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. Author manuscript; available in PMC: 2013 May 15.
Published in final edited form as: Biochem J. 2013 May 15;452(1):111–119. doi: 10.1042/BJ20130147

Figure 4. Bak is necessary and sufficient for ceramide synthase activity, in vitro.

Figure 4

A, Bak deficient mitochondria are severely deficient in CerS activity. Increasing amounts of mitochondria isolated from wild-type (wt) or Bak deficient (Bak KO) baby mouse kidney (BMK) cells were incubated with C17-sphingosine for 15 minutes. Lipids were extracted and the conversion of C17-sphingosine into C17-ceramide was quantitated by HPLC-MS/MS. Data shown are the combination of biological triplicates done in duplicate. B, Soluble Bak, but not Bax, activates CerS on Bak KO microsomes. Microsomes isolated from Bak KO BMK cells (10 μg) were incubated with increasing concentrations of cleaved. soluble Bak (cBak, closed circles) or soluble Bax (open circles) for 15 mm and the activity of CerS was determined. C, Activated Bak is a more potent activator of CerS, in vitro. Microsomes from Bak KO BMK cells (10 μg) were incubated with recombinant Bak (5 ng), cleaved, active Bid (tBid) (10 ng), or Bak and tBid (5 ng Bak; 10 ng tBid). Reactions were incubated with C17-sphingosine for 15 minutes and the conversion to C17-ceramide was quantitated by HPLC-MS/MS. D. Baby mouse kidney cells established from wild-type mice (WT BMK) were treated with 2.67 μM of ABT-263 (ABT) or vehicle (DMSO) for 2 and 8 hours. Lipids were extracted and the total cellular levels of individual ceramide species were quantitated by HPLC-MS/MS. E, Baby mouse kidney cells established from Bak KO mice (BAK KO BMK) were treated with 2.67 μM of ABT-263 (ABT) or vehicle (DMSO) for 2 and 8 hours. Lipids were extracted and the total cellular levels of individual ceramide species were quantitated by HPLC-MS/MS. C, Heavy membranes were isolated from WT (closed circles) or Bak KO (open circles) BMK cells and incubated with increasing concentrations of ABT-263. The relative increase in the conversion of C17-sphingosine to C17-ceramide is indicated, as compared to vehicle treated heavy membranes from the respective genotype of BMK cell. Dashed line indicates no fold change. Each point represents biological triplicates done in duplicate. ***, p=<0.0005; **, p=<0.005; *, p=<0.05.