Figure 1. PI3KC2b interacts with Dbl GEF for Rho GTPases. (A) Lysates of NIH3T3 cells stably overexpressing wild-type Glu-PI3KC2b (NIH3T3-C2b-WT) were immunoprecipitated (IP) with an anti-C2b, anti- Glu (EE) tag, anti-Dbl or control IgG antibody. PI3KC2b interaction with the endogenous Dbl, as well as Grb2 and Src was detected by immunoblotting (IB) with the indicated antibodies. (B) Complex of endogenous PI3KC2b and Dbl was immunoprecipitated from the whole cell extract (WCE) of the parental NIH3T3 cells and IMR5 neuroblastoma human cancer cells with anti-Dbl, anti-C2b and control IgG antibody. Samples were subjected to western blot analysis with the indicated antibodies. In the case of IMR5 cells Triton 0.5% and Brij96 1% lysis buffers were used, while NIH3T3 cells were lysed with Triton 1%. Asterisk indicates PI3KC2b. (C) Interaction between the PI3KC2b and Dbl was examined by the pull-down (PD) of PI3KC2b GST-fused N-terminal and C2 C-terminal domains in HEK293 cells transfected with HA-proto- Dbl (Dbl), pcDNA3 empty vector (V) or untransfected control (-). Complex formation was shown by immunoblotting with anti-Dbl antibody. Asterisk indicates ectopically expressed (HA-tag) Dbl. (D) Role of PI3KC2b in RhoA Activation Interaction between the Dbl and Grb2 was examined by the GST-Grb2 pull-down in HA-proto-Dbl (Dbl) transfected and untransfected (-) HEK293 cells. Pull-down samples were subjected to western blot analysis with indicated antibodies. All experiments were repeated at least two times with similar results. doi:10.1371/journal.pone.0044945.g001 Transient Transfections HEK293 cells were transiently transfected with a Ca2+ phosphate protocol [7], while NIH3T3 and COS-1 cells were transfected with Lipofectamine or Lipofectamine 2000 (Invitrogen) respectively, according to the manufacturer’s instructions. Growth Factor Stimulations To investigate Erk and Akt signalling downstream of EGFR and PDGFR in NIH3T3 cells stably expressing PI3KC2b wild-type (WT) and dominant-negative (DN) were grown to confluency in a six-well plate and starved overnight in DMEM containing 0.5% FCS and penicillin/streptomycin/L-glutamine. Cells were then stimulated with the EGF (1 nM) and PDGF (1 nM) for 10 min. at 37uC, as indicated in the figure legends. Cellular lysates were prepared as described below. Stimulation of cells with PDGF and LPA for investigation of RhoA and Rac1 activity is described below in the section of RhoGTPases activation assay. SDS/PAGE and Western-blot Analysis NIH3T3-C2b-WT and –DN cellular lysates were prepared with RIPA buffer (50 mM Tris.Cl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10% glycerol, 10 mM leupeptin, 10 mM pepstatin A, 1 mM phenylmethylsulfonylfluorid [PMSF], 5 mM iodoacetamide, 5 mM benzamidine, 2 mM sodium orthovanadate [http://en. wikipedia.org/wiki/Chemical_formulaNa3VO4] and 10 mM so- dium fluoride [NaF]). In the case of NIH3T3, COS1 and HEK293 cells lysis buffer was changed to Triton 1% (50 mM Tris.Cl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.5% NP-40) supplemented with 76concen- trated stock of protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (1 mM NaF, 1 mM Na3VO4, 10 mM b- glycerophosphate). For the studies of endogenous complex formation in IMR5 cells Triton 0.5% and Brij96 1% (50 mM Tris.Cl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Brij96) lysis buffers supplemented with 76concentrated stock of protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (1 mM NaF, 1 mM Na3VO4, 10 mM b-glycerophos- phate) were used, as indicated in the figure legends. Prior to lysis cells were washed once with ice-cold phosphate-buffered saline (PBS), lysed on ice for 20 min., and centrifuged at 16,0006g for 25 min. at 4uC. The supernatants were collected and normalized for protein content by using the BCA (bicinchoninic acid) protein assay kit (Pierce). 2x or 5x SDS sample buffer (50 mM Tris.HCl, pH 6.8, 2%/5% SDS, 10% glycerol, 200 mM DTT, and 0.25% bromophenol blue) was then added to the samples, followed by denaturation for 3 min. at 100uC and analysed by SDS/PAGE. The gels were transferred to a hydrophobic PVDF membrane (Hybond-P; Amersham Biosciences) by electrophoresis. The membranes were then blocked in 16 PBS, 5% (w/v) BSA (phospho-specific antibodies) or 16 PBS/3% non-fat dry milk (all other antibodies) for 16 h at 4uC. The membranes were incubated with primary antibodies (diluted according to the manufacturer’s protocol) for 16 h at 4uC. After washing in 16 PBS and 0.1% (w/ v) Tween 20, the immunoblots were incubated with donkey anti- rabbit IgG or sheep anti-mouse IgG secondary antibodies (1:10000 dilution) coupled with horseradish peroxidase (Amer- PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44945