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. 2001 Nov 20;98(24):13884–13888. doi: 10.1073/pnas.241358198

Figure 5.

Figure 5

Requirement of both caspase-8 and -10 for Fas signaling. (A) I9.2 cells were transfected with 12 μg of caspase-8, caspase-10L, or caspase-10S plus 12 μg of vector control, mutant caspase-8, mutant caspase-10L, or mutant caspase-10S by electroporation. Four micrograms of GFP plasmid was included in each transfection. The cells were incubated with 30 μg/ml of FLAG-tagged FasL or TRAIL plus 1 μg/ml of anti-FLAG Ab (Alexis). After culture at 37°C for 24 h, GFP+ cells negative for annexin V staining were quantitated by flow cytometry, and the percentage of cell loss was calculated (13). Less than 5% transfected cells were positive for annexin V staining without FasL or TRAIL treatment. (B) I9.2 cells were transfected with caspase-8, -10, or control vector plus GFP. Between 55–65%, cells were GFP+ by flow cytometry analysis (data not shown). The cells were treated with 0.1 μg/ml of FLAG-tagged FasL plus 1 μg/ml of anti-FLAG Ab (Alexis), and cell lysates were used for Western blot analysis of RIP (Upper) and caspase-3 (Lower).