Polyclonal anti tyrosine phosphorylated src py 418

Cultured cells were fixed with 1% paraformaldehyde in PBS for 30 min and treated with 0.05% Triton X-100 in PBS for 5 min for permeabilization. The cells were then treated with 10% normal goat serum for 1 h at room temperature, and stained with monoclonal anti-FAK (1:100) (Takara, Tokyo, Japan), monoclonal anti-alpha-actinin (Sigma), anti-paxillin (BD Biosciences), polyclonal anti-tyrosine-phosphorylated FAK (pY-418; BioSource) antibody, or polyclonal anti-tyrosine-phosphorylated Src (pY-418; BioSource) for 60 min. After washing in PBS for 20 min, the fixed specimens were incubated with fluorescein-conjugated goat anti-rabbit or anti-mouse IgG. Some samples were double stained with anti-FAK. Some samples were stained with FITC (Cytoskeleton) or rhodamine-labeled phalloidin (Molecular Probes) for staining of actin filaments. Samples were then observed by TIRFM.

Monoclonal anti- alpha-actinin (Sigma, St. Louis, MO, USA), monoclonal anti-paxillin (BD Biosciences, Franklin Lakes, NJ, USA), polyclonal anti-focal adhesion kinase (FAK: Takara, Shiga, Japan), polyclonal anti-tyrosine-phosphorylated Src (pY-418) (BioSource, San Jose, CA, USA) and polyclonal anti-tyrosine-phosphorylated FAK (pY-397) antibodies (BioSource) were purchased from the sources shown. FITC-labeled phalloidin for staining of actin filaments was also purchased from Cytoskeleton (Denver, CO, USA). FITC (Cytoskeleton) and rhodamine labeled-phalloidin (Molecular Probes, Eugene, OR, USA) for staining of actin filaments were also purchased from the sources shown.