Fig. 4.

Caspase-mediated cleavage of GCLC does not alter GCL holoenzyme formation in vitro. Murine GCLC-His or GCLM-His fusion proteins were synthesized in BL21(DE3) E. coli in the absence or presence of pGEX2T-caspase-3 (Deak et al., 1998) and purified as previously described (Krzywanski et al., 2004). Purified full-length and caspase-cleaved GCLC-His were incubated in the absence or presence of increasing molar equivalents of purified recombinant GCLM-His as indicated and GCL holoenzyme formation was assessed by native gel electrophoresis and immunoblotting for GCLC.