Anti pkcβii antibody

Pure mitochondria (100 µg) from the CA2-4, DG region of control and ischemic gerbils (I/R 1 h and I/R 96 h) were incubated in lysis buffer (20 mM Tris HCl pH 7.5, 1% Triton, 0.5% NP-40, 0.1 mM CaCl₂, 1 mM PMSF, phosphatase and protease inhibitor cocktail (Sigma)) for 30 min at 4 °C and centrifuged (12,000g, 15 min, 4 °C). The supernatants were incubated (2 h, RT) with anti-PKCβII antibody (Abcam) attached to magnetic Dynabeads Protein G (Novex, Life Technologies) to achieve purification of endogenous PKCβII from the mitochondrial fraction. After extensive washing, the beads conjugated to PKCβII were incubated with 200 µM ATP (Cell Signaling) and 0.1 mg/ml Histone H1 (Millipore) for 30 min at 37 °C. The reaction mixture was then transferred to another tube and boiled in 5× SDS protein sample buffer (5 min, 100 °C). Samples were separated by SDS-PAGE and analyzed with anti-phospho-Histone H1 (Upstate) and anti-Histone H1 (Abcam) antibodies.

To establish whether changes in PKCβII immunoreactivity are due to its translocation to mitochondria following I/R injury, hippocampi from the control, as well as the groups termed ischemic (I/R 1 h + saline) and ischemic treated with βIIV5-3 peptide, were used in the isolation of the particulate (P2) and soluble (S2) fractions (obtained from the CA2-4, DG regions of hippocampi). Obtained fractions were separated by SDS-PAGE and analyzed by western blot with anti-PKCβII antibody (Abcam). Equal protein loading was confirmed by GAPDH level (mouse monoclonal anti-GAPDH, Santa Cruz).