Figure 4. PI3KC2b regulates RhoA and Rac1 activity. (A) PI3KC2b up-regulates RhoA activity. NIH3T3-V, -C2b-DN and –C2b–WT cells were grown in 10% FCS or serum-starved for 24 h and stimulated with 1 mM LPA for 2 min. Cell lysates were equalized for protein content and subjected to GST-rhotekin pull-down as measure of RhoA activity. GTP- bound and total RhoA were detected with an anti-RhoA antibody. (B) Role of PI3KC2b in RhoA Activation PI3KC2b mediates RhoA-dependent serum response factor (SRF) activation. NIH3T3-V, -C2b-DN and -C2b-WT cells were transiently transfected with the SRF luciferase reporter plasmid p3DA-Luc and pRL- TK control construct. Cells were maintained in 0.5% FCS for 24 h prior to stimulation with 15% FCS for 7 h. Luciferase activity was normalized to pRL-TK control. Figure shows mean 6 SD of three independent experiments. (C) PI3KC2b up-regulates Rac1 activity. NIH3T3-V, -C2b-DN and -C2b-WT cells were grown in 10% FCS. Cell lysates were equalized for protein content and subjected to GST-PAK CRIB pull-down as measure of Rac-1 activity. GTP-bound and total Rac1 were detected with anti-Rac1 antibody. Densitometry was performed and signal intensities were normalized against total Rac1. Data are mean with SD from two independent experiments. *, p,0.05. (D) PI3KC2b mediates Rac1-dependent platelet derived growth factor (PDGF) activation. NIH3T3-V, -C2b-DN and -C2b-WT cells were serum-starved for 24 h and stimulated with 1 nM PDGF for 2 min. Cell lysates were equalized for protein content and subjected to GST-PAK CRIB pull-down as measure of Rac-1 activity. GTP-bound and total Rac1 were detected with anti-Rac1 antibody. Densitometry was performed and signal intensities were normalized against total Rac1. Data are mean with SD from two independent experiments. *, p,0.05. A representative western blot is shown. Activation of JNK was assessed in the cell lines treated with PDGF by immunoblotting with phospho-specific SAPK/JNK antibody. WT1, WT2 and DN1-3 indicate individual clones. doi:10.1371/journal.pone.0044945.g004 reaction was started by the addition of 1 mM GTPcS and 50 mg/ ml purified recombinant RhoA and incubated for 30 min at room temperature. The samples were then placed on ice and supernatants collected. Active RhoA was isolated from the supernatants by using GST-rhotekin, as described above and samples were analysed by SDS-PAGE and western blot. Pull-down Assays for PI3KC2b/Dbl/Grb2 Interaction After 48 h of culture in 10% FCS medium, untransfected and transfected with HA-proto-Dbl or pcDNA3 empty vector HEK293 cells were washed with ice-cold PBS and lysed on ice for 20 min. in Triton 1% (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.5% NP-40, 1 mM DTT added before use) lysis buffer supplemented with 76concentrated stock of protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (1 mM NaF, 1 mM Na3VO4 and 10 mM b-glycerophosphate). The crude lysates were centri- fuged at 16,0006g for 25 min. at 4uC. Protein concentration was determined (BCA, Pierce) and equal amounts of lysates were immediately supplemented with GST-NT-C2b, GST-(CT)C2- C2b or GST-Grb2 immobilized on gluthatione beads. The tubes were incubated with constant rotation for 2 h at 4uC. The beads were thereafter washed four times with ice-cold lysis buffer. SDS- PAGE sample buffer was added. The samples were heated at 95uC for 3 min. and subjected to SDS-PAGE and western blot analysis for detection of interaction with Dbl. In the case of Dbl domains interaction with PI3KC2b, COS-1 cells were co-transfected with wild-type PI3KC2b and GST- tagged Dbl domains (NT-, spectrin-, onco-, PH) or pcDNA3 epmty vector and HA-proto-Dbl as control. 48 h post-transfection cells were washed with ice-cold PBS and lysed on ice for 20 min. in Triton 1% lysis buffer supplemented with protease and phosphatase inhibitors (as described above). The crude lysates were centrifuged at 16,0006g for 25 min. at 4uC. Protein concentration was determined (BCA, Pierce) and equal amounts of lysates were supplemented with gluthatione-sepharose beads (GE Healthcare). The tubes were incubated with constant rotation for 2 h at 4uC. The beads were thereafter washed four times with ice-cold lysis buffer. SDS-PAGE sample buffer was added. The samples were heated at 95uC for 3 min. and subjected to SDS- PAGE and western blot analysis. PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e44945