Ephb4

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).

Y- and A-TSPC plated and cultured on 20 μg/ml collagen 1-coated glass slides (BD Bioscience, USA) for 48 h were fixed with 4% paraformaldehyde (Merck, Germany) or 7% formalin/PHEM (6 mM PIPES, 25 mM HEPES, 10 mM EGTA, 3 mM MgCl2, pH 6.1, all Sigma–Aldrich) solutions. Then, cells were permeabilized with 0.1% Triton X100 (Sigma–Aldrich) and blocked with 3% BSA (Millipore, USA). Primary antibodies against EphA4 (Abnova, Germany), EphB2 (Biomol, Germany), EphB4 (Thermo scientific), EFNB1 and EFNB2 (both Sigma–Aldrich) were applied overnight at 4°C. Next, secondary Alexa Flour 488-conjugated antibodies and DAPI were used (all Life technologies). For negative control, cell-seeded slides were incubated only with the secondary antibody and DAPI. For F-actin staining, formalin/PHEM fixed cells were incubated with phalloidin-AF546 (Life technologies) for 40 min at room temperature. Photomicrographs were taken with Axiocam MRm camera on AxiovertS100 microscope (Carl Zeiss, Germany). Staining experiments were repeated two independent times for all three Y- and A-TSPC donors (n = 6).