with 10% (v/v) heat-inactivated FCS and penicillin/streptomycin. LUHMES cells were cultivated according to Schildknecht et al. [19]. Production of scTNFR2 and TNC-scTNFR2 HEK293T cells were transfected with scTNFR2 or TNC- scTNFR2 expression constructs, using lipofectamine 2000 (Invitro- gen). Stable cells were selected with Zeocin (Invitrogen). For protein production, cells were expanded in T175 culture flasks. When grown to 90% confluence, cells were cultivated for a total of 8 days with Opti-MEM (Invitrogen, USA). Medium was changed after 2 days. Supernatant was centrifuged (720g; five minutes) and stored at 4uC. TNF variants were purified by immobilized metal ion chromatography (IMAC). For this purpose, supernatant was loaded onto a column containing Ni-NTA agarose (Qiagen, Hilden, Germany) and unbound proteins were washed away using IMAC wash buffer (50 mM sodium-phosphate-buffer, 10–30 mM imidazole). Bound proteins were eluted with IMAC elution buffer (50 mM sodium-phosphate-buffer, 100 mM imidazole) in frac- tions of 1 ml. The purity of the protein in the eluted fractions was analyzed by SDS-PAGE and pure fractions were pooled and dialyzed (cut-off 4–6 kDa, Roth) against PBS overnight at 4uC. Protein concentration was determined by measuring the absor- bance at 280 nm. SDS-PAGE and Immunoblot Cells were lysed in homogenization buffer (10 mM HEPES pH 7.5, 1.5 mM MgCl2, 1.5 mM KCl, 1% NP-40, 0.2 mM PMSF, 20 mM ß-glycerophosphate and 100 mM Na3VO4) at 4uC for 30 min. Lysates were centrifuged (2 min at 9600 g) and protein concentration of supernatants were determined using the BCA method (Pierce, Bonn, Germany). 20 mg total protein or 2 mg (SDS-PAGE) and 1 mg (immunoblot) respectively of the TNF muteins were denatured in Laemmli buffer and resolved by 8% SDS-PAGE (100 V; 90 minutes). For Coomassie staining of proteins the gel was incubated in Coomassie staining solution for 60 minutes at room temperature and destained. For immunoblot analyses the proteins were transferred onto nitrocellulose mem- branes (semidry blot; 1.5 mA/cm2 gel for 90 minutes). Non- specific protein binding was blocked with 5% skimmed milk powder solution in PBS/0.1% Tween20 for 30 minutes at room temperature and the membrane was incubated overnight at 4uC using specific antibodies. After incubation with anti-rabbit HRP- conjugated secondary antibodies for 90 minutes at room temper- ature the signals were detected by enhanced chemiluminescence (Super Signal, Pierce, Rockford, IL). Immunoprecipitation Approximately 56106 R2 MEF were seeded in T75 cell culture flask (Greiner) and cultivated overnight. Cells were stimulated as indicated and homogenized with 200 ml IP lysis buffer (10 mM TRIS pH 7.4, 100 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.5% NP-40, 100 mM Na3VO4, 0.2 mM PMSF, 20 mM ß-glycero-phosphate, Roche complete protease inhibitor cocktail) for 30 minutes on ice. The lysate was centrifuged (2 minutes; 9600g; 4uC) and the supernatant was incubated with 5 mg TNFR2-specific antibody MR2-1 (Hbt) for 2 hours at 4uC. The immunocomplexes were precipitated with protein G agarose (75 ml; Pierce) for two hours at 4uC. The precipitates were washed four times with IP washing buffer (10 mM TRIS pH 7.4, 100 mM KCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40) and dissolved in 100 ml PBS, supplemented with Laemmli buffer. To elute bound proteins, the precipitates were boiled for five minutes at 95uC. Neuroregeneration by a TNFR2 Agonist 30 ml of the eluted proteins were separated by SDS-PAGE and analyzed by immunoblot analysis. HPLC The oligomerization state of the TNF variants under native conditions was analyzed by HPLC-SEC. 50 ml sample (0.1 to 0.5 mg/ml) was applied to a BioSep-SEC-S2000 7.86300 column (Phenomenex, Aschaffenburg, Germany) equilibrated with PBS and eluted at a flow rate of 0.5 ml/min. For determining the size of recombinant proteins, standard proteins (29 kDa, carbonic anhydrase; 67 kDa, bovine serum albumin (BSA); 200 kDa, b- amylase; 443 kDa, apoferritin; 669 kDa, thyroglobulin) were run under the same conditions. Cell survival assays Crystal violet. Mouse fibroblasts or Kym-1 cells (16104 cells/well) were grown in 96-well flat bottom cell culture plates overnight. If indicated, the cells were stimulated with 80M2 (1 mg/ ml) for 30 minutes. Then the cells were incubated with different concentrations of TNF variants for 24 hours at 37uC. The cells were washed with PBS and incubated with crystal violet (20% methanol; 0.5% crystal violet) for 20 minutes to stain viable cells. The dye was washed away under rinsing water and the cells were air-dried. Crystal violet was resolved with methanol and the optical density at 550 nm was determined. Each sample was analyzed in triplicates and data were analyzed using the software Microsoft Excel and GraphPad Prism 4. MTT. Fully differentiated LUHMES cells cultivated in 96- well plates were stimulated with H2O2 with or without LY294002 (25 mM). After one hour cells were washed with medium and regenerated in medium with or without TNC-scTNFR2 (100 ng/ ml). After 24 hours, the medium was removed carefully and 60 ml MTT solution (1,25 mg/ml) diluted 1:6 in DMEM (1% FCS) was added. Cells were incubated for 90 minutes at 37uC. Then 150 ml acidic isopropanol was added and cells were dissolved. Absorbance was measured at 550 nm. Differentiated LUHMES cells were stimulated with 6-OHDA. After 1, 2 or 4 hours TNC-scTNFR2 (100 ng/ml) was added and cells were cultured for a total time of 20 hours. Cell viability was determined as described above. Each sample was analyzed in triplicates and cell viability was calculated using the software Microsoft Excel and GraphPad Prism 4. TUNEL. Fully differentiated LUHMES cells cultivated on cover slips were stimulated with H2O2. After one hour, the cells were washed with medium and regenerated for the indicated times in medium with or without TNC-scTNFR2 (100 ng/ml). At point in time zero, the cells were washed with PBS, fixed with PBS/ 4%PFA for 20 minutes at 37uC and permeabilized with PBS/ 0.1% TritonX-100 for five minutes at room temperature. Labeling with the TUNEL reaction mix was performed due to the recommendations of the manufacturer (Roche Applied Science, Mannheim, Germany). Coverslips were mounted with Fluoromount G (Southern Biotech, Birmingham, AL). The fluorescence was analyzed by wide-field fluorescence microscopy (CellObserver, Carl Zeiss). Fluorescence microscopy For immunofluorescence analyses LUHMES cells were culti- vated on PLO-fibronectin-coated cover slips and differentiated for 72 hours as described in [19]. R2 MEF and Kym-1 cells (36104 cells/well) were cultivated overnight on Lab-Tek 8-well chamber glass slides (Thermo, Schwerte, Germany) coated with 10 mg/ml poly-D-lysine (Sigma) for one hour at 37uC. If indicated in the legend, R2 MEFs were transiently transfected with pTRAF2- PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27621