Fig. 2.

Cord blood serum (CBS) directly mediates α-cleavage of neuron-specific amyloid precursor protein (APP₆₉₅), but this activity is not mediated by a disintegrin and metalloproteinase domain-containing protein (ADAM) and tumor necrosis factor-α converting enzyme (TACE). (A) Human recombinant full-length APP₆₉₅ tagged with C-terminal MYC/DDK (fAPP₆₉₅, OriGen, 100 ng) was incubated with 5% CBS, inact CBS, or aged blood serum (AgBS) at 37 °C for 5 h in the presence or absence of a protease inhibitor cocktail (PI, 1 X), TACE (ADAM17) inhibitor (TAPI-0, 1 µM), or ADAM inhibitor (GM6001, 1 µM). Lanes 1 to 4 represent CBS (1), inact CBS (2), AgBS (3), and fetal bovine serum (FBS; 4) sample only control without substrate fAPP₆₉₅. Lane 5 represents fAPP₆₉₅ (5) substrate only control without any serum sample. Lanes 6 to 11 represent substrate fAPP₆₉₅ with CBS (6), inact CBS (7), CBS with PI (8), CBS with TAPI-0 (9), CBS with GM6001 (10), and AgBS (11). Lanes 12 to 14 represent (lanes 12 to 14; PI (12), TAPI-0 (13), and GM6001 (14) inhibitor and substrate control, respectively, without any serum sample. The reaction mixtures were subjected to soluble amyloid precursor protein α (sAPPα) Western blot (WB) analysis using 2B3 antibody (top panel) and total APP using 6E10 (an anti-Aβ_1-17 antibody; lower panel). sAPP₇₇₀ refers to the endogenous α-secretase cleavage product of CBS or AgBS, whereas sAPP₆₉₅ refers to the α-secretase cleavage product of fAPP₆₉₅. (B) 100 ng of fAPP₆₉₅ was incubated with 5% CBS for 1, 5, or 24 h, or 5% FBS or inact. FBS for 24 h, and then subjected to sAPPα and total APP WB analysis using 2B3 (top panel) and 6E10 (lower panel), respectively. sAPP₇₇₀ refers to the endogenous α-secretase cleavage product of CBS or AgBS, whereas sAPP₆₉₅ refers to the α-secretase cleavage product of fAPP₆₉₅.