Halt protease inhibitor cocktail

ADC-7 β-lactamase
was expressed as previously described^{10 (link)} and purified using cation exchange chromatography.^{32 (link)} The expression plasmids for the other ADCs (-30/-33/-162/-212/-219)
were constructed in pET28a vectors by GenScript. For the purification
of all ADCs, cell pellets were suspended in 25 mM 3-(N-morpholino)propanesulfonic acid (MOPS buffer), pH 6.5, with 1×
HALT protease inhibitor cocktail (Sigma) and DNase I (50 Units). The
solution was sonicated for 4 × 30 s intervals on ice. The lysate
was centrifuged at 15,000 rpm at 4 °C for 20 min. The cell-free
extract was then loaded onto a carboxymethyl-cellulose column by gravity
flow at 4 °C (5 mL resin per gram of cell pellet). The column
was washed with 100 mL of 25 MOPS, pH 6.5 at a flow rate of 0.3 mL/min
followed by elution with a linear gradient of 0–0.5 M NaCl
in 25 MOPS, pH 6.5. The fractions containing ADC were collected, pooled,
and then dialyzed in 2 × 5 L of 25 MOPS, pH 6.5 at 4 °C.
The dialyzed ADC was concentrated to at least 10 mg/mL using an Amicon
Ultra centrifugal filter unit with Ultra-10 membrane (Millipore).
The concentration of ADCs was determined using the A₂₈₀ with an extinction coefficient of 46,300 M^–1 cm^–1, as calculated for the expressed residues 24–383
of all ADC variants by the ProtParam tool on the ExPASy bioinformatics
portal.³³

Cells were lysed with RIPA buffer including Halt Protease Inhibitor Cocktail (mouse myofibroblasts) or sodium vanadate (catalog #S6508, Sigma) and complete mini (Roche, Penzberg, Germany #11836170001; human lung fibroblasts). After storage at −80°C, cell lysates were sonicated and processed for immunoblot analysis (as described in “in vivo” methods).

To verify the occurrence of TNF-α elevation in our mTBI model and define its time dependence, mice were subjected to mTBI and brains were removed at specific times thereafter (1 to 18 h; n = 4 to 5 per time). The right cortex was immediately frozen in liquid nitrogen and homogenized with appropriate protease inhibitors (Halt Protease Inhibitor Cocktail; Sigma-Aldrich, St. Louis, MO, USA). The samples were then quantified for TNF-α levels by ELISA assay (BioLegend, San Diego, CA, USA).

Western blotting was performed as per the methodology described in previous reports [49 (link),50 (link),51 (link)]. The cells were lysed using an M-PER Mammalian Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL, USA) containing a Halt Protease Inhibitor Cocktail (Sigma Aldrich, St. Louis, MO, USA). CM was mixed with four volumes of cold acetone and incubated at −20 °C for 1 h, followed by centrifugation at 10,000× g for 20 min. The pellet was washed with ethanol and resuspended in the SDS sample buffer, and the protein extracts (10 μg) were separated using SDS-PAGE (10% acrylamide gel). Information about the primary antibodies is listed in Table S1. The HRP-conjugated secondary antibody was purchased from GE Healthcare (Buckinghamshire, UK) and the immunoreactive bands on the membrane were visualized using ImmunoStar Reagents (Fujifilm Wako) and the LAS-4000 image analyzer (Fuji Photo Film Co., Tokyo, Japan). β-actin and CD63 were used as loading controls for total cell lysate and sEVs lysate, respectively. Densitometric analysis was performed using ImageJ 1.52a software. The protein level of HSP70 and TGF-β was normalized by that of the loading controls and the data are presented as the fold change. The uncropped blots and molecular weight markers are shown in Supplemental Materials.