Fig. 1. Purification of PIDDosome components for in vitro analysis. (A) Domain organizations of PIDDosome components, PIDD, RAIDD, and caspase-2. RAIDD interacts with PIDD via a DD:DD interaction (black arrow) and with caspase-2 via a CARD:CARD interaction (black arrow). LRR, Leucine-Rich Repeats; ZU5, Zo-1 and UNC-5 protein containing domain; DD, Death Domain; CARD, Caspase Recruiting Domain. (B) His-tag affinity purification of full-length RAIDD. Lane 1, marker; Lane 2, supernatant of the cell lysate; Lane 3, pellet of the cell lysate; Lane 4, flow through after incubation with Ni-NTA beads; Lane 5, washing; Lanes 6-8, imidazole eluted fraction. (C) His-tag affinity purification of RAIDD CARD. Lane 1, marker; Lane 2, supernatant of the cell lysate; Lane 3, pellet of the cell lysate; Lane 4, flow through after incubation with Ni-NTA beads; Lane 5, washing; Lanes 6-8, imidazole eluted fraction. (D) His-tag affinity purification of RAIDD DD. Lane 1, marker; Lane 2, supernatant of the cell lysate; Lane 3, pellet of the cell lysate; Lane 4, flow through after incubation with Ni-NTA beads; Lane 5, washing; Lanes 6-8, imidazole eluted fraction. (E) His-tag affinity purification of PIDD DD. Lane 1, marker; Lane 2, supernatant of the cell lysate; Lane 3, pellet of the cell lysate; Lane 4, flow through after incubation with Ni-NTA beads; Lane 5, washing; Lanes 6-8, imidazole eluted fraction. (F) His-tag affinity purification of caspase-2 CARD. Lane 1, marker; Lane 2, supernatant of the cell lysate; Lane 3, pellet of the cell lysate; Lane 4, flow through after incubation with Ni-NTA beads; Lane 5, washing; Lanes 6-8: imidazole eluted fraction.