Chk1 and 14‐3‐3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest Experimental procedures of the SILAC assay to identify binding.

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Chk1 and 14‐3‐3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest Experimental procedures of the SILAC assay to identify binding partners of E2F7 and E2F8. 14‐3‐3 ε interacts with wild‐type but not alanine mutant E2F7 and E2F8 in vitro. HEK 293T cells were transfected as indicated, and lysates were precipitated with GFP‐Trap beads and immunoblotted with antibodies directed against GFP or Myc. Inputs confirm similar expression of Myc‐14‐3‐3 ε in all samples. 14‐3‐3 ζ interacts with endogenous phosphorylated E2F7 and E2F8 in vivo. HEK 293T cells were transfected with Myc‐14‐3‐3 ζ and treated with or without HU. Lysates were precipitated with anti‐Myc antibodies and immunoblotted with antibodies directed against the Chk1 phosphorylation site of E2F7S410 or E2F8S396 (asterisks indicate the signal). Inputs confirm similar expression of Myc‐14‐3‐3 ζ in all samples. 14‐3‐3 ζ does not alter the nuclear localization of E2F7/8. U2OS cells were transfected with Myc‐14‐3‐3 ζ with either empty vector, EGFP wild‐type E2F7 or E2F8 followed by immunofluorescence staining. DAPI was used to stain nuclear DNA. Scale bar: 5 μm. Chromatin immunoprecipitation (ChIP) demonstrated that 14‐3‐3 proteins bind to the E2F7/8 target gene promoters. HEK cells were transfected with either PEI reagent alone (control) or indicated plasmids. 48 h after transfection, cells were harvested for ChIP assay and followed by qPCR. Histogram represents the enrichment ratio (bound/input) in E2Fs target gene promoters. A primer set designed against a distal region in the E2F1 gene served as a negative control. Validation of E2F7 and E2F8 knockdown efficiency of experiments shown in Fig F. E2F7 and E2F8 mRNA levels was determined by qPCR. Data information: In (D, F), data represent mean ± SEM (n = 3); **P < 0.01 (Student's t‐test).Source data are available online for this figure. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 37, Issue: 5, First published: 23 January 2018, DOI: (10.15252/embj.201797877)