Tenev et al. 2006
Supplementary Information:
Suppl. Info Fig. 1. (A) The viral IAPs CpGV-IAP3, OpIAP3 and HcNPV-IAP3 fail to be
cleaved by drICE. TAP-tagged IAPs were expressed in 293T cells and purified using tandem
affinity purification. V5/His-tagged drICE was expressed in, and purified from, 293T cells
cellular extracts using nickel beads. Immobilised active drICE was subsequently incubated
with the indicated purified IAPs (lanes 2, 4,6 and 8). IAPs were monitored using western blot
analysis with biotinylated-calmodulin, which detects the calmodulin-binding protein that is part
of the TAP tag. DIAP1 (lane 1 and 2) was used as positive control in this study. Note, in this
assay, the protein A portion of the TAP tag was removed by TEV protease to reduce the size
of proteins, facilitating the visualization of N-terminal cleavage. Purification of active drICE
was verified by immunoblot analysis with anti-V5 antibodies. (B and C) Viral IAPs fail to stably
associate with the Drosophila effector caspases drICE (B) or Ag- and Sf-caspase (C). TAPtagged proteins were expressed in 293T cells and purified using IgG Sepharose beads.
Subsequently, immobilised and purified TAP-tagged proteins were used as affinity reagents to
purify the indicated active effector caspases from cellular extracts. Expression of drICE, Agor Sf-caspase and their co-purification with the indicated TAP-tagged proteins was
determined by immunoblot analysis using the indicated antibodies. Purification of TAP-tagged
proteins was verified by immunoblotting with an antibody (anti-TAP) recognising the TAP tag
(bottom panels). DIAP1 was included in this study as positive control.
Suppl. Info Fig. 2. Co-migration approach to identify the cleavage site in AgIAP3 (A-C) and
SfIAP (D-E). (A) Schematic diagram depicting the putative AgIAP3 cleavage sites tested. The
protein fragments, starting with P1’, were generated through the Ub-fusion technique. Ubspecific proteases (UBP) cleave these fusion proteins at the C-terminus of the Ub moiety,
yielding the test proteins
17LP-, 20NK-
and
25DD-AgIAP3,
which start at the putative P1’
position. (B) Co-migration assay. TAP-tagged Ub-IAP-BIR1 fragments were expressed in
293T cells and purified using tandem affinity purification. For lanes 2, 4 and 6, WT-AgIAP3BIR1 was purified and subsequently incubated with apoptotic lysate from Sua4.1 cells.
AgIAP3-BIR1 co-migrated with the cleaved fragment, while
17LP-AgIAP3-BIR1
20NK-
migrated
slower and 25DD-AgIAP3-BIR1 faster. (C) Moreover, mutation of D24 to A or E didn’t abrogate
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Tenev et al. 2006
caspase-mediated cleavage of AgIAP3, ruling out D24 as the caspase cleavage site. (D)
Schematic diagram depicting the putative SfIAP cleavage sites tested. (E)
migrated with the cleaved fragment of WT-SfIAP-BIR1, while
slower and
87TF-SfIAP-BIR1
84NH-SfIAP
81KT-SfIAP-BIR1
co-
migrated
faster. For lanes 2, 4 and 6, WT-SfIAP-BIR1 was purified and
subsequently incubated with drICE.
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