Biochemistry
Cortical cultures were stimulated with 1 µg/ml clustered (see above) ephrin-A5-Fc and/or BDNF (10 ng/ml) in NMEM/B27 medium for the indicated time periods. Phosphatase inhibitors were applied for 15 mins prior to ephrin-A5-Fc and/or BDNF stimulation: sodium vanadate (200 µM; Sigma), okadaic acid (200 nM; Calbiochem), PhosStop (1x; Roche). Cells were lysed in 100 mM Tris pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Triton-X-100, 0.1% SDS and protease inhibitors (Roche). Samples were resolved on 10-12% SDS-PAGE, followed by transfer on PVDF membranes (Amersham). After blocking, first antibodies were applied overnight at 4°C: rabbit anti-ERK (1∶1000; Cell Signaling), rabbit anti-P-ERK (1∶1000; Cell Signaling), mouse anti-GAPDH (1∶50000), rabbit anti-TrkB (1∶1000; Santa Cruz), rabbit anti-P-TrkB (1∶1000; Cell Signaling), mouse anti-EphA4 (1∶750; BD Transduction Laboratories), P-EphA3 (1∶10000, gift of Dr. M. Greenberg, Harvard Medical School, Boston). Detection of first antibodies involved horseradish-peroxidase conjugated secondary antibodies (1∶5000) and the ECL Western Blotting Substrate (Pierce).