Radioactive in vitro kinase assay

In vitro kinase assays were essentially carried out as before (Gat et al., 2019). 1 μM of full-length UPF1 was mixed with 50 nM SMG1-8-9, 10mM MgCl₂ and 2mM DTT in 1xPBS. The reaction was started by adding 0.5mM ATP and 0.06 μM of γ-³²P-labeled ATP. The reaction was incubated at 30°C and samples were taken at different time points to follow phosphorylation over time. The samples were immediately quenched by adding SDS-containing sample buffer and initially analyzed by SDS gel electrophoresis followed by Coomassie-staining. Phosphoproteins were subsequently detected using autoradiography and a Typhoon FLA7000 imager (GE Healthcare).