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. 2002 May;160(5):1887–1894. doi: 10.1016/S0002-9440(10)61135-2

Figure 4.

Figure 4.

GCLC is a direct target for caspase-mediated cleavage at AVVD499G. A: 35S-labeled in vitro translated wild-type GCLC(WT) or mutant GCLC(D499A) proteins were incubated in the absence or presence of soluble extract from bacteria expressing active recombinant caspase-3 (casp3) and Ac-DEVD-CHO (10 μmol/L) for 1 hour at 30°C as indicated. Radiolabeled proteins were resolved by SDS-PAGE and visualized by fluorography. Arrows denote full-length 73-kd GCLC protein and the 60-kd and 13-kd cleavage fragments. B: HeLa cells were transiently transfected with pcDNA3.1-His vector, pcDNA3.1-His-GCLC(WT) or pcDNA3.1-His-GCLC(D499A) and 24 hours post-transfection were treated for 6 hours with either TNF (30 ng/ml) (T) or αFas antibody (100 ng/ml) (F) in the presence of cycloheximide (10 μg/ml). His-tagged GCLC proteins were recovered by metal affinity chromatography and analyzed by immunoblotting with anti-GCLC antisera. Data presented are from a representative experiment performed at least three times with similar results.