Role of PI3KC2b in RhoA Activation Figure 5. The assembly of the Dbl - PI3KC2b complex is not modulated by PI3K activity or cell stimulation with EGF or PDGF. (A) HEK293 cells were transfected with vectors encoding Dbl in combination with Myc-PI3KC2b WT or DN, or empty vector. Immunoprecipitates prepared with anti-Dbl or anti-Myc tag antibodies were analysed by western blot with the antibodies indicated. (B) Lysates from NIH3T3-V, -C2b-WT or -C2b-DN cells were immunoprecipitated with anti-Dbl antibodies and analysed by western blot. (C) Serum-starved NIH3T3-C2b-WT cells were stimulated with EGF (20 ng/ml) or PDGF (20 ng/ml) for the indicated lengths of time. Immunoprecipitates prepared with anti-Glu (EE) tag antibodies were analysed by western blot with the antibodies indicated. (D) HEK293 cells were transfected with vectors encoding Dbl in combination with Myc- PI3KC2b WT or DN, or empty vector. Immunoprecipitates prepared with anti-Dbl antibodies were analysed for GEF activity towards recombinant RhoA. doi:10.1371/journal.pone.0044945.g005 In Vitro Binding Assay To test the PI3KC2b association to Dbl spectrin- and PH- domains an in vitro binding assay, which utilized GST-tagged Dbl spectrin- and PH- domains and GST alone immobilized on gluthatione-speharose beads was performed. Immobilized proteins were incubated in vitro with soluble Grb2- and NT-C2b- domain in 1% Brij 96 (50 mM Tris.Cl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Brij96) lysis buffer supplemented with 76concentrated stock of protease (Complete Mini, Roche) and phosphatase inhibitors (1 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM b-glycerophosphate). Soluble Grb2- and NT-C2b-domain were obtained from GST-fused Grb2 and GST- N-terminal PI3KC2b domain as a result of digestion with thrombin. Following constant rotation for 2 h, at 4uC, the complexes were washed 3x with Brij 96 1% lysis buffer. SDS- PAGE sample buffer was added and the samples were heated at 95uC for 5 min. and subjected to SDS-PAGE followed by western blot analysis. Cell Proliferation Cell counting experiments based on Trypan Blue exclusion were performed. NIH3T3-V, -C2b-WT, -C2b-DN cells were grown in 12-well plates at a density 20 cells/ml in the presence of either 10% or 0.5% FCS. After 48 h cells were washed once with phosphate-buffered saline (PBS) and trypsinized. When needed clumps within the cell suspension were disaggregated by passing the well content 5 times through a 20-gauge needle and the number of viable cells was determined by Trypan Blue exclusion, using a haemocytometer. Experiment was performed in triplicate. Wound Healing Assay For wound healing assays, NIH3T3-V, -C2b-WT, -C2b-DN cells were plated in 12-well culture plates in complete medium and grown to confluency. Where indicated, confluent cells were serum- deprived overnight and treated with 10 nM PDGF. A wound was done by making a scratch on the cells monolayer with a 200 ml tip. The migration rate was monitored for 16 h by phase contrast microscopy (Leica DM IRBE Inverse, Widefield) equipped with a temperature controller. The wounded cell monolayers were photographed and quantitative analysis was prepared for each experiment. The wounded area was defined in each image by positioning lines in correspondence of the original scratch and wound closure distance was analysed by micrometer measure- ments. Cell Death (Anoikis) Assay NIH3T3-V, NIH3T3-C2b-WT, NIH3T3-C2b-DN cells or NIH3T3 and NIH3T3-C2b-WT cells transiently transfected (Lipofectamine, Invitrogen) with pcDNA3 empty vector, PI3KC2b-WT, constitutively active or dominant-negative form PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e44945