Fig. 6

APE1 regulates YAP1 stability through β-TrCP ubiquitinase. A-B FLO-1 (A) and OE33 (B) cells with or without APE1 transient knockdown were treated with cycloheximide (CHX, 100 µg/mL) for the indicated timepoints. The levels of APE1 and YAP1 were determined using western blot. β-actin was used as a loading control. C FLO-1 and OE33 cells with or without APE1 transient knockdown were treated or not with MG132 (10 µM) for 16 h. The levels of APE1 and YAP1 were evaluated using western blot. β-actin was used as a loading control. D Western blot was used to analyze APE1 and β-TrCP expression following transient APE1 knockdown (si-APE1) in FLO-1 and OE33 cells. β-actin was used as a loading control. E FLO-1 and OE33 cells were transfected with wild type APE1 (flag APE1), immunoprecipitated with flag antibody and immunoblotted with β-TrCP and APE1 antibodies. F-G Representative images of proximity ligation assay for APE1 and β-TrCP in FLO-1 and OE33 cells (F) and YAP1 and β-TrCP in OE33 cells with and without APE1 knockdown (G). DAPI (blue) was used for nuclear staining. H In vitro ubiquitination assay in FLO-1 and OE33 cells transfected with HA-ubiquitin plasmid then treated or not with APE1 redox inhibitor E3330 (100 µM, overnight) followed by MG132 treatment (10 µM for 6 h). YAP1 antibody was used for immunoprecipitation and samples were then blotted with anti-ubiquitin and anti-YAP1