Structured Review

Santa Cruz Biotechnology raf 1 antibody
Potential role for 14-3-3 in <t>Raf-1</t> maturation. 14-3-3 may play a critical role in Raf-1 maturation. Raf-1 molecules may exist in dynamic equilibrium between phosphorylated and unphosphorylated forms. Raf-1 can autophosphorylate itself at S621, but in the absence of 14-3-3, this phosphorylation is rapidly lost in the cell, presumably via the action of a phosphatase. The binding of 14-3-3 to this site protects the phosphorylation from phosphatase activity and is proposed to stabilize a kinase-competent conformation in Raf. This 14-3-3-bound form of CT-Raf possesses constitutive activity, requiring no additional activation events. In the context of the full-length molecule, the binding of 14-3-3 to the pS621 site is proposed to result in a preactivated molecule whose kinase activity is repressed due to interactions with the amino-terminal domains. This form of the kinase would be competent to bind Ras and become activated, or derepressed, at the plasma membrane.
Raf 1 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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raf 1 antibody - by Bioz Stars, 2020-07
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1) Product Images from "14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity"

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

Journal: Molecular and Cellular Biology

doi:

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

Techniques Used: Binding Assay, Activity Assay, Activation Assay

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

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

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

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

2) Product Images from "Dual farnesyl and geranylgeranyl transferase inhibitor thwarts mutant KRAS-driven patient-derived pancreatic tumors"

Article Title: Dual farnesyl and geranylgeranyl transferase inhibitor thwarts mutant KRAS-driven patient-derived pancreatic tumors

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-18-3399

Effects of accumulating KRAS in the cytosol on binding to RAF-1 and KSR and on RAF-1 kinase activity. A , FGTI-2734, but not FTI-2148 or GGTI-2418, inhibits membrane localization of GFP mutant KRAS-CVIM in human cancer cells. MiaPaCa2, H460, Calu6, A549, and DLD1 cells were grown on glass coverslips and infected with lentiviruses of GFP-tagged mutant KRAS-CVIM, GFP-tagged 185 Cys to Ser mutant KRAS that cannot be prenylated (GFP-mutant KRAS-SVIM), and corresponding empty vector (GFP-tagged-EV) and treated with vehicle control (DMSO) or FGTI-2734, FTI-2148, or GGTI-2418. Cells were fixed and viewed through a confocal microscope. B , FGTI-2734 inhibits RAF-1 kinase activity and binding to KSR but not to KRAS. MiaPaCa2 cells were treated with FGTI-2734 at 30 μM and processed for RAF-1 immunoprecipitation, followed by KRAS and KSR immunoblotting as well as RAF-1 kinase assay in the immunoprecipitates using recombinant inactive MEK1 as a substrate and western blotting with an antibody to phosphorylated Serines 217 and 221as described under Methods. C , The cytosolic CAAX mutant KRASG12V-SVIM binds RAF-1 but does not activate it. MiaPaCa2 cells expressing FLAG-tagged KRASG12V-CVIM or FLAG-tagged KRASG12V-SVIM were processed for coimmunoprecipitation and RAF-1 kinase assays as described above.
Figure Legend Snippet: Effects of accumulating KRAS in the cytosol on binding to RAF-1 and KSR and on RAF-1 kinase activity. A , FGTI-2734, but not FTI-2148 or GGTI-2418, inhibits membrane localization of GFP mutant KRAS-CVIM in human cancer cells. MiaPaCa2, H460, Calu6, A549, and DLD1 cells were grown on glass coverslips and infected with lentiviruses of GFP-tagged mutant KRAS-CVIM, GFP-tagged 185 Cys to Ser mutant KRAS that cannot be prenylated (GFP-mutant KRAS-SVIM), and corresponding empty vector (GFP-tagged-EV) and treated with vehicle control (DMSO) or FGTI-2734, FTI-2148, or GGTI-2418. Cells were fixed and viewed through a confocal microscope. B , FGTI-2734 inhibits RAF-1 kinase activity and binding to KSR but not to KRAS. MiaPaCa2 cells were treated with FGTI-2734 at 30 μM and processed for RAF-1 immunoprecipitation, followed by KRAS and KSR immunoblotting as well as RAF-1 kinase assay in the immunoprecipitates using recombinant inactive MEK1 as a substrate and western blotting with an antibody to phosphorylated Serines 217 and 221as described under Methods. C , The cytosolic CAAX mutant KRASG12V-SVIM binds RAF-1 but does not activate it. MiaPaCa2 cells expressing FLAG-tagged KRASG12V-CVIM or FLAG-tagged KRASG12V-SVIM were processed for coimmunoprecipitation and RAF-1 kinase assays as described above.

Techniques Used: Binding Assay, Activity Assay, Mutagenesis, Infection, Plasmid Preparation, Microscopy, Immunoprecipitation, Kinase Assay, Recombinant, Western Blot, Expressing

3) Product Images from "Cyclic AMP/PKA-dependent paradoxical activation of Raf/MEK/ERK signaling in polycystin-2 defective mice treated with Sorafenib"

Article Title: Cyclic AMP/PKA-dependent paradoxical activation of Raf/MEK/ERK signaling in polycystin-2 defective mice treated with Sorafenib

Journal: Hepatology (Baltimore, Md.)

doi: 10.1002/hep.25872

Sorafenib inhibited the kinase activity of B-Raf but not of Raf-1 in Pkd2cKO cholangiocytes
Figure Legend Snippet: Sorafenib inhibited the kinase activity of B-Raf but not of Raf-1 in Pkd2cKO cholangiocytes

Techniques Used: Activity Assay

4) Product Images from "Raf-1, Actin Dynamics, and Abelson Tyrosine Kinase in Human Airway Smooth Muscle Cells"

Article Title: Raf-1, Actin Dynamics, and Abelson Tyrosine Kinase in Human Airway Smooth Muscle Cells

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2012-0315OC

Interaction of Raf-1 with actin in HASM and in vitro . ( A ) Representative immunoblots illustrating the presence of Raf-1 in cytoskeletal actin (F-actin). Blots of supernatant (S) and pellet (P) fractions from HASM cells were probed with use of Raf-1 antibody
Figure Legend Snippet: Interaction of Raf-1 with actin in HASM and in vitro . ( A ) Representative immunoblots illustrating the presence of Raf-1 in cytoskeletal actin (F-actin). Blots of supernatant (S) and pellet (P) fractions from HASM cells were probed with use of Raf-1 antibody

Techniques Used: In Vitro, Western Blot

Stimulation with PDGF results in an increase in the association of Raf-1 with F-actin. HASM cells were stimulated with different concentration of PDGF for 10 minutes. Equal amounts of F-actin from the cells were separated by SDS-PAGE and transferred to
Figure Legend Snippet: Stimulation with PDGF results in an increase in the association of Raf-1 with F-actin. HASM cells were stimulated with different concentration of PDGF for 10 minutes. Equal amounts of F-actin from the cells were separated by SDS-PAGE and transferred to

Techniques Used: Concentration Assay, SDS Page

Effects of latrunculin A (LAT-A) and nocodazole (Noc) on the association of Raf-1 with F-actin and Raf-1 translocation in HASM cells. ( A and B ) Blots of cytoskeletal actin from unstimulated and PDGF-stimulated HASM cells in the absence or presence of
Figure Legend Snippet: Effects of latrunculin A (LAT-A) and nocodazole (Noc) on the association of Raf-1 with F-actin and Raf-1 translocation in HASM cells. ( A and B ) Blots of cytoskeletal actin from unstimulated and PDGF-stimulated HASM cells in the absence or presence of

Techniques Used: Translocation Assay

Treatment with platelet-derived growth factor (PDGF) induces the spatial translocation of Raf-1 in human airway smooth muscle (HASM) cells. HASM cells were stimulated with PDGF (10 ng/ml) for 10 minutes or left unstimulated. The cellular localization
Figure Legend Snippet: Treatment with platelet-derived growth factor (PDGF) induces the spatial translocation of Raf-1 in human airway smooth muscle (HASM) cells. HASM cells were stimulated with PDGF (10 ng/ml) for 10 minutes or left unstimulated. The cellular localization

Techniques Used: Derivative Assay, Translocation Assay

Knockdown and rescue of Abl affect the interaction of Raf-1 with actin and Raf-1 redistribution induced by PDGF. ( A ) Representative immunoblots illustrating the role of Abl in the association of Raf-1 with F-actin. To assess the role of Abl, control cells,
Figure Legend Snippet: Knockdown and rescue of Abl affect the interaction of Raf-1 with actin and Raf-1 redistribution induced by PDGF. ( A ) Representative immunoblots illustrating the role of Abl in the association of Raf-1 with F-actin. To assess the role of Abl, control cells,

Techniques Used: Western Blot

5) Product Images from "RKIP Regulates MAP Kinase Signaling in Cells with Defective B-Raf Activity"

Article Title: RKIP Regulates MAP Kinase Signaling in Cells with Defective B-Raf Activity

Journal: Cellular signalling

doi: 10.1016/j.cellsig.2013.02.005

Raf-1 signaling is inhibited by RKIP in B-Raf deficient cells (A) Immortalized wild-type (RKIP+/+) and RKIP −/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1) or control siRNA (siCon). After 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP and Raf-1 were detected by western blot. (B) RKIP was stably depleted in WT and Raf-1−/− MEFs by shRNA. MEFs were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) for 5 min. pERK1/2, ERK1/2, RKIP, and Raf-1 were detected by western blot. (C) Immortalized wild-type and RKIP−/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1), B-Raf siRNA (siB-Raf), double B-Raf and Raf-1 siRNA (siRaf-1+siB-Raf) or control siRNA (siCon); after 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP, Raf-1 and B-Raf were detected by western blot. *, p
Figure Legend Snippet: Raf-1 signaling is inhibited by RKIP in B-Raf deficient cells (A) Immortalized wild-type (RKIP+/+) and RKIP −/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1) or control siRNA (siCon). After 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP and Raf-1 were detected by western blot. (B) RKIP was stably depleted in WT and Raf-1−/− MEFs by shRNA. MEFs were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) for 5 min. pERK1/2, ERK1/2, RKIP, and Raf-1 were detected by western blot. (C) Immortalized wild-type and RKIP−/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1), B-Raf siRNA (siB-Raf), double B-Raf and Raf-1 siRNA (siRaf-1+siB-Raf) or control siRNA (siCon); after 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP, Raf-1 and B-Raf were detected by western blot. *, p

Techniques Used: Western Blot, Stable Transfection, shRNA

Loss of RKIP rescues ERK activation in B-Raf knockout MEFs that express kinase-dead B-Raf mutants (A) RKIP was stably depleted in WT and B-Raf −/− MEFs by shRNA. MEFs were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) for 5 min. pERK1/2, ERK1/2, RKIP, B-Raf and Raf-1 were detected by western blot. *, p
Figure Legend Snippet: Loss of RKIP rescues ERK activation in B-Raf knockout MEFs that express kinase-dead B-Raf mutants (A) RKIP was stably depleted in WT and B-Raf −/− MEFs by shRNA. MEFs were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) for 5 min. pERK1/2, ERK1/2, RKIP, B-Raf and Raf-1 were detected by western blot. *, p

Techniques Used: Activation Assay, Knock-Out, Stable Transfection, shRNA, Western Blot

Related Articles

Produced:

Article Title: Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding
Article Snippet: .. Phospho-MEK, phospho-ERK, MEK, and ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA); FLAG antibody from Sigma-Aldrich (St. Louis, MO); glutathione S -transferase (GST) antibody was from Upstate Biotechnology (Lake Placid, NY); HSC-70 and Raf-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); heat-shock protein (HSP)-70 antibody was from Affinity Bioreagents (Golden, CO); and hemagglutinin (HA) and myc epitope tag antibodies were produced from the 12CA5 and 9E10 hybridoma cell lines, respectively. .. EGF was purchased from Sigma-Aldrich; U0126 MEK inhibitor was from Promega (Madison, WI); and protein phosphatase 1-γ was from Calbiochem (San Diego, CA).

Immunoprecipitation:

Article Title: Cyclic AMP/PKA-dependent paradoxical activation of Raf/MEK/ERK signaling in polycystin-2 defective mice treated with Sorafenib
Article Snippet: .. WT and Pkd2cKO cell lysates were immunoprecipitated overnight by gentle rotation at 4°C with an anti B-Raf or an anti Raf-1 antibody (Santacruz Biotechnology, Santa Cruz, CA) covalently coupled to protein A/G Plus agarose beads. ..

Article Title: Dual farnesyl and geranylgeranyl transferase inhibitor thwarts mutant KRAS-driven patient-derived pancreatic tumors
Article Snippet: .. The cell lysates (1.5~2 mg, 1000 μl each) were used for immunoprecipitation with RAF-1 antibody (Santa Cruz, # sc-7267) or anti-Flag M2 affinity gel (Sigma, #A2220) for 3 hours at 4°C. .. The protein bound beads were washed 2 times with Cell Lysis Buffer and 2 times with kinase buffer (25 mM Tris (pH 7.5), 5 mM β-Glycerolphosphate, 10 mM MgCl2, 1 mM DTT, 0.1 mM Na3VO4).

other:

Article Title: An Hsp90 co-chaperone protein in yeast is functionally replaced by site-specific posttranslational modification in humans
Article Snippet: The Cdk4 and Raf-1 antibodies were purchased from Santa Cruz Biotechnology.

Article Title: Endosomal Targeting of MEK2 Requires RAF, MEK Kinase Activity and Clathrin-Dependent Endocytosis
Article Snippet: RAF-1 antibodies were purchased from Santa Cruz Biosciences, and B-RAF antibodies were kindly provided by Dr R Nemenoff (University of Colorado Denver Health Sciences Center, UCDHSC).

Article Title: RKIP Regulates MAP Kinase Signaling in Cells with Defective B-Raf Activity
Article Snippet: Raf-1 antibody (Cat No: sc133) and B-Raf antibody (Cat No: sc-5284) and goat anti-mouse IgG-HRP (Cat No: sc-2005) were purchased from Santa Cruz.

Lysis:

Article Title: 14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity
Article Snippet: .. After the lysates were cleared, immunoprecipitates were prepared with Raf-1 antibody (Santa Cruz Biotechnology) and protein A-Sepharose and washed six times in NP-40 lysis buffer containing 0.1% SDS. .. The Raf-1 immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose, and the phosphoproteins were visualized by phosphorimaging.

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    Santa Cruz Biotechnology c anti raf 1 antisera
    Inhibition of <t>raf-1</t> kinase activity by p38. ATDC5 cells were stimulated with IGF-I (100 ng/ml) for 15 min; cell lysates immunoprecipitated for endogenous raf-1 protein were incubated with or without recombinant (His) 6 -p38 protein for 30 min, then kinase
    C Anti Raf 1 Antisera, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c anti raf 1 antisera/product/Santa Cruz Biotechnology
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Santa Cruz Biotechnology raf 1
    Effect of azaspirene on the autophosphorylation of KDR/Flk-1, phosphorylation of <t>Raf-1</t> and disruption of Raf-1 complexes.
    Raf 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/raf 1/product/Santa Cruz Biotechnology
    Average 93 stars, based on 97 article reviews
    Price from $9.99 to $1999.99
    raf 1 - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

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

    Journal: Molecular Endocrinology

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

    doi: 10.1210/me.2012-1063

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

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

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

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

    Journal: Molecular Endocrinology

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

    doi: 10.1210/me.2012-1063

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

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

    Techniques: Activity Assay, Isolation, Immunoprecipitation

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

    Journal: Cancer science

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

    doi: 10.1111/j.1349-7006.2008.00890.x

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

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

    Techniques:

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

    Journal: Toxicological Sciences

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

    doi: 10.1093/toxsci/kfr118

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

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

    Techniques: Expressing, Transfection

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

    Journal: Journal of Cell Science

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

    doi: 10.1242/jcs.177543

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

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

    Techniques: Immunoprecipitation, Transfection