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. 2000 Oct 30;151(3):483–494. doi: 10.1083/jcb.151.3.483

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© 2000 The Rockefeller University Press

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.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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graphic file with name JCB0006120.f6ab.jpg — Bcl-2 but not active Akt blocks cytochrome c release. (A) Active Akt fails to inhibit cytochrome c release induced by C2-ceramide. Vector control line or two independent active Akt lines were treated with 30 μM C2-ceramide for the indicated period. Cells were then fractionated and equal amounts of cytosolic protein were loaded into each lane. The blot was probed with an mAb to cytochrome c (top). For the internal control, the blots were stripped and reprobed with antibody to α-tubulin (middle). The cytosolic fraction was confirmed to be without mitochondria with an mAb to COX IV (bottom; M represents mitochondria as a positive control for COX IV). Data are from a representative experiment performed three times. Note the delay in cytochrome c release is due to cells being treated with 30 μM C2-ceramide in this experiment rather than 50 μM C2-ceramide as used in experiments shown in Fig. 2 C. (B) Quantification of cytochrome c release. Bars: white, pCR vector; black, myr-Akt clone A7; stippled, myr-Akt clone B10. Data represent mean ± SEM of three independent experiments. (C) Bcl-2 inhibits cytochrome c release induced by C2-ceramide. Vector control line or two independent Bcl-2 lines were treated with 30 μM C2-ceramide for the indicated period. Cells were then fractionated and equal amounts of cytosolic protein were loaded into each lane. The blot was probed with an mAb to cytochrome c (top). For the internal control, the blots were stripped and reprobed with antibody to α-tubulin (middle). The cytosolic fraction was confirmed to be without mitochondria with an mAb to COX IV (bottom; M represents mitochondria as a positive control for COX IV). Data are from a representative experiment performed three times. (D) Quantification of cytochrome c release. Bars: white, pCMV vector; black, hBcl-2 clone 98-1; stripped, hBcl-2 clone 98-2. Data represent mean ± SEM of three independent experiments.

graphic file with name JCB0006120.f6cd.jpg — Bcl-2 but not active Akt blocks cytochrome c release. (A) Active Akt fails to inhibit cytochrome c release induced by C2-ceramide. Vector control line or two independent active Akt lines were treated with 30 μM C2-ceramide for the indicated period. Cells were then fractionated and equal amounts of cytosolic protein were loaded into each lane. The blot was probed with an mAb to cytochrome c (top). For the internal control, the blots were stripped and reprobed with antibody to α-tubulin (middle). The cytosolic fraction was confirmed to be without mitochondria with an mAb to COX IV (bottom; M represents mitochondria as a positive control for COX IV). Data are from a representative experiment performed three times. Note the delay in cytochrome c release is due to cells being treated with 30 μM C2-ceramide in this experiment rather than 50 μM C2-ceramide as used in experiments shown in Fig. 2 C. (B) Quantification of cytochrome c release. Bars: white, pCR vector; black, myr-Akt clone A7; stippled, myr-Akt clone B10. Data represent mean ± SEM of three independent experiments. (C) Bcl-2 inhibits cytochrome c release induced by C2-ceramide. Vector control line or two independent Bcl-2 lines were treated with 30 μM C2-ceramide for the indicated period. Cells were then fractionated and equal amounts of cytosolic protein were loaded into each lane. The blot was probed with an mAb to cytochrome c (top). For the internal control, the blots were stripped and reprobed with antibody to α-tubulin (middle). The cytosolic fraction was confirmed to be without mitochondria with an mAb to COX IV (bottom; M represents mitochondria as a positive control for COX IV). Data are from a representative experiment performed three times. (D) Quantification of cytochrome c release. Bars: white, pCMV vector; black, hBcl-2 clone 98-1; stripped, hBcl-2 clone 98-2. Data represent mean ± SEM of three independent experiments.