Versatility of the newly generated MultiSite GatewayTM vector set. (A) Inducibility of the pGAL1 promoter. Arabidopsis genes At4g32295 and At3g24150 were expressed in yeast under the control of the pGAL1 promoter and fused with an NLS-FLAG-HIS tag using the pMG416 vector. After total protein extraction, expression was verified in inducing (+) and non-inducing (−) medium through immunoblot analysis with anti-FLAG antibodies. (B) Functionality of newly developed C-terminal translational fusion epitope tags. Yeasts were transformed with pMG425-ADH1::JAZ12:−NLS-FLAG-HIS, pMG423-GPD::COI1-C:−NLS-V5 where COI1-C is a truncated version of COI1, or pMG423-GPD::At2g26000: NLS-c-myc. Expression of the different constructs was verified with immunoblot analysis of total protein extracts with anti-FLAG, anti-V5 and anti-c-myc antibodies, respectively. (C) Interaction of proteins in the trimeric JAZ3-TPL-NINJA complex in Y3H assays. Transformed yeasts were spotted in 10-fold and 100-fold dilutions on control medium (−3: SD-Leu-Trp-Ura) and selective medium (−4: SD-His-Leu-Trp-Ura). Gene constructs in the pGBKT7 and pGADT7 vectors carry the DNA-binding domain or the transcription activation domain, respectively, in contrast to constructs expressed in pMG426, which do not carry DNA-binding or transcription activation domains. (D) Verification of expression of NINJA: NLS-FLAG-HIS in the Y3H set-ups shown in panel C. After total protein extraction, NINJA: NLS-FLAG-HIS was detected through immunoblot analysis with anti-FLAG antibodies. Arrowheads indicate the fusion protein.
Nagels Durand et al. BMC Molecular Biology 2012 13:30 doi:10.1186/1471-2199-13-30