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. 2008 Nov 17;183(4):681–696. doi: 10.1083/jcb.200803129

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© 2008 Gonzalvez et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 4. — CL-deprived mitochondria efficiently release cytochrome c and Smac/Diablo after treatment with recombinant tBid protein. (A) The indicated cells were transfected with either empty vector control (EGFPN1) or with a plasmid encoding either full-length Bid protein fused to GFP (BidGFP) or a fused tBid-GFP protein (tBidGFP). Cell death was assessed by PI exclusion in GFP-positive cells by FACS analysis. Error bars represent ± standard deviation. (B) A cytochrome c–releasing assay of mitochondria isolated from control (DB037) or Barth syndrome–derived lymphoblastoid cells (DB105.2 and DB105.3). Mitochondria were either left untreated (u) or treated with increasing amounts of tBid (0.05, 0.1, 0.2, 0.5, 1, 5, 10, and 50 nM). Triton X-100 was used as a positive control for maximal cytochrome c release. In all cases, cytochrome c was efficiently released to the supernatant buffer when mitochondria were treated with tBid concentrations of 0.5 nM or higher. (C) Mitochondria were isolated from control-transfected (shCont1) or tafazzin knockdown (shTaz2) HeLa cells, and were either left untreated (u) or treated with increasing amounts of tBid (0.05, 0.1, 0.25, 0.5, and 5 nM). Cytochrome c and Smac/Diablo were analyzed in the supernatant (Sup) and cytochrome c, and tBid (translocated to the mitochondria) and subunit 6 of complex I (as mitochondrial loading control) were analyzed in the mitochondrial pellet (Mito). Efficient release of cytochrome c and Smac/Diablo from the mitochondria as well as the translocation of tBid to the mitochondria were identified at tBid concentrations equal to 0.1 nM or higher. (D and E) Mitochondria were isolated from control or tafazzin-deficient lymphoblastoid cells (D) or HeLa cells (E), and Bak oligomerization on the mitochondria was analyzed after treatment without (u) or with increasing amounts of tBid, as indicated in B and C. BMH (protein cross-linker) or DMSO (control solvent) were added after incubation with tBid. No higher molecular weight forms of Bak were observed in the absence of BMH, even when the maximal tBid concentration was added (E, right). Asterisks indicate multimers of the Bak protein.