Figure 1

Analysis of cytosolic split-TEV reporters to monitor the dimerization of 14-3-3 proteins. (a) The flow-chart depicts the transcription-coupled reporter systems (GV-ER/GV-2ER used in combination with a GV-dependent reporter construct) and the proteolysis-only reporter systems (RedERnuc/LucER). Whereas the readout via the transcription-coupled reporter systems is generated by two steps, namely proteolytic cleavage and transcriptional activation, the proteolysis-only reporter systems require the proteolytic activation only. Transcription-coupled reporters are inactivated transcription factors; proteolysis-only reporters are inactivated final reporter proteins. tevS, TEV protease cleavage site; GV, artificial transcription factor composed of the DNA-binding domain of Gal4 from yeast and the transactivation domain VP16 from herpes simplex virus; DsRednuc, nuclear variant of DsRed; Luciferase, firefly luciferase moiety; ERT2, modified ligand binding domain of the estrogen receptor. (b, c) Luciferase (a) and FACS (b) assays showing the dimerization of the 14-3-3ε isoform with all cytosolic reporters. NIH-3T3 cells were transfected with the indicated combinations of constructs. GCN4cc-N-TEV was used as a control.