Phosphatase inhibitor cocktail 2 3

Flash frozen brain tissue was obtained from 37 of the 86 CTE cases as previously described [9 (link)]. Frozen tissue was collected from identical regions in Broadman area 8/9. Briefly, freshly prepared, ice cold 5 M Guanidine Hydrochloride in Tris-buffered saline (20 mM Tris-HCl, 150 mM NaCl, pH 7.4 TBS) containing 1:100 Halt protease inhibitor cocktail (Thermo Scientific) and 1:100 Phosphatase inhibitor cocktail 2 & 3 (Sigma) was added to the brain tissue at 5:1 and homogenized with Qiagen Tissue Lyser LT, at 50 Hz for 5 min. Lysate was diluted according to manufacture protocol and spun down at 17,000 g, 4 °C, for 15 min. Supernatant was investigated using a PSD-95 ELISA (MSD #K250QND) and run according to manufactures protocols. Plates were analyzed with an MSD SECTOR S 600 Imager, and results were reported as arbitrary values. Values appeared normally distributed on visual inspection and then were converted to z-scores with a mean of zero and standard deviation of one.

Cells were harvested using 1x RIPA buffer (Cell Signaling Technology) with protease inhibitor (cOmplete Mini, EDTA-free, Roche (Basel, Switzerland)) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2 &3, Sigma-Aldrich; PMSF, Roche). Twenty μg of protein per sample was loaded onto NuPAGE Novex Bis-Tris 4–12% Protein Gels (Invitrogen) for electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membranes (0.45 μm, Millipore (Darmstadt, Germany)). Membranes were blocked in 5% non-fat dry milk (BioRad Laboratories (Hercules, CA)) for 1 hour at room temperature and then incubated with appropriate primary antibodies overnight at 4°C. Secondary antibodies and an ECL kit from GE Healthcare Life Sciences (Pittsburgh, PA) were employed to generate chemiluminescent signals. All immunoblot data represent triplicate repeats. Densitometry analysis was performed using National Institutes of Health (NIH) ImageJ software [17 (link)].

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).

For 2D culture, cells were seeded at 5 × 10⁵ cells per well in a six well plate with regular media. After 24 h the regular media was removed and cells were washed twice with PBS, and serum-free media was added. Conditioned (serum-free) media and cell lysate were collected after 24 h. For 3D culture, cell washing and refresh with conditioned medium was conducted repeated at day 8, following sample collection at day 9. For 3D cultures, cells were also harvested from lrECM for immunoprecipitation as previously described [25 (link)]. Melanoma cells and normal human melanocytes were lysed in Mammalian Protein Extraction Reagent (Thermo Scientific, Waltham, MA), 1× protease inhibitor cocktail (Calbiochem, Billerica, MA) and 1× phosphatase inhibitor cocktail 2 & 3 (Sigma-Aldrich, St. Louis, MO) followed by centrifugation (13,200 rpm, 4 °C, 5 min), after which the supernatants were stored at -80 °C until use. Protein concentrations were determined with the Pierce^® BCA Protein Assay kit (Thermo Scientific), using BSA as the concentration standard. Extracted proteins were then resolved using 4–12 % Bis-Tris Gels (Life Technologies) and subsequently transferred to PVDF membranes (Life Technologies) for immunoblotting.

We transfected HEK-293T cells with plasmid DNAs encoding, respectively, human suPAR1 (C-terminal Myc/Flag tag), ACE2 (C-terminal C9 tag), SARS-CoV-2 spike (C-terminal C9 tag) and integrin aV subunit (C-terminal V5-HIS tag) fusion proteins using Fugene 6 reagent (Promega, E2692). Thirty-six hours after transfection, cells were harvested and cell pellets were resuspended in cell lysis buffer with Protease inhibitor cocktail (Roche, 04693116001) and Phosphatase Inhibitor Cocktail 2 & 3 (Sigma, P5726 and P0044). Co-immunoprecipitation was performed using a Pierce Protein A/G Agarose (Thermo Scientific, 20421) or according to the manufacturer’s instructions. Briefly, the Myc mouse monoclonal antibody (Sigma, M4439), mouse anti-V5 antibody (Invitrogen, 46-075) was added to the precleared cell lysate, respectively and was rotated overnight at 4 °C, then, incubated with A/G Agarose slurry for 4 h. Samples were analyzed by immunoblotting with anti-C9 antibody (Abnova, PAB26959, 1:1000) to detect spike protein or ACE2, while mouse anti-Flag antibody (Sigma, F1804, 1:1000) was applied to detect suPAR1. The presence of integrin aV subunit was detected by monoclonal anti-His antibody (Invitrogen, MA1-21315, 1:1000). Experiments were repeated three times.