Product: Phospho-C-RAF (Ser301) Antibody
Catalog: AF0047
Source: Rabbit
Application: WB, IHC, IF/ICC, ELISA(peptide)
Reactivity: Human, Mouse, Rat
Mol.Wt.: 73kD; 73kD(Calculated).
Uniprot: P04049
RRID: AB_2834093

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Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IF/ICC 1:100-1:500, IHC 1:50-1:200, ELISA(peptide) 1:20000-1:40000
*The optimal dilutions should be determined by the end user.
Reactivity:
Human,Mouse,Rat
Clonality:
Polyclonal
Specificity:
Phospho-C-RAF (Ser301) Antibody detects endogenous levels of C-RAF only when phosphorylated at Sersine 301.
RRID:
AB_2834093
Cite Format: Affinity Biosciences Cat# AF0047, RRID:AB_2834093.
Purification:
The antibody is from purified rabbit serum by affinity purification via sequential chromatography on phospho-peptide and non-phospho-peptide affinity columns.
Storage:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
Alias:

Fold/Unfold

c Raf; C-raf; C-Raf proto-oncogene, serine/threonine kinase; CMD1NN; Craf 1 transforming gene; cRaf; Craf1 transforming gene; EC 2.7.11.1; kinase Raf1; Murine sarcoma 3611 oncogene 1; NS5; Oncogene MIL; Oncogene RAF1; OTTHUMP00000160218; OTTHUMP00000207813; OTTHUMP00000209389; Protein kinase raf 1; Proto-oncogene c-RAF; Raf 1; Raf 1 proto oncogene serine/threonine kinase; RAF; Raf proto oncogene serine/threonine protein kinase; RAF proto-oncogene serine/threonine-protein kinase; RAF-1; RAF1; RAF1_HUMAN; Similar to murine leukemia viral (V-raf-1) oncogene homolog 1; TRANSFORMING REPLICATION-DEFECTIVE MURINE RETROVIRUS 3611-MSV; v raf 1 murine leukemia viral oncogene homolog 1; v-raf murine sarcoma viral oncogene homolog 1; v-raf-1 murine leukemia viral oncogene-like protein 1; vraf1 murine leukemia viral oncogene homolog 1;

Immunogens

Immunogen:

A synthesized peptide derived from human C-RAF around the phosphorylation site of Ser301.

Uniprot:
Gene(ID):
Expression:
P04049 RAF1_HUMAN:

In skeletal muscle, isoform 1 is more abundant than isoform 2.

Description:
Raf-1 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 Raf-1 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.
Sequence:
MEHIQGAWKTISNGFGFKDAVFDGSSCISPTIVQQFGYQRRASDDGKLTDPSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSNIRQLLLFPNSTIGDSGVPALPSLTMRRMRESVSRMPVSSQHRYSTPHAFTFNTSSPSSEGSLSQRQRSTSTPNVHMVSTTLPVDSRMIEDAIRSHSESASPSALSSSPNNLSPTGWSQPKTPVPAQRERAPVSGTQEKNKIRPRGQRDSSYYWEIEASEVMLSTRIGSGSFGTVYKGKWHGDVAVKILKVVDPTPEQFQAFRNEVAVLRKTRHVNILLFMGYMTKDNLAIVTQWCEGSSLYKHLHVQETKFQMFQLIDIARQTAQGMDYLHAKNIIHRDMKSNNIFLHEGLTVKIGDFGLATVKSRWSGSQQVEQPTGSVLWMAPEVIRMQDNNPFSFQSDVYSYGIVLYELMTGELPYSHINNRDQIIFMVGRGYASPDLSKLYKNCPKAMKRLVADCVKKVKEERPLFPQILSSIELLQHSLPKINRSASEPSLHRAAHTEDINACTLTTSPRLPVF

PTMs - P04049 As Substrate

Site PTM Type Enzyme
S12 Phosphorylation
K18 Methylation
S29 Phosphorylation P28482 (MAPK1)
T31 Phosphorylation
Y38 Phosphorylation
S43 Phosphorylation P17252 (PRKCA) , P17612 (PRKACA)
T49 Phosphorylation
K53 Ubiquitination
K65 Ubiquitination
S211 Phosphorylation
T213 Phosphorylation
S220 Phosphorylation
R223 Methylation
R231 Methylation
Y232 Phosphorylation
S233 Phosphorylation P17612 (PRKACA) , P17252 (PRKCA)
T234 Phosphorylation
T239 Phosphorylation
T242 Phosphorylation
S244 Phosphorylation
S247 Phosphorylation
S250 Phosphorylation
S252 Phosphorylation
S257 Phosphorylation
T258 Phosphorylation
S259 Phosphorylation O95835 (LATS1) , P17612 (PRKACA) , P31749 (AKT1) , Q13131 (PRKAA1) , P04049 (RAF1)
T260 Phosphorylation
S267 Phosphorylation
T268 Phosphorylation P04049 (RAF1)
T269 Phosphorylation Q8IVT5 (KSR1)
S283 Phosphorylation
S285 Phosphorylation
S287 Phosphorylation
S289 Phosphorylation P28482 (MAPK1) , P27361 (MAPK3)
S291 Phosphorylation
S294 Phosphorylation
S295 Phosphorylation
S296 Phosphorylation P27361 (MAPK3) , P28482 (MAPK1)
S301 Phosphorylation P28482 (MAPK1) , P27361 (MAPK3)
T303 Phosphorylation
S306 Phosphorylation
T310 Phosphorylation
T324 Phosphorylation
S338 Phosphorylation Q02750 (MAP2K1) , P04049 (RAF1) , O75914 (PAK3) , P53350 (PLK1) , Q13153 (PAK1) , Q16512 (PKN1)
S339 Phosphorylation P53350 (PLK1) , O75914 (PAK3) , Q16512 (PKN1) , Q13153 (PAK1)
Y340 Phosphorylation P12931 (SRC)
Y341 Phosphorylation P12931 (SRC)
T353 Phosphorylation
S357 Phosphorylation
S359 Phosphorylation
T362 Phosphorylation
K365 Ubiquitination
K367 Ubiquitination
K378 Ubiquitination
Y458 Phosphorylation
K462 Ubiquitination
K470 Ubiquitination
S471 Phosphorylation
T481 Phosphorylation
T491 Phosphorylation
K493 Ubiquitination
S494 Phosphorylation
S497 Phosphorylation P05771 (PRKCB) , P05129 (PRKCG) , P17252 (PRKCA)
S499 Phosphorylation P17252 (PRKCA)
K575 Ubiquitination
S612 Phosphorylation
S619 Phosphorylation P05129 (PRKCG) , P05771 (PRKCB) , P17252 (PRKCA)
S621 Phosphorylation P17612 (PRKACA) , P04049 (RAF1) , Q13131 (PRKAA1)
S624 Phosphorylation
T638 Phosphorylation
T640 Phosphorylation
S642 Phosphorylation P28482 (MAPK1)

PTMs - P04049 As Enzyme

Substrate Site Source
O14974 (PPP1R12A) T696 Uniprot
O43306 (ADCY6) S732 Uniprot
O43306 (ADCY6) S734 Uniprot
O43306 (ADCY6) S738 Uniprot
O43306 (ADCY6) S742 Uniprot
P01106 (MYC) T8 Uniprot
P04049 (RAF1) S259 Uniprot
P04049-1 (RAF1) T268 Uniprot
P04049 (RAF1) S338 Uniprot
P04049-1 (RAF1) S621 Uniprot
P28482-1 (MAPK1) S29 Uniprot
P45379 (TNNT2) T213 Uniprot
Q02750 (MAP2K1) S218 Uniprot
Q02750-1 (MAP2K1) S222 Uniprot
Q02750-1 (MAP2K1) T286 Uniprot
Q02750-1 (MAP2K1) T292 Uniprot
Q02750-1 (MAP2K1) T386 Uniprot
Q05639 (EEF1A2) S21 Uniprot
Q13118 (KLF10) T93 Uniprot
Q92934 (BAD) S75 Uniprot
Q92934 (BAD) S99 Uniprot
Q92934 (BAD) S118 Uniprot

Research Backgrounds

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.

PTMs:

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 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. Dephosphorylation at Ser-338 by PPP5C results in an activity decrease.

Methylated at Arg-563 in response to EGF treatment. This modification leads to destabilization of the protein, possibly through proteasomal degradation.

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.

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Tissue Specificity:

In skeletal muscle, isoform 1 is more abundant than isoform 2.

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 LZTR1. 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). Probably forms a complex composed of chaperones HSP90 and HSP70, co-chaperones CDC37, PPP5C, TSC1 and client protein TSC2, CDK4, AKT, RAF1 and NR3C1; this complex does not contain co-chaperones STIP1/HOP and PTGES3/p23. 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 (By similarity). 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, PPP1R12A, PKB/AKT1, PPP2CA, PPP2R1B, SPRY2, SPRY4, CNKSR1/CNK1, KSR2 and PHB/prohibitin. Interacts with ROCK2 (By similarity). In its active form, interacts with PRMT5. Interacts with FAM83B; displaces 14-3-3 proteins from RAF1 and activates RAF1. Interacts with PDE8A; the interaction promotes RAF1 activity. Interacts with MFHAS1. Interacts with GLS.

Family&Domains:

Belongs to the protein kinase superfamily. TKL Ser/Thr protein kinase family. RAF subfamily.

Research Fields

· Cellular Processes > Transport and catabolism > Autophagy - animal.   (View pathway)

· Cellular Processes > Cell growth and death > Apoptosis.   (View pathway)

· Cellular Processes > Cell growth and death > Cellular senescence.   (View pathway)

· Cellular Processes > Cellular community - eukaryotes > Focal adhesion.   (View pathway)

· Cellular Processes > Cellular community - eukaryotes > Gap junction.   (View pathway)

· Cellular Processes > Cellular community - eukaryotes > Signaling pathways regulating pluripotency of stem cells.   (View pathway)

· Cellular Processes > Cell motility > Regulation of actin cytoskeleton.   (View pathway)

· Environmental Information Processing > Signal transduction > MAPK signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > ErbB signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > Ras signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > Rap1 signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > cGMP-PKG signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > cAMP signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > FoxO signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > Sphingolipid signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > Phospholipase D signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > mTOR signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > PI3K-Akt signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > Apelin signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > Jak-STAT signaling pathway.   (View pathway)

· Human Diseases > Drug resistance: Antineoplastic > EGFR tyrosine kinase inhibitor resistance.

· Human Diseases > Drug resistance: Antineoplastic > Endocrine resistance.

· Human Diseases > Substance dependence > Alcoholism.

· Human Diseases > Infectious diseases: Bacterial > Tuberculosis.

· Human Diseases > Infectious diseases: Viral > Hepatitis C.

· Human Diseases > Infectious diseases: Viral > Hepatitis B.

· Human Diseases > Infectious diseases: Viral > Influenza A.

· Human Diseases > Infectious diseases: Viral > Human papillomavirus infection.

· Human Diseases > Cancers: Overview > Pathways in cancer.   (View pathway)

· Human Diseases > Cancers: Overview > Proteoglycans in cancer.

· Human Diseases > Cancers: Overview > MicroRNAs in cancer.

· Human Diseases > Cancers: Specific types > Colorectal cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Renal cell carcinoma.   (View pathway)

· Human Diseases > Cancers: Specific types > Pancreatic cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Endometrial cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Glioma.   (View pathway)

· Human Diseases > Cancers: Specific types > Prostate cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Melanoma.   (View pathway)

· Human Diseases > Cancers: Specific types > Bladder cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Chronic myeloid leukemia.   (View pathway)

· Human Diseases > Cancers: Specific types > Acute myeloid leukemia.   (View pathway)

· Human Diseases > Cancers: Specific types > Non-small cell lung cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Breast cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Hepatocellular carcinoma.   (View pathway)

· Human Diseases > Cancers: Specific types > Gastric cancer.   (View pathway)

· Human Diseases > Cancers: Overview > Central carbon metabolism in cancer.   (View pathway)

· Human Diseases > Cancers: Overview > Choline metabolism in cancer.   (View pathway)

· Organismal Systems > Immune system > Chemokine signaling pathway.   (View pathway)

· Organismal Systems > Circulatory system > Vascular smooth muscle contraction.   (View pathway)

· Organismal Systems > Development > Axon guidance.   (View pathway)

· Organismal Systems > Immune system > Natural killer cell mediated cytotoxicity.   (View pathway)

· Organismal Systems > Immune system > T cell receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > B cell receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > Fc epsilon RI signaling pathway.   (View pathway)

· Organismal Systems > Immune system > Fc gamma R-mediated phagocytosis.   (View pathway)

· Organismal Systems > Nervous system > Long-term potentiation.

· Organismal Systems > Nervous system > Neurotrophin signaling pathway.   (View pathway)

· Organismal Systems > Nervous system > Serotonergic synapse.

· Organismal Systems > Nervous system > Long-term depression.

· Organismal Systems > Endocrine system > Insulin signaling pathway.   (View pathway)

· Organismal Systems > Endocrine system > Progesterone-mediated oocyte maturation.

· Organismal Systems > Endocrine system > Estrogen signaling pathway.   (View pathway)

· Organismal Systems > Endocrine system > Melanogenesis.

· Organismal Systems > Endocrine system > Prolactin signaling pathway.   (View pathway)

· Organismal Systems > Endocrine system > Thyroid hormone signaling pathway.   (View pathway)

· Organismal Systems > Endocrine system > Oxytocin signaling pathway.

· Organismal Systems > Endocrine system > Relaxin signaling pathway.

References

1). Zhou H et al. Curcumin promotes cell cycle arrest and apoptosis of acute myeloid leukemia cells by inactivating AKT. Oncol Rep 2021 Apr;45(4):11. (PubMed: 33649826) [IF=4.136]

Application: WB    Species: Human    Sample: ML-2 cells

Figure 1. Influence of curcumin (CCM) on protein phosphorylation, as detected via antibody array and western blotting. (A) Scatter plot demonstrating the relative expression levels of the 55 phosphorylated proteins. ML-2 cells were treated with or without CCM for 6 h. After treatment, a phosphorylation antibody array was used for detecting the relative levels of phosphorylation. The red dots represent upregulated proteins, the blue dots represent downregulated proteins and the grey dots represent non-differentially phosphorylated proteins. (B) Most enriched KEGG pathways of the 18 differentially phosphorylated proteins. (C) PPI network of the 18 differentially phosphorylated proteins. (D) Western blot analysis of phosphorylation levels of AKT and its interacting proteins in ML-2 cells after treatment with increasing concentrations of CCM for 6 h. CCM, curcumin; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein-protein interaction.

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