Figure 1.

Localization of caspase-4 in SK-N-SH and HeLa cells. (a–l) SK-N-SH cells (a–f) or HeLa cells (g–l) were stained with anti–caspase-4 and anti-KDEL antibodies (a and g, caspase-4, green; b and h, KDEL, red; c and i, overlapping, yellow), or with anti–caspase-4 antibody and Mitotracker (d and j, caspase-4, green; e and k, Mitotracker, red; f and l, overlapping, yellow), and observed under a confocal microscope as described in Materials and methods. Anti-KDEL antibody detects both GRP78 and GRP94 (ER markers), whereas Mitotracker stains the mitochondria. (m–o) HeLa cells were transfected with a caspase-4–GFP fusion gene, and were stained with ER-tracker (m, caspase-4, green; n, ER-tracker, blue; o, overlapping, blue-green), and observed under a nonconfocal fluorescence microscope. Bars, 5 μm. (p and q) Immunoelectron microscopic analysis was performed for SK-N-SH cells as described in Materials and methods. Photograph shown in panel q is the enlarged image of a part of photograph p. Gold grains showed the immunoreactivity of caspase-4, and blue and red arrows showed the ER and mitochondria, respectively. Bars: (p) 200 nm; (q) 90 nm. (r) Gold grains observed on indicated organelles in immunoelectron microscopic analysis were counted and displayed. (s) Biochemical fractionation was performed as described in Materials and methods, and analyzed by Western blotting using the indicated antibodies. Lamin B1, nuclear marker; cytochrome c, mitochondrial marker; presenilin-1 NH₂-terminal fragment (PS1 NTF), microsomal marker; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cytosolic marker.