A-B. Overexpression and knockdown of PLD3 produce opposing effects on extracellular Aβ levels. N2A cells stably expressing hAPP695-WT were transiently transfected with vectors containing no insert (pcDNA3), human PLD3-WT, scrambled shRNA (Origene), or mouse PLD3 shRNA (Origene) for 48 hours. Cell media were analyzed with Aβ40 and Aβ42 ELISAs and corrected for total intracellular protein. Aβ levels were then expressed relative to pcDNA3. Graphs represent the mean±SEM. A) Overexpression of human PLD3 produces significantly less extracellular Aβ42 and Aβ40. “*”, p<0.0001. B). Knockdown of endogenous PLD3 cells produces significantly more extracellular Aβ42 and Aβ40. “*”, p<0.002. C) Members of the PLD protein family have different effects on APP processing. HEK293T cells were transiently transfected with vectors containing hAPP-WT and an empty vector (pcDNA3), PLD1, PLD2, or PLD3-WT or PLD1, PLD2, PLD3 carrying a dominant negative mutation.. Left panel, PLD3 affects full-length APP levels. Cell lysates were extracted in non-ionic detergent, analyzed by SDS-PAGE and immunoblotting with antibodies to the myc-tag on APP (9E10) or β-tubulin. Middle (Aβ42) and right (Aβ40) panel, cell media were analyzed with Aβ40 and Aβ42 ELISAs and corrected for total intracellular protein. Graphs represent the mean±SEM. “*”, p<0.01, different from pcDNA3; “ο”, p=0.002, different from PLD1-WT; “•”, p<0.0001, different from PLD2-WT. Images are representative of at least three replicate experiments.