Role of PI3KC2b in RhoA Activation Figure 2. PI3KC2b binds with high affinity to the spectrin- and PH- Dbl domain. (A) Schematic representation of Dbl mutant constructs, which were kind gift of Prof. Danny Manor from the School of Medicine at Case Western Reserve University, Cleveland, USA. (B) COS-1 cells co- transfected with Myc-PI3KC2b and different GST-fused Dbl domains (N-, spectrin-, onco-, PH-) in pEBG vector were subjected to gluthatione- sepharose beads affinity-purification and immunobotted with indicated antibodies. Experiment was repeated at least two times with similar results. PC – positive control; COS-1 cells co-transfected with PI3KC2b and HA-proto-Dbl, and immunoprecipitated with anti-Dbl antibody. doi:10.1371/journal.pone.0044945.g002 sham Biosciences) for 1 h at room temperature. After washing of the immunoblots, chemiluminescence was used for detection, using either the ECLH (enhanced chemiluminescence) western blotting detection reagents (Amersham Biosciences) or Super- Signal West Femto Maximum Sensitivity Substrate (PIERCE) according to the manufacturer’s protocol. Immunoprecipitations NIH3T3-C2b-WT, NIH3T3 parental and IMR5 cells were grown to confluence in 10-cm dishes, washed with ice cold phosphate-buffered saline (PBS) before lysis on ice for 20 min. in 0.5–1 ml of lysis buffer (as indicated in the previous section). Insoluble material was removed by centrifugation for 30 min. at 16,0006g in 4uC. Immunoprecipitation was performed for 2 h at 4uC with primary antibodies (diluted according to the manufac- turer’s instructions). IP Matrix (ExactaCruz, Santa Cruz Biotech- nology) or Protein A- or G-Sepharose 4 Fast Flow beads (GE Healtcare) were then added, and the incubation was continued for 1 h at 4uC. The immunoprecipitates were washed three times in the lysis buffer and resuspended in 2x SDS sample buffer (50 mM Tris.HCl, pH 6.8, 2% SDS, 10% glycerol, 200 mM DTT, and 0.25% bromophenol blue). Samples were denatured for 3 min. at 100uC and analysed by SDS/PAGE and western blot. Production of GST-Fusion Proteins pGEX-2T expression vectors encoding GST-PAK-CRIB, GST-rhotekin, GST-RhoA, GST-Grb2, GST-PI3KC2b N-termi- nal and C2 C-terminal domains were transformed into Escherichia coli BL21 cells. The incubation proceeded at 37uC until OD600 reached 0.5. Protein expression was induced with 0.2 mM isopropyl-b-D-thiogalactopyranoside (IPTG) and incubated at 30uC for 3–4 h. The bacteria were collected by centrifugation and resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 10 mM leupeptin, 10 mM pepstatin and 1 mM PMSF). In the case of GST-Grb2, GST-PI3KC2b N-terminal and C2 C-terminal domains lysis buffer was changed to: 50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA pH 8.0, 1% Triton supplemented with 76concentrated stock of protease inhibitors (Complete Mini, Roche), 1 mM PMSF, 10 mM leupeptin, 10 mM pepstatin, 5 mM benzamidin, 1 mM DTT, 1 ml/ml aprotinin, 1 mM NaF and 1 mM Na3VO4. Bacteria were thereafter sonicated on ice six times for 10 s or 3 times for 45 sec with 1 min. intervals (in the case of GST-Grb2, GST-PI3KC2b N-terminal and C2 C-terminal domains). The lysed cells were clarified by centrifugation at 30,0006g for 30 min. at 4uC. GST-fusion proteins were affinity- purified from the resulting supernatants by addition of glutathione- sepharose beads (Amersham Biosciences) followed by incubation at 4uC for 60 min. The glutathione-sepharose beads were collected by centrifugation at 8056g at 4uC followed by four washes with washing buffer (50 mM Tris-HCl pH 7.5, 0.5% Triton X-100, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 10 mM leupeptin, and 0.1 mM PMSF. After the final wash, the beads containing the GST-fusion proteins were resuspended in the washing buffer supplemented with 10% glycerol. The purity of the GST-fusion proteins was assayed by SDS-PAGE. GST-tagged NT-, spectrin-, onco- and PH- Dbl mutant constructs, described previously in [31], were expressed in COS- 1 cells, lysed with HEPES buffer as in [31] and purified with glutathione-sepharose beads (GE Healthcare). Beads containing the GST-fusion proteins were washed 3–46with ice-cold HEPES buffer and 2x with washing buffer (50 mM Tris pH 7.5, 150 mM NaCl). Next, they were resuspended in the washing buffer supplemented with 1 mM DTT and 50% glycerol and stored in 220uC. PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44945