1. Autoproteolytic Activation of Pro-Caspases by Oligomerization
Xiaolu Yang, Howard Y Chang, David Baltimore 
Molecular Cell 
Volume 1, Issue 2, Pages (January 1998)
DOI: /S (00)

2. Figure 1
Apoptotic Activity of FKBP Fusions of Full-Length and Mutant Pro-Caspase-8
(A) Schematic diagram of the FKBP fusions of pro-caspase-8. (Fkp), FK506 binding protein FKBP12; (DED), death effector domain; (p18) and (p12), subunits that form caspase-8; (D), aspartic acids at the cleavage sites for the generation of p18 and p12; (*), the active site cysteine-to-serine mutation. c-Src myristylation signal (M) and HA and FLAG tags are also indicated.
(B) Apoptosis induced by pro-caspase-8 fusion proteins. HeLa cells were transfected with μg of each Fkp-Casp8 plasmid and 0.25 μg of pRK-crmA as indicated. Percentages of specific apoptosis were determined as described in the Experimental Procedures.
Molecular Cell 1998 1, DOI: ( /S (00) )

3. Figure 2
Oligomerization of the Protease Domain of Pro-Caspase-8 Induces Apoptosis
(A) Caspase-8(180)-induced apoptosis requires oligomerization and intrinsic protease activity. HeLa cells were transfected with μg of Fkp3, Fkp3-Casp8(180), or Fkp3-Casp8(180, C360S), together with 0.25 μg of pRK-crmA or μg of Fkp3 as indicated. Treatment of AP1510 (concentration indicated) and FK506 (50 nM) was done for 10 hr as described in the Experimental Procedures.
(B) Pro-caspase processing is required for oligomerization-induced apoptosis. HeLa cells were transfected with μg of Fkp3, Fkp3-Casp8(206), or Fkp3-Casp8(217). AP1510 treatment was done as described in (A).
Molecular Cell 1998 1, DOI: ( /S (00) )

4. Figure 3
Apoptotic Activity of Pro-Caspase Fusions with the Fas Extracellular Domain
(A) Schematic diagram of pro-caspase-1, -3, and -8 fusions with the murine Fas extracellular domain. The fusion constructs contained either pro-caspase-8 (amino acids 182–479), full-length murine pro-caspase-1, or full-length human pro-caspase-3—as well as the corresponding catalytic Cys-to-Ser mutations (not shown)—fused to the extracellular and transmembrane domain of murine Fas (FasEC). The leader peptide (L) and transmembrane domain (TM) of murine Fas, FLAG tag, and the large and small subunits of each caspase are indicated.
(B) FasEC-mediated oligomerization of pro-caspase-8 activates its apoptotic activity. HeLa cells were transfected with FasEC (25 ng), FasEC-Casp8(182) (25 ng), FasEC-Casp8(182, C360S) (125 ng), or pEBB-mFas (250 ng), together with pRK-crmA (250 ng) as indicated. Jo2 treatment and X-gal staining are as described in the Experimental Procedures.
(C) FasEC-mediated oligomerization activates the apoptotic activity of pro-caspase-1 but not pro-caspase-3. Experiments were done as described above. The amount of plasmids used for each transfection was 12.5 ng for FasEC and FasEC-Casp1; 125 ng for FasEC-Casp1(C284S), FasEC-Casp3, and FasEC-Casp3(C163S); and 250 ng for pRK-crmA.
Molecular Cell 1998 1, DOI: ( /S (00) )

5. Figure 4 Pro-Caspase Processing in Transfected Cells
(A) Pro-caspase processing induced by oligomerization. 293T cells were transfected with 1 μg of the indicated Fkp3-fusion expression constructs. Vehicle or 1 mM AP1510 was added 11 hr after transfection, and cell extracts were made after the indicated times and immunoblotted for FLAG.
(B) Processing in the presence of crmA. 293T cells were cotransfected with 1 μg of Fkp3-Casp8(180) and 1 μg of pRK5-crmA. Drug treatment, cell extracts, and FLAG immunoblot were performed as in (A).
Molecular Cell 1998 1, DOI: ( /S (00) )

6. Figure 5 Pro-Caspase Processing in a Cell-Free System
(A) Pro-caspase processing induced by oligomerization. The processing reaction was carried out with in vitro-translated, 35S-labeled FKBP fusions as described in Experimental Procedures and visualized by SDS–PAGE and autoradiography.
(B) Time course of pro-caspase processing. The deduced domain structure of the indicated bands is shown on the right.
Molecular Cell 1998 1, DOI: ( /S (00) )