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. 2019 Apr 12;11(4):523. doi: 10.3390/cancers11040523

Figure 2.

Figure 2

USF2 is phosphorylated at Ser155 and Ser222 by CDK5. (A) In silico predicted CDK5 phosphorylation sites in human USF2 (B) Purified GST-tagged USF2 variants with point mutations at the predicted CDK5 phosphorylation sites were phosphorylated with recombinant human CDK5 in the presence of [32P]-γ-ATP for 20 min at 30 °C. Proteins were separated by 10% SDS-PAGE and radioactivity was detected by autoradiography. The lower panel shows the recombinant USF2 proteins on a Coomassie-stained gel performed as a loading control. (C) PC3 cells were cotransfected with pFR-5Gal4-RE-Luc, an expression vector for CDK5 and constructs expressing WT or mutant pcDNA6-Gal4-USF2 (1–231) or the appropriate empty Gal4 vector. The luciferase activity was calculated as fold induction compared to the Gal4-USF2 (1–231)-WT luciferase activity after subtracting the values from the empty Gal4 expression vector. *, significant differences control vs. CDK5.