Fig. 2. (A) Human tumor cell lysate from H1299 cells were immunoprecipitated with protein G beads linked with anti-PAK4, anti-GEF-H1 and anti-PAK4 preimmune sera. Bead eluates were analysed by western blotting using anti-GEF-H1 antiserum (upper panel). The same beads were subject to a kinase reaction and separated by SDS-PAGE and autoradiographed to detect incorporation of [-³²P]ATP (lower panel). (B) H1299 cells were stained for PAK4 (FITC) and the Golgi marker ß-COP (Texas Red) and F-actin (Coumarin-Phalloidin). The merge shows co-localization of PAK4 with Golgi-like structures. (C) PAK4 (FITC) localizes to the Golgi (arrow a), and MT-like structures (arrow b) (I and V). GEF-H1 (Texas Red) localizes to the cytoplasm (arrow c) and co-stains with PAK4 on Golgi and MT structures (I and II). Staining for phosphorylated GEF-H1 (Marina Blue) shows localization to MTs surrounding the nucleus, cytoplasm and Golgi-like structures (compare III and IV). ß-tubulin (FITC) staining was used to show MTs of the cytoskeleton throughout the cell (IV). In the presence of DSP crosslinking PAK4 is more concentrated in the Golgi and some MT-like structures (compare V and VIII). GEF-H1 staining retains its localization to all compartments consistent with conventional crosslinking (compare VI with II). Phosphorylated GEF-H1 is clearly cytoplasmic bearing no spatial relationship to MTs, although MTs are still present (compare VII with VIII). Briefly, for DSP treatment, H1299 cells on coverslips were removed from growth medium and incubated in D-PBS (Dulbecco's PBS) containing 1 mM DSP at 37°C. After 20 minutes the coverslips were incubated in MTSB (containing 50 mM glycine) to quench the DSP and stabilize the MTs. Washes and further fixation is described in Materials and Methods.