P5318

Experiments were performed according to the Dutch regulation and approved by the Committee for Animal Welfare of Maastricht University. Ldlr^−/− mice on a C57BL/6 background were housed under standard conditions and given free access to food and water. Female 12 week-old Ldlr^−/− mice were fed either regular chow or a high-fat, high-cholesterol (HFC) diet (SAFE, Augy, France) for 3 weeks and were divided in three groups (n = 11 for each group). The HFC diet contained 21% milk butter, 0.2% cholesterol, 46% carbohydrates and 17% casein. To examine whether proteolytic inhibition of CTSD would decrease hepatic inflammation, Ldlr^−/− mice were injected intraperitoneally with pepstatin A (PepA; 50 µg/g body weight; P5318, Sigma, Zwijndrecht, the Netherlands), a proteolytic inhibitor of aspartyl proteases. Mice were injected with DMSO (8%) or PepA two times every week. An overview of all experimental groups is depicted in Supplementary Fig. S1. Collection of blood and tissue specimens, fluorescence-activated cell sorting (FACS), liver histology, plasma alanine transaminase levels (ALT), RNA isolation, cDNA synthesis and qPCR were determined as described previously^{4 (link), 15 , 41 , 42 (link)}.

For each genotype, 15 brains from third-instar larvae were dissected in PBS, 1% protease inhibitor cocktail and extracted in 100 μl of cold protein extract buffer (50 mM trisHCl pH 7.4 (200923A, Euromedex), 50 mM NaCl (27810, Prolabo), 1 mM EDTA (E9884, Sigma), 0.25% DOC (D6750, Sigma), 0.1% SDS (EU0660, Euromedex), 1 mM PMSF (P7626, Sigma), 1‰, DTT (D0632), 1% of protease inhibitor solution (1 mM benzamidine-HCl (B6506, Sigma), 0,1 mg ml⁻¹ phenanthroline (131377, Sigma), 1 mg ml⁻¹ aprotinin (A6103, Sigma), 1 mg ml⁻¹ leupeptin (L2884, Sigma), 1 mg ml⁻¹ pepstatin A (P5318, Sigma)). Protein extracts were separated by SDS–polyacrylamide gel electrophoresis with either a 10% BIS-TRIS or a 3-8% TRIS-Glycine NuPAGE gel (Invitrogen) and then transferred on a nitrocellulose membrane (Protran BA83, Whatman). Membranes were probed with antibodies anti-Fzy (1:1,000, a gift from J. Raff lab) and anti-α-tubulin (DM1Α) (1:1,000, Sigma-Aldrich).