Mab425

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).

SCs were plated in PDL-coated 6-well plates at a density of 3.0 × 10⁵ cells/well and cultured in complete medium until ~85% confluent. Cells were treated with various proteins and reagents, alone, simultaneously or preincubated as noted, including: TNFα (0–1 nM); SC EVs (10 μg); TACE-EVs (10 μg); TNFR1-neutralizing monoclonal antibody (2.5 or 10 μg, R&D Systems, MAB425); a neutralizing antibody to TNFR2 (2, 4 or 8 μg; MA5–29837, Invitrogen); or IgG (10 μg; Sigma Aldrich, 06–255). In some cases, SCs were electroporated using the Rat Neuron Nucleofector Amaxa Kit (Lonza Biosciences) and incubated with siRNA to silence TNFR2 expression (siTNFR2; M-094191–01-0010, Dharmacon). Control cells were transfected with non-targeting control (NTC) siRNA (NTC; D-001810–10-05, Dharmacon). Cells were treated with TNFα. Cell extracts were prepared in RIPA buffer, and sodium orthovanadate. The protein concentration was determined by BCA. An equivalent amount of cellular protein (10–20 μg) was subjected to SDS-PAGE and immunoblot analysis. Membranes were incubated with primary antibodies that recognize phospho-p38 MAPK (rabbit polyclonal 1:1000, Cell Signaling, 9211 s) and total p38 MAPK (1:10000, Cell Signaling, 9212 s).