Exogenous and endogenous NO donors induce S-nitrosylation of BACE1 in cultured PRCN neurons. (A) Nitrosylation of BACE1 by SNOC was detected with biotin-switch assays: After exposure to SNOC (100 μM) for 30 min, to detect S-nitrosylated cystein residues, the free cysteine residues of BACE1 were first masked by methylthiolation with MMTS. Nitrosothiols were then selectively reduced by ascorbate to reform free thiol groups, which reacted with biotin-HPDH. Subsequently, biotin-HPDH conjugated proteins can be precipitated by streptavidin beads. In this experiment, MMTS was not added, to serve as a positive control, since all the cysteine residues in BACE1 can react with biotin-HPDP (last lane). Samples not treated with ascorbate (third lane) were used as a negative control due to the lack of reactive cysteine residues to biotin-HPDH. (B) Effect of Ca2++ ionophore, ionomycin (1 μM), on SNO-BACE1 was examined at various time points (0, 5, 10, 15, and 30 min) by biotin-switch assay.