Kinetic analysis of UV exposure revealed a timedependent reversible accumulation of XPCGFP in the majority of the cells. In addition, immunoprecipitation studies using an antiGFP antiserum showed a significant increase in time after UV irradiation of the amount of precipitated fusion protein. Monitoring individual cells in time after UV irradiation corroborated these findings. Analyses of XPCGFP expression after UV irradiation and treatment with proteasome inhibitor CBZLLL.The precipitates were analyzed by immunoblotting using polyclonal antiXPC antiserum.The amount of expressed XPCGFP was also visualized by immunoblot analysis of the total cell lysate with antiXPC or antiGFP antibodies, and with a monoclonal antibody against the p subunit of TFIIH as a loading control. XPCGFP expression was confirmed by immunoblot analysis of the total cell lysate using antiXPC or antiGFP antibodies, and antip as a loading control detected in the h samples after UV, inducing mainly strand breaks and interstrand crosslinks, respectively, failed to provoke detectable XPC accumulation failed to boost fluorescence.White arrows indicate the scratch mark on glass coverslips for proper orientation.Numbers represent the same living cells before and after UV exposure.White arrows indicate the scratch on glass coverslips for proper comparison.The numbers represent the corresponding living cells on coverslips before and after NAAAF treatment.No induction of XPCGFP fluorescence was observed: instead, preincubation with DRB. Similar to UV irradiation and NAAAF, all cells displayed a striking XPCGFP accumulation in time. Longer exposure to CBZLLL further increased XPCGFP levels, as demonstrated by immunoblot analysis. Therefore, we examined whether XPC is also subject to and subsequent immunoblot analysis with XPC antibodies from cells exposed to UVC light or CBZLLL revealed a clear timedependent accumulation of XPCGFP. Interestingly, in addition to the expected XPCGFP protein band, a number of slowermigrating XPC species were identified ubiquitination products of XPCGFP.Unfortunately, none of the different antiubiquitin antisera used was sensitive enough to detect those species above background, precluding unequivocal assessment of the identity of these XPC modifications.The binding of XPC to DNA damage per se might protect it from proteolysis.Alternatively or in addition, DNA damage may trigger a specific response such as XPC modification that inhibits degradation.To explore the mechanism by which hXPCGFP is stabilized, we used a recently developed method for induction of DNA damage in a restricted part of the nucleus.Upon UV irradiation, only at the position of pores is UV damage induced, as detected with antibodies that specifically recognize CPD and PP lesions.These locations attract all NER proteins tested thus far, including XPC. Cells were fixed at different time points after UV to allow <a href="https://www.ncbi.nlm.nih.gov/pubmed/27337968"></a>
simultaneous immunostaining with antibodies against various proteins and GFP fluorescence microscopy. Nonirradiated nuclei and undamaged regions within partly irradiated nuclei serve as internal controls.Very rapidly and antiHA immunostaining revealed high local accrual of hXPCGFP and the p subunit of TFIIH. If XPC stabilization only occurs when bound to the damage, we expect an increase in fluorescent signal predominantly at the damaged sites.On the other hand, with an overall stabilization of hXPC, it is expected that in time a concomitant increase of fluorescence over the entire nucleus would be observed in comparison to undamaged nuclei.