Aprotinin

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.

Mice fed a HFD were fasted for 4 h for fasting blood glucose measurements, or before an ITT or OFTT. ITT was performed subcutaneously at a 0.75 mUi/g body weight dose. Tail vein blood glucose was recorded prior to the assay and every 15 min for 2 h with a glucometer. OFTT was performed with olive oil at a dose of 5 µL/g body weight. Blood was sampled prior to oil gavage and after 15 and 30 min. Plasma was recovered by adding a 10% (vol/vol) anticoagulant mix to blood composed of 0.02 mg/µL EDTA (Sigma-Aldrich), 20% (vol/vol) DPP4 (Millipore), 10% (vol/vol) protease inhibitor cocktail (P8340, Sigma-Aldrich), 5 KIU/µL Aprotinin (AD0153, Bio Basic), and 0.2 M NaCl followed by immediate centrifugation at 3000 × g, 4 °C for 10 min. Total GIP (EMD Millipore, EZRMGIP-55K), GLP-1 (Crystal Chem, 81508), and resistin (ab205574) were measured using ELISA from plasma samples according to the manufacturer’s instructions.