H 250 sc 7150

T265-2c cells were plated in 100mm dishes at densities described above in the Cell Culture section and drug treatments were performed after 24h. To account for loss of total cell number resulting from cytotoxic effects of BH3 mimetic treatment, twice as many 100mm dishes were used for each treatment condition versus untreated and each dish of respective cells was combined after fixation. ChIP using antibodies against PARP1 (Santa Cruz H-250 [sc-7150]) (12μg/ml) or rabbit IgG (Santa Cruz sc-2027) (12μg/ml) was performed using the ChIP-IT Express Enzymatic kit from Active Motif (Carlsbad, CA #53009) per the manufacturer's instructions. DNA was amplified using primers flanking the putative PARP1 [“A/GNNA/TCAAA” [53 (link)] binding site within the CXCL12 promoter. Negative control primers were obtained from Active Motif (#103708).
Primer sequence:
Forward: 5′-GAATCTCCCGTCCCACTCC-3′
Reverse: 5′-GCCGAGCCTCAGTTTCCT-3′

HEK293 stably expressed WT-PARP1-GFP or SNP-PARP1-GFP were treated with elevated doses of Olaparib (0, 0.01, and 0.1 µM) for 30′ at 37 °C. Lysates were subjected to SDS-PAGE gel and the blotted membrane was incubated with anti-PARP1 rabbit antibody (H-250) (sc-7150) (Santa Cruz) and with anti-GAPDH rabbit (ab8245). Secondary Peroxidase Affinity Pure Goat Anti-Rabbit IgG (H+L) (111-035-144; Jackson ImmunoResearch) was used soon after.