Pgex 4t 1 vector

To generate GST-tagged N- and C-terminal BTG3 truncation constructs, the corresponding regions of BTG3 were amplified by PCR and cloned into the BamHI and XhoI sites of the pGEX4T-1 vector (Amersham Biosciences, Piscataway, NJ, USA). The plasmids expressing GST-tagged AKT full-length and truncation mutants were generated by PCR amplification of the corresponding coding regions from pcDNA3-myr-HA-AKT (kindly provided by Jeffrey J-Y Yen, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan) and cloned between the EcoRI and XhoI sites of pGEX4T-1. For mammalian expression of HA-AKT, the full-length AKT was amplified by PCR from pcDNA3-myr-HA-AKT and cloned between the EcoRI and XhoI sites of the pcDNA3-HA vector. The myristoylation (myr) sequence was removed to allow dynamic regulation of AKT in cells. All sequences amplified by PCR were verified by DNA sequencing.

To deplete Luciferase (control), PAK1 and MEC-17 from D723H cells, we respectively used Eurogentec synthesized siRNA 5′-CGUACGCGGAAUACUUCGA(dT)(dT)3′, siRNA 5′-GCCCGAUUGCUUCAAACA(dT)(dT)3′ targeting nt310-328 from ATG of PAK1 and siRNA 5′-CCACACCAACUGGCCAUUGA (dT)(dT)3′, targeting nt478-497 from ATG of MEC-17. For mammalian cell transfection we used pCMV6M-PAK1 and pCMV6M-PAK1-K299R (Myc-PAK1-KD) that were gifts from Dr. J. Chernoff and are now commercially available (Addgene plasmids #12209 and # 12210). For recombinant PAK1 production in E. Coli, we used pMBP-X-PAK1-Cter (described in [44 (link)]). For MEC-17 production we started from a full length mouse MEC-17 cDNA in sPort–CMV vector (gift from Dr. J. Gaertig, University of Georgia, Athens, USA). Full-length MEC-17, its N-ter (aa 2-186) or its C-ter (aa 192-421) domains were amplified by PCR and subcloned into pGEX-4T-1 vector to add an N-terminal GST tag (Amersham). Site directed mutagenesis was performed using the QuickChange Site-directed mutagenesis kit (Agilent Technologies) on pGEX-4T-1-mMEC-17FL, all mutations were verified by sequencing. Oligonucleotide sequences used for PCRs are given in Table 1.

LvRab11, 14-3-3ES and 14-3-3EL were cloned into the pET-28a (+) vector (Novagen) that already contained an N-terminal 6xHis tag. The protein was expressed in Escherichia coli strain BL21 (DE3) and purified using HisPur Ni–NTA Superflow Agarose (Thermo Scientific) following the manufacturer’s instructions. GST-fusion 14-3-3ε proteins were subcloned into the pGEX-4T-1 vector (Amersham Biosciences). The recombinant GST-14-3-3EL and GST-14-3-3ES proteins were produced in E. coli strain BL21 and purified using Glutathione Sepharose 4 Fast Flow (GE Healthcare) following the manufacturer’s instructions. Purified recombinant proteins were analysed by SDS-PAGE. The protein concentration was measured with a dye-binding assay by the Bradford method and stored at − 20 °C.

For use in the pull-down assay, the glutathione S-transferase (GST) fusion protein containing the Ras binding domain (GST-RBD) of Raf1 was prepared as follows. The cDNA encoding the Ras binding domain (RBD) of Raf1 was amplified by PCR and then subcloned into the pGEX-4T-1 vector at the BamHI-XhoI sites (Amersham Biosciences, Piscataway, NJ, USA). The resulting plasmid was used to transform BL23 Escherichia coli cells. GST-RBD was purified from the cell lysate by extraction of the supernatant fraction with glutathione-agarose beads. The pull-down assay was carried out by incubating a mixture consisting of 485 μL of AGS cell extract, 10 μg of GST-RBD and 15 μL of glutathione-agarose beads at 4 °C for 45 min. The beads were separated from the mixture by centrifugation, washed three times with lysis buffer, and then resuspended in SDS-sample buffer for Western blot analysis. Anti-Ras antibody was used to measure the total Ras in the mixture whereas anti-H-Ras antibody (sc-520, Santa Cruz Biotechnology) was used to measure the activated Ras.

The full-length ORFs of FaGT2* and FaGT5 were obtained from cDNA of ripe fruit of F.×ananassa cv. Elsanta by amplification with primers introducing BamHI/NotI (FaGT2*) and EcoRI/XhoI (FaGT5) restriction sites (Supplementary Table S1 at JXB online). The genes were subcloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). The sequence of FaGT2 was synthesized by Eurofins Genomics (Ebersberg, Germany), already flanked by BamHI and NotI restriction sites, and subcloned into the pEX-K4 vector. Subsequently, the genes were cloned into the pGEX-4T-1 vector (Amersham Bioscience, Freiburg, Germany) in-frame with the N-terminal tag. Sequencing of the complete insert (Eurofins Genomics) confirmed the identity of the cloned sequences. The full-length ORF of FvGT2 was amplified from cDNA of turning fruits (stage 3) of F. vesca and cloned into pGEX4T-1 as a BamHI/NotI fragment. The full-length ORF of RiGT2 was amplified from cDNA of turning fruits (stage 3) of R. idaeus ‘Tulameen’ and cloned into pGEX4T-1 as a BamHI/EcoRI fragment. Primer sequences to amplify RiGT2 were obtained from Judson Ward (personal communication). All constructs were verified by DNA sequencing.

Recombinant proteins were expressed as fusion proteins either with a glutathione S-transferase (GST)-tag using the pGEX 4T1 vector (Amersham Bioscience) or with a maltose binding protein (MaBP)-tag using the pIH-vector (kindly provided by Kim Williamson, Loyola University Chicago). Cloning into the pGEX4T1 vector was mediated by EcoRI/NotI restriction sites added at the ends of PCR-amplified gene fragments and cloning into the pIH-vector was mediated by EcoRI/SalI restriction sites. Primers used for cloning are listed in (Table S1). Recombinant proteins were expressed in E. coli BL21 (DE3) RIL according to the manufacturer’s protocol (Stratagene). GST-fusion proteins were purified from bacterial extracts using glutathione-sepharose according to the manufacturer’s protocol (GE Healthcare). MaBP-tagged recombinant proteins were purified using amylose resin (New England Biolabs) as described previously [35] (link) with following modifications of the procedure: pelleted bacteria were directly resuspended in lysis buffer containing complete, EDTA-free protease inhibitor cocktail (Roche), incubated on ice for 20 min and homogenized by 4 min of sonication (50 cycles/50% intensity). DNAse treatment was not deployed. Amylose-bound fusion protein was eluted during batch purification according to the manufacturer’s protocol.