Mab531

Unconjugated and Alexa 647-conjugated anti-mouse uPAR antibody were from R&D Systems (MAB531 and FAB531R, respectively); TLR4 antibody (MAB27591) was from R&D Systems; LPS (L2887) was from Sigma, Biotin-LPS and PAM3CSK4 were from Invivogen; Soluble mouse uPAR was from CinoBiologicals. Human IL-6 and IL-8 ELISAs were from Thermofisher Scientific. Mouse inflammation CBA kit was from BD Biosciences. Mouse and human uPAR siRNA and non-sence siRNA control duplexes were from Santa Cruz Biotechnology. Fluorescein isothiocyanate (FITC) was from Sigma.

Protein concentration in the human serum/mouse plasma samples was determined by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Equal amounts (30 μg) of protein from each samples were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel was equilibrated in transfer buffer at RT, and the protein was transferred onto polyvinylidene difluoride membranes (Millipore Immobilon-P, Sigma, St. Louis, MO) for 2.5 h at 4 °C in transfer buffer. The membranes were then blocked with 2 % bovine serum albumin (BSA) of TBST at 4 °C overnight. The next day, membranes were washed with TBST, and blots were individually incubated with antibodies against the human uPAR (MAB807, R&D) or mouse uPAR (MAB531, R&D) overnight at 4 °C, followed by incubation with a goat anti-rabbit antibodyfor 30 min at RT. Bindings were visualized with the Western Lightning Chemiluminescence Reagent Plus (Perkin-Elmer Life Sciences, Boston, MA) and exposed to Kodak film (Rochester, NY). Five individual samples at indicated time points were randomly selected for testing.