Fig. 9. Rac1b activation is dependent on PI 3-kinase. Time course of Rac1 and Rac1b activation by laminin 5 matrix. GST-PAK-CRIB specifically pulls down GTP-loaded Rac. Lysates were prepared from HT-29 cells cultured on laminin 5 matrix (LN5) for various time periods (0-72 hours) or for 72 hours on plastic (PL). The amount of Rac1-GTP (upper panel) and Rac1b-GTP (upper middle panel) were estimated by pulling down the GTP-loaded proteins with GST-PAK-CRIB, followed by a western blot analysis with antibodies specific for Rac1 and Rac1b. Samples of the whole-cell lysates were also fractionated to determine the total levels of Rac1 and Rac1b (lower middle panel). Expression of actin was used as a loading control (lower panel). (B) Analysis of Rac1b activation upon inhibition of PI 3-kinase by LY294002: HT-29 cells were grown for 24 hours on laminin 5 matrix in the presence of increasing concentrations of LY294002 (0-100 µM). Then, the level of Rac1b-GTP was estimated as described above. Lower panel: the band densitometry was determined and the ratio of Rac1b-GTP to total Rac1b was used to allow quantification on the histogram. Results are representative of four experiments. ^**P<0.01, as determined by Student's t-test. (C) Subcellular distribution of Rac1b: HT-29 cells grown for 24 hours on plastic or laminin 5 matrix were fixed and stained with anti-Rac1b antibodies before confocal microscopy analysis on optical sections. Bars, 16 µm.