Antibodies to Focal Adhesion Kinase (FAK)

Introduction

Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor protein tyrosine kinase involved in integrin-mediated control of cell behaviour.

Integrins are the major cell surface receptors for extracellular matrix molecules, which play critical roles in a variety of biological processes. Focal adhesion kinase (FAK) has been discovered as a substrate for Src and it is a key protein in the signal transduction pathways triggered by integrins. FAK plays a central role in cell spreading, differentiation, migration, cell death and acceleration of the G1 to S phase transition of the cell cycle. Aggregation of FAK with integrins and cytoskeletal proteins in focal contacts has been proposed to be responsible for FAK activation and autophosphorylation by integrins in cell adhesion.

Interaction with integrin and focal adhesion kinase (FAK) regulates the cancer cell adhesion and invasion into extracellular matrix (ECM). In addition, phosphorylation of FAK correlates with the increase of cell motility and invasion. Adhesion and spreading of cancer cells on a variety of ECM proteins, including collagen type IV (Coll IV), leads to an increase in tyrosine phosphorylation and activation of FAK. FAK regulation includes phosphorylation at multiple tyrosine and serine residues. Phosphorylation of tyrosine generally is associated with positive regulation and growth promotion, however, dephosphorylation at these sites occurs as cells enter mitosis (M-Phase of the cell cycle).

In contrast, serine phosphorylation either remains high or is increased as cells enter mitosis and may play a role in focal adhesion disassembly.

Antibody Panel to Focal Adhesion Kinase (FAK)

Acris Antibodies offers a range of polyclonal antibodies to FAK and phoshorylated FAK (serine or tyrosine phosphospecific antibodies). All products are suitable for Western blot application, some of these antibodies can be used for Immunohistochemistry and / or Immunofluorescence (for more details see pictures and table below.)

Fig.1: Peptide competition experiment using anti-FAKpSer732- antibody Cat. No. BP7066:

Extracts prepared from PC12 cells treated with nocozadole were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer for one hour at room temperature, then incubated with FAK [pSer732] antibody for two hours at room temperature in a 1% Milk-TBST buffer, following prior incu¬bation with: no peptide (1), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphoserine containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal method.

The data show that only the peptide corresponding to FAK [pSer732] blocks the antibody signal, thereby demonstrating the specificity of the antibody.

Fig.2:Peptide competition experiment using anti-FAKpTyr861 antibody Cat. No. BP7073:

Extracts prepared from CEF cells expressing FAK and plated on fibronectin were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then were incubated with the FAK [pTyr861] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: (1) the Phospho¬peptide immunogen, (2) a generic phosphotyrosine containing peptide, (3) the non-phosphorylated peptide corresponding to the phosphopeptide, and (4) no peptide.

After washing, membranes were incubated with goat F(ab’)2 antirabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal method.

The data show that only the peptide corresponding to FAK [pTyr861] blocks the antibody signal, thereby demonstrating the specificity of the antibody.

Antibody Panel to FAK

Focal Adhesion Kinase (FAK) –antibody.

Literature

Sawai H, Okada Y, Funahashi H, Matsuo Y, Takahashi H, Takeyama H, Manabe T. Activation of focal adhesion kinase enhances the adhesion and invasion of pancreatic cancer cells via extrcellular signal-regulated kinase-1/2 signaling pathway activation. Molecular Cancer 2005; 4:37.

Eliceiri, B.P., et al. (2002) Src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta5 in vascular endothelial growth factor signaling. J. Cell Biol. 157(1):149-160.

Hauck, C.R., et al. (2002) v-Src SH3-enhanced interaction with focal adhesion kinase at beta 1 integrin-containing invadopodia promotes cell invasion. J. Biol. Chem. 277(15):12487-12490.

Guan JL. Role of focal adhesion kinase in integrin signalling. Int J Biochem Cell Biol. 1997 Aug-Sep; 29(8-9): 1085-96.

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