PDE3A active site mutants impair DNMDP sensitivity.
A, A2058 cells. Top, 72-h CellTiter-Glo assay. Bottom, anti-PDE3A immunoblot analysis confirming construct expression. B, HeLa cells. Top, 72-h CellTiter-Glo assay. Bottom, anti-PDE3A immunoblot analysis confirming construct expression. C (left), active-site mutations (purple), but not 44-amino acid catalytic domain insert mutations (blue), abolish binding of PDE3A in 200 μg of cell lysate from transduced A2058 cells to resin-linked DNMDP. Right, the isolated V5-tagged PDE3A catalytic domain fragment (amino acids 668–1141) in 200 μg of cell lysate from transduced A2058 cells binds resin-linked DNMDP. Treq or T, trequinsin; i, input; p, pellet.