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. 2010 Oct 27;123(22):3863–3871. doi: 10.1242/jcs.073627

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Fig. 5. — HIPK2 phosphorylates ATF1 at Ser198. (A) Wild-type Flag-HIPK2 and various ATF1 mutants (Ser63, Thr99, Ser164, Ser198 to Ala) were co-transfected into HEK293 cells and nuclear fractions were subjected to western blotting with anti-ATF1 antibody, anti-HIPK2 antibody or anti-lamin B antibody. ATF1 and retarded ATF1 are indicated with arrowheads. (B) Ten nanograms of His-tagged recombinant ATF1 wt, Ser198Ala (198A) or Ser63Ala (63A) was incubated with 4 ng recombinant HIPK2 (kinase domain aa 165–564) or PKA in the presence of 5 μCi γ-³²P-ATP at 30°C for 20 minutes. Samples were loaded on 10% SDS-PAGE and phosphorylated bands were detected by autoradiograph. Comparable loading of the proteins was verified by gel Silver staining (bottom).