Figure 4

(a) Interaction between XIAP and different Cdc42 mutants was tested via an in vitro GST Pulldown assay. With GST protein as a control, two different concentrations of cleaved Cdc42 mutants were used to test the binding to GST tagged XIAP (b) In vitro ubiquitination of Cdc42 by XIAP. Purified recombinant Cdc42Q61L was subjected to in vitro ubiquitination by XIAP and cIAP1 recombinant proteins (protocol described in Materials and Methods). (c) HeLa cells were transfected with XIAP siRNA for 48 h and treated with MG132 for 6 h. The cells were lysed in RIPA buffer and His-TUBE immobilized on Ni-NTA beads were employed to enrich the ubiquitinated proteins. The samples were loaded onto a gel and the presence of Cdc42 was monitored by immunoblots. The efficiency of XIAP knockdown was tested in the lysates control. * denotes an unspecific band. (d) Gel slices of the in vitro ubiquitination reaction were subjected to mass spectrometric analysis to determine the Lysine(s) responsible for the ubiquitination. Inset shows the gel slices taken for the analysis as well as a comparison between the sequences of Rac1 and Cdc42 (e) Mouse embryonic fibroblasts (MEFs) were cultured and lysed to check for Cdc42 levels. Control MEFs and XIAP knockout MEFs stably complemented with different XIAP constructs were used for this experiment. * denotes lower exposure of Cdc42