Skip to main content
. 2008 Sep 16;105(38):14453–14458. doi: 10.1073/pnas.0807549105

Fig. 2.

Fig. 2.

Cyt b is cleaved during FAS-induced apoptosis and its C-terminal part is released into the cytoplasm. (A) Intracellular distribution of Cyt b in DF-1 cells left untreated (control) or treated with FAS-agonistic antibody in the absence or presence on the proteosomal inhibitor MG-132 (PI). Cytoplasmic and mitochondrial fractions prepared either 1 or 2 h after treatment were analyzed by Western blotting with antibodies against Cyt b, proliferating cell nuclear antigen (PCNA) (cytoplasmic marker) and Cyt c. Full-length (30 kDa) and cleaved (15 kDa) Cyt b are indicated by arrows. (B) Mitochondrial and cytoplasmic fractions were prepared from HeLa cells left untreated (control) or treated with FAS antibody at the indicated times after treatment (0.5–4 h). Fractions were analyzed by Western blotting for Cyt b (full-length, 30 kDa, and cleaved, 15 kDa), GRP75 (mitochondrial marker), PCNA (cytoplasmic marker), and Cyt c. (C) Western blot analysis of HeLa cells expressing full-length Cyt b, Cyt b512–1146 (C-term), or the portion of Cyt b corresponding to GSE F21 in the cytoplasm. All recombinant proteins contained a C-terminal FLAG tag and were detected using anti-FLAG antibodies. (D) Full-length Cyt b and Cyt b512–1146 are toxic when expressed in the cytoplasm. HeLa cells were infected with lentiviruses directing expressing of EGFP (negative control), full-length Cyt b, the C-terminal half of Cyt b (Cyt b512–1146), the N-terminal half of Cyt b or the portion of Cyt b corresponding to GSE F21 as indicated below each bar. Cell survival was measured by methylene-blue staining 72 h after infection and is shown relative to the EGFP control (set at 100%). Here and in (E), the error bars indicate standard deviation of three independent experiments. (E) Quantitation of cells with subG1 DNA content. FACS analysis was performed on HeLa cells 72 h after infection with lentiviral Cyt b constructs as in (D). (F) Time course of caspase induction in HeLa cells expressing cytoplasmic Cyt b. Activation of caspases 3/7 and 9/6 were measured using the fluorogenic substrates DEVD-AMC (filled circles) and LEHD-AMC (open circles), respectively. AMC fluorescence resulting from substrate cleavage was measured at the indicated times after substrate addition.