Fig. 4. Inhibition of caspase 3 is due to a block of caspase activation. Activation of caspase 3 was detected by immunoblot (A,B) and immunofluorescence (C) analysis. Upon activation by upstream caspases, caspase 3 is proteolytically cleaved from the 32 kDa inactive zymogen to a 17-20 kDa and 11kDa active form. Following stimulation of apoptosis using STS for 8 hours (A), the active form of caspase 3 is detected as an 18 kDa band in uninfected cells (A, Uninfected, arrowhead). This conversion is blocked in identically treated T.-gondii-infected cells (A, Infected) where the 18 kDa band is not actively generated. Identical results are obtained using TNF-mediated apoptosis in the presence of cycloheximide (B). (C) Immunofluorescence microscopy of STS-treated (150 nM for 6 hours) uninfected and infected 3T3 fibroblasts using an antibody specific for the active form of caspase 3 (a,d). Toxoplasma-infected cells were detected using an antibody against the secreted antigen GRA3 (b,e). STS treatment causes morphological changes in both infected and uninfected cells (c,f). Notably, uninfected cells (lacking GRA3 labeling) primarily contain activated caspase 3 (c,f, black boxes), whereas those that are infected rarely label with the antibody against activated caspase 3 (c,f, white boxes). In the presented experiment, 15% of uninfected cells and 4% of infected cells exhibited staining with antibody against activated caspase 3 above the background level. Scale bar, 10 µm.