Product: Phospho-FAK (Tyr925) Antibody
Catalog: AF3619
Description: Rabbit polyclonal antibody to Phospho-FAK (Tyr925)
Application: IHC
Reactivity: Human, Mouse, Rat
Mol.Wt.: 119kD(Calculated).
Uniprot: Q05397
RRID: AB_2846933

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Product Info

Source:
Rabbit
Application:
IHC 1:50-1:200
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

Reactivity:
Human,Mouse,Rat
Clonality:
Polyclonal
Specificity:
Phospho-FAK (Tyr925) Antibody detects endogenous levels of FAK only when phosphorylated at Tyr925.
RRID:
AB_2846933
Cite Format: Affinity Biosciences Cat# AF3619, RRID:AB_2846933.
Conjugate:
Unconjugated.
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

FADK 1; FADK; FAK related non kinase polypeptide; FAK1; FAK1_HUMAN; Focal adhesion kinase 1; Focal adhesion Kinase; Focal adhesion kinase isoform FAK Del33; Focal adhesion kinase related nonkinase; FRNK; p125FAK; pp125FAK; PPP1R71; Protein phosphatase 1 regulatory subunit 71; Protein tyrosine kinase 2; Protein-tyrosine kinase 2; Ptk2; PTK2 protein tyrosine kinase 2;

Immunogens

Immunogen:

A synthesized peptide derived from human FAK around the phosphorylation site of Tyr925.

Uniprot:
Gene(ID):
Expression:
Q05397 FAK1_HUMAN:

Detected in B and T-lymphocytes. Isoform 1 and isoform 6 are detected in lung fibroblasts (at protein level). Ubiquitous.

Sequence:
MAAAYLDPNLNHTPNSSTKTHLGTGMERSPGAMERVLKVFHYFESNSEPTTWASIIRHGDATDVRGIIQKIVDSHKVKHVACYGFRLSHLRSEEVHWLHVDMGVSSVREKYELAHPPEEWKYELRIRYLPKGFLNQFTEDKPTLNFFYQQVKSDYMLEIADQVDQEIALKLGCLEIRRSYWEMRGNALEKKSNYEVLEKDVGLKRFFPKSLLDSVKAKTLRKLIQQTFRQFANLNREESILKFFEILSPVYRFDKECFKCALGSSWIISVELAIGPEEGISYLTDKGCNPTHLADFTQVQTIQYSNSEDKDRKGMLQLKIAGAPEPLTVTAPSLTIAENMADLIDGYCRLVNGTSQSFIIRPQKEGERALPSIPKLANSEKQGMRTHAVSVSETDDYAEIIDEEDTYTMPSTRDYEIQRERIELGRCIGEGQFGDVHQGIYMSPENPALAVAIKTCKNCTSDSVREKFLQEALTMRQFDHPHIVKLIGVITENPVWIIMELCTLGELRSFLQVRKYSLDLASLILYAYQLSTALAYLESKRFVHRDIAARNVLVSSNDCVKLGDFGLSRYMEDSTYYKASKGKLPIKWMAPESINFRRFTSASDVWMFGVCMWEILMHGVKPFQGVKNNDVIGRIENGERLPMPPNCPPTLYSLMTKCWAYDPSRRPRFTELKAQLSTILEEEKAQQEERMRMESRRQATVSWDSGGSDEAPPKPSRPGYPSPRSSEGFYPSPQHMVQTNHYQVSGYPGSHGITAMAGSIYPGQASLLDQTDSWNHRPQEIAMWQPNVEDSTVLDLRGIGQVLPTHLMEERLIRQQQEMEEDQRWLEKEERFLKPDVRLSRGSIDREDGSLQGPIGNQHIYQPVGKPDPAAPPKKPPRPGAPGHLGSLASLSSPADSYNEGVKLQPQEISPPPTANLDRSNDKVYENVTGLVKAVIEMSSKIQPAPPEEYVPMVKEVGLALRTLLATVDETIPLLPASTHREIEMAQKLLNSDLGELINKMKLAQQYVMTSLQQEYKKQMLTAAHALAVDAKNLLDVIDQARLKMLGQTRPH

PTMs - Q05397 As Substrate

Site PTM Type Enzyme
A2 Acetylation
Y5 Phosphorylation P09619 (PDGFRB) , P08581 (MET) , P00533 (EGFR)
T13 Phosphorylation
S16 Phosphorylation
T20 Phosphorylation
T24 Phosphorylation
S29 Phosphorylation
R65 Methylation
K70 Ubiquitination
Y148 Phosphorylation
Y155 Phosphorylation
Y194 Phosphorylation P09619 (PDGFRB) , P12931 (SRC) , P00533 (EGFR) , P51813 (BMX) , P08581 (MET)
K199 Ubiquitination
K216 Ubiquitination
S248 Phosphorylation
Y347 Phosphorylation
K364 Ubiquitination
S372 Phosphorylation
T386 Phosphorylation
S390 Phosphorylation
S392 Phosphorylation
T394 Phosphorylation
Y397 Phosphorylation P12931 (SRC) , Q05397 (PTK2) , P08581 (MET) , P46108 (CRK) , A0A059VC25 (FAK) , P09769 (FGR)
T406 Phosphorylation
Y407 Phosphorylation P08581 (MET) , Q05397 (PTK2) , Q14289 (PTK2B) , P12931 (SRC)
T408 Phosphorylation
Y441 Phosphorylation P51813 (BMX) , P12931 (SRC)
S443 Phosphorylation
T460 Phosphorylation
S461 Phosphorylation
S463 Phosphorylation
K467 Acetylation
T474 Phosphorylation
T503 Phosphorylation
S517 Phosphorylation
S568 Phosphorylation
Y570 Phosphorylation
S574 Phosphorylation
T575 Phosphorylation
Y576 Phosphorylation P08581 (MET) , P06241 (FYN) , P07949 (RET) , Q05397 (PTK2) , A0A059VC25 (FAK) , P51813 (BMX) , P07947 (YES1) , P09769 (FGR) , P12931 (SRC)
Y577 Phosphorylation P12931 (SRC) , A0A059VC25 (FAK) , P51813 (BMX) , P07949 (RET) , P08581 (MET) , Q05397 (PTK2)
S580 Phosphorylation
S677 Phosphorylation
K684 Ubiquitination
S695 Phosphorylation P31749 (AKT1)
T700 Phosphorylation P31749 (AKT1)
S702 Phosphorylation
S705 Phosphorylation
S708 Phosphorylation
S716 Phosphorylation
Y720 Phosphorylation
S722 Phosphorylation P49840 (GSK3A)
S726 Phosphorylation
S732 Phosphorylation Q13464 (ROCK1) , Q00535 (CDK5)
Y742 Phosphorylation P12931 (SRC) , P51813 (BMX)
S759 Phosphorylation
Y761 Phosphorylation
T805 Phosphorylation
S840 Phosphorylation
S843 Phosphorylation Q9UQM7 (CAMK2A)
S850 Phosphorylation
Y861 Phosphorylation Q05397 (PTK2) , P07949 (RET) , P08581 (MET) , Q13882 (PTK6) , P16591 (FER) , P12931 (SRC) , P51813 (BMX) , P07947 (YES1)
S887 Phosphorylation
S892 Phosphorylation
S893 Phosphorylation
Y898 Phosphorylation P51813 (BMX) , P12931 (SRC)
S910 Phosphorylation Q00535 (CDK5) , P27361 (MAPK3) , P28482 (MAPK1)
T914 Phosphorylation
S920 Phosphorylation
K923 Ubiquitination
Y925 Phosphorylation P12931 (SRC) , P16591 (FER) , P09769 (FGR) , P51813 (BMX) , P07947 (YES1) , P07949 (RET) , P08581 (MET)
T929 Phosphorylation
T963 Phosphorylation
T967 Phosphorylation
T971 Phosphorylation
K1000 Ubiquitination
Y1007 Phosphorylation P12931 (SRC) , P51813 (BMX)
K1017 Acetylation

PTMs - Q05397 As Enzyme

Substrate Site Source
O00401 (WASL) Y256 Uniprot
O43707 (ACTN4) Y4 Uniprot
O43707 (ACTN4) Y31 Uniprot
O43707 (ACTN4) Y265 Uniprot
O75962-1 (TRIO) Y2796 Uniprot
P07949 (RET) Y905 Uniprot
P12814-1 (ACTN1) Y12 Uniprot
P23634-6 (ATP2B4) Y1176 Uniprot
P26010 (ITGB7) Y753 Uniprot
P26010 (ITGB7) Y758 Uniprot
P29353 (SHC1) Y315 Uniprot
P49023 (PXN) Y31 Uniprot
P49023 (PXN) Y118 Uniprot
P51813 (BMX) Y40 Uniprot
P56945 (BCAR1) Y410 Uniprot
P56945-1 (BCAR1) Y664 Uniprot
P56945-1 (BCAR1) Y666 Uniprot
P63000 (RAC1) Y64 Uniprot
Q05397-1 (PTK2) S390 Uniprot
Q05397 (PTK2) Y397 Uniprot
Q05397 (PTK2) Y407 Uniprot
Q05397 (PTK2) Y576 Uniprot
Q05397 (PTK2) Y577 Uniprot
Q05397-1 (PTK2) S850 Uniprot
Q05397 (PTK2) Y861 Uniprot
Q05397-1 (PTK2) Y925 Uniprot
Q14451 (GRB7) Y188 Uniprot
Q14451 (GRB7) Y338 Uniprot
Q658W2 (DKFZp666O0110) S412 Uniprot
Q658W2 (DKFZp666O0110) Y419 Uniprot
Q658W2 (DKFZp666O0110) S872 Uniprot
Q658W2 (DKFZp666O0110) Y947 Uniprot
Q99961 (SH3GL1) Y315 Uniprot
Q9H5V8 (CDCP1) Y734 Uniprot
Q9H9S0 (NANOG) Y35 Uniprot
Q9H9S0 (NANOG) Y174 Uniprot
Q9Y2X7 (GIT1) Y321 Uniprot

Research Backgrounds

Function:

Non-receptor protein-tyrosine kinase that plays an essential role in regulating cell migration, adhesion, spreading, reorganization of the actin cytoskeleton, formation and disassembly of focal adhesions and cell protrusions, cell cycle progression, cell proliferation and apoptosis. Required for early embryonic development and placenta development. Required for embryonic angiogenesis, normal cardiomyocyte migration and proliferation, and normal heart development. Regulates axon growth and neuronal cell migration, axon branching and synapse formation; required for normal development of the nervous system. Plays a role in osteogenesis and differentiation of osteoblasts. Functions in integrin signal transduction, but also in signaling downstream of numerous growth factor receptors, G-protein coupled receptors (GPCR), EPHA2, netrin receptors and LDL receptors. Forms multisubunit signaling complexes with SRC and SRC family members upon activation; this leads to the phosphorylation of additional tyrosine residues, creating binding sites for scaffold proteins, effectors and substrates. Regulates numerous signaling pathways. Promotes activation of phosphatidylinositol 3-kinase and the AKT1 signaling cascade. Promotes activation of MAPK1/ERK2, MAPK3/ERK1 and the MAP kinase signaling cascade. Promotes localized and transient activation of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), and thereby modulates the activity of Rho family GTPases. Signaling via CAS family members mediates activation of RAC1. Recruits the ubiquitin ligase MDM2 to P53/TP53 in the nucleus, and thereby regulates P53/TP53 activity, P53/TP53 ubiquitination and proteasomal degradation. Phosphorylates SRC; this increases SRC kinase activity. Phosphorylates ACTN1, ARHGEF7, GRB7, RET and WASL. Promotes phosphorylation of PXN and STAT1; most likely PXN and STAT1 are phosphorylated by a SRC family kinase that is recruited to autophosphorylated PTK2/FAK1, rather than by PTK2/FAK1 itself. Promotes phosphorylation of BCAR1; GIT2 and SHC1; this requires both SRC and PTK2/FAK1. Promotes phosphorylation of BMX and PIK3R1. Isoform 6 (FRNK) does not contain a kinase domain and inhibits PTK2/FAK1 phosphorylation and signaling. Its enhanced expression can attenuate the nuclear accumulation of LPXN and limit its ability to enhance serum response factor (SRF)-dependent gene transcription.

PTMs:

Phosphorylated on tyrosine residues upon activation, e.g. upon integrin signaling. Tyr-397 is the major autophosphorylation site, but other kinases can also phosphorylate this residue. Phosphorylation at Tyr-397 promotes interaction with SRC and SRC family members, leading to phosphorylation at Tyr-576, Tyr-577 and at additional tyrosine residues. FGR promotes phosphorylation at Tyr-397 and Tyr-576. FER promotes phosphorylation at Tyr-577, Tyr-861 and Tyr-925, even when cells are not adherent. Tyr-397, Tyr-576 and Ser-722 are phosphorylated only when cells are adherent. Phosphorylation at Tyr-397 is important for interaction with BMX, PIK3R1 and SHC1. Phosphorylation at Tyr-925 is important for interaction with GRB2. Dephosphorylated by PTPN11; PTPN11 is recruited to PTK2 via EPHA2 (tyrosine phosphorylated). Microtubule-induced dephosphorylation at Tyr-397 is crucial for the induction of focal adhesion disassembly; this dephosphorylation could be catalyzed by PTPN11 and regulated by ZFYVE21. Phosphorylation on tyrosine residues is enhanced by NTN1 (By similarity).

Sumoylated; this enhances autophosphorylation.

Subcellular Location:

Cell junction>Focal adhesion. Cell membrane>Peripheral membrane protein>Cytoplasmic side. Cytoplasm>Cell cortex. Cytoplasm>Cytoskeleton. Cytoplasm>Cytoskeleton>Microtubule organizing center>Centrosome. Nucleus.
Note: Constituent of focal adhesions. Detected at microtubules.

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

Detected in B and T-lymphocytes. Isoform 1 and isoform 6 are detected in lung fibroblasts (at protein level). Ubiquitous.

Subunit Structure:

Interacts (via first Pro-rich region) with CAS family members (via SH3 domain), including BCAR1, BCAR3, CASS4 and NEDD9. Interacts with GIT1. Interacts with SORBS1. Interacts with ARHGEF28. Interacts with SHB. Interacts with PXN and TLN1. Interacts with STAT1. Interacts with DCC. Interacts with WASL. Interacts with ARHGEF7. Interacts with GRB2 and GRB7 (By similarity). Component of a complex that contains at least FER, CTTN and PTK2/FAK1. Interacts with BMX. Interacts with TGFB1I1. Interacts with STEAP4. Interacts with ZFYVE21. Interacts with ESR1. Interacts with PIK3R1 or PIK3R2. Interacts with SRC, FGR, FLT4 and RET. Interacts with EPHA2 in resting cells; activation of EPHA2 recruits PTPN11, leading to dephosphorylation of PTK2/FAK1 and dissociation of the complex. Interacts with EPHA1 (kinase activity-dependent). Interacts with CD4; this interaction requires the presence of HIV-1 gp120. Interacts with PIAS1. Interacts with ARHGAP26 and SHC1. Interacts with RB1CC1; this inhibits PTK2/FAK1 activity and activation of downstream signaling pathways. Interacts with P53/TP53 and MDM2. Interacts with LPXN (via LD motif 3). Interacts with MISP. Interacts with CIB1 isoform 2. Interacts with CD36. Interacts with EMP2; regulates PTK2 activation and localization. Interacts with DSCAM (By similarity).

Family&Domains:

The Pro-rich regions interact with the SH3 domain of CAS family members, such as BCAR1 and NEDD9, and with the GTPase activating protein ARHGAP26.

The carboxy-terminal region is the site of focal adhesion targeting (FAT) sequence which mediates the localization of FAK1 to focal adhesions.

Belongs to the protein kinase superfamily. Tyr protein kinase family. FAK subfamily.

Research Fields

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

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

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

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

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

· Human Diseases > Infectious diseases: Bacterial > Bacterial invasion of epithelial cells.

· Human Diseases > Infectious diseases: Parasitic > Amoebiasis.

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

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

· Human Diseases > Cancers: Overview > Transcriptional misregulation in cancer.

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

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

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

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

· Organismal Systems > Immune system > Leukocyte transendothelial migration.   (View pathway)

References

1). Single-cell transcriptomics reveals ependymal subtypes related to cytoskeleton dynamics as the core driver of syringomyelia pathological development. iScience, 2023 (PubMed: 37275526) [IF=5.8]

Application: IF/ICC    Species: Human    Sample: EPCs

Figure 5 Dynamic changes in spinal cord ependymal populations post-op for syringomyelia (A–D) We performed further subtype analysis of the EPCs, the UMAP of which revealed two main distinct subtypes. Subtypes were annotated using a combination of canonical markers, transcription factor expression, and GO enrichment analyses of the DEGs (A). Cell cycle assignment showed that cells in Cluster 5 were mainly in the S phase (B). In general, ependymal cells showed high expression of the EPC-specific marker Foxj1 in the dot plot and violin plot (C). Cells are colored by the expression value of canonical marker genes, the motor ciliary ependymal subpopulations (with featured genes of Cfap43 and Sncg), and the glial ependymal subpopulations (with a higher expression of GFAP and Rnd3). Values are log-transformed normalized expression counts (D). (E, F, and H) The differentiation and development trajectory of EPCs was constructed by the diffusion map of pseudotime analysis. Pseudotime trajectory analysis corresponding to the differentiation of EPCs from Cluster 0 to other clusters. Cluster 5 (proliferating) between Cluster 0 in the early state and the other clusters. Cells are colored by cell types, pseudotime, and cell states, as indicated, from the left to the right panels (E). A ridge plot was drawn based on the pseudotime analysis to simulate the trajectory of cell clusters in cell differentiation and the proportions of ependymal-lineage subtypes at each time point from the top to the bottom (F). GO biological process terms associated with the top DEGs among two different cell subtypes (H). (G) Immunofluorescence shows the expression pattern of Sox2, Foxj1, and Nestin during syringomyelia development. EPCs (Foxj1+) in the CC area represented the EPCs with colocalization of Sox2 and Nestin. During the syringomyelia formation with the CC expansion, the syringomyelia was in a moniliform shape, and there were obvious separations between adjacent dilated compartments. The EPCs are irregularly distributed throughout the central canal due to moniliform-like dilatation. The stacked cells remained as EPCs (Foxj1+) in the stenosis, resembling a tunneling effect and showing that the CC was still communicating, but the monolayer of cells was organized in the significant dilatated area and even disrupted in some regions. The cytoskeleton outlined by Netin also became gradually disorganized or even disordered at post-op 6 weeks.

2). RIOK3 promotes pancreatic ductal adenocarcinoma cell invasion and metastasis by stabilizing FAK. Heliyon, 2023 (PubMed: 35982848) [IF=4.0]

Application: WB    Species: Human    Sample: pancreatic cancer tissues

Figure 1. Knockdown of RIOK3 significantly inhibits FAK activation in PDAC cells. Using lentiviral transfection, PANC-1 cells with stable knockdown of RIOK3 were created, followed by RNA extraction and sequencing. Up-regulated (red) and down-regulated (blue) mRNAs were characterized using volcano plots (A). Differential genes (| Log2FC | > 0.5, FDR ˂ 0.05) were used to perform a KEGG pathway enrichment analysis (B). The Log2 FC of all mRNA expression was used in GSEA (C). The spearman correlation coefficients of RIOK3 with all other genes in the TCGA-PDAC transcriptome database were used in GSEA (D). Western blot assay and quantitative analysis of the protein expression of RIOK3, FAK, Phospho-FAK(Tyr397), Phospho-FAK(Tyr925), Integrin 1, Integrin 4, and vinculin in cells (E). The qRT-PCR method was used to detect RIOK3 and FAK mRNA expression (F). Representative IHC staining of RIOK3 and Phospho-FAK(Tyr925) in pancreatic cancer tissues and correlation analysis between RIOK3 and Phospho-FAK(Tyr925) based on H-Score (G). R represents Pearson correlation coefficient. Analysis of the ratio of phospho-FAK/FAK in Figure 1E (H). The data shown represent the means (SD) of biological triplicates. *p < 0.05, **p < 0.01, ANOVA test. The uncropped images of (E) were referred to in supplementary Figure 1.

Application: IHC    Species: Human    Sample: pancreatic cancer tissues

Figure 1. Knockdown of RIOK3 significantly inhibits FAK activation in PDAC cells. Using lentiviral transfection, PANC-1 cells with stable knockdown of RIOK3 were created, followed by RNA extraction and sequencing. Up-regulated (red) and down-regulated (blue) mRNAs were characterized using volcano plots (A). Differential genes (| Log2FC | > 0.5, FDR ˂ 0.05) were used to perform a KEGG pathway enrichment analysis (B). The Log2 FC of all mRNA expression was used in GSEA (C). The spearman correlation coefficients of RIOK3 with all other genes in the TCGA-PDAC transcriptome database were used in GSEA (D). Western blot assay and quantitative analysis of the protein expression of RIOK3, FAK, Phospho-FAK(Tyr397), Phospho-FAK(Tyr925), Integrin 1, Integrin 4, and vinculin in cells (E). The qRT-PCR method was used to detect RIOK3 and FAK mRNA expression (F). Representative IHC staining of RIOK3 and Phospho-FAK(Tyr925) in pancreatic cancer tissues and correlation analysis between RIOK3 and Phospho-FAK(Tyr925) based on H-Score (G). R represents Pearson correlation coefficient. Analysis of the ratio of phospho-FAK/FAK in Figure 1E (H). The data shown represent the means (SD) of biological triplicates. *p < 0.05, **p < 0.01, ANOVA test. The uncropped images of (E) were referred to in supplementary Figure 1.

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