HEK 293T cells were transiently transfected with TNF-FLAG or Fc fusion constructs. Fc fusion proteins were purified from culture supernatant using protein A Sepharose (Sigma). Following Fc-fusion protein pre-incubation with TNF-FLAG, immunoprecipitation was performed using protein A Sepharose. An Fc fusion of the first cysteine-rich domain of DR5 (DR5.CRD1-Fc) was used as a negative control for TNF binding.
