In vivo interaction of caspase-8L with FADD in MCF-7 cell lines.
MCF-7 cells (2 × 107 cells) were transiently transfected with 4 μg empty vector (pcDNA3.1/HisA, Invitrogen) and His-tagged caspase-8L expression vector using LipofectAMINE (Life Technologies) as described in “Materials and methods.” Eighteen hours after transfection, cells were lysed and cleared by centrifugation. The supernatants were incubated with Ni-NTA Magnetic Agarose Beads, and Ni-NTA beads were collected using a magnetic separator. After washing, the proteins bound to His-tagged protein were eluted with SDS-PAGE buffer and fractionated by SDS-PAGE. In the upper column, eluates were blotted with anti-FADD antibody. In the middle column, eluates were blotted with anti-HisG antibody to confirm the binding of His-tagged caspase-8L to the Ni-NTA beads. In the bottom column, cell lysates of transfected MCF-7 cells were analyzed by SDS-PAGE and blotted with anti-FADD antibody. NS indicates nonspecific band.
