• Western blot analysis of extracts of HeLa and 293T cells, using Phospho-Raf1-S621 antibody (STJ22366) at 1:1000 dilution. HeLa cells were treated by EGF (100ng/mL) for 30 minutes after serum-starvation overnight. 293T cells were treated by PMA/TPA (200nM) for 30 minutes or treated by EGF (25ug/mL) for 30 minutes after serum-starvation overnight. Secondary antibody: HRP Goat Anti-rabbit IgG (H+L) at 1:10000 dilution. Lysates/proteins: 25ug per lane. Blocking buffer: 3% BSA.
  • Immunoprecipitation analysis of 200 Mu g extracts of HeLa cells, using 3 Mu g Phospho-Raf1-S621 polyclonal antibody (STJ22366). Western blot was performed from the immunoprecipitate using Phospho-Raf1-S621 polyclonal antibody (STJ22366) at a dilution of 1:1000. HeLa cells were treated by EGF (100 ng/mL) at 37 °C for 30 minutes after serum-starvation overnight.
  • Immunoprecipitation analysis of 200ug extracts of HeLa cells, using 3 ug Phospho-Raf1-S621 polyclonal antibody (STJ22366). Western blot was performed from the immunoprecipitate using Phospho-Raf1-S621 polyclonal antibody (STJ22366) at a dilition of 1:1000. HeLa cells were treated by EGF (100 ng/mL) at 37 °C for 30 minutes after serum-starvation overnight.
  • Western blot analysis of lysates from HeLa and 293T cells, using Phospho-Raf1-S621 Rabbit polyclonal antibody (STJ22366) at 1:1000 dilution. HeLa cells were treated by EGF (100ng/mL) for 30 minutes after serum-starvation overnight. 293T cells were treated by PMA/TPA (200nM) for 30 minutes or treated by EGF (25 Mu g/mL) for 30 minutes after serum-starvation overnight. Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) (STJS000856) at 1:10000 dilution. Lysates/proteins: 25 Mu g per lane. Blocking buffer: 3% BSA.

Anti-Phospho-RAF1-S621 antibody (STJ22366)

SKU:
STJ22366

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Host: Rabbit
Applications: WB/IP
Reactivity: Human
Note: STRICTLY FOR FURTHER SCIENTIFIC RESEARCH USE ONLY (RUO). MUST NOT TO BE USED IN DIAGNOSTIC OR THERAPEUTIC APPLICATIONS.
Short Description: Rabbit polyclonal antibody anti-Phospho-Raf1-S621 is suitable for use in Western Blot and Immunoprecipitation research applications.
Clonality: Polyclonal
Conjugation: Unconjugated
Isotype: IgG
Formulation: PBS with 0.02% Sodium Azide, 50% Glycerol, pH7.3.
Purification: Affinity purification
Dilution Range: WB 1:500-1:2000
IP 1:50-1:100
Storage Instruction: Store at-20°C for up to 1 year from the date of receipt, and avoid repeat freeze-thaw cycles.
Gene Symbol: RAF1
Gene ID: 5894
Uniprot ID: RAF1_HUMAN
Immunogen: A synthetic phosphorylated peptide around S621 of human Phospho-Raf1-S621 (NP_002871.1).
Immunogen Sequence: SASEP
Tissue Specificity In skeletal muscle, isoform 1 is more abundant than isoform 2.
Post Translational Modifications 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.
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.
Protein Name Raf Proto-Oncogene Serine/Threonine-Protein Kinase
Proto-Oncogene C-Raf
Craf
Raf-1
Database Links Reactome: R-HSA-2672351
Reactome: R-HSA-392517
Reactome: R-HSA-430116
Reactome: R-HSA-5621575
Reactome: R-HSA-5673000
Reactome: R-HSA-5674135
Reactome: R-HSA-5674499
Reactome: R-HSA-5675221
Reactome: R-HSA-6802946
Reactome: R-HSA-6802948
Reactome: R-HSA-6802952
Reactome: R-HSA-6802955
Reactome: R-HSA-9649948
Reactome: R-HSA-9656223
Reactome: R-HSA-9726840
Reactome: R-HSA-9726842
Reactome: R-HSA-9732724
Cellular Localisation Cytoplasm
Cell Membrane
Mitochondrion
Nucleus
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
Alternative Antibody Names Anti-Raf Proto-Oncogene Serine/Threonine-Protein Kinase antibody
Anti-Proto-Oncogene C-Raf antibody
Anti-Craf antibody
Anti-Raf-1 antibody
Anti-RAF1 antibody
Anti-RAF antibody

Information sourced from Uniprot.org

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