Adam9

Western blotting was performed as described previously (15 (link)). All the types of cells were lysed by RIPA buffer with cocktail protease and phosphatase inhibitors (Thermo Fisher Scientific). Total protein extracts (40 μg) were subjected to western blotting analysis with antibodies against the following proteins: ADAM9 (R&D system); p-EGFR (Tyr1068), EGFR, p-MAPK (Tyr202/204), MAPK, GAPDH, p-AKT (Ser473), AKT, p-IKKα/β (Ser176/180), IKKβ, p-NF-κBp65 (Ser536), NF-κBp65, p-IκBα (Ser32), IκBα, and β-actin (Cell Signaling, Massachusetts, USA).
Total RNA was extracted from all the types of cells by using TRIzol Reagent (Thermo Fisher Scientific) according to the vendor's instruction. The cDNA synthesis was achieved by using PrimeScript reverse transcription reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan). Quantitative PCR was performed with ADAM9 and GAPDH primers by using SYBR Green PCR Master Mix (Roche, Baden-Württemberg, Germany) in a real-time PCR System (Applied Biosystems 7500, Thermo Fisher Scientific). Primer sequences were as follows: ADAM9, 5′-CCTCGGGGACCCTTCGTGT-3′ and 5′-ATCCCATAACTCGCATTCTCTAAA-3′; GAPDH, 5′-CCACCCATGGCAAATTCCATGGCA-3′ and 5′-TCTAGACGGCAGGTCAGGTCCACC-3′. Quantitative analysis of RT-qPCR was achieved by the 2^−ΔΔCt method.

Sections (5 μm) were cut from formalin-fixed paraffin-embedded specimens and mounted on Superfrost slides (Menzel Glaser, Lower Saxony, Germany) and gradually rehydrated after deparaffinization by xylene. Antigen retrieval was achieved by super heating in microwave oven with pH 6 citrate buffer for 15 min after boiling. The primary antibody ADAM9 (R&D System, Minnesota, USA) was diluted to 1:50 in Antibody Diluent (Dako, Denmark). After incubation at room temperature for 30 min, antigen-antibody reaction was detected by using anti-goat HRP-DAB tissue staining kit (R&D System). The slides were then counterstained with haematoxylin. For the negative control, the primary antibody was omitted. The immunohistochemistry staining results were evaluated by two experienced pathologists. The ADAM9 IHC intensity was classified into 5 grades (0 = negative, 1 = weak, 2 = moderate, 3 = strong, and 4 = prominent staining). The ADAM9 staining percentage was calculated by quantifying stained cells. ADAM9 expression level was calculated by applying the following formula: Expression level = ADAM9 staining intensity x ADAM9 staining percentage.

To determine the level of sANPEP in the plasma of LPS-injected mice (sacrificed 72 h after intraperitoneal injection of 1 mg/kg LPS, male) and 5xFAD mice (8 months of age, male), blood samples were collected from the vena cava using EDTA tubes and shaken gently. To separate plasma from the blood, samples were centrifuged (1500g, 15 min, 4 °C), and then, the plasma was transferred into a new tube. Samples (1:100) were diluted and assessed using a Mouse ANPEP ELISA Kit (MyBioSource) according to the manufacturer’s instructions. To measure the levels of human sANPEP in the clinical samples, plasma (1:10,000) and CSF (1:10) were first diluted. ELISA kits for human ANPEP (R&D Systems), TNFAIP6 (MyBioSource), MXRA8 (Cusabio), PEDF (Cloud-Clone Corp), ADAM9 (R&D Systems), and C1NH (R&D Systems) were used for the assay according to the manufacturer’s instructions. The level of human IL-1β in the conditioned media was determined using a human IL-1β ELISA Kit (R&D Systems) following the manufacturer’s instructions. The absorbance was read at 450 and 540 nm using a microplate reader (Molecular Devices).

TACE activity was measured in SLE patient serum as well as from supernatants of stimulated monocytes with oxLDL and DNA-IC. Where indicated, anti-LOX-1 antibody (50 μg/mL) or TAPI-1 inhibitor (100 μM) were used to pre-treat cells for 30m. TACE activity was measured by using a synthetic peptide substrate containing the cleavage site (Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Ser-Arg-NH2) primarily for ADAM17 and related enzymes such as ADAM8, ADAM9 and ADAM10 (R&D Systems, Inc., MN, USA). The cleavage site by ADAM17 and ADAM10 is the peptide bond between Ala and Val. 10 μL of each sample (serum or supernatant) was added to 90 μL of 25 mM Tris buffer, pH 8.0. The substrate was then added at a final concentration of 10 μM in a total of 100 μL reaction mixture for 1h. Fluorescence units (FU) were measured using a fluorescent microplate reader, Spectramax (Molecular Devices, CA, USA), with an excitation wave length at 320 nm and an emission wavelength at 405 nm. TACE activity results were normalized to total protein concentration of serum samples or supernatant. Enzymatic activity of TACE is represented as FU/min/μg.