PBA Enhances APP Proteolysis Figure 3. Tunicamycin inhibits APP proteolysis and induces ER stress UPR signaling. The NAG cells were treated with titrated doses of the N-glycosylation inhibitor tunicamycin, which inhibits c-secretase cleavage of APPGV16 (top graph). Increasing doses of tunicamycin induced elevated levels UPR activation, demonstrated by elevated levels of phospho-PERK (pPERK blot) and phospho-eIF2a (p-eIF2 a) levels. Tunicamycin elevated phospho-p38 (pp38 blot) and phospho- JNK (pp54/JNK, pp46JNK blot) levels in parallel with increased UPR signaling and decreased APPGV16 proteolysis. doi:10.1371/journal.pone.0009135.g003 ADP ribosylation factor guanosine triphosphate hydrolase (Arf- GTPase), which results in the dissolution of the Golgi apparatus [33]. The NAG cells were treated for 18–20 hours and employed in transactivation assays, to measure gamma-secretase mediated APPGV16 cleavage, and in western blots examining ER stress Figure 4. Brefeldin A inhibits APP proteolysis and induces ER stress UPR signaling. Brefeldin A inhibited c-secretase cleavage of APPGV16 (top graph). Western blots show that 0.5 mg/ml Brefeldin A induces activation of PERK (pPERK blot) and eIF2a (p-eIF2 a). Increases in phospho-p38 (pp38 blot) and phospho-JNK (pp54/JNK, pp46JNK blot) levels are also observed at the same concentration of BFA which fosters increased UPR signaling and diminished c-secretase dependent APPGV16 cleavage. doi:10.1371/journal.pone.0009135.g004 markers. As markers for ER stress induction, we examined PKR- like ER kinase (PERK) and eukaryotic translation initiation factor 2a (eIF2a) phosphorylation—two critical components of UPR signaling. As ER stress culminates in the activation of the pro- apoptotic kinases JNK and p38 [34], we included phospho-JNK and phospho-p38 in our examination. PLoS ONE | www.plosone.org 5 February 2010 | Volume 5 | Issue 2 | e9135