Fig. 8. Imp7 and Imp9 but not Imp3 directly interact with JNK/p38 MAPKs. (A) In vitro binding assay demonstrate a direct Imp7or Imp9 interaction with JNK1/2 and p38α/β. Imps 3, 7 and 9 were IPed from either stimulated or untreated cells (NT) and extensively washed with RIPA buffer following LiCl (0.5 M) and buffer A. To examine association, 500 ng of each indicated GST-MAPK were incubated with the indicated IPed Imps (2 hr with rotation) following washing and resuspention with sample buffer. Interacting MAPKs were detected using Western blotting with the indicated Abs. (B) Imp3 is not required for Imp7 or Imp9 interactions with MAPKs. SiRNAs of Imp3 and scrambled si of Imp3 that served as control (Si SCR) were transfected to HeLa cells, as described. Cell extracts were then subjected to CoIP with the indicated Abs. The amount of CoIPed MAPKs, Imp and input Imps were detected by Western blotting with the indicated Abs. To confirm the efficiency of the siRNA, cells were transfected with the indicated siRNAs, and then subjected to a Western blot analysis with the indicated Abs (lower panels). The experiments were reproduced three times.