Fig. 4. CRL4 is activated by NEDD8 in response to TOP1-DPCs and target TOP1-DPCs for ubiquitylation.

a HCT116 cells were transfected with the indicated Myc-tag cullin overexpression plasmids plus RBX1-FLAG overexpression plasmid for 48 h before CPT treatment (20 μM, 30 min). The cells were subjected to the DUST assay for immunodetection of ubiquitylated TOP1-DPC and total TOP1-DPC using anti-ubiquitin (Ub) and anti-TOP1 antibodies. Total DNA was detected using anti-DNA antibody as loading control. The order of DNA slot blots has been altered, as indicated by the line, and that uncropped labeled blots can be found in the Source Data file. b In vitro ubiquitylation assay with recombinant TOP1-DPC (generated using a suicidal DNA substrate¹⁴) and CUL4A-RBX1 complex. TOP1 was tested for ubiquitin conjugation in the presence of Ub E1, Ub E2 UbcH5a and the indicated concentrations of CUL4A-RBX1 complex. Reaction products were separated by SDS-PAGE and monitored by IB using anti-ubiquitin antibody. c HCT116 cells were transfected with CUL4A siRNA or Myc-CUL4A and RBX1-FLAG overexpression plasmids (CRL4) for 48 h before PEV pre-treatment (10 μM, 1 h) then co-treatment with CPT (20 μM, 30 min). The cells were subjected to DUST assay for immunodetection of ubiquitylated TOP1-DPC and total TOP1-DPC using anti-ubiquitin and anti-TOP1 antibodies. The order of DNA slot blots has been altered, as indicated by the line, and that uncropped labeled blots can be found in the Source Data file. d HCT116 cells WT, CUL4B KO cells and CUL4B KO cells replenished with Myc-CUL4B overexpression plasmid were pre-treated with BTZ (1 μM, 4 h) or PEV (10 μM, 4 h) then co-treated with CPT (20 μM, 30 min). The cells were then subjected to DUST assay for immunodetection of ubiquitylated TOP1-DPC and total TOP1-DPC using anti-ubiquitin and anti-TOP1 antibodies. The order of DNA slot blots has been altered, as indicated by the line, and that uncropped labeled blots can be found in the Source Data file. e Myc-CUL4A WT or K705R (KR) overexpressing HCT116 cells were transfected with HA-NEDD8 overexpression plasmid, followed by treatments with indicated inhibitors. Immunoprecipitation (IP) using anti-Myc tag antibody was performed after the treatments. IP samples and cell lysates (input) were subjected to immunoblotting (IB) with indicated antibodies. f Myc-CUL4B WT or K859R (KR) overexpressing HCT116 cells were transfected with HA-NEDD8 overexpression plasmid, followed by treatments with indicated inhibitors. IP using anti-Myc tag antibody was performed after the treatments. IP samples and input were subjected to IB with indicated antibodies. g RBX1-FLAG overexpressing HCT116 cells were transfected with indicated CUL4 overexpression plasmids, followed by CPT treatment (20 μM, 30 min) for DUST assay for immunodetection of ubiquitylated TOP1-DPC and total TOP1-DPC using anti-ubiquitin and anti-TOP1 antibodies. The order of DNA slot blots has been altered, as indicated by the line, and that uncropped labeled blots can be found in the Source Data file.