p104 Binds to Rac1 and Reduces Its Activity during Myotube Differentiation of C2C12 Cell

Figure 3

p104 was specifically associated with Rac1. (a) p104 interacted with CrkII as well as Rac1. After immunoprecipitation was performed with the p104 antibody, the presence of CrkII and Rac1 in the precipitated immune complex was analyzed by Western blot with anti-CrkII and anti-Rac1 antibodies, respectively. (b) CrkII and Rac1 bind to distinct regions of p104. GST-fused p104 I, II, and III proteins linked to glutathione-Sepharose beads were incubated with mouse brain extract and then bound proteins were analyzed by Western blot with antibodies against CrkII and Rac1. (c) p104 directly interacts with CrkII through the proline-rich region. Mouse brain extract was incubated with GST-p104 (WT, wild type), GST-2mp (second proline-rich motif mutant), and GST-3mp (third proline-rich motif mutant) immobilized onto glutathione-Sepharose beads. Bound proteins were analyzed by Western blot with an anti-CrkII antibody. A coomassie brilliant blue stained SDS-PAGE gel showed that equal amounts of fusion proteins were used.