Rabbit anti histone h4

25 or 50 μL of protein extracts was mixed with sample buffer (125 mM Tris–HCl, pH 6.8; 2 % SDS, w/v; 20 % glycerol, w/v; 20 µg/µL bromophenol blue and 5 % β-mercaptoethanol), boiled for 5 min, separated by electrophoresis on a 8 % SDS-PAGE gel and blotted onto a BioTrace™ pure nitrocellulose membrane (Pall Corporation, Pensacola, FL, USA). Membranes were blocked with Tris-buffered saline (TBS) containing 0.1 % Tween-20 and 5 % (w/v) non-fat dry milk at room temperature for 1 h, and incubated with (i) mouse anti-pADPr (170–70 kDa) (3H2844) (sc-71848, Santa Cruz Biotechnology); (ii) goat anti-PARP-1 (113 kDa) (sc-F0908, Santa Cruz Biotechnology); (iii) Rabbit anti-p65 (65 kDa) (sc-372, Santa Cruz Biotechnology); (iv) Mouse anti-iKB-α (35–41 kDa) (sc-1643, Santa Cruz Biotechnology); (v) Mouse anti-β-actin (42 kDa) (Sigma A5316); (vi) Rabbit anti-histone H4 (13–15 kDa) (Abcam 1261) and (vii) mouse anti-α-tubulin (45 kDa) (Sigma T8203). Membranes were washed and incubated with HRP-conjugated secondary antibody in TBS at room temperature. The immune complexes were visualized with an enhanced chemiluminescent substrate, according to the instructions of the manufacturer (Perkin Elmer Life Sciences, Boston, MA), captured by a ChemiScope 3400 (Clinx Sciences Instruments Co, Ltd). Reactive bands were quantified by densitometric analysis with Photoshop software.

For SDS-PAGE and western blotting 18% SDS were run at 200 V, constant current. SDS polyacrylamide gels were stained with Coomassie Instant Blue (Expedeon). For western blotting, proteins were transferred onto nitrocellulose membrane (GE Healthcare) by wet transfer at 200 mA for 2 h. Membranes were blocked in 5% milk. Primary antibodies were rabbit anti-phospho-histone H3 threonine 3 (H3T3ph) (Millipore) and rabbit anti-phospho-histone H3 serine 10 (H3S10ph) (Cell Signalling Technologies), which were used at 1 : 1000 dilution, and rabbit anti-histone H4 (Abcam), which was used at 1 : 5000 dilution. The secondary antibody was IRDye 800CW goat anti-rabbit (Licor) which was used at 1 : 10 000 dilution. The membranes were imaged using a Licor Odyssey infrared imaging scanner.