Peptide Competition: Extracts prepared from CEF cells expressing Pyk2 and plated on fibronectin were resolved by SDS-PAGE on a 10% Tris glycine gel. The proteins were then transferred to nitrocellulose. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4^oC, then were incubated with 0.50 µg/mL ab4800 antibody, following prior incubation with: no peptide (1), the non-phosphopeptide corresponding to phosphopeptide immunogen (2), a generic phosphotyrosine containing peptide (3), the phosphopeptide corresponding to tyrosine 397 on FAK (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar method. The data show that only the phosphopeptide corresponding to this site blocks the antibody signal, therefore demonstrating the specificity of ab4800 antibody for this phosphorylated residue. Peptide Competition: Extracts p

Figure shows Pyk2 staining: Normal mouse mammary (NmuMG) cells were treated with TGFbeta and then grown to confluence, fixed and permeabilized. ab4800 was identified using a selective PSSA followed by incubation with a Cy3-conjugated anti-rabbit secondary antibody (red). F actin was visualized with Oregon Green-conjugated phalloiden (green) and paxillin staining is indicated in blue. The merged image indicates colocalization (purple) of ab4800, F-actin and paxillin in focal adhesion structures.