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Figure 3 | BMC Developmental Biology

Figure 3

From: Cleavage of Armadillo/beta-catenin by the caspase DrICE in Drosophilaapoptotic epithelial cells

Figure 3

Arm is cleaved by caspases in vitro. (A) Cleavage of Arm and Lamin Dmo by embryonic extracts in vitro. 35S-labeled Arm (lane a-c) or Lamin Dmo (d-f) was incubated with protein extracts of wildtype embryos (wt extract; c, f) or extracts prepared from embryos obtained from th109 heterozygous flies (1/4 of the embryos are homozygous for th109 indicated as th109 extract; b, e). The th109 extract contains proteolytic activity that leads to quantitative degradation of Lamin Dmo (e) and Arm (b). 35S-labeled Arm appears as a range of bands suggesting premature termination during in vitro translation. Arm cleavage results in proteolytic fragments with the apparent Mr between 90 kDa and 70 kDa suggesting N-terminal cleavage of all isoforms produced in vitro (bracket in b). Addition of the pan-caspase inhibitor z-VAD-FMK completely prevents degradation of Arm (a) and Lamin Dmo (d). (B) In vitro cleavage of Arm by recombinant drICE, but not DCP-1. 35S-labeled Arm was incubated with 0.5 μg and 1 μg recombinant drICE (b+c) or DCP1 (d, e), respectively. drICE cleavage of Arm leads to stable proteolytic products with the Mr of 90 kDa and 70 kDa, the 70 kDa product again likely represents a cleaved isoform produced by premature termination of the substrate (lower stars in b and c). (C) drICE and DCP1 are both active in vitro. (a) In vitro translated, S35-methionine labelled Arm is cleaved by drICE resulting into the 90 kDa Arm cleavage product. (b) Cleavage of S35-methionine labelled Arm can be prevented by addition of z-VAD-FMK to the lysate prior to incubation with drICE. (c) S35-methionine labelled Lamin Dmo is cleaved by drICE into 20 kDa and 50 kDa fragments (d) DCP1 cleaves Lamin Dmo into 20 kDa, 50 kDa and 55 kDa fragments. Another caspase3 (and -7) related caspase Decay [55] is unable to cleave Lamin Dmo (lane e).

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