1. Volume 2, Issue 5, Pages 1051-1058 (September 2009)
New GATEWAY vectors for High Throughput Analyses of Protein–Protein Interactions by Bimolecular Fluorescence Complementation 
Gehl Christian , Waadt Rainer , Kudla Jörg , Mendel Ralf-R. , Hänsch Robert  
Molecular Plant 
Volume 2, Issue 5, Pages (September 2009)
DOI: /mp/ssp040
Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Visualization of Protein–Protein Interaction Using the New GATEWAY Vectors.
The single cLSM pictures represent the BiFC-signal (BiFC), the far-red fluorescent protein eqFP611 (eqFP611), the chlorophyll auto-fluorescence (Chl), and the merged pictures (Merge) imaged in the channel-mode of the cLSM with identical settings of the microscope in A, B, and C (laser power, pinhole, detector gain, magnification, etc.). The Lambda-mode was used to detect the spectral signature and the intensity (gray scale from 0 to 255) of the BiFC-signal; the small insert shows the chosen area. The fusion constructs used are symbolized on top. Protein–protein interaction of Cnx6 and Cnx7 is shown, representing the directly cloned constructs pVYNE(R)–Cnx6 and pCYCE(R)–Cnx7 (A), the GATEWAY-clones pEXP–CYCE(R)–Cnx6 and pEXP–VYNE(R)–Cnx7 (B), pEXP–SCYCE(R)–Cnx6 and pEXP–SCYNE(R)–Cnx7 (D), and pEXP–VYCE(R)–Cnx6 plus pEXP–VYNE(R)–Cnx7 (E). As negative controls, the GW vectors pEXP–SCYCE(R)–Cnx6 plus pEXP–T14-3c–VYNE (C) and pEXP–SCYCE(R)–Cnx6 plus pEXP–Nia2–VYNE (F) were used, which show nearly no BiFC signal. For direct comparison of the BiFC fluorescence, the expression control eqFP611 was used (A–C). Bars represent 50 μm.
Molecular Plant 2009 2, DOI: ( /mp/ssp040)
Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

3. Figure 2 A New Set of Binary BiFC–GATEWAY Vectors.
Binary GATEWAY vectors to study protein–protein-interaction using bimolecular fluorescence complementation (BiFC): pDEST–GWVYNE (Venus aa 1–173), pDEST–VYNE(R)GW (Venus aa 1–173), pDEST–GWSCYNE (SCFP3A aa 1–173), pDEST–SCYNE(R)GW (SCFP3A aa 1–173), pDEST–GWVYCE (Venus aa 156–239), pDEST–VYCE(R)GW (Venus aa 156–239), pDEST–GWSCYCE (SCFP3A aa 156–239), and pDEST–SCYCE(R)GW (SCFP3A aa 156–239) carry a sub-fragment of SCFP3A or Venus in N- or C-terminal fusion to the GATEWAY-cassette and kanamycin selection in E. coli and A. tumefaciens. The constructs p(MAS)DEST–GWSCYCE and p(MAS)DEST–SCYCE(R)GW under the control of the MAS-promoter should be used in the case of a stronger protein expression in the mcBiFC competition reaction. The arrows mark the possible interactions and the numbers represent the emission wavelength. Relevant restriction sites and linker proteins are indicated: c-myc, c-myc epitope tag; FLAG, FLAG epitope tag; HA, hemagglutinin epitope tag.
Molecular Plant 2009 2, DOI: ( /mp/ssp040)
Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

4. Figure 3 Visualization of Protein–Protein Interaction Using mcBiFC.
The mcBiFC suitability of the new vectors was investigated using the constructs p(MAS)EXP–SCYCE(R)–Cnx6, pEXP–VYNE(R)–Cnx7, and pEXP–SCYNE(R)–Cnx6. In (A), a schematic overview is given to show the possible interaction and the expected fluorescence emission. In (B), the fluorescence is presented in the merged picture (top) and the Lambda signature of the scanned cells (bottom). Bars represent 50 μm.
Molecular Plant 2009 2, DOI: ( /mp/ssp040)
Copyright © 2009 The Authors. All rights reserved. Terms and Conditions