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. Author manuscript; available in PMC: 2018 Sep 1.
Published in final edited form as: DNA Repair (Amst). 2017 Jul 5;57:91–97. doi: 10.1016/j.dnarep.2017.06.029

Figure 1. Identification of NTHL1 as the enzyme in nuclear extracts responsible for excision of thymine glycol (Tg) from DNA. (A).

Figure 1

Nuclear extracts were prepared from cells expressing shRNA against NTHL1 (N.E. shNTHL1), and from cells expressing an off-target control shRNA (N.E. shGFP). 5.9 ug of each extract was incubated with 5 nM of 35 base pair Tg containing DNA fragment in an EDTA-containing buffer. Reactions were quenched after 0.5, 30 or 60 min. in formamide/20 mM EDTA, and products were fractionated using 12% polyacrylamide gels. Parallel reactions using 80 nM giant mimivirus NEI protein (MV NEI) and 20 nM human NTHL1 demonstrate that the NEI-generated β-δ-elimination product can be readily distinguished from the NTHL1-generated β-elimination product. (B) Western blot showing a ~5-fold decrease in NTHL1 abundance in MCF10A cells expressing shRNA against endogenous NTHL1 (shNTHL1) as compared to MCF10A cells expressing an off-target control shRNA (shGFP).