Cortical neurons and brain cortex from siah2+/+ control mice and mice exposed to tMCAO were homogenized in lysis buffer containing 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1% Triton X-100, 100 mM NaF, 100 mM Na3VO4, 5 μg/ml aprotinin, 10 μg/ml leupeptin, and 2 μg/ml pepstatin. One milligram of lysate was precleared using protein A/Gplus (Santa Cruz, Dallas, TX) for 1 h at 4 °C with constant rotation and centrifuged for 2 min at 8000 rpm. The cell lysates (1 mg) were cleared by centrifugation at 15,000 x g for 15 min and were immunoprecipitated with anti-Siah2 mouse antibody (Sigma-Aldrich, 1:100). An aliquot of cell lysate (100 μg) or immunoprecipitates were resolved by SDS-PAGE gel and transferred onto nitrocellulose membrane. Immunoblot analysis was performed using anti NCX3 and VDAC antibodies, as previously described [1 (link)]. Chemio-luminescent (ECL) signals were quantified by Chemi Doc Imaging System (Biorad).
Regarding lysates from brain tissue, 1 mg of precleared lysate was immunoprecipitated with an anti-Siah2 mouse antibody (Sigma-Aldrich, 1:100) using the same experimental procedure described above. Finally, total lysates or immunoprecipitates were resolved by SDS-PAGE gel and transferred to a nitrocellulose membrane. Immunoblot analysis was performed using anti-NCX3 and anti-VDAC antibodies.
Regarding lysates from brain tissue, 1 mg of precleared lysate was immunoprecipitated with an anti-Siah2 mouse antibody (Sigma-Aldrich, 1:100) using the same experimental procedure described above. Finally, total lysates or immunoprecipitates were resolved by SDS-PAGE gel and transferred to a nitrocellulose membrane. Immunoblot analysis was performed using anti-NCX3 and anti-VDAC antibodies.