Figure 5. 14-3-3θ mutant that cannot bind Bax is not protective against rotenone.

a) Lysates from stable cells overexpressing either full-length 14-3-3θ or a C-terminally deleted mutant 14-3-3θ (aa1-239) were immunoprecipitated with a polyclonal rabbit antibody against Bax and then immunoblotted with a monoclonal mouse antibody against V5. Blot was reprobed with anti-Bax antibody to verify Bax pulldown (bottom blot). Lysate lanes on right in the Bax blot are shown at a different exposure time than the immunoprecipitant lanes from the same gel. Considerably much less mutant 14-3-3θ was immunoprecipitated with Bax compared to full-length 14-3-3θ. b) Vector control, full-length 14-3-3θ, or mutant 14-3-3θ cells were treated with rotenone for 48 hours. Cell death was assessed by LDH release. While full-length 14-3-3θ cells showed decreased cell death in response to rotenone, cells overexpressing mutant 14-3-3θ showed no protection against rotenone compared to vector control cells. Error bars reflect SEM. Results reflect three independent experiments with at least two replicates per experiment. ***p<0.001 (Bonferroni's multiple comparison test). n.s.  =  non-significant.