6614 Nucleic Acids Research, 2012, Vol. 40, No. 14 Figure 4. PNKP levels and activity in MCSZ lymphoblastoid cell extracts. (A) Western blot analysis of MCSZ LCL. Samples of the indicated LCLs (2 105 cells) were fractionated by SDS–PAGE, transferred to nitrocellulose and immunoblotted for PNKP. Quantiﬁcation is shown on the right. The signal intensity of PNKP was quantiﬁed using Aida Image Analyser v.427 and normalized using the signal intensity of actin. Data are the mean ± SEM of six experiments. Note that, while not included here, PNKP protein levels in MC22601 were similar to its sibling LCL, MC22602. (B) Reduced DNA 30-phosphatase activity in MCSZ LCL extract. 25 nM single-stranded substrate harbouring a 30-phosphate terminus (top) was incubated with the indicated LCL extract (2.5 mg total protein) for the indicated time periods at 30 C. Reaction products were fractionated by denaturing PAGE and detected by phosphorimaging. (C) Reduced DNA 30-phosphatase activity in MCSZ LCL extract. 25 nM duplex substrate harbouring a 1-bp gap and 30-phosphate terminus (top) was incubated with the indicated LCL extract (1, 2.5, or 7.5 mg total protein) for 60 min at 30 C. Reaction products were analysed as above. Note that DNA 30-phosphatase activity is indicated by conversion of 17-P to 17-OH and DNA gap ﬁlling by conversion of 17-OH to 18-OH. (D) Reduced DNA 50-kinase activity in MCSZ LCL extract. 25 nM duplex substrate harbouring a nick and 50-hydroxyl terminus (top) was incubated with the indicated LCL extract (1, 2.5 or 7.5 mg total protein) for 60 min at 30 C. Reaction products were analysed as above. Note that DNA 50-kinase activity is indicated indirectly in this experiment by appearance of 43-bp DNA ligation product.