Fig. 3

TRAF4 promotes K63-linked ubiquitination of CHK1 is required for CHK1 activation. a In vivo CHK1 ubiquitination by TRAF4 in 293T cells transfected with various constructs. b CHK1 ubiquitination by TRAF4 is K63-linked. An in vivo ubiquitination assay was performed in 293T cells transfected with various constructs and treated with 5-Fu. c In vivo CHK1 ubiquitination by TRAF4 mutants. TRAF4 WT, TRAF4 (DM-RING) mutant, or TRAF4 (C18A) mutant were co-transfected into 293T cells with various constructs, and WCEs were subjected to an in vivo ubiquitination assay. d In vitro CHK1 ubiquitination by TRAF4. e Endogenous CHK1 ubiquitination by TRAF4 in HT29 cells. TRAF4-WT or TRAF4-knockout HT29 cells were treated with 5-Fu for 24 h, and WCEs were immunoprecipitated and subjected to ubiquitination analysis. f CHK1 ubiquitination in TRAF4-null cells is rescued by exogenous WT TRAF4. TRAF4-knockout HT29 cells were transfected with various constructs and treated with 5-Fu, and WCEs were immunoprecipitated for IB. g CHK1 phosphorylation in TRAF4-null cells is rescued by exogenous WT TRAF4. TRAF4-knockout HT29 cells were transfected with various constructs and treated with different doses of 5-Fu, and WCEs were immunoblotted. *Non-specific signal