Figure 3.

Subcellular distribution of procaspase-8L. (A) KB cells were homogenized in isotonic buffer and the P1 nuclear (500 × g pellet), heavy membrane (HM) (9,000 × g pellet), LM (100,000 × g pellet), and cytosolic S-100 fractions were separated by differential centrifugation. The fractions were analyzed for the presence of procaspase-8L, procaspase-8, the ER marker, calnexin, and the mitochondrial marker, cytochrome c oxidase subunit IV (CoxIV), by immunoblotting with the respective antibodies. (B) Sucrose density gradient fractions of rodent liver membranes were analyzed for the presence of procaspase-8L, BAP31, and calnexin as in A. (C) Procaspase-8L is peripherally associated with the cytosolic face of the light membranes. (Upper) The LM fraction from KB cells was subjected to extraction with 0.5M NaCl or alkali (0.1 M NaCO₃, pH 11.5) and, after centrifugation, the membrane pellets (P; lanes 1 and 3) and supernatants (lanes 2 and 4) were analyzed as in A. (Lower) The LM fraction was incubated with (+) or without (−) trypsin and the integrity of procaspase-8L, calnexin (ER transmembrane protein), and BiP (ER luminal protein) assessed by immunoblotting. TritonX-100 was added to 1% in one reaction to demonstrate that BiP was trypsin sensitive following solubilization of the LM microsomal fraction.