Cathepsin s

Cell pellets were suspended with RIPA lysis buffer with complete inhibitor cocktail (Roche, Basel, Switzerland), quantified with Bradford reagent (Sigma-Aldrich, St Louis, MO, USA), run on standard 10% SDS PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes for immunoblot (IB). The following antibodies were used for IB: β-actin (Santa Cruz Biotechnology, Dallas, Texas, USA; cat#47778), cathepsin S (Abcam, Cambridge, UK cat#134157), cathepsin B (Abcam cat# ab58802); Secondary antibodies: Anti-Mouse-alkaline-phosphatase (AP)-conjugated (Sigma-Aldrich, cat#, A3562), Anti-rabbit-AP conjugated (Sigma-Aldrich, cat#, A3687). All IBs were scanned at high resolution and band intensity was determined using Image J software (NIH, USA). After subtracting the background, each band was normalized to the corresponding housekeeping protein (that is, β-actin) appearing on the blot. Band intensity was presented in arbitrary units (A.U.) adjacent to the representative IB.

TREM-1 high and low FACSorted cells were lysed in RIPA buffer (0.15M NaCl, 0.05M TRIS pH 7.5, 1% NP40, 0.5% DCA, 0.1% SDS, 0.1 mM EDTA), run on 15% SDS-PAGE gels under reducing conditions and transferred to an Immobilon-P PVDF Membrane (Millipore, Burlington, MA). The membrane was blocked by incubation in 5% non-fat milk (Nutricia, Wageningen, The Netherlands) in TBST (TBS + 0.1% Tween-20) for 2 hours at room temperature (RT) and subsequently incubated in LC3 (Cell Signaling, 4108S) or Cathepsin S (Abcam, ab18822) antibodies in 2% milk/TBST overnight at 4° C. After incubation membranes were washed 3 times with TBST, incubated with HRP conjugated secondary antibodies (1:2000, DAKO) in 2% milk/PBST for 2 hours at RT. Expression was detected by Lumilight Plus (Roche, Woerden, The Netherlands). Afterwards blots were stripped in stripping buffer (ThermoFischer Scientific) for 10 min at RT and incubated with β-actin (clone AB1978, Sigma, Deisenhofen, Germany) as a loading control. The Optical Density (OD) of the protein bands was determined using ImageJ software.

Recombinant human cytokines TNF, IFN-γ and IL-17A/F were obtained from R&D Systems. Anti-human lipocalin-2, elafin, cathepsin S and cathepsin V antibodies were obtained from Abcam (Toronto, ON, Canada). Anti-human actin antibody was obtained from Millipore (Burlington, MA, USA). HRP-linked purified anti-rabbit IgG and anti-mouse IgG-secondary antibodies were obtained from Cell Signaling Technology, distributed by New England Biolabs (Pickering, ON, Canada).