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. 1999 Apr 13;96(8):4552–4557. doi: 10.1073/pnas.96.8.4552

Figure 4.

Figure 4

Defective CD95 signaling in ALPS patient cells. (A) Analysis of CD95 signal complex formation in ALPS patient cell lines. The top immunoblots show caspase-8 (pro-Casp-8), FADD, and CD95 proteins immunoprecipitated from either normal EBV-transformed lymphocytes (lanes 1, 2) or representative ALPS patients’ cells (Pt 3:T225P, Pt 6:A241D, Pt 26:D244V, Pt 29:R234Q, and Pt 31:R234P) after incubation either with (+) or without (−) anti-CD95 Apo-1 MAb treatment as indicated. The caspase-8 immunoprecipitate blot is intentionally overexposed to show the residual levels of caspase 8 associated with CD95 in ALPS patients’ cells. Equivalent levels of CD95 were precipitated as shown. The bottom immunoblot shows caspase-8 levels in total cell lysates from the same samples. (B) Kinetic analysis of CD95-stimulated caspase activity in extracts from EBV-immortalized B cell lines from a normal control (WT) or from the ALPS patients as indicated. Caspase activity was determined by measuring the fluorescence of the AMC group released from DEVD-AMC tetrapeptide substrate. The fluorescence units of the assay are based on an arbitrary scale standardized to control extracts. These data are representative of three experiments.