Mutagenesis of the Bak Membrane Anchor Figure 3. The Bax C-segment converts Bak to a semi-cytosolic protein. (A) C-terminal sequence of Bak/Bax chimeras. (B) Bak containing the Bax C-terminus or C-segment retains stability and function. bak2/2bax2/2MEFs expressing Bak or Bak/Bax chimeras were left untreated or treated with UV or etoposide. Percentage cell death is expressed as the mean 6 SEM from three independent experiments. Statistical significance for treatment when compared to Bak; **p,0.01. Upper panel is a western blot of cell lysates immunoblotted for Bak and for b-actin as a loading control. (C) Bak containing the Bax C-terminus or C-segment retains stability. Cells from (B) were incubated with cycloheximide and cell lysates immunoblotted for Bak, and for b-actin as a loading control. (D) Bak containing the Bax transmembrane domain or C-segment locates to both cytosolic and membrane fractions. Cells from (B) were left untreated or treated with UV, separated into cytosolic and membrane fractions and immunoblotted for Bak or Bax, and for the cytosolic marker HSP70. (E) Bak/Bax chimeras in the membrane fraction are mostly membrane-integrated. Cells were treated as in (D) and the membrane fractions treated with sodium carbonate to extract peripherally attached proteins. Samples were immunoblotted for Bak or Bax. (F) Bak/Bax chimeras retain ability to change conformation and oligomerize during apoptosis. Cells were treated as in (D) and the cell lysates exposed to oxidant (CuPhe), run on non-reducing SDS-PAGE, and immunoblotted for Bak. MX, non-activated intramolecular crosslinked monomer; M, non-crosslinked monomer; D, intermolecular crosslinked dimers; T, crosslinked trimers. (G) Following UV, oligomerized Bak/ BaxCS is mainly at mitochondria. Cells were treated as in (D), separated into cytosolic and membrane fractions, run on non-reducing SDS-PAGE and immunoblotted for Bak. Results are representative of two or more independent experiments. doi:10.1371/journal.pone.0031510.g003 PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e31510