Total and phospho jnk

Total protein from Y- and A-TSPC (clustered EphA4 and EphB2, and Fc-control) was isolated with RIPA-buffer (0.1% SDS, 1% Na-DOC, 1% Triton X-100, 50mM Tris-HCl pH8.2, 150mM NaCl, 10mM EDTA, 20mM NaF, 1mM Na₃VO₄) supplemented with complete protease inhibitors (Roche). Proteins (20 μg) were separated on SDS gels, transferred onto PVDF membrane and blocked with 5% skim milk (Merck) for 1 h at room temperature. Primary antibodies against human EphA4 (Abnova), EphB2 (Biomol), EphB4 (Thermo scientific), EFNB1 and EFNB2 (both Sigma–Aldrich); phospho-FAK (Thermo scientific), total FAK, total and phospho-ERK1/2; total and phospho-Akt; total and phospho-p38; total and phospho-Jnk (all Cell Signaling, USA), and GAPDH (Merck) were applied overnight at 4°C. Then, membranes were incubated with corresponding secondary HRP-conjugated antibodies (Cell Signaling) for 1 h at room temperature and consequently with ECL solution (GE Healthcare, USA). Photomicrographs were taken on ImageQuant LAS 4000 mini (GE Healthcare) as band intensities were quantified with ImageProPlus4 software program (Media Cybernetics, USA). Western blot experiments were preformed two independent times with all three Y- and A-TSPC donors (n = 6).