Figure 6
From: Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35
MaviP35 is cleaved by DRONC but does not inhibit DRONC activity in vitro. (a) Yeast were transformed with the indicated expression plasmids. Suspensions containing equivalent concentrations of each transformant were serially diluted, and 5 μl of each dilution were spotted onto inducing and repressing plates. A total of 400 ng of FLAG-tagged MaviP35 (b) or AcP35 (c) were incubated with 0, 15, 150 or 1500 nM of DRONC or DRICE, then processing was monitored by anti-FLAG immunoblotting. (d) Drosophila Kc167cells were co-transfected with a plasmid encoding either eGFP or DRICEC211A-eGFP, together with either an empty vector or plasmid directing expresion of AcP35-F, MaviP35-FLAG or DIAP1. Transfectants were incubated in media containing or lacking 1 μM actinomycin D (ActD) fo 12 h. Lysates were subjected to anti-GFP or anti-FLAG immunoblotting, or coomassie staining. (e) The structure of the DRICEC211A–eGFP fusion protein is shown. (f) Cleavage of Ac-TQTD-AFC by purified recombinant DRONC is plotted as the change in RFU per minute, in the presence of 1.6 μM of FLAG-tagged MaviP35, AcP35 or truncated CED-9
