Anti eps8

Antibodies used were as follows: anti-Eps8, anti-paxillin, anti-GM130 and anti-p130Cas antibodies (BD Transduction Laboratories, NJ), anti-FAK, anti-pSrc Y416, anti-Src (clone 36D10), anti-LC3B, anti-GAPDH, anti-pPaxillin Y118 and anti-β-actin antibodies (Cell Signaling Technologies, Danvers, MA), as well as anti-FAK antibody conjugated to agarose (clone 4.47) (Millipore, Billerica, MA) and anti-LC3B for immunoprecipitations (MBL, Woburn, MA). The anti-TagCGYFP antibody was purchased from Evrogen (Cambridge, UK). TRITC–phalloidin was purchased from Life Technologies (Paisley, UK). Horseradish-peroxidase-conjugated secondary antibodies against rabbit or mouse IgG were purchased from Cell Signaling Technologies. Dasatinib was obtained from Bristol Myers Squibb (Princeton, NJ). The pEGFP-Eps8 1–821 and pEGFP-Eps8 1–535 constructs were a generous gift from Giorgio Scita (FIRC Institute of Molecular Oncology, Milan, Italy).

Dissection and preparation of mouse cochleas were performed as previously described [7 (link)]. Eyes obtained from deeply anaesthetized adult macaques perfused with paraformaldehyde, were dissected and processed as previously described [8 (link)]. Retinal cryosections were incubated in 10% bovine serum albumin in phosphate buffered saline (PBS) for 1 hour, incubated with the appropriate primary antibody overnight at 4°C, rinsed in PBS, incubated with the appropriate secondary antibody for 1 hour at room temperature, and rinsed again in PBS. The primary antibodies used were anti-EPS8 (BD Transduction Laboratories), anti-synaptophysin (MAB368, Millipore), anti-whirlin (G-8, Santa Cruz Biotechnology, Inc) mouse monoclonal antibodies, and anti-EPS8 (M-238, Santa Cruz Biotechnology, Inc) rabbit polyclonal antibody. Secondary antibody was Alexa Fluor 488 goat anti-rabbit IgG. TRITC-phalloidin (Sigma-Aldrich) and DAPI (1 μg/ml; Sigma-Aldrich) were used to label F-actin and cell nuclei, respectively.