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Figure S4. Subcellular distribution of endogenous and exogenous hICAD in HeLa cells after biochemical fractionation. Nuclear (nu), microsomal (mi), and cytosolic (cy) fractions of HeLa cells were isolated with differential centrifugation from parental cells (a) or from cells transiently expressing ICAD-C-myc and CAD-N-HA (b). Cells were homogenized in sucrose solution (0.25 M sucrose, 10 mM Hepes, 1 mM EDTA, 1 mM DTT, pH 6.8, supplemented with protease inhibitors; 10 mg/ml leupeptin and pepstatin, 1 mM PMSF, 2 mg/ml iodoacetamide, and 100 µg/ml wheat germ agglutinin to inhibit transport via the nuclear pore complex), either by nitrogen cavitation (under 200 Psi for 3 min), or three cycles of freezing in liquid nitrogen and thawing at 0°C. Nuclei were sedimented by centrifugation (1,500 g, 5 min at 4°C). Microsomes were separated from the postnuclear supernatant by ultracentrifugation (100,000 g, 50 min at 4°C). Nuclear and microsomal proteins were extracted in RIPA buffer (150 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100, 0.1% SDS, and 0.5% deoxycholate at pH 8, supplemented with the protease inhibitor cocktail). Equal amounts of protein were immunoblotted from the nuclear, cytosolic, and microsomal fractions with the indicated antibodies and enhanced chemiluminescence as described in Materials and Methods.