Bt210 010

To covalently stabilize non-covalent complexes, we used the bifunctional cross-linker, bis (sulfosuccinimidyl) suberate (BS³) (Thermo Fisher, 21580) as previously described (Crookston & Gonias, 1994 (link)). Rat SC EVs (10 μg) were incubated with TNFα (0.5 nM, R&D Systems, BT210–010) and then with 5 mM BS³ for 10 min at 37°C. TNFα (0.5 nM) was treated with BS³ in the absence of SC EVs as a control. All samples were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk (Bio-Rad, 170–6404) in 10 mM Tris–HCl, 150 mM NaCl, pH 7.5, 0.1% Tween 20 (TBS-T) for 90 min at room temperature. Blots were probed with primary TNFα-specific antibody (TNFα mouse monoclonal 1:500, Santa Cruz, sc-133192) (1:1000) overnight at 4°C. Subsequently, the membranes were washed with TBS-T and incubated with secondary mouse HRP-conjugated antibodies (1:2000; Cell Signaling Technology, 7076S) in TBS-T for 1 h at room temperature. Signals were detected by ECL (GE Healthcare).

To measure TNFα-binding, different amounts of SC EVs in 3 μl final volume were dotted onto nitrocellulose membranes and allowed to air dry for 1 h at room temperature. The membranes were then blocked with 5% milk in TBS-T and subsequently incubated with 1 nM of biotinylated TNFα (R&D, BT210–010) in 0.1% BSA and TBS-T overnight at 4°C and then with streptavidin-HRP-conjugated antibody (R&D DY998). Binding was detected by enhanced chemiluminescence (GE Healthcare). In separate studies, membranes with immobilized EVs were blocked with 5% milk in TBS in the absence of 0.1% (vol/vol) Tween-20, to avoid EV permeabilization, for 1 h at room temperature. Subsequently, membranes were incubated with primary antibodies against TNFR1 (rat monoclonal 1:1000; R&D Systems, MAB425) and then with HRP-conjugated secondary antibody in 5% milk with TBS followed by detection and imaging with ECL (GE Healthcare).