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

Figure EV2. Chk1‐dependent phosphorylation does not cause stabilization or subcellular relocalization of atypical E2Fs.

Figure EV2

  1. Protein expression of endogenous and exogenous atypical E2Fs in the HeLa/TO cell lines. EGFP‐tagged wild‐type, alanine and aspartic mouse constructs were incorporated in the HeLa/TO‐inducible system by single colony selection. Cells were then treated either without (Veh) or with (Dox) doxycycline for 24 h. Exogenous E2F7 was measured by anti‐GFP antibody, and endogenous E2F7 was measured by anti‐human E2F7 antibody. For E2F8, both exogenous and endogenous forms were detected by a single E2F8 antibody. Asterisks indicate specific detections from the antibodies.
  2. Protein expression of endogenous E2F7, E2F8, phos‐Chk1S345, and total Chk1 in U2OS cells treated with cycloheximide (CHX), in the presence or absence of etoposide.
  3. E2F7 and E2F8 proteins are localized in the nucleus. Inducible cell lines were treated with doxycycline and etoposide for 16 h, followed by isolation of cytosolic and nuclear protein fractions. HDAC1 expression was used as an indicator for enrichment of nuclear proteins.
  4. Immunofluorescence showing the localization of E2F7, E2F8, and MDC1 in response to camptothecin treatment for 8 h in U2OS cells. E2F7, E2F8, and MDC1 were labeled with GFP, and DNA damage sites are detected with an antibody against γ‐H2AX. DNA was stained with DAPI. Scale bar: 5 μm.

Source data are available online for this figure.