Figure 5.

GAPDH activated Ape1. WT and R3 SMCs were grown until confluence and treated with H₂O₂ (110 μM, 16 h). Cytosolic and nuclear fractions of WT and R3 cells were separated with NE-PER nuclear and cytoplasmic separation kit. A) Samples were immunoblotted with Ab for GAPDH, α-tubulin (cytosolic marker), and p53 (nuclear marker). B) Quantitative data show relative GAPDH amount in cytosolic and nuclear fraction (n = 3). *P < 0.05, ^¶P < 0.01, ^§P < 0.005. C, D) Ape1 activity in nuclear fraction of WT and R3 cells. C) The 5′-endonuclease activity of Ape1 was quantified by measuring the incision of a 26-mer FAM-labeled oligonucleotide substrate that contained a single synthetic AP site. Ape1 endonuclease reaction mix contained 300 ng cell lysate and 1 μM FAM-labeled Ape1 substrate. Products were separated on 15% PAGE/Urea gels. D) Endonuclease activity was calculated as the relative amount of the 13-mer oligo product with the unreacted 26-mer substrate [product/(product + substrate)]. Data from a representative experiment are shown (n = 5). NS, not significant. *P < 0.05, ^§P < 0.005.