Fig. 2

CHK1 activation is regulated by TRAF4. a TRAF4 knockout reduces CHK1 phosphorylation in 5-Fu-treated CRC cells. HT29 and SW620 cells expressing sgCtrl or sgTRAF4 were treated with 5-Fu for 24 h. WCEs were subjected to IB analysis. b TRAF4 knockout inhibits CHK1 phosphorylation in UV-treated CRC cells. HT29 and SW620 cells expressing sgCtrl or sgTRAF4 were treated with UV. WCEs were collected at various time points and subjected to IB analysis. c TRAF4 overexpression enhances CHK1 phosphorylation in 5-Fu-treated FHC cells. IB analysis of immortalized colon epithelial cell FHC transfected with vector control or Flag-TRAF4 constructs and treated with 5-Fu. d TRAF4 knockout reduces CHK1 kinase activity ex vivo. TRAF4-WT or TRAF4-knockout HT29 cells were treated with 5-Fu for 24 h, and the WCEs were prepared and immunoprecipitated with the CHK1 antibody. The immunoprecipitated CHK1 was incubated with recombinant CDC25C and subjected to an in vitro kinase assay. e The IF analysis of CHK1 phosphorylation in 5-Fu-treated HT29 cells. ***p < 0.001. Scale bar, 10 μm. f, g TRAF4 knockout compromises DNA repair in 5-Fu-treated HT29 cells. TRAF4-WT or TRAF4-knockout HT29 cells were treated with 5-Fu for 24 h, then maintained in 5-Fu-free medium and subjected to IF (f) or IB (g) analysis at various time points with γ-H2AX antibody. ***p < 0.001. Scale bar, 5 μm