Bas ip ms 2340

Ten micrograms MACC1-GFP from Nanotrap IP were incubated with 500-ng recombinant MEK1 S218D S222D (Thermo Fisher) and 0.3-µM γP³² ATP (Perkin Elmer, Rodgau, Germany) at 37 °C in 80-mM HEPES pH 7.5, 4-mM MgCl₂, 4-mM MnCl₂, 1.6-mM DTT, and 70-µg/ml PEG_{20 000} for 15, 30, 45, and 60 min. The reaction was competed with 1000 × cold ATP or inhibited with 1.4-µM AZD6244. Heat-inactivated samples were separated with 10% polyacrylamide gels. The gels were dried and developed with imaging plates (BAS-IP MS 2340, Fujifilm, Japan).

For the kinase assay, DCLK1 was isolated from HEK293T cells after lentiviral transduction. To isolate the protein, Nanotrap-based (ChromoTek, Martinsried, Germany) Co-IP for GFP was performed following manufacturers recommendations. Cleared lysates of 10⁷ cells were incubated with Nanotrap-coupled beads for 1 h. After washing, the protein-loaded beads were directly used for the kinase assay. Five-hundred nanograms of recombinant MACC1 (Origene, Rockville, MD, USA) was incubated at 37 °C with 10 µg isolated DCLK1 in kinase buffer containing 0.3 µM γP³² ATP (Perkin Elmer, Rodgau, Germany), 80 mM HEPES pH 7.5, 4 mM MgCl₂, 4 mM MnCl₂, 1.6 mM DTT, and 70 µg/mL PEG_20,000 for 15, 30, 45, and 60 min. Samples were heat-inactivated and separated on 10% polyacrylamide gels. The gels were dried on Whatman paper and bands were visualized with imaging plates (BAS-IP MS 2340, Fujifilm, Japan) using an Amersham Typhoon FLA 7000 (GE Healthcare, Freiburg, Germany).