FIGURE 3.

Estimation of phosphorylation efficiency using radiolabeling and SDS-PAGE. Phosphorylation was measured using radiolabeling. Kinesin was phosphorylated by JNK3 in the presence of [γ-³²P]ATP. The result of the reaction was run through an SDS-polyacrylamide gel to separate kinesin from JNK3, which autophosphorylates. The resulting intensity of the kinesin band of known concentration was compared with bands of [γ-³²P]ATP standards of known concentration to calculate the percentage of kinesin that had been phosphorylated. A, SDS-PAGE gel of radiolabeled kinesin-1. *, [γ-³²P]ATP standards corresponding to labeling efficiencies varying from 6 to 100% (arrow). JNK3 also autophosphorylates (**). B, ATP standard band intensity as a function of P32 labeling of kinesin (black line) and kinesin band intensity (red line).