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. 2012 Mar 19;196(6):681–688. doi: 10.1083/jcb.201107050

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© 2012 Bergink et al.

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Figure 2. — RAD23A and RAD23B do not accumulate/immobilize at DNA damage and are not immobilized upon UV treatment. (A and B) Analysis of RAD23B-YFP and RAD23B at local damaged DNA spots. Cells were fixed between 30 and 60 min after local UV irradiation. (A) RAD23B-YFP (identified by the YFP fluorescence) does not accumulate at local damaged sites, whereas its complex partner XPC (identified by XPC antibodies) does. The nuclei are counterstained with DAPI (blue). (B) Comparative immunofluorescence on a mixture of WT and DKO MEFs, recognized by bead-labeled cells with small and large beads, respectively (DAPI-derived fluorescence mixed with transmitting light; left). Endogenous RAD23B (identified by RAD23B antibodies) fails to accumulate at local DNA damage (see arrows), as is indicated by the accumulation of the TFIIH subunit p62 (αp62). The specificity of the RAD23B antibody is illustrated by the virtual absence of staining in the DKO cells (small beads; left), whereas a clear signal is visible in WT cells (large beads; left). (C) FRAP analysis of RAD23B-YFP before and after UV-induced DNA damage. RAD23B-YFP does not immobilize after UV treatment. Mobilities were measured between 30 and 60 min after UV-C (16 J/m²) treatment.