Fig. 2. Oxidative stress induces APE1 methylation and mitochondrial translocation.

In all co-IP assays, the loading percentage (to whole cell lysate) is 1% for input and 12.5% for IP samples. HeLa cells were treated with 100 μM H₂O₂ (a) or 10 μM menadione (b) for 2 h. An anti-arginine specific antibody was used to detect methylated APE1 (MeAPE1). (a) Immunoprecipitation of HeLa lysates with (right panel) or without (left panel) H₂O₂ treatment, using an anti-APE1 antibody. (b) Relative MeAPE1 levels compared to input showed significant elevation after H₂O₂ treatment. (p = 0.0126). (c) Immunoprecipitation of HeLa lysates with (right) or without (left) menadione treatment, using an anti-APE1 antibody. (d) Relative MeAPE1 levels compared to input showed significant increase after menadione treatment. (p = 0.024). (e) HeLa cells were treated with 100 μM H₂O₂ or 60 mM NAC (N-Acetyl-L-cysteine) for 2 h. Immunoprecipitation of HeLa lysates of untreated cells, H₂O₂ treated cells and NAC treated cells, using an anti-APE1 antibody. Anti-arginine specific antibody (ASYM24) was used to detect methylated APE1 (meAPE1) and anti-Tom 20 antibody was used to detect co-IPed Tom20. (f) bar chart for (e). Relative MeAPE1 levels showed that H₂O₂ treatment significantly increased compared to untreated group (p < 0.001). NAC treated group showed significant decrease comparing to untreated group (p < 0.001). (g) Subcellular fractionation of HeLa cells and immunoprecipitation of N (nucleus), C (cytosol), and M (mitochondria) fractions. The cytoplasmic protein extracts used for IP contained mitochondria from the nuclei-cytoplasm fractionation. Mitochondria was isolated in an independent experiment to achieve equivalent APE1 reading in western blot. GAPDH, cytosol control; mtHSP70 (mitochondrial HSP70), mitochondria control; Lamin A/C, nucleus control. (h) Bar chart for mitochondrial relative meAPE1 level in (g). p < 0.001.