Figure 4

The N-terminus of Arm is cleaved by drICE. (A) Schematic representation of the domain structure of Arm. Arm repeats are indicated as red boxes. Putative caspase cleavage sites are indicated in green boxes. Blue bars depict the extent of the antigens used to produce Arm^Nterm (monoclonal antibody 7A1) and Arm^Central, respectively. (B) Cleavage of in vitro translated, ³⁵S-methionine labelled mutated forms of Arm. As control full length Arm was incubated with buffer (control). Mutated variants of Arm (D⁸⁸A, D¹²³A, D¹⁷⁰A, D¹⁷²A and D⁷⁵⁵A) were incubated with recombinant drICE. Upon incubation with drICE stable products were identified of all of the Arm mutants except for D⁸⁸A. The cleavage products have a M_r of approximately 90 kDa and are marked as Arm^ΔN. Full length Arm (Arm^Full, M_r 110 kDa) is reduced except for D⁸⁸A, which remains stable. (C) Western blot of embryo extracts of th¹⁰⁹ homozygous embryos (- Arm^D88A) and th¹⁰⁹ homozygous embryos expressing Arm^D88A from a transgene (+ Arm^D88A); expression of Arm^D88A was achieved using the Gal4/UAS system with Gal4 expressed under the control of a maternal beta-tubulin promoter (mat15::Gal4). Hand selected embryos of either genotype were subjected to Western blotting using Arm^Nterm antibody; the blot was stripped and re-probed using an anti-Actin antibody for loading control; note that Actin remains stable in th¹⁰⁹ apoptotic cells.