(a) Schematic representation of the C-terminal fragments of human IQGAP1 used

GRD, Ras GTPase-activating protein–related domain (binding site for GTP-Rac1/Cdc42); CC, coiled-coil domain predicted by COILS (version 2.2); RasGAP_C, RasGAP C terminus; Sec3 BD, minimal overlapping region of nine independent IQGAP1 clones isolated in a yeast two-hybrid screen using human Sec3 as bait (Fig. S1, available at ). (b) Lysates of HeLa cells transfected with HA-tagged human Sec3, Sec8, Exo70, or Exo84 were incubated with the indicated C-terminal fragment of IQGAP1 fused with GST immobilized on beads, and bound proteins were analyzed by immunoblotting with anti-HA antibody. 1% of lysates was loaded as a control (input). The bottom panel shows the different GST-IQGAP1 fragments separated by SDS-PAGE and stained with Coomassie Brilliant blue (CBB). Arrowheads indicate intact GST fusion proteins. (c) Effect of Sec3/Sec8 depletion on binding of the exocyst to IQGAP1-CTer2. HeLa cells were treated for 48 h with Sec3- or Sec8-specific siRNA alone or in combination as indicated and were further transfected with a construct encoding HA-tagged human Exo70 for 18 h. Lysates were prepared and incubated with GST-IQGAP1-CTer2, and bound proteins were analyzed by immunoblotting with anti-HA (top) or anti-Sec8 (middle) antibodies. The bottom panel shows GST and GST-IQGAP1-CTer2 proteins separated by SDS-PAGE and stained with Coomassie Brilliant blue. Residual levels of knocked down proteins and of proteins bound to GST-IQGAP1/CTer2 are indicated underneath the blots with respect to the level of mock-treated cells based on densitometric analysis. The efficiency of Sec3 depletion with siSec3 is documented in Fig. S2 b. Molecular masses are indicated in kilodaltons.<p><b>Copyright information:</b></p><p>Taken from "The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA"</p><p></p><p>The Journal of Cell Biology 2008;181(6):985-998.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426946.</p><p>