Diisopropylfluorophosphate dfp

Mgp^+/− mice on the C57BL/6J background^{23 (link)} were obtained from Dr. Cecilia Giachelli, University of Washington, with the permission of Dr. Gerard Karsenty, Columbia University. Cdh5^Cre (B6.Cg-Tg(Cdh5-cre)7Mlia/J) and Sox2^flox/flox (Sox2tm1.1Lan/J) mice were obtained from the Jackson Laboratory. Genotypes were confirmed by PCR^{21 (link), 24 (link), 25 (link)}, and experiments were performed with generations F4–F6. Littermates were used as wild type controls. All mice were fed a standard chow diet (Diet 8604, HarlanTeklad Laboratory). The studies were reviewed and approved by the Institutional Review Board and conducted in accordance with the animal care guideline set by the University of California, Los Angeles. The investigation conformed to the National Research Council, Guide for the Care and Use of Laboratory Animals, Eighth Edition (Washington, DC: The National Academies Press, 2011). Diisopropylfluorophosphate (DFP) (Sigma-Aldrich) and serpina1 (Origene) were injected via tail vein or retro-orbital injection (20–50 ng/g, daily) as in previous studies^{26 (link), 27 (link)}. Injections in Mgp^−/− mice were started at 2 weeks of age, and continued for 2–4 weeks.

Protein expression and phosphorylation of target proteins were analyzed by western blotting. Murine neutrophils were stimulated at an MOI of 1:1 with fungal particles. Following incubation for 30 min at 37°C, cells were lysed with RIPA buffer (Sigma, St. Louis, MO, USA) or a nuclear/cytosol fractionation kit (ThermoFisher Scientific, Waltham, MA, USA). All lysis buffers contain final 2.5mM Diisopropyl fluorophosphate (DFP) (Sigma, St. Louis, MO, USA), 1x protease inhibitor cocktail (Roche, Basel, Switzerland) and 1x phosphatase inhibitor cocktail (ThermoFisher Scientific, Waltham, MA, USA). A total of 40 μg lysate was loaded on 4–12% SDS-PAGE gels (Invitrogen) and transferred to 0.2 μm PVDF membranes (Bio-Rad, Hercules, CA, USA). Protein lysates were equally loaded, and expression of each target protein and its phosphorylated form was measured by GelDoc imaging system (Bio-Rad, Hercules, CA, USA) and analyzed in ImageJ (NIH). Results are expressed in arbitrary units (a.u), which is a relative number for phospho-form of the target protein compared to the total expression of the target protein, normalizing using the unstimulated WT negative control as 1.

ApoE^−/− (B6.129P2-ApoEtm1Unc/J) mice were obtained from the Jackson Laboratory. Genotypes were confirmed by PCR (24 (link)) and experiments were performed with generations F4-F6. All mice were fed a standard chow diet (Diet 8604, HarlanTeklad Laboratory). At 8 to 10 weeks of age, all ApoE^−/− mice were switched to a high-fat/high-cholesterol diet (Western diet) (Research Diets, New Brunswick, NJ, diet #D12108, containing 21% fat, 1.25% cholesterol) for 16 weeks. Diisopropylfluorophosphate (DFP) (Sigma-Aldrich), serpina1 (Origene) or Sox2 shRNA were injected via tail vein or retro-orbital injection (20–50 ng/g, daily) as in previous studies (13 (link)). Injections in ApoE^−/− mice were started at 16 to 18 weeks of age, and continued for 8 weeks. The studies were reviewed and approved by the Institutional Review Board and conducted in accordance with the animal care guideline set by the University of California, Los Angeles. The investigation conformed to the National Research Council, Guide for the Care and Use of Laboratory Animals, Eighth Edition (Washington, DC: The National Academies Press, 2011).

Native BChE and mutated variants were purified from mouse serum collected after gene transfer of enzyme cDNA by adeno-associated viral vector (AAV 2/8, 1×10¹³ viral genomes delivered by tail-vein injection). After two weeks to reach maximal expression, the mice were euthanized by pentobarbital and blood was collected from the vena cava and heart. BChE protein was then enriched to ~ 90% purity by ammonium sulfate fractionation and separation on procainamide Sepharose affinity columns as previously described [24 (link)]. Certain experiments utilized >98% pure human BChE (O. Lockridge, Univ. Nebraska, Omaha, NE). Enzyme active sites were titrated with di-isopropyl fluorophosphate (DFP, Sigma) to determine final molar enzyme concentration as described previously [25 (link)].