In vitro kinase assays

MST1 Kinase Enzyme System, including Axltide peptide (KKSRGDYMTMQIG) as MST1 substrate, with ADP-Glo reagent (V4153, Promega) was used according to manufacturer’s instructions to measure ADP production in kinase reactions. The reaction buffer (40 mm Tris, pH 7.5—20 mm magnesium chloride—0.1 mg/ml BSA—50 μm dithiotreitol) was supplemented with 2.5 µm manganese chloride to enhance tyrosine kinase activity. Recombinant MST1 kinase (3 ng) was incubated with 0–10 ng of recombinant kinase domain of FGFR4 (P3054, Thermo Fisher Scientific), together with 1 µm XMU-MP-1 (MST1/2 inhibitor) as an assay control. The kinase reactions with final 50 μm ATP concentration were incubated at room temperature for 1 h on ProxiPlate (PerkinElmer, Waltham, MA, USA), ADP-Glo reagent was added for 40 min, followed by addition of kinase detection reagent for 30 min’ incubation before reading the luminescence with EnSight plate reader (PerkinElmer). Experiment was repeated three times. For in vitro kinase assay detection by immunoblotting, 40 ng of MST1 kinase and 0–100 ng recombinant kinase domain of FGFR4 were incubated together for 30 min at 30 °C, with or without 1 µm XMU-MP-1 or 100 nm BLU9931 (FGFR4 inhibitor) using the abovementioned buffers and MST1 substrate. Proteins in the kinase reaction were denatured and reduced using SDS-PAGE sample buffer (0.12 m Tris-HCl pH 6.8, 0.02% bromophenol blue, 4% SDS, 50% glycerol) supplemented with 10% (v/v) β-mercaptoethanol, and subjected to immunoblotting for detecting phosphorylation of MST1 and FGFR4. Experiment was repeated two times.