Fig. 6. FL but not 993 TIAM1 activates signaling downstream of Rac. A, COS cells were transfected with JNK/pEBG encoding GST-JNK and the indicated TIAM1 constructs or constitutively active Rac (Rac61L). GST-JNK was purified using glutathione-agarose beads and subjected to in vitro kinase assays using GST-ATF2 as substrate. Phosphorylated GST-ATF2 was visualized by autoradiography. Data are representative of three independent experiments. B, TIAM1 constructs were cotransfected with a SRE-luciferase reporter construct into NIH3T3 cells. Cells were starved for 24 h and then assayed for luciferase activity. Data are presented as fold activation over cotransfection with empty vector and represent the average of three independent experiments performed in duplicate. For both A and B, TIAM1 immunoblots were performed to ensure comparable expression of the TIAM1 mutants (data not shown).