Fig. 6.

ToxB elicits translocation of a GFP-Bax fusion protein to mitochondria and conformational activation of endogenous Bax in CGNs. (a) On day 5 in vitro, CGNs were transfected with either GFP alone or a GFP-Bax fusion protein using a helium-powered gene gun, as described in Experimental procedures. At 48 h post-transfection, cells were exposed for 24 h to either vehicle (Veh; 2 μg/mL BSA in PBS) or ToxB (40 ng/mL). CGNs were then fixed in paraformaldehyde and nuclei were stained with DAPI. The expressed GFP-Bax fusion protein had a diffuse distribution in the predominantly healthy CGNs incubated with Veh (upper panels). In contrast, cells exposed to ToxB demonstrated marked nuclear condensation and fragmentation, and GFP-Bax staining that was punctate and consistent with localization to mitochondria (middle panels). In CGNs transfected with GFP alone, ToxB still induced substantial apoptosis; however, GFP maintained a distribution that encompassed the entire cell unlike the more localized distribution of GFP-Bax under these conditions (lower panels). The images shown are representative of results from two experiments. Transfected cells in each panel are indicated by the arrows. Scale bar, 10 μm. (b) The areas demarcated by the boxes in (a) have been enlarged 300% to enhance the visualization of GFP-Bax or GFP. (c) Untransfected CGNs were incubated with either Veh or ToxB for 24 h and cells were fixed and immunostained with a monoclonal antibody (clone 6A7) that specifically detects the active conformation of Bax. Active endogenous Bax was visualized in a punctate mitochondrial distribution using a Cy3-conjugated anti-mouse secondary antibody (indicated by the arrows). Scale bar, 10 μm.