FIGURE 2.

TOPK phosphorylates Prx1 in vitro and UVB induces phosphorylation of TOPK in RPMI7951 melanoma cells. A, His-Prx1 and His-Prx3 fusion proteins were purified from BL21 bacteria using Ni-NTA-agarose beads and purity was confirmed by Coomassie Blue R-250 staining. B, in vitro kinase assay to determine whether TOPK phosphorylates Prx1 or Prx3. Results are visualized by autoradiography with [γ-³²P]ATP. C, different human melanoma cell lines were screened by Western blot to determine total protein abundance of endogenous TOPK, Prx1, or Prx2. β-Actin verified equal protein loading. D, time-dependent UVB-induced phosphorylation of TOPK (Thr-9). RPMI7951 cells were harvested at various times after UVB (4 kJ/m²) treatment. Phosphorylation of TOPK (Thr-9) or nonphosphorylated TOPK was detected. Data for C and D are representative of three independent experiments.