Recombinant human EIF2AK2 (PKR) protein (Thermo Fisher Scientific), human PLK-2 (Thermo Fisher Scientific), MKK4 (ProQinase), or JNK2 (ProQinase) were resuspended in kinase activity buffer (100 mM Hepes, pH 7.5, 20 mM MgCl2, and 2 mM EGTA).
For subsequent autoradiography analysis, recombinant kinases were incubated for 1 h at 37°C with ATP (8:1 mixture of nonlabeled and (γ-32)-labeled ATP, 10 μM final concentration) in a 1:50 ratio with recombinant human α-syn wt or α-syn S87A/S129A. Each sample was subjected to SDS-PAGE, stained at room temperature (RT) using Coomassie Brilliant Blue, destained overnight (ON) at RT and left for 1 h at RT in a 35% EtOH, 3.5% glycerol solution prior to drying. The dried gel was photographed on Fuji LAS-3000 Intelligent Dark Box (Fujifilm, Japan) and developed using standard autoradiography.
For subsequent MS analysis, recombinant human α-syn wt or α-syn S87A/S129A was incubated with Recombinant human EIF2AK2 (PKR) protein (Thermo Fisher Scientific) or MKK4 protein (ProQinase) in kinase activity buffer for 18 h at 37°C in a 50:1 ratio in the presence of pure, nonlabeled ATP.
For subsequent autoradiography analysis, recombinant kinases were incubated for 1 h at 37°C with ATP (8:1 mixture of nonlabeled and (γ-32)-labeled ATP, 10 μM final concentration) in a 1:50 ratio with recombinant human α-syn wt or α-syn S87A/S129A. Each sample was subjected to SDS-PAGE, stained at room temperature (RT) using Coomassie Brilliant Blue, destained overnight (ON) at RT and left for 1 h at RT in a 35% EtOH, 3.5% glycerol solution prior to drying. The dried gel was photographed on Fuji LAS-3000 Intelligent Dark Box (Fujifilm, Japan) and developed using standard autoradiography.
For subsequent MS analysis, recombinant human α-syn wt or α-syn S87A/S129A was incubated with Recombinant human EIF2AK2 (PKR) protein (Thermo Fisher Scientific) or MKK4 protein (ProQinase) in kinase activity buffer for 18 h at 37°C in a 50:1 ratio in the presence of pure, nonlabeled ATP.