Cell-free γ-secretase assay for ephrin-B1 cleavage. A) Cell-assay using membranes from mock- or ephrin-B1-transfected COS cells. Samples incubated at 4 degree (4), 37 degree (37) or 37 degree with DAPT (D) were analyzed by immunoblotting by anti-ephrin-B antibody (Upper panel). Same fractions without incubation were indicated as "C". ephrin-B1 FL, ephrin-B-CTF and de novo generated ICD were shown by black arrow, arrowhead and asterisk, respectively. Endogenous APP (Lower panel) C-terminal stubs (white arrowhead) were cleaved by γ-secretase to generate AICD (double asterisks). B) Cell-free assay of membranes from wild-type MEF (MEFwt) and MEF lacking Psen1 and Psen2 (DKO). ephrin-B-CTF and de novo generated ICD were indicated by arrowhead and asterisk.