phospho raf 1 Search Results


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  • 90
    Thermo Fisher anti phospho raf1
    Anti Phospho Raf1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rat monoclonal antibody against phospho ser338 raf 1
    Rat Monoclonal Antibody Against Phospho Ser338 Raf 1, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    raf 1  (Abcam)
    94
    Abcam raf 1
    Altered <t>RAF1</t> Phosphorylation in HD Models Is Rescued by RRAS Inhibition. (A) Ratio of phospho-S338 to total RAF1 is increased in ST Hdh Q111/Q111 cells due to a reduced level of total RAF1 (n = 3). (B) Enhanced phospho-S338/total RAF1 in transiently transfected HEK293T cells (n = 3). (C) The R6/2 mouse model of Huntington's disease has elevated ratios of phospho-S338 to total RAF1 in regions of the brain affected by the disease (n = 2). **p
    Raf 1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co phospho raf1
    Proposed actions of PP5 in cardiomyocytes. Under basal conditions, PP5 activity towards titin N2Bus and MAPK/ERK family member <t>Raf1</t> is low and the relatively high distensibility of N2Bus results in relatively low titin-based passive tension (left side). The strain-dependent mechanosensor connecting MAPKs to N2Bus via FHL-1 functions normally, as downstream signaling from Raf1 to ERK2 is enabled. When PP5 expression is increased (as in failing hearts) and PP5 becomes activated through interaction with Hsp90, Ca 2+ /S100 protein, arachidonic acid (aa), or long chain fatty acid-CoA esters (LCACE), the phosphatase translocates to the I-band mechanosensor at N2Bus (right side). Thus, N2Bus (previously phosphorylated by ERK2, PKA, PKG, or CaMKII) is dephosphorylated, which reduces its distensibility and increases titin-based passive tension; the mechanosensor is now less sensitive. Raf-1 is also dephosphorylated and signaling to ERK2 is disabled, such that the mechanosensor function is additionally compromised. The process is embedded in signaling pathways activated via G-protein coupled receptor (GPCR) and Ras, and it can be reversed when PP5 is deactivated. (Molecules that have a color code were studied here, those with no color/white background were inferred from the literature)
    Phospho Raf1, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho raf 1
    Effects of <t>Raf-1</t> kinase inhibition on PE-induced MLC 20 ( a ) and MYPT1 ( b ) phosphorylation in rat VSMCs. Left and right panels show concentration- and time-dependent effects of PE, respectively. Cultured cells were pretreated with vehicle or GW5074 (10 μ M ) for 30 min before stimulated with 10 μ M PE. Treated cells were processed as described in Methods. The phosphorylation levels of MLC 20 or MYPT1 were determined by immunoblotting. Top panels show representative blots of phospho-MLC 20 or phospho-MYPT1 and β-actin, bottom panels are the summary of densitometric results. Results were normalized against β-actin and expressed as folds change relative to control. Data represent means of 3–5 independent experiments. * p
    Phospho Raf 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phospho s621 raf1
    Effects of <t>Raf-1</t> kinase inhibition on PE-induced MLC 20 ( a ) and MYPT1 ( b ) phosphorylation in rat VSMCs. Left and right panels show concentration- and time-dependent effects of PE, respectively. Cultured cells were pretreated with vehicle or GW5074 (10 μ M ) for 30 min before stimulated with 10 μ M PE. Treated cells were processed as described in Methods. The phosphorylation levels of MLC 20 or MYPT1 were determined by immunoblotting. Top panels show representative blots of phospho-MLC 20 or phospho-MYPT1 and β-actin, bottom panels are the summary of densitometric results. Results were normalized against β-actin and expressed as folds change relative to control. Data represent means of 3–5 independent experiments. * p
    Phospho S621 Raf1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho raf1 s338
    Pak1 stimulates phosphorylation of Ser112 on BAD via <t>Raf-1.</t> (A) Effects of kinase inhibitors on BAD S112 phosphorylation. 293T cells were co-transfected with expression vectors encoding full length (FL) WT BAD and Pak1 (KD or T423E). 24 hr after transfection, cells were starved for 16 hr and treated with GW5074 (5 µM), PD098059 (20 µM), Rapamycin (5 µM) or H89 (5 µM) for 2.5 hr as indicated. Equal amounts of proteins were used for Western blot to assess BAD phosphorylation at S112 or S111. The cell lysates were also subjected to immunoblotting with anti-BAD, anti-Myc, anti-phospho-ERK and anti-ERK antibodies. (B) Effects of inhibitors on forskolin stimulated activation of BAD phosphorylation. 293T cells were transfected with full length (FL) WT BAD for 24 hr, starved for 16 hr and treated with vehicle (DMSO), GW5074 (5 µM), or H89 (5 µM) for 2.5 hr prior to forskolin (50 µM) treatment. Equal amounts of proteins were used for Western blot to assess BAD phosphorylation at S112. The cell lysates were also subjected to immunoblotting with anti-BAD.
    Phospho Raf1 S338, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti raf1 phospho s338 antibody
    Pak1 stimulates phosphorylation of Ser112 on BAD via <t>Raf-1.</t> (A) Effects of kinase inhibitors on BAD S112 phosphorylation. 293T cells were co-transfected with expression vectors encoding full length (FL) WT BAD and Pak1 (KD or T423E). 24 hr after transfection, cells were starved for 16 hr and treated with GW5074 (5 µM), PD098059 (20 µM), Rapamycin (5 µM) or H89 (5 µM) for 2.5 hr as indicated. Equal amounts of proteins were used for Western blot to assess BAD phosphorylation at S112 or S111. The cell lysates were also subjected to immunoblotting with anti-BAD, anti-Myc, anti-phospho-ERK and anti-ERK antibodies. (B) Effects of inhibitors on forskolin stimulated activation of BAD phosphorylation. 293T cells were transfected with full length (FL) WT BAD for 24 hr, starved for 16 hr and treated with vehicle (DMSO), GW5074 (5 µM), or H89 (5 µM) for 2.5 hr prior to forskolin (50 µM) treatment. Equal amounts of proteins were used for Western blot to assess BAD phosphorylation at S112. The cell lysates were also subjected to immunoblotting with anti-BAD.
    Anti Raf1 Phospho S338 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho raf 1 ser259
    Pak1 stimulates phosphorylation of Ser112 on BAD via <t>Raf-1.</t> (A) Effects of kinase inhibitors on BAD S112 phosphorylation. 293T cells were co-transfected with expression vectors encoding full length (FL) WT BAD and Pak1 (KD or T423E). 24 hr after transfection, cells were starved for 16 hr and treated with GW5074 (5 µM), PD098059 (20 µM), Rapamycin (5 µM) or H89 (5 µM) for 2.5 hr as indicated. Equal amounts of proteins were used for Western blot to assess BAD phosphorylation at S112 or S111. The cell lysates were also subjected to immunoblotting with anti-BAD, anti-Myc, anti-phospho-ERK and anti-ERK antibodies. (B) Effects of inhibitors on forskolin stimulated activation of BAD phosphorylation. 293T cells were transfected with full length (FL) WT BAD for 24 hr, starved for 16 hr and treated with vehicle (DMSO), GW5074 (5 µM), or H89 (5 µM) for 2.5 hr prior to forskolin (50 µM) treatment. Equal amounts of proteins were used for Western blot to assess BAD phosphorylation at S112. The cell lysates were also subjected to immunoblotting with anti-BAD.
    Phospho Raf 1 Ser259, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology phospho raf 1
    Effect of PP5 inhibition on IL-2/IL-4 phosphorylation of <t>Raf-1</t> (Ser338) and Erk1 and Erk 2 kinases (Thr202 and Tyr204, respectively). Whole cell lysates from eosinophils were cultured in the presence of IL-2/IL-4 and control or PP5 siRNA for 15 min and 36 h were analyzed for phosphorylation of Raf-1 and Erk1 and Erk2 kinases.
    Phospho Raf 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti phospho raf 1
    Knockdown (KD) of Plk1 attenuates the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, but not <t>Raf-1</t> phosphorylation. a Representative immunoblots illustrating the effects of Plk1shRNA on Plk1 expression. Blots of HASM cells infected with lentiviruses encoding control shRNA or Plk1 shRNA were probed with antibodies against Plk1 and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells producing Plk1 shRNA were normalized to ratios obtained from cells producing control shRNA. Values are mean ± SE ( n = 4). *Significantly lower Plk1/GAPDH ratios in cells producing Plk1 shRNA compared with cells producing control shRNA ( P
    Anti Phospho Raf 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam phospho raf1
    Knockdown (KD) of Plk1 attenuates the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, but not <t>Raf-1</t> phosphorylation. a Representative immunoblots illustrating the effects of Plk1shRNA on Plk1 expression. Blots of HASM cells infected with lentiviruses encoding control shRNA or Plk1 shRNA were probed with antibodies against Plk1 and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells producing Plk1 shRNA were normalized to ratios obtained from cells producing control shRNA. Values are mean ± SE ( n = 4). *Significantly lower Plk1/GAPDH ratios in cells producing Plk1 shRNA compared with cells producing control shRNA ( P
    Phospho Raf1, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc anti phospho ser338 raf 1
    Knockdown (KD) of Plk1 attenuates the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, but not <t>Raf-1</t> phosphorylation. a Representative immunoblots illustrating the effects of Plk1shRNA on Plk1 expression. Blots of HASM cells infected with lentiviruses encoding control shRNA or Plk1 shRNA were probed with antibodies against Plk1 and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells producing Plk1 shRNA were normalized to ratios obtained from cells producing control shRNA. Values are mean ± SE ( n = 4). *Significantly lower Plk1/GAPDH ratios in cells producing Plk1 shRNA compared with cells producing control shRNA ( P
    Anti Phospho Ser338 Raf 1, supplied by Upstate Biotechnology 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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    Millipore anti phospho raf 1
    Deletion of CEK1 triggers Syk, ERK, and <t>Raf-1</t> phosphorylation eliciting IκB degradation on hDCs and induces dectin-1-dependent AP-1 activation on transfected cells. (A) Western blot of cell lysates from hDCs cocultured with live C. albicans strains
    Anti Phospho Raf 1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore phospho c raf 1 y341
    Deletion of CEK1 triggers Syk, ERK, and <t>Raf-1</t> phosphorylation eliciting IκB degradation on hDCs and induces dectin-1-dependent AP-1 activation on transfected cells. (A) Western blot of cell lysates from hDCs cocultured with live C. albicans strains
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    Millipore phospho raf 1
    Deletion of CEK1 triggers Syk, ERK, and <t>Raf-1</t> phosphorylation eliciting IκB degradation on hDCs and induces dectin-1-dependent AP-1 activation on transfected cells. (A) Western blot of cell lysates from hDCs cocultured with live C. albicans strains
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    Santa Cruz Biotechnology phospho raf 1 ser338
    Deletion of CEK1 triggers Syk, ERK, and <t>Raf-1</t> phosphorylation eliciting IκB degradation on hDCs and induces dectin-1-dependent AP-1 activation on transfected cells. (A) Western blot of cell lysates from hDCs cocultured with live C. albicans strains
    Phospho Raf 1 Ser338, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc anti phospho raf 1 mab
    Effect of S. enterica serovar Typhimurium ( S.ty ) porins on <t>Raf-1</t> (A) and MEK1/2 (B) activation after different stimulation times. Proteins from cell lysates (3 × 10 6 cells/ml) were analyzed by SDS-15% PAGE and immunoblotted with a Raf-1- or MEK1/2-phosphospecific antibody. Shifts in band mobility on SDS-PAGE due to phosphorylation were obtained with anti-Raf-1, anti-MEK1, and anti-MEK2. Fold increases in Raf-1 and MEK1/2 activation, compared to unstimulated cells (control), are shown below each lane for each blot. BSA represents a control of an unspecific stimulus.
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    Millipore phospho raf1
    Activation of MEK1 and <t>Raf1</t> by the active form of ARF1. A , activation of MEK1 by ARF1Q71L. pMEK1 , phosphorylated MEK1; Ctrl , control. B , activation of Raf1 by ARF1Q71L. pRaf1 , phosphorylated Raf1. C , expression of the dominant-negative mutant Raf1K375M,
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    Millipore anti phospho raf1
    Effect of brucine on protein expressions of KDR, PKCα, PLCγ-1 and <t>Raf1</t> in LoVo cells. Cells were treated with VEGF (50 ng/ml) for 30 min. before extracting proteins with RIPA lysis buffer. Results were quantified by densitometry analysis of the bands form and then normalization to GAPDH protein. ( A ) Effect of brucine on protein expressions. ( B ) Quantitation data of ( A ). Quantitation data showed brucine decreased the phosphorylation levels of KDR, PKCα, PLCγ-1 and Raf1 in a dose-dependent manner compared to the untreated control. Data represent the means ± SD ( n = 3) with * P
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    Image Search Results


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

    Journal: PLoS Genetics

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

    doi: 10.1371/journal.pgen.1003042

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

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

    Techniques: Inhibition, Transfection

    Proposed actions of PP5 in cardiomyocytes. Under basal conditions, PP5 activity towards titin N2Bus and MAPK/ERK family member Raf1 is low and the relatively high distensibility of N2Bus results in relatively low titin-based passive tension (left side). The strain-dependent mechanosensor connecting MAPKs to N2Bus via FHL-1 functions normally, as downstream signaling from Raf1 to ERK2 is enabled. When PP5 expression is increased (as in failing hearts) and PP5 becomes activated through interaction with Hsp90, Ca 2+ /S100 protein, arachidonic acid (aa), or long chain fatty acid-CoA esters (LCACE), the phosphatase translocates to the I-band mechanosensor at N2Bus (right side). Thus, N2Bus (previously phosphorylated by ERK2, PKA, PKG, or CaMKII) is dephosphorylated, which reduces its distensibility and increases titin-based passive tension; the mechanosensor is now less sensitive. Raf-1 is also dephosphorylated and signaling to ERK2 is disabled, such that the mechanosensor function is additionally compromised. The process is embedded in signaling pathways activated via G-protein coupled receptor (GPCR) and Ras, and it can be reversed when PP5 is deactivated. (Molecules that have a color code were studied here, those with no color/white background were inferred from the literature)

    Journal: Nature Communications

    Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

    doi: 10.1038/s41467-017-02483-3

    Figure Lengend Snippet: Proposed actions of PP5 in cardiomyocytes. Under basal conditions, PP5 activity towards titin N2Bus and MAPK/ERK family member Raf1 is low and the relatively high distensibility of N2Bus results in relatively low titin-based passive tension (left side). The strain-dependent mechanosensor connecting MAPKs to N2Bus via FHL-1 functions normally, as downstream signaling from Raf1 to ERK2 is enabled. When PP5 expression is increased (as in failing hearts) and PP5 becomes activated through interaction with Hsp90, Ca 2+ /S100 protein, arachidonic acid (aa), or long chain fatty acid-CoA esters (LCACE), the phosphatase translocates to the I-band mechanosensor at N2Bus (right side). Thus, N2Bus (previously phosphorylated by ERK2, PKA, PKG, or CaMKII) is dephosphorylated, which reduces its distensibility and increases titin-based passive tension; the mechanosensor is now less sensitive. Raf-1 is also dephosphorylated and signaling to ERK2 is disabled, such that the mechanosensor function is additionally compromised. The process is embedded in signaling pathways activated via G-protein coupled receptor (GPCR) and Ras, and it can be reversed when PP5 is deactivated. (Molecules that have a color code were studied here, those with no color/white background were inferred from the literature)

    Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr202 /Tyr204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

    Techniques: Activity Assay, Expressing

    PP5 and phospho-titin in PP5-overexpressing TG mouse hearts a Expression level of PP5 (left) and phospho-Raf1 S338 (right) in PP5 TG vs. WT mouse hearts by western blot. mean ± s.e.m., n = 4 hearts/group (age 5–6 months); duplicate analysis/group. b Sarcomeric localization of PP5 in PP5 WT and TG hearts by immunogold electron microscopy. Bars, 500 nm. Bar graph shows average number of gold particles counted in 50-µm 2 -sized regions-of-interest (ROI), either on the sarcomeric I-band or elsewhere in the cardiomyocyte (‘Not on I-band’). Data are mean ± s.e.m., n = 5 ROIs from 2 hearts/group. c PP5 localization in cardiomyocytes from PP5 TG and WT mouse hearts by indirect immunofluorescence. PP5 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK (titin) antibody (secondary antibody: FITC-conjugated IgG). Bars, 2 µm (main) and 1 µm (insets). d PP5 overexpression specifically decreases titin phosphorylation at N2Bus in PP5 TG vs. WT hearts. Total titin phosphorylation measured by ProQ Diamond/Sypro Ruby staining (upper left), site-specific titin phosphorylation detected by western blot using antibodies to P-S3991, P-S4043, P-S4080 (all N2Bus; right panels), P-S2080 (titin Z/I junction), and P-S12742 (PEVK region). Phospho-titin signals were normalized to total titin signals detected by WB using a panel of sequence-specific antibodies (Pan). Means were indexed to those of control (WT) groups. Data are mean ± s.e.m., n = 4 hearts/group, samples analyzed in triplicate. e Localization of phospho-N2Bus P-S3991 in cardiomyocytes from PP5 TG and WT hearts by indirect immunofluorescence. Anti-N2Bus P-S3991 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK antibody (secondary antibody: FITC-conjugated IgG). Bars, 2 µm (main) and 1 µm (insets). f Sarcomeric localization of phospho-N2Bus P-S3991 in PP5 WT and TG hearts by immunogold electron microscopy. Bars, 500 nm (main) and 100 nm (insets). Bar graph shows average number of gold particles counted in 50-µm 2 -sized regions-of-interest (ROI), either on the sarcomeric I-band or elsewhere in the cardiomyocyte (‘Not on I-band’). Data are mean ± s.e.m., n = 5 ROIs from 2 hearts/group. In a and d , bar graphs show relative signal changes indexed to the respective controls. In a , b , d , and f , * p

    Journal: Nature Communications

    Article Title: Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes

    doi: 10.1038/s41467-017-02483-3

    Figure Lengend Snippet: PP5 and phospho-titin in PP5-overexpressing TG mouse hearts a Expression level of PP5 (left) and phospho-Raf1 S338 (right) in PP5 TG vs. WT mouse hearts by western blot. mean ± s.e.m., n = 4 hearts/group (age 5–6 months); duplicate analysis/group. b Sarcomeric localization of PP5 in PP5 WT and TG hearts by immunogold electron microscopy. Bars, 500 nm. Bar graph shows average number of gold particles counted in 50-µm 2 -sized regions-of-interest (ROI), either on the sarcomeric I-band or elsewhere in the cardiomyocyte (‘Not on I-band’). Data are mean ± s.e.m., n = 5 ROIs from 2 hearts/group. c PP5 localization in cardiomyocytes from PP5 TG and WT mouse hearts by indirect immunofluorescence. PP5 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK (titin) antibody (secondary antibody: FITC-conjugated IgG). Bars, 2 µm (main) and 1 µm (insets). d PP5 overexpression specifically decreases titin phosphorylation at N2Bus in PP5 TG vs. WT hearts. Total titin phosphorylation measured by ProQ Diamond/Sypro Ruby staining (upper left), site-specific titin phosphorylation detected by western blot using antibodies to P-S3991, P-S4043, P-S4080 (all N2Bus; right panels), P-S2080 (titin Z/I junction), and P-S12742 (PEVK region). Phospho-titin signals were normalized to total titin signals detected by WB using a panel of sequence-specific antibodies (Pan). Means were indexed to those of control (WT) groups. Data are mean ± s.e.m., n = 4 hearts/group, samples analyzed in triplicate. e Localization of phospho-N2Bus P-S3991 in cardiomyocytes from PP5 TG and WT hearts by indirect immunofluorescence. Anti-N2Bus P-S3991 antibody (secondary antibody: Cy3-conjugated IgG), counterstained with anti-PEVK antibody (secondary antibody: FITC-conjugated IgG). Bars, 2 µm (main) and 1 µm (insets). f Sarcomeric localization of phospho-N2Bus P-S3991 in PP5 WT and TG hearts by immunogold electron microscopy. Bars, 500 nm (main) and 100 nm (insets). Bar graph shows average number of gold particles counted in 50-µm 2 -sized regions-of-interest (ROI), either on the sarcomeric I-band or elsewhere in the cardiomyocyte (‘Not on I-band’). Data are mean ± s.e.m., n = 5 ROIs from 2 hearts/group. In a and d , bar graphs show relative signal changes indexed to the respective controls. In a , b , d , and f , * p

    Article Snippet: Antibodies to the following proteins were used: PP5 (target, N-terminal of human PP5; Cell Signaling, #2289; polyclonal, rabbit; 1:2000), PP5c (target, rat PP5 amino acids 36–238; 3/PP5; BD Biosciences, 611021; monoclonal, mouse; 1:2000), GAPDH (target, full-length protein corresponding to human GAPDH; Abcam, #ab9484; monoclonal, mouse; 1:2000), PP1α (target, peptide corresponding to the N-terminal sequence of human PP1α; Cell Signaling, #2582; polyclonal, rabbit; 1:1000), PP2a (α + β isoform; target, peptide corresponding to amino acids at the C-terminus of human PP2A catalytic subunit; Cell Signaling, #2038; polyclonal, rabbit; 1:1000), phospho-Raf1 (Ser338; target, phosphopeptide corresponding to residues 333–345 of human Raf-1; Merck, 05–538; monoclonal, mouse; 1:1000), Hsp90 (target, peptide surrounding Asn300 of human Hsp90; C45G5; Cell Signaling, #4877S; monoclonal, rabbit; 1:1000), ERK1/2 (target, peptide corresponding to a sequence in the C-terminus of rat p44 MAP kinase; Cell Signaling, #9102; polyclonal, rabbit; 1:1000) and phospho-ERK1/2 (target, phosphopeptide corresponding to residues surrounding Thr202 /Tyr204 of ERK/MAPK; Biaffin, #AB-pERK-100; polyclonal, rabbit; 1:1000).

    Techniques: Expressing, Western Blot, Electron Microscopy, Immunofluorescence, Over Expression, Staining, Sequencing

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

    Journal: Journal of Vascular Research

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

    doi: 10.1159/000277726

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

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

    Techniques: Inhibition, Concentration Assay, Cell Culture

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

    Journal: Journal of Vascular Research

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

    doi: 10.1159/000277726

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

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

    Techniques:

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

    Journal: Journal of Vascular Research

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

    doi: 10.1159/000277726

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

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

    Techniques: Inhibition, Concentration Assay

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

    Journal: Journal of Vascular Research

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

    doi: 10.1159/000277726

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

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

    Techniques: Concentration Assay, Cell Culture

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

    Journal: Journal of Vascular Research

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

    doi: 10.1159/000277726

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

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

    Techniques: Inhibition, Cell Culture

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

    Journal: Journal of Vascular Research

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

    doi: 10.1159/000277726

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

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

    Techniques: Inhibition, Cell Culture

    Erbin disrupts the interaction between KSR1 and RAF1. (A) 293T cells transfected with Flag-RAF1 and CFP-KSR1 in the presence or absence of Myc-Erbin co-expression were lysed and immunoprecipitated with Flag-affinity beads. The presence of KSR1 and Erbin in the immunoprecipitates were detected by Western blot. The arrow indicates the endogenous KSR1 co-immunoprecipitated by Flag-RAF1. (B) The relative amount of KSR1 co-immunoprecipitated with RAF1 in the presence or absence of Myc-Erbin co-expression was quantified and expressed graphically (n=3, * p

    Journal: Cancer research

    Article Title: Erbin suppresses KSR1-mediated RAS/RAF signaling and tumorigenesis in colorectal cancer

    doi: 10.1158/0008-5472.CAN-17-3629

    Figure Lengend Snippet: Erbin disrupts the interaction between KSR1 and RAF1. (A) 293T cells transfected with Flag-RAF1 and CFP-KSR1 in the presence or absence of Myc-Erbin co-expression were lysed and immunoprecipitated with Flag-affinity beads. The presence of KSR1 and Erbin in the immunoprecipitates were detected by Western blot. The arrow indicates the endogenous KSR1 co-immunoprecipitated by Flag-RAF1. (B) The relative amount of KSR1 co-immunoprecipitated with RAF1 in the presence or absence of Myc-Erbin co-expression was quantified and expressed graphically (n=3, * p

    Article Snippet: The phospho-AKT (p-AKT for Ser473), pan-Akt, phospho-RAF1 (p-RAF1 for Ser338), total RAF1, phospho-MEK1/2 (p-MEK for Ser217/221), total MEK1/2, phospho-ERK1/2 (p-ERK for Thr202/Tyr204), total ERK1/2, and E-cadherin antibodies were from Cell Signaling.

    Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot

    Pak1 stimulates phosphorylation of Ser112 on BAD via Raf-1. (A) Effects of kinase inhibitors on BAD S112 phosphorylation. 293T cells were co-transfected with expression vectors encoding full length (FL) WT BAD and Pak1 (KD or T423E). 24 hr after transfection, cells were starved for 16 hr and treated with GW5074 (5 µM), PD098059 (20 µM), Rapamycin (5 µM) or H89 (5 µM) for 2.5 hr as indicated. Equal amounts of proteins were used for Western blot to assess BAD phosphorylation at S112 or S111. The cell lysates were also subjected to immunoblotting with anti-BAD, anti-Myc, anti-phospho-ERK and anti-ERK antibodies. (B) Effects of inhibitors on forskolin stimulated activation of BAD phosphorylation. 293T cells were transfected with full length (FL) WT BAD for 24 hr, starved for 16 hr and treated with vehicle (DMSO), GW5074 (5 µM), or H89 (5 µM) for 2.5 hr prior to forskolin (50 µM) treatment. Equal amounts of proteins were used for Western blot to assess BAD phosphorylation at S112. The cell lysates were also subjected to immunoblotting with anti-BAD.

    Journal: PLoS ONE

    Article Title: p21-Activated Kinase 1 (Pak1) Phosphorylates BAD Directly at Serine 111 In Vitro and Indirectly through Raf-1 at Serine 112

    doi: 10.1371/journal.pone.0027637

    Figure Lengend Snippet: Pak1 stimulates phosphorylation of Ser112 on BAD via Raf-1. (A) Effects of kinase inhibitors on BAD S112 phosphorylation. 293T cells were co-transfected with expression vectors encoding full length (FL) WT BAD and Pak1 (KD or T423E). 24 hr after transfection, cells were starved for 16 hr and treated with GW5074 (5 µM), PD098059 (20 µM), Rapamycin (5 µM) or H89 (5 µM) for 2.5 hr as indicated. Equal amounts of proteins were used for Western blot to assess BAD phosphorylation at S112 or S111. The cell lysates were also subjected to immunoblotting with anti-BAD, anti-Myc, anti-phospho-ERK and anti-ERK antibodies. (B) Effects of inhibitors on forskolin stimulated activation of BAD phosphorylation. 293T cells were transfected with full length (FL) WT BAD for 24 hr, starved for 16 hr and treated with vehicle (DMSO), GW5074 (5 µM), or H89 (5 µM) for 2.5 hr prior to forskolin (50 µM) treatment. Equal amounts of proteins were used for Western blot to assess BAD phosphorylation at S112. The cell lysates were also subjected to immunoblotting with anti-BAD.

    Article Snippet: Rabbit polyclonal antibodies against BAD, phospho-Ser112, phospho-Ser136, ERK, phospho-ERK, phospho-c-Raf (Raf-1) (Ser338) were from Cell Signaling Technology (Beverly, MA).

    Techniques: Transfection, Expressing, Western Blot, Activation Assay

    Pak1 inhibition reduced BAD S111 and S112 phosphorylation. (A) BAD S111 phosphorylation was observed in a lung cancer cell line H358 and the MPNST cell lines 90-8 and STS26. (B) Effects of protein kinase inhibitors on BAD S111 and S112 phosphorylation. The MPNST cells, ST88-14, were starved and treated with vehicle (DMSO), IPA3 (20 µM), GW5074 (5 µM), or H89 (5 µM) for 15 hr. Equal amounts of proteins were used for Western blot to assess BAD phosphorylation at S111and S112. The cell lysates were also subjected to immunoblotting with anti-BAD. As a control, Raf-1 phosphorylation at S338 was examined. (C) Model showing that Pak1 phosphorylates BAD at S111 directly, but most of its protective effects are mediated through Raf-1 and phosphorylation of S112. S111 also appears to facilitate phosphorylation at S112 because mutating it reduces phosphorylation by Pak1, but not phosphorylation by PKA. S136 is the most important phosphorylation site for the BAD/14-3-3 complex formation as previously observed. The enhanced Bcl-2 binding by the triple mutant is likely the result of the release of BAD from 14-3-3 sequestering in addition to the enhanced Bcl-2 association.

    Journal: PLoS ONE

    Article Title: p21-Activated Kinase 1 (Pak1) Phosphorylates BAD Directly at Serine 111 In Vitro and Indirectly through Raf-1 at Serine 112

    doi: 10.1371/journal.pone.0027637

    Figure Lengend Snippet: Pak1 inhibition reduced BAD S111 and S112 phosphorylation. (A) BAD S111 phosphorylation was observed in a lung cancer cell line H358 and the MPNST cell lines 90-8 and STS26. (B) Effects of protein kinase inhibitors on BAD S111 and S112 phosphorylation. The MPNST cells, ST88-14, were starved and treated with vehicle (DMSO), IPA3 (20 µM), GW5074 (5 µM), or H89 (5 µM) for 15 hr. Equal amounts of proteins were used for Western blot to assess BAD phosphorylation at S111and S112. The cell lysates were also subjected to immunoblotting with anti-BAD. As a control, Raf-1 phosphorylation at S338 was examined. (C) Model showing that Pak1 phosphorylates BAD at S111 directly, but most of its protective effects are mediated through Raf-1 and phosphorylation of S112. S111 also appears to facilitate phosphorylation at S112 because mutating it reduces phosphorylation by Pak1, but not phosphorylation by PKA. S136 is the most important phosphorylation site for the BAD/14-3-3 complex formation as previously observed. The enhanced Bcl-2 binding by the triple mutant is likely the result of the release of BAD from 14-3-3 sequestering in addition to the enhanced Bcl-2 association.

    Article Snippet: Rabbit polyclonal antibodies against BAD, phospho-Ser112, phospho-Ser136, ERK, phospho-ERK, phospho-c-Raf (Raf-1) (Ser338) were from Cell Signaling Technology (Beverly, MA).

    Techniques: Inhibition, Western Blot, Binding Assay, Mutagenesis

    Effect of PP5 inhibition on IL-2/IL-4 phosphorylation of Raf-1 (Ser338) and Erk1 and Erk 2 kinases (Thr202 and Tyr204, respectively). Whole cell lysates from eosinophils were cultured in the presence of IL-2/IL-4 and control or PP5 siRNA for 15 min and 36 h were analyzed for phosphorylation of Raf-1 and Erk1 and Erk2 kinases.

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

    Article Title: Cytokine-Induced Glucocorticoid Resistance from Eosinophil Activation: Protein Phosphatase 5 Modulation of Glucocorticoid Receptor Phosphorylation and Signaling

    doi: 10.4049/jimmunol.1601029

    Figure Lengend Snippet: Effect of PP5 inhibition on IL-2/IL-4 phosphorylation of Raf-1 (Ser338) and Erk1 and Erk 2 kinases (Thr202 and Tyr204, respectively). Whole cell lysates from eosinophils were cultured in the presence of IL-2/IL-4 and control or PP5 siRNA for 15 min and 36 h were analyzed for phosphorylation of Raf-1 and Erk1 and Erk2 kinases.

    Article Snippet: Polyclonal antibodies against phosphorylated GCR (S203, S211, S226) were from Abcam (Cambridge, MA); and antibodies against GCR, FPR2, Annexin 1, PP5, GILZ, phospho-Raf-1 and MKP1 were from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Inhibition, Cell Culture

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

    Journal: Respiratory Research

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

    doi: 10.1186/s12931-015-0257-8

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

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

    Techniques: Western Blot, Expressing, Infection, shRNA

    Deletion of CEK1 triggers Syk, ERK, and Raf-1 phosphorylation eliciting IκB degradation on hDCs and induces dectin-1-dependent AP-1 activation on transfected cells. (A) Western blot of cell lysates from hDCs cocultured with live C. albicans strains

    Journal: Infection and Immunity

    Article Title: Candida albicans ?-Glucan Exposure Is Controlled by the Fungal CEK1-Mediated Mitogen-Activated Protein Kinase Pathway That Modulates Immune Responses Triggered through Dectin-1 ▿-Mediated Mitogen-Activated Protein Kinase Pathway That Modulates Immune Responses Triggered through Dectin-1 ▿ †

    doi: 10.1128/IAI.00989-09

    Figure Lengend Snippet: Deletion of CEK1 triggers Syk, ERK, and Raf-1 phosphorylation eliciting IκB degradation on hDCs and induces dectin-1-dependent AP-1 activation on transfected cells. (A) Western blot of cell lysates from hDCs cocultured with live C. albicans strains

    Article Snippet: ERK1/2, Syk, and Raf-1 activation was detected by using antibodies specific for anti-phospho-p44/42-MAPK/anti-p44/42-MAPK (Cell Signaling), for anti-phospho-Syk (Calbiochem)/anti-Syk MAb (Upstate), and for anti-phospho-Raf-1 (Tyr340/341)/anti-Raf-1 Abs (Upstate, Millipore).

    Techniques: Activation Assay, Transfection, Western Blot

    Effect of S. enterica serovar Typhimurium ( S.ty ) porins on Raf-1 (A) and MEK1/2 (B) activation after different stimulation times. Proteins from cell lysates (3 × 10 6 cells/ml) were analyzed by SDS-15% PAGE and immunoblotted with a Raf-1- or MEK1/2-phosphospecific antibody. Shifts in band mobility on SDS-PAGE due to phosphorylation were obtained with anti-Raf-1, anti-MEK1, and anti-MEK2. Fold increases in Raf-1 and MEK1/2 activation, compared to unstimulated cells (control), are shown below each lane for each blot. BSA represents a control of an unspecific stimulus.

    Journal: Infection and Immunity

    Article Title: Porins from Salmonella enterica Serovar Typhimurium Activate the Transcription Factors Activating Protein 1 and NF-?B through the Raf-1-Mitogen-Activated Protein Kinase Cascade

    doi: 10.1128/IAI.70.2.558-568.2002

    Figure Lengend Snippet: Effect of S. enterica serovar Typhimurium ( S.ty ) porins on Raf-1 (A) and MEK1/2 (B) activation after different stimulation times. Proteins from cell lysates (3 × 10 6 cells/ml) were analyzed by SDS-15% PAGE and immunoblotted with a Raf-1- or MEK1/2-phosphospecific antibody. Shifts in band mobility on SDS-PAGE due to phosphorylation were obtained with anti-Raf-1, anti-MEK1, and anti-MEK2. Fold increases in Raf-1 and MEK1/2 activation, compared to unstimulated cells (control), are shown below each lane for each blot. BSA represents a control of an unspecific stimulus.

    Article Snippet: For Western blotting analysis, the following phosphorylated antibodies were used: phospho-p44/42 MAPK (Thr202/Tyr204) E10 monoclonal antibody (MAb) (isotype, mouse IgG1) (anti-p44/42) (New England Biolabs, Beverly, Mass.), which detects doubly phosphorylated threonine 202 and tyrosine 204 of p44 and p42 MAPKs (ERK1 and ERK2) and is produced by immunizing mice with a synthetic phospho-Thr202 and phospho-Tyr204 peptide corresponding to residues around Thr202/Tyr204 of human p44 MAPK; rabbit polyclonal phospho-MEK1/2 antibody (anti-p-MEK1/2) (New England Biolabs), which detects MEK1/2 only when activated by phosphorylation at Ser217 and Ser221 and does not cross-react with other related family members; anti-phospho-Raf-1 MAb (anti-p-Raf-1) (Upstate Biotechnology, Lake Placid, N.Y.), which recognizes Raf-1 phosphorylated at Ser338 and Tyr341 and does not bind to inactive Raf-1 or Raf-1 peptides phosphorylated only at Ser338 or Tyr341; anti-phospho-p38 (New England Biolabs), which is a rabbit polyclonal antibody raised against a peptide mapping at the amino terminus of p38 of mouse origin identical to the corresponding human sequence and directed against Thr180- and Tyr182-phosphorylated p38; and phospho-JNK antibody (anti-p-JNK) (Santa Cruz Biotechnology, Inc.), which is a mouse IgG1 MAb raised against a peptide corresponding to a short amino acid sequence phosphorylated on Thr183 and Tyr185 of JNK of human origin.

    Techniques: Activation Assay, Polyacrylamide Gel Electrophoresis, SDS Page

    Activation of MEK1 and Raf1 by the active form of ARF1. A , activation of MEK1 by ARF1Q71L. pMEK1 , phosphorylated MEK1; Ctrl , control. B , activation of Raf1 by ARF1Q71L. pRaf1 , phosphorylated Raf1. C , expression of the dominant-negative mutant Raf1K375M,

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of ?2B-Adrenergic Receptor-mediated Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Activation by ADP-ribosylation Factor 1 *

    doi: 10.1074/jbc.M111.267286

    Figure Lengend Snippet: Activation of MEK1 and Raf1 by the active form of ARF1. A , activation of MEK1 by ARF1Q71L. pMEK1 , phosphorylated MEK1; Ctrl , control. B , activation of Raf1 by ARF1Q71L. pRaf1 , phosphorylated Raf1. C , expression of the dominant-negative mutant Raf1K375M,

    Article Snippet: Antibodies were purchased from the following companies: ARF1 from StressGen (Ann Arbor, MI); ARF3 from BD Transduction Laboratories; phospho-Raf1 (Ser-338) from Upstate Biotech Millipore (Lake Placid, NY); GFP, phospho-ERK1/2, and Raf1 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); HA-fluorescein (3F10) from Roche Diagnostics; ERK1/2, MEK1, and phospho-MEK1 (Thr-286) from Cell Signaling Technology, Inc. (Beverly, MA).

    Techniques: Activation Assay, Expressing, Dominant Negative Mutation

    Effect of brucine on protein expressions of KDR, PKCα, PLCγ-1 and Raf1 in LoVo cells. Cells were treated with VEGF (50 ng/ml) for 30 min. before extracting proteins with RIPA lysis buffer. Results were quantified by densitometry analysis of the bands form and then normalization to GAPDH protein. ( A ) Effect of brucine on protein expressions. ( B ) Quantitation data of ( A ). Quantitation data showed brucine decreased the phosphorylation levels of KDR, PKCα, PLCγ-1 and Raf1 in a dose-dependent manner compared to the untreated control. Data represent the means ± SD ( n = 3) with * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Brucine suppresses colon cancer cells growth via mediating KDR signalling pathway

    doi: 10.1111/jcmm.12108

    Figure Lengend Snippet: Effect of brucine on protein expressions of KDR, PKCα, PLCγ-1 and Raf1 in LoVo cells. Cells were treated with VEGF (50 ng/ml) for 30 min. before extracting proteins with RIPA lysis buffer. Results were quantified by densitometry analysis of the bands form and then normalization to GAPDH protein. ( A ) Effect of brucine on protein expressions. ( B ) Quantitation data of ( A ). Quantitation data showed brucine decreased the phosphorylation levels of KDR, PKCα, PLCγ-1 and Raf1 in a dose-dependent manner compared to the untreated control. Data represent the means ± SD ( n = 3) with * P

    Article Snippet: Anti-phospho-KDR (Tyr1175 ) was from Cell Signaling (Cell Signaling Tech., Danvers, MA, USA), anti-phospho-PKCα (Tyr658 ) and anti-phospho-PLCγ-1 (Tyr771 ) were from Upstate (Upstate, Temecula, CA, USA), anti-phospho-Raf1 (Tyr340/Tyr341 ) was from Millipore (Millipore, Billerica, MA, USA).

    Techniques: Lysis, Quantitation Assay

    Effect of brucine on mRNA expressions of KDR, PKCα, PLCγ and Raf1 in LoVo cells. Relative ratio is shown, where KDR, PKCα, PLCγ and Raf1 signals were normalized to GAPDH signal. Data represent the means ± SD ( n = 3) with * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Brucine suppresses colon cancer cells growth via mediating KDR signalling pathway

    doi: 10.1111/jcmm.12108

    Figure Lengend Snippet: Effect of brucine on mRNA expressions of KDR, PKCα, PLCγ and Raf1 in LoVo cells. Relative ratio is shown, where KDR, PKCα, PLCγ and Raf1 signals were normalized to GAPDH signal. Data represent the means ± SD ( n = 3) with * P

    Article Snippet: Anti-phospho-KDR (Tyr1175 ) was from Cell Signaling (Cell Signaling Tech., Danvers, MA, USA), anti-phospho-PKCα (Tyr658 ) and anti-phospho-PLCγ-1 (Tyr771 ) were from Upstate (Upstate, Temecula, CA, USA), anti-phospho-Raf1 (Tyr340/Tyr341 ) was from Millipore (Millipore, Billerica, MA, USA).

    Techniques:

    Effect of brucine on normal cells and cells transfected with siRNAs of KDR, PKCα, PLCγ and Raf1. ( A – D ) Knockdown quantification of RT-PCR on KDR, PKCα, PLCγ and Raf1; ( E ) The effect of brucine on cells proliferation of normal cells and knockdown cells. Data are expressed as means ± SD ( n = 3).

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Brucine suppresses colon cancer cells growth via mediating KDR signalling pathway

    doi: 10.1111/jcmm.12108

    Figure Lengend Snippet: Effect of brucine on normal cells and cells transfected with siRNAs of KDR, PKCα, PLCγ and Raf1. ( A – D ) Knockdown quantification of RT-PCR on KDR, PKCα, PLCγ and Raf1; ( E ) The effect of brucine on cells proliferation of normal cells and knockdown cells. Data are expressed as means ± SD ( n = 3).

    Article Snippet: Anti-phospho-KDR (Tyr1175 ) was from Cell Signaling (Cell Signaling Tech., Danvers, MA, USA), anti-phospho-PKCα (Tyr658 ) and anti-phospho-PLCγ-1 (Tyr771 ) were from Upstate (Upstate, Temecula, CA, USA), anti-phospho-Raf1 (Tyr340/Tyr341 ) was from Millipore (Millipore, Billerica, MA, USA).

    Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction