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. 2018 Jan 23;37(5):e97877. doi: 10.15252/embj.201797877

Figure 1. Chk1 phosphorylates atypical E2Fs.

Figure 1

  1. Putative Chk1 substrate motifs in human or mouse E2F7 and E2F8 proteins, and known human Chk1 substrate motifs.
  2. Schematic overview of the putative Chk1 phosphorylation motifs on mouse E2F7 and E2F8.
  3. Kinase assay showing that Chk1 phosphorylates mouse E2F7 and E2F8 in vitro. HEK cells were transfected with EGFP‐tagged wild‐type and mutant versions of E2F7 and E2F8, lysed, and precipitated with GFP‐Trap beads. Precipitated proteins were incubated with radiolabeled 32P‐ATP, in the presence or absence of recombinant active Chk1 and loaded onto SDS–PAGE gel.
  4. Chk1 phosphorylates atypical E2Fs in vivo. 24 h before treatment, siRNA against Chk1 was transfected to HeLa/TO cells. Then, cells were treated either without doxycycline (Veh) or with doxycycline (Dox) for 16 h to induce the over‐expression of wild‐type E2F7 and E2F8. UCN‐01 was added to the cells simultaneously to inhibit Chk1 kinase. Whole‐cell lysates were harvested for immunoblot.

Source data are available online for this figure.