Invitrolon pvdf filter paper membranes

HT1080 cells (~1 × 10⁷) were treated with NOR (0, 50, 100, 250, or 500 μM) for 3 h at 37 °C. Chromosomal DNA, along with any covalent DPCs, was extracted and quantified as described above. From each sample, 50 μg of DNA was subjected to neutral thermal hydrolysis (1 h at 70 °C) to release protein-guanine conjugates from the DNA backbone. The proteins were resolved by NuPAGE Novex 12% Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred to Invitrolon PVDF filter paper membranes (0.45 μm pore size, Life Technologies, Carlsbad, CA). The membranes were blocked for 4 h in Tris-buffered saline-Tween 20 (TBST) containing 5% (w/v) bovine serum albumin. Following blocking, the membranes were incubated with the primary antibodies against vimentin, nucleophosmin, prohibitin-2, matrin-3, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), poly-(ADP-ribose) polymerase 1 (PARP), and histone-H4 overnight at 4 °C. The membranes were rinsed with TBST and incubated with the corresponding alkaline phosphatase-conjugated antibody for 1 h at room temperature. The gels were developed using SIGMA Fast BCIP/NBT (Sigma, St. Louis, MO) according to manufacturer’s protocol.

Cardiomyocyte proteins (50 μg) were resolved by NuPAGE Novex 12% Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred to Invitrolon PVDF filter paper membranes (0.45 μm pore size, Life Technologies, Carlsbad, CA). The membranes were blocked for 1 h in Tris-buffered saline-Tween 20 (TBST) containing 5% (w/v) blotting grade blocker (BioRad, Hercules, CA). Following blocking, the membranes were incubated with the primary antibodies against matrin-3, superoxide dismutase [Mn], mitochondrial (SOD2), pyruvate dehydrogenase E1 component subunit beta, mitochondrial (PDH-1Eβ), troponin-T (cTni), and actin overnight at 4 °C. The membranes were rinsed with TBST and incubated with the corresponding alkaline phosphatase-conjugated antibody for 1 h at room temperature. The membranes were developed using the Clarity Western ECL substrate (BioRad) and visualized on an Odyssey 2800 Fc imager (Li-COR, Lincoln, NE) with Image Studio version 3.1 software (Li-COR). To confirm the identity of the protein constituents from cardiomyocyte DPCs, 5 μg of DNA from one LAD ligation/reperfusion and sham surgery samples were enzymatically digested to yield a nucleoside-protein conjugate. Nucleoside-protein conjugates were resolved and immunoblotted as described above.