Psen1

Primary antibodies for EGFR (rabbit, Cell Signaling), NOTCH1 (rabbit, Cell Signaling), AKT (rabbit, Cell Signaling), PAX6 (rabbit, COVANCE), PTEN (A2B1, mouse, Santa Cruz), NES (mouse, EMD), Gamma Secretase (Ab kit for NCSTN), PSEN1 (rabbit, Cell Signaling), SPARC (mouse, Santa Cruz), AGAP2 (rabbit, Abnova), ANGTP1 (rabbit, Abnova), ANGTP2 (mouse, Abnova), CHIL31 (rabbit, Abnova), and EFEMP1 (rabbit, Abnova) were diluted 1:500 or 1000, and for GFAP (C-19, rabbit, Dako) and ACTB (IgM-specific mouse, Millipore) were diluted 1:10,000 for immunoblotting as described previously [11 (link)].
Gelatin zymography and Matrigel invasion assays were performed following procedures described previously [11 (link)]. For the invasion assay in this study, 5×10⁵ cells in 500 μl DMEM/F12 were loaded in 2-3 replications on Matrigel (1 μg/ml)-coated trans-well in 12-well plates (8μm; Fisher Scientific), and 1 ml DMEM/F12 was added to the bottom chamber. For 51B, addition of a small amount of bovine serum (final 0.05%) in the medium of the bottom chamber was applied to enhance migration of cells in penetration through the transwell-filter after degrading the Matrigel, but not to interfere cell-mediated matrix degradation by proteases within serum. Images of invasion were taken 48 hours later and cells were counted.

Cells were analyzed by western blotting using 4–12% SDS gradient gels (NP0322PK2; Invitrogen) and the following antibodies were used: APP (Abcam, ab32136, Cambridge, UK), BACE1 (Abcam, ab183612), PSEN1 (Cell Signaling Technology, D39D1, Danvers, MA, USA), cleaved Notch1 (Cell Signaling Technology, 4147), Aβ1-42 (Abcam, ab201060), and anti-β-actin (Abcam, ab8227). Electrophoreses were performed using 15% Tris-Glycine SDS gels for Aβ and 12% Tris-Glycine SDS gradient gels for PSEN1 in 12-well gel, and the resulting bands were transferred by western blotting. The protein concentration of the loading sample was 30 μg. Immunoreactivity was analyzed by chemiluminescence (GE Healthcare, Piscataway, NJ, USA). The protein marker ranges from 10–180 kDa were used (Tris-glycine-SDS running buffer) (GeneDireX, PM006-0500, Taoyuan City, Taiwan). The transfer buffer was 25% Methanol and 10% TG-SDS buffer (Avantor, 0783-5L, Radnor, PA, USA). The setup time and voltage are 90 min and 400 mA by gel transfer to PVDF member. The chemiluminescence signal was observed using a digital image analyzer (Fuji Film Inc, LAS-3000, Tokyo, Japan).

The proteins in whole brain lysates was quantified using the BCA protein assay kit (Thermo Scientific) and separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane in transfer buffer^{19 (link)}. The membrane was incubated for 1 h in blocking solution (6% skim milk) at room temperature, and incubated overnight with primary antibodies such as NeuN (Millipore, Billerica, MA, USA), synaptophysin (Abcam, Cambridge, UK), Iba1 (Novus, Littelton, CO, USA), APP (22C11, Milipore), PSEN1 (Cell signaling, Danver, MA, USA), Tau (H-150, Santa Cruz, Dallas, TX, USA), or GAPDH (Bioworld Technology, Inc., St. Louis Park, MN, USA) (Table 1)^{19 (link)}. After being washed in Tris-buffered saline with Tween 20 (TBS-T) three times and incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h, the membrane was visualized using an Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500). The immunoblots were imaged using blue detection medical X-ray film (Agfa, Mortsel, Belgium) and densitometric analyses were performed by using ImageJ software (v1.4.3.67, NIH).