Supplementary Figure 2: UbcH5c does not induce dimerization of cIAP1.

(a) cIAP1-B3R responds to SMAC mimetics by dimerizing, as detected by native gel electrophoresis. Lane 1 shows a native molecular weight marker (Native Mark, Invitrogen) with molecular weights indicated at left. Lane 2 shows monomeric cIAP1 (64 μM). Lane 3 shows dimerized cIAP1 in the presence of AVPW (1 mM). Binding of the bivalent SMAC mimetic BV6 (1 mM) induces stronger and more compact dimerization because it can engage two BIR3 domains simultaneously. Lanes 4-7 demonstrate that UbcH5c (64 μM) does not affect the dimerization state of cIAP1. Lanes 8-10 demonstrate that the gel mobility of UbcH5c is not affected by the SMAC mimetics. All samples contain 1% DMSO. (b) cIAP1-B3R (64 μM) was incubated with UbcH5c at 0 to 128 μM. No detectable dimer is formed. cIAP1 bound to BV6 is included as a dimerization control.