FIG. 4.

Effect of LPS on Cas-1 expression. Spleens and livers of C3H/HeN mice were isolated at 0, 3, and 8 h after treatment with 75 μg (□) and 500 μg (▪) of LPS. Cas-1 activity was determined in spleen extracts prepared by the method of Thornberry (35). Enzyme activity was calculated as follows: (maximum OD₄₀₅/microgram of protein) × 10,000. Each point represents the mean ± SE of at least two animals. (A) Reactions with enzyme preparation alone and with enzyme plus substrate plus 10 μM Cas-1 inhibitor were used as controls. Total RNA was analyzed by RT-PCR for cas-1 and GAPDH mRNA levels, and the cas-1 mRNA levels in the livers (B) and spleens (C) of mice treated with 75 μg (□) and 500 μg (▪) of LPS were plotted as the ratio of Cas-1 to GAPDH. Spleen extracts containing equal amounts of protein were separated on an SDS-7.5% polyacrylamide gel, transferred to Immobilon-P membrane, and immunostained with polyclonal anti-Cas-1 antibody (1:1,000). Blot represents two separate mice per time point per treatment group. Lanes 1 and 2, zero hour; lanes 3 and 4, 75 μg of LPS at 3 h; lanes 5 and 6, 75 μg of LPS at 8 h; lanes 7 and 8, 75 μg of LPS at 15 h; lanes 9 and 10, 500 μg of LPS at 3 h; lanes 11 and 12, 500 μg of LPS at 8 h; lanes 13 and 14, 500 μg of LPS at 15 h. (D) The figure represents a single representative Western blot containing samples from the spleens of mice treated with 75 and 500 μg of LPS.