Presenilin 1

HeLa cells were transfected with 25 or 50 nM pre-miRs (Applied Biosystems, USA), 2.5 ng/cm²; pRL control vector, and 50 ng/cm²; pGL3_HSV TK_3′UTR hNCSTN WT or C460T (rs1043329), T623G (rs113810300) or delCA515–516 (rs141849450) mutant variant plasmids. Twenty-four hours post-transfection, cells were lyzed, and luciferase activity was measured according to the manufacturer’s instructions (Promega, USA). For western blots, cells were lyzed in RIPA buffer [50 mM Tris HCl, 1% NP40, 0.9% NaCl, 0.25% Na-deoxycholate, 1 mM EDTA, 1× proteinase inhibitors (Roche, Basel, Switzerland), 1 mM PMSF, 1 mM Na₃VO₄, and 1 mM NaF], mixed with LDS sample buffer (Invitrogen, Carlsbad, CA, USA) containing 5% β-mercapto-ethanol and boiled at 95°C for 8 min. Crude extracts (10 μg) were immunoblotted with the Aph-1a [clone B80.2, kind gift from B. De Strooper (Nyabi et al., 2003 (link))], Pen2 (Cell Signaling, clone D2G6), Nicastrin (Cell Signaling, clone D38F9), Presenilin 1 (Millipore, clone PS1-loop), and Gapdh (Millipore, clone 6c5) antibodies. Membranes were detected using the ECL detection kit (Millipore, Billerica, MA, USA). Quantifications were performed using the Multi Gauge software (FUJIFILM, Minato-ku, Tokyo, Japan).