Caffeine

NPE was prepared as described previously (30 (link)). Briefly, sperm chromatin was incubated in interphase egg extract to form nuclei. Nuclei were then harvested and genomic material was removed by ultracentrifugation to create a soluble nuclear protein extract. For all reactions, NPE was supplemented with an ATP regeneration mix (6.5 mM phosphocreatine, 0.65 mM ATP, and 1.6 μg/ml creatine phosphokinase) and 1 mM DTT. All incubations were performed at 21°C. Extracts were placed at 21°C for 10 min prior to the addition of 2.5–10 ng/μl plasmid DNA, which represents the reaction start time. Where indicated, reactions were supplemented with 5 ng/uL pCON or pDSB, 5 mM caffeine (Fisher), 10–100 μM VE-821 (Abcam), 10–100 μM KU-5593 (Abcam), 20 μM ubiquitin vinyl sulfone (Fisher), 30 μM JQ1 (Sigma), or 40–400 nM recombinant BRCA1-BARD1. The endogenous level of BRCA1-BARD1 in NPE is estimated to be ∼40 nM. All reactions were performed at least two times with representative or averaged data shown. Where indicated, data were graphed with error bars representing ±1 standard deviation and P values determined using a two-tailed t-test: P < 0.05 (*), P < 0.005 (**), P < 0.0005 (***), P ≥ 0.05 (not significant, n.s.).

LC–MS‐Grade water, methanol (MeOH), acetonitrile (ACN), formic acid (FA), and acetic acid (HOAc) for the electrospray solvent were purchased from Fisher Scientific (Nazareth, PA, USA). Caffeine was also purchased from Fisher Scientific (Hampton, NH, USA). Theophylline, riboflavin, glutathione (GSH), benzoic acid, sorbic acid, MRFA (peptide), and polyethylene glycol (PEG) were purchased from Sigma Aldrich (St. Louis, MO, USA). Homo‐glutathione (hGSH) was purchased from Bachem (Bubendorf, Switzerland). Stable isotope‐labeled glutathione (SIL‐GSH, ¹³C₂¹⁵N) and stable isotope‐labeled Caffeine (SIL‐Caffeine, ¹³C₃) were purchased from Cambridge Isotope Laboratories, Inc (Tewksbury, MA, USA). Splash Lipidomix Mass Spec Standard (Splashmix) was purchased from Avanti Polar Lipids (Birmingham, AL, USA). Well plates (100 μl) were purchased from Brand GMBH & CO KG (Wertheim, Germany).