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3 protocols using rucaparib

1

Antibodies and Inhibitors for Cancer Research

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Antibodies used in this study are as follows: tubulin (#T5168) and Flag (#F3165) were purchased from Sigma-Aldrich (St. Louis, MO); PARP1 (#sc-7150) for Western blotting from Santa Cruz Biotechnology (Santa Cruz, CA); PARP1 (#9532) for immunoprecipitation (IP), MET (#8198), phosphorylated MET (#3077), phosphorylated EGFR for IHC (#3777) and EGFR (#4267) from Cell Signaling Technology (Danvers, MA); and phosphorylated EGFR for Western blotting (#5650), Ki67 (#21700), cleaved caspase 3 (#2302), γ-H2AX (#140498), and PAR (#14460) from Abcam (Cambridge, MA); 8-hydroxy-2′-deoxy guanosine (8-OHdG) from Genox Corporation (Baltimore, MD). The mouse phospho-Y907-PARP1 antibody was generated against a phosphorylated synthetic peptide (ADMVSKSAN-Yp-CHTSQGD) at China Medical University as described previously (13 (link)). The following inhibitors were used in this study: MET kinase inhibitor crizotinib (#C-7900) was purchased from LC Laboratories (Woburn, MA); EGFR inhibitor gefitinib (#S1025) and erlotinib (#S1023) from Selleck Chemicals (Houston, TX); PARP inhibitors ABT-888 (veliparib, #CT-A888) and AG014699 (rucaparib, #CT-AG01) from ChemieTek (Indianapolis, IN). Hydrogen peroxide (#216763) and sodium arsenite solution (#35000) were obtained from Sigma-Aldrich.
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Synthesis and Storage of PARP Inhibitors

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Olaparib (AZD2281; Chemietek), niraparib (MK4827; Chemietek), rucaparib (AG014699; Chemietek), and AZD5363 (Active Biochem) were dissolved in DMSO at a concentration of 10 mM and stored at −20 °C. 6-alkyne (6Yn)- and 2-alkyne (2Yn)-adenosine probes were synthesized in-house by the chemistry unit of the Chemical Biology Core of the Moffitt Cancer Center. Drug dilutions were made in DMSO.
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3

PARP Inhibitor Preparation Protocol

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Olaparib (AZD2281; Chemietek), niraparib (MK4827; Chemietek), rucaparib (AG014699; Chemietek), Veliparib (CT-A888; Chemietek), talazoparib (BMN673; Cayman Chemical and Moffitt Cancer Center) and adavosertib (MK1775; Chemietek) were dissolved in DMSO at a concentration of 10 mM and stored at −20°C. (Rac)-i-Talazoparib (2) was made in house using the chemistry unit of the Chemical Biology Core of the Moffitt Cancer Center. Drug dilutions were made in DMSO as necessary.
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