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. 2012 Mar 19;7(3):e31510. doi: 10.1371/journal.pone.0031510

Figure 2. Basic residues in the C-segment are necessary for Bak stability and function.

Figure 2

(A) C-terminal sequence of Bak variants indicating which basic residues in the C-segment that were substituted with serine. (B) Substitution of basic residues in the C-segment reduces Bak proapoptotic function. bak−/−bax−/− MEFs expressing Bak, BakRRS, BakRSS or BakSSS were left untreated, or treated with UV or etoposide for 24 h. Percentage cell death is expressed as the mean ± SEM from three independent experiments. Statistical significance for treatment when compared to Bak; *p<0.05, **p<0.01. Upper panel is a western blot of cell lysates immunoblotted for Bak, and for β-actin as a loading control. (C) Substitution of basic residues in the C-segment destabilizes Bak. Cells from (B) were incubated with cycloheximide for up to 24 h and cell lysates immunoblotted for Bak, and for β-actin as a loading control. Note that due to low expression of BakSSS, 4-fold total protein was loaded onto the gels. (D) Substitution of basic residues does not prevent targeting to membranes. Cells from (B) were left untreated or treated with UV, separated into cytosolic and membrane fractions, and immunoblotted for Bak and for the cytosolic marker HSP70. Results are representative of two or more independent experiments.