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. 2013 Mar 15;24(6):748–756. doi: 10.1091/mbc.E11-08-0740

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© 2013 Iwasaki et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Caspase-3–mediated cleavage of MYPT1 during apoptosis. (A) Cleavage of endogenous MYPT1 in apoptotic cells. HeLa cells were treated with dimethyl sulfoxide (DMSO) or TNF/CHX for 3 h. Total cell lysates were immunoblotted with anti-MYPT1 antibody. (B) Inhibition of MYPT1 cleavage. HeLa cells were transiently transfected with V5-MYPT1-WT. At 24 h after transfection, the cells were treated with DMSO, TNF/CHX, TNF/CHX zVAD-FMK, or TNF/CHX/IETD-CHO for 3 h. Total cell lysates were immunoblotted using anti-V5 antibody (top panel), cleaved PARP antibody (middle panel), or BID antibody (bottom panel). (C) Identification of MYPT1-cleaving caspase. FLAG-MYPT1 was treated with caspase-3 or caspase-8 at 30˚C for 1 h in vitro. Cleavage of MYPT1 was analyzed by immunoblot assay using the anti-FLAG antibody. Asterisk indicates the caspase-8–cleaved form of MYPT1. (D) Schematic representation of MYPT1. ANK, ankyrin repeat; MBD, myosin-binding domain. The caspase-3 cleavage site TQTD884 was identified by amino acid sequencing. D884A and (1-884) indicate the caspase-3 cleavage site mutant and the caspase-3–cleaved form of MYPT1 (lacking the C-terminal end), respectively. (E) In vivo cleavage of MYPT1 at Asp-884. HeLa cells transfected with V5-MYPT1-WT, V5-MYPT1-D884A, or V5-MYPT1-(1-884) were treated with DMSO or TNF/CHX for 3 h. Total cell lysates were immunoblotted using anti-V5 antibody (top panel) or anti–α-tubulin antibody (bottom panel). Positions of the full-length and caspase-3–cleaved MYPT1 are indicated using filled and open arrowheads, respectively. Positions of molecular weight standards are indicated in kilodaltons on the left.