Fig. 4. Drice regulates Diap2 levels and ubiquitination of Dredd and Kenny.

A Adult Canton^S flies were fed 50 μM Z-DEVD-FMK, their intestines were dissected, lysed and subjected to western blotting analysis with α-Diap2, α-K63 and α-Actin antibodies, n = 3. The relative protein levels of full-length Diap2 and K63-linked ubiquitin chains were quantified. B Canton^S and Diap2^7c flies were fed 50 μM Z-DEVD-FMK and lysed. Ubiquitin chains were isolated with GST-TUBE under denaturing conditions and the samples were analysed by western blotting with α-K63, α-Diap2, α-Drice and α-Actin antibodies, n = 3. The relative protein level of K63-Ub chains was quantified. Drosophila S2 cells were transfected with empty vector, Diap2^WT, Drice^WT, Drice^C211A, V5-tagged Dredd (C) or with HA-tagged Kenny (D), where after the cells were treated with 20 µM Z-DEVD-FMK. Ubiquitin chains were isolated with GST-TUBE at denaturing conditions and the samples were analysed by western blotting with α-V5, α-HA, α-Diap2, α-K63 and α-Actin antibodies, n = 3. The relative protein level of ubiquitinated Dredd or Kenny was quantified. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001.