Leupeptin

Human histone proteins were expressed and purified as previously described^{17 (link)}. Fpr4 NPL domain (and deletion constructs), Fpr4 FKBP domain (WT, F323Y surface charge mutants A-D) and Fpr1 were expressed in E. coli BL21 (DE3) as N-terminal 6His tag fusion proteins using the pETHis1a expression vector. Expressed proteins were purified with Ni-NTA agarose resin (Qiagen, cat#30210) using lysis buffer (50 mM Tris pH 7.5, 300 mM NaCl, 10 mM Imidazole, 5% glycerol, 1 ug/mL each of leupeptin (Bio Basic, cat#LDJ691), pepstatin (Bio Basic, cat#PDJ694), aprotinin (Bio Basic, cat#AD0153) and 2mM PMSF (Sigma, cat#P7626)), wash buffer (50 mM Tris pH 7.5, 300 mM NaCl, 20 mM imidazole and 5% glycerol) and elution buffer (50 mM Tris pH 7.5, 300 mM NaCl, 250 mM imidazole and 5% glycerol). Elutions containing purified protein were combined and dialyzed overnight at 4C against 1X TBS +5% glycerol. Site-directed mutagenesis to generate catalytic null Fpr4 F323Y was performed as per standard protocols. Histone H1 was purchased from Roche. DNA corresponding to Fpr4 FKBP mutants A-D was synthesized by Genscript. Mutagenesis to generate Fpr4 NL ΔA2 and ΔA1/B1 was performed using a Q5 Site-Directed Mutagenesis Kit (NEB, cat#E0554S) using primers whose sequences are available upon request.

Samples were lysed with 0.1% NP40 buffer supplemented with Leupeptin (10 μg/ml; BioBasic), Aprotinin (10 μg/ml; Sigma), and PMSF (1 mM; BioBasic). Samples were analyzed by 10% SDS-PAGE and then transferred to a PVDF membrane. Primary antibodies were applied and incubated over-night at 4°C using dilutions specified above. Proteins were detected via treatment with Perkin-Elmer Enhanced Chemiluminscence reagent/ECL Western Gel Substrate (Perkin Elmer) and quantified using FlourChem HD2 software (AlphaInnotech; Perkin Elmer).