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. 2010 Dec 24;30(3):556–568. doi: 10.1038/emboj.2010.346

Figure 1.

Figure 1

Fis1-induced apoptosis requires Bap31, but activated Bap31 does not depend on Fis1. (A) The percentage of apoptosis induced by Fis1 or Bax (as a control) was measured at different time points with propidium iodide staining of the cells by FACS. For Bax transfections, the amount of plasmid was 1/5 of Fis1 to match the percentage of apoptosis. (B) EYFP was fused to the C-terminus of human Bap31 to generate Bap31–EYFP. Its cleavage (indicated by triangles) can be determined by the production of EYFP plus an eight amino-acid extension (top panel). Cells were transfected and harvested at the indicated time periods (bottom panel). For Bax transfections, the amount of plasmid used was as in A. Bap31–EYFP cleavage was revealed by immunoblotting. Cell lysates from GFP-transfected cells were used as a size indicator. p.t.: post transfection. (C) Cells were co-transfected with Fis1 and Bap31–EYFP with or without zVAD-fmk (50 μM) and harvested at the indicated time points p.t. The cleavage of Bap31–EYFP was determined by immunoblotting as in B. (D) Cells were transfected either with β-gal or Fis1 and harvested 27 h p.t. and the cleavage of the endogenous Bap31 was revealed by immunoblotting. Note that the blots shown were from the same membrane with the same exposure time. (E) Cells were transfected with Bap31 siRNA (si) or control siRNA (co) and harvested on days 1 to 5. Downregulation of Bap31 was analysed by western blot. (F) Cells were transfected with the indicated genes on day 3 after siRNA transfection targeting Bap31 (or a control) and the percentage of subG1 population was quantified on day 5. (G) Cells were transfected with shRNA against Fis1 (pSuper Fis1) or an empty pSuper vector and harvested at the indicated time points. The vector-transfected cells were collected on day 5. Fis1 downregulation was determined by western blot. (H) Cells were transfected either with pSuper Fis1 or pSuper vector. On day 4 after shRNA transfection, cells were transfected with β-gal or p20Bap31 and the percentage of the subG1 cell population measured on day 5. All the histograms are presented as means±s.d.