Fig. 1.
(A) Full length 55-kDa is the predominant form of soluble TNFR1 present in human serum and lung epithelial lining fluid. Samples of proteins (60 μg) of serum and BALF from healthy research volunteers, or medium from HUVEC, were separated by SDS/PAGE, transferred to nitrocellulose, and reacted with a murine IgG2b monoclonal antibody (H-5) against the extracellular domain of human TNFR1. A recombinant human TNFR1 extracellular domain (rhTNFR1) is shown as a positive control. (B) Characterization of HUVEC-derived soluble TNFR1 as the full-length 55-kDa receptor by sequential centrifugation. Samples of proteins (60 μg) from the pellets [200 × g (P1), 500 × g (P2), 1,200 × g (P3), 10,000 × g (P4) or 100,000 × g (P5)], and the 100,000 × g supernatant (S5) from HUVEC medium, were separated by SDS/PAGE, transferred to nitrocellulose, and reacted with the H-5 anti-TNFR1 antibody. Also shown are total cellular (T) and membrane (M) proteins. Nitrocellulose membranes were stripped and reacted with antibodies against rab5 and LAMP-1. (C) Characterization of HUVEC-derived soluble TNFR1. Sequential centrifugations were performed as described in B. Samples of proteins (60 μg) from the pellets [100,000 × g (P5), 175,000 × g (P6)] and supernatants [100,000 × g (S5), 175,000 × g (S6)] from HUVEC medium were separated by SDS/PAGE, transferred to nitrocellulose, and reacted with the H-5 anti-TNFR1 antibody. These immunoblots are representative of three experiments demonstrating similar results. (D) Characterization of HUVEC-derived soluble TNFR1. Samples of proteins (40 μg) from HUVEC lysates (T) and medium that had been precleared of cells and debris by sequential centrifugation at 1,200 × g and 10,000 × g (S4) were separated by SDS/PAGE, transferred to nitrocellulose, and reacted with the H-5 anti-TNFR1 antibody. C-S4 denotes a 30-fold concentration of the S4 supernatant. (E) Confirmation of TNFR1 in HUVEC-derived secreted vesicles. Samples of proteins (60 μg) from the 175,000 × g pellet (P6) were separated by SDS/PAGE, transferred to nitrocellulose, and reacted with the H-5 (EC 1) and the clone 16805.21 (EC 2) murine monoclonal antibodies directed against the TNFR1 extracellular domain and the C-20 goat polyclonal antibody directed against the TNFR1 intracytoplasmic domain. (F) Characterization of the TNFR1 signaling complex I in HUVEC-derived exosome-like vesicles. Samples of proteins (60 μg) from whole cell lysates (T) and the 175,000 × g pellet (P6) were separated by SDS/PAGE, transferred to nitrocellulose and reacted with antibodies against SODD, TRADD, RIP, and TRAF2.