Peptides and antibodies
A biotinylated substrate peptide mimicking the sequence around Thr212 of the tau protein (Biotin-Ttds-GSRSRT PSLPTPP-NH2, Thr212 is underlined, where Ttds is a proprietary linker) was selected from the Single Kinase Substrate Peptides commercially available from JPT Peptide Technologies (Berlin, Germany). For use in standard curves, a phosphorylated version was custom synthesized by Eurogentec (Seraing, Belgium). The optimized substrate peptide DYRKtide and the rabbit antibody directed against the C-terminus of human DYRK1B have been described previously [26, 34]. The following antibodies were obtained from commercial sources: rabbit anti TAU(pT212) antibody (Cat. No. 44-740G; Invitrogen, Carlsbad, CA), mouse monoclonal antibodies for phosphotyrosine (PY99, Santa Cruz Biotechnology, Santa Cruz, CA), FLAG epitope (FLAG-M2, Sigma, Munich, Germany) and DYRK1A (clone 7D10; Abnova, Taipei City, Taiwan), and goat GFP-specific antibody (Rockland, Gilbertsville, PA).
Preparation of mouse heart and Xenopus oocyte lysates
Hearts from TW-104 mice were weighed and 2 ml of lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.5% NP-40, 15% glycerol, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 10 μg/ml aprotinin, 10 μg/mL pepstatin, 10 μg/mL leupeptin, 1 μL PMSF) were added per 400 mg of tissue. The hearts were cut into small pieces, subjected to 10 strokes of medium rpm (stage 6 out of 10) in a Potter Elvehjem homogenizer, and the homogenates were then sonified for 20 s. After centrifugation (21,000 g, 4°C, 10 minutes), the supernatant was taken and stored at -80°C. The lysates were cleared by pre-incubation for 30 minutes with 30 μL protein G agarose (EZview™ Red Protein G Affinity Gel, Sigma, Munich, Germany) and subsequent centrifugation before the supernatant was taken for immunoprecipitations. Follicle cell-free oocytes of oogenesis stages V or VI from Xenopus laevis [35] were kindly provided by the members of the Schmalzing group in the Institute of Pharmacology. The oocytes were lysed in 4 volumes of lysis buffer by up-and-down pipetting, and the lysates were cleared by repeated centrifugation to remove lipids and insoluble debris.
Immunoprecipitation
For the immunoprecipitation from heart lysates, 1 μg monoclonal anti DYRK1A or 1 μg anti-FLAG antibody were added to a volume of lysate containing 1 mg of total protein. For the oocyte experiments, the amount of lysate derived from a volume of 100 μl settled oocytes was used for immunoprecipitation with either 1 μg anti DYRK1A antibody, 1 μl anti DYRK1B serum [26] or 1 μl non-reacting serum (preimmune serum of the same rabbit used to raise the DYRK1B-specific serum).
Immuncomplexes were captured by over-night incubation with protein G agarose at 4°C in an end-over-end rotator. After washing the agarose beads twice with TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA) supplemented with 0.1% Igepal CA-630, twice with TBS without Igepal and once with kinase buffer, bound immunocomplexes were assayed for in vitro-kinase activity (see below). In some experiments, the affinity matrix was divided equally between two test tubes during the last washing step to allow for the direct comparison of radioactive and non-radioactive assay.
Cell culture and transfection
HeLa cells were cultured in Quantum 101 Medium for HeLa cells (PAA Laboratories, Pasching, Austria) and COS7 cells in DMEM medium supplemented with 10% fetal calf serum (PAA) at 37°C in a humidified 5% CO2 atmosphere. Transient transfections were performed using the FuGENE HD transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany). Expression clones for GFP-DYRK1A and GFP-DYRK1B (p69 splicing variant) have been described previously [26, 36]. An expression clone for untagged full length xDYRK1B (IMAGE clone 4724858) was obtained from Imagenes (Berlin, Germany).
Western blotting
After washing the immunoprecipitates with TBS, bound proteins were eluted with Laemmli sample buffer, separated by SDS-PAGE (7%) and blotted onto nitrocellulose membranes. Immunodetection with commercial antibodies was performed according to the supplier's instructions. The DYRK1B antiserum was used at a dilution of 1:5000 for 2 h at room temperature, and the TBS for the washes following incubation with the primary antibody was supplemented with 0.1% SDS to reduce non-specific binding [26]. Western blots were developed using horseradish peroxidase-coupled secondary antibodies and chemiluminescence detection. The Clean-Blot IP Detection Reagent (Thermo Scientific, Rockford, IL) was used as a secondary antibody when proteins were immunodetected with the same antibody used for immunoprecipitation.
Kinase assays
Recombinant glutathione S-transferase (GST) fusion proteins of rat DYRK1A-ΔC and human DYRK2 were prepared as described previously [34, 36]. One unit of kinase activity was defined as the amount of enzyme that catalyzed the phosphorylation of 1 nmol DYRKtide per min at 30°C in kinase buffer (25 mM Hepes, pH 7.0, 5 mM MgCl2, 0.5 mM dithiothreitol).
To determine the assay sensitivity (Figure 2), reactions were performed in a total volume of 10 μl with variable amounts of the recombinant kinase, 50 μM tau207-219 and 100 μM ATP at 30°C for 30 min. Reactions were stopped by addition of 10 μl 10 mM EDTA and stored at -20°C until analysis by ELISA.
To prepare phosphorylated tau207-219 for the use in the ELISA (Figure 1), we aimed to achieve maximal phosphorylation by incubating 2.5 nmol tau207-219 with an excess of active kinase (4 mU/μL GST-DYRK1A-ΔC) in the presence of 0.82 mM ATP. The reaction mixture was incubated for 2 hours, and the same amount of kinase was supplemented after one hour. Total incorporation of phosphate was determined in a parallel reaction that was incubated in the presence of [γ-33P]ATP under identical conditions. All other kinase assays were incubated for 30 min at 30°C.
Immunocomplex kinase assays were carried out in a total reaction volume of 50 μl in kinase buffer, 100 μM ATP and 50 μM tau207-219 at 30°C for 30 min and stopped by addition of 50 μL 10 mM EDTA. Samples were stored at -20°C until evaluation by ELISA. Radiometric assays were run in the presence of [γ-33P]ATP (~ 2000 cpm/pmol). Incorporation of 33P into the biotinylated tau peptide was determined by spotting aliquots of the reaction mix onto a streptavidin membrane (SAM2 Biotin Capture Membrane, Promega, Mannheim, Germany), which was then washed as described by the manufacturer and evaluated by scintillation counting. Radiometric immunocomplex kinase assays with the substrate peptide DYRKtide (at 100 μM) were run with 10 μM ATP as described before [37].
Direct Sandwich ELISA
MaxiSorp F96 MicroWell™ Plates (Nunc, Langenselbold, Germany) were coated with 100 ng/well TAU(pT212) antibody at 4°C overnight. To keep the antibody consumption low, we used 100 ng/well of capturing antibody for coating in the standard protocol, although 200 ng/well gave a slightly higher readout (1.2-fold). After blocking for 1 hour with 200 μl/well 2% (w/v) bovine serum albumin (fraction V, modified Cohn, Calbiochem) in phosphate buffered saline (PBS) (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4), the antigen was added in a volume of 100 μL/well and the plates were incubated for 2 h at room temperature. Bound antigen was detected with streptavidin-HRP conjugate (Pierce, Rockford, IL; 100 μL/well, 2 h at room temperature; 25 ng/ml) and subsequent incubation with 100 μL/well TMB (3,3',5,5'-tetramethylbenzidine) substrate solution until colour development was observed. Other detection reagents (HRP-linked anti-biotin antibody from CST; strepavidin-HRP from R&D systems) were less sensitive, and higher concentration of the streptavidin-HRP conjugate resulted in higher background in empty wells. The reaction was then stopped by adding 50 μl/well 1 M HCl, and plates were read at 450 nm (reference wavelength 620 nm). After each incubation step, plates were washed 4 times with PBS, pH 7.4, 0.1% Tween-20. Antibody and antigen were diluted in PBS.
ELISA format based on streptavidin-coated plates
Reacti-Bind™ Streptavidin High Binding Capacity Coated 96-Well Plates (Pierce; Rockford, IL, USA) were loaded with the indicated concentrations of the biotinylated peptides (100 μL/well) and incubated at RT for 2 h. After washing, bound phosphopeptide was detected with rabbit anti TAU(pT212) antibody (1:16.000) and HRP-linked goat anti rabbit IgG (Fc) (Pierce) (1:40.000) and TMB substrate as described above. PBS with 2% BSA and 0.1% Tween-20 was used for plate washing and for dilution of antigen and antibodies.
Data evaluation and curve fitting
Standard curves of phosphorylated tau peptide and the graphical representation of the time course shown in Figure 4 were generated by non-linear curve fitting using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).