Figure 2.

Src kinase activity modulates Fas-induced apoptosis and Caspase-8 activity. (A) HeLa cells were transiently transfected with SrcY527F or with the empty vector as control. Immunoprecipitated Caspase-8 was separated by SDS–PAGE gel and analyzed by immunoblotting with anti-phosphotyrosine and with anti-Caspase-8 antibodies. The arrow indicates Caspase-8. (B) At 24 h after transfection, HeLa cells, transiently transfected with the indicated constructs, were treated with 250 ng/ml anti-Fas and 1 μg/ml cycloheximide. Apoptosis was determined by counting Hoechst-stained fragmented nuclei in GFP-positive cells. The data are presented as the mean value±s.d. of five independent experiments. (C) HeLa cells were transfected with the indicated expression constructs. Apoptosis was induced as in Figure 2B and Caspase-8 activity was analyzed in protein extract by measuring the hydrolysis of the Caspase-8 substrate Ac-IETD-pNA. Data are presented as the mean value±s.d. of three independent experiments.