Fig. 5. TIAM1 993 cannot localize to the plasma membrane TIAM1 or activate Rac in vivo. A, HMN1 cells were transfected with the indicated TIAM1 constructs. TIAM1 peptides were visualized by indirect immunofluorescence. B, COS cells were transfected with the indicated TIAM construct and then starved for 24 h. Cells were lysed in the presence of GST-PBD and precipitated using glutathione-agarose beads. Whole cell lysates (Total Rac) and GST-PBD pulldowns (Active Rac) were subjected to immunoblotting with anti-Rac antibody; whole cell lysates were also immunoblotted with anti-TIAM1 to confirm comparable expression of TIAM1 constructs (data not shown).