HEK 293T cells were transfected with the indicated N-terminally HA-tagged TNFR1-EYFP fusion protein constructs. Culture supernatants or anti-TNFR1 antibodies (R&D Systems; Santa Cruz Biotechnology, Inc.) were used to coat Nunc™ microtitre plates. HA-tagged TNFR1 proteins were detected using biotinylated anti-HA.11 (clone 16B12; Covance) or biotinylated anti-TNFR1 antibodies (R&D Systems; Santa Cruz Biotechnology, Inc.) and Europium-conjugated Streptavidin (Perkin Elmer). For Western blots, HA-tagged TNFR1 proteins detected using anti-HA.11 (clone 16B12; Covance) and IRDye® 800CW goat-anti-mouse IgG secondary antibody, and visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences).
