGN-HA-RMND5A (+) were prepared and analyzed by Western blot. The top shows a representative analysis using c-Raf, HA (hemagglutinin), RMND5A, and β-actin antibodies to detect endogenous c-Raf, exogenous HA-RMND5A, endogenous RMND5A, and β-actin, respectively. Below, relative endogenous c-Raf protein levels were quantified by normalizing c-Raf to β-actin, and comparing values to wild-type HEK293 when set to a value of 1. Quantifications are shown with error bars indicating SD. p

    Techniques Used: CRISPR, Transfection, Western Blot

    2) Product Images from "Apoptosis of human melanoma cells induced by inhibition of B-RAFV600E involves preferential splicing of bimS"

    Article Title: Apoptosis of human melanoma cells induced by inhibition of B-RAFV600E involves preferential splicing of bimS

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2010.48

    Bim S has a critical role in PLX4720-induced apoptosis of mutant B-RAF melanoma cells. ( a ) Left panel: Mel-RMu and Mel-CV cells (B-RAF V600E ) were transfected with the control or Bim siRNA. After 24 h, cells were treated with PLX4720 for 16 h. Total RNA was isolated and subjected to real-time PCR analysis for Bim mRNA expression. The relative abundance of mRNA expression in cells transfected with the control siRNA before treatment was arbitrarily designated as 1. Right panel: Mel-RMu and Mel-CV cells were transfected with the control or Bim siRNA. After 24 h, cells were treated with PLX4720 for a further 72 h. Apoptosis was quantitated by the propidium iodide (PI) method. The data shown are the mean±S.E. of three individual experiments. ( b ) Mel-RMu cells were transfected with the control, Bim EL , or Bim S siRNA. After 24 h, cells were treated with PLX4720 (10 μ M) for 16 h. Total RNA was isolated and subjected to real-time PCR analysis for Bim EL (left panel) and Bim S (right panel) mRNA expression. The levels of the expression of individual species in cells transfected with the control siRNA without treatment were arbitrarily designated as 1. The data shown are the mean±S.E. of three individual experiments. ( c ) Whole cell lysates from Mel-RMu cells treated as in b were subjected to western blot analysis of Bim and GAPDH (as a loading control). The arrowhead points to nonspecific bands. The data shown are representative of three individual western blot analyses. ( d ) Mel-RMu cells with Bim EL or Bim S knocked down as in b were treated with PLX 4720 at 10 μ M for 72 h. Apoptosis was quantitated by the PI method. The data shown are the mean±S.E. of three individual experiments. ( e ) Left panel: Mel-RMu and Mel-CV cells were transfected with pCMV6-AC-GFP or pCMV6-AC-Bim S -GFP. After 24 h, whole cell lysates were subjected to western blot analysis of Bim S -GFP using an antibody against GFP. Western blot analysis of GAPDH was then performed as a loading control. Right panel: Mel-RMu and Mel-CV cells were transfected with pCMV6-AC-GFP or pCMV6-AC-Bim S -GFP. After 24 h, mitochondrial fractions were subjected to western blot analysis of Bim S -GFP using an antibody against GFP. Western blot analysis of COX IV was then performed as a loading control. ( f ) Left panel: Representative flow cytometric histograms of measurement of apoptosis using PE-conjugated Annexin-V in Mel-RMu and Mel-CV cells transfected with pCMV6-AC-GFP or pCMV6-AC-Bim S -GFP. PE-positive cells were quantitated in gated GFP-positive cell populations. The numbers represent percentages of positive cells. Right panel: Mel-RMu and Mel-CV cells were transfected with pCMV6-AC-GFP or pCMV6-AC-Bim S -GFP for indicated time periods. Apoptotic cells were quantitated with PE-conjugated Annexin-V in gated GFP-positive cell populations. The data shown are representative of two experiments. ( g ) Mitochondrial fractions from Mel-RMu and Mel-CV cells treated with PLX4720 (10 μ M) for indicated time periods were subjected to western blot analysis of Bim and COX IV (as a control). The data shown are representative of three individual western blot analyses
    Figure Legend Snippet: Bim S has a critical role in PLX4720-induced apoptosis of mutant B-RAF melanoma cells. ( a ) Left panel: Mel-RMu and Mel-CV cells (B-RAF V600E ) were transfected with the control or Bim siRNA. After 24 h, cells were treated with PLX4720 for 16 h. Total RNA was isolated and subjected to real-time PCR analysis for Bim mRNA expression. The relative abundance of mRNA expression in cells transfected with the control siRNA before treatment was arbitrarily designated as 1. Right panel: Mel-RMu and Mel-CV cells were transfected with the control or Bim siRNA. After 24 h, cells were treated with PLX4720 for a further 72 h. Apoptosis was quantitated by the propidium iodide (PI) method. The data shown are the mean±S.E. of three individual experiments. ( b ) Mel-RMu cells were transfected with the control, Bim EL , or Bim S siRNA. After 24 h, cells were treated with PLX4720 (10 μ M) for 16 h. Total RNA was isolated and subjected to real-time PCR analysis for Bim EL (left panel) and Bim S (right panel) mRNA expression. The levels of the expression of individual species in cells transfected with the control siRNA without treatment were arbitrarily designated as 1. The data shown are the mean±S.E. of three individual experiments. ( c ) Whole cell lysates from Mel-RMu cells treated as in b were subjected to western blot analysis of Bim and GAPDH (as a loading control). The arrowhead points to nonspecific bands. The data shown are representative of three individual western blot analyses. ( d ) Mel-RMu cells with Bim EL or Bim S knocked down as in b were treated with PLX 4720 at 10 μ M for 72 h. Apoptosis was quantitated by the PI method. The data shown are the mean±S.E. of three individual experiments. ( e ) Left panel: Mel-RMu and Mel-CV cells were transfected with pCMV6-AC-GFP or pCMV6-AC-Bim S -GFP. After 24 h, whole cell lysates were subjected to western blot analysis of Bim S -GFP using an antibody against GFP. Western blot analysis of GAPDH was then performed as a loading control. Right panel: Mel-RMu and Mel-CV cells were transfected with pCMV6-AC-GFP or pCMV6-AC-Bim S -GFP. After 24 h, mitochondrial fractions were subjected to western blot analysis of Bim S -GFP using an antibody against GFP. Western blot analysis of COX IV was then performed as a loading control. ( f ) Left panel: Representative flow cytometric histograms of measurement of apoptosis using PE-conjugated Annexin-V in Mel-RMu and Mel-CV cells transfected with pCMV6-AC-GFP or pCMV6-AC-Bim S -GFP. PE-positive cells were quantitated in gated GFP-positive cell populations. The numbers represent percentages of positive cells. Right panel: Mel-RMu and Mel-CV cells were transfected with pCMV6-AC-GFP or pCMV6-AC-Bim S -GFP for indicated time periods. Apoptotic cells were quantitated with PE-conjugated Annexin-V in gated GFP-positive cell populations. The data shown are representative of two experiments. ( g ) Mitochondrial fractions from Mel-RMu and Mel-CV cells treated with PLX4720 (10 μ M) for indicated time periods were subjected to western blot analysis of Bim and COX IV (as a control). The data shown are representative of three individual western blot analyses

    Techniques Used: Mutagenesis, Transfection, Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Flow Cytometry

    3) Product Images from "Raf-independent, PP2A-dependent MEK Activation in Response to ERK Silencing"

    Article Title: Raf-independent, PP2A-dependent MEK Activation in Response to ERK Silencing

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2009.05.082

    MEK activation after ERK1/2 silencing is independent of raf
    Figure Legend Snippet: MEK activation after ERK1/2 silencing is independent of raf

    Techniques Used: Activation Assay

    4) Product Images from "Endosomal Targeting of MEK2 Requires RAF, MEK Kinase Activity and Clathrin-Dependent Endocytosis"

    Article Title: Endosomal Targeting of MEK2 Requires RAF, MEK Kinase Activity and Clathrin-Dependent Endocytosis

    Journal: Traffic (Copenhagen, Denmark)

    doi: 10.1111/j.1600-0854.2008.00788.x

    Effect of A-RAF/RAF-1 siRNA on MEK2–GFP recruitment to endosomes
    Figure Legend Snippet: Effect of A-RAF/RAF-1 siRNA on MEK2–GFP recruitment to endosomes

    Techniques Used:

    5) Product Images from "Splicing factor hnRNP A2 activates the Ras-MAPK-ERK pathway by controlling A-Raf splicing in hepatocellular carcinoma development"

    Article Title: Splicing factor hnRNP A2 activates the Ras-MAPK-ERK pathway by controlling A-Raf splicing in hepatocellular carcinoma development

    Journal: RNA

    doi: 10.1261/rna.042259.113

    Sensitivity of HCC cells to MEK inhibitor–induced apoptosis correlates with hnRNP A1 and A2 levels. ( A ) Cells were lysed, and protein levels of hnRNP A2/B1, hnRNP A1/A1b, A-Raf FL and total and phosphorylated MEK1/2 and ERK1/2 were examined using
    Figure Legend Snippet: Sensitivity of HCC cells to MEK inhibitor–induced apoptosis correlates with hnRNP A1 and A2 levels. ( A ) Cells were lysed, and protein levels of hnRNP A2/B1, hnRNP A1/A1b, A-Raf FL and total and phosphorylated MEK1/2 and ERK1/2 were examined using

    Techniques Used:

    Related Articles

    Nucleic Acid Electrophoresis:

    Article Title: Regulation of c-Raf Stability through the CTLH Complex
    Article Snippet: .. Samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on either 8% or 10% acrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked in 5% non-fat dry milk in Tris Buffered Saline with Tween 20 (TBS-T) or in Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE, USA), and hybridized with the following antibodies: c-Raf (C-12, 1:500, Santa Cruz), HA (HA-7, 1:1000, Sigma–Aldrich, St. Louis, MO, USA), RMND5A (NBP1–92337, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (I-19, 1:2000, Santa Cruz), RanBPM (5M, 1:2000, Bioacademia, Osaka, Japan), GST (B-14, 1:500, Santa Cruz), A-Raf (1:500, Cell Signaling Technology, Danvers, MA, USA, #4432), B-Raf (1:1000, Cell Signaling Technology #9434), phospho-T202/Y204-ERK1/2 (1:2000, Cell Signaling #4370), ERK1/2 (1:1000, Cell Signaling #9102), phospho-S217/221-MEK1/2 (1:2000, Cell Signaling #9154), MEK1/2 (1:1000, Cell Signaling #9122). .. Following antibody incubations, blots were developed using either Western Lightning Enhanced Chemiluminescence (ECL) Substrate (Perkin Elmer Inc., Waltham, MA, USA) or Clarity Western ECL Substrate (Bio-Rad Laboratories Inc., Hercules, CA, USA).

    other:

    Article Title: Raf-independent, PP2A-dependent MEK Activation in Response to ERK Silencing
    Article Snippet: Antibodies used were as follows: ERK1/2, p-ERK1/2(thr202/tyr204), MEK1/2, p-MEK1/2(ser217/221), A-Raf, B-Raf, C-Raf, p-A-Raf(ser299), p-B-Raf(ser445), p-C-Raf(ser338) (Cell Signaling), p-p38(thr180/tyr182) and p-JNK(thr183/tyr185) (Santa Cruz) and α-tubulin (Sigma).

    Article Title: HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold
    Article Snippet: Specific proteins were detected using primary antibodies to the following: EGFR, the A isoform of RAF (A-RAF), RAF-1 (S338), the B isoform of RAF (B-RAF), ERK1/2, phospho-ERK1/2, MEK1/2, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Cell Signaling); Shoc2 (Proteintech); hemagglutinin (HA; SydLabs Inc.); tag red fluorescent protein (tRFP; Evrogen); glutathione S -transferase (GST), M-Ras, RAF-1, and phospho-MEK1/2 (Santa Cruz); HUWE1 (Bethyl Laboratories Inc.); ubiquitin (Covance); PP1c (Millipore); and cyclin D1 (kindly provided by Mark Evers).

    Article Title: Regulation of Erk1/2 activation by osteopontin in PC3 human prostate cancer cells
    Article Snippet: Reagents Monoclonal rabbit anti-phospho-p44/42MAPK (Erk1/2) (Thr202/Tyr204), anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-phospho-c-Raf (Ser338), anti-p44/42MAPK (Erk1/2), anti-B-Raf, polyclonal rabbit anti-phospho-p38MAPK (Thr180/Tyr182), anti-phospho-c-Raf (Ser259), anti-phospho-c-Raf (Ser289/296/301), anti-phospho-A-Raf (Ser299), anti-phospho-B-Raf (Ser445), anti-p38MAPK, anti-SAPK/JNK, anti-A-Raf, and anti-c-Raf were purchased from Cell Signaling Technology (Danvers, MA).

    Article Title: Endosomal Targeting of MEK2 Requires RAF, MEK Kinase Activity and Clathrin-Dependent Endocytosis
    Article Snippet: Antibodies to MEK1/2, MEK2, ERK1/2, pERK1/2, pMEK1/2 and A-RAF were purchased from Cell Signaling Technology.

    SDS Page:

    Article Title: Regulation of c-Raf Stability through the CTLH Complex
    Article Snippet: .. Samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on either 8% or 10% acrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked in 5% non-fat dry milk in Tris Buffered Saline with Tween 20 (TBS-T) or in Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE, USA), and hybridized with the following antibodies: c-Raf (C-12, 1:500, Santa Cruz), HA (HA-7, 1:1000, Sigma–Aldrich, St. Louis, MO, USA), RMND5A (NBP1–92337, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (I-19, 1:2000, Santa Cruz), RanBPM (5M, 1:2000, Bioacademia, Osaka, Japan), GST (B-14, 1:500, Santa Cruz), A-Raf (1:500, Cell Signaling Technology, Danvers, MA, USA, #4432), B-Raf (1:1000, Cell Signaling Technology #9434), phospho-T202/Y204-ERK1/2 (1:2000, Cell Signaling #4370), ERK1/2 (1:1000, Cell Signaling #9102), phospho-S217/221-MEK1/2 (1:2000, Cell Signaling #9154), MEK1/2 (1:1000, Cell Signaling #9122). .. Following antibody incubations, blots were developed using either Western Lightning Enhanced Chemiluminescence (ECL) Substrate (Perkin Elmer Inc., Waltham, MA, USA) or Clarity Western ECL Substrate (Bio-Rad Laboratories Inc., Hercules, CA, USA).

    Blocking Assay:

    Article Title: Regulation of c-Raf Stability through the CTLH Complex
    Article Snippet: .. Samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on either 8% or 10% acrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked in 5% non-fat dry milk in Tris Buffered Saline with Tween 20 (TBS-T) or in Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE, USA), and hybridized with the following antibodies: c-Raf (C-12, 1:500, Santa Cruz), HA (HA-7, 1:1000, Sigma–Aldrich, St. Louis, MO, USA), RMND5A (NBP1–92337, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (I-19, 1:2000, Santa Cruz), RanBPM (5M, 1:2000, Bioacademia, Osaka, Japan), GST (B-14, 1:500, Santa Cruz), A-Raf (1:500, Cell Signaling Technology, Danvers, MA, USA, #4432), B-Raf (1:1000, Cell Signaling Technology #9434), phospho-T202/Y204-ERK1/2 (1:2000, Cell Signaling #4370), ERK1/2 (1:1000, Cell Signaling #9102), phospho-S217/221-MEK1/2 (1:2000, Cell Signaling #9154), MEK1/2 (1:1000, Cell Signaling #9122). .. Following antibody incubations, blots were developed using either Western Lightning Enhanced Chemiluminescence (ECL) Substrate (Perkin Elmer Inc., Waltham, MA, USA) or Clarity Western ECL Substrate (Bio-Rad Laboratories Inc., Hercules, CA, USA).

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    Cell Signaling Technology Inc ser601 phosphorylated b raf
    Fig. 1. Activation of <t>B-Raf</t> by oncogenic Ras requires both Thr598 and <t>Ser601</t> phosphorylation. ( A ) Sequence alignment of Raf kinase activation loop (residues 593–622 for human B-Raf). Residues subjected to site-directed mutagenesis are indicated by arrows. An asterisk denotes the protein kinase C phosphorylation sites. ( B ) Oncogenic Ras-induced kinase activation of B-Raf and its mutants. Fifty nanograms of HA-tagged pcDNA3 (vector control, lane 1), HA-tagged B-Raf (lanes 2--4), B-RafT598A (lane 5 and 6), B-RafS601A (lanes 7 and 8), B-RafS613A (lanes 9 and 10), B-RafAA (lanes 11 and 12) or B-RafED (lanes 13 and 14) were transiently transfected in COS cells alone (open bars) or with 100 ng of oncogenic Ras (HRasV12, solid bars), as indicated at the top of the panel. The kinase was immunoprecipitated and its activity, reflected by phosphorylation of GST–Elk1 (pElk1), was measured using the coupled assay (see Materials and methods). The upper panel shows autoradiographs of pElk1 that are representative of five independent experiments. The intensity of pElk1 bands was quantified by PhosphorImager (lower panel); results were subtracted from the reading of lane 1 (background) and expressed as fold increase with respect to the cells transfected with wild-type B-Raf without stimulation (lane 2). Lane 4* denotes that GST–MEK was omitted in the assay as a control. Results are mean ± SD from three independent experiments. Western blot of the immunoprecipitated kinase is shown in the middle panel, indicating equivalent protein loadings. ( C ) Activation of B-RafED by oncogenic Ras. COS cells were transfected with vector (lane 1), 50 ng of wild-type B-Raf (lanes 2 and 3), 5 ng of B-RafED (lanes 4 and 5), 25 ng of B-RafED (lanes 6 and 7) or 50 ng of B-RafED (lanes 8 and 9) in the presence or absence of HRasV12, as indicated at the top of the panel. Results of kinase assay and western blotting of B-Raf using α-HA were representative of two separate experiments. ( D ) Carbachol-induced kinase activation of B-Raf and its mutants. Cells were co-transfected with the above DNA constructs and hM3. After 24 h, cells were starved in FBS-free medium for 5 h followed by stimulation with carbachol for 5 min. Kinase immunoprecipitation, kinase assay and quantitation of results are as described in (B).
    Ser601 Phosphorylated B Raf, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phosphorylated raf 1 antibodies
    Insulin activates <t>Raf-1</t> and ERK in transformed MIN6 cells. Panel A, ERK phosphorylation in MIN6 cells treated with low glucose (5 m m ) or high glucose (25 m m ) for 15 min with or without somatostatin (Soma, 1 μ m ; n = 3). Panel B, Insulin
    Phosphorylated Raf 1 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc raf 1
    <t>B-Raf/Raf-1</t> heterodimerization in QTRRE cells
    Raf 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fig. 1. Activation of B-Raf by oncogenic Ras requires both Thr598 and Ser601 phosphorylation. ( A ) Sequence alignment of Raf kinase activation loop (residues 593–622 for human B-Raf). Residues subjected to site-directed mutagenesis are indicated by arrows. An asterisk denotes the protein kinase C phosphorylation sites. ( B ) Oncogenic Ras-induced kinase activation of B-Raf and its mutants. Fifty nanograms of HA-tagged pcDNA3 (vector control, lane 1), HA-tagged B-Raf (lanes 2--4), B-RafT598A (lane 5 and 6), B-RafS601A (lanes 7 and 8), B-RafS613A (lanes 9 and 10), B-RafAA (lanes 11 and 12) or B-RafED (lanes 13 and 14) were transiently transfected in COS cells alone (open bars) or with 100 ng of oncogenic Ras (HRasV12, solid bars), as indicated at the top of the panel. The kinase was immunoprecipitated and its activity, reflected by phosphorylation of GST–Elk1 (pElk1), was measured using the coupled assay (see Materials and methods). The upper panel shows autoradiographs of pElk1 that are representative of five independent experiments. The intensity of pElk1 bands was quantified by PhosphorImager (lower panel); results were subtracted from the reading of lane 1 (background) and expressed as fold increase with respect to the cells transfected with wild-type B-Raf without stimulation (lane 2). Lane 4* denotes that GST–MEK was omitted in the assay as a control. Results are mean ± SD from three independent experiments. Western blot of the immunoprecipitated kinase is shown in the middle panel, indicating equivalent protein loadings. ( C ) Activation of B-RafED by oncogenic Ras. COS cells were transfected with vector (lane 1), 50 ng of wild-type B-Raf (lanes 2 and 3), 5 ng of B-RafED (lanes 4 and 5), 25 ng of B-RafED (lanes 6 and 7) or 50 ng of B-RafED (lanes 8 and 9) in the presence or absence of HRasV12, as indicated at the top of the panel. Results of kinase assay and western blotting of B-Raf using α-HA were representative of two separate experiments. ( D ) Carbachol-induced kinase activation of B-Raf and its mutants. Cells were co-transfected with the above DNA constructs and hM3. After 24 h, cells were starved in FBS-free medium for 5 h followed by stimulation with carbachol for 5 min. Kinase immunoprecipitation, kinase assay and quantitation of results are as described in (B).

    Journal: The EMBO Journal

    Article Title: Activation of B-Raf kinase requires phosphorylation of the conserved residues Thr598 and Ser601

    doi: 10.1093/emboj/19.20.5429

    Figure Lengend Snippet: Fig. 1. Activation of B-Raf by oncogenic Ras requires both Thr598 and Ser601 phosphorylation. ( A ) Sequence alignment of Raf kinase activation loop (residues 593–622 for human B-Raf). Residues subjected to site-directed mutagenesis are indicated by arrows. An asterisk denotes the protein kinase C phosphorylation sites. ( B ) Oncogenic Ras-induced kinase activation of B-Raf and its mutants. Fifty nanograms of HA-tagged pcDNA3 (vector control, lane 1), HA-tagged B-Raf (lanes 2--4), B-RafT598A (lane 5 and 6), B-RafS601A (lanes 7 and 8), B-RafS613A (lanes 9 and 10), B-RafAA (lanes 11 and 12) or B-RafED (lanes 13 and 14) were transiently transfected in COS cells alone (open bars) or with 100 ng of oncogenic Ras (HRasV12, solid bars), as indicated at the top of the panel. The kinase was immunoprecipitated and its activity, reflected by phosphorylation of GST–Elk1 (pElk1), was measured using the coupled assay (see Materials and methods). The upper panel shows autoradiographs of pElk1 that are representative of five independent experiments. The intensity of pElk1 bands was quantified by PhosphorImager (lower panel); results were subtracted from the reading of lane 1 (background) and expressed as fold increase with respect to the cells transfected with wild-type B-Raf without stimulation (lane 2). Lane 4* denotes that GST–MEK was omitted in the assay as a control. Results are mean ± SD from three independent experiments. Western blot of the immunoprecipitated kinase is shown in the middle panel, indicating equivalent protein loadings. ( C ) Activation of B-RafED by oncogenic Ras. COS cells were transfected with vector (lane 1), 50 ng of wild-type B-Raf (lanes 2 and 3), 5 ng of B-RafED (lanes 4 and 5), 25 ng of B-RafED (lanes 6 and 7) or 50 ng of B-RafED (lanes 8 and 9) in the presence or absence of HRasV12, as indicated at the top of the panel. Results of kinase assay and western blotting of B-Raf using α-HA were representative of two separate experiments. ( D ) Carbachol-induced kinase activation of B-Raf and its mutants. Cells were co-transfected with the above DNA constructs and hM3. After 24 h, cells were starved in FBS-free medium for 5 h followed by stimulation with carbachol for 5 min. Kinase immunoprecipitation, kinase assay and quantitation of results are as described in (B).

    Article Snippet: Antibodies specifically recognizing Thr598 or Ser601 phosphorylated B-Raf were prepared by immunizing rabbits with synthetic phosphopeptides (Cell Signaling Technologies, Boston, MA).

    Techniques: Activation Assay, Sequencing, Mutagenesis, Plasmid Preparation, Transfection, Immunoprecipitation, Activity Assay, Western Blot, Kinase Assay, Construct, Quantitation Assay

    Fig. 2. Tryptic phosphopeptide mapping of B-Raf. ( A ) Metabolic labeling and immunoprecipitation of B-Raf and B-RafAA. COS1 cells grown on a 60 mm plate were transfected with wild-type B-Raf (lane 1), B-Raf + HRasV12 (lane 2) or B-RafAA + HRasV12 (lane 3), labeled with [ 32 P]orthophosphate, and immunoprecipitated with anti-HA antibody. Immunocomplexes were separated by SDS–PAGE and visualized by antoradiography. Positions of size markers are indicated in kilodaltons (KD) on the right. An immunoblot of the above immunoprecipitation using anti-HA antibody is shown at the bottom. ( B , C and D ) Phosphopeptide mappings for wild-type B-Raf, B-Raf + HRasV12 and B-RafAA + HRasV12, respectively. The 32 P-labeled bands were excised from the membrane, digested with trypsin and analyzed by two-dimensional thin-layer electrophoresis. The directions for electrophoresis (E, from cathode to anode) and TLC (T) are indicated in the lower left corner of each panel. Arrows point to positions of phosphopeptides for references between different panels. Dashed circles indicate phosphopeptides present in (C) that are missing in (D). Open arrowheads indicate the Ras-induced phosphopeptide not affected in B-RafAA. ( E ) Phosphorylation of Thr598 and Ser601 determined by specific anti-phospho Thr598 and Ser601 antibodies. COS cells were transfected with pcDNA (vector, lane 1), wild-type B-Raf (lane 2), co-transfected with wild-type B-Raf and HRasV12 (lane3), B-RafAA (lane 4) or co-transfected B-RafAA and HRasV12 (lane 5). Immunoprecipitates of B-Raf and B-RafAA were isolated and resolved by SDS–PAGE, transferred to membrane, and blotted with anti-phospho Thr598 (top panel) or anti-phospho Ser601 antibody (middle panel). Protein loading was examined by blotting using HA antibody (bottom panel).

    Journal: The EMBO Journal

    Article Title: Activation of B-Raf kinase requires phosphorylation of the conserved residues Thr598 and Ser601

    doi: 10.1093/emboj/19.20.5429

    Figure Lengend Snippet: Fig. 2. Tryptic phosphopeptide mapping of B-Raf. ( A ) Metabolic labeling and immunoprecipitation of B-Raf and B-RafAA. COS1 cells grown on a 60 mm plate were transfected with wild-type B-Raf (lane 1), B-Raf + HRasV12 (lane 2) or B-RafAA + HRasV12 (lane 3), labeled with [ 32 P]orthophosphate, and immunoprecipitated with anti-HA antibody. Immunocomplexes were separated by SDS–PAGE and visualized by antoradiography. Positions of size markers are indicated in kilodaltons (KD) on the right. An immunoblot of the above immunoprecipitation using anti-HA antibody is shown at the bottom. ( B , C and D ) Phosphopeptide mappings for wild-type B-Raf, B-Raf + HRasV12 and B-RafAA + HRasV12, respectively. The 32 P-labeled bands were excised from the membrane, digested with trypsin and analyzed by two-dimensional thin-layer electrophoresis. The directions for electrophoresis (E, from cathode to anode) and TLC (T) are indicated in the lower left corner of each panel. Arrows point to positions of phosphopeptides for references between different panels. Dashed circles indicate phosphopeptides present in (C) that are missing in (D). Open arrowheads indicate the Ras-induced phosphopeptide not affected in B-RafAA. ( E ) Phosphorylation of Thr598 and Ser601 determined by specific anti-phospho Thr598 and Ser601 antibodies. COS cells were transfected with pcDNA (vector, lane 1), wild-type B-Raf (lane 2), co-transfected with wild-type B-Raf and HRasV12 (lane3), B-RafAA (lane 4) or co-transfected B-RafAA and HRasV12 (lane 5). Immunoprecipitates of B-Raf and B-RafAA were isolated and resolved by SDS–PAGE, transferred to membrane, and blotted with anti-phospho Thr598 (top panel) or anti-phospho Ser601 antibody (middle panel). Protein loading was examined by blotting using HA antibody (bottom panel).

    Article Snippet: Antibodies specifically recognizing Thr598 or Ser601 phosphorylated B-Raf were prepared by immunizing rabbits with synthetic phosphopeptides (Cell Signaling Technologies, Boston, MA).

    Techniques: Labeling, Immunoprecipitation, Transfection, SDS Page, Electrophoresis, Thin Layer Chromatography, Plasmid Preparation, Isolation

    Insulin activates Raf-1 and ERK in transformed MIN6 cells. Panel A, ERK phosphorylation in MIN6 cells treated with low glucose (5 m m ) or high glucose (25 m m ) for 15 min with or without somatostatin (Soma, 1 μ m ; n = 3). Panel B, Insulin

    Journal: Endocrinology

    Article Title: Acute Insulin Signaling in Pancreatic Beta-Cells Is Mediated by Multiple Raf-1 Dependent Pathways

    doi: 10.1210/en.2009-0678

    Figure Lengend Snippet: Insulin activates Raf-1 and ERK in transformed MIN6 cells. Panel A, ERK phosphorylation in MIN6 cells treated with low glucose (5 m m ) or high glucose (25 m m ) for 15 min with or without somatostatin (Soma, 1 μ m ; n = 3). Panel B, Insulin

    Article Snippet: Insulin receptor and phosphorylated Raf-1 antibodies were from Cell Signaling and phosphorylated insulin receptor, Raf-1, and insulin antibodies were from Stressgen (Ann Arbor, MI), BD Biosciences, and Linco Research (St. Charles, MO), respectively.

    Techniques: Transformation Assay

    Insulin promotes Raf-1 and ERK activation in mouse islets. A, Acute insulin signaling stimulation for 15 min in primary mouse islets with 0.2 and 200 n m insulin resulted in a loss of inhibitory phosphorylation of Raf-1 (pRaf-1) at serine 259. B, Mouse

    Journal: Endocrinology

    Article Title: Acute Insulin Signaling in Pancreatic Beta-Cells Is Mediated by Multiple Raf-1 Dependent Pathways

    doi: 10.1210/en.2009-0678

    Figure Lengend Snippet: Insulin promotes Raf-1 and ERK activation in mouse islets. A, Acute insulin signaling stimulation for 15 min in primary mouse islets with 0.2 and 200 n m insulin resulted in a loss of inhibitory phosphorylation of Raf-1 (pRaf-1) at serine 259. B, Mouse

    Article Snippet: Insulin receptor and phosphorylated Raf-1 antibodies were from Cell Signaling and phosphorylated insulin receptor, Raf-1, and insulin antibodies were from Stressgen (Ann Arbor, MI), BD Biosciences, and Linco Research (St. Charles, MO), respectively.

    Techniques: Activation Assay

    Insulin promotes Raf-1 translocation to the mitochondria. A, Insulin (0.2 n m ) promoted Raf-1 mitochondrial localization compared with serum free in mouse pancreatic β-cell (n = 3). Scale bars , 10 μm. B, A plot of Pearson correlation

    Journal: Endocrinology

    Article Title: Acute Insulin Signaling in Pancreatic Beta-Cells Is Mediated by Multiple Raf-1 Dependent Pathways

    doi: 10.1210/en.2009-0678

    Figure Lengend Snippet: Insulin promotes Raf-1 translocation to the mitochondria. A, Insulin (0.2 n m ) promoted Raf-1 mitochondrial localization compared with serum free in mouse pancreatic β-cell (n = 3). Scale bars , 10 μm. B, A plot of Pearson correlation

    Article Snippet: Insulin receptor and phosphorylated Raf-1 antibodies were from Cell Signaling and phosphorylated insulin receptor, Raf-1, and insulin antibodies were from Stressgen (Ann Arbor, MI), BD Biosciences, and Linco Research (St. Charles, MO), respectively.

    Techniques: Translocation Assay

    Endogenous Bcl-2 family members and Raf-1 form protein-protein interactions in pancreatic β-cell. A, Immunofluorescence imaging of endogenous Raf-1 and Bad in primary human and mouse β-cells. Pearson correlation r values between Raf-1

    Journal: Endocrinology

    Article Title: Acute Insulin Signaling in Pancreatic Beta-Cells Is Mediated by Multiple Raf-1 Dependent Pathways

    doi: 10.1210/en.2009-0678

    Figure Lengend Snippet: Endogenous Bcl-2 family members and Raf-1 form protein-protein interactions in pancreatic β-cell. A, Immunofluorescence imaging of endogenous Raf-1 and Bad in primary human and mouse β-cells. Pearson correlation r values between Raf-1

    Article Snippet: Insulin receptor and phosphorylated Raf-1 antibodies were from Cell Signaling and phosphorylated insulin receptor, Raf-1, and insulin antibodies were from Stressgen (Ann Arbor, MI), BD Biosciences, and Linco Research (St. Charles, MO), respectively.

    Techniques: Immunofluorescence, Imaging

    B-Raf/Raf-1 heterodimerization in QTRRE cells

    Journal: Molecular carcinogenesis

    Article Title: Transcriptional and Post-translational Modifications of B-Raf in Quinol-Thioether Induced Tuberous Sclerosis Renal Cell Carcinoma

    doi: 10.1002/mc.22366

    Figure Lengend Snippet: B-Raf/Raf-1 heterodimerization in QTRRE cells

    Article Snippet: Each IP and total cell lysate (TCL) from QTRRE cells were immunoblotted for B-Raf , Raf-1 , or 14-3-3 isoforms ( ) protein expression.

    Techniques:

    PAK1 regulated MEK1 activity independent of its kinase activity. A , HeLa, SW480, HT-29, NIH3T3, and IEC-6 cells were transfected with PAK1 WT or PAK1 K299R plasmid for 18 h. Cell lysates were immunoblotted with antibodies to p-B-RAF (Ser-445), p-C-RAF (Ser-338),

    Journal: The Journal of Biological Chemistry

    Article Title: p21-Activated Kinase 1 (PAK1) Can Promote ERK Activation in a Kinase-independent Manner *

    doi: 10.1074/jbc.M112.426023

    Figure Lengend Snippet: PAK1 regulated MEK1 activity independent of its kinase activity. A , HeLa, SW480, HT-29, NIH3T3, and IEC-6 cells were transfected with PAK1 WT or PAK1 K299R plasmid for 18 h. Cell lysates were immunoblotted with antibodies to p-B-RAF (Ser-445), p-C-RAF (Ser-338),

    Article Snippet: The following primary antibodies were used: PAK1, C-RAF, MEK1, p-B-RAF (Ser-445), p-C-RAF (Ser-338), p-MEK1/2 (Ser-217/Ser-221), p-ERK1/2 (Thr-202/Tyr-204) (all from Cell Signaling Technology), and tubulin (Abcam, Cambridge, MA).

    Techniques: Activity Assay, Transfection, Plasmid Preparation