|Detection of Human Caspase‑8 by Western Blot. Western blot shows lysates of Jurkat human acute T cell leukemia cell line untreated (-) or treated (+) with 1 mM staurosporine (STS) for for 3 hours. PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human/Mouse Caspase‑8 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1650), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). For additional reference Recombinant Human Caspase-8 (Catalog # 705‑C8) was included. Specific bands were detected for Caspase‑8 precursor at approximately 57-60 kDa (as indicated). In STS-treated samples, specific bands were detected for Caspase-8 p41/43 subunit at approximately 41 and 43 kDa (as indicated) and Caspase-8 p18, p14, and p10 subunits at approximately 18 kDa, 14 kDa, and 10 kDa, respectively (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 4.|
|Detection of Human Caspase‑8 by Simple WesternTM. Simple Western lane view shows lysates of Jurkat human acute T cell leukemia cell line untreated (-) or treated (+) with 1 mM Staurosporine (STS) for 3 hours, loaded at 0.2 mg/mL. Specific bands were detected for Caspase‑8 at approximately 58-62 kDa (as indicated) using 5 µg/mL of Rabbit Anti-Human/Mouse Caspase‑8 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1650). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.|
Caspase-8 (Cysteine-aspartic acid protease 8/Casp8a; also named MCH5, FLICA and MACH alpha 1) is a 28 kDa member of the peptidase C14A family of enzymes (1, 2, 3). It is widely expressed and is considered an initiating caspase for the apoptotic cascade (4). Caspase-8 acts on a wide variety of substrates, including procaspases-3, 4, 6, 7, 9 and 10, c-FLIPL and procaspase-8 itself (1, 5, 6). Human procaspase-8a is a 54‑56 kDa, 479 amino acid (aa) protein (4, 7, 8, 9). It contains two N-terminal death domains (aa 1‑177), followed by a catalytic site that utilizes His317Gly318 plus Cys360. Normally, it is an inactive, cytosolic monomer (1, 10, 11). But following death-domain (DD) containing receptor oligomerization, Caspase-8 is recruited to the death-inducing signaling complex (DISC) that forms around the death domains of the oligomerized receptor (12). FADD/CAP-1 is recruited first, followed by procaspase-8/CAP-4 and, possibly, c-FLIPL and procaspase-10 (12). The recruitment, or concentration, of procaspase-8 induces homodimerization. This act alone is sufficient for activation. However, the activity level is modest at best, and appears to be directed towards either itself, or c-FLIPL, which is known to form a functional heterodimer with procaspase-8 (5, 11). When directed towards itself, autocleavage occurs first between Asp374Ser375, generating a 43 kDa (p43) N-terminal (aa 1‑374) and an 11 kDa C-terminal (aa 375 - 479) fragment. The C-terminus is further cleaved between Asp384Leu385 to generate a mature p10 subunit (aa 385‑479). The p43 subunit is next cleaved twice, once between Asp216Ser217, and again between Asp210Ser211 to generate a 26 kDa DD-containing prodomain (aa 1‑210) with an additional 18 kDa mature p18 subunit (aa 217‑374) (12). p18 and p10 noncovalently associate to form a 28 kDa heterodimer, which subsequently associates with another p18:p10 heterodimer to form an active, mature Caspase-8 molecule. This leaves the DISC to act on downstream apoptotic procaspases. In the event procaspase-8 comes to the DISC complexed with c-FLIPL, c-FLIPL will be cleaved by procaspase-8, generating a p43 fragment that is analogous to the Caspase-8 p43 subunit. This fragment, however, appears not to be an intermediate in a proteolytic cascade. Rather, it serves as a functional subunit, interacting with TRAF2 and activating NF kappa B. This may account for many of the nonapoptotic activities associated with Caspase-8 (5, 6, 13). Mature human and mouse Caspase-8a heterodimers are 73% aa identical (14).
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