Involvement of ER-localized Bak in the generation of reticular swelling. (A) Perivacuolar localization of AU-Bak, AU-Bcl-XL, and AU-Bak-ΔBH3. 293T cells were transfected with the indicated constructs along with GFP. 36 h after transfection cells were fixed, stained with an anti-AU mAb followed by an anti–mouse-Cy3 (red) antibody, and mounted for confocal microscopy. N and V denote nuclei and vacuolae, respectively. (B) A version of Bak-ΔBH3 specifically targeted to the ER (Bak-ΔBH3-cb5) retains the capacity to induce ER swelling, whereas mitochondrial targeting (Bak-ΔBH3-ActA) results in a decreased activity. 293T or HeLa cells (as shown) were transfected with the indicated expression plasmids along with GFP. Z-VAD.fmk (100 μM) was added 1 h after transfection to reduce death. 36 h after transfection, cytoplasmic vacuolization was scored as in Fig. 1 B.