FIGURE 5.

Rnd3 is not efficiently stabilized by all Rnd-interacting domains. A, schematic represents constructs used in 293T cell expression and Rnd interaction studies. Sequence alignment shows the highly conserved Raf1-like RBDs in mouse Syx (AAU04953) and RGS14 (NP_006471). B, p190B RhoGAP amino acid 382–910 is the minimum Rnd3-binding region. 293T cells were co-transfected with SBP-FLAG Rnd3 and HA or HA-GST p190B RhoGAP constructs. The purified protein complex was immunoblotted to visualize bound p190B RhoGAP. C, binding of Rnd isoforms to Plexin B1 was much weaker compared with Syx and p190 RhoGAP. 293T cells were transfected with expression vectors encoding SBP-FLAG Rnd1, Rnd2, or Rnd3 together with the indicated constructs and assessed for interaction. D, 293T cells overexpressing Rnd3 and the indicated plasmids were treated for 3 h with cycloheximide, lysed, and immunoblottted.