SUPPLEMENTARY NOTE 2. Supplememtary Note 2, Wehr et al., Monitoring Regulated Protein-Protein Interactions Using Split-TEV 1

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1 SUPPLEMENTARY NOTE 2 A recombinase reporter system for permanent reporter activation We made use of the Cre-loxP recombinase system for a maximal amplification and complete kinetic uncoupling within single cells. Cre has already been described to measure signal transduction in mammalian cells 1. The objective here was to introduce a molecular switch to transfer finally a transient TEV activity into a long lasting and amplified output leading to the permanent, inheritable activation of a reporter gene in living cells (Fig. 1, 2). In a first step, we generated a series of PC12 cell lines which stably integrated the components of the Crereporter system. In one cell line (PC ), no Cre background activity was detectable. Upon transfection with the synthetic transcription factor of the yeast Gal4 DNA binding moiety fused to the transactivation domain of the Herpes Simplex VP16 protein (referred to as GV), we observed the activation of the co-integrated Cre dependent GFP reporter plasmid (Stop-EGFP) with a high signal to noise ratio (Fig. 2a). The transient expression of the soluble transcription factor GV as well as the TEV-dependent cleaved TM-GV resulted in the permanent activation of the reporter in PC cells (Fig. 2b, c). To test the Crereporter system in an interaction event, we measured the constitutive GABA-B receptor 1 and 2 coiled-coil domain (GBR1cc and 2cc) mediated interactions in a mammalian two-hybrid approach in the stable PC cells (Fig. 1b) 2. Using the well characterized phosphorylation dependent interaction of the transcription factor CREB with the CBP-KIX adaptor domain, we could also prove the usefulness of the Cre-reporter system to monitor stimulus dependent interactions in the nucleus of mammalian cells (Fig. 1c) 3. We quantified the TEV/TM-GV dependent activation of the PC endogenous Cre-reporter system with FACS that clearly showed the tightness and feasibility of the cell line to report TEV activity with almost no detectable background (Fig. 2d). In parallel, the applicability of the coupled Cre-reporter system was tested in transient transfections (Fig. 2e). To yield an optimal signal to noise ratio, the relative amounts of the Gal4-dependent Cre reporter (G5- Cre) to the Cre-dependent STOP-Reporter (either Stop-Luci or Stop-EGFP) has to be determined for each cell line (see METHODS below). Finally, we used the rapamycin regulated FRB-FKBP interaction to monitor in a Split-TEV assay for the dose-response and time-dependence with the Cre-reporter system in PC cells (Fig. 2f, g). In summary, highly sensitive Cre-recombinase based reporter systems can be combined with Split-TEV assays. Principally, transient TEV activity can be transferred into the permanent and inheritable activation of reporter genes in living cells. Supplememtary Note 2, Wehr et al., Monitoring Regulated Protein-Protein Interactions Using Split-TEV 1

2 Fig. 1: Two-hybrid assay using a Cre-recombinase based reporter system to monitor transient proteinprotein interactions in the nucleus of cultured mammalian cells. (a) Schematic drawing of the Crerecombinase based two-hybrid assay. Interacting protein(fragment)s (X and Y) are expressed as Gal4-DNA binding domain or VP16 transactivation domain fusion proteins (GX and VY), respectively. Upon stimulation (symbolized by the red star), e.g. the X domain becomes phosphorylated (P) leading to an interaction with Y and a subsequent activation of the Gal4 dependent Cre recombinase reporter gene. Even if the interaction was transient, the Cre activity leads to a permanent activation of the second Cre-dependent reporter driven by a constitutive promoter, such as the CMV-Promoter. Boxed are the elements of the bacteriophage P1 Cre recombinase (Cre) dependent reporter system that consists of two elements: 1) G5-Cre, a construct for the Gal4- dependent (UAS enhancer) expression of Cre 2) Stop-Luci or Stop-EGFP, a CMV-driven expression construct consisting of a transcriptional stop cassette (STOP) flanked by Cre specific recombination sites (loxp). (b) Twohybrid assay performed in PC cells monitoring the constitutive interaction of the coiled-coiled domains from GBR1 and GBR2, respectively. GBR1 and -2 coiled-coiled (cc) domains are expressed as a VP16 fusion (V-GBR1cc and V-GBR2cc) and as Gal4 fusion proteins (G-GBR1cc and G-GBR2cc). FACS analysis was performed 48h post-transfection. (c) Two-hybrid assay performed in PC cells monitoring the forskolin induced interaction of CREB with the KIX domain from the co-activator CREB-Binding-Protein (CBP) expressed as Gal4 (G-CREB) and VP16 (V-CBP-KIX) fusion proteins in PC cells. Cells were transfected, forskolin-stimulated for 8h and FACS analysis was performed 48h later. In the single transfection with G-CREB, a stimulus-dependent increase in GFP positive cells could be observed, reflecting possibly the activity of endogenous CBP or other transcriptional co-activators. This forskolin induced effect becomes much more pronounced in G-CREB V-CBP-KIX co-transfections. Supplememtary Note 2, Wehr et al., Monitoring Regulated Protein-Protein Interactions Using Split-TEV 2

3 Fig. 2: Cre recombinase reporter system for Split-TEV. (a) PC cells, containing stably integrated G5- Cre and Stop-EGFP, were transfected with an empty expression vector (MOCK, top) or with GV (bottom). EGFP expression was monitored 48 h after transfection by fluorescence microscopy. The small inlets show the same region visualized with phase optics showing that a comparable number of cells are depicted. Upon transfection with GV, a high number of green fluorescent PC cells are visible. After transfection of a control vector, which is not capable of activating the Cre reporter system, no green cells are visible. (b) PC cells were transfected with the inactivated, membrane bound TEV dependent transcriptional activator with TM-GV only (top) or TEV and TM-GV (bottom). EGFP expression was monitored 48 h after transfection by fluorescence microscopy. The small inserts show the number of cells in phase contrast. The TM-GV reporter shows no detectable background activity but becomes activated upon co-transfection with TEV. (c) Once induced, the Cre reporter system is permanently activated. GFP fluorescence of cell clones grown at low density for four weeks after transient transfection with GV is shown (top). All cells of the two clones depicted do express GFP at a high level. At the bottom the corresponding phase bright image are depicted. The constitutive activation of the GFP reporter indicates the uncoupling of the activation event (in this case the transient expression of GV) and the detection. (d) Quantitative FACS analysis of PC cells harbouring stably integrated the elements of the Cre reporter system (G5-Cre and Stop-EGFP) transfected with the indicated combinations of expression plasmids. The relative number of green cells counted is depicted, with GV set to 100%; the membrane bound TM-GVno-tevS and TM-GV (which includes the TEV cleavage site) transcription factors show no detectable background over the control (mock = empty expression plasmid). Only the TEV dependent reporter TM-GV becomes activated upon proteolytic cleavage. (e) TEV assays performed in native Supplememtary Note 2, Wehr et al., Monitoring Regulated Protein-Protein Interactions Using Split-TEV 3

4 PC12 cells transfected with various amounts of G5-Cre (as indicated) and constant amount of a Cre-dependent firefly luciferase reporter (Stop-Luci). (f, g) Split-TEV assays performed in PC cells with TM-FRB-N- TEV-GV and FKBP-C-TEV to monitor the dose-response and kinetic analysis of the rapamycin induced FKBP- FRB interaction. (f) The addition of the interaction inhibitor FK506 significantly reduces the signal obtained with all Rapamycin concentrations as indicated. (g) Rapamycin was applied 12h post transfection for the indicated times, followed by 3x PBS washes, FACS assays were performed 24 h later. METHODS Generation of stable cell lines Stable PC12 cells were generated upon electroporation of linearized Gal4-dependent Cre recombinase G5-Cre plasmids with linearized expression vector fragments conferring resistance to Hygromycin B (Calbiochem) (PGK-Hygro-pA); the G5-Cre fragment was used with a 10-fold molar excess over PGK-Hygro-pA. Stable clones were selected in PC12 medium containing 300 µg/ml Hygromycin B for four weeks and were subsequently separated to obtain sub-clonal cell lines. Clonal lines were monitored for absence of constitutive Cre recombinase activity by transfection with a CMV-driven EGFP expression vector containing a 5 located loxp-flanked PGK-neo-pA transcriptional stop-cassette ( Stop- EGFP ). Clones without constitutive Cre-activity were tested for Gal4-VP16 (GV) dependent induction. The clone with the best re-induction (PC12#20) was used for further experiments. PC12#20 cells were subsequently transfected with linearized plasmid constructs Stop-GFP and Stop-BlasR (CMV-driven Blasticidin resistance conferring expression vector containing a 5 located loxp-flanked ZeoR-pA transcriptional stop-cassette; ZeoR confers resistance to phleomycin-related drugs). Cells were selected for four weeks in medium supplemented with 100 µg/ml Zeocin (Invitrogen); resistant and EGFP negative clones were separated and further analyzed for GV dependent EGFP activation. The sub-clone PC12#20.4 was further expanded and used throughout the study unless not otherwise indicated. Cre-recombinase reporter gene assays To optimize the Cre-based luciferase assays for PC12 cells, varying amounts of G5-Cre and Stop-Luci reporter plasmids were transiently co-transfected with TM-GV and TM-TEV expression plasmids. The combined use of 50 ng G5-Cre with 200 ng of Stop-LUCI provided the highest signal to noise ratio when co-transfected with either 100 ng GV or 100 ng of each TM-GV and TM-TEV per well. Supplememtary Note 2, Wehr et al., Monitoring Regulated Protein-Protein Interactions Using Split-TEV 4

5 FACS Analysis For FACS analysis, single cell suspensions of transfected cell were prepared by trypsin treatment and thorough pipetting in PBS containing 5mM EDTA. Before analysis, cells were filtered through 50 µm meshes and kept on ice. GFP and DsRed analysis was performed with standard filter settings, gates were adjusted with negative and positive control samples. All experiments were repeated at least three times or were performed with three independent replicates for each data point. Results were given either as relative percentage of positive cells or total accumulated fluorescence (average fluorescence times scored events). FACS measurements were performed with a FACS-Aria (Becton-Dickinson). Errors are given as SEM. References 1. Mattheakis, L.C., Olivan, S.E., Dias, J.M. & Northrop, J.P. Expression of cre recombinase as a reporter of signal transduction in mammalian cells. Chem Biol 6, (1999). 2. Grunewald, S. et al. Importance of the gamma-aminobutyric acid(b) receptor C- termini for G-protein coupling. Mol Pharmacol 61, (2002). 3. Deisseroth, K. & Tsien, R.W. Dynamic multiphosphorylation passwords for activitydependent gene expression. Neuron 34, (2002). Supplememtary Note 2, Wehr et al., Monitoring Regulated Protein-Protein Interactions Using Split-TEV 5