Fig. 3. The CIM of CSB mediates the recruitment of CSA to DNA damage-stalled RNAPIIo.

a A schematic representation of CSB and the CSB^ΔCIM mutant. b Western blot analysis of U2OS (FRT) and CSB-KO complemented with either GFP-CSB^WT or GFP-CSB^ΔCIM (n = 2). c Co-IP of GFP-CSB^WT and GFP-CSB^ΔCIM on the combined soluble and chromatin fraction. d Endogenous RNAPII Co-IP in GFP-CSB^WT and GFP-CSB^ΔCIM cell lines. See also Supplementary Fig. 6e for additional Co-IP data. e Clonogenic Illudin S survival of WT and CSB-KO cell lines and the GFP-tagged CSB rescue cell lines. Each symbol represents the mean of an independent experiment (n = 2), each of which is based on  two technical replicates. Note that the same survival data for WT, CSB-KO and CSB-KO + GFP-CSB is also shown in Fig. 1e. f In vitro ubiquitylation of recombinant Xenopus laevis (xl) and Homo sapiens (hs) CSB variants with recombinant xlCRL4^CSA, E1, E2, ubiquitin, and ATP. At indicated times, in vitro ubiquitylation reactions were stopped and blotted with anti-FLAG (top three panels) or anti-xlCSA (bottom panel) antibodies. See also Supplementary Fig. 6d. g Immobilized recombinant CSB variants were incubated with Xenopus laevis nucleoplasmic extract (NPE), recovered, and blotted with anti-FLAG (top panel) or anti-xlCSA (bottom panel) antibody. At least two independent replicates of each IP and in vitro ubiquitylation experiment were performed obtaining similar results. Source data are provided as a Source Data file.