Rabbit anti fak

PEC lysates were prepared with RIPA extraction buffer containing phosphatase inhibitors and protease inhibitors (Roche). Equal amounts of proteins were loaded onto sodium dodecyl sulfate–polyacrylamide electrophoresis gels for separation and transferred onto poly(vinylidene difluoride) membranes. The membranes were blocked with milk and probed with different antibodies: rat anti-CD9 (BD Pharmingen, 553758, 1:1000), rabbit anti-phospho-PDGF receptor-ß Y1009 (Cell Signaling Technology, 4549; 1:1000), rabbit anti-PDGFRβ (Cell Signaling, 3169; 1:1000), rabbit anti-integrin ß1 (Millipore, 04-1109, 1:1000), rabbit anti-CASP3 (Cell Signaling Technology, 9662; 1:1000), rabbit anti-phospho EGFR Y1068 (Cell Signaling Technology, 2234; 1:1000), rabbit anti EGFR (Cell Signaling Technology, 2232; 1:1000), rabbit anti-phospho-FAK Y397 (Cell Signaling 3283, 1:1000), rabbit anti-FAK (Millipore, 06-543, 1:1000). Membranes were then probed with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, 7074 and 7076; 1:2000; Jackson Immunoresearch, 706-036-148; 1:1000) and bands were visualized by enhanced chemiluminescence (Clarity Western ECL substrate; Bio-Rad, 170–5061). A LAS-4000 imaging system (Fuji, LAS4000, Burlington, NJ, USA) was used to reveal bands and densitometric analysis was used for quantification. Uncropped blots are shown in Supplementary Figs 18–22.

To proof the reduction of the pFAK level by FAK-inhibitor 14, cells dissected from whole E14 brains were cultured 2 DIV at 37°C and 5% CO₂ in cell culture medium supplemented with 1 mM, 3 mM or 7.5 mM FAK-inhibitor 14. The medium was renewed after 1 DIV. Then cells were lysed in STEN buffer (150 mM NaCl, 50 mM Tris, 2 mM EDTA, and 0.2% NP-40) including broad spectrum-protease inhibitor (Sigma) and phosphatase inhibitor (PhosStop, Roche). Lysates were separated on NuPAGE 4–12% Bis-Tris Gels (life technologies) following the manufacturer’s instructions and transferred to nitrocellulose membranes. Membranes were blocked in TBS-T buffer (300 mM NaCl, 10 mM Tris, pH 7.6, and 0.1% Tween20) containing 5% milk-powder for 30 min and then incubated with the primary antibody over night at room temperature. Antibodies used were mouse anti-actin (Santa Cruz, 1:1000); rabbit anti-pFAK (pY397) (Invitrogen; 1:500); rabbit anti-FAK (Millipore; 1:1000). After washing in TBS-T, membranes were incubated for 1 h at room temperature with biotin-conjugated secondary antibodies (goat anti-mouse, Sigma; 1:400 and goat anti-rabbit, Vector; 1:400). Membranes were washed again in TBS-T and the signal was detected using DAB detection kit (Vector laboratories) following the manufacturer’s instructions. The total FAK amount was normalized to actin and pFAK bands were then normalized to the normalized FAK.