RhoGDI1 interacts with inactive RhoA and Rac1 and upon CCK stimulation both complexes are dissociated.

<p><b>A)</b> Total lysates were incubated with GST-CA-RhoA, GST-DN-RhoA, GST-CA-Rac1 and GST-DN-Rac1. GST-empty vector was used as a control. The beads were eluted with elution buffer containing reduced glutathione, and then, total lysates, as well as elutes were subjected to Western-blotting for RhoGDI1. A representative immunoblot for RhoGDI1 shows that only GST-DN-RhoA and GST-DN-Rac1 are able to interact with RhoGDI1. Comparable amount of GST-fusion proteins was shown by Western-blot using anti-GST antibody. <b>B)</b> Isolated pancreatic acini were stimulated with 300 pM CCK for 5 min. A cytosolic fraction was obtained using differential centrifugation and co-immunoprecipitated with anti-RhoGDI1. <i>Left:</i> representative immunoblots for RhoA and RhoGDI1 shows that upon CCK stimulation, RhoA is released from RhoGDI1. Comparable amount of immunoprecitated RhoGDI1 was confirmed by Western-blotting using anti-RhoGDI1 antibody. <i>Right:</i> quantitative analysis of RhoGDI1 associated with RhoA. Data are representative of 4 independent experiments; the dissociation levels were normalized to control = 1.0 in each assay and expressed as fold change.Values are means ± SE. *: p<0.05 vs control. <b>C)</b><i>Top</i>: Acini were stimulated with CCK (300 pM) for 5 min. A representative immunoblot for Rac1 and Cdc42 shows Rac1, but not Cdc42, is associated with RhoGDI1. <i>Bottom</i>: Acini were stimulated with CCK (300 pM) for 5, 15 or 30 min. A representative immunoblot for RhoA and Rac1 shows that RhoA is dissociated from RhoGDI1 faster than Rac1 (5 min vs 15 min). Comparable amount of immunoprecitated RhoGDI1 was confirmed by Western-blotting using anti-RhoGDI1 antibody. Data are representative of 3 independent experiments; the dissociation levels were normalized to control = 1.0 in each assay and expressed as fold change.