Adam10

The paraffin-embedded specimens were dewaxed in dimethylbenzene and hydrated in gradient alcohol. The sections were treated according to the following protocols: Endogenous peroxidase removed (incubation in 3% H₂O₂ for 30 min), antigen retrieved (about 95°C for 45 min), blocking with 10% goat serum (at room temperature for 30 min), primary antibody incubation overnight at 4°C, HRP-conjugated secondary antibodies incubation for 30 min at 37°C, DAB color reaction, hematoxylin staining, differentiation, dehydration and transparency. The dilution of primary antibodies was 1∶50 for ADAM-8, ADAM-10, ADAM-11 (Santa Cruz Biotechnology, Dallas, TX, USA), 1∶200 for ADAM-12 (Abgent, Suzhou, China), ADAM-15 and ADAM-17 (R&D Systems, Minneapolis, MN, USA). Two pathologists who were blinded to the clinical diagnosis performed the staining scores. In the event of a disputed result, consensus was reached by discussion. A semi-quantitative scoring system was used to grade the samples according to the percentage of positive staining: grade 0 (negative), grade 1 (weak positive, positive expression was less than 10%), grade 2 (moderate positive, positive expression was between 10% and 60%) and grade 3 (strong positive, positive expression was greater than 60%) [20] (link).

Brain (cortex and hippocampus regions) was homogenized in RIPA lysis buffer containing protease inhibitors as described [31 (link)]. Equal amounts of sample protein were separated on Tris–HCl polyacrylamide SDS gels and transferred to polyvinylidene fluoride membranes. Blots were placed in blocking solution with 10% non-fat milk in PBS with 0.05% Tween-20 (PBS-T) for 1 h, followed by incubation with various primary antibodies with 5% non-fat milk in PBS-T for 3 h at room temperature or overnight at 4 °C. Primary antibodies include BACE1 (Santa Cruz), ADAM10 (Santa Cruz), ADAM17 (Millipore Sigma), Presenilin-1 (PS-1) (Novus Biologicals) and APP [to detect both full-length APP and C-terminal fragments (CTFs); CT695, ThermoFisher]. Blots were washed with PBS-T, incubated with horseradish peroxidase-conjugated secondary antibodies, and then developed with Super Signal® West Pico chemiluminescent Substrate (Thermo Scientific).

Cells were lysed using RIPA buffer (Biosolution, Korea), phenylmethylsulfonyl fluoride (Sigma), protease inhibitor, and phosphatase inhibitor cocktail 2/3 (Sigma) and then centrifuged at 25,000 x g for 30 min. Cell extracts were quantified using the BCA protein assay kit (Pierce Biotechnology, USA) according to the manufacturer's instructions. The proteins (20-30 μg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (GE Healthcare, UK) for western blot analyses. The transferred membrane was blocked with 1x Tris-buffered saline with Tween 20 (Sigma) (TBST) containing 5% skim milk (BD Biosciences) for 1 h and then incubated with primary antibodies ADAM10 (Santa Cruz), ADAM17 (Abcam, USA), β-arrestin (Bethyl, USA), and CXCR6 (GeneTex) in 1x TBST containing 1% skim milk at 4°C overnight. The membrane was washed three times with 1x TBST for 10 min, then incubated with secondary anti-rabbit (Cell Signaling) and anti-mouse (Santa Cruz) antibodies in 1x TBST containing 1% skim milk for 45 min. The membrane was washed three times with 1x TBST for 15 min and was visualized with enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, USA) and imaged by ChemiDoc (Bio-Rad Laboratories, USA).