Phospho-RAF1 (S301) Antibody

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Description

Definition of Phospho-RAF1 (S301) Antibody

The Phospho-RAF1 (S301) Antibody is a research-grade immunoglobulin designed to detect the phosphorylation of Raf-1 kinase at serine residue 301 (S301). Raf-1 is a critical serine/threonine kinase in the Ras-MAPK/ERK signaling cascade, which regulates cellular growth, differentiation, and apoptosis. The phosphorylation at S301 is part of a regulatory mechanism that modulates Raf-1 activity, with evidence suggesting it plays a role in both activation and feedback regulation .

Mechanism of Raf-1 Phosphorylation at S301

Phosphorylation at S301 is mediated by extracellular signal-regulated kinase (ERK)-1/2, part of a positive feedback loop in the MAPK cascade . This modification:

  • Stabilizes active Raf-1: Enhances kinase activity and sustains signaling through MEK/ERK .

  • Regulates Raf-1 localization: Phosphorylation at S301, alongside other sites (e.g., S338, S339), influences mitochondrial and nuclear translocation .

  • Counterbalances inhibitory modifications: ERK-mediated phosphorylation at S301/S289/S296 counteracts inhibitory phosphorylation by PKA/AKT at S259 .

Role in Signal Transduction

  • Positive Feedback Regulation: ERK phosphorylates Raf-1 at S301, creating a self-sustaining loop that amplifies MAPK signaling .

  • Cancer Implications: Hyperactivation of Raf-1 via S301 phosphorylation is linked to oncogenic transformation and resistance to targeted therapies .

Experimental Validation

  • Western Blot: Detects a 73–74 kDa band corresponding to phosphorylated Raf-1. Signal is abolished by lambda-phosphatase treatment .

  • ELISA: Quantifies phospho-S301 Raf-1 levels in cell lysates, with Jurkat cells treated with PMA as a positive control .

  • IHC: Visualizes phosphorylated Raf-1 in paraffin-embedded tissues, such as human tonsil sections .

Key Insights from Research Studies

  • Feedback Regulation: ERK phosphorylates Raf-1 at S301, creating a positive feedback loop that sustains MAPK signaling .

  • Oncogenic Potential: Hyperphosphorylation at S301/S338/S339 correlates with Raf-1 activation in cancer cells, promoting proliferation and survival .

  • Therapeutic Targeting: Inhibitors of Raf-1 phosphorylation (e.g., MEK inhibitors) are under investigation for cancer therapy .

Product Specs

Buffer
Liquid in PBS containing 50% glycerol, 0.5% BSA and 0.02% sodium azide.
Form
Liquid
Lead Time
Generally, we can ship the products within 1-3 business days after receiving your order. Delivery time may vary depending on the purchase method or location. Please consult your local distributors for specific delivery time.
Synonyms
c Raf antibody; C-raf antibody; C-Raf proto-oncogene, serine/threonine kinase antibody; CMD1NN antibody; Craf 1 transforming gene antibody; cRaf antibody; Craf1 transforming gene antibody; EC 2.7.11.1 antibody; kinase Raf1 antibody; Murine sarcoma 3611 oncogene 1 antibody; NS5 antibody; Oncogene MIL antibody; Oncogene RAF1 antibody; OTTHUMP00000160218 antibody; OTTHUMP00000207813 antibody; OTTHUMP00000209389 antibody; Protein kinase raf 1 antibody; Proto-oncogene c-RAF antibody; Raf 1 antibody; Raf 1 proto oncogene serine/threonine kinase antibody; RAF antibody; Raf proto oncogene serine/threonine protein kinase antibody; RAF proto-oncogene serine/threonine-protein kinase antibody; RAF-1 antibody; RAF1 antibody; RAF1_HUMAN antibody; Similar to murine leukemia viral (V-raf-1) oncogene homolog 1 antibody; TRANSFORMING REPLICATION-DEFECTIVE MURINE RETROVIRUS 3611-MSV antibody; v raf 1 murine leukemia viral oncogene homolog 1 antibody; v-raf murine sarcoma viral oncogene homolog 1 antibody; v-raf-1 murine leukemia viral oncogene-like protein 1 antibody; vraf1 murine leukemia viral oncogene homolog 1 antibody
Target Names
Uniprot No.

Target Background

Function
RAF1, a serine/threonine-protein kinase, acts as a crucial regulatory link between membrane-associated Ras GTPases and the MAPK/ERK cascade. This vital regulatory role acts as a switch influencing cell fate decisions, encompassing proliferation, differentiation, apoptosis, survival, and oncogenic transformation. The activation of RAF1 initiates a mitogen-activated protein kinase (MAPK) cascade that involves 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'. It phosphorylates adenylyl cyclases: ADCY2, ADCY5, and ADCY6, leading to their activation. It further phosphorylates PPP1R12A, resulting in the inhibition of phosphatase activity. RAF1 also phosphorylates TNNT2/cardiac muscle troponin T. It can promote NF-kB activation and inhibit signal transducers involved in motility (ROCK2), apoptosis (MAP3K5/ASK1 and STK3/MST2), proliferation, and angiogenesis (RB1). RAF1 can also protect cells from apoptosis by translocating to the mitochondria where it binds BCL2 and displaces BAD/Bcl2-antagonist of cell death. It regulates Rho signaling and migration and is essential for normal wound healing. RAF1 plays a role in the oncogenic transformation of epithelial cells through repression of the TJ protein, occludin (OCLN), by inducing the up-regulation of a transcriptional repressor SNAI2/SLUG, which subsequently down-regulates OCLN. RAF1 restricts caspase activation in response to specific stimuli, including Fas stimulation, pathogen-mediated macrophage apoptosis, and erythroid differentiation.
Gene References Into Functions
  1. Functional analysis supported the pathogenicity of the RAF1 and RIT1 variants of uncertain significance (VUSs), while the significance of two VUSs in A2ML1 remained unclear. PMID: 29402968
  2. This report presents the second familial case of Noonan syndrome due to a germline p.S427G substitution in RAF1, without any occurrence of malignant tumor. This may suggest that carrying a germline mutation in the RAF1 oncogene might not be associated with an increased risk of tumor development. Notably, RAF1 mutations have been observed as somatic events in many types of cancer. PMID: 30204961
  3. Data indicate that Raf-1 proto-oncogene, serine-threonine kinase (RAF1) is a negative regulator of hepatocarcinogenesis. PMID: 28000790
  4. We report a patient with an inherited RAF1-associated Noonan syndrome, presenting with an antenatally diagnosed abnormality of skull shape, bilateral subdural haematomas of unknown cause, delayed myelination, and polymicrogyria. PMID: 27753652
  5. Raf1 may serve as a novel prognostic factor and potential target for improving the long-term outcome of non-small cell lung cancer (NSCLC). PMID: 29484414
  6. Results provide evidence that RAF1 binding to SPRY4 is regulated by miR-1908 in glioma tumors. PMID: 29048686
  7. High RAF1 expression is associated with malignant melanoma. PMID: 28677804
  8. Two premature neonates with progressive biventricular hypertrophy found to have RAF1 variants in the CR2 domain, are reported. PMID: 28777121
  9. Data indicate connector enhancer of kinase suppressor of Ras 1 protein (CNK1) as a molecular platform that controls c-raf protein (RAF) and c-akt protein (AKT) signaling and determines cell fate decisions in a cell type- and cell stage-dependent manner. PMID: 27901111
  10. CRAF is a bona fide alternative oncogene for BRAF/NRAS/GNAQ/GNA11 wild-type melanomas. PMID: 27273450
  11. Authors evaluated the expression of known targets of miR-125a and found that sirtuin-7, matrix metalloproteinase-11, and c-Raf were up-regulated in tumor tissue by 2.2-, 3-, and 1.7-fold, respectively. Overall, these data support a tumor suppressor role for miR-125a. PMID: 28445974
  12. Overexpression of ciRS-7 in HCT116 and HT29 cells led to the blocking of miR-7 and resulted in a more aggressive oncogenic phenotype, and ciRS-7 overexpression permitted the inhibition of miR-7 and subsequent activation of EGFR and RAF1 oncogenes. PMID: 28174233
  13. miR-497 could serve as a tumor suppressor and a potential early diagnostic marker of gastric cancer by targeting Raf-1 proto-oncogene. PMID: 28586056
  14. RAF1 may have a role in survival in hepatocellular carcinoma and indicate whether sorafenib should be used as a postoperative adjuvant. PMID: 26981887
  15. Mutational activation of Kit-, Ras/Raf/Erk- and Akt- pathways indicate the biological importance of these pathways and their components as potential targets for therapy. PMID: 27391150
  16. Results indicate that des-gamma-carboxy prothrombin (DCP) antagonizes the inhibitory effects of Sorafenib on hepatocellular carcinoma (HCC) through activation of the Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways. PMID: 27167344
  17. DiRas3 binds to KSR1 independently of its interaction with activated Ras and RAF. PMID: 27368419
  18. RhoA/ROCK and Raf-1/CK2 pathway are responsible for TNF-alpha-mediated endothelial cytotoxicity via regulation of the vimentin cytoskeleton. PMID: 28743511
  19. Although Raf-1 gene is not mutated, an abnormality of Raf-1 kinase feedback regulation enhances its antiapoptotic function, and Raf-1 can still be a pharmaceutical target to increase chemotherapy or radiotherapy sensitivity in these cancer cells. PMID: 27841865
  20. RAF1 plays a critical role in maintaining the transformed phenotype of CRC cells, including those with mutated KRAS. PMID: 27670374
  21. This finding suggests that stringent assemblage of Hsp90 keeps CRAF kinase equipped for participating in the MAPK pathway. Thus, the role of Hsp90 in CRAF maturation and activation acts as a limiting factor to maintain the function of a strong client like CRAF kinase. PMID: 27703006
  22. Oncogenic NFIA:RAF1 fusion activation of the MAPK pathway is associated with pilocytic astrocytoma. PMID: 27810072
  23. IGF2BP2 as a post-transcriptional regulatory mRNA-binding factor, interfering with Raf-1 degradation by miR-195, that contributes to Colorectal carcinogenesis. PMID: 27153315
  24. Data show that when microRNA miR-125b was over-expressed in THP-1 macrophages, the expression of Raf1 proto-oncogene serine/threonine protein kinase (RAF1) was reduced to promote the apoptosis of macrophages. PMID: 27363278
  25. Data show that Griffipavixanthone (GPX), a dimeric xanthone isolated from Garcinia esculenta, is a B-RAF and C-RAF inhibitor against esophageal cancer cells. PMID: 26646323
  26. Up-regulation of Raf-1 is associated with triple-negative breast cancer. PMID: 26513016
  27. This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3zeta protein and gives structural insights responsible for phosphorylation-mediated protein binding. PMID: 26295714
  28. a model that CD166 regulates MCAM through a signaling flow from activation of PI3K/AKT and c-Raf/MEK/ERK signaling to the inhibition of potential MCAM ubiquitin E3 ligases, betaTrCP and Smurf1. PMID: 26004137
  29. Suggest an interrelated kinase module involving c-Raf/PI3K/Lyn and perhaps Fgr functions in a nontraditional way during retinoic acid-induced maturation or during rescue of RA induction therapy using inhibitor co-treatment in RA-resistant leukemia cells. PMID: 25817574
  30. Abnormal activation of the Ras/MAPK pathway may play a significant role in the development of pulmonary vascular disease in the subset of patients with Noonan syndrome and a specific RAF1 mutation. PMID: 25706034
  31. Raf-1 may be an important biomarker in predicting the prognosis of chordoma patients. PMID: 25755752
  32. In the presence of Raf1, the RasQ61L mutant has a rigid switch II relative to the wild-type and increased flexibility at the interface with switch I, which propagates across Raf-Ras binding domain. PMID: 25684575
  33. Besides mediating the anticancer effect, pDAPK(S308) may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal preclinical model that is worthy of clinical translation. PMID: 26100670
  34. DJ-1 directly binds to the kinase domain of c-Raf to stimulate its self-phosphorylation, followed by phosphorylation of MEK and ERK1/2 in EGF-treated cells. PMID: 26048984
  35. Truncated RAF1 and BRAF proteins, recently described as products of genomic rearrangements in gastric cancer and other malignancies, have the ability to render neoplastic cells resistant to RTK-targeted therapy. PMID: 25473895
  36. Our study demonstrated that miR-455-RAF1 may represent a new potential therapeutic target for colorectal carcinoma treatment. PMID: 25355599
  37. This approach identified 18 kinase and kinase-related genes whose overexpression can substitute for EGFR in EGFR-dependent PC9 cells, and these genes include seven of nine Src family kinase genes, FGFR1, FGFR2, ITK, NTRK1, NTRK2, MOS, MST1R, and RAF1. PMID: 25512530
  38. Aberrant expression of A-, B-, and C-RAF, and COT is frequent in PTC; increased expression of COT is correlated with recurrence of PTC. PMID: 25674762
  39. Authors demonstrate that the N-terminus of human Raf1 kinase (hRaf11-147aa) binds with human RKIP (hRKIP) at its ligand-binding pocket, loop "127-149", and the C-terminal helix by nuclear magnetic resonance experiments. PMID: 24863296
  40. Including several anti-apoptotic Bcl-2 family members and c-Raf. PMID: 24969872
  41. These data suggest that miR-7-5p functions as a tumor suppressor gene to regulate glioblastoma microvascular endothelial cell proliferation potentially by targeting the RAF1 oncogene. PMID: 25027403
  42. A novel mechanism for response was discovered whereby high expression level of CAV-1 at the plasma membrane disrupts the BRaf/CRaf heterodimer and thus inhibits the activation of the MAPK pathway during dasatinib treatment. PMID: 24486585
  43. Results show that ubiquitination and levels of RAF-1 is controlled by both Shoc2 and HUWE1. PMID: 25022756
  44. Raf-1/JNK /p53/p21 pathway may be involved in apoptosis, and NFkappaB1 may play a possible role in inhibiting apoptosis. PMID: 22282237
  45. The higher expression of RAF1 mRNA and the activation of AKT/ERK proteins in vinorelbine-resistant non-small cell lung cancer cell lines may confer resistance to vinorelbine. PMID: 24427333
  46. Analysis of RAF1 mutations in cohorts of South Indian, North Indian, and Japanese patients with childhood-onset dilated cardiomyopathy. PMID: 24777450
  47. Expression of miR-195 or knockdown of Raf-1 can similarly reduce tumor cell survival. PMID: 23760062
  48. We hypothesize a potential direct or indirect role for SRC, RAF1, PTK2B genes in neurotransmission and in central nervous system signaling processes. PMID: 24108181
  49. We identified multiple C-RAF mutations that produced biochemical and pharmacologic resistance in melanoma cell lines. PMID: 23737487
  50. ARAF seems to stabilize BRAF:CRAF complexes in cells treated with RAF inhibitors and thereby regulate cell signaling in a subtle manner to ensure signaling efficiency. PMID: 22926515

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Database Links

HGNC: 9829

OMIM: 164760

KEGG: hsa:5894

STRING: 9606.ENSP00000251849

UniGene: Hs.159130

Involvement In Disease
Noonan syndrome 5 (NS5); LEOPARD syndrome 2 (LPRD2); Cardiomyopathy, dilated 1NN (CMD1NN)
Protein Families
Protein kinase superfamily, TKL Ser/Thr protein kinase family, RAF subfamily
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.
Tissue Specificity
In skeletal muscle, isoform 1 is more abundant than isoform 2.

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