Supplementary information
Supplementary Figure 1. OCIAD2 overexpression does not affect -and -secretase.
(A) OCIAD2 overexpression does not affect -secretase activity in SH-SY5Y cells. SH-SY5Y cells were
transfected with pCtrl or pOCIAD2 for 24 h and cell extracts were subjected to -secretase specific fluoregenic
substrate assay. Bars represent mean value ± SD (n = 3). (B) HEK293T cells were transfected with pCtrl or
pOCIAD2-HA for 48 h and cell extracts were analyzed by Western blotting. (C) OCIAD2 overexpression does
not affect α-secretase activity in HEK-APP695 cells. HEK-APP695 cells were transfected with pCtrl or pOCIAD2HA for 48 h and then incubated in serum-free DMEM for another 6 h. The harvested media was concentrated
and separated by SDS-PAGE for Western blotting and the signals on the blot were quantified by densitometry
analysis Bars represent mean value ± SD (n = 3). (D) OCIAD2 overexpression does not affect protein level of
-secretase (ADAM10 or TACE). After transfection of HEK293T cells with either pCtrl or pOCIAD2-HA for
24 h, cell lysates solubilized by RIPA buffer were analyzed by Western blotting.
Supplementary Figure 2. Expression analysis of OCIAD2 and its effect on the stabilization of NCT.
(A) Effect of OCIAD2 overexpression on PEN2 and APH-1. After transfection of HEK293T cells with pCtrl or
pOCIAD2 for 48 h, cell lysates solubilized in 1% CHAPS lysis buffer were analyzed by Western blotting. (B)
Expression analysis of OCIAD2 in cell lines. RIPA-soluble cell lysates were prepared from 5 different cell lines
and analyzed by Western blotting. (C) The level of NCT protein is reduced by OCIAD2 knockdown. After
transfection of HeLa cells with pshCtrl or pshOCIAD2 for 72 h, cell lysates were analyzed by Western blotting.
(D) Effects of OCIAD2 on mRNA levels of the NCT. After transfection of HEK293T cells with pCtrl,
pOCIAD1-HA or pOCIAD2-HA for 24 h, total RNA was purified and analyzed by RT-PCR using synthetic
primers for NCT, OCIAD1, OCIAD2 and -ACTIN. (E) OCIAD2 knockdown reduces the stability of NCT
protein. After co-transfection of HEK-APP695 cells with pNCT-V5 and either pshCtrl or pshOCIAD2 for 57 h,
cells were treated with 30 μM cycloheximide (CHX) for the indicated times. Cell lysates solubilized in RIPA
buffer were analyzed by Western blotting. (F) Effect of OCIAD2 knockdown on SGK1. HEK-APP695 cells were
transfected with pshCtrl or pshOCIAD2 for 72 h and cell extracts were separated by SDS-PAGE and analyzed
by Western blotting. (G) Lack of ERK1/2 activation in OCIAD2 knockdown cells. After transfection of HEKAPP695 cells with pshCtrl or pshOCIAD2 for 72 h, cell lysates were analyzed by Western blotting.
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Supplementary Figure 3. Interaction of OCIAD2 with -secretase components.
(A, B) Interaction of OCIAD2 with -secretase components. After transfection of HEK293T cells with
pOCIAD2 and pHA-PEN2, or pOCIAD2-HA and either pAPH1-FLAG or pPS1 for 36 h, cell extracts were
analyzed by IP assays using anti-HA antibody. *N.S, nonspecific signal (A). Cell lysates of HEK-APP695 cells
were analyzed by IP assay using anti-OCIAD2 antibody. The immunoprecipitates were proved by Western
blotting (B).
Supplementary Figure 4. No stimulatory effects of OCIAD2 on NICD generation.
OCIAD2 overexpression does not affect the cleavage of Notch to generate NICD. This is control blots of Figure
4A. After boiling, Aph1-FLAG and PS1 were detected with higher molecular weight (> 130 kDa).
Supplementary Figure 5. Interaction of OCIAD2 with NCT via C-terminal region.
(A) Schematic diagram of OCIAD2 C-terminal deletion mutants. (B) OCIAD2 N142 mutant interacts with NCT.
HEK293T cells were co-transfected with pNCT-V5 and pOCIAD2 deletion mutant for 36 h. Cell extracts were
then analyzed by IP assays using anti-HA antibody. The immunoprecipitates and WCL were proved by Western
blotting. (C) Limited digestion of OCIAD2 by protease. Heavy membrane fraction was isolated from HEKAPP695 cells by centrifugation and incubated with the increasing concentrations of proteinase K for 7 min. The
reaction samples were subjected to Western blot analysis. (D) OCIAD2 interacts with NCT via cytoplasmic
region. HEK293T cells were co-transfected with pNCT-V5, pOCIAD2-HA and either pEGFP or pEGFPNCTC19 for 30 h. Cell extracts were then analyzed by IP assays using anti-HA antibody and Western blotting.
(E, F) Ectopic expression of NCTC19 decreased AICD generation. HEK-APP695 cells were transfected with
pEGFP or pEGFP-NCTC19 for 24 h and cell extracts were subjected to Western blotting (E). Crude membrane
fraction from the harvested sample (E) was prepared by centrifugation.. The membrane fraction was
resuspended and incubated at 37°C for 2 h with or without 100 M Comp. E and separated by SDS-PAGE for
Western blotting (F).
Supplementary Figure 6. OCIAD2 is found in the mitochondria and the MAM.
(A) Immunocytochemical analysis showing subcellular localization of OCIAD2 in the mitochondria and MAM.
HeLa cells were co-transfected with pOCIAD2-GFP/-RFP and pER-RFP, pMito-RFP, pRab5-GFP or pLamp12
GFP for 36 h and fluorescence signals were observed under confocal fluorescence microscope (upper 4 panels).
HeLa cells were transfected with pOCIAD2-RFP for 36 h and then subjected to immunocytochemical analysis
using anti-FACL4 antibody (lower panel). (B) Schematic diagram of ER-/Mito (Mitochondria)-targeting
OCIAD2 construct. (C, D) ER-targeted OCIAD2 activates -secretase activity to increase AICD production.
HEK293T cells were transfected with pCtrl, pER-OCIAD2-HA or pMito-OCIAD2-HA for 48 h and 1%
CHAPS-soluble cell lysates were subjected to SDS-PAGE and analyzed by Western blotting (C). HEK-APP695
cells were transfected with pCtrl, pMTS-OCIAD2-HA or pER-OCIAD2-HA for 48 h and cell lysates were
subjected to Western blotting (D).
Supplementary Figure 7. Induction of OCIAD2 expression by oxidative stress.
Induction of OCIAD2 expression by H2O2. SH-SY5Y cells were treated with 200 M H2O2 for the indicated
times and cell extracts were analyzed by Western blotting.
Supplementary table 1. AD patient brain information.
*PMI : Post-mortem interval (h)
** - : None, + : Mild, ++ : Moderate, +++ : Abundent/Severe
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