Figure 2.

SNX1 and SNX2 are cleaved by recombinant caspases. (a) Cleavage assays were performed using 25 nM recombinant SNX1 or SNX2 protein and physiological concentrations (10 or 50 nM) of the indicated initiator (left panels) or executioner (right panels) caspases for an hour at 37 °C in the appropriate buffer as described in the Materials and Methods section. (b) Similar assays as in (a) were performed using twofold serial dilutions of the indicated recombinant caspases. The highest concentration of enzyme used and assay time are indicated in the black wedges. Proteins were analyzed by immunoblotting using the indicated antibodies. The rate k of full-length SNX disappearance was obtained from the equation k=ln 2/tE, in which t is time and E is caspase concentration at 50% protein cleavage (indicated by an arrow on each blot).³⁹ Because of the high efficacy of caspase-8 at cleaving SNX2, a different range of concentrations was used (bottom panel) to estimate the cleavage rate k. Closed and open arrowheads indicate full-length and cleaved fragments, respectively. Asterisks indicate nonspecific proteins recognized by SNX2 antibody.