Role of PI3KC2b in RhoA Activation Figure 3. Kinase-dependent PI3KC2b effect on cytoskeletal rearrangements. (A) Cell lysates of NIH3T3 cells stably expressing empty vector (NIH3T3-V), kinase-dead (D1213A) dominant-negative Myc-PI3KC2b (NIH3T3-C2b-DN) and wild-type Glu-PI3KC2b (NIH3T3-C2b-WT) were analysed for the recombinant PI3KC2b expression by immunoblotting with the indicated antibodies (EE: Glu-tag, 9E10: Myc-tag). (B) Confocal images of NIH3T3-V, -C2b-WT and –C2b-DN cells grown on cover slips for 24 h in 10% FCS and stained with Alexa Fluor 555 dye to localise F-actin, and an anti-PI3KC2b antibody follwed by FITC-labelled anti-rabbit antibody, to localize the kinase. WT1, WT2 and DN1, DN2 indicate individual clones. Scale bar represents 40 mm. doi:10.1371/journal.pone.0044945.g003 Rho GTPases Activation Assay NIH3T3-V, -C2b-WT, -C2b-DN cells were grown in 10% FCS to confluence or they were made quiescent by culturing them in serum-free medium (0.1%) for 24 h. After starvation they were stimulated either with 1nm PDGF or 1 mM LPA for 2 min., as indicated in the figure legends. Afterwards, the cells were washed with ice-cold PBS supplemented with 1 mM MgCl2 and immediately lysed in ice-cold Triton 1% lysis buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2, 10 mM leupeptin, 10 mM pepstatin and 1 mM PMSF). The cells were rapidly scraped off the plates and the crude lysates were transferred to pre- chilled Eppendorf tubes and centrifuged at 16,0606g for 5 min. at 4uC. Protein concentration was determined (Bradford assay, Pierce) and equal amounts of lysates were immediately supple- mented with GST-PAK-CRIB or GST-rhotekin for detection of activated Rac1 and RhoA, respectively. GST-fusion protein (20– 30 mg) on glutathione beads was added to the supernatants and the tubes were incubated with constant rotation at 4uC for 30 min. The beads were thereafter washed 4x with ice-cold washing buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, mM leupeptin, 10 mM pepstatin and 1 mM PMSF). SDS-PAGE sample buffer was added. Samples were heated at 95uC for 3 min. and subjected to SDS-PAGE and western blot analysis. GEF Activity Aassays HEK293 cells were transiently transfected with plasmids as described above. Cell lystaes were prepared as above and immunoprecipitated with anti-Dbl antibodies and Protein A- Sepharose beads. The immunoprecipitates were washed twice with lysis buffer and once with GEF reaction buffer (20 mM Tris- HCl pH 7.6, 0.5% Triton X-100, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT) and resuspended in GEF reaction buffer on ice. The PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e44945