Fig. 2. Caspase-8 binding to FADD during induction of Fas- or Ras-mediated apoptosis. In A, lysates containing an equal amount of total proteins from Jurkat, Jurkat/Fasm (I4.1), or Jurkat/FADDm (I2.1), with or without expressing v-ras, were immunoprecipitated and immunoblotted with an anti-FADD Ab directed against either the COOH or NH2 terminus of the protein (upper two panels). The same blots were reprobed with an anti-actin Ab for the determination of equal loading of samples (lower two panels). In B, after treatment with either high-dose PMA (500 nM for 24 h) or an anti-Fas Ab (for 30 min), lysates from Jurkat cells or mutant Jurkat cell lines without (panels 1 and 3 from the top) or with (panels 2 and 4) ectopic expression of v-ras were prepared. Upper two panels, after normalization for protein concentration, the samples were immunoprecipitated with the anti-FADD Ab directed against the NH2 terminus of the protein, and the immunocomplexes were separated on a SDS-PAGE gel. After transfer of the proteins to nitrocellulose, immunoblotting was performed using an anti-caspase-8 Ab. Lower two panels, the same blots were reprobed with the anti-FADD Ab for determination of even loading of samples.