Histone h1

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.

Synthesis of PAR was performed as previously reported^{20 (link)}. Briefly, 50 units of purified human PARP-1 (High Specific Activity hPARP-1, Trevigen) were incubated in a mixture containing 100 mM Tris-HCl pH 8, 10 mM MgCl₂, 2 mM dithiothreitol, 2.5 μg of DNase I-activated calf thymus DNA (Trevigen) and 200 mM NAD+ (Sigma-Aldrich) for 45 min at 30 °C. The reaction was stopped by adding ice-cold trichloroacetic acid (TCA) to a final concentration of 20% (w/v). PARs were detached from proteins by incubation in 50 mM NaOH and 10 mM EDTA for 1 hr at 60 °C. After adjustment of pH to 7.5, PAR were purified by phenol/chloroform extraction as described^{20 (link)}.
For the study of non-covalent interaction of PAR with TRF-1, graded concentrations of purified His-hTRF1 protein were immobilized directly by slot-blotting on nitrocellulose membranes. Histone H1 (Millipore) was used as positive control in the PAR binding assay. Subsequently, filters were incubated with PAR diluted in TBS-T (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20) for 1 hr at room temperature. After high-stringency salt washes, protein bound PAR were detected using the anti-PAR monoclonal antibody (mouse Mab ALX-804220).

Embelin was purchased from Sigma-Aldrich (St. Louis, MO). A 100 mM solution of Embelin was prepared in dimethyl sulfoxide, stored as small aliquots at -20°C and then diluted as needed in cell culture medium. A 8M solution of lithium chloride was obtained from Sigma-Aldrich and diluted as needed in cell culture medium. FBS and RPMI 1640 medium were purchased from HyClone (Logan, UT). The Neon transfection system, JC-1 assay kit and COX-4 antibody were from Life Technologies (Carlsbad, CA). The bicinchoninic acid (BCA) protein assay kit and ECL kit were from Thermo Scientific (Rockford, IL). Antibodies against phospho-Akt (Ser 473), GSK-3β, phospho-GSK-3β, or Bcl-2 were from Cell Signaling Technology (Beverly, MA). Antibodies against Total-Akt, VDAC1, Bcl-xL, Bax, β-catenin, cyclin D1, GAPDH or goat anti-rabbit IgG-Texas Red and Ultra Cruz mounting medium were from Santa Cruz Biotechnology (Santa Cruz, CA). Cyclooxygenase-2 (COX-2), Mcl-1, cytochrome c, AIF, β-catenin, c-myc or histone H1 antibodies were from Millipore Co. (Bedford, MA). All other chemicals were of the highest purity or molecular biology grade and were obtained from commercial sources.