Rabbit RAF1 Polyclonal Antibody | anti-RAF1 antibody

Phospho-RAF1 (Tyr341) Antibody

Cat. Num. AAA7133390

• Gene Names: RAF1; NS5; CRAF; Raf-1; c-Raf
• Reactivity: Human, Mouse, Rat
• Applications: ELISA, Western Blot, Immunohistochemistry
• Purity: Antibodies were produced by immunizing rabbits with synthetic phosphopeptide and KLH conjugates. Antibodies were purified by affinity-chromatography using epitope-specific phosphopeptide. Non-phospho specific antibodies were removed by chromatography usin
• Synonyms: RAF1; Polyclonal Antibody; Phospho-RAF1 (Tyr341) Antibody; C-RAF; C-Raf; CRAF; RAF-1; anti-RAF1 antibody

• Host: Rabbit
• Reactivity: Human, Mouse, Rat
• Clonality: Polyclonal
• Isotype: IgG
• Purity/Purification: Antibodies were produced by immunizing rabbits with synthetic phosphopeptide and KLH conjugates. Antibodies were purified by affinity-chromatography using epitope-specific phosphopeptide. Non-phospho specific antibodies were removed by chromatography usin
• Form/Format: Liquid; Rabbit IgG In Phosphate Buffered Saline (without Mg2+ and Ca2+), pH 7.4, 150mM NaCl, 0.02% Sodium Azide and 50% Glycerol.

• Applicable Applications for anti-RAF1 antibody: ELISA (EIA), Western Blot (WB), Immunohistochemistry (IHC)
• Application Notes: WB: 1:500-1:1000; IHC: 1:50-1:100
• Immunogen: Peptide sequence around phosphorylation site of tyrosine 341 (S-Y-Y(p)-W-E) derived from Human C-RAF.
• Immunogen Species: Human
• Conjugate: Non-conjugated
• Preparation and Storage: Upon receipt, store at -20 degree C or -80 degree C. Avoid repeated freeze.

Western Blot (WB)

(Western blot analysis of extracts from Jurkat cells treated with Paclitaxel using Raf1 (Phospho-Tyr341) Antibody.The lane on the right is treated with the antigen-specific peptide.)

Immunohistochemistry (IHC)

(Immunohistochemical analysis of paraffin-embedded human pancreas tissue using Raf1 (Phospho-Tyr341) antibody (left)or the same antibody preincubated with blocking peptide (right).)

Product Categories/Family for anti-RAF1 antibody

Signal Transduction

NCBI and Uniprot Product Information

• NCBI GI #: 4506401
• NCBI GeneID: 5894
• NCBI Accession #: NP_002871.1
• NCBI GenBank Nucleotide #: NM_002880.3
• UniProt Accession #: P04049; Q15278; Q9UC20; B0LPH8; B2R5N3
• Molecular Weight: 73,052 Da
• NCBI Official Full Name: RAF proto-oncogene serine/threonine-protein kinase
• NCBI Official Synonym Full Names: v-raf-1 murine leukemia viral oncogene homolog 1
• NCBI Official Symbol: RAF1
• NCBI Official Synonym Symbols: NS5; CRAF; Raf-1; c-Raf
• NCBI Protein Information: RAF proto-oncogene serine/threonine-protein kinase; Oncogene RAF1; proto-oncogene c-RAF; raf proto-oncogene serine/threonine protein kinase
• UniProt Protein Name: RAF proto-oncogene serine/threonine-protein kinase
• Protein Family: Rik1-associated factor
• UniProt Gene Name: RAF1
• UniProt Synonym Gene Names: RAF; cRaf
• UniProt Entry Name: RAF1_HUMAN

NCBI Description

This gene is the cellular homolog of viral raf gene (v-raf). The encoded protein is a MAP kinase kinase kinase (MAP3K), which functions downstream of the Ras family of membrane associated GTPases to which it binds directly. Once activated, the cellular RAF1 protein can phosphorylate to activate the dual specificity protein kinases MEK1 and MEK2, which in turn phosphorylate to activate the serine/threonine specific protein kinases, ERK1 and ERK2. Activated ERKs are pleiotropic effectors of cell physiology and play an important role in the control of gene expression involved in the cell division cycle, apoptosis, cell differentiation and cell migration. Mutations in this gene are associated with Noonan syndrome 5 and LEOPARD syndrome 2. [provided by RefSeq, Jul 2008]

Uniprot Description

Function: Serine/threonine-protein kinase that acts as a regulatory link between the membrane-associated Ras GTPases and the MAPK/ERK cascade, and this critical regulatory link functions as a switch determining cell fate decisions including proliferation, differentiation, apoptosis, survival and oncogenic transformation. RAF1 activation initiates a mitogen-activated protein kinase (MAPK) cascade that comprises a sequential phosphorylation of the dual-specific MAPK kinases (MAP2K1/MEK1 and MAP2K2/MEK2) and the extracellular signal-regulated kinases (MAPK3/ERK1 and MAPK1/ERK2). The phosphorylated form of RAF1 (on residues Ser-338 and Ser-339, by PAK1) phosphorylates BAD/Bcl2-antagonist of cell death at 'Ser-75'. Phosphorylates adenylyl cyclases: ADCY2, ADCY5 and ADCY6, resulting in their activation. Phosphorylates PPP1R12A resulting in inhibition of the phosphatase activity. Phosphorylates TNNT2/cardiac muscle troponin T. Can promote NF-kB activation and inhibit signal transducers involved in motility (ROCK2), apoptosis (MAP3K5/ASK1 and STK3/MST2), proliferation and angiogenesis (RB1). Can protect cells from apoptosis also by translocating to the mitochondria where it binds BCL2 and displaces BAD/Bcl2-antagonist of cell death. Regulates Rho signaling and migration, and is required for normal wound healing. Plays a role in the oncogenic transformation of epithelial cells via repression of the TJ protein, occludin (OCLN) by inducing the up-regulation of a transcriptional repressor SNAI2/SLUG, which induces down-regulation of OCLN. Restricts caspase activation in response to selected stimuli, notably Fas stimulation, pathogen-mediated macrophage apoptosis, and erythroid differentiation. Ref.11 Ref.16 Ref.17 Ref.24 Ref.25 Ref.28 Ref.33

Catalytic activity: ATP + a protein = ADP + a phosphoprotein.

Cofactor: Binds 2 zinc ions per subunit.

Enzyme regulation: Regulation is a highly complex process involving membrane recruitment, protein-protein interactions, dimerization, and phosphorylation/dephosphorylation events. Ras-GTP recruits RAF1 to the membrane, thereby promoting its activation. The inactive conformation of RAF1 is maintained by autoinhibitory interactions occurring between the N-terminal regulatory and the C-terminal catalytic domains and by the binding of a 14-3-3 protein that contacts two phosphorylation sites, Ser-259 and Ser-621. Upon mitogenic stimulation, Ras and PPP2R1A cooperate to release autoinhibition and the subsequent phosphorylation of activating sites: Ser-338, Tyr-341, Thr-491, and Ser-494, yields a fully active kinase. Through a negative feedback mechanism involving MAPK1/ERK2, RAF1 is phosphorylated on Ser-29, Ser-43, Ser-289, Ser-296, Ser-301 and Ser-642 by MAPK1/ERK2, which yields an inactive, desensitized kinase. The signaling-competent conformation of RAF1 is finally re-established by the coordinated action of PIN1, a prolyl isomerase that converts pSer and pThr residues from the cis to the trans conformation, which is preferentially recognized and dephosphorylated by PPP2R1A. Activated by homodimerization and heterodimerization (with BRAF). Also regulated through association with other proteins such as KSR2, CNKSR1/CNK1, PEBP1/RKIP, PHB/prohibitin and SPRY4. PEBP1/RKIP acts by dissociating RAF1 from its substrates MAP2K1/MEK1 and MAP2K2/MEK2. PHB/prohibitin facilitates the displacement of 14-3-3 from RAF1 by activated Ras, thereby promoting cell membrane localization and phosphorylation of RAF1 at the activating Ser-338. SPRY4 inhibits Ras-independent, but not Ras-dependent, activation of RAF1. CNKSR1/CNK1 regulates Src-mediated RAF1 activation. Ref.14 Ref.15 Ref.18 Ref.21 Ref.35 Ref.41

Subunit structure: Monomer. Homodimer. Heterodimerizes with BRAF and this heterodimer possesses a highly increased kinase activity compared to the respective homodimers or monomers. Heterodimerization is mitogen-regulated and enhanced by 14-3-3 proteins. MAPK1/ERK2 activation can induce a negative feedback that promotes the dissociation of the heterodimer. Forms a multiprotein complex with Ras (M-Ras/MRAS), SHOC2 and protein phosphatase 1 (PPP1CA, PPP1CB and PPP1CC). Interacts with Ras proteins; the interaction is antagonized by RIN1. Weakly interacts with RIT1. Interacts (via N-terminus) with RGS14 (via RBD domains); the interaction mediates the formation of a ternary complex with BRAF, a ternary complex inhibited by GNAI1

By similarity. Interacts with STK3/MST2; the interaction inhibits its pro-apoptotic activity. Interacts (when phosphorylated at Ser-259) with YWHAZ (unphosphorylated at 'Thr-232'). Interacts with MAP2K1/MEK1 and MAP2K2/MEK2

By similarity. Interacts with MAP3K5/ASF1 (via N-terminus) and this interaction inhibits the proapoptotic function of MAP3K5/ASK1. Interacts with PAK1 (via kinase domain). The phosphorylated form interacts with PIN1. The Ser-338 and Ser-339 phosphorylated form (by PAK1) interacts with BCL2. Interacts with PEBP1/RKIP and this interaction is enhanced if RAF1 is phosphorylated on residues Ser-338, Ser-339, Tyr-340 and Tyr-341. Interacts with ADCY2, ADCY5, ADCY6, DGKH, RCAN1/DSCR1, ROCK2, PPP1R12A, PKB/AKT1, PPP2CA, PPP2R1B, SPRY2, SPRY4, CNKSR1/CNK1, KSR2 and PHB/prohibitin. In its active form, interacts with PRMT5. Ref.7 Ref.11 Ref.14 Ref.15 Ref.16 Ref.17 Ref.18 Ref.21 Ref.24 Ref.25 Ref.26 Ref.28 Ref.30 Ref.31 Ref.35 Ref.41

Subcellular location: Cytoplasm. Cell membrane. Mitochondrion. Nucleus. Note: Colocalizes with RGS14 and BRAF in both the cytoplasm and membranes. Phosphorylation at Ser-259 impairs its membrane accumulation. Recruited to the cell membrane by the active Ras protein. Phosphorylation at Ser-338 and Ser-339 by PAK1 is required for its mitochondrial localization. Retinoic acid-induced Ser-621 phosphorylated form of RAF1 is predominantly localized at the nucleus. Ref.19 Ref.28 Ref.40 Ref.41

Tissue specificity: In skeletal muscle, isoform 1 is more abundant than isoform 2. Ref.9

Post-translational modification: Phosphorylation at Thr-269, Ser-338, Tyr-341, Thr-491 and Ser-494 results in its activation. Phosphorylation at Ser-29, Ser-43, Ser-289, Ser-296, Ser-301 and Ser-642 by MAPK1/ERK2 results in its inactivation. Phosphorylation at Ser-259 induces the interaction with YWHAZ and inactivates kinase activity. Dephosphorylation of Ser-259 by the complex containing protein phosphatase 1, SHOC2 and M-Ras/MRAS relieves inactivation, leading to stimulate RAF1 activity. Phosphorylation at Ser-338 by PAK1 and PAK7/PAK5 and Ser-339 by PAK1 is required for its mitochondrial localization. Phosphorylation at Ser-621 in response to growth factor treatment stabilizes the protein, possibly by preventing proteasomal degradation. Phosphorylation at Ser-289, Ser-296, Ser-301, Ser-338 and Ser-621 are somehow linked to the methylation potential of cells. Treatment of cells with HGF in the presence of the methylation inhibitor 5'-methylthioadenosine (MTA) results in increased phosphorylation at Ser-338 and Ser-621 and decreased phosphorylation at Ser-296, Ser-301 and Ser-338.Methylated at Arg-563 in response to EGF treatment. This modification leads to destabilization of the protein, possibly through proteasomal degradation. Ref.7

Involvement in disease: Noonan syndrome 5 (NS5) [MIM:611553]: A syndrome characterized by facial dysmorphic features such as hypertelorism, a downward eyeslant and low-set posteriorly rotated ears. Other features can include short stature, a short neck with webbing or redundancy of skin, cardiac anomalies, deafness, motor delay and variable intellectual deficits.Note: The disease is caused by mutations affecting the gene represented in this entry. Ref.52 Ref.53 Ref.55LEOPARD syndrome 2 (LEOPARD2) [MIM:611554]: A disorder characterized by lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonic stenosis, abnormalities of genitalia, retardation of growth, and sensorineural deafness.Note: The disease is caused by mutations affecting the gene represented in this entry. Ref.52

Sequence similarities: Belongs to the protein kinase superfamily. TKL Ser/Thr protein kinase family. RAF subfamily.Contains 1 phorbol-ester/DAG-type zinc finger.Contains 1 protein kinase domain.Contains 1 RBD (Ras-binding) domain.