Protease inhibitor mixture and phosphatase inhibitors

The peri-infarct cortices, pyramid, and spinal cord tissues (n = 5/group) were washed with ice-cold PBS and lysed on ice in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.25% Na-deoxycholate, and 0.1% SDS) containing a protease inhibitor mixture and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Thirty micrograms of soluble protein were subjected to SDS–PAGE and electrotransferred onto a PVDF membrane (26 (link)). Specific protein bands were detected using specific Eph/ephrin antibodies (Supplementary Table 1) and enhanced chemiluminescence (Pierce, Rockford, IL, USA).

B cells, CD4⁺ T cells, and CD8⁺ T cells were collected in RIPA lysis buffer (1% Triton X-100, Sigma-Aldrich, St. Louis, MO), 20 mmol/L Tris, pH 7.5, 137 mmol/L NaCl, 1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 10% glycerol, 1.5 mmol/L MgCl₂, and protease inhibitor mixture and phosphatase inhibitors (Roche Products Limited, Welwyn Garden City, UK). Lysates were sonicated and centrifuged at 4°C. Per lane, whole-cell lysate was separated on 12% sodium dodecyl sulfate–acrylamide gels and transferred on Immobilon polyvinylidene difluoride membranes (Millipore). The membranes were probed with primary antibodies (Table 4) overnight at 4°C and incubated for 1 hour with secondary peroxidase-conjugated antibodies. For detecting the apoptotic pathway, anti–caspase 3, anti-Bcl-2 Associated X Protein, and anti-Bcl2 (Table 4) were used. Chemiluminescent signals then were developed with Lumiglo reagent (Cell Signaling Technology) and detected by the ChemiDoc XRS gel documentation system (Bio-Rad).