Anti parp1 antibody

Rec. human PARP1 (1 μg) was incubated with 10 μg anti-PARP1 antibody (FI-23) (36 ) or with mouse IgG control (Santa Cruz) for 1 h at 4°C in 300 μl IP buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate). 10 μl Protein G sepharose beads (Sigma-Aldrich) were added and incubated for 2 h at 4°C, while rotating. Indicated samples were subjected to auto-PARylation of PARP1 by adding 10 mM MgCl₂, 7.7 μg/ml self-annealed oligonucleotide GGAATTCC, 1 mM DTT and 100 μM NAD⁺. Samples were incubated for 5 min at 4°C, while rotating. After centrifugation at 2400 g for 20 s, beads were washed with 500 μl wash buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% NP-40, 0.05% deoxycholate), followed by a second washing step, using 250 μl IP buffer. After centrifugation and removal of the supernatant, 300 μl IP buffer were added, together with 1 mM DTT and 23 pmol of rec. p53. Incubation took place for 1 h at 4°C while rotating. Beads were washed twice for 5 min in 500 μl IP buffer and once for 5 min in 500 μl wash buffer while rotating. Proteins were eluted from beads by adding 22 μl of 2 × SDS sample buffer, incubating 5 min at 95°C and subjected to SDS-PAGE followed by Western blotting. The rabbit polyclonal anti-PARP1 antibody H250 (Santa Cruz) and the rabbit polyclonal anti-p53 antibody FL-393 (Santa Cruz) were used for immunodetection.

Western blot was performed as described previously [17] (link). Finally, protein bands were visualized by ECL or ECL prime detection reagents (GE Healthcare). The primary antibodies used in this study were: anti-human GRP78 polyclonal antibody (1:1000, Santa Cruz), anti-human GALNT6 polyclonal antibody (1:1000, Sigma-Aldrich), anti-Flag M2 monoclonal antibody (1:1000, Sigma-Aldrich), anti-HA monoclonal antibody (1:1000, Roche), anti-PARP-1 antibody (1:1000, Santa Cruz), anti-caspase 7 (1:1000, Cell signaling), anti-PERK (1:1000, Cell signaling), anti-IRE1α (1:1000, Cell signaling), anti-ATF6 (1:1000, Cell signaling), and anti-β-actin monoclonal antibody (1:10,000, Sigma-Aldrich). The secondary antibodies were goat anti-rabbit, anti-rat, and anti-mouse IgG-HRP secondary antibodies (1:10,000~ 1:30,000, Santa Cruz). Intensity of protein band was quantified by ImageJ software as previously described [8] (link).