TNFα-induced Fas is exposed to the cell surface and enhances FasL-induced DISC formation. A. SK-N-AS cells were treated with 100 ng/ml TNFα for the indicated times, and cell surface levels of Fas protein were assessed by cell surface biotinylation and subsequent Western blot. Below, quantification of total and cell surface Fas expression normalized vs. matching α-Tubulin controls. B. DISC (Death inducing signaling complex) formation assay in SK-N-AS cells pre-treated or not with 100 ng/ml TNFα and treated for 30 min with 2.5 μg/ml Fc:hFasL. The DISC proteins were immunoprecipitated by pulling down Fc:hFasL with protein G-Sepharose and analyzed by Western blot.