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. 2011 Nov 25;31(1):14–28. doi: 10.1038/emboj.2011.423

Figure 4.

Figure 4

IAPs can bind to Rac1 in vitro and in vivo. (A) Endogenous interaction between Rac1 and IAPs. HeLa cells grown in 10 cm dishes were stimulated with HGF (100 ng, 15 h) and lysed with appropriate buffer. Endogenous Rac1 was then immunoprecipitated and the immunocomplexes were resolved in an SDS–PAGE and western blotted for co-precipitating XIAP and c-IAP1. (B) In-vitro binding assays with purified proteins showing XIAP and c-IAP1 binding to GST–Rac1 in a GTP/GDP-independent manner. The GST–Rac1 is pretreated with GTPγS and GDP to analyse the dependency of nucleotides in binding with IAPs. The nucleotide loading was confirmed by PAK1-PBD pull-down experiments, which revealed the GTP-specific binding. (C) The interaction between Myc-tagged XIAP or XIAP (1–240) and HA–Rac1 is tested after expressing them in 293T cells. The unspecific binding of antibodies in immunoprecipitation experiments was always checked with respective isotype controls. Figure source data can be found in Supplementary data.