Fig. 2.

Caspase-1 is essential for activation of caspase-7, but not caspase-3, in response to microbial stimuli. A, macrophages from wild type and caspase-1^−/− mice were stimulated with LPS for 3 h and then pulsed with ATP for 30 min, stimulated with LPS alone, pulsed with ATP alone, or infected with wild type S. typhimurium (Salm) or a flagellin-deficient mutant (Salm flib/c−) for 1 h. Extracellular bacteria were washed away, and macrophages were further incubated for 2 h in medium containing gentamycin. Cell extracts were immunoblotted with antibodies against caspase-1 (CASP1), caspase-3 (CASP3), and caspase-7 (CASP7). Black arrows indicate full-length caspases, and white arrows mark the large subunits of activated caspase-1, -3, and -7. Results are representative of at least three independent experiments. B and C, macrophages were left untreated, stimulated with LPS for 3 h and then pulsed with ATP, or infected with S. typhimurium (Salm) for 1 h. Cell lysates were incubated with biotin-DEVD-fmk, and streptavidin-bound complexes were precipitated as described under “Experimental Procedures.” Cell lysates and streptavidin-purified complexes were immunoblotted for caspase-3 (B) and caspase-7 (C). Results are representative of three experiments. CTRL, control; WT, wild type; IP, immunoprecipitation.