SUPPLEMENTARY NOTE 3. Supplememtary Note 3, Wehr et al., Monitoring Regulated Protein-Protein Interactions Using Split-TEV 1

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1 SUPPLEMENTARY NOTE 3 Fluorescent Proteolysis-only TEV-Reporters We generated TEV reporter that allow visualizing TEV activity at the membrane and in the cytosol of living cells not relying on the addition of exogenous substrates. All fluorescent proteolysis-only reporters are based on the red fluorescent protein from Discosoma sp. reef coral (see Methods). TM-Rednuc reports TEV activity at the membrane through translocation of the fluorescent signal from the membrane to the nucleus (Fig. 1a). Counting the TEV-dependent nuclear translocation of the fluorescent signal reveals a high signal to noise ratio (Fig. 1b). TEV cleavage of RedERnuc in the cytosol induces tetramerization of the previously inactive DsRednuc monomer thereby gaining fluorescent properties and accumulating in the nucleus (Fig. 2a). The applicability of the technique was also verified for its use in primary cultured cortical neurons and astrocytes (Fig. 2b), proving that both the TEV activity and the artificial reporter were tolerated in these primary cells. When DsRed was used instead of DsRednuc, a decrease in cellular fluorescence intensity was observed demonstrating the benefits of the nuclear accumulation (data not shown). TEV-dependent cleavage of RedERnuc was verified by western blotting (Fig. 2c). FACS analysis of RedERnuc activation revealed a high signalto-noise ratio (Fig. 2d). RedERnuc displayed elevated levels of background fluorescence (which was not localized to the nucleus) only in few cells of aberrant morphology, likely to be stressed or dying cells. Treatment of RedERnuc transfected COS1 cells with 1 µm 4-OHT also resulted in a red nuclear staining, but untreated cells showed no fluorescence suggesting that the ERT2 domains can both mask efficiently the NLS and the fluorescent activity of the DsRednuc moiety (Fig. 3). RedERnuc constructs equipped only with one ERT2 domain are already fluorescently active without adding 4-OHT, with the signal localized to the cytoplasm (data not shown). In summary, fluorescent proteolysis-only TEV reporters allow to visualize TEV activity at the membrane and in the cytosol of single living cells. The use of these reporters may be particularly valuable for primary cells (or other cells) that are difficult to transfect at increased efficiency. Since these reporters are autonomous and solely rely on proteolytic activation, they offer the possibility to analyze TEV activity with microscopy at the single cell level. Supplememtary Note 3, Wehr et al., Monitoring Regulated Protein-Protein Interactions Using Split-TEV 1

2 Fig. 1: Fluorescent Proteolytic Reporter at the membrane. (a) COS1 cells were transfected with TM- Rednuc reporter constructs alone or in combination with TM-TEV constructs. Whereas the uncleaved TM- Rednuc reporter clearly localized to the membrane, TEV protease mediated activation caused a nuclear staining. Nuclei were counterstained with a Hoechst nuclear dye. Phase, phase contrast optics. (b) Quantification of the TM-Rednuc activation as depicted in (a) by manual counting. Supplememtary Note 3, Wehr et al., Monitoring Regulated Protein-Protein Interactions Using Split-TEV 2

3 Fig. 2: Fluorescent Proteolytic Reporter in the cytosol. (a) COS1 cells were transfected with RedERnuc reporter constructs alone or in combination with TEV protease constructs (left panels). Upon TEV cotransfection, fluorescent activity accumulates in the nucleus. (b) RedERnuc reporter activation in primary cultured cortical neurons (left panels) and astrocytes (right panels). TEV protease-mediated activation of the RedERnuc reporter results in red fluorescence of the nuclei. Nuclei were counterstained with Dapi. Phase, phase contrast optics. Neurons were identified with MAP2-, astroglia with GFAP-staining. (c) Biochemical verification of RedERnuc cleavage. COS1 cells were transiently transfected with RedERnuc reporter constructs (left) or in combination with TEV protease constructs (center) and analyzed with an α-her antibody. Asterisk, full-length RedERnuc reporter protein; arrowhead, band representing a singly cleaved reporter resulting in either DsRednuc-tevS-ERT2 (DsRednuc-ER) or ERT2-tevS-DsRednuc (ER-DsRednuc); arrow, band representing the ERT2 (ER) domains from a doubly cleaved reporter. Calculated protein (in kda): RedERnuc, 103.8; DsRednuc- ER, 67.4; ER-DsRednuc, 67.1; ER, (d) Quantitative analysis of RedERnuc reporter activation. COS1 cells were transfected with expression constructs as indicated and FACS-analyzed events were counted per sample and the total fluorescence was calculated. Supplememtary Note 3, Wehr et al., Monitoring Regulated Protein-Protein Interactions Using Split-TEV 3

4 Fig. 3: Functionality of ERT2 domains in living cells. COS-1 cells were transiently transfected with a CMVdriven RedERnuc construct in the presence or absence of a CMV-driven cytosolic TEV protease construct. Upon addition of 1 µm 4-hydroxytamoxifen (4-OHT) to the regular cell culture medium, the entire reporter protein translocated into the nuclear compartment and additionally became fluorescently active (center). In contrast to the 4-OHT mechanism, the TEV protease cleaved off the flanking ERT2 domains, enabling the exposition of the NLS of the DsRednuc moiety, finally leading to the nuclear red fluorescence. Phase, phase contrast optics. METHODS Cloning of fluorescent reporters TM-Rednuc and RedERnuc The ORF of the red fluorescent protein from Discosoma sp. reef coral (DsRed-Express from Clontech) including a nuclear localization sequence (NLS) was amplified by PCR, sequence verified and sub-cloned into the final expression plasmids. Primary neuronal cell culture Primary hippocampal and cortical neurons were prepared from E17 old embryos. After preparation of the hippocampus and the cortex, both tissues were incubated in HBSS (supplemented with 10 mm Hepes, NaHCO 3 and 1x penicillin/streptomycin) containing 0.5x trypsin-edta and 0.1 mg/ml DNAseI for 10 min at 37 C. The cells were further dissociated by gentle trituration until they were homogenously dispersed in the medium. The cells were pelleted (800 rpm for 10 min) and resuspended in Neurobasal medium supplemented with B27-supplement (1:50, Gibco), L-glutamine (1:100, 2 mm), penicillin (100 U/ml), streptomycin (100 µg/ml), MK-801 (10 µm, Tocris), NGF (0.1ng/µl, Promega) and β-fgf Supplememtary Note 3, Wehr et al., Monitoring Regulated Protein-Protein Interactions Using Split-TEV 4

5 (0.1 ng/µl, PeproTech). Finally, cells were counted and plated for immunocytochemistry or luciferase assays (24-well plate: 10 5 cells/well; 96-well plate: 6x10 4 /well). Primary astroglial cell culture Primary astrocytes were prepared from both hemispheres and the midbrain of E15 to E17 old mice embryos. After removal of the meninges, the tissues were incubated in 0.5% trypsin- EDTA for 10 min at 37 C. Subsequently, cells were washed twice in HBSS and triturated in 10 ml pre-warmed BME medium for thorough dissociation. Cells were counted and seeded onto PLL-coated cell culture flasks, with an approximate amount of cells from 3 4 brains per flask. Flasks were incubated at 37 C and 5% CO 2. Between the 4 th and 6 th day of incubation neurons were killed with AK358 (kindly provided by J. Trotter) and complement (Linaris) which were both applied for 30 min. Thereafter, cells were washed twice with medium. At day 8, half of the medium was exchanged. At day 11, microphages were washed off by gently rocking the flask with subsequent change of the medium. At day 14, oligodendrocytes were removed by shake off. Astrocytes remain seated as continuous layer and were grown in DMEM supplemented with 10 % fetal calf serum, 1% L-glutamine (2mM), 1% penicillin (100 U/ml) and streptomycin (100 µg/ml). After 1 or 2 days astrocytes were splitted for further culturing and/or plated on coverslips for transfection experiments. Immunocytochemistry Cells were fixed in 4% paraformaldehyde for 5 min and washed 3 times with PBS before permeabilizing them with 0.1% Triton-X-100 in PBS for 5 min. To block nonspecific staining, coverslips were incubated with 5% horse serum in PBS for 30 min and washed twice with PBS. Cells were then incubated for 1h with primary antibodies diluted in PBS and 2% horse serum. The following antibodies were used: monoclonal mouse α-map2 (Chemicon, 1:500) and polyclonal rabbit α-gfap (Dako, 1:250). Cells were washed 3 times with PBS and incubated with the appropriate secondary antibody for 1 h (Cy2, 1:200, Jackson Immuno Research). Cells were again washed 3 times with PBS, nuclei were counterstained with Dapi (1:3000) and again washed 3 times with PBS. Finally, coverslips were mounted on glass slides with Polyaquamount (Polysciences). Fluorescent images were obtained using a Leica DM XRA microscope and a Jenaoptik C14 Resolution camera. Supplememtary Note 3, Wehr et al., Monitoring Regulated Protein-Protein Interactions Using Split-TEV 5