Fig. 3.

OCIAD2 interacts with NCT through its C-terminal region. a Exogenous OCIAD2 interacts with NCT. After transfection of HEK293T cells with pNCT-V5 and pOCIAD2-HA for 36 h, cell extracts were analyzed by IP assays using anti-NCT (left) or anti-HA (right) antibody followed by western blotting. H.C heavy chain of immunoglobulin. b Co-localization of OCIAD2 with γ-secretase complex in microsomal membrane fraction. After transfection of HEK293T cells with pOCIAD2-HA for 24 h, microsomal membrane fraction prepared by centrifugation and solubilized in 1 % CHAPSO was subjected to glycerol velocity gradient fractionation assay. Each fraction (top, fraction 1; bottom, fraction 12) was analyzed by western blotting. c Endogenous OCIAD2 interacts with NCT. Cell lysates of HEK-APP₆₉₅ cells were analyzed by IP assays using either anti-OCIAD2 (left) or anti-NCT (right) antibody. d Schematic diagram of OCIAD2 and its deletion mutants. e The C-terminal region of OCIAD2 is required for its interaction with NCT. HEK293T cells were transfected with pOCIAD2 deletion mutant for 48 h and cell extracts were then analyzed by IP assay using anti-HA antibody. The immunoprecipitates and whole cell lysates were proved by western blotting. f Effects of OCIAD2 mutants on AICD generation. HEK-APP₆₉₅ cells were transfected with pOCIAD2 or pOCIAD2 deletion mutant for 48 h and cell lysates were analyzed by western blotting. g Effects of OCIAD2 mutants on the amount of γ-secretase components. HEK293T cells were transfected with pOCIAD2 or pOCIAD2 deletion mutant for 48 h and cell lysates were analyzed by western blotting. h Effects of OCIAD2 mutants on γ-secretase reporter activity. HEK293T cells were transfected with pC99-GVP, pUAS-Luciferase, pβ-galactosidase, and either pOCIAD2 or pOCIAD2 deletion mutant with or without 3 μM Comp. E for 24 h. Cell extracts were subjected to luciferase assay. The luciferase activity was normalized by β-galactosidase activity. Bars mean ± SD (n = 3). *P < 0.05, ***P < 0.001